1

Tissue Preparation
 INTRODUCTION
o Artifacts
 FIXATION
 EMBEDDING
 STAINING
 SECTIONING

INTRODUCTION

Most fresh tissue specimens are colorless and squishy. They provide little useful information.
For scientific or diagnostic purposes, tissue specimens must undergo substantial alteration in
preparation for viewing under a microscope.

There are four steps in tissue preparation.

 Fixation stabilizes and preserves the tissue.

 Embedding converts the tissue into a solid form which can be sliced
("sectioned").
 Sectioning (slicing) provides the very thin specimens needed for microscopy.
 Staining provides visual contrast and may help identify specific tissue
components.

Most basic histology texts offer a minimal account of basic histological technique. For routine
examination of tissues, you probably don't need to know much more.

Artifacts. Be aware that each step of tissue preparation introduces artifacts by altering or
distorting the natural appearance of cells.

Some artifacts are unavoidable. Fixation, by its very nature, kills cells and stabilizes dynamic
cell processes. Enzyme activity is usually altered. Ions and small molecules are usually washed
away.

Some artifacts are intentional, most notably the colors added by staining. The pink and purple
colors of H&E staining can become so familiar that they appear "normal", as if they were the
natural colors of tissues.
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Still other artifacts are accidental. Cells may shrink or swell during fixation. Extracellular
spaces may be distorted by compression or stretching. Ripples and wrinkles can be introduced
during cutting and handling of sections.

Unintended artifacts can be minimized by optimal procedures -- but optimal procedures are often
impractical, especially with human specimens. Ideal tissue preparation preserves cells in a form
that resembles the living state, but this ideal is seldom practical with clinical specimens. Often,
especially in post mortem (autopsy) material, cells have been dead and deteriorating for several
hours before fixation. Therefore, certain artifacts must be appreciated as part of the normal
appearance of tissue specimens.

The process of sectioning can introduce still other artifacts.

The most common mode of routine tissue preparation involves fixation with buffered
formaldehyde, embedding in paraffin, sectioning into slices about 5 micrometers in thickness,
and staining with hematoxylin and eosin.

Modern cell biology uses many tools to reveal cell structures and functions that are not apparent
on routine H&E slides. Many of these involve sophisticated reagents based on the specificity of
enzymes, immunological antibodies, or gene sequences to label and localize specific proteins or
other molecules. Some textbooks present additional detail.

FIXATION
Fresh tissue samples must be preserved for future examination. This process is called fixation,
and the resulting specimen is described as fixed.

Boiling an egg and pickling a cucumber represent examples of fixation, in which heat or
chemistry stabilizes the organic materials.

A variety of chemicals can be used for fixing histological specimens. Routine fixation often uses
a solution of formaldelhyde (formalin) to react with proteins and other organic molecules to
stabilize cell structures. This solution is buffered and osmotically balanced to minimize
shrinkage, swelling, and other collateral damage.

Ideally, fixation should be accomplished extremely quickly to minimize post-mortem changes in

cell structure. Since fixation rate is limited by diffusion, ideal tissue preservation requires that
fixative be delivered as closely as possible to each cell. Rapid delivery of fixative can be
accomplished either by perfusion or by immersion.

Perfusion involves the delivery of fixative through the circulatory system of living tissue, by
direct injection into a major artery. Such a procedure is commonly used with experimental
animals but is obviously impractical for obtaining clinical specimens from patients.
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Successful fixation by immersion requires very small samples. However, surgical removal of
very small tissue samples often entails incidental mechanical damage, especially with punch
biopsies.

These constraints on ideal fixation mean that tissue quality may vary across a specimen, with
possible distortion near edges (especially with needle or punch biopsies) and with variation in
fixation quality (and attendant staining character) in deeper areas (into which fixative diffuses
more slowly).

An alternative to chemical fixation is freezing, followed by direct sectioning of the frozen

specimen.

Frozen sections are seldom as "pretty" as well-fixed specimens, but they do have certain
advantages. Because frozen sections do not require hours for the normal schedule of fixation and
embedding, they can provide immediate diagnostic information to a surgeon in the operating
room. Frozen sections can also permit analysis of small diffusable molecules or of enzyme
activity whose presence would be lost during chemical fixation.

EMBEDDING and SECTIONING

After fixation, tissue specimens are routinely embedded in a solid material which will support
very thin sectioning.

To embed a tissue sample, tissue water is replaced first by solvents (such as alcohol and xylene)
and then with a liquid such as melted wax (paraffin) or epoxy solution which can be
subsequently solidified by cooling or polymerization.

Sectioning is the production of very thin slices from a tissue sample. The tool used for
sectioning is called a microtome (tom = to cut, as in appendectomy). A microtome may be as
simple as razor blade, or it may be a complex machine costing several tens of thousands of
dollars (for producing the ultrathin sections needed for electron microscopy).

 Most laboratory microtomes have the essential machinery of a

baloney-slicer:
o a cutting edge (which may be a razor, a heavy knife, a
piece of broken glass, or a finely sharpened diamond),
o a specimen holder,
o a screw to advance the specimen toward the blade (ultramicrotomes may
use carefully-controlled thermal expansion in lieu of a screw),
o a crank, such that each turn of the crank raises the specimen, advances it,
and then lowers it across the blade.
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Sections for routine light microscopy are typically 5-10µm (micrometers, microns) in thickness.
Exceptionally thin sections may less than 2µm thick. For electron microscopy, sections are
typically 50-100 nanometers (millimicrons) in thickness.

Sectioning necessarily reduces the specimen to a two-dimensional representation.

Reconstructing the three-dimensional structure of the original sample requires either the
"stacking" of multiple images from serial sections, or else judicious use of imagination (3-D
visualization). A very small amount of three-dimensional information may be directly visualized
under the microscope, by focussing up and down through the thickness of the specimen.

For a further account of 3-D visualization, see here.

Sectioning can certain introduce artifacts.

Among the commonest artifacts, and most distracting for a beginner, are wrinkles. To
appreciate why wrinkles form, imagine trying to lay a sheet of wet tissue paper (representing the
slice from the sample) flat onto a table (representing the microscope slide). Even with great care,
wrinkles sometimes appear. Sometimes wrinkles are "forced" when the tissue section stretches
unevenly around structures of differing consistencies.

Another sectioning-related artifact is the disappearance of small structures which fall out of their
proper place on the specimen, and the occasional reappearance of such structures at other
inappropriate locations. This happens most often when the process of slicing separates a part
which is attached only outside the plane of section, such as a hair shaft within a hair follicle.
Except in the case of perfect lengthwise slices, the hair shaft will be cut into an oval slice that is
not attached to the sides of the hair follicle and may therefore come out (leaving the follicle
apparently empty) and then alight somewhere else (as an odd oval structure anywhere on the
slide).

Yet other common artifacts are scratches and "chatter". Scratches are caused by flaws or dirt
on the cutting edge, and appear as straight slashes or ragged tears across the specimen. "Chatter"
is the visible record of knife vibration. The the process of slicing sometimes induces vibrations
in the knife edge, which then cause variations in thickness (ripples) in the section. These appear
as narrow parallel bands, usually evenly spaced, across a tissue specimen. They are often most
evident in areas of smooth texture, such as the colloid in thyroid follicles.

STAINING

Most cells are essentially transparent, with little or no intrinsic pigment.

Even red blood cells, packed with hemoglobin, appear nearly colorless when
unstained, unless packed into thick masses. Stains are used to confer contrast,
to make tissue components visibly conspicuous. Certain special stains, which
bind selectively to particular components, may also be used to identify those
structures. But the essential function for staining is simply to make structures easier to see.
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NOTE that all stain color is artifactual and does not represent the natural color of the tissue.
The same structures may have very different colors with different stains. For example, collagen
is pink with H&E but blue or green with trichrome. You should generally use specific aspects of
actual structure (location, size, shape, texture) to identify cells and tissues, rather than color.
Color can offer additional information if used wisely, but is unreliable by itself.

H&E stain

Routine histology uses the stain combination of hematoxylin and eosin,

commonly referred to as H&E.

Hematoxylin is a basic stain with deep purple or blue color. Structures that are stained by basic
stains are described as basophilic ("base-loving"). Chromatin (i.e., cell nuclei) and ribosomes
are basophilic. With H&E staining, basophilic structures are stained purple.

Eosin is an acidic stain with a red color. Structures stained by acid stains are described as
acidophilic (or eosinophilic) and include collagen fibers, red blood cells, muscle filaments,
mitochondria. With H&E staining, acidophilic structures are stained red or pink.

 Note that, basophilic cell structures are NOT necessarily acidic; they only
happen to stain with basic stains. Likewise for acidophilic structures,
which are NOT necessarily basic. Many tissue staining properties are
determined by the complex chemistry of proteins and other
macromolecules after interactions with fixatives and other processing
agents, and defy simple analysis.
 Also note that absolute color intensity on H&E-stained slides can be quite
variable, with the same cell structure appearing red on one slide, pink on
another, and possibly even blue on yet another. Relative stain intensity on
the same slide is a more reliable indicator of acidophilic/basophilic quality
than is absolute color, but this can also vary, especially between edges and
center of a section.

Remember that nuclei are not really purple and collagen is not really pink. All such stain colors
are artifacts, albeit intentional ones.

If an H&E slide shows any colors other than purple/blue and red/pink -- such as yellow or brown
-- the additional color is probably due to an intrinsic pigment such as melanin.

Some cell structures do not stain well with aqueous dyes and so routinely appear clear. This is
especially so for those which are hydrophobic, containing fat. Included in this category are
adipocytes, myelin around axons, and cell membranes of the Golgi apparatus.
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Trichrome stain

Trichrome uses three dyes (hence the name), including one that is
specific for the extracellular protein collagen. Depending on the
particular stain combination, a trichrome stain may color collagen
fibers sky-blue or bright green. The principle use for trichrome is to
differentiate collagen from other eosinophilic structures, such as muscle fibers.

Trichrome stains can be especially useful for highlighting an accumulation of scar tissue, as in
glomerulosclerosis of the kidney (see WebPath) or cirrhosis of the liver.

PAS (Periodic acid Schiff) is used for

glycogen, glycoproteins (such as mucus), and
basement membranes (which contain
glycoprotein).

Other stains. Be aware that many other stain techniques exist, for special cases. Some of these
are classical procedures can yield beautiful results but depend on mysterious art and alchemy for
success. Other, more-modern techniques have been rationally designed to exploit recent
developments in molecular biology.

In the "classic" category are a number of stains based on metal salts.

A silver-based stain that demonstrates reticular fibers and basement membranes

is especially useful for diagnosing certain pathologies of kidney glomeruli.

A variety of silver stains have been very powerful for research into the central
nervous tissue. Their only common feature is that silver grains form a dark precipitate on
selected structures, with empirical variables determining which structures are visualized.

Some cells have traditional names based on their demonstration with certain stains, such as the
"argentaffin cells" (cells with an affinity for silver) and "chromaffin cells" (cells with an affinity
for chromium) of the gastrointestinal tract.

In the "modern" category are stains based on the application of particular molecules that can be
selectively stained using radioactive labels, enzyme reactions or specific antigen binding. The
techniques of autoradiography, enzyme histochemistry and immunocytochemistry often
require sections of frozen rather than fixed tissue.
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Classification of Tissue Types

This page is part of the section about the structure and function of different Tissue Types and
indicates how the tissues mentioned in this section may be described in relation to each other,
e.g. adipose tissue, areolar tissue, blood tissue, bone tissue etc. are all different types of
"connective tissue".
To read about the individual tissue types, see the links listed on the left.

There are Four (4) Basic Types of Animal Tissue:

T Epithelial Connective Muscular Nervous

y Tissue Tissue Tissue Tissue
p
e

o
f
T
i
s
s
u
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:

F Covers body Binds and Enables Enables

u surfaces and Supports body movement of responses to
n lines body parts structures within stimuli and
c cavities the body and coordinates
t movement of bodily functions
i the entire
o person/animal
n
s

o
f
t
y
p
e
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o
f
t
i
s
s
u
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:

Each of the tissue-types listed in the panel on the left falls into one of the four categories
above.
However, the four "Basic Types" of animal tissues can be sub-divided further as each includes
several different sub-types of the tissue, each being specialised to meet specific needs and/or
perform particular tasks.

1. Epithelial Tissue

Epithelial tissue exists in many forms and can be classified or sub-divided in different ways.

Types of Epithelial Tissue:

Covering and Lining Epithelial Types of Epithelial Tissue (in this classification)
Tissue
Classification by Cell Shape:
 Squamous
 Cuboidal
 Columnar
 Transitional
Classification by Arrangement of Layers:
 Simple Epithelium  Simple squamous epithelium,
 Simple cuboidal epithelium,
 Nonciliated simple columnar epithelium,
 Ciliated simple columnar epithelium.
 Stratified Epithelium  Stratified squamous epithelium,
 Stratified cuboidal epithelium
 Stratified columnar epithelium,
 Transitional epithelium.
 Pseudostratified  Pseudostratified columnar epithelium.
columnar Epithelium
Glandular Epithelial Tissue
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 Endocrine Glands  (Tissue of) Endocrine Glands

 Exocrine Glands  (Tissue of) Exocrine Glands

Each of the sub-divisions of epithelial tissue identified above can be described in terms of its
structure (using both text and diagrams), location, and function within the body.

2. Connective Tissue

Connective tissues serve the general purpose of supporting and connecting the tissues of the
body, and vary considerably in structure and composition. Teaching materials (incl. textbooks
and websites) sub-divide this tissue category in various different ways - hence it is useful to be
aware of variations and overlap in classifications and terminology.

Types of Connective Tissue:

Embryonic Connective Tissue

 Mesenchyme
 Mucous connective tissue
Mature Connective Tissue
Loose Connective Tissue:
 Areolar Tissue
 Adipose Tissue
 Reticular Tissue
Dense Connective Tissue:
 Dense Regular Connective Tissue
(White Fibrous Tissue)
 Dense Irregular Connective Tissue
 Elastic Connective Tissue
(Yellow Elastic Tissue)
Cartilage Tissue:
 Hyaline Cartilage
 Fibrocartilage
 Elastic Cartilage
Bone (Osseous) Tissue:
 Compact Bone
 Spongy Bone
Blood Tissue:
 Erythrocytes
 Thrombocytes
 Leucocytes
Lymphatic Tissue:
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 Lymph

3. Muscular Tissue
There are three (3) types of muscular tissue:

Skeletal located throughout the body and under conscious (i.e. "voluntary") control, main
Muscle function movement of the structures of the body, and the body as a whole, e.g.
(Tissue): by walking, running, etc..
Cardiac which is found only in the heart and is important for effective blood-flow through
Muscle the heart.
(Tissue):
Smooth involuntary muscle tissue located around the walls of many internal structures
Muscle such as the stomach and intestines and important for aiding the passage of
(Tissue): materials/fluids through those structures.

4. Nervous Tissue

Nervous tissue consists of two (2) main types of cells:

Nerve Cells (also known as Neurons or Neurones) -

whose purpose is to transmit (electrical) nerve impulses that move information
around the body.
Neuroglia (also known as simply Glia) -
which support and protect nerve cells, depending on the particular type of glia.
Examples of types of glia include astrocytes, ependymal cells, microglial cells,
oligodendrocytes and Schwann cells.

Further information about each of the basic types of animal tissue is included on the pages
indicated.

Junctions
Adhering Junctions
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Epithelial cells are held together by strong anchoring (adherens) junctions.

There are two types of adherens junctions:

zonula adherens - which contain actin filaments

macula adherens (desmosomes) which contain intermediate filaments.

The zonula adherens junction lies below the tight junction (occluding junction). In the gap
between the two cells, there is a protein called E-cadherin - a cell membrane glycoprotein. The
cadherins from adjacent cells interact to 'zipper' up the two cells together.

Inside the cell, E-cadherin binds to catenin, which in turn binds to other proteins (vinculin, alpha
actinin) in a protein complex with actin filaments (microfilaments, shown here in red).

The actin filaments tend to be arranged circumferentially around the cell, into what is called a
'marginal' band. This marginal band can contract, and deform the shape of cells held together in
this way - this is thought to be key in the morphogenesis of epithelial cells, in forming ducts for
example.

Tight junctions
These are regions in which two cells are very tightly connected together, and they will prevent
some molecules from passing across an epithelium. The borders of two cells are fused together,
often around the whole perimeter of each cell, forming a continuous belt like junction known as
a tight junction or zonula occludens (zonula = latin for belt). Transmembrane proteins from each
cell membrane interlock across the intercellular space, all around the cell, in this belt (black lines
in the diagram).
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The permeability of tight junctions varies from site to site, and are often can be selectively leaky.
For example, these junctions are important in the gut, in acting as a selective diffusion barrier,
preventing diffusion of water soluble molecules. They also act to restrict the localisation of
membrane bound proteins. (For more, see the section on the gut)

from Molecular Biology of the Cell

Desmosomes and Hemidesmosomes

Desmosomes connect two cells together. A desmosome is also known as a spot desmosome or
macula adherens (macula = latin for spot), because it is circular or spot like in outline, and not
belt- or band shaped like adherens junctions.

Desmosomes are particularly common in epithelia that need to withstand abrasion (see skin).
Desmosomes are also found in cardiac cells, but the intermediate filament in this case is desmin,
not keratin (which is found in epithelial cells).

The picture shows an EM of a desmosome formed between two cells.Notice the phase dense
material between the two cell membranes, which is mad up of transmembrane linker
glycoproteins (e.g. demosgleins and desmocollins - which are cadherin proteins). Also notice the
intermediate filaments running from the desmosome into the cytoplasm.

Other proteins run across the membrane into the intracellular space, to connect the two cells
together. These 'transmembrane linker' proteins are called desmoglein and desmocollin, which
are types of cadherin.
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This diagram shows a desmosome. It is made up of a dense cytoplasmic plaque, to which the
intermediate filaments attach.

Hemidesmosomes
These look similar to desmosomes, but are different functionally, and in their content. The
connect the basal surface of epithelial cells via intermediate filaments to the underlying basal
lamina. The transmembrane proteins of hemidesmosomes are not cadherins, but another type of
protein called integrin.

This electron micrograph shows a Hemidesmosome (H), and two of the three layers of the
underlying basal lamina. LL - lamina densa, LD - lamina lucida. Integrins in the plasma
membrane link the cell to the extra-cellular matrix.

from Wheater's Functional Histology.

Gap Junctions.
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Gap junctions are the most widespread of all cell junctions in animal tissues. Gap junctions thus
couple cells electrically and metabolocially, enabling cells to communicate with each other
directly. Gap junctions can open and close in response to changes in calcium levels, and pH. Gap
junctions form in a narrow gap of 2-4nm, between two adjacent cells.

The picture shows an EM (A) of two gap junctions between two cells, and a freeze fracture EM
(B) of the particles on the cytoplasmic face of the plasma membrane.

A group of protein molecules called connexins form a structure called a connexon (each particle
in B above is a connexon). When connexons (blue in the diagram to the right) from two adjacent
cells (red and yellow in the diagram) align, they form a continuous channel between them.

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Epithelia and Glands

EPITHELIA

Epithelia are tissues consisting of closely apposed cells without intervening intercellular
substances. Epithelia are avascular, but all epithelia "grow" on an underlying layer of vascular
connective tissue. The connective tissue and the epithelium are separated by a basement
membrane. Epithelium covers all free surfaces of the body. Epithelium also lines the large
internal body cavities, where it is termed mesothelium. Furthermore, the internal surfaces of
blood and lymph vessels are lined by epithelium, here called endothelium.
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Epithelia are classified on the basis of the number of cell layers and the shape of the cells in the
surface layer.

 If there is only one layer of cells in the epithelium, it is designated simple.

 If there are two or more layers of cells, it is termed stratified.
 Cells in the surface layer are, as a rule, described according to their height as squamous
(scale- or plate-like), cuboidal or columnar.

Simple Epithelia

Simple squamous epithelium

This type is composed of a single layer of flattened, scale- or plate-like cells. It is quite
common in the body. The large body cavities and heart, blood vessels and lymph vessels
are typically lined by a simple squamous epithelium. The nuclei of the epithelial cells are
often flattened or ovoid, i.e. egg-shaped, and they are located close to the centre of the
cells.

Simple cuboidal epithelium

Cells appear cuboidal in sections perpendicular to the surface of the epithelium. Viewed
from the surface of the epithelium they look rather like small polygons. Simple cuboidal
epithelium occurs in small excretory ducts of many glands, the follicles of the thyroid
gland, the tubules of the kidney and on the surface of the ovaries.
Can there be "low cuboidal" epithelia?

Simple columnar epithelium

The cells forming a simple columnar epithelium are taller than they are wide. The nuclei
of cells within the epithelium are usually located at the same height within the cells -
often close to the base of the cells. An example is the simple columnar epithelium which
lines the internal surface of the gastrointestinal tract (GIT) from the cardia of the stomach
to the rectum.
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? Identifying Epithelia

The outlines of individual

epithelial cells are not always
visible, and it may be difficult to
identify the shape of the cells.

It is often helpful to look at the

shape, location and spacing of the
nuclei in the epithelium, which
together will allow a very good
guess at the shape of the cells
forming the epithelium.

How many cell layers seem to be

visible in a section depends very
much on the angle between the
plane of the section plane the
surface of the epithelium.

Oblique sections of epithelium

will be visible in almost all slides
of organs in which epithelium
lines a surface with a very
irregular profile. A single surface
is usually not lined by several
types of epithelia.

The number of epithelial cell

layers will usually be the smallest
number of layers visible
anywhere along the surface lined
by the epithelium.

Suitable Slides
simple squamous epithelium: any section containing blood vessels, sections of organs
which include an outer lining (or serosa) of the organs, or sections of kidney (parietal
blades of Bowman's capsules) - H&E, trichrome
simple cuboidal epithelium: sections of ovaries (epithelium lining the surface), thyroid
gland (follicles), kidney (tubules) or large glands (e.g. parotid gland) with well preserved
small ducts
simple columnar epithelium: sections of the small intestine (duodenum, jejunum or
ileum), uterus (uterine glands), liver (large bile ducts) or gall bladder - H&E, trichrome
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Sublingual Gland, Human, H&E

Blood vessels are probably
present in all sections you will
ever see. With very few
exceptions, they are lined by a
simple squamous epithelium. The
individual epithelial cells are
extremely flattened and form a
much larger part of the surface
than individual cells in cuboidal
or columnar epithelia. The nuclei
of the squamous epithelial cells
are also flattened and often stain
darkly. Not every epithelial cell
nucleus will be included in the
plane of the section, and if the
vessel is very small (e.g. a
capillary), there may not be any
visible nuclei in the epithelial
lining.
Capillaries and other small
vessels are easily deformed
during tissue processing, and the
epithelium of larger vessels may
be damaged or look corrugated. It
may therefore take a little more
patience than you expect to find a
"good" simple squamous
epithelium.
Draw a small vessel with its
epithelial lining, label the
features visible in your drawing
and include a suitable scale.
Duodenum, Rat, H&E and Ileum,
Human - H&E
The small intestines are lined by
a simple columnar epithelium.
Most of the epithelial cells
(enterocytes) are involved in the
absorption of components of the
digested food in the lumen of the
intestines. Complex folds of the
intestinal lining increase the
surface area available for
absorption. The plane of the
section will therefore often pass
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at an oblique angle through the

epithelium. The epithelium may
look stratified where this
happens. Scan along the
epithelium until you find a spot
where it is cut perpendicular to its
surface, i.e. where it looks like a
simple columnar epithelium.
Mucus producing goblet cells are
a second cell type of this
epithelium. Mucus stains only
weakly or not at all in H&E
stained sections. Round, light
"hollows" in the epithelium
represent the apical cytoplasm of
the goblet cells, which is filled
with mucin-containing secretory
vesicles.
Microvilli extend from the apical
surface of epithelial cells into the
intestinal lumen. They increase
surface area by a factor of ~20
and thereby facilitate absorption.
Together, the microvilli are
visible as a light red band along
the apical limit of the epithelium,
i.e. the side of the epithelium
facing the lumen of the intestine.
This band is call the brush
border.
Draw and label the epithelium.
Include goblet cells in your
drawing.

Stratified Epithelia

Stratified squamous epithelium

Stratified squamous epithelia vary in thickness depending on the number of cell layers
present. The deepest cells, which are in contact with the basement membrane, are
cuboidal or columnar in shape. This layer is usually named the basal cell layer, and the
cells are called basal cells. Basal cells are mitotically active and replace the cells of the
epithelium which are lost by "wear and tear". The basal cell layer is followed by layers of
cells with polyhedral outlines. Close to the surface of the epithelium, cells become more
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flattened. At the surface of the epithelium, cells appear like flat scales - similar to the
epithelial cells of simple squamous epithelia.
Remember that it is the shape of the cell which form the surface of the epithelium which
gives the name to the epithelium.

Stratified cuboidal and columnar epithelia

are not common. A two-layered cuboidal epithelium is, for example, seen in the ducts of
the sweat glands. Stratified columnar epithelia are found in the excretory ducts of the
mammary gland and the main excretory duct of the large salivary glands.

Suitable Slides
stratified squamous epithelium: sections of the oesophagus, tongue or vagina - H&E, van
Gieson, trichrome
stratified cuboidal epithelium: skin (excretory ducts of sweat glands) - H&E
stratified columnar epithelium: sections of the parotid gland or mammary gland - H&E
Oesophagus, human - H&E
The oesophagus is lined by a
stratified squamous epithelium
consisting of many cell layers.
Basal cells often form a well
defined layer at the border of the
epithelium to the underlying
connective tissue. The underlying
connective tissue forms finger-
like extensions towards the lumen
of the oesophagus, which are
called papillae. The border
between epithelium and
connective tissue may appear
quite irregular because of the
papillae. This irregular border
aids in anchoring of the
epithelium to the connective
tissue. If these extensions are not
cut exactly along their long axis,
they may look like isolated small
islands of connective tissue and
blood vessels within the
epithelium.
Draw the stratified squamous
epithelium of the oesophagus and
label your drawing. Try to draw a
little schematic illustration which
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shows how the plane of section

would effect the appearance of
the connective tissue extensions.

Parotid Gland, Human - H&E

Stratified columnar epithelia are found in the largest excretory ducts of some glands. The parotid
gland, a large salivary gland, is one of them. Several epithelial types are found in the duct system
of the parotid. The smallest ducts, which are embedded in the secretory tissue (intralobular
ducts), are lined by cuboidal or columnar epithelia. Small ducts, which are embedded in
connective tissue located between areas of secretory tissue (interlobular ducts), are lined by
columnar or pseudostratified epithelia. These ducts finally coalesce to form the main excretory
duct of the parotid which is lined by a stratified columnar epithelium.
Draw the stratified columnar epithelium seen in the largest ducts and label your drawing.

Pseudostratified and Transitional Epithelia

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These two types of epithelia are difficult to classify using the shape of the cells in the surface
layer and the number of the cell layers as criteria.

Transitional epithelium

Transitional epithelium is found exclusively in the excretory urinary passages (the renal
calyces and pelvis, the ureter, the urinary bladder, and part of the urethra).

The shape of the cells in the surface layer of a transitional epithelium varies with the
degree of distension of the organs whose lumen is lined by this type of epithelium. In the
'relaxed' state of the epithelium, it seems to be formed by many cell layers. The most
basal cells have a cuboidal or columnar shape. There are several layers of polyhedral
cells, and, finally, a layer of superficial cells, which have a convex, dome-shaped luminal
surface. In the distended state of the epithelium only one or two layers of cuboidal cells
are followed by a superficial layer of large, low cuboidal or squamous cells. In the
distended state the epithelium will resemble a stratified squamous epithelium.

Pseudostratified columnar
epithelium

All cells of this type of

epithelium are in contact
with the basement
membrane, but not all of
them reach the surface of
the epithelium. Nuclei of
the epithelial cells are
typically located in the
widest part of the cell.
Consequently, the nuclei
of cells which do or do
not reach the surface of
the epithelium are often
located at different
heights within the
epithelium and give the
epithelium a stratified
appearance. The
epithelium will look
stratified but it is not -
hence its name
"pseudostratified".
Pseudostratified columnar
epithelia are found in the
excretory ducts of many
glands.
 22

Suitable Slides
transitional epithelium: sections of ureter or bladder - H&E
pseudostratified epithelium: sections of the trachea - H&E
Bladder, Monkey - H&E
At a first glance a transitional
epithelium looks like a stratified
cuboidal epithelium. Several
rows of nuclei appear to be
topped by a layer of dome-shaped
cells which bulge into the lumen
of the ureter. The shape of the
surface cells and the number of
rows change if the bladder is
distended. The number of rows
decreases. This decrease should
tell us that many of the nuclei
located in different layers of the
epithelium belong to cells which
are all in contact with the
basement membrane. With
distension, the shape of the cells
in the surface layer will become
squamous.
Draw the epithelium and label the
features you can see. Add a
simple schematic drawing of how
you expect the epithelium to look
like if the ureter is distended.

It has not yet been resolved if all

the epithelial cells are in contact
with the basement membrane.
Some texts consider transitional
epithelium as a specialised
stratified epithelium while others
group it with pseudostratified
epithelia. Maybe it is best to also
consider it 'transitional' in this
regard.
 23

Trachea, Human - H&E

At least two, sometimes three
rows of nuclei are seen in the
pseudostratified columnar
epithelium lining the trachea. The
nuclei belong to cells which are
all in contact with the basement
membrane. The epithelial lining
of the trachea is also one of the
few examples of a basement
membrane clearly visible in H&E
stained sections. Epithelial cells
can be ciliated or they can be
goblet cells (unicellular exocrine
glands). Basal cell regenerate
other cell types of the epithelium.
Capillaries and small vessels are
visible in the connective tissue
beneath the epithelium.
A ciliated pseudostratified
columnar epithelium with goblet
cells is a characteristic feature of
parts of the respiratory system,
where it is call respiratory
epithelium. It contains several
cell types in addition to ciliated,
goblet and basal cells.
Draw the epithelium at high
magnification and label your
drawing.

Special Cytological Features of Epithelia

Basement membrane or basal lamina

Epithelia are separated from the underlying connective tissue by an extracellular supporting layer
called the basement membrane. The basement membrane is composed of two sublayers. The
basal lamina (about 80 nm thick) consists of fine protein filaments embedded in an amorphous
matrix. Membrane proteins of the epithelial cells are anchored in the basal lamina, which is also
produced by the epithelial cells. The major components of the basal lamina are two glycoproteins
- laminin and (usually type IV) collagen. The reticular lamina consists of reticular fibres
embedded in ground substance. The fibres of the reticular lamina connect the basal lamina with
the underlying conective tissue. The components of the reticular lamina are synthesised by cells
of the connective tissue underlying the epithelium.
 24

In addition to its function as support of the epithelium, the basal lamina acts as a selectively
permeable filter between epithelium and connective tissue.

Unless special stains are used, the basement membrane is rarely visible using light microscopy.
You can read more about reticular fibres and ground substance on the Connective Tissues page.

Specialisations of the apical surface

Microvilli and stereocilia are finger- or thread-shaped extensions of the epithelial cells. Their
main function is to increase the surface area of epithelial cells. They are typically found in
epithelia active in absorption. Microvilli contain actin filaments, which are in contact with the
terminal web of the cell . The only difference between microvilli and stereocilia is their
length. Microvilli are much shorter than stereocilia. Stereocilia are, despite their name ("cilia"),
not actively moving structures.

Using light microscopy, stereocilia are difficult to discern from cilia.

Specialisations of the lateral and basal surfaces

Connective tissue is responsible for the structural integrity of most organs. As mentioned above,
it is absent from epithelia. Instead, tissue integrity as well as the barrier function of epithelia is
taken care of by extensive cell-to-cell contacts between epithelial cells. These functions are
mediated by several specialisations in the lateral and basal parts of the cell membranes of the
epithelial cells.

Desmosomes

are specialisations of the lateral cell membranes which mediate cell adhesion. Proteins
inserted into the cell membrane of the adjacent cells form a protein-'zipper' linking the
cells. Fibers of the cytoskeleton attach to the cytoplasmic side of the desmosome to
stabilise the area of contact. Hemi-desmosomes mediate the attachment of the epithelial
cells to the basement membrane.
A group of glycoproteins (cadherins) inserted into the opposing plasma membranes
mediate cell-to-cell adhesion at desmosomes and also at the adhesion zones or patches
mentioned below. Integrins, another group of proteins, allow the cell to attach to the
matrix proteins of the basal lamina.

Intermediate junctions (zonula adherens)

are structurally not as well-characterised as desmosomes. An intermediate junction

typically appears as a close and consistent apposition (15-20 nanometers) of the cell
membranes near the apical cell surface. Intermediate junctions surround the entire cell.
Again, fibres of the cytoskeleton insert into the cytoplasmic side of this membrane
specialisation. Patches of adhesion resemble intermediate junctions structurally, but form
more localized, patch- or strip-like contacts between neighbouring cells. They are found
scattered over the lateral surfaces of the epithelial cell.
 25

The above mentioned membrane specialisations mediate cell-adhesion but are less well suited to
support one of the essential functions of epithelia - the isolation of the interior of the body from
the outside world. A tight junction (zonula occludens) between epithelial cells mediates this
aspect of epithelial function.

Proteins inserted into the cell membranes of adjacent cells 'stitch' the membranes of the
cells together and provide an effective barrier to the diffusion of substances from the
outside of the epithelium (called luminal side if the epithelium covers the surface of a
tubular structure). Several "rows of stitches" may be found. Their number depends on the
demand to reduce diffusion across the epithelium. Each of these rows reduces diffusion
by about a factor 10 of what it was 'before'.

GLANDS

are cells or aggregations of cells whose function is secretion.

 Exocrine glands release the secretory product via a system of ducts that opens upon one
of the surfaces of the body which are in contact with the external world (skin,
gastrointestinal tract etc.).

 Endocrine glands release their secretory product (typically hormones) into the spaces
between the secretory cells (extracellular space) from which it enters the bloodstream.

Both endocrine and exocrine glands are developmentally derived from epithelia, which form a
down-growth into the underlying connective tissue. The cells forming this down-growth then
develop the special characteristics of the mature gland. Exocrine glands maintain the connection
with the surface epithelium, whereas the connection is lost by endocrine glands.

Classification of Exocrine Glands

Exocrine glands may be classified according to cell number, and/or the shape and branching
pattern of their secretory portions and ducts.

Unicellular Glands

consist of a single secretory cell. In mammals the only example of unicellular exocrine
glands are goblet cells, which occur in the epithelium of many mucous membranes.
Goblet cells secrete the glycoprotein mucin, which by the uptake of water is converted
into a slimy substance, mucus.
 26

Multicellular glands

The simplest form of a

multicellular gland is a secretory
epithelial sheath - a surface
epithelium consisting entirely of
secretory cells (e.g. the
epithelium lining the inner
surface of the stomach, where the
mucous secretion protects the
stomach wall from the acidic
contents of the stomach). Other
multicellular glands have their
secretory portion embedded in
the connective tissue underlying
the epithelium. The secretion is
either discharged directly from
the secretory portion onto the
epithelium or reaches the
epithelium via a duct system that
consists of non-secretory cells.

The secretory portion may have a

variety of shapes. Secretory cells
may form

 tubes in tubular
glands,
 acini in acinar
glands or
 alveoli in alveolar
glands.

Combinations exist - the pancreas is a tubulo-acinar gland, in which each section of the secretory
system has a specialized function.
The precursors of digestive enzymes are produced by the acinar cells. Tubular cells secrete the
alkaline bicarbonate solution which eventually neutralizes the acidic contents of the stomach that
are released into the duodenum.

Multicellular glands with an unbranched excretory duct are called simple. We talk about a
compound gland when the excretory duct is branched. Finally, the part of the gland consisting of
secretory cells is branched in a (surprise!) branched gland.
 27

The classification scheme may appear somewhat elaborate - but there are many exocrine glands
around. All of them can be identified and described by this scheme, and some ideas about their
function can be derived from this description.

Suitable Slides
unicellular exocrine glands (goblet cells): sections of intestines (duodenum, jejunum,
ileum or colon) or trachea - H&E
secretory epithelial sheath: stomach - H&E
straight tubular glands: sections of stomach (principal glands) or colon (intestinal glands)
- van Gieson, H&E
coiled tubular glands: sections of skin (sweat glands) - see lab section on the
Integumentary System page.
Colon, Human - van Gieson
Straight tubular glands extend
from the surface of the colon into
the underlying connective tissue.
Although they are present
throughout the intestines they are
largest in the colon and, because
of the smooth inner surface of the
colon, they often show in good
longitudinal or transverse
sections. The lumen of the glands
is narrow and surrounded by
secretory cells of several types,
which include goblet cells. The
connective tissue beneath the
epithelium and surrounding the
glands in the colon contains more
cells than the connective tissue
beneath other epithelia that were
considered on this page. This is a
characteristic feature of the
epithelia in the digestive system.
Glands cut at slightly oblique
angles will connect to the lumen
outside of the plane of the
section.
If possible, draw both
longitudinally and transversely
sections intestinal glands. Include
part of the surrounding
connective tissue and surface
 28

epithelium.

Secretory Mechanisms

The secretory cells can release their secretory products by one of three mechanisms.

Merocrine secretion
corresponds to the process of exocytosis. Vesicles open onto the surface of the
cell, and the secretory product is discharged from the cell without any further
loss of cell substance.
Apocrine secretion
designates a mechanism in which part of the apical cytoplasm of the cells is
lost together with the secretory product. The continuity of the plasma
membrane is restored by the fusion of the broken edges of the membrane, and
the cell is able to accumulate the secretory product anew. This mechanism is
used by apocrine sweat glands, the mammary glands and the prostate.

Holocrine
secretion designates the breakdown and discharge of the entire secretory cell.
It is only seen in the sebaceous glands of the skin.

There are two additional mechanisms by which secretory cells can release their products. Lipid
soluble substances may diffuse out of the secretory cell (e.g. steroid hormone-producing
endocrine cells). Transporters (membrane proteins) may actively move the secretory product
across the plasma membrane (e.g. the acid producing parietal cells of the gastric glands). These
secretory mechanisms may not involve any light microscopically visible specialisations of the
cell.

Histological Structure of Large Exocrine Glands

The relationship between the secretory tissue (parenchyma) of glands and the supporting
connective tissue is similar in most larger glands. Externally the entire gland is surrounded by a
layer of dense connective tissue, the capsule. Connective tissue sheets (septa) extend from the
capsule into the secretory tissue and subdivide the gland into a number of lobes. Thinner
connective tissue septa subdivide the lobes into a number of lobules. Reticular connective tissue
(hardly visible in H&E stained sections) surrounds and supports the secretory units of the glands
(alveoli, acini etc.) and the initial parts of the excretory ducts if present.

Blood and lymph vessels as well as nerves penetrate the capsule and form a delicate network
between the secretory units and the initial parts of the duct system.
 29

The main excretory duct conveys the secretory product to one of the external surfaces of the
body. Other parts of the duct system are named according to their relation to the lobes and
lobules of the gland.

 Lobar ducts are are large branches of the main duct which extend to the lobes of the
gland. They may be called
 Interlobar ducts if they are found in the connective tissue surrounding the lobes.
Interlobar ducts branch and give rise to
 Interlobular ducts, which are found in the connective tissue surrounding the individual
lobules of the gland. Branches of the interlobular ducts enter the lobules and are now
called
 Intralobular ducts. The terminal branches of the duct system, which connect intralobular
ducts with the secretory units of the gland, are called
 Intercalated ducts.

The appearance of the different portions of the duct system is quite variable from gland to gland
and may allow the identification of the gland. Quite often, the appearance of parts of the duct
system also permits some deductions about their functions.

? Note that lobes and lobules are defined by their relationship to each other. Many small lobules
may form one large lobe. Neither size nor the spatial relationship between different parts of the
tissue can be unequivocally determined in a single, two-dimensional section of the tissue. Lobes
and interlobar ducts may therefore be difficult to distinguish from lobules and interlobular ducts.

Suitable Slides
alveolar gland: lactating mammary gland H&E - see lab section on the Female
Reproductive System page.
serous and mucous acinar glands: sections of parotid gland, sublingual gland or tongue
(lingual salivary glands) - H&E
 30

Parotid Gland, Human - H&E

Find an area of secretory tissue at
low magnification, and scan over
this area at high magnification.
Within the lobules and between
the acini of the parotid you can
find two types of ducts. Since
they are both located within the
lobules they are both intralobular
ducts. Striated ducts are lined by
a simple tall columnar
epithelium. Intercalated ducts are
lined by a simple cuboidal
epithelium and connect
individual acini to the striated
ducts.
Try to capture the features of the
acini, intercalated and striated
ducts in one compound drawing
which shows how they connect to
each other. Label your drawing.

Parotid Gland, Human and Sublingual Gland, Human - H&E

Many secretory cells and the secretory structures formed by them belong to one of two
morphologically distinct forms: serous or mucous. Serous secretions have a low viscosity, i.e.
they are rather "watery". Mucous secretions have a high viscosity, i.e. they are rather "slimy".
The apical cytoplasm of the cells forming serous acini is usually well-stained. Secretory vesicles
are visible in the apical cytoplasm in well-preserve tissue. The nuclei are round or slightly ovoid
and located in the basal cytoplasm of the cells. The bluish color of the basal cytoplasm reflects
the presence of large amounts of rough endoplasmatic reticulum.
The contents of the secretory vesicles in the apical cytoplasm of cells forming mucous acini are
only weakly stained. These empty-looking vesicles give the apical cytoplasm of mucus-
producing cells a distinct "foamy" or "frothy" appearance. The nuclei of mucous cells appear
darker and smaller than the nuclei of serous cells. They also seem to be "pressed" against the
basal limit of the cells and may look flattened with an angular ("edgy") outline. Glands
containing mucous acini (e.g. the sublingual glands) are called mucous glands. Glands
containing serous acini (e.g. the parotid glands) are called serous glands. If both types of acini
are present the gland is muco-serous.
Identify and draw serous and mucous acini at high magnification. Label your drawing. Make
 31

sure that the features which characterise serous and mucous acini are visible in your drawing - if
necessary use a little artistic freedom.

page content and construction: Lutz Slomianka

last updated: 6/08/09
 32

School of Anatomy and Human Biology - The University of Western

Australia

Blue Histology - Connective Tissues

CONNECTIVE TISSUE

Connective tissue consists of cells separated by varying amounts of extracellular substance. In

connective tissues cells typically account for only a small fraction of the tissue volume. The
extracellular substance consists of fibres which are embedded in ground substance containing
tissue fluid. Fibres in connective tissue can be divided into three types: collagen fibres, reticular
fibres and elastic fibres.

Extracellular Substance

Collagen fibres

Collagen fibres are the dominant fibre type in most connective tissues. The primary function of
collagen fibres is to add strength to the connective tissue.

The thickness of the fibres varies from ~ 1 to 10 µm. Longitudinal striations may be visible in
thicker fibres. These striations reveal that the fibres are composed of thinner collagen fibrils (0.2
to 0.5 µm in diameter). Each of these fibrils is composed of microfibrils, which are only visible
using electron microscopy.

Microfibrils are assemblies of tropocollagen, which, in turn, is an spiral-like assembly of three

collagen molecules (triple helix). The organisation of the tropocollagen within the microfibrils is
highly regular. A small gap (60 nm wide) is found between the subsequent tropocollagens which
form the microfibrils. Staining solutions used in electron microscopy tend to fill in these gaps,
and the alignment of the gaps gives the microfibrils a cross-striated appearance (with 68 nm
intervals) in EM images .

Coarse collagen fibres are formed by type I tropocollagen.

There are many different tropocollagen types around (currently named type I to XXI). These
types differ in their content of the amino acids hydroxyproline and hydroxylysine. They also
differ in the amount of carbohydrates attached to the collagen molecules. The different types of
tropocollagen give the fibres the structural and functional features which are appropriate for the
organ in which the fibres are found. Types I, II and III are the major fibre-forming
tropocollagens. Tropocollagen type IV is an important structural component of the basal lamina.
 33

A tensile force of several hundred kg/cm2 is necessary to tear human collagen fibres. The fibres
stretch by only 15-20%.

Reticular fibres

Reticular fibres are very delicate and form fine networks instead of thick bundles. They are
usually not visible in histological sections but can be demonstrated by using special stains. For
example, in silver stained sections reticular fibres look like fine, black threads - coarse collagen
fibres appear reddish brown in the same type of preparation.

Because of their different staining characteristics, reticular fibres were initially thought to be
completely different from collagen fibres. Cross-striations with the same periodicity as in coarse
collagen fibres are however visible using electron microscopy. We now know that reticular fibres
consist of collagen - although the main type of tropocollagen found in reticular fibres, type III, is
different from that of the coarse collagen fibres.

Reticular fibres give support to individual cells, for example, in muscle and adipose tissue.

Suitable Slides
sections of liver, spleen or lymph nodes - reticulin
 34

Liver - Reticulin Stain

The liver is one of the organs in
which the cells are supported by a
network of reticular fibres. They
appear as fine black lines in this
silver stained preparation. The
fibres surround the individual
sheets of liver cells (hepatocytes)
and are the only fibrous
connective tissue component
supporting the cells. While
providing support, the fine, open
meshwork of reticular fibres
facilitates the exchange of
substances between the
hepatocytes and the blood, which
circulates in the irregularly
shaped blood vessels (sinusoids)
between the hepatocytes.
Reticular fibres are also present
in the connective tissue
surrounding the larger vessels,
which penetrate the parenchyma
of the liver.
Draw reticular fibres as they
surround a nice piece of a row of
liver cells at high magnification -
include a suitable scale and label
your drawing.

Blood will not be visible in some

types of preparations and the
sinusoids appear empty.

Elastic fibres

Elastic fibres are coloured in fresh tissues - they are light yellow - but this colouration is only
visible if large amounts of elastic fibres are present in the tissue, for example, in the elastic
ligaments of the vertebral column. Special stains are necessary to show elastic fibres in tissue
sections.
Resorcin fuchsin is one of these stains, which gives the elastic fibres a dark violet colour.

Light microscopy does not reveal any substructure in the elastic fibres. Electron microscopy
shows that elastic fibres consist of individual microfibrils, which are embedded in an amorphous
 35

matrix. The matrix accounts for about 90% of the fibre and is composed of the protein elastin.
Neither the elastin nor the microfibrils are collagens.

Elastic fibres can be stretched to about 150% of their original length. They resume their original
length if the tensile forces applied to the elastic fibres are relaxed.

Elastin is a somewhat odd protein in that its amino acid sequence does not determine a specific
three-dimensional structure of the molecule. Instead, elastin remains unfolded as a "random
coil". Elastin molecules are cross-linked to each other by desmosin and isodesmosin links, which
are only found between elastin molecules. Tensile forces straighten the cross-linked mesh of
elastin coils.

Suitable Slides
sections of blood vessels or skin - elastin

Skin, human - elastin & van Gieson or Artery, human - elastin & eosin
Like reticular fibres, elastic fibres require special stains to be visualized. Typically elastic fibres
will appear as fine, dark violet and gently undulating fibres in the tissue. Elastic fibres can form
membranes - not unlike the collagen membrane in the basal lamina of epithelia. This is the case
at some levels in the walls of blood vessels.
Collagen and elastic fibres intermingle in the dermis, i.e. the connective tissue beneath the
epithelium of the skin. Immediately beneath the epithelium both fibre types are relatively fine -
they appear much thicker in the deeper parts of the dermis. At least the internal elastic lamina
should be visible in the smaller arteries which course through the dermis.
A combination with a second stain is necessary to visualize other tissue components. Colours
visible in the sections will depend on the method used in combination with the elastin stain.
Eosin gives an even pink or red colour to many tissue components. Nuclei of cells remain
unstained without the inclusion of the haematoxylin in the staining solutions.
Identify the artery and the vein in the section. Their walls contain large amounts of elastic fibres.
Which one contains more elastic fibres - artery or vein?
Blood vessels: draw a small section of the wall of a vessel, preferably an artery, at high
magnification. Identify elastic laminae, fine and coarse elastic fibres in your drawing.
Skin: draw a small section of the dermis - preferablyof a part of the dermis where both the very
fine and the coarse fibres are visible.
 36

Ground substance

Ground substance is found in all cavities and clefts between the fibres and cells of connective
tissues. Water, salts and other low molecular substances are contained within the ground
substance, but its main structural constituent are proteoglycans.
Ground substance is soluble in most of the solvents used to prepare histological sections and
therefore not visible in ordinary sections.

Proteoglycans are responsible for the highly viscous character of the ground substance.
Proteoglycans consist of proteins (~5%) and polysaccharide chains (~95%), which are covalently
linked to each other. The polysaccharide chains belong to one of the five types of
glycosaminoglycans, which form the bulk of the polysaccharides in the ground substance.

Hyaluronan (or hyaluronic acid) is the dominant glycosaminoglycan in connective tissues. The
molecular weight (MW) of hyaluronic acid is very high (~ MW 1,000,000 ). With a length of
about 2.5 µm hyaluronan is very large. Hyaluronan serves as a "backbone" for the assembly of
 37

other glycosaminoglycans in connective and skeletal tissue, which results in even larger
molecule complexes (MW 30,000,000 - 200,000,000).
Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the
vitreous body of the eye.

The remaining four major glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan
sulfate and heparan sulfate. These glycosaminoglycans attach via core- and link-proteins to a
backbone formed by the hyaluronic acid. The coiled arrangement of the hyaluronan and other
attached glucosaminoglycans fills a roughly spherical space with a diameter of ~0.5 µm. This
space is called a domain. Neighbouring domains overlap and form a more or less continuous
three-dimensional molecular sieve in the interstitial spaces of the connective tissues.

The large polyanionic carbohydrates of the glycosaminoglycans bind large amounts of water and
cations. The bound water in the domains forms a medium for the diffusion of substances of low
molecular weight such as gases, ions and small molecules, which can take the shortest route, for
example, from capillaries to connective tissue cells. Large molecules are excluded from the
domains and have to find their way through the spaces between domains.

The restricted motility of larger molecules in the extracellular space inhibits the spread of
microorganisms through the extracellular space. A typical bacterium ( 0.5 x 1 µm) is essentially
immobilised in the meshwork formed by the domains. The pathogenicity of a bacterium is
indeed to some extent determined by its ability to find its way through the mesh, and some of the
more invasive types produce the enzyme hyaluronidase, which depolymerises hyaluronic acid.

The components of the ground substance, collagen, elastic and reticular fibres are synthesised by
cells of the connective tissues, the fibrocytes.

Connective Tissue Cells

Connective tissue cells are usually divided into two groups based on their ability to move within
the connective tissue. Fibrocytes (or fibroblasts) and fat cells are fixed cells. Macrophages,
monocytes, lymphocytes, plasma cells, eosinophils and mast cells are wandering cells.

Fibrocytes

Fibrocytes are the most common cell type in connective tissues. They are the "true" connective
tissue cells. Usually only their oval, sometimes flattened nuclei are visible in LM sections. The
cytoplasm of a resting (i.e. inactive) fibrocyte does not contain many organelles. This situation
changes if the fibrocytes are stimulated, for example, by damage to the surrounding tissue. In this
case the fibrocyte is transformed into a fibroblast, which contains large amounts of the organelles
which are necessary for the synthesis and excretion of proteins needed to repair the tissue
damage (Which ones?). Fibrocytes do not usually leave the connective tissue. They are,
however, able to perform amoeboid movement.
 38

The terms fibrocyte and fibroblast refer here to the inactive and active cells respectively - at
times you will see the two terms used as synonyms without regard for the state of activity of the
cell.

Reticular cells

Reticular cells are usually larger than an average fibrocyte. They are the "fibrocytes" of reticular
connective tissue and form a network of reticular fibres, for example, in the lymphoid organs.
Their nuclei are typically large and lightly stained (H&E) and the cytoplasm may be visible
amongst the cells which are housed within the network of reticular fibres.

Adipocytes

Fat cells or adipocytes are fixed cells in loose connective tissue. Their main function is (what
surprise!) the storage of lipids. If "well fed" the cytoplasm only forms a very narrow rim around
a large central lipid droplet. The flattened nucleus may be found in a slightly thickened part of
this cytoplasmic rim - if it is present in the section, which may not be the case since the diameter
of an adipocyte (up to 100 µm) is considerable larger than the thickness of typical histological
sections. A "starving" adipocyte may contain multiple small lipid droplets and gradually comes
to resemble a fibrocyte.

Lipid storage/mobilisation is under nervous (sympathetic) and hormonal (insulin) control.

Adipocytes also have an endocrine function - they secrete the protein leptin which provides brain
centers which regulate appetite with feedback about the bodies fat reserves.
Leptin deficiency in experimental animals results in obesity.

Adipocytes are very long-lived cells. Their number is determined by the number of
preadipocytes (or lipoblast) generated during foetal and early postnatal development.

Macrophages

Macrophages arise from

precursor cells called monocytes.
Monocytes originate in the bone
marrow from where they are
released into the blood stream.
They are actively mobile and
leave the blood stream to enter
connective tissues, where they
differentiate into macrophages.
Macrophages change their
appearance depending on the
demand for phagocytotic activity.
Resting macrophages may be as
numerous as fibrocytes.
Resting macrophages are difficult
 39

to distinguish from fibrocytes in

H&E stained sections.

Suitable Slides
sections of liver or lymph nodes - carbon injected animal - trichrome or H&E

sections of lung - H&E

In the lung, macrophages "patrol" the respiratory surfaces and ingest airborne particles
which settle there. They "retire" to the connective tissue of the lung were they can be
identified by the accumulations of fine particles in their cytoplasm.

sections containing irregular connective connective tissues - Alcian blue & van Gieson
In Alcian blue & van Gieson stained sections macrophages may appear as intensely
green, roundish cells with a dark nucleus.
Liver, rabbit - ink injected,
trichrome
Macrophages are usually difficult
to distinguish from other cell
types in connective tissues. One
way to visualize them is to inject
an experimental animal with very
fine carbon particles.
Macrophages which come into
contact with the circulating
particles will phagocytose some
of them. In sections the particles
will be visible as dark, black-
brown accumulations in the
cytoplasm of the macrophages.
Draw a few macrophages in situ.

Macrophages found in the liver

are also called Kupffer cells.
They adhere to the epithelial
lining of the liver sinusoids, i.e.
blood filled spaces between the
liver cells. Blood will not be
visible in some types of
preparations and the sinusoids
appear empty.

Once you have identified

macrophages, go hunting for
some good collagen - in this
trichrome stains the collagen
 40

fibres will appear green(ish).

Typically you will see them only
in the connective tissue
surrounding larger blood vessels.
Improve your knowledge on
epithelia and look out for ducts
lined by a simple cuboidal or
columnar epithelium. What is
flowing in these ducts?

Mast cells

Mast cells are - like macrophages, lymphocytes and eosinophils - in demand when something
goes wrong in the connective tissue. Quite a few of them are present in healthy connective tissue
as they stand on guard and monitor the local situation. The cytoplasm of mast cells is filled by
numerous large vesicles. Mast cells discharge the contents of these vesicles if they come in
contact with antigens, for example, proteins on the surface of an invading bacterium or, in
allergic reactions, in response to antigens found, for example, on the surface of pollen grains.

The most prominent substances contained in the vesicles are heparin and histamine. They
increase blood flow in close by vessels and the permeability of the vessel walls to plasma
constituents and other white blood cells. By facilitating access to the area, mast cells facilitate an
immune response to the antigen which triggered the release histamine and heparin.

Other connective tissue cells

Lymphocytes and plasma cells

Lymphocytes are usually small cells (6 - 8 µm). Their nuclei are round and stain very dark. The
cytoplasm forms a narrow rim around the nucleus and may be difficult to see. There are many of
them in the connective tissue underlying the epithelia of the gastrointestinal tract but usually
much fewer in other connective tissues. Again, this situation may change - in this case with
immunological reactions. Some lymphocytes may differentiate into plasma cells. Plasma cells
are lymphocytes which produce antibodies. To accommodate the necessary organelles for this
function the size of the cytoplasm increases dramatically and the cells become basophilic.
Plasma cells can occasionally be spotted in the loose connective tissue present in sections.
Like eosinophilic cells and monocytes, lymphocytes are white blood cells. More information
about these cell types can be found on the Blood page.

Eosinophilic cells

Eosinophilic cells are typically rounded or oval, large cells, which contain large amounts of
bright red granules in their cytoplasm. They originate, like the monocytes, in the bone marrow.
They enter connective tissues early in inflammatory reactions, where they phagocytose antigen-
 41

antibody complexes. Their numbers in healthy connective tissue vary with location, but a few of
them can usually be found.

Mesenchymal cells

During development, mesenchymal cells give rise to other cell types of the connective tissue. A
small number of them may persist into adulthood. Mesenchymal cells are smaller than fibrocytes
and difficult to detect in histological sections. They may regenerate blood vessels or smooth
muscle which have been lost as a consequence of tissue damage.

Suitable Slides
sections of tongue, skin, mesentery or other sections containing epithelia and / or loose
connective tissue - toluidine blue, cresyl violet

Mast Cells, Tongue - toluidine

blue and
Mesentry, Rat - cresyl violet
Mast cells are relatively frequent
in the connective tissue benath
the epithelium and between the
muscle fibres of the tongue. In
most connective tissue cells and
the muscle fibres only the
nucleus is stained by the toluidine
blue. The cytoplasm of the mast
cells is however filled with dark,
blue / violet grains which
represent their secretory vesicles.
At low magnification mast cells
stand out as large, dark dots
among smaller and lighter stained
nuclei and among the very
weakly stained remaining
connective tissue components.
Draw a few mast cells in situ and
label both the mast cells and
some of the surrounding tissue
components.

Connective Tissue Types

 42

Loose connective tissue and dense connective tissues

These two tissues are distinguished according to the relative amounts of fibres they contain.
Dense connective tissues are completely dominated by fibres. They are subdivided according to
the spatial arrangement of the fibres in the tissue.

In dense irregular connective tissue the fibres do not show a clear orientation within the tissue
but instead form a densely woven three-dimensional network. A good example is the dermis of
the skin.

We talk about regular dense connective tissue if the fibres run parallel to each other. Good
examples of regular dense connective tissue are tendons, ligaments and the fasciae and
aponeuroses of muscles.

Loose connective tissue is relatively cell rich, soft and compliant. It is also rich in vessels and
nerves. It is best understood as a kind of generalised connective tissue in which all connective
tissue cell types may occur. Loose connective tissue may occur in some special variants: mucous
connective tissue, reticular connective tissue and adipose tissue.

Suitable Slides
sections of tendons or ligaments - van Gieson, H&E
 43

Muscle-Tendon Junction, rat -

van Gieson
In van Gieson stained
preparations collagen stains dark
red while other tissue
components appear in varying
shades of grey (nuclei) and
yellow (cytoplasm). Areas of
dense regular connective tissue
are usually easy to identify in
these preparations. Coarse
collagen fibres are aligned with
each other with only very narrow
opens spaces between them. Like
in most other connective tissues,
there will be only a few cells
between the fibres. Their
cytoplasm is difficult to identify
but the nuclei can be seen
scattered among the collagen
fibres. Nuclei are often elongated,
and their long axis runs parallel
to the course of the collagen
fibres
Sketch part of the regular dense
connective tissue.

Suitable Slides
sections of skin or non-lactating mammary gland - H&E, van Gieson, trichrome
 44

Non-lactating Breast - H&E

Dense irregular connective tissue
forms the dermis of the skin, i.e.
the layer of connective tissue
immediately below the
epithelium lining the surface of
the skin. Beneath the skin
forming the mammae (nipples),
dense connective tissue areas are
very extensive. This tissue
surrounds the resting mammary
gland. Both the high density of
collagen fibres and the their
irregular distribution are easily
visible. Again, only a very small
fraction of the tissue is taken up
by cells. Like in van Gieson
stained preparations, their
cytoplasm is often not visible in
H&E stained sections. Dark spots
scattered between the collagen
fibres represent the nuclei of the
cells.
Draw part of the connective
tissue including some fibrocytes.

The excretory ducts of the

mammary glands are called
lactiferous ducts. They are lined
by a quite nice stratified
columnar epithelium. If you are
working with a section of non-
lactating breast look for the
lactiferous ducts in the
connective tissue.

Reticular connective tissue

Reticular connective tissue consists of reticular cells and the network of reticular fibres formed
by them. Most connective tissues contain reticular fibres, but only in reticular connective tissue
are they the dominant fibre type. In a number of tissues and organs, reticular connective tissue
forms the structural framework in which the cells of the organ are suspended. The open
 45

meshwork of fine fibres is particularly useful in tissues and organs in which diffusion and / or
cell movements are functionally important, for example, in the liver, lymph nodes and the spleen.

Adipose tissue

Adipose tissue is essentially loose connective tissue containing large numbers of adipocytes.
There are two types of adipose tissue, which derive their names from the colour of the tissue
(white or brown) and the number of lipid droplets found in the adipocytes.

 Adipocytes of white, unilocular adipose tissue contain one large lipid droplet.
 Adipocytes of brown, multilocular adipose tissue contain many lipid droplets.

White adipose tissue does not only function in the storage of lipids. For example, in the palms of
the hands, on the plantar surface (sole) of the feet and in the gluteal region (buttocks) it has a
structural, cushioning function. In these regions, accumulations of adipocytes are surrounded by
strong connective tissue fibres. Also, the distribution of white adipose tissue is different in males
and females and is part of the secondary sexual characteristics. The storage and mobilisation of
lipids does require quite some metabolic activity of the tissue. Consequently, adipose tissue has a
rich supply of capillaries.

Brown adipose tissue occurs mainly during development and may account for 2 - 5 % of the
body weight in a newborn. In adult individuals most of the brown fat has further differentiated
into white fat. Adipocytes in brown fat contain plenty of mitochondria. A very rich capillary
supply and the cytochromes found in the mitochondria give the tissue its characteristic colour. A
protein (UCP-1 or thermogenin) found in these mitochondria decouples the oxidation of fatty
acids from the generation of ATP. Instead, these cells generate heat.

The location of the brown fat reflects its heat-generating function. It is located in the axilla
(armpits), between the shoulder blades, in the region of the neck and along large blood vessels.
The heat generated by the brown fat warms the blood which supplies nearby organs or which re-
enters the trunk from the limbs.

Suitable Slides
white adipose tissue: sections of skin - H&E, trichrome, van Gieson
Section are rarely prepared to show just white adipose tissue. The hypodermis, i.e.
lightest and loosest appearing layer, of skin sections will typically contain large areas of
adipose tissue. Other good candidates are bone sections which contain yellow bone
marrow or sections of lymph nodes which are often embedded in adipose tissue. Small
spots of adipose tissue should be present in many other sections.
brown adipose tissue: sections of brown adipose tissue or kidney - trichrome, H&E
Brown adipose tissue is often located in the connective tissue close to the renal hilus or in
the renal sinus of sections which contain the entire kidney of small laboratory animals.
Look for an indentation in the outline of the kidney, which corresponds to its hilus.
 46

Thick Skin - H&E

In well-preserved thin sections,
the adipocytes form a mosaic of
rounded or slightly angular white
tiles, which correspond to the
locations of the lipid droplets,
separated from each other by
darker seams, which correspond
to the cytoplasm of the
adipocytes and the sparse
connective tissue components
between them. Because of the
large size of the adipocytes you
will only rarely see it "typical"
signet ring-like appearance of the
cells.
Although the tissue my be a little
distorted, thicker sections give a
good three dimensional feel of
the adipose tissue. The
cytoplasmic rims of the
adipocytes form thin veils which
enclose the open spaces which
were occupied by the lipid
droplets. Oval adipocyte nuclei
are often located close to the
corners at which the adipocytes
meet.
Draw two or three adipocytes at
high magnification and a survey
image which illustrates the
appearance of adipose tissue at
low magnification.
 47

Kidney - trichrome
In the renal sinus, islands of
brown adipose tissue are often
surrounded by white adipose
tissue, which emphasises the
different appearances of the two
tissue types. In brown adipose
tissue, the nuclei of adipocytes
are round and located more or
less centrally in a cytoplasm
which, after the extraction of
lipids during tissue preparation,
looks very frothy. Cell borders
can be difficult to identify.
Capillaries are very frequent.
Sketch the appearance of the
brown adipose tissue. Contrast
the characteristic features of
white and brown adipose if both
types are present side by side.

Mesenchymal connective tissue

Mesenchyme forms the undifferentiated "filling" of the early embryo. It consists of mesenchymal
cells, which interconnect by slender cell processes. Mesenchymal cells have stem cell properties,
i.e. they are able give rise to other cell and tissues types. The wide extracellular space between
the mesenchymal cells is occupied by ground substance, which can be stained with dyes that also
stain mucin - hence the alternative name of this tissue type: mucoid connective tissue. Collagen
or reticular fibres may not be visible at all or form a loose network between the cells. With fetal
development, mesenchyme forms the connective tissue between and within the developing
tissues and organs. Mucoid connective tissue also forms a compliant cushion around the vessels
of the umbilical cord, where it is also called Wharton's jelly.

In adult humans, mesenchymal connective tissue is only found in the dental pulp.
 48

Suitable Slides
sections of umbilical cord, tooth (pulp), or sections of embryonic and early foetal
development - H&E, Azan or Alcian blue & van Gieson
section usable for "intramembranous ossification" during foetal development will contain
areas of mucoid connective tissue around the developing bone.

Umbilical Cord, Human - H&E and

Foetal Kidney, Human - H&E
Within the umbilical cord you will be able to identify three large vessels and their walls. Mucoid
connective tissue fills the space between the vessels and the simple squamous epithelium lining
the surface of the umbilical cord. Note the very fine appearance of the collagen fibres and the
lack of apparent specialisations in this type of connective tissue.
The number of cells and appearance of the collagen fibres vary depending on the precise location
of the tissue. In some locations, mucoid connective tissues will contain a large number of cells
and only a few, very delicate collagen fibres. Examples are dental pulp and the mucoid
connective tissue which is found between the developing tubuli and glomeruli of the foetal
kidney.
A small drawing should be sufficient to capture the appearance of the tissue.
 49

page content and construction: Lutz Slomianka

last updated: 6/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology-Adipose Tissue

Adipose Tissue
 50

Cells which accumulate fat are often present in loose connective tissue, either
singly or in small groups. When they are present in large numbers we call such
tissue adipose (fatty) tissue. layers of fatty tissue may be present under the skin
(in what anatomically we call superficial fascia), under serous membranes
(especially the peritoneum) and even within certain organs. Groups of cells are
usually separated from each other by fine partitions of loose connective tissue.

Adipose tissue in the adult human appears white or yellowish in colour. In foetal
life and in the newborn there is another variety of fat that is brownish in colour.
The brown colour is in fact due to blood vessels. Brown fat is also present in
adult animals of species which hibernate.

We consider the commoner variety, white fat, first.

When a cell accumulates fat in the cytoplasm, small "droplets" of fat grow into
a single large droplet. This pushes the remaining cytoplasm and the nucleus to
one corner of the cell. During preparation of H & E slides the fat is dissolved and
the cell appears empty, with a very thin rim of cytoplasm and the nucleus along
the edge. This appearance is described as "signet ring" appearance. (If you have
a historical or biblical movie you may have seen how the ring of a king carries
his seal which can be "impressed" on writing material by punching it!).

Adipose tissue is essentially loose connective tissue containing large numbers of

adipocytes. There are two types of adipose tissue, which derive their names from
the colour of the tissue (white or brown) and the number of lipid droplets found
in the adipocytes.

1. Adipocytes of white, unilocular adipose tissue contain one large lipid

droplet.
2. Adipocytes of brown, multilocular adipose tissue contain many lipid
droplets.

White adipose tissue does not only function in the storage of lipids. For example,
in the palms of the hands, on the plantar surface (sole) of the feet and in the
gluteal region (buttocks) it has a structural, cushioning function. In these
 51

regions, accumulations of adipocytes are surrounded by strong connective tissue

fibres. Also, the distribution of white adipose tissue is different in males and
females and is part of the secondary sexual characteristics. The storage and
mobilisation of lipids does require quite some metabolic activity of the tissue.
Consequently, adipose tissue has a rich supply of capillaries.

Brown adipose tissue occurs mainly during development and may account for 2 -
5 % of the body weight in a newborn. In adult individuals most of the brown fat
has further differentiated into white fat. Adipocytes in brown fat contain plenty
of mitochondria. A very rich capillary supply and the cytochromes found in the
mitochondria give the tissue its characteristic colour. A protein (UCP-1 or
thermogenin) found in these mitochondria decouples the oxidation of fatty acids
from the generation of ATP. Instead, these cells generate heat.

The location of the brown fat reflects its heat-generating function. It is located in
the axilla (armpits), between the shoulder blades, in the region of the neck and
along large blood vessels. The heat generated by the brown fat warms the blood
which supplies nearby organs or which re-enters the trunk from the limbs.

. A "starving" adipocyte may contain multiple small lipid droplets and gradually
comes to resemble a fibrocyte.

Lipid storage/mobilisation is under nervous (sympathetic) and hormonal

(insulin) control. Adipocytes also have an endocrine function - they secrete the
protein leptin which provides brain centers which regulate appetite with
feedback about the bodies fat reserves.
Leptin deficiency in experimental animals results in obesity.

Adipocytes are very long-lived cells. Their number is determined by the number
of preadipocytes (or lipoblast) generated during foetal and early postnatal
development.

Suitable Slides
 52

white adipose tissue: sections of skin - H&E, trichrome, van Gieson

Section are rarely prepared to show just white adipose tissue. The hypodermis, i.e.
lightest and loosest appearing layer, of skin sections will typically contain large
areas of adipose tissue. Other good candidates are bone sections which contain
yellow bone marrow or sections of lymph nodes which are often embedded in
adipose tissue. Small spots of adipose tissue should be present in many other
sections.
brown adipose tissue: sections of brown adipose tissue or kidney - trichrome, H&E
Brown adipose tissue is often located in the connective tissue close to the renal
hilus or in the renal sinus of sections which contain the entire kidney of small
laboratory animals. Look for an indentation in the outline of the kidney, which
corresponds to its hilus.

Thick Skin - H&E

In well-preserved thin
sections, the adipocytes form
a mosaic of rounded or
slightly angular white tiles,
which correspond to the
locations of the lipid
droplets, separated from
each other by darker seams,
which correspond to the
cytoplasm of the adipocytes
and the sparse connective
tissue components between
them. Because of the large
size of the adipocytes you
will only rarely see it
"typical" signet ring-like
appearance of the cells.
Although the tissue my be a
little distorted, thicker
sections give a good three
dimensional feel of the
 53

adipose tissue. The

cytoplasmic rims of the
adipocytes form thin veils
which enclose the open
spaces which were occupied
by the lipid droplets. Oval
adipocyte nuclei are often
located close to the corners
at which the adipocytes
meet.
Draw two or three
adipocytes at high
magnification and a survey
image which illustrates the
appearance of adipose tissue
at low magnification.
 54

Kidney - trichrome
In the renal sinus, islands of
brown adipose tissue are
often surrounded by white
adipose tissue, which
emphasises the different
appearances of the two
tissue types. In brown
adipose tissue, the nuclei of
adipocytes are round and
located more or less
centrally in a cytoplasm
which, after the extraction of
lipids during tissue
preparation, looks very
frothy. Cell borders can be
difficult to identify.
Capillaries are very frequent.
Sketch the appearance of the
brown adipose tissue.
Contrast the characteristic
features of white and brown
adipose if both types are
present side by side.

The pictures here (at different magnifications) show both types of fat in the same
section.
 55

In the picture on the left, brown fat is at the top

left and white fat at bottom right.
In the picture on the right, white fat is at top left.
In this picture also note the blue line drawn to
show the two parts separately and single cells in
circles.
Aren't the pictures self-explanatory?

Figure 5 demonstrates adipose tissue in a whole mount

of mesentery using a lipid soluble Sudan stain. The
lipids are stained red. A faint edge of cytoplasm can be
seen around the fat droplets at the right. Fibroblast and
other nuclei can be seen within the mesentery. In your
paraffin embedded slides, all the fat has been removed
and the fat cells appear as empty circles, in which a
peripheral nucleus (signet ring) can often be identified.

A different kind of adipose tissue is known as brown or multilocular fat. Brown fat
contains fat droplets of varying sizes. The cells are smaller than those of white fat,
with an eccentric but not flattened nucleus. Brown fat has a very limited
distribution in adult humans, but is found in many animals. In hibernating animals,
the oxidation of brown fat warms the blood flowing through it during arousal from
hibernation. Human newborns, whose large surface to volume ratio can result in
 56

heat loss, also have alot of brown fat. Most of it disappears during the first decade
of life.

Figure 6 shows a section of brown fat from a gerbil. Fat

droplets of varying size lie within the cells. The cells
are tightly packed and boundaries are not always
distinct. The lower third of the field of view is occupied
by a pair of white fat cells.

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology –
Skeletal Tissues – Cartilage

CARTILAGE

 is a specialised type of connective tissue.

 consists, like other connective tissues, of cells and extracellular components.
 does, unlike other connective tissues, not contain vessels or nerves.
 is surrounded by a layer of dense connective tissue, the perichondrium.

Cartilage is rather rare in the adult humans, but it is very important during development
because of its firmness and its ability to grow rapidly. In developing humans, most of
the bones of the skeleton are preceded by a temporary cartilage "model". Cartilage is
 57

also formed very early during the repair of bone fractures.

Types of Cartilage

1. Hyaline Cartilage

Hyaline
cartilage
develops, like
other types of
connective
tissue, from
mesenchymal
cells. From
about the fifth
foetal week
precursor cells
become
rounded and
form densely
packed cellular
masses,
chondrification
centres. The
cartilage-
forming cells,
chondroblasts,
begin to secrete
the
components of
the
extracellular
matrix of
cartilage. The
extracellular
matrix consists
of, ground
substance
(hyaluronan,
chondroitin
sulfates and
keratan
sulfate) and
tropocollagen,
which
 58

polymerises
extracellularly
into fine
collagen fibres.

Tropocollagen type II is the dominant form in collagen fibres of almost all types of
cartilage.

As the amount of matrix increases the chondroblasts become separated from each other
and are, from this time on, located isolated in small cavities within the matrix, the
lacunae. Concurrently the cells differentiate into mature cartilage cells, chondrocytes
.

Growth occurs by two mechanisms

 Interstitial growth - Chondroblasts within the existing cartilage

divide and form small groups of cells, isogenous groups, which
produce matrix to become separated from each other by a thin
partition of matrix. Interstitial growth occurs mainly in immature cartilage.
 Appositional growth - Mesenchymal cells surrounding the cartilage in the deep
part of the perichondrium (or the chondrogenic layer) differentiate into
chondroblasts. Appositional growth occurs also in mature cartilage.

Like all protein-producing cells, chondroblasts contain plenty of rough endoplasmatic

reticulum while they produce matrix. The amount of rough endoplasmatic reticulum
decreases as the chondroblasts mature into chondrocytes. Chondrocytes fill out the
lacunae in the living cartilage.

The matrix appears structureless because the collagen fibres are too fine to be resolved
by light microscopy (~20nm), and because they have about the same refractive index as
the ground substance. Collagen accounts for ~ 40% of the dry weight of the matrix.

The matrix near the isogenous groups of chondrocytes contains larger amounts and
different types of glycosaminoglycans than the matrix further away from the
isogenous groups. This part of the matrix is also termed territorial matrix or capsule.
In H&E stained sections the territorial matrix is more basophilic, i.e. it stains darker.
The remainder of the matrix is called the interterritorial matrix. Fresh cartilage
contains about 75% water which forms a gel with the components of the ground
substance. Cartilage is nourished by diffusion of gases and nutrients through this gel.

Suitable Slides
sections of the trachea or larynx - H&E, van Gieson

Trachea, cat, H&E and Trachea, cat, van Gieson

 59

Both stains are equally well suited to look at the organisation of hyaline cartilage. The
van Gieson method stains collagen red. The cartilage appears as a wide red zone
underneath the epithelium and loose connective tissue, which line the lumen of the
trachea. The staining may appear a little lighter close to the lacunae. This lighter stained
zone defines the territorial matrix surrounding the lacunae and chondrocytes. Colour
intensities appear reversed in the H&E stained section. The two compartments of the
matrix are usually better defined than in van Gieson stained sections. The interterritorial
matrix appears very light; the territorial matrix is somewhat darker. Groups of
chondrocytes surrounded by these lighter (van Gieson) or darker (H&E) staining zones
belong to the same isogenous group. A layer of dense connective tissue surrounding the
cartilage and blending with it is the perichondrium.
The isogenous groups may form small "squares" (e.g. four chondrocytes, separated by
thin cartilage membranes, in a 2x2 arrangement) or short columns (e.g. four
chondrocytes in a 1x4 arrangement).
Draw a small section of the cartilage and identify in your drawing territorial matrix,
interterritorial matrix, isogenous groups, and chondrocytes. Think about how the spatial
arrangement of chondrocytes in the isogenous group may reflect patterns of cell
divisions.
 60

2. Elastic Cartilage

 occurs in the epiglottic cartilage, the corniculate and cuneiform cartilage of

the larynx, the cartilage of the external ear and the auditory tube.
 corresponds histologically to hyaline cartilage, but, in addition, elastic
cartilage contains a dense network of delicately branched elastic fibres.

Suitable Slides
Sections of the epiglottis - elastin

Epiglottis, human, elastin

Preparations of the epiglottis are usually dominated by the cartilage surrounded by
varying amounts of connective tissue and epithelia. The appearance of the cartilage (in
 61

this preparation a blue-green colour) will depend on the method used to show tissue
components other than elastic fibres. Although the matrix appears blue-green, the
typical organisation of cartilage is readily visible. Within the green matrix you can see
the fine elastic fibres which give this cartilage its elastic properties. The elastic fibres
may form dense masses in which individual fibres are difficult to distinguish. The
staining of these masses of fibres may appear more reddish than dark-violet.
A change of the colour of the stain in intensely stained tissue areas is called
"metachromatic staining".
Draw and label a small section of elastic cartilage.

Depending on the quality of tissue preservation on your slide, it may be possible to

identify the types of epithelia present in the section. It wouldn't hurt trying.

3. Fibrous Cartilage
 62

 is a form of connective tissue transitional between dense connective tissue and

hyaline cartilage. Chondrocytes may lie singly or in pairs, but most often they
form short rows between dense bundles of collagen fibres. In contrast to other
cartilage types, collagen type I is dominant in fibrous cartilage.
 is typically found in relation to joints (forming intra-articular lips, discs and
menisci) and is the main component of the intervertebral discs.
 merges imperceptibly into the neighbouring tissues, typically tendons or
articular hyaline cartilage. It is difficult to define the perichondrium because
of the fibrous appearance of the cartilage and the gradual transition to
surrounding tissue types.

Suitable Slides
sections of intervertebral discs or articular discs - H&E, van Gieson

Fibrous Cartilage, Intervertebral Disc, sheep, H&E and Articular Disc, rabbit, H&E
The fibrous cartilage forming the intervertebral discs varies in appearance from the
center of the disc (the nucleus pulposus) the the periphery of the disc (the annulus
fibrosus). Centrally, the fibrous matrix is very loose. The jelly-like consistency of the
central part allows the intervertebral discs to function as a shock absorber. Towards the
periphery, the fibrous matrix is organised into layers. It is often visible that the fibres of
different layers are oriented at angles to each other - similar to the orientation of the
thread in radial tires. Chondrocytes are very flattened in the periphery and may be
difficult to find.
Midway between periphery and center of the intervertebral disc, chondrocytes are
scattered singly or in small isogenous groups in the dense fibrous matrix of the
cartilage. If you take a close look at the cells you will see that their appearance actually
resembles that of chondrocytes in other types of cartilage - their characteristic
appearance distinguishes fibrous cartilage preparations from connective tissues. The
very regular arrangement of the fibres in the articular disc may initially let you guess at
dense regular connective tissue. Isogenous groups of chondrocytes again identify the
tissue as fibrous cartilage.
Draw a small section of the fibrous cartilage, including (if possible) a group of
chondrocytes.
 63

4. Articular Cartilage

 is a specialised form of hyaline cartilage.

 transforms the articulating ends of the bones into lubricated, wear-proof,
slightly compressible surfaces, which exhibit very little friction.
 is not surrounded by a perichondrium and is partly vascularised.
 is, depending on the arrangement of chondrocytes and collagenous fibres,
divided into several zones:

Tangential layer

Chondrocytes are rather small and flattened parallel to the surface. The most
superficial part (lamina splendens) is devoid of cells. Collagen fibres in the
matrix of the tangential layer are very fine. They run parallel to the surface of
 64

the cartilage.

Similar to the collagen fibres of the skin, the general orientation of collagen
fibres in articular cartilage is determined by tensile and compressive forces at
the articulating surfaces.

Transitional zone

The chondrocytes are slightly larger, are round and occur both singly and in
isogenous groups. Collagen fibres take an oblique course through the matrix of
the transitional zone.

Radial zone

Fairly large chondrocytes form radial columns, i.e. the stacks of cells are
oriented perpendicular to the articulating surface. The course of the collagen
fibres follows the orientation of the chondrocyte columns.

Calcified cartilage layer

It rests on the underlying cortex of the bone. The matrix of the calcified
cartilage layer stains slightly darker (H&E) than the matrix of the other layers.

The main source of nourishment for articular cartilage is the synovial fluid, which
fills the joint cavity. Additional small amounts of nutrients are derived from blood
vessels that course through the calcified cartilage close to the bone.
Living chondrocytes have been found in small pieces of cartilage floating in the joint
cavity after damage to the articular cartilage.

Osteoarthritis, the slow progressive degeneration of articular cartilage, is the most

common joint disease. It may be caused by persistent and abnormally high loads on
the joint surfaces, which initially result in the loss of proteoglycans and chondrocytes
from the articulating surface of the cartilage. Subsequently, the cartilage may crack
(fibrillate), erode and expose the underlying bone.

Suitable Slides
Sections of large joints - H&E
Layers are difficult to identify in the articular cartilage of small joints.

Articular Cartilage, bovine, H&E

The layers of articular cartilage are easiest to identify in large joints. Note the changing
orientations of the lacunae and isogenous groups at different depth in the cartilage. The
changing orientations of chondrocytes and isogenous groups reflect the orientations of
the collagen fibres in the matrix. The fibres are not visible in the slide. The darker hue
 65

of the cartilage close to the bone is caused by the calcification of the cartilage.
Draw the articular cartilage at low magnification. Indicate in your drawing the preferred
orientations of lacunae and isogenous groups and the expected orientation of collagen
fibres.

Degeneration and Regeneration of Cartilage

Due to the fairly poor access of nutrients to the chondrocytes they may atrophy in
deep parts of thick cartilage. Water content decreases and small cavities arise in the
matrix, which often leads to the calcification of the cartilage. This further
compromises nutrition. The chondrocytes may eventually die, and the cartilage is
gradually transformed into bone.

Chondrogenic activity of the perichondrium is limited to the period of active growth

before adulthood. Although chondrocytes are able to produce matrix components
throughout life, their production can not keep pace with the repair requirements after
 66

acute damage to hyaline or articular cartilage. If these cartilages are injured after the
period of active growth, the defects are usually filled by connective tissue or fibrous
cartilage. The extracellular matrix of these "repair tissues" is only poorly integrated
with the matrix of the damaged cartilage.

Fortunately, cartilage is rather well suited for transplantation - the metabolism of the
chondrocytes is rather slow, the antigenic power of cartilage is low, and it is difficult,
if not impossible, for antibodies or cells of the immune system to diffuse through the
matrix into the cartilage.

page content and construction: Lutz Slomianka

last updated: 6/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology
Skeletal Tissues - Bone

BONE

Bone is the main component of the skeleton in the adult

human. Like cartilage, bone is a specialized form of dense
connective tissue. Bone gives the skeleton the necessary rigidity
to function as attachment and lever for muscles and supports
the body against gravity.

Two types of bone can be distinguished macroscopically:

 67

 Trabecular bone
(also called
cancellous or
spongy bone)
consists of
delicate bars and
sheets of bone,
trabeculae,
which branch
and intersect to
form a sponge
like network.
The ends of long
bones (or
epiphyses)
consist mainly of
trabecular bone.
 Compact bone
does not have
any spaces or
hollows in the
bone matrix that
are visible to the
eye. Compact
bone forms the
thick-walled
tube of the shaft
(or diaphysis) of
long bones,
which surrounds
the marrow
cavity (or
medullary
cavity). A thin
layer of compact
bone also covers
the epiphyses of
long bones.
 68

Bone is, again like

cartilage, surrounded
by a layer of dense
connective tissue, the
periosteum. A thin
layer of cell-rich
connective tissue, the
endosteum, lines the
surface of the bone
facing the marrow
cavity. Both the
periosteum and the
endosteum possess
osteogenic potency.
Following injury, cells
in these layers may
differentiate into
osteoblasts (bone
forming cells) which
become involved in the
repair of damage to the
bone.

Histological Organisation of Bone

Compact Bone

Compact bone consists almost entirely of extracellular substance,

the matrix. Osteoblasts deposit the matrix in the form of thin
sheets which are called lamellae. Lamellae are microscopical
structures. Collagen fibres within each lamella run parallel to
each other. Collagen fibres which belong to adjacent lamellae
run at oblique angles to each other. Fibre density seems lower at
the border between adjacent lamellae, which gives rise to the
lamellar appearance of the tissue. Bone which is composed by
lamellae when viewed under the microscope is also called
lamellar bone.
 69

In the process of the deposition of the matrix, osteoblasts become

encased in small hollows within the matrix, the lacunae. Unlike
chondrocytes, osteocytes have several thin processes, which
extend from the lacunae into small channels within the bone
matrix , the canaliculi. Canaliculi arising from one lacuna may
anastomose with those of other lacunae and, eventually, with
larger, vessel-containing canals within the bone. Canaliculi
provide the means for the osteocytes to communicate with each
other and to exchange substances by diffusion.

In mature compact bone most of the individual lamellae form

concentric rings around larger longitudinal canals (approx. 50
µm in diameter) within the bone tissue. These canals are called
Haversian canals. Haversian canals typically run parallel to
the surface and along the long axis of the bone. The canals and
the surrounding lamellae (8-15) are called a Haversian system
or an osteon. A Haversian canal generally contains one or two
capillaries and nerve fibres.

Irregular areas of interstitial lamellae, which apparently do not

belong to any Haversian system, are found in between the
Haversian systems. Immediately beneath the periosteum and
endosteum a few lamella are found which run parallel to the
inner and outer surfaces of the bone. They are the circumferential
lamellae and endosteal lamellae.

A second system of canals, called Volkmann's canals,

penetrates the bone more or less perpendicular to its surface.
These canals establish connections of the Haversian canals with
the inner and outer surfaces of the bone. Vessels in Volkmann's
canals communicate with vessels in the Haversian canals on the
one hand and vessels in the endosteum on the other. A few
communications also exist with vessels in the periosteum.

Trabecular Bone

The matrix of trabecular bone is also deposited in the form of

lamellae. In mature bones, trabecular bone will also be
lamellar bone. However, lamellae in trabecular bone do not form
 70

Haversian systems. Lamellae of trabecular bone are deposited on

preexisting trabeculae depending on the local demands on bone
rigidity.

Osteocytes, lacunae and canaliculi in trabecular bone resemble

those in compact bone.

? Note the distinction between macroscopic (visible to the eye)

and microscopic (only visible under the microscope) appearance
when the bone is named. Lamellar bone forms both trabecular
bone and compact bone, which are the two macroscopically
recognizable bone forms.

Suitable Slides
sections of compact bone (usually part of the diaphysis of
a long bone) - ground (unstained), Schmorl stained or
H&E

Compact bone, human - Schmorl stain

Lamellae which run parallel to the surface of the bone are visible
both on the outer, convex surface of the bone (circumferential
lamellae) and on the inner, concave surface of the bone facing
the marrow cavity (endosteal lamellae). The surface formed by
the endosteal lamellae is often more irregular than the surface
formed by the circumferential lamellae. The space between these
two sets of lamellae is filled by Haversian systems and
interstitial lamellae. Only few of the Haversian systems are
"textbook" circular. Osteocyte lacunae are visible between the
lamellae. Canaliculi become visible at high magnification
(illustrated in the ground section below).
You should be able to see, draw and identify Haversian systems,
interstitial and circumferential lamellae and/or endosteal
lamellae.
 71

Compact bone, human - ground (unstained)

The osteocyte lacunae are the main feature of the ground section.
They are visible as elongated black spots in the bone matrix.
Canaliculi, radiate from the lacunae into the surrounding bone
matrix. Some lamellae are visible in the ground section. There is
actually no distinct border between most lamellae, but our brain
can use the elongated osteocyte lacunae and their orientation to
"reconstruct" the lamellae. Volkman's canals connect to a few of
the Haversian canals.
This is one of the cases where it may pay off to close down the
iris diaphragm. The canaliculi should stand out more clearly if
you do so. Remember to open the diaphragm afterwards!
Draw a small section of the a Haversian system at high
magnification and identify in your drawing lacunae, canaliculi
and (if visible) lamellae.
 72

Suitable Slides
sections of part of a vertebra or an epiphysis of a long
bone - H&E, van Gieson
Sections prepared to show articular cartilage will often
also contain trabecular bone in the epiphysis below the
articular cartilage.

Articular cartilage, bovine - H&E

Thin sheets and bars of bone, trabeculae, are visible at low
magnification. Although they may appear as individual pieces in
sections, they form an interconnected meshwork in the living
bone. The spaces between the trabeculae, the marrow cavity of
the epiphysis, is filled by either red bone marrow or yellow bone
marrow. At high magnification, elongated osteocyte lacunae,
which in well preserved tissue still contain osteocytes, are visible
in the matrix. If the H&E stain also turned out well, it should be
visible that the matrix of the trabecular bone is formed by
 73

lamellae. Canaliculi are present but hard to identify in most H&E

stained sections. Haversian systems are not present in the
trabeculae.
In trabecular bone obtained from young individuals, in which the
bone is still growing, small areas of calcified cartilage are
occasionally seen in the bone trabeculae. They are remnants of
the cartilage scaffold on which bone matrix was deposited during
the formation of the trabeculae. With the reorganisation of bone
such areas will eventually be lost.
Draw trabecular bone at high and low magnification. Make sure
that it is visible that the trabecular bone is also lamellar bone.
Include enough detail of the marrow as you think you will need
to identify this type of preparation.

Bone Matrix and Bone Cells

Bone Matrix
 74

Bone matrix consists of collagen fibres (about 90% of the

organic substance) and ground substance. Collagen type I is
the dominant collagen form in bone. The hardness of the
matrix is due to its content of inorganic salts (hydroxyapatite;
about 75% of the dry weight of bone), which become deposited
between collagen fibres.

Calcification begins a few days after the deposition of organic

bone substance (or osteoid) by the osteoblasts. Osteoblasts are
capable of producing high local concentration of calcium
phosphate in the extracellular space, which precipitates on the
collagen molecules. About 75% of the hydroxyapatite is
deposited in the first few days of the process, but complete
calcification may take several months.

Bone Cells

1. Osteoprogenitor cells (or stem cells of bone)

are located in the periosteum and endosteum. They are

very difficult to distinguish from the surrounding
connective tissue cells. They differentiate into

2. Osteoblasts (or bone forming cells).

Osteoblasts may form a low columnar "epitheloid layer" at

sites of bone deposition. They contain plenty of rough
endoplasmatic reticulum (collagen synthesis) and a large
Golgi apparatus. As they become trapped in the forming
bone they differentiate into

3. Osteocytes.

Osteocytes contain less endoplasmatic reticulum and are

somewhat smaller than osteoblasts.

4. Osteoclasts
 75

are very large

(up to 100 µm),
multi-nucleated
(about 5-10
visible in a
histological
section, but up to
50 in the actual
cell) bone-
resorbing cells.
They arise by the
fusion of
monocytes
(macrophage
precursors in the
blood) or
macrophages.
Osteoclasts
attach
themselves to
the bone matrix
and form a tight
seal at the rim of
the attachment
site. The cell
membrane
opposite the
matrix has deep
invaginations
forming a ruffled
border.
Osteoclasts
empty the
contents of
lysosomes into
the extracellular
space between
the ruffled
border and the
 76

bone matrix. The

released
enzymes break
down the
collagen fibres
of the matrix.
Osteoclasts are
stimulated by
parathyroid
hormone
(produced by the
parathyroid
gland) and
inhibited by
calcitonin
(produced by
specialised cells
of the thyroid
gland).
Osteoclasts are
often seen within
the indentations
of the bone
matrix that are
formed by their
activity
(resorption bays
or Howship's
lacunae).

Formation of Bone

Bones are formed by two mechanisms: intramembranous

ossification (bones of the skull, part of the mandible and
clavicle) or endochondral ossification.

1. Intramembranous Ossification
 77

Intramembranous ossification occurs within a membranous,

condensed plate of mesenchymal cells. At the initial site of
ossification (ossification centre) mesenchymal cells
(osteoprogenitor cells) differentiate into osteoblasts. The
osteoblasts begin to deposit the organic bone matrix, the osteoid.
The matrix separates osteoblasts, which, from now on, are
located in lacunae within the matrix. The collagen fibres of the
osteoid form a woven network without a preferred orientation,
and lamellae are not present at this stage. Because of the lack of
a preferred orientation of the collagen fibres in the matrix, this
type of bone is also called woven bone. The osteoid calcifies
leading to the formation of primitive trabecular bone.

Further deposition and calcification of osteoid at sites where

compact bone is needed leads to the formation of primitive
compact bone.

? Note the distinction between macroscopic and microscopic

appearance when the bone is named. We again have the two
macroscopically different forms of bone - trabecular bone and
compact bone - but their early developmental ("primitive") forms
consist of woven bone.

Through subsequent reorganisation the primitive compact and

trabecular bone is converted into mature compact and trabecular
bone. During reorganisation and growth, woven bone will, in
time, be replaced by lamellar bone.

Intramembranous ossification does not require the existence of

a cartilage bone model.

Suitable Slides
sections of the developing mandible (or some other bones
of the skull) or clavicle - H&E, van Gieson
Sections prepared to show endochondral ossification (see
below) may be an alternative if no specifically prepared
slides of intramembranous ossification are available. The
periosteal collar, i.e. the manchette of bone forming
 78

around the diaphysis of the cartilage model of the bone, is

formed by a mechanism similar to intramembranous
ossification and results in the deposition of woven bone.

Mandible, intramembranous ossification - H&E

The developing bone will in sections usually be associated with a
number of other tissues which develop in close association with
it. In case of the mandible, there can be developing teeth, the
tongue, skin and salivary glands.
The first job - best done at low magnification - is to find the
developing bone. It should look like a coarse meshwork
(trabecular bone) of pink tissue surrounding patches of much
lighter or unstained tissue. Lamellae are not visible (woven
bone) and the lacunae are larger than lacunae in mature bone.
Ossification centres appear as areas of a gradual transition from
connective tissue to bone. Light, pinkish bone matrix is found
between the osteoblasts.
Depending on the state of development of the bone, it is
occasionally possible to find bone trabeculae which are lined by
a layer of osteoblasts. These osteblasts are depositing the first
lamellae on the already existing trabeculae. The trabeculae will
therefore have a core of woven bone, which is surrounded by
lamellar bone. Compare the shapes, sizes and frequencies of
lacunae in lamellar and woven bone if both types of bone are
present.
Draw a part of trabecular bone consisting of woven bone and an
ossification centre. Label the features in your drawings.
 79

2. Endochondral Ossification

Most bones are formed by the transformation of cartilage

"bone models", a process called endochondral ossification. A
periosteal bud invades the cartilage model and allows
osteoprogenitor cells to enter the cartilage. At these sites, the
cartilage is in a state of hypertrophy (very large lacunae and
chondrocytes) and partial calcification, which eventually leads to
the death of the chondrocytes.
Invading osteoprogenitor cells mature into osteoblasts, which use
the framework of calcified cartilage to deposit new bone. The
bone deposited onto the cartilage scaffold is lamellar bone. The
initial site of bone deposition is called a primary ossification
centre. Secondary ossification centres occur in the future
epiphyses of the bone.
A thin sheet of bone, the periosteal collar, is deposited around
the shaft of the cartilage model. The periosteal collar consists of
woven bone.
 80

Close to the zone of ossification, the cartilage can usually be

divided into a number of distinct zones :

1. Reserve cartilage, furthest away from the zone of

ossification, looks like immature hyaline cartilage.
2. A zone of chondrocyte proliferation contains longitudinal
columns of mitotically active chondrocytes, which grow in
size in
3. the zone of cartilage maturation and hypertrophy.
4. A zone of cartilage calcification forms the border between
cartilage and the zone of bone deposition.

Primary and secondary ossification centres do not merge before

adulthood. Between the diaphysis and the epiphyses a thin sheet
of cartilage, the epiphyseal plate, is maintained until adulthood.
By continuing cartilage production, the epiphyseal plate provides
the basis for rapid growth in the length of the bone. Cartilage
production gradually ceases in the epiphyseal plate as maturity is
approached. The epiphyseal plate is finally removed by the
continued production of bone from the diaphyseal side.

Bone formation and bone resorption go hand in hand during the

growth of bone. This first deposited trabecular bone is removed
(By which cells?) as the zone of ossification moves in the
direction of the future epiphyses. This process creates the
marrow cavity of the bones. Simultaneously, bone is removed
from the endosteal surface and deposited on the periosteal
surface of the compact bone which forms the diaphysis. This
results in a growth of the diameter of the bone.

Suitable Slides
sections of bones during the early stages of their
development - H&E, van Gieson

Foetal vertebral column, human - H&E

Hold the section against a light background. The cartilage
models will stain very light, and the outlines of the skeletal
structures they will form can often be identified. If an
 81

ossification centre is present, it will appear as a darker area

within the cartilage model. The zonation of the cartilage should
be visible in all ossification centres. How much bone is present
depends on how far ossification has proceeded. The newly
formed bone trabeculae will often consist of lamellar bone with a
more or less extensive core of darkly staining calcified cartilage.
The lamellar organisation of the bone may not be visible.
How the ossification centre exactly will look also depends on the
plane of the section in relation to the ossification centre.
Draw the ossification centre/zone, include the adjacent cartilage
and identify the zones within it in your drawing.

Suitable Slides
sections of the epiphyseal disc of growing bones - H&E
 82

Growing bone, rabbit -

H&E
The cartilage model
has almost entirely
been transformed into
bone. The only
remaining cartilage is
found in the epiphyseal
disk. Zones of cartilage
proliferation,
hypertrophy and
calcification are visible
at high magnification,
but only on one side of
the epiphyseal disk -
towards the diaphysis,
which increases in
length as the cartilage
generated by the
epiphyseal disc is
transformed into bone.
Osteoclasts may be
found on the newly
formed trabeculae or
associated with parts of
the cartilage scaffold.
Draw the region of the
epiphyseal disks.
Identify in your
drawing the epiphyseal
disk and the bone of
the epiphysis and
diaphysis. Indicate the
direction of cartilage
proliferation in the
epiphyseal disk and the
direction of bone
growth.
 83

more about .... Bone

Reorganisation and Dynamics
Restoration of Bone by Lis Mosekilde, M.D.,
D.Med.Sc.

Changes in the size and shape of bones during the period of

growth imply some bone reorganisation. Osteoblast and
osteoclast constantly deposit and remove bone to adjust its
properties to growth-related demands on size and/or changes of
tensile and compressive forces.

Although the reorganisation of bone

may not result in macroscopically
visible changes of bone structure, it
continues throughout life to mend
damage to bone (e.g. microfractures)
and to counteract the wear and tear
occurring in bone. Osteoclasts and
osteoblasts remain the key players in this process. Osteoclasts
"drill" more or less circular tunnels within existing bone matrix.
Osteoblasts deposit new lamellae of bone matrix on the walls of
these tunnels resulting in the formation of a new Haversian
system within the matrix of compact bone. Parts of older
Haversian systems, which may remain between the new ones,
represent the interstitial lamellae in mature bone. Capillaries and
nerves sprout into new Haversian canals.

Restorative activity continues in aged humans (about 2% of the

Haversian systems seen in an 84 year old individual contained
lamellae that had been formed within 2 weeks prior to death!).
However, the Haversian systems tend to be smaller in older
individuals and the canals are larger because of slower bone
deposition. If these age-related changes in the appearance of the
Haversian systems are pronounced they are termed osteopenia or
senile osteoporosis. The reduced strength of bone affected by
osteoporosis will increase the likelihood of fractures.
 84

page content and construction: Lutz Slomianka

last updated: 5/08/09

School of Anatomy and Human Biology - The

University of Western Australia

Blue Histology - Blood

BLOOD
Blood is sometimes considered to be a fluid connective tissue because of
the mesenchymal origin of its cells and a low ratio of cells to liquid
intercellular substance, the blood plasma. In human adults about 5 liter
of blood contribute 7-8 % to the body weight of the individual. The
contribution of red blood cells (erythrocytes) to the total volume of the
blood (haematocrit) is about 43%.

Erythrocytes are the dominant (99%) but not the only type of cells in the
blood. We also find leukocytes and, in addition, blood platelets.
Erythrocytes, leukocytes and blood platelets are also being referred to as the
formed elements of the blood. Erythrocytes and blood platelets perform their functions
exclusively in the blood stream. In contrast, leukocytes reside only
temporarily in the blood. Leukocytes can leave the blood
stream through the walls of capillaries and venules and enter
either connective or lymphoid tissues.

Erythrocytes
 85

Erythrocytes do not contain a nucleus. They do contain

haemoglobin, which fills almost the entire cytoplasm. Erythrocytes are
unable to move actively, but they are remarkably elastic and can withstand
deformation. They are typically biconcave disks although their shape is
influenced by osmotic forces. The average diameter of the disk is ~7 µm.
Since erythrocytes can be found in the vast majority of histological sections
- in small numbers even in perfused tissues - they will often allow us to
estimate the size of other structures or cells. Mature erythrocytes do not
contain organelles, and their cytoplasm looks fairly homogenous - even in
the EM
At high magnification some granularity may be visible in EM images.
The granular appearance is caused by haemoglobin molecules.
Foetal erythrocytes (up to the 4th month of gestation) are larger than
"adult" erythrocytes, and they are nucleated. The later feature they
share with erythrocytes of other animal classes (e.g. amphibia and
birds).

Functions

Erythrocytes function in the transport of oxygen. Haemoglobin, the

oxygen binding protein in erythrocytes, contributes about 30% of the
weight of an erythrocyte. The lifespan of an erythrocyte in the
bloodstream is 100-120 days.
About 5×1011 erythrocytes are formed/destroyed each day.

Suitable Slides
blood smear - Leishman, Wright's, Giemsa or May-
Grünwald-Giemsa stains
? Where to look for cells in a
blood smear
The density of cells varies
across the smear. Cells will be
"heaped and piled" close to the
point were the drop of blood was
placed on the slide. White blood
cells appear shrunken, and
some types are difficult to
distinguish from each other.
 86

There are fewer cells close to

the tip of the smear. In this
region, white blood cells are
sometimes damaged and
erythrocytes may be deformed.
The best area to look at is
between these two regions.
Where it is located exactly and
how wide it is will depend on the
smear, but the middle of the
smear is a good starting point.

Blood Smear, human - Leishman

stain
How does the shape of the
erythrocyte facilitate its
function? How would you expect
an erythrocyte to look like if it is
in an extracellular fluid of very
low or very high osmotic
pressure?
Identify and draw a few
erythrocytes.

It is a good idea to do one large

composite drawing for all types of
blood cells.

Leukocytes

Leukocytes can be further subdivided into granular leukocytes, i.e.

neutrophils, basophils and eosiniphils, and non-granular leukocytes, i.e.
monocytes and lymphocytes.

In healthy individuals the relative numbers of circulating leukocyte types

 87

are quite stable. A differential leukocyte count would typically produce the
following cell frequencies (numbers in parentheses are the range of
normal frequencies reported in different texts):

 ~ 60% neutrophils (50% - 70%)

 ~ 3% eosinophils (>0% - 5%)
 ~ 0.5% basophils (>0% - 2%)
 ~ 5% monocytes (1% - 9%)
 ~ 30% lymphocytes (20% - 40%)

Changes in their relative numbers indicate that something abnormal is

happening in the organism. A larger than usual number of neutrophils
(neutrophilia) would indicate e.g. an acute or chronic infection. The
number of basophils and eosinophils may increase (eosinophilia or
basophilia) as a consequence of e.g. allergic disorders.

Granular Leukocytes

Granular leukocytes are all approximately the same size - about 12-15 µm
in diameter. Their nuclei form lobes, and nucleoli cannot be seen. The
number of nuclear lobes varies according to cell type. All granulocytes are
motile.

The term granulocytes refers to the presence of granules in the cytoplasm

of these cells. The granules correspond to secretory vesicles and lysosomes.
Specific granules are the granules which are only found in one particular
type of granulocytes.

Neutrophil granulocytes (or neutrophils)

have a very characteristic nucleus. It is divided into 3-5 lobes

which are connected by thin strands of chromatin. The
number of lobes increases with cell age. Up to 7 lobes can be
found in very old neutrophils (hypersegmented cells).

Neutrophils (like all other granulocytes, monocytes and

lymphocytes) contain all the organelles that make up a typical
cell. In addition to the usual complement of organelles, they
also contain two types of granules . Primary granules (or
A granules) contain lysosomal enzymes and are likely to be
 88

primary lysosomes, although they are larger (0.4 µm) than the
"ordinary" primary lysosome. Secondary granules (or B
granules), the specific granules of the neutrophils, contain
enzymes with strong bactericidal actions. The specific
granules of neutrophils stain only weakly if they are at all
visible - they are "neutral", hence the term neutrophil.

Functions

Neutrophils play a central role in inflammatory processes. Large

numbers invade sites of infection in response to factors (e.g.
cytokines) released by cells which reside at an infection site.
Neutrophils are the first wave of cells invading infection sires.
Receptors in their plama membrane allow them to recognise foreign
bodies, e.g. bacteria, and tissue debris, which they begin to
phagocytose and destroy. The phagocytotic activity of neurophils is
further stimulated if invading microorganisms are "tagged" with
antibodies (or opsonised). Neutrophils cannot replenish their store of
granules. The cells die once their supply of granules has been
exhausted. Dead neutrophils and tissue debris are the major
components of pus. Their lifespan is only about one week.

Lost neutrophils are quickly replenished from a reserve population

in the bone marrow. Because they are younger, their nuclei have
fewer lobes than the "average" neutrophil. A high proportion of
neutrophils, with few nuclear lobes indicates a recent surge in their
release from the bone marrow.

Suitable Slides
see lab section on erythrocytes
 89

Blood Smear, human -

Leishman stain
Neutrophil granulocytes
are easy to find. They
are the most frequent
type of white blood
cells, and the complex
shape of their nucleus
identifies them
unequivocally. In darkly
stained smears it is
possible to see some
faintly purple, very
small granules in the
cytoplasm. These
granules represent the
primary granules of
neutrophils.
Have a close look at
the nucleus of a
number of neutrophils,
and make a qualified
guess at the gender of
the individual, which
donated blood for the
slides.
Identify and draw one
or two neutrophil
granulocytes.

How the neutrophils

and other leukocytes
exactly will look like
depends somewhat on
how the stain turned
out. In some batches
(labeled B2 in the
images) nuclei are
dark and crisp, and
 90

the cytoplasm is well

demarcated. In other
batches (labeled B1 in
the images) nuclei and
cytoplasm are lighter
and their boundaries
are less well defined.
While the morphology
appears clearer in the
darker stained
smears, it will usually
be more difficult to
identify eosinophils
and basophils (see
below).

Eosinophil granulocytes (or eosinophils)

Their nucleus usually has only two lobes. Almost all of the
cytoplasm appears filled with the specific granules of the
eosinophils. As the term "eosinophil" indicates, these granules are
not neutral but stain red or pink when eosin or a similar dye is used
in the staining process. Aside from the usual complement of
organelles eosinophils contain some large rounded vesicles (up to 1
µm) in their cytoplasm . These granules correspond to the
eosinophilic grains that we see in the light microscope. The specific
granules contain, in addition to enzymes that otherwise are found in
lysosomes, an electron-dense, proteinaceous crystal. This crystal is
composed of major basic protein (MBP).

Functions

The presence of antibody-antigen complexes stimulates the immune

system. Eosinophils phagocytose these complexes and this may prevent
the immune system from "overreacting". Their granules also contain
the enzymes histaminase and arylsufatase. These enzymes break down
histamine and leukotrienes, which again may dampen the effects of their
release by basophils or mast cells. MBP, which can also function as a
cytotoxin, and its release by eosinophils may be involved in the response
 91

of the body against parasitic infections, which are accompanied by an

increase in the number of eosinophils.

Suitable Slides
see lab section on erythrocytes

Blood Smear, human - Leishman stain

Eosinophils and basophils are the only cell types present in normal blood
which initially may be difficult to distinguish - in particular in darker
smears. If you see them side by side in your drawing the difference between
them should become apparent. Chances are 6:1 that the you find an
eosinophil before you find a basophil. The two lobes of the nucleus of
eosinophils are usually well-defined and of about equal size. The nucleus is
embedded in a cytoplasm crowded with granules, which seem to form a
solid mass in the cell. The 2-3 nuclear lobes of basophils are not as well
defined as those of eosinophils, granules are not as numerous as in
eosinophils, and pretty much all of them can be identified "as individuals"
rather than the dense mass they form in eosinophils.
Note that eosinophils and basophils are much easier to distinguish in
B1. In B2, the difference in the staining of their of granules is not as
pronounced, and the nuclei do not stand out as clear as in B1.
Identify, draw and label an eosinophil and a basophil.
 92

Basophil granulocytes (or basophils)

Basophilic granulocytes have a 2 or 3 lobed nucleus. The lobes are

usually not as well defined as in neutrophilic granulocytes and the
nucleus may appear S-shaped. The specific granules of basophils are
stained deeply bluish or reddish-violet. Their colour corresponds
closely to the colour of the nucleus which sometimes is
difficult to see amongst or behind the granules. The granules
are not as numerous as those in eosinophils. The specific granules of
basophils (about 0.5 µm) appear quite dark in EM pictures. They
contain heparin, histamine lysosomal enzymes and leukotrienes
(the later correspond to the slow-reacting substance of
anaphylaxis or SRS-A).

Functions

Heparin and histamine are vasoactive substances. They dilate the blood
vessels, make vessel walls more permeable and prevent blood
coagulation. As a consequence, they facilitate the access of other
lymphocytes and of plasma-borne substances of importance for the
 93

immune response (e.g. antibodies) to e.g. a site of infection. The

release of the contents of the granules of basophils is receptor-
mediated. Antibodies produced by plasma cells (activated B-
lymphocytes; see below) bind to Fc-receptors on the plasma
membrane of basophils. If these antibodies come into contact with
their antigens, they induce the release of the contents of the basophil
granules.

Non-granular leukocytes

Monocytes

These cells can be slightly larger than granulocytes (about 12-18

µm in diameter). Their cytoplasm stains usually somewhat stronger
than that of granulocytes, but it does not contain any structures
which would be visible in the light microscope using most
traditional stains (a few very fine bluish gains may be visible in
some monocytes). The "textbook" monocyte has a C-shaped
nucleus. Monocytes contain granules (visible in the EM) which in
appearance and content correspond to the primary granules of
neutrophils, i.e. the granules correspond to lysosomes.

Functions

Once monocytes enter the connective tissue they differentiate into

macrophages. At sites of infection macrophages are the dominant
cell type after the death of the invading neutrophils. The
phagocytose microorganisms, tissue debris and the dead neutrophils.
Monocytes also give rise to osteoclasts, which are able to dissolve bone.
They are of importance in bone remodelling.

Suitable Slides
see lab section on erythrocytes

Blood Smear, human - Leishman stain

Monocytes and lymphocytes definitely look much prettier in darker stained
smears (B2) than in lighter ones (B1) - mainly because of a clearer
 94

distinction between cytoplasm and nucleus. The cell is very likely to be a

lymphocyte if the nucleus is round and surrounded by a narrow rim of
cytoplasm. The C-shaped nucleus of a textbook monocyte may not be easy
to find. The nuclei will vary from a peanut- to a "fat" S-shape in smears.
Whatever the shape of the nucleus, it is usually not lobed, and it is bound, at
least on its concave side, by a wide rim of non-granular cytoplasm. Note
also the light area of monocyte cytoplasm which is often visible close to the
concave surface of the nucleus. The Golgi apparatus is located in the area.
The Golgi apparatus does not stain as well as the remainder of the
cytoplasm and leaves a light "impression" - the phenomenon is also
called a "negative image".
Identify and draw lymphocytes and a monocyte.

Lymphocytes

These cells are very variable in size. The smallest may be smaller
than erythrocytes (down to ~5 µm in diameter) while the largest may
reach the size of large granulocytes (up to 15 µm in diameter). How
much cytoplasm is discernible depends very much on the size of the
lymphocyte. In small ones, which are the majority of lymphocytes in
 95

the blood, the nucleus may appear to fill the entire cell. Large
lymphocytes have a wider rim of cytoplasm which surrounds the
nucleus. Both the nucleus and the cytoplasm stain blue (and
darker than most other cell types in the blood) . The typical
lymphocyte only contains the usual complement of cellular
organelles. The appearance of lymphocytes may change drastically
when they are activated (see below).

Functions

Most lymphocytes in the blood stream belong to either the group of

B-lymphocytes (~5%) or the group of T-lymphocytes (~90%). Unless
they become activated, the two groups can not easily be
distinguished using routine light or electron microscopy.
Upon exposure to antigens by antigen-presenting cells (e.g.
macrophages) and T-helper cells (one special group of T-lymphocytes)
B-lymphocytes differentiate into antibody producing plasma cells. The
amount of cytoplasm increases and RER fills a large portion of the
cytoplama of plama cells . T-lymphocytes represent the "cellular
arm" of the immune response (cytotoxic T cells) and may attack foreign
cells, cancer cells and cells infected by e.g. a virus.

T-lymphocytes and B-
lymphocytes form the vast
majority of lymphocytes in
the blood stream, but they
do not add up to 100%,
and they usually are small
lymphocytes. The much
less frequent medium-
sized or large lymphocytes
may represent e.g.

 natural killer
(Nk-) cells
which belong
to the group
of large
granular
 96

lymphocytes,
or
 haemopoietic
stem cells of
which a few
will be
circulating in
the blood
stream.

Blood Platelets (or thrombocytes)

Blood platelets do not contain a nucleus. Unlike erythrocytes, which

also lack a nucleus, the blood platelets of mammals have never been
nucleated cells. Instead, blood platelets are fragments of the cytoplasm
of very large thrombocyte precursor cells, megakaryocytes. Like other
cells involved in the formation in blood cells, megakaryocytes are
found in the bone marrow.

Platelets are about 3 µm long but appear somewhat smaller in the

microscope. This is because their cytoplasm is divided into two zones:
and outer hyalomere, which hardly stains, and an inner granulomere,
which contains bluish staining granules. These granules are
usually not individually visible with the highest magnification
on your microscope, and the granulomere appears more or
less homogeneously blue. In addition to different types of
vesicles (i.e. the granules), mitochondria, ribosomes, lysosomes and
a little ER are present in the thrombocyte granulomere. Different types
of vesicles contain either serotonin (electron-dense delta granules;
few) or compounds important for blood coagulation (alpha granules -
they also contain platelet-derived growth factor (PDGF) which
may play a role in the repair of damaged tissue). The hyalomere
contains cytoskeletal fibres, which include actin and myosin.

Functions

Platelets assist in haemostasis, the arrest of bleeding. Serotonin is a

potent vasoconstrictor. The release of serotonin from thrombocytes,
which adhere to the walls of a damaged vessels, is sufficient to close
 97

even small arteries. Platelets, which come into contact with

collagenous fibers in the walls of the vessel (which are not usually
exposed to the blood stream), swell, become "sticky" and
activate other platelets to undergo the same transformation. This
cascade of events results in the formation of a platelet plug (or
platelet thrombus). Finally, activating substances are released from
the damaged vessel walls and from the platelets. These substances
mediate the conversion of the plasma protein prothrombin into
thrombin. Thrombin catalyzes the conversion of fibrinogen into fibrin,
which polymerizes into fibrils and forms a fibrous net in the arising
blood clot. Platelets captured in the fibrin net contract leading to clot
retraction, which further assists in haemostasis.

Blood coagulation is a fairly complex process, which involves

a large number of other proteins and messenger substances.
Deficiencies in any one of them, either inherited or acquired,
will lead to an impairment of haemostasis.

Suitable Slides
blood platelets (thrombocytes) : see lab section on
erythrocytes
megakaryoblasts and megakaryocytes: see lab section on
haemopoiesis
 98

Blood Smear, human - Leishman

stain
In lightly stained smears (B1),
blood platelets will appear like light
blue, fairly ill-defined specks
between the other blood cells. In
darker smears (B2), you will be
able to see that the blue specks are
formed by an accumulation of
small bluish grains, the granules of
the blood platelets.
Identify and include a platelet in
one of your other drawings.

Red Bone Marrow, rabbit - H&E

The marrow cavity of this bone
is filled with red bone marrow.
H&E is not the method of choice
for looking at haemopoietic
cells, but a few of the numerous
named types or broader groups
can actually be recognized.
Precursors of platelets are the
haemopoietic cells easiest to find in
red bone marrow. The very dark
and large megakaryoblast and the
even larger but light
megakaryocytes are clearly visible
even at low magnifications. Note
that adipocytes are present also in
the red bone marrow.
Identify and draw a
megakaryocyte and
megakaryoblast.
 99

Haemopoiesis

During foetal development, the formation of blood cells (haemopoiesis)

commences in wall of the yolk sac. After the second month of foetal
development, the liver, and, slightly later, the spleen, become the dominant
sites of haemopoiesis. From the 6th month, and dominating from the
7th month onwards, the formation of blood cells occurs in bone
marrow, which is the major site of formation blood cells in normal
adult humans.

Yellow bone marrow, which harbours mainly adipocytes, dominates in the

hollow of the diaphysis of adult long bones. Haemopoiesis occurs in red bone
marrow, which is typically found between the trabeculae of spongy bone in the
epiphysis of adult long bones. Both age and demands on haemopoiesis may
effect the relative amounts of red and yellow bone marrow. Haemopoietic cells
surround the vascular sinusoids and are supported by reticular connective
tissue. In addition to the endothelial cells of the sinusoids and the reticulocytes
of the connective tissue, macrophages are frequent in red bone marrow.
 100

Haemopoietic Cells

The basis of haemopoiesis is a small population of self-replicating stem cells,

which ultimately can generate all types of blood cells. Their progeny may
develop into either lymphocytic stem cells or pluripotent haemal stem
cells (colony-forming unit - stem cell - CFU-S). The latter type gives rise
to stem cells which can form the major groups of blood cells other than
lymphocytes. Depending on their progeny it is possible to differentiate

 burst-forming unit of the erythroid line (BFU-E),

 colony-forming unit - granulocytes and macrophages (CFU-G/M), and
 colony-forming unit - megakaryocytes (CFU-Mk).

Erythrocytes
The first
identifiable stage of
erythropoiesis is the
proerythroblast - a
large, slightly
basophilic cell,
which contains a
large, lightly
stained nulceus.
Proerythroblasts
proliferate to
generate a sequence
of cells which show
a gradual decrease
in size and
condensation of
their chromatin.
They are named
after changes in
the staining
characteristic of
their cytoplasm
(basophilic
erythroblast,
polychromatophili
 101

c and
orthochromic
normoblasts). The
nucleus is finally
extruded from the
normoblast. The
cell enters
circulation as a
reticulocyte, which
still contains some
organelles.
Reticulocytes
remain for a few
days in either the
bone marrow or the
spleen to mature to
erythrocytes.
In some blood
smears
reticulocytes may
be recognisable
because of a very
slight basophilic
staining - either
homogeneous or
in the form of a
basophilic
stippling.
Granulocytes
Myeloblast appear light-microscopically similar to proerythroblast.
They proliferate to generate promyelocytes. Promyelocytes begin to
accumulate nonspecific granules, but they are still able to divide.
The maturation of their progeny, the myelocytes, is characterised by
the accumulation of specific granules and changes in nuclear
morphology. Metamyelocytes have a C-shaped nucleus.

Blood Platelets (Thrombocytes)

are, as mentioned above, fragments of the cytoplasm of
megakaryocytes. Megakaryocytes are very large cells (up to 160 µm
 102

in diameter), which contain very large, highly lobulated, polyploid

nuclei. Megakaryocytes are in turn the product of the differentiation
of basophilic megakaryoblasts.

Precursors of blood cells which are usually only found in the bone marrow
can be found in peripheral blood in a variety of pathological conditions.
If a Rh-negative mother has been immunised by erythrocytes of a
Rh-positive foetus, a condition called Erythroblastosis fetalis may
develop during subsequent pregnancies. It would show itself in the
foetus or newborn by the presence of erythrocyte precursors in
peripheral blood - although other, more severe symptoms should be
obvious. Chronic myeloid leukemia is another condition - in this case
showing itself by the presence of all types of granulocyte precursors
in peripheral blood.

The nomenclature employed for haemopoietic cells (but not the

number of stages recognized) is somewhat variable across texts.
Note also that these cell types refer to stages of development along
a morphologically more or less continuous spectrum.

Suitable Slides
sections of red bone marrow - H&E or a bone marrow smear
- Leishman, Wright's, Giemsa or May-Grünwald-Giemsa
stains

Red Bone Marrow, rabbit - H&E

Most of the haemopoietic cells visible will be of the erythroblastic
line. The only cell type of this line which is easy to distinguish in H&E
stained sections are normoblasts.
A very condensed nucleus is seen in late (orthochromic) normoblast.
Granulocyte and erythrocyte precursors will mostly intermingle, but may be
distinguished by nuclear morphology and/or size. A bent nucleus is found in
metamyelocytes - this shape is very pronounced in the last, immature form
of neutrophils, which are also called stab or band cells. If the cell (1) is
large, with a distinct "clearing" in the otherwise pink cytoplasm and (2) has
an ovoid or slightly indented nucleus, it is likely to be a myelocyte. Cells
with large light nuclei and almost unstained cytoplasm are either
reticulocytes or macrophages.
Macrophages are frequently associated with normoblasts, and
 103

together these cells form erythroblastic islands. The name for

macrophages in these islands, nurse cells, may tell you a bit about
their function in addition to the scavenging of the expelled nuclei.
Identify normoblasts, myelocytes and metamyelocytes and include them in
your drawing of the megakaryocyte/blast.

If you still have some time and are desperate to get frustrated try to
hunt up a nice basophilic erythroblast - a basophilic cell with
homogeneously staining nucleus that is somewhat smaller than the
nuclei of granulocyte precursors.

<HRpage content and construction: Lutz Slomianka

last updated: 1/10/06
 104

School of Anatomy and Human Biology - The University of

Western Australia

Blue Histology - Muscle

MUSCLE

Motion, as a reaction of multicellular organisms to changes in the internal

and external environment, is mediated by muscle cells.

The basis for motion mediated by muscle cells is the conversion of

chemical energy (ATP) into mechanical energy by the contractile apparatus
of muscle cells. The proteins actin and myosin are part of the contractile
apparatus. The interaction of these two proteins mediates the contraction of
muscle cells. Actin and myosin filaments, each composed of many action
and myosin molecules, form myofibrils arranged parallel to the direction of
cellular contraction.

A further specialisation of muscle cells is an excitable cell membrane which

propagates the stimuli which initiate cellular contraction.

Three structurally and functionally distinct types of muscle are found in

vertebrates:

1. smooth muscle,
2. skeletal muscle and
3. cardiac muscle.

Smooth Muscle

 Smooth muscle consists of spindle shaped cells of variable size.

The largest smooth muscle cells occur in the uterus during pregnancy
(12x600 µm). The smallest are found around small arterioles (1x10 µm).
 Smooth muscle cells contain one centrally placed nucleus.
The chromatin is finely granular and the nucleus contains 2-5 nucleoli.
 The innervation of smooth muscle is provided by the autonomic nervous
system.
 Smooth muscle makes up the visceral or involuntary muscle.

Structure of smooth muscle

 105

In the cytoplasm, we find longitudinally oriented bundles of the

myofilaments actin and myosin. Actin filaments insert into attachment
plaques located on the cytoplasmic surface of the plasma membrane. From
here, they extend into the cytoplasm and interact with myosin filaments.
The myosin filaments interact with a second set of actin filaments which
insert into intracytoplasmatic dense bodies. From these dense bodies further
actin filaments extend to interact with yet another set of myosin filaments.
This sequence is repeated until the last actin filaments of the bundle again
insert into attachment plaques .

In principle, this organisation of bundles of myofilaments, or myofibrils,

into repeating units corresponds to that in other muscle types. The repeating
units of different myofibrils are however not aligned with each other, and
myofibrils do not run exactly longitudinally or parallel to each other
through the smooth muscle cells. Striations, which reflect the alignment of
myofibrils in other muscle types, are therefore not visible in smooth muscle.

Smooth endoplasmatic reticulum is found close to the cytoplasmatic surface

of the plasma membrane. Most of the other organelles tend to accumulate in
the cytoplasmic regions around the poles of the nucleus. The plasma
membrane, cytoplasm and endoplasmatic reticulum of muscle cells are
often referred to as sarcolemma, sarcoplasm, and sarcoplasmatic reticulum.

During contraction, the tensile force generated by individual smooth muscle

cells is conveyed to the surrounding connective tissue by the sheath of
reticular fibres. These fibres are part of a basal lamina which surrounds
muscle cells of all muscle types. Smooth muscle cells can remain in a
state of contraction for long periods. Contraction is usually slow and may
take minutes to develop.

Origin of smooth muscle

Smooth muscle cells arise from undifferentiated mesenchymal cells. These

cells differentiate first into mitotically active cells, myoblasts, which
contain a few myofilaments. Myoblasts give rise to the cells which will
differentiate into mature smooth muscle cells.

Types of smooth muscle

Two broad types of smooth muscle can be distinguished on the basis of the
type of stimulus which results in contraction and the specificity with which
individual smooth muscle cells react to the stimulus:

1. The multiunit type represents functionally independent smooth muscle

 106

cells which are often innervated by a single nerve terminal and which
never contract spontaneously (e.g. smooth muscle in the walls of blood
vessels).
2. The visceral type represents bundles of smooth muscle cells connected by
GAP junctions, which contract spontaneously if stretched beyond a
certain limit (e.g. smooth muscle in the walls of the intestines).

Suitable Slides

Sections of the intestines (duodenum, jejunum, ileum or colon) - H&E

Jejunum, baboon - H&E

The outer part of the tube forming the intestines consists of two layers of
smooth muscle - one circular layer and one longitudinal layer. If you look at
the tissue close to the border between the two layers of smooth muscle, you
will be able to see both longitudinally sectioned smooth muscle cells and
transversely sectioned smooth muscle cells. The smooth muscle cells are
much longer than their nuclei. Transversely sectioned smooth muscle cells
may not have their nuclei in the plane of the section.
Occasionally you will find small nerves between the two muscle layers,
and, if you are lucky and/or patient, you will also see some very large nuclei
in this region. These nuclei belong to peripheral nerve cells (ganglion cells
of the myenteric plexus), which regulate the contraction of the muscle
around the gastrointestinal tract.
Draw a small area which contains both longitudinally sectioned and
transversely sectioned smooth muscle at high magnification.
 107

? The only tissues which perhaps could be confused with smooth muscle are
dense regular connective tissues and peripheral nerves. Both the number of
nuclei and their shapes clearly distinguish smooth muscle from dense
regular connective tissues. Nuclei are much more frequent and larger in
smooth muscle, and they are very elongated if cut longitudinally. Peripheral
nerves will be surrounded by a capsule of cells and connective tissue - the
perineurium. The thickness of longitudinally cut nerve fibres is constant
while smooth muscle cells are spindle shaped. Also, axon and nodes of
Ranvier should be visible in peripheral nerves

Skeletal Muscle

 Skeletal muscle consists of very long tubular cells, which are also called
muscle fibres.
 108

The average length of skeletal muscle cells in humans is about 3 cm

(sartorius muscle up to 30 cm, stapedius muscle only about 1 mm). Their
diameters vary from 10 to 100 µm.
 Skeletal muscle fibres contain many peripherally placed nuclei.
Up to several hundred rather small nuclei with 1 or 2 nucleoli are located
just beneath the plasma membrane.
 Skeletal muscle fibres show in many preparations characteristic cross-
striations. It is therefore also called striated muscle.
 Skeletal muscle is innervated by the somatic nervous system.
 Skeletal muscle makes up the voluntary muscle.

Structure of skeletal muscle

Muscle fibres in skeletal muscle occur in bundles, fascicles, which make up

the muscle. The muscle is surrounded by a layer of connective tissue, the
epimysium, which is continuous with the muscle fascia. Connective tissue
from the epimysium extends into the muscle to surround individual
fascicles (perimysium). A delicate network of loose connective tissue
composed of fine collagenous and reticular fibres (endomysium) is found
between the muscle fibres of a fascicle. Finally, each muscle fibre is
surrounded by a basement membrane.

Origin of skeletal muscle

The myoblasts of all skeletal muscle fibres originate from the paraxial
mesoderm. Myoblasts undergo frequent divisions and coalesce with the
formation of a multinucleated, syncytial muscle fibre or myotube. The
nuclei of the myotube are still located centrally in the muscle fibre. In the
course of the synthesis of the myofilaments/myofibrils, the nuclei are
gradually displaced to the periphery of the cell.

Satellite cells are small cells which are closely apposed to muscle fibres
within the basal lamina which surrounds the muscle fibre. Their nuclei are
slightly darker than those of the muscle fibre. Satellite cells are believed to
represent persistent myoblasts. They may regenerate muscle fibres in case
of damage.

Suitable Slides

Sections of skeletal muscle, tongue or upper oesophagus - H&E

 109

Tongue,
Skeletal
Muscle,
human -
H&E
Skeletal
muscle in the
tongue is
arranged in
bundles
which
typically run
at right
angles to
each other.
Both
longitudinally
and
transversely
cut skeletal
muscle fibres
are present.
In both
section
planes you
can see that
the nuclei are
located in the
periphery of
the muscle
fibre.
Myofibrils
may be
visible as
very fine dots
in some of
the
transversely
muscle fibres.
Striations
formed by the
A- and I-
Bands of the
sarcomeres
are visible in
 110

longitudinally
cut fibres. Z-
lines and H-
bands can be
identified in
well-
preserved
tissue.
Details of the
sarcomeres
stand out
more clearly
if you close
the iris
diaphragm of
the
microscope.
Remember to
open the
diaphragm
after you
have seen the
striations
clearly !
In the
connective
tissue
between the
muscle fibres,
the
endomysium,
numerous
capillaries
supply the
muscle with
oxygen and
nutrients.
Draw a small
section of
longitudinal
and
transversely
cut skeletal
muscle at
high
 111

magnification
.

The muscle
surrounding
the upper
one-third of
the
oesophagus is
skeletal
muscle.
Smooth
muscle
surrounds its
lower one-
third. In
section of the
middle of the
esophagus it
is usually
possible to
identify both
muscle types
and their
appearances
can be
compared.
 112

The Contractile Apparatus of Skeletal Muscle

The spatial relation between the filaments that make up the myofibrils
within skeletal muscle fibres is highly regular. This regular organisation of
the myofilaments gives rise to the cross-striation, which characterises
skeletal and cardiac muscle. Sets of individual "stria" correspond to the
smallest contractile units of skeletal muscle, the sarcomeres. Rows of
sarcomeres form the myofibrils (), which extend throughout the length of
the skeletal muscle fibre.

Depending on the distribution and interconnection of myofilaments a

number of "bands" and "lines" can be distinguished in the sarcomeres :
 113

I-band -
actin
filaments,
A-band -
myosin
filaments
which may
overlap with
actin
filaments,
H-band -
zone of
myosin
filaments
only (no
overlap with
actin
filaments)
within the
A-band,
Z-line - zone
of
apposition
of actin
filaments
belonging to
two
neighbourin
g
sarcomeres
(mediated
by a protein
called alpha-
actinin),
M-line -
band of
connections
between
myosin
filaments
(mediated
by proteins,
e.g.
myomesin,
M-protein).
 114

The average length of a sarcomere is about 2.5 µm (contracted ~1.5 µm,

stretched ~3 µm).

The protein titin extends from the Z-line to the M-line. It is attached to the
Z-line and the myosin filaments. Titin has an elastic part which is located
between the Z-line and the border between the I- and A-bands. Titin
contributes to keeping the filaments of the contractile apparatus in
alignment and to the passive stretch resistance of muscle fibres.
Other cytoskeletal proteins interconnect the Z-lines of neighbouring
myofibrils. Because of this connection, the A- and I-bands of neighbouring
myofibrils lie side-by-side in the muscle fibre. These cytoskeletal proteins
also connect the Z-lines of the peripheral myofibrils to the sarcolemma.

Muscle-Tendon Junction

At the muscle-tendon junction, the end of a muscle fibre forms deep

invaginations, which increase its surface area. The basement membrane of
the muscle fibre extends into these invagination and, so do the collagen
fibrils of the tendons. The actin filaments of the last sarcomeres extend into
cytoplasmic specialisations associated with zonula adherens-like membrane
specialisations. Instead of interconnecting two cells, the cell membrane is
here anchored to the basement membrane of the muscle cell. The basement
membrane is, in turn, connected to the collagen fibrils of the tendons.

Excitation and Contraction of Skeletal Muscle

The area of
contact
between the
end of a
motor nerve
and a skeletal
muscle cell is
called the
motor end
plate. Small
branches of
the motor
nerve form
contacts
(boutons)
with the
muscle cell
in a roughly
 115

eliptical area.
The
excitatory
transmitter at
the motor
end plate is
acetylcholine
. The space
between the
boutons and
the muscle
fibre is called
primary
synaptic
cleft.
Numerous
infoldings of
the
sarcolemma
in the area of
the motor
end plate
form
secondary
synaptic
clefts. Motor
end plates
typically
concentrate
in a narrow
zone close to
the middle of
the belly of a
muscle. The
excitable
sarcolemma
of skeletal
muscle cells
will allow
the stimulus
to spread,
from this
zone, over
the entire
muscle cell.
 116

The spread of excitation over the sarcolemma is mediated by voltage-gated

ion channels.

Invaginations of the sarcolemma form the T-tubule system which "leads"

the excitation into the muscle fibre, close to the border between the A- and
I-bands of the myofibrils. Here, the T-tubules are in close apposition with
cisternae formed by the sarcoplasmatic reticulum. This association is called
a triad. The narrow gap between the T-tubule and the cisternae of the
sarcoplasmatic reticulum is spanned by proteins which mediate the
excitation-contraction coupling.

Proteins in the sarcolemma which forms the wall of the T-tubule

(dihydropyridine (DHP) receptors) change conformation, i.e. they change
their shape, in response to the excitation travelling over the sarcolemma.
These proteins are in touch with calcium channels (ryanodine receptors)
which are embedded in the membrane of the cisternae of the sarcoplasmatic
reticulum. The change in the shape of the proteins belonging to the T-tubule
opens the calcium channels of the sarcoplasmatic reticulum. Calcium can
now move from stores in the sarcoplasmatic reticulum into the cytoplasm
surrounding the myofilaments.

Sites of interaction between actin and myosin are in resting muscle cells
"hidden" by tropomyosin. Tropomyosin is kept in place by a complex of
proteins collectively called troponin. The binding of calcium to troponin-C
induces a conformational change in the troponin-tropomyosin complex
which permits the interaction between myosin and actin and, as a
consequence of this interaction, contraction.

ATP-dependent calcium pumps in the membrane of the sarcoplasmatic

reticulum typically restore the concentration of Ca to resting levels within
30 milliseconds after the activation of the muscle fibre.

Types of Skeletal Muscle

Skeletal muscle cells respond to stimulation with a brief maximal

contraction - they are of the twitch type. Individual muscles fibres cannot
maintain their contraction over longer periods. The sustained contraction of
a muscle depends on the "averaged" activity of often many muscles fibres,
which individually only contract for a brief period of time.
The force generated by the muscle fibre does depend on its state of
contraction at the time of excitation. Excitation frequency and the
mechanical summation of the force generated is one way to graduate the
force generated by the entire muscle. Another way is the regulation of the
 117

number of muscle fibres which contract in the muscle. Additional motor

units, i.e. groups of muscle fibres innervated by one motor neurone and its
branches, are recruited if their force is required. The functional properties of
the muscle can be "fine-tuned" further to the tasks the muscle performs by
blending functionally different types of muscle fibres:

Type I fibres (red fibres)

Red muscles contain predominantly (but not exclusively) red muscle

cells. Red muscle fibres are comparatively thin and contain large amounts of
myoglobin and mitochondria. Red fibres contain an isoform of myosin with low
ATPase activity, i.e. the speed with which myosin is able to use up ATP. Contraction
is therefore slow. Red muscles are used when sustained production of force is
necessary, e.g. in the control of posture.

Type II fibres

White
muscle
cells, which
are
predominan
tly found in
white
muscles, are
thicker and
contain less
myoglobin.
ATPase
activity of
the myosin
isoform in
white fibres
is high, and
contraction
is fast. Type
IIA fibres ctsy M. Müntener
(red)
contain
many
mitochondri
a and are
available for
both
sustained
 118

activity and
short-
lasting,
intense
contractions
. Type
IIB/IIX
fibres
(white)
contain only
few
mitochondri
a. They are
recruited in
the case of
rapid
acceleration
s and short
lasting
maximal
contraction.
Type
IIB/IIX
fibres rely
on
anaerobic
glycolysis
to generate
the ATP
needed for
contraction.

Skeletal muscle fibres do not contract spontaneously. Skeletal muscle fibres

are not interconnected via GAP junctions but depend on nervous
stimulation for contraction. All muscle fibres of a motor unit are of the
same type.

Fibre type is determined by the pattern of stimulation of the fibre, which, in

turn, is determined by the type of neuron which innervates the muscle. If the
stimulation pattern is changed experimentally, fibre type will change
accordingly. This is of some clinical / pathological importance. Nerve fibres
have the capacity to form new branches, i.e. to "sprout", and to re-innervate
muscle fibres, which may have lost their innervation as a consequence of an
acute lesion to the nerve or a neurodegenerative disorder. The type of the
muscle fibre will change if the type of stimulation provided by the sprouting
 119

nerve fibre does not match with the type of muscle. The process of
reinnervation and type adjustment may result in fibre type grouping within
the muscle, i.e. large areas of the muscle are populated by muscle fibres of
one type.

Muscle Spindles
Muscle
spindles are
sensory
specializatio
n of the
muscular
tissue. A
number of
small
specialised
intrafusal
muscle fibres
(nuclear bag
fibres and
nuclear chain
fibres) are
surrounded
by a capsule
of
connective
tissue. The
intrafusal
fibres are
innervated
by efferent
motor nerve
fibres.
Afferent
sensory
nerve fibres
surround the
intrafusal
muscle
fibres.
If the muscle
is stretched,
the muscle
fibres in the
 120

muscle
spindle are
stretched,
sensory
nerves are
stimulated,
and a change
in
contraction
of the
muscle is
perceived.
Different
types of
intrafusal
fibres and
nerve
endings
allow the
perception of
position,
velocity and
acceleration
of the
contraction
of the
muscle.
The
contraction
of the
intrafusal
fibres, after
stimulation
by the
efferent
nerve fibres,
may
counteract or
magnify the
changes
imposed on
the muscle
spindle by
the
surrounding
 121

muscle. The
intrafusal
fibres and
the efferent
nerves can in
this way set
the
sensitivity
for the
sensory
nerve ending
in the muscle
spindle.

Cardiac Muscle

 Cardiac muscle, the myocardium, consists of muscle cells,

cardiomyocytes, with one centrally placed nucleus.
Nuclei are oval, rather pale and located centrally in the muscle cell which
is 10 - 15 µm wide.
 Cardiac muscle cells exhibit cross-striations.
 Cardiac muscle cells excitation is mediated by rythmically active modified
cardiac muscle cells.
 Cardiac muscle is innervated by the autonomic nervous system, which
adjusts the force generated by the muscle cells and the frequency of the
heart beat.
 Cardiac muscle is for these reasons also called involuntary striated
muscle.

Structure of cardiac muscle

The ultrastructure of the contractile apparatus and the mechanism of

contraction largely correspond to that seen in skeletal muscle cells.
Although equal in ultrastructure to skeletal muscle, the cross-striations in
cardiac muscle are less distinct, in part because rows of mitochondria and
many lipid and glycogen droplets are found between myofibrils.

In contrast to skeletal muscle cells, cardiac muscle cells often branch at

acute angles and are connected to each other by specialisations of the cell
membrane in the region of the intercalated discs. Intercalated discs
invariably occur at the ends of cardiac muscle cells in a region
corresponding to the Z-line of the myofibrils (the last Z-line of the
myofibril within the cell is "replaced" by the intercalated disk of the cell
 122

membrane). In the longitudinal part of the cell membrane, between the

"steps" typically formed by the intercalated disk, we find extensive GAP
junctions.

T-tubules are typically wider than in skeletal muscle, but there is only one
T-tubule set for each sarcomere, which is located close to the Z-line. The
associated sarcoplasmatic reticulum is organised somewhat simpler than in
skeletal muscle. It does not form continuous cisternae but instead an
irregular tubular network around the sarcomere with only small isolated
dilations in association with the T-tubules.

Cardiac muscle does not contain cells equivalent to the satellite cells of
skeletal muscle. Therefore cardiac muscle cannot regenerate.

Suitable Slides

Sections of cardiac muscle - Alizarin Blue, Whipf's polychrome, iron

haematoxylin, H&E
 123

Cardiac
Muscle,
human - H&E
Use a low
magnification
to find a part
of the tissue
in which the
cardiac
muscle cells
are cut
longitudinally
. At high
magnification
you should
see striations
and the large
nuclei of the
cardiac
muscle cells.
If you follow
the course of
individual
cardiac
muscle cells
you will note
fine, darker
lines which
seem to cross
the fibres.
These are the
intercalated
discs which
connect the
individual
muscle cells
mechanically
and permit
the
conduction of
electrical
impulses
between the
cells ... how?
 124

A light streak
of cytoplasm
is often
visible
extending
from the
poles of the
nucleus. This
part of the
cytoplasm
does not
contain
myofibrils,
and it appears
very light in
transversely
cut cardiac
muscle cells.
Myofibrils
are often
visible in
transversely
cut cells.
Their visible
separation
reflects the
large
numbers of
mitochondria
located
between
them. Also,
the large
number
capillaries
reflect the
need of a
good blood
supply to the
constantly
active muscle
cells.

Draw
longitudinally
 125

cut cardiac
muscle cells
which show
all the
features
mentioned.
Label the
features in
your drawing,
and include
an suitable
scale.

Excitation in cardiac muscle

A number of specialised structures, which are composed of modified

cardiac muscle cells, ensure that the contraction of the atria and ventricles
takes place in the order that is most appropriate to the pumping function of
the heart. The excitation of the myocardium originates from the sinuatrial
node, which is located in the wall of the right atrium lateral to the opening
of the superior vena cava into the atrium. The sinuatrial node initiates the
contraction of atrial myocardium. Excitation also reaches the
atrioventricular node at the base of the interatrial septum. The myocardium
of the atria and ventricles are separated from each other by a zone of
connective tissue, the fibrous body of the heart. The fibrous body prevents
the spread of excitation from atrial muscle cells to those of the ventricles.

A system of modified cardiac muscle cells, Purkinje fibres, has developed,

which conduct stimuli faster than ordinary cardiac muscle cells (2-3 m/s vs.
0.6 m/s). A bundle of Purkinje fibres extends from the atrioventricular node,
pierces the fibrous body, divides into left and right bundles, and travels,
beneath the endocardium, towards the tip (apex) of the heart. Branches of
the bundle contact ordinary cardiac muscle cells by way of specialisations
similar to intercalated discs. Purkinje fibres contain large amounts of
glycogen but fewer myofibrils than ordinary cardiac muscle cells.
Myofibrils are mainly located in the periphery of the cell. Purkinje fibres
are also thicker than ordinary cardiac muscle cells.

Modified muscle cells in nodal tissue (nodal muscle cells or P cells; P ~

pacemaker or pale-staining) of the heart exert the pacemaker function that
drives the Purkinje cells. The rhythm generated by the nodal muscle cells
can be modified by the autonomic nervous system, which innervates the
nodal tissue and accelerates (sympathetic) or decelerates (parasympathetic)
 126

heart rate.

Suitable Slides

Sections of cardiac muscle (interventricular septum) - Whipf's

polychrome, iron haematoxylin, H&E

Purkinje Fibre, sheep - Whipf's polychrome

Cardiac muscle cells in this preparation have a red-violet appearance. Much
of the connective tissue looks light blue, striations of cardiac muscle cells
are visible. Intercalated discs may be more difficult to find, but nuclei stand
out very clearly. Bundles of Purkinje fibres are present in areas of
connective tissue between areas of "normal" cardiac muscle tissue and
beneath the endocardium. Purkinje fibres appear as a chain of light blue
profiles with a red rim. Browse through the tissue at low magnification and
change to high magnification when you suspect the presence of Purkinje
fibres. The red rim is formed by the contractile filaments. They are
displaced to the periphery of the cells and can sometimes be used to define
the outline of individual cells. The nuclei are large, but the cells are even
larger and you will not see a nucleus in each cell.
Draw a Purkinje fibre at high magnification. Try to include a bit of
"normal" cardiac muscle and a suitable scale.
 127

<hr size=2 width="100%" align=center>

page content and construction: Lutz Slomianka

last updated: 6/08/09
 128

The nervous system consists of all nervous tissue in the body. It is divided
anatomically into the central nervous system and the peripheral nervous
system.

Central Nervous System (CNS)

The CNS consists of the brain (encephalon), which is enclosed in the skull,
and the spinal cord, which is contained within the vertebral canal. Nervous
tissue of the CNS does not contain connective tissue other than that in the
meninges and in the walls of large blood vessels. Collagenous fibers or
fibrocytes/blasts are consequently not observed, which is quite unlike other
tissues. Because of the absence of connective tissue, fresh CNS tissue has a
very soft, somewhat jelly-like consistency. The two major classes of cells
that make up the nervous tissue are nerve cells, neurones, and supporting
cells, glia.

Neurones

The vast majority of neurones is generated before birth. Persisting stem

cells give rise to a small number of new neurones throughout the lifetime of
mammals, including humans. The permanent addition of neurones may be
important for the maintenance and plasticity of some parts of the CNS, but
it is insufficient to replace neurones that die because of disease or acute
damage to the CNS. Neurones are "intended" to last a lifetime. Mature
neurones are not mitotically active, i.e. they do not divide.

Neurones are generally large cells. Neural activity and its control require
the expression of many genes, which is reflected in the large and light
nuclei of most neurones. The keys to the understanding of the function of a
neurone lies in (1) the shape of the neurone and, in particular, its processes,
(2) the chemicals the neurone uses to communicate with other neurones
(neurotransmitters) and (3) the ways in which the neurone may react to the
neurotransmitters released by other neurones.
 129

The shape of
the neurone
and its
processes

Neurones have
long processes,
which extend
from the part
of the cell
body around
the nucleus,
the perikaryon
or soma. The
processes can
be divided into
two
functionally
and
morphologicall
y different
groups,
dendrites and
axons.
Dendrites are
part of the
receptive
surface of the
neurone. As a
rule neurones
have one to
several
primary
dendrites,
which emerge
from the
perikaryon.
Primary
dendrites may
divide into
secondary,
tertiary etc.
dendrites.
Dendrites can
be smooth, or
 130

they can be
studded with
small,
mushroom-
shaped
appendages,
which are
called spines.

Each neurone
has as a rule
one axon, and
never more
than one axon
which emerges
from the
perikaryon or
close to the
trunks of one
of the primary
dendrites. The
point of origin
of the axon
from the
perikaryon is
the axon
hillock. The
axon may, like
the dendrites,
branch as it
travels through
the nervous
tissue to its
destination(s).
The axon is the
"transmitting"
process of the
neurone.
 131

The axon forms small,

bulb-shaped swellings
called boutons at the
ends (terminal boutons)
or along the course
(boutons en passant) of
its branches. Synapses
are morphologically
specialised contacts
between a bouton
formed by one neurone,
the presynaptic neurone,
and the cell surface of
another neurone, the
postsynaptic neurone.
Synaptic vesicles
contain the
neurotransmitters.
Synaptic vesicles
typically accumulate
close to the site of
contact between the
bouton and the
postsynaptic neurone.
The release of the
neurotransmitter from
the synaptic vesicles into
the synaptic cleft, i.e. the
space between the
bouton and the
postsynaptic neurone,
mediates the transfer of
information from the
pre- to the postsynaptic
neurone.

The shape and orientation of the dendritic tree of the neurone determines
the amount and type of information that may reach the neurone. The course
of its axon determines to which neurones this information may be passed
on. The location of the neurone within the CNS determines to which major
system the neurone belongs.

There are several hundred functionally different areas, i.e. groups of

neurones, in the CNS. Based on their location, the shape of their dendritic
tree and the course of their axon, several thousand types of neurones can be
 132

distinguished in the CNS.

Transmitters

Neurotransmitters either excite or inhibit the postsynaptic neurone. The

most prominent excitatory transmitter in the CNS is L-glutamate. The most
prominent inhibitory transmitter in the CNS is GABA (gamma-amino
butyric acid). Other "main" neurotransmitters are e.g. dopamine, serotonin,
acetylcholine, noradrenaline and glycine. Each neurone uses only one of the
main transmitters, and this transmitter is used at all synaptic boutons that
originate from the neurone.
One or more of the "minor" transmitters (there are several dozens of them -
such as cholecystokinin, endogenous opioids, somatostatin, substance P)
may be used together with a main transmitter.

The molecular machinery which is needed to mediate the events occurring

at excitatory synapses differs from that at inhibitory synapses. Differences
in the morphological appearances of the synapses accompany the functional
differences.

Receptors

Usually there exists a multitude of receptors which are all sensitive to one
particular neurotransmitter. Different receptors have different response
properties, i.e. they allow the flux of different ions over the plasma
membrane of the neurone or they may address different second messenger
systems in the postsynaptic neurones. The precise reaction of the neurone to
the various neurotransmitters released onto its plasma membrane at the
synapses is determined by the types of receptors expressed by the neurone.

Suitable Slides

sections of spinal cord - H&E, luxol fast blue/cresyl violet

(LFB/CV), toluidine blue, Giemsa

Thoracic Spinal Cord, sheep - LFB/CV

Most neurones have a light, large nucleus with a distinct nucleolus. The
cytoplasm of many neurones contains fairly large amounts of rough
endoplasmatic reticulum, which may aggregate within the cytoplasm of the
neurone to form Nissl-bodies. Nissl-bodies are prominent in motor neurones
located in the ventral horn of the grey matter of the spinal cord. The neurites
are difficult to identify in most types of stained sections. Only the most
proximal segments of the primary dendrites are seen clearly. The size of the
 133

perikaryon depends on the level of activity of the neurone and the length of
the processes which the neurone has to support. An usable range for the size
of the perikaryon would be 15 - 50 µm, although much smaller and much
larger neuronal perikarya exist.
Draw the spinal cord at low magnification and indicate the distribution of
grey matter and white matter. Find a nice group of neurones in the grey
matter and draw them at a high magnification. Finally, have a look at the
white matter and identify the nuclei of glial cells. You will find similar
nuclei also in the grey matter.

Thoracic spinal Cord - H&E, silver stain

These slides show the same major features as the LFB/CV stained sections.
Try to identify neurones (primary dendrites, Nissl-bodies) and glial cell
nuclei in the H&E stained section. Part of the cytoskeleton of neurones is
(like the reticular connective tissue fibers) argyrophilic, i.e. they "love"
silver and can be stained by silver stains. Aside from the neurones and their
processes, fine fibrils are visible in the neuropil. Many of the fibrils
represent axons travelling in the grey and white matter of the spinal cord.
It should not be necessary to prepare separate drawings for these slides.
Make sure that you can identify the main structural features in all
preparations.
 134

Glia

CNS tissue contains several types of non-neuronal, supporting cells,

neuroglia.

 Astrocytes
(or
astroglia)
are star-
shaped
cells. Their
processes
are often in
contact
with a
blood
vessel
(perivascul
ar foot
 135

processes).
Astrocytes
provide
physical
and
metabolic
support to
the
neurones
of the
CNS. They
participate
in the
maintenanc
e of the
compositio
n of the
extracellul
ar fluid.
Although
not
themselves
directly
involved in
the process
of
communic
ation
between
neurones,
they may
be
involved in
the
removal of
transmitter
s from
synapses
and the
 136

metabolis
m of
transmitter
s.
Astrocytes
are also the
scar-
forming
cells of the
CNS.

 Oligodendrocytes (or oligoglia) have fewer and shorter processes.

Oligodendrocytes form myelin sheath around axons in the CNS and
are the functional homologue of peripheral Schwann cells.
Oligodendrocytes may, in contrast to Schwann cells in the
periphery, form parts of the myelin sheath around several axons.

 Microglia are small cells with complex shapes. Microglia are, in

contrast to neurones and the other types of glial cells, of mesodermal
origin. They are derived from the cell line which also gives rise to
monocytes, i.e. macrophage precursors which circulate in the blood
stream. In the case of tissue damage, microglia differentiate into
phagocytotic cells.

 The
ventricl
es of
the
brain
and the
central
canal of
the
spinal
cord are
lined
with
ependy
 137

mal
cells.
The
cells are
often
cilated
and
form a
simple
cuboida
l or low
column
ar
epitheli
um. The
lack of
tight
junction
s
between
ependy
mal
cells
allows a
free
exchang
e
between
cerebro
spinal
fluid
and
nervous
tissue.
Ependy
mal
cells
can
speciali
 138

se into
tanycyt
es,
which
are
rarely
ciliated
and
have
long
basal
process
es.
Tanycyt
es form
the
ventricu
lar
lining
over the
few
CNS
regions
in
which
the
blood-
brain
barrier
is
incompl
ete.
They do
form
tight
junction
s and
control
the
 139

exchang
e of
substan
ces
between
these
regions
and
surroun
ding
nervous
tissue
or
cerebro
spinal
fluid.

Many glial cells do express neurotransmitter receptors, but they do not form
synapses with neurones. Neuronal activity may regulate glial function by a
spillover of transmitter from synaptic sites, which are typically surrounded
by fine processes of glial cells. Glial cells may also communicate with each
other via GAP junctions.

Suitable Slides

sections of the forebrain - toluidine blue, Giemsa, luxol fast

blue/cresyl violet

Forebrain, Cortex, mouse - Giemsa and Forebrain, Hippocampus, mouse -

Giemsa
Most glial cells are much smaller than neurones. Their nuclei are generally
much smaller than neuronal nuclei, and they rarely contain an easily visible
nucleolus. Other aspects of their morphology are variable. The glial
cytoplasm is, if visible at all, very weakly stained. Different types of glial
cells cannot be easily distinguished by their appearance in this type of
preparation. Most of the small nuclei located in the white matter of the
CNS, where they may form short rows, are likely to represent
oligodendrocytes.
Browse through the sections at low or medium magnification and try to get
 140

a feeling for the structural diversity visible in the section available to you -
parts of the section that look different from others are very likely to have
different functions.
Find a spot that appears interesting (or least boring) to you and sketch its
structure at low magnification. Choose a spot for high magnification, and
draw some of the visible neurones and glial cells. Note the difference in the
size and number of glial cells and neurones.

Peripheral Nervous System (PNS)

The PNS comprises all nervous tissue outside the brain and spinal cord. It
consists of groups of neurones (ganglion cells), called ganglia, feltworks of
nerve fibres, called plexuses, and bundles of parallel nerve fibres that form
the nerves and nerve roots. Nerve fibres, which originate from neurones
within the CNS and pass out of the CNS in cranial and spinal nerves, are
called efferent or motor fibers. Nerve fibres which originate from nerve
 141

cells outside the CNS but enter the CNS by way of the cranial or spinal
nerves are called afferent or sensory nerve fibres.

The principal neurotransmitters in the PNS are acetylcholine and

noradrenalin.

Peripheral Nerves

Afferent, sensory fibres enter the spinal cord via the dorsal roots, while
efferent, motor fibres leave the spinal cord via the ventral roots. Dorsal and
ventral roots merge to form the spinal nerves, which consequently contain
both sensory and motor fibres. As the spinal nerves travel into the periphery
they split into branches and the exact composition of the nerve in terms of
motor and sensory fibres is, of course, determined by the structures the
nerve will innervate.

One nerve fibre consists of an axon and its nerve sheath. Each axon in the
peripheral nervous system is surrounded by a sheath of Schwann cells. An
individual Schwann cell may surround the axon for several hundred
micrometers, and it may, in the case of unmyelinated nerve fibers, surround
up to 30 separate axons. The axons are housed within infoldings of the
Schwann cell cytoplasm and cell membrane, the mesaxon .

In the case of myelinated

nerve fibres, Schwann
cells form a sheath around
one axon and surround
this axon with several
double layers (up to
hundreds) of cell
membrane. The myelin
sheath formed by the
Schwann cell insulates the
axon, improves its ability
to conduct and, thus,
provides the basis for the
fast saltatory transmission
of impulses. Each
Schwann cell forms a
myelin segment, in which
the cell nucleus is located
approximately in the
middle of the segment.
 142

The node of Ranvier is

the place along the course
of the axon where two
myelin segments abut.

Fibre types in peripheral nerves:

 Type A fibres (myelinated) are 4 - 20 µm in diameter and conduct

impulses at high velocities (15 - 120 m per second). Examples:
motor fibers, which innervate skeletal muscles, and sensory fibres.
 Type B fibres (myelinated) are 1 - 4 µm in diameter and conduct
impulses with a velocity of 3 - 14 m per second. Example:
preganglionic autonomic fibres.
 Type C fibres (unmyelinated) are 0.2 - 1 µm thick and conduct
impulses at velocities ranging from 0.2 to 2 m per second.
Examples: autonomic and sensory fibres.

Peripheral nerves contain a considerable amount of connective tissue. The

entire nerve is surrounded by a thick layer of dense connective tissue, the
epineurium. Nerve fibres are frequently grouped into distinct bundles,
fascicles, within the nerve. The layer of connective tissue surrounding the
individual bundles is called perineurium. The perineurium is formed by
several layers of flattened cells, which maintain the appropriate
microenvironment for the nerve fibres surrounded by them. The space
between individual nerve fibres is filled by loose connective tissue, the
endoneurium.

Fibrocytes, macrophages and mast cells are present in the endoneurium.

Nerves are richly supplied by intraneural blood vessels, which form
numerous anastomoses. Arteries pass into the epineurium, form arteriolar
networks in the perineurium and give off capillaries to the endoneurium.

Suitable Slides

sections of peripheral nerve - H&E, osmium or plucked

preparations of peripheral nerve - osmium

Peripheral Nerve, cat - osmium

Which structures can be recognized in peripheral nerves depends on the
stain that has been used in the preparation. Osmium gives a black color to
lipids. In osmium stained preparations it is possible to observe the myelin
 143

sheath surrounding the axon. A good impression of the different sizes of the
nerve fibres may be obtained. The axon is usually not well preserved. It
may only form a little dark spot somewhere within the dark ring which
represents the myelin sheath. Lipid droplets in fat cells, which can be found
in the connective tissue around nerves, stand out as large (much larger than
the nerve fibres), round, homogeneously stained areas.
Draw the nerve at low magnification (you may include some of the stained
lipid droplets) and a small section of it at high magnification.

Peripheral Nerve, rat - H&E

In longitudinal H&E stained sections it is possible to identify the axon
running in its myelin sheath, nodes of Ranvier and Schwann cell nuclei.
Components of the connective tissue elements, which accompany the nerve,
should be visible and identifiable in both longitudinal and transverse
sections. H&E stained and transversely cut preparations give a good picture
of the axon in the middle of a ring-like structure (sometimes fussy), which
represents the remains of the myelin sheath. Due to their small size and the
 144

lack of a myelin sheath, type C fibres are very difficult to detect in either
osmium or H&E stains.
Draw part of the longitudinally and transversely sectioned nerve at high
magnification. Include Schwann cell nuclei, myelin sheath, axons and, if
possible, nodes of Ranvier.

Ganglia

Ganglia are aggregations of nerve cells (ganglion cells) outside the CNS.
Cranial nerve and dorsal root ganglia are surrounded by a connective tissue
capsule, which is continuous with the dorsal root epi- and perineurium.
Individual ganglion cells are surrounded by a layer of flattened satellite
cells. Neurones in cranial nerve and dorsal root ganglia are pseudounipolar.
They have a T-shaped process. The arms of the T represent branches of the
neurite connecting the ganglion cell with the CNS (central branch) and the
periphery (peripheral branch). Both branches function as one actively
conducting axon, which transmits information from the periphery to the
CNS. The stem is connected to the perikaryon of the ganglion cell and is the
only process originating from it. Ganglion cells in dorsal root ganglia do not
receive synapses. Their function is the trophic support of their neurites.
 145

Early in development two processes emerge from the perikaryon of dorsal

root ganglion cells, which merge in the course of development. These
ganglion cells are therefore also called pseudounipolar neurones. Two
processes emerge from the perikaryon of bipolar neurones. The majority of
CNS neurones are multipolar, i.e. more than two processes (but only one
axon) emerge from their perikaryon.

Autonomic ganglia do contain synapses, and the ganglion cells within them
do have dendrites. They receive synapses from the first neurone of the two-
neurone chain, which characterises most of the efferent connections of the
autonomic nervous system. The second neurone is the ganglion cell itself.
Some autonomic ganglia are embedded within the walls of the organs which
they innervate (intramural ganglia - e.g. GIT and bladder).

Suitable Slides

sections of dorsal root ganglia and autonomic ganglia - H&E,

toluidine blue, Giemsa, luxol fast blue/cresyl/violet (LFB/CV)

Dorsal Root Ganglion, cat - H&E and Autonomic Ganglion - H&E

Ganglion cells will typically be several times larger than other cells in the
ganglia. The perikaryon is very large and surrounds a large and light
nucleus. Only the cells immediately surrounding the ganglion cells as one
flattened layer are satellite cells. With a lot of luck you may see the process
of a ganglion cell as it passes out of the capsule of satellite cells. Ganglion
cells are of course in contact with other parts of the nervous system and
with the peripheral tissues which they innervate. Consequently, nerve fibers
will be visible close to or within the ganglion.
Sketch the appearance of the spinal ganglion section at low magnification.
Draw a small section of the spinal ganglion and peripheral ganglion at high
magnification. Label ganglion cells, satellite cells and, if visible, nerve
fibres and connective tissue elements.
 146

<hr page content and construction: Lutz Slomiankalast updated: 1/10/06

School of Anatomy and Human Biology - The University of

Western Australia

Blue Histology - Vascular System

VASCULAR SYSTEM

The cardiovascular system is concerned with the transport of

blood and lymph through the body. It may be divided into four
major components: the heart, the macrocirculation, the
microcirculation and the lymph vascular system.

Essentially, the macrocirculation comprises all vessels, both

arteries and veins, that would be visible to the eye. The vessels
 147

of the macrocirculation supply and drain a network of fine

vessels interposed between them, the capillaries. This network is
also called the capillary bed. Water and other components of the
blood plasma which exude from the blood vessels form the
interstitial fluid, which is returned to the circulation by the lymph
vascular system.

General Structure of Blood Vessels

You have already seen blood vessels of various sizes and types
in preparations available in other lab sessions, and you should be
aware that the histological appearances of vessels of different
sizes (arterioles vs. arteries) and different types (arteries vs.
veins) are different from each other. These differences are the
result of quantitative variations of a common structural pattern
that can be seen in all blood vessels with the exception of
capillaries, i.e. the division of the walls of the blood vessels into
three layers or tunics.

The tunica intima

delimits the
vessel wall
towards the
lumen of the
vessel and
comprises its
endothelial
lining (typically
simple,
squamous) and
associated
connective
tissue. Beneath
the connective
tissue, we find
the internal
elastic lamina,
 148

which delimits
the tunica
intima from

the tunica media.

The tunica
media is
formed by a
layer of
circumferential
smooth muscle
and variable
amounts of
connective
tissue. A
second layer of
elastic fibers,
the external
elastic lamina,
is located
beneath the
smooth muscle.
It delimits the
tunica media
from

the tunica adventitia,

which consist
mainly of
connective
tissue fibres.
The tunica
adventitia
blends with the
connective
tissue
surrounding the
 149

vessel. The
definition of
the outer limit
of the tunica
adventitia is
therefore
somewhat
arbitrary.

Variations of Vessel Wall Structure

Arteries

All arterial vessels originate with either the pulmonary trunk

(from the right ventricle) or the aorta (from the left ventricle).
Specialisations of the walls of arteries relate mainly to two
factors: the pressure pulses generated during contractions of the
heart (systole) and the regulation of blood supply to the target
tissues of the arteries. The tunica media is the main site of
histological specialisations in the walls of arteries.

Vessels close to the heart (aorta, pulmonary trunk and the larger
arteries that originate from them) are

Elastic arteries

The tunica intima of elastic arteries is thicker than in

other arteries. A layer of loose connective tissue beneath
the endothelium (subendothelial connective tissue) allows
the tunica intima to move independently from other layers
as the elastic arteries distend with the increase in systolic
blood pressure. Distension of the walls is facilitated by
concentric fenestrated lamellae of elastic fibres in a thick
tunica media. In adult humans, about 50 elastic lamellae
are found in the tunica media of the aorta. The energy
 150

stored in the elastic fibres of the tunica media allows

elastic arteries to function as a "pressure reservoir" which
forwards blood during ventricular relaxation (diastole).
Smooth muscle cells and collagen fibres are present
between the layers of elastic fibres. Both fibre types are
produced by the smooth muscle cells. Each elastic lamella
forms together with interlamellar fibres and cells a
lamellar unit. The external elastic lamina is difficult to
discern from other layers of elastic fibres in the tunica
media. The tunica adventitia appears thinner than the
tunica media and contains collagen fibres and the cell
types typically present in connective tissue.

The walls of these large arteries are so thick that their

peripheral parts cannot derive enough oxygen and
nutrients from the blood of the vessel that they form.
Larger vessels are therefore accompanied by smaller blood
vessels which supply the tunica adventitia and, in the
largest vessels, the outer part of the tunica media of the
vessel wall. The vessels are called vasa vasorum. In
macroscopic preparations vasa vasorum are visible as fine
dark lines on the surface of the larger arteries.

Suitable Slides
sections of the aorta - H&E, elastin

Aorta, human - H&E , elastin & van Gieson

The thin endothelial lining of the aorta corresponds to that of
other vessels. The flattened cells are easily damaged during
preparation and it may be difficult to identify the endothelium.
The subendothelial layer of connective tissue is characterised by
a lower density of cells, i.e. fewer nuclei, a fibrous appearance of
the tissue and the absence of well-defined elastic layers. Because
the lamellae of elastic fibers diffract light differently from the
remaining tissues they should also be visible in H&E stained
sections. Elastic lamellae become visible in the tunica media.
The majority of cells in the tunica media are smooth muscle
 151

cells. Smooth muscle cells and collagen fibres are found between
the layers of elastic fibres. If you scan the periphery of the aorta
you may find small blood vessels, the vasa vasorum, in the
tunica adventitia and penetrating into the outer part of the tunica
media.
Draw the aorta at low magnification and label the three tunics.
Draw part of the tunica media at high magnification and identify
collagen fibres, layers of elastic fibres and smooth muscle cell
nuclei in your drawing.

The diameter of individual arteries decreases as we follow them

further into the periphery. However, their total diameter
increases, which leads to a fall in blood pressure. Also, the
properties of the elastic arteries have to some extent evened out
differences in diastolic and systolic blood pressure. The amount
of elastic fibres in the tunica media decreases with these
physiological changes. We now find a type of arteries which are
termed
 152

Muscular arteries

The tunica intima is thinner than in elastic arteries.

Subendothelial connective tissue other than the internal
elastic lamina is often difficult to discern. The internal
elastic lamina forms a well defined layer. The tunica
media is dominated by numerous concentric layers of
smooth muscle cells. Fine elastic fibres and and a few
collagen fibres are also present. The external elastic
lamina can be clearly distinguished although it may be
incomplete in places. The thickness and appearance of the
tunica adventitia is variable.

The basic structure of the walls of arteries does not change much
as we come to the next type of arterial vessels. Size is used to
differentiate them from muscular arteries.

Arterioles

are arterial vessels with a diameter below 0.1 - 0.5 mm

(different values in different textbooks). Endothelial cells
are smaller than in larger arteries, and the nucleus and
surrounding cytoplasm may 'bulge' slightly into the lumen
of the arteriole. The endothelium still rests on a internal
elastic lamina, which may be incomplete and which is not
always well-defined in histological sections. The tunica
media consists of 1-3 concentric layers of smooth muscle
cells. It is difficult to identify an external elastic lamina or
to distinguish the tunica adventitia from the connective
tissue surrounding the vessel.
The smooth muscle of arterioles and, to some extent, the
smooth muscle of small muscular arteries regulate the
blood flow to their target tissues. Arterioles receive both
sympathetic and parasympathetic innervation. The final
branching of the arterioles finally gives rise to the
capillary network (microcirculation).
 153

Suitable Slides
sections of arteries - H&E or elastin (in combination with
other stains)
Sections of small muscular arteries and arterioles are
present in many sections, and the basic features of their
structure are usually visible - even in smaller arteries.
Large muscular arteries often have their "own section" in
teaching collections.
Artery - H&E and
elastin & eosin
Identifying muscular
arteries in sections is
rather straight forward.
There are two easily
recognizable features
which distinguish these
arteries from veins. If
two vessels have a
similarly sized lumen,
the walls of arteries
will be much thicker
and more compact than
the wall of veins. At
high magnification, the
internal elastic lamina
forms a pink streak
immediately below the
endothelial cell lining
in arteries and even
arterioles, while it is
difficult to identify in
veins.
The layer of
subendothelial
connective tissue is
very thin, and the
endothelium seems to
rest on the internal
elastic lamina. Smooth
 154

muscle cell nuclei are

frequent in the tunica
media. The external
elastic lamina stains
similar to the internal
elastic lamina, but it is
thicker and appears
fibrous instead of
forming a continuous
band. Collagen fibres
and a few connective
tissue cell nuclei are
visible in the tunica
adventitia.
If you close the iris
diaphragm of the
microscope, the elastic
layers will stand out
very clearly, but
remember to open the
diaphragm once you
have seen them.
 155

In addition to the inner

and outer elastic
laminae, elastin stains
will show fine elastic
fibres in the tunica
media and coarse
elastic fibres between
the collagen fibres of
the tunica adventitia.
The appearance of
other structures will
depend on the stain
used together with the
elastin stain. Eosin, the
E in H&E, gives a pink
colour to both collagen
fibres and the
cytoplasm of cells.
Nuclei are not stained
if the H is omitted from
the H&E.
Draw either one large
composite image
containing the three
tunics and the cellular
and fibrous elements
which form the tunics.
Alternatively, you can
draw a low power
overview and
supplement it with high
magnification
illustrations of the
individual tunics.
Focus on an H&E
stained section.
 156

Capillaries

The sum of the diameters of all capillaries is significantly larger than that of the
aorta (by about three orders of magnitude), which results in decreases in blood
pressure and flow rate. Also, capillaries are very small vessels. Their
diameter ranges from 4-15 µm. The wall of a segment of capillary may be
formed by a single endothelial cell. This results in a very large surface to
volume ratio. The low rate of blood flow and large surface area facilitate the
functions of capillaries in

1. providing nutrients and oxygen to the surrounding tissue, in

2. the absorption of nutrients, waste products and carbon dioxide,
and in
3. the excretion of waste products from the body.

These functions are also facilitated by a very simple organisation of the wall
of capillaries. Only the tunica intima is present, which typically only
consists of the endothelium, its basal lamina and an incomplete layer of
cells surrounding the capillary, the pericytes. Pericytes have contractile
properties and can regulate blood flow in capillaries. In the course of
vascular remodelling and repair, they can also differentiate into endothelial
and smooth muscle cells.

Three types of capillaries can be distinguished based on features of the

endothelium.

Continuous capillaries

are formed by "continuous" endothelial cells and basal lamina. The endothelial cell and
basal lamina do not form openings, which would allow substances to pass the capilla
wall without passing through both the endothelial cell and the basal lamina. Both
endothelial cells and the basal lamina can act as selective filters in continuous capilla

Fenestrated capillaries

The endothelial cell body forms small openings called fenestrations, which allow compo
of the blood and interstitial fluid to bypass the endothelial cells on their way to or fro
the tissue surrounding the capillary. The fenestrations may represent or arise from
pinocytotic vesicles which open onto both the luminal and basal surfaces of the cell. T
extent of the fenestration may depend on the physiological state of the surrounding ti
i.e. fenestration may increase or decrease as a function of the need to absorb or secre
The endothelial cells are surrounded by a continuous basal lamina, which can act as
 157

selective filter.

Discontinuous capillaries

are formed by fenestrated endothelial cells, which may not even form a complete layer of c
The basal lamina is also incomplete. Discontinuous capillaries form large irregularly
shaped vessels, sinusoids or sinusoid capillaries. They are found where a very free
exchange of substances or even cells between bloodstream and organ is advantageou
(e.g. in the liver, spleen, and red bone marrow).

Suitable Slides
Sections of any well preserved tissue - H&E, Whipf's polychrome
cardiac and skeletal muscle, glands or the papillary layer of the skin
contain dense capillary beds.
Cardiac Muscle, sheep -
Whipf's polychrome
Large numbers of
capillaries are present in
almost all tissues. At least
a few dozen cross sections
are present in every sqr.
mm of section of poorly
vascularised tissues. There
may be thousands in highly
vascularised tissues.
However, a "good"
capillary is not that easy to
find because of their small
size and because the
capillary walls are very
thin, which often leads to
the collapse of the capillary
during tissue preparation.
Cardiac muscle is highly
vascularised. Each muscle
cell is surrounded by one
or more capillaries. The
capillaries roughly follow
the course of the muscle
cells. To find capillaries in
transverse and longitudinal
sections it is easiest to first
find areas in which the
muscle cells have been cut
 158

in these planes. Only one

or two red blood cells fit
side by side in the
capillary. A single
endothelial cell forms the
wall around the entire
circumference of a segment
of the capillary.
Endothelial cell nuclei are
therefore not always
visible, and some red blood
cells are only surrounded
by a fine line representing
the capillary wall.
Identify and draw a few
capillaries. Include some of
the surrounding tissue
features - maybe a cardiac
muscle cell, a venule or
arteriole - as a scale.

Veins

The walls of veins are thinner than the walls of arteries, while their diameter
is larger. In contrast to arteries, the layering in the wall of veins is not very
distinct. The tunica intima is very thin. Only the largest veins contain an
appreciable amount of subendothelial connective tissue. Internal and
external elastic laminae are absent or very thin. The tunica media appears
thinner than the tunica adventitia, and the two layers tend to blend into each
other. The appearance of the wall of veins also depends on their location.
The walls of veins in the lower parts of the body are typically thicker than those
of the upper parts of the body, and the walls of veins which are embedded in
tissues that may provide some structural support are thinner than the walls
of unsupported veins.

Venous vessels originate from the capillary network which coalesce into the
smallest venous vessels the

Venules.

They are larger than capillaries. Small venules are surrounded by pericytes.
A few smooth muscle cells may surround larger venules. The venules
merge to form
 159

Small to medium-sized veins

which contain bands of smooth muscle in the tunica media. The tunica
adventitia is well developed. In some veins (e.g. the veins of the
pampiniform plexus in the spermatic cord) the tunica adventitia contains
longitudinally oriented bundles of smooth muscle.
Aside from most veins in the head and neck, small to medium-sized veins
are also characterised by the presence of valves. The valves are formed by
loose, pocket-shaped folds of the tunica intima, which extend into the
lumen of the vein. The opening of the pocket will point into the direction
of blood flow towards the heart. One to three (usually two) pockets form
the valve. Blood flowing towards heart will pass the pockets. If the flow
reverses, blood will fill the pockets which will occlude the lumen of the
vein and prevent the return of blood into the part of the vein preceding the
valve. The ability of the valves to prevent backflow depends to some extent on
the state of contraction (tone) of the smooth muscle in the wall of the vein.

The largest veins of the abdomen and thorax

do contain some subendothelial connective tissue in the tunica intima, but

both it and the tunica media are still comparatively thin. Collagen and
elastic fibres are present in the tunica media. The tunica adventitia is very
wide, and it usually contains bundles of longitudinal smooth muscle. The
transition from the tunica adventitia to the surrounding connective tissue is
gradual. Valves are absent.
Vasa vasorum are more frequent in the walls of large veins than in that of
the corresponding arteries - probably because of the lower oxygen tension
in the blood contained within them.

Suitable Slides
sections of veins - H&E, van Gieson or elastin (in combination with
other stains)
Like arteries, veins and venules are present in many sections. The
basic features of their structure are however more difficult to
identify because of the thin walls of veins - in particular in small
veins. It is best to resort to sections of large veins, which together
with large muscular arteries often have their "own section" in
teaching collections.
 160

Vein, human - H&E

The tunica intima is very
narrow and the internal
elastic lamina is difficult to
identify - even in elastin
stained sections. A few
elastic fibres below the
endothelium form only a
very thin and incomplete
internal elastic lamina.
Smooth muscle is present
in the tunica media, but it
is organised less regular
than in the artery. The
tunica media is, again as
compared to the artery,
very thin and there is no
sharp border between the
tunica media and the tunica
adventitia. The tunica
adventitia of the largest
veins contains coarse
collagen fibres, elastic
fibres and longitudinal
bundles of smooth muscle.
Small and medium sized
veins will not contain
smooth muscle in the
tunica adventitia.
Draw part of the wall of
the vein, label the tunics
and indicate the presence
of smooth muscle, fibres
and their types.

Suitable Slides
sections containing small to medium sized veins - H&E, van Gieson
or trichrome
Unless a specifically prepared slide is available, I would recommend
looking at skin slides, in which I have found quite a few nice valves
in the veins located at the border between dermis and hypodermis.
 161

Vein Valve - H&E

Unless the section has been
specifically prepared to
illustrate valves, you will
have to search for a while -
probably through several
sections. If a vein with
valves is present in the
section, it should be easy to
identify. Valves are only
found in small to medium-
sized veins. You should see
one or two bands of tissue
in the lumen of the vein.
Each band is formed by
two apposing layers of
tunica intima. The bands
may share their origin from
the inner aspect of the wall
of the vein or they may
have separate origins.
Folding of the tissue bands
forming the valves is
variable.
Draw the vein and valve
and label the tunics which
you can identify. Try to
indicate the position of the
section in the schematic
drawing of the valve.
You can observe the
function of vein valves. Put
your finger firmly on one
of the veins which are
visible on the back of your
hand. Use another finger to
stroke over the vein in the
direction of the wrist. The
vein will empty and not
backfill (closed valves!)
until you release the first
finger.

Additional Specialisations of Vessels

 162

Small arteries and veins often form anastomosing networks, which provides
routes for alternative blood supply and drainage if one of the vessels should
become occluded because of pathological or normal physiological
circumstances. Some arteries are however the only supply of blood to their
target tissues. These arteries are call end arteries. Tissues which are
supplied by end arteries die if the arteries become occluded.

The segments of the kidney and the heads of the gastrocnemius muscle are
examples of tissues supplied by end arteries.

Arteries and veins may also form arteriovenous shunts, which can shunt the
blood flow that otherwise would enter the capillary network between the vessels .
These shunts usually contain specialisations of the smooth muscle in the
region of the shunt. Arteriovenous shunts are frequently seen in the blood
supply of distal parts of the limbs and the nose (thermoregulation) and in the
blood supply of endocrine organs.

Lymphatic Vessels

Parts of the blood plasma will exude from the blood vessels into the
surrounding tissues because of transport across the endothelium or because
of blood pressure and the fenestration of some capillaries (this process is
partly counteracted by the higher osmotic pressure of the blood). The fluid
entering tissues from capillaries adds to the interstitial fluid normally found
in the tissue. The surplus of liquid needs to be returned to the circulation.
Lymph vessels are dedicated to this unidirectional flow of liquid, the lymph.
Three types of lymph vessels can be distinguished based on their size and
morphology.

Lymph capillaries

are somewhat larger than blood capillaries and very irregularly

shaped. They begin as blind-ending tubes in connective tissue. The
basal lamina is almost completely absent and the endothelial cells do
not form tight junctions, which facilitates the entry of liquids into
the lymph capillary. Temporary openings in the endothelial lining of
the lymph capillaries also allow the entry of larger particles into the
lymph capillaries (lipid droplets, which are absorbed from the lumen
of the gut do not enter blood capillaries, but enter the circulation via
lymph vessels which are found in the villi of the ileum and
jejunum). Lymph capillaries merge to form
 163

Lymph collecting vessels

which are larger and form valves but otherwise appear similar to
lymph capillaries. The lymph is moved by the compression of the
lymph vessels by surrounding tissues. The direction of lymph flow
is determined by the valves. Lymph vessels empty intermittently
into lymph nodes from which the lymph continues in efferent lymph
vessels.

Only very little lymph is returned from the limbs if they are
immobilized, which illustrates the importance of muscular action in
lymph transport. This is also the reason for immobilizing limbs that
are either infected or that have been bitten by venomous Australians.
The effect can also be observed after long intercontinental flight
when you may feel that your shoes and socks are just about one
number too small. Finally, impeded lymph drainage is one of the
problems associated with surgery which requires the removal of
lymph nodes and which thereby interrupts the lymph collecting
vessels.

Eventually the lymph collecting vessels merge to form

Lymph ducts

which contain one or two layers of smooth muscle cells in their wall
(some textbooks call this layer the tunica media of lymph vessels).
They also form valves. The walls of the lymph ducts are less flexible
in the region of the attachment of the valves to the wall of the duct,
which may give a beaded appearance to the lymph ducts. Peristaltic
contractions of the smooth muscle contribute to the movement of
lymph towards the heart in addition to the compression of the ducts
by surrounding tissues.

The largest lymph duct of the body, the thoracic duct, drains lymph
from the lower half and upper left quadrant of the body and empties
the lymph into the circulation by merging with the vascular system
close to the junction of the left internal jugular and subclavian veins.
That it is the largest lymph duct does not mean that it is a large
vessel when compared to the large arteries and veins. It actually is
not much larger (about 5mm in diameter) than one of the superficial
forearm veins.

Suitable Slides
 164

Sections of small intestine - H&E

Jejunum, baboon - H&E
At low magnification you will
see villi extending into the
lumen of the jejunum. Find
villi which are cut
longitudinally and change to
medium magnification. In
some of the villi you will see
fairly large open spaces,
which are surrounded by a
layer of flattened endothelial
cells. They should not contain
any red blood cells - if they do
you are looking at a capillary,
which also should be
somewhat smaller. These
openings represent the blind
end of lymph capillaries
which originate in the villi.
Their name, lacteals, is
derived from the milky
appearance of the lymph. This
appearance is caused by
suspended lipid droplets
which enter these lymph
capillaries.

Draw a villus containing a

lymph capillary. Focus on the
features of the lymph capillary
and the surrounding
connective tissue. Include, if
possible, a blood capillary in
your drawing.

The jejunum slide is also good

for revision. You should be
able to find columnar
epithelium, goblet cells,
smooth muscle, small nerves,
a few ganglion cells and, of
course, lots of loose
connective tissue and
 165

bloodvessels

Histology of the Heart

The major part of the heart is constituted of
cardiac muscle. This kind of muscle is found only
in the heart and in the tunica media of the
terminal parts of the venae cavae as they enter the
heart. The layer of the heart consisting of cardiac
muscle is called the myocardium. Its inner surface
of the myocardium is lined with endocardium,
and the outer surface with epicardium.

The Myocardium:

Cardiac muscle is similar to skeletal muscle in

many ways. Both types appear striated as a result
of the arrangement of the actin and myosin
filaments in the sarcomeres of the muscle fibres.
The sarcolemma (plasma membrane) of both
types has invaginations called T-tubules that
spread depolarization throughout the cell. The
signal is transmitted to the terminal cisternae of
the sarcoplasmic reticulum, which are adjacent to
the T-tubules. The release of calcium from the
cisternae allows contraction to take place. The
mechanism of muscle contraction (sliding
filament) is the same in the cardiac and skeletal
muscle.

There are differences in cardiac and skeletal

muscle observable at the light microscope and
ultrastructural level. Cardiac muscles fibres are of
smaller diameter (about 15 micrometers) than
most skeletal muscle fibres (10-100 micrometers).
Cardiac muscle fibres are formed by individual
muscle cells with one or two centrally placed
 166

nuclei, while skeletal muscle fibres are

multinucleated protoplasmic units in which the
nuclei are peripherally located. Cardiac muscle
fibres branch and anastomose, skeletal muscle
fibres do not. Cardiac muscle fibres are arranged
in a linear array, each fibre is about 85-100
micrometers long. The junction between two
cardiac muscle cells, called an intercalated disk, is
another distintguishing feature. The intercalated
disk is made up of three types of cell junctions:
fascia adherentes, desmosomes and gap junctions.

At the ultrastructural level, the arrangement of T-

tubules is more regularly organized in skeletal
muscle, and they are found at the A-I junction, in
contrast to the Z-line in cardiac muscle. T-tubules
are usually associated with two terminal cisternae
(triad) in skeletal muscle, versus one (diad) in
cardiac muscle. The cisternae of skeletal muscle
are much larger than those of cardiac muscle.
Cardiac muscle is more vascularized and has
more abundant mitochondria than does skeletal
muscle (40% of volume vs. 2%), it also contains
glycogen granules between the myofibrils.
Physiologically, cardiac muscle is intrinsically
rhythmic (contracts without outside stimulation)
although it is regulated through nervous and
hormonal mechanisms. The rate of cardiac muscle
contraction is set by the sinoatrial node, whose
intinsic rhythm is the most rapid.

Cardiac
muscle in
longitudinal
section

Figure 1 shows
cardiac muscle in
longitudinal
section. (It is taken
from slide 94.)
The striations can be seen along the length of the
muscle fibres. [The striations are easier to see
when looking through the microscope, they are
not as obvious on these scanned computer
 167

images.] The nuclei of the cardiac muscle cells lie

in the middle of the cells. In a good face-view
section, the nucleolus is well-stained and the rest
of the nucleus has a delicate pattern. The
myofibrils separate to bypass the nucleus, and
there is often a perinuclear region in which no
striations are seen. This region contains
cytoplasmic organelles not directly involved in
contraction.

Each muscle fibre is surrounded by an

endomysium of delicate connective tissue with a
rich capillary network. Although the reticular
fibres of the endomysium are not usually seen,
you will see the nuclei of fibroblasts between the
muscle fibres and also many capillaries running
alongside them. Fibroblast nuclei tend to be more
flattened and darker staining than those of cardiac
muscle cells and are of course peripherally
located. [In your sections, you won’t be able to
identify each nucleus. Find a good cardiac muscle
nucleus with the features described above, then
look for ones that look similar.]

Intercalated disks appear as slightly darker lines

perpendicular to the length of the cardiac muscle
fibres. Depending upon the preparation and the
staining, intercalated disks can be obvious or
barely identifiable.

Branching in
cardiac muscle
fibres

Figure 2 shows
another
longitudinal
section of cardiac
muscle (from slide
23). In this section,
several cardiac fibres are seen branching.

Cross section of cardiac muscle

Figure 3 shows cardiac muscle fibres in cross

 168

section. The cut ends of the myofibrils appear

stippled. When
cross sections of
myofibrils appear
irregular, it
probably indicates
an area of
branching. The
nuclei of cardiac
fibres are found
near the middle of
the cross section,
sometimes a paler perinuclear region can be seen.
Connective tissue runs between bundles of muscle
cells, these bundles may become more widely
separated during tissue preparation. Fibroblast
nuclei will be found within the connective tissue
or at the periphery of a muscle fibre (since each
muscle fibre is also individually wrapped in
endomysium). Many capillaries can be seen
among the cardiac muscle fibres. The small empty
circles among the muscle fibres are all capillaries
(except for those larger than one RBC, they
represent pre- or post-capillary vessels). The
thickened areas of the capillary walls are
endothelial cell nuclei. A large blood vessel,
containing RBCs, is wrapped around a muscle
bundle to the right of the figure, and a much
smaller vessel (but too large to be a capillary), is a
bit to the left of the large vessel. The endothelial
cell nucleus in the smaller vessel is prominent.

The heart has a "skeleton" , which is the site for

the origin and insertion of cardiac muscle. It
consists of a fibrous ring surrounding each of the
four orifices (aortic, pulmonary, tircuspid and
mitral). The heart valves are attached to the
cardiac skeleton.

Impulses originating in the sinoatrial node (SA

node or pacemaker) pass along the cardiac muscle
fibres of the atria and along internodal tracts of
modified muscle fibres to the atrioventricular
node (AV node) near the tricuspid valve. The AV
node provides the only bridge between atrial and
 169

ventricular muscle. From the AV node, impulses

pass across the fibrous skeleton of the heart to the
ventricles via the AV bundle of His. The bundle
of His divides into a right and left branch (the
latter with 2 fascicles) which travel along the
ventricular septum to the apex of the heart and
then reverse their direction.

The branches of the bundle of His give off fibres,

called Purkinje fibres, which are modified cardiac
muscle cells with a diameter about twice that of
regular fibres (30 vs. 15 micrometers). Purkinje
fibres contain fewer myofibrils than regular
cardiac muscle fibres and have large
concentrations of glycogen. Their nuclei tend to
be surrounded by a large perinuclear space with
the myofibrils well toward the periphery of the
muscle fibre. Purkinje fibres are much faster
conducting than regular cardiac muscle fibres,
with which they make contact via gap junctions.
The impulse initiated in the SA node cause the
atria to contract first and expel blood into the
ventricles. The impulse is also carried along the
internodal fibres to the AV node, bundle of His
and its branches and then to the Purkinje fibres.
Contraction of the ventricles begins at the apex
and continues in a wavelike fashion toward the
base, forcing blood into the aorta and pulmonary
trunk.

Longitudional section of Purkinje fibers

Figure 4 shows
Purkinje fibres in
longitudinal
section. Because
of the lower
density of
myofibrils,
Purkinje fibres
appear paler than
regular cardiac
muscle fibres (a few of which can be seen at the
bottom of the figure). A prominent perinuclear
region is seen around several nuclei, and
 170

intercalated disks are evident. As in other cardiac

muscle, capillaries are abundant. A capillary can
be seen branching from a larger vessel near the
middle, top third of the figure.

The Purkinje fibres shown in Figure 4 were

scanned from slide 8. In this slide, collagen stains
blue (Masson trichromic). This blue might be
helpful in guiding you to the Purkinje fibres when
you look at the slides under your microscope, as
they are found in a layer of CT called the
subendocardial layer (see below). (Little CT was
visible in Figure 4, it would have been toward the
top of the figure beyond the field of view).

Cross section
of Purkinje fibres

Figure 5 (also
from slide 8)
shows Purkinje
fibres in cross or
oblique section
embedded in the
CT of the subendocardial layer. The wide
diameter of the fibres and the large perinuclear
region devoid of myofibrils can be seen clearly.

Regular
cardiac fibres in
cross section

Figure 6 is also
scanned from slide
8, at exactly the
same
magnification as
Figure 5. Figure 6
shows regular cardiac muscle fibres in cross
section. It can be seen that the fibres are of
smaller diameter, stain more darkly because of a
higher density of myofibrils, and have a smaller
perinuclear region than the Purkinje fibres of
Figure 5.
 171

The Endocardium:

The endocardium lies on the luminal side of the

myocardium. Its inner surface is covered with
endothelial cells – the squamous epithelium lining
the inside of the heart and blood vessels. Beneath
the endothelium is a layer of fairly loose, well-
vascularized connective tissue, this becomes a bit
denser closer to the myocardium. The thickness of
the endocardium varies inversely with the
thickness of the myocardium. In other words, it is
thicker in the atria than in the ventricles, as the
muscular walls are more substantial in the
ventricles. The layer of CT closest to the
myocardium is slightly looser and is called the
subendocardial layer. It contains veins and nerves,
as well as the Purkinje fibres when present.

Figures 7 and 8 show the endocardial layer of the

atrium and ventricle, respectively. At this
magnification, the nuclei of the endothelial cells
are barely distinguishable. The difference in the
thickness of the endocardium between the atrium
and ventricle is evident, both figures were
scanned at the same magnification. Part if the
myocardium is visible in both figures. No veins or
nerves (or Purkinje fibres) are seen in the fields of
view.

Endocardium of Endocardium of
atrium ventricle

The Epicardium:
 172

The epicardium is the delicate, inner visceral

layer of the pericardium. We do not see the outer,
fibroelastic parietal layer of the pericardium on
our slides. The outer part of the epicardium is
lined with mesothelium: the epithelium lining the
walls and contents of the closed cavities of the
body, such as the thoracic, pericardial and
abdominal cavities. Large blood vessels and
nerves are found in the epicardium, and adipose
tissue can be abundant.

Low power
view of
epicardium

Figure 9 shows a
low power view
of the epicardium
of the ventricle.
Part of the
myocardium is
also visible. In the field of view shown here, there
is a large amount of adipose tissue within the
connective tissue of the epicardium. A nerve
bundle and several blood vessels can also be seen.
The mesothelial lining (at the top) is not really
distinguishable.

Higher
power view of the
epicardium

A higher power
view of the
epicardium is
shown in Figure
10. The nerve is
the same as the
one in Figure 9 and can be used for orientation.
Blood vessels are more easily identified (brighter
red due to RBCs). The nuclei of the mesothelial
cells can be distinguished (albeit with difficulty)
at this magnification.
 173

<hr size=2 width="100%" align=center>

page content and construction: Lutz Slomianka

last updated: 6/08/09

" Profit, good friends, I beseech you, by my example.

It will save you from many troubles of the vexing
sort. Cultivate a superiority to reason, and see how
you pare the claws of all the sensible people when
they try to scratch you for your own good!"

Wilkie Collins, from "The Moonstone"

 174

School of Anatomy and Human Biology - The University of Western

Australia
Blue Histology - Lymphoid Tissues I

LYMPHOID TISSUES

Lymphoid (or lymphatic) tissues, which mainly consist of dense accumulations of

lymphocytes, are widely distributed in the body. Lymphoid tissues are typically located
at sites that provide a possible route of entry of pathogens and/or sites that are liable to
infections. Epithelia delimit all other tissues from the "outside world", and it is not
surprising that lymphoid tissues are often found near them. Such lymphoid tissues are
grouped together as epithelium-associated lymphoid tissues. Depending on their precise
location these lymphoid tissues may be referred to as e.g. mucosa-associated lymphoid
tissue (MALT) or bronchus-associated lymphoid tissue (BALT). The tonsils or Peyer's
patches are examples of mucosa-associated lymphoid tissues. Lymphoid tissues
represent the sites of proliferation and differentiation of lymphocytes.

Lymphoid organs may be defined as anatomical "entities" which consists chiefly of

lymphoid tissues. The thymus is a primary lymphoid organ in that it supplies other
lymphoid organs and tissues with T-lymphocytes. Inserted into the blood and lymph
vascular system, the spleen and lymph nodes (secondary lymphoid organs) monitor the
internal environment of the body.

Thymus

The thymus is situated in the upper parts of the thorax, behind the sternum and the
upper four costal cartilages, in the anterior and superior mediastina. The size of the
thymus changes in the course of life. It weighs about 10-15 g at birth and reaches its top
weight (about 30-40 g) at puberty. After puberty a progressive involution (see below)
occurs, which leaves a middle-aged person with a thymus weighing about 10 g. The
thymus consists of a right and left lobe which are joined by connective tissue.

The thymus is enclosed by a thin connective tissue capsule from which numerous septa
extend into the thymus subdividing the two lobes into numerous lobules (about 0.5 -2
mm in diameter). Blood vessels enter and leave the thymus via the connective tissue
septa. Each lobulus is divided into a darker peripheral zone, the cortex, and a lighter,
 175

central zone, the medulla. Medullary tissue is continuous from lobule to lobule
throughout each lobe.

Reticular cells and macrophages are present in addition to the lymphocytes, which are
the dominant cell type within the lobules.

Reticular cells

are quite abundant. Their cytoplasm is eosinophilic, and their large, ovoid and
light nuclei may contain one or two nucleoli. The cells are branched, and their
slender processes are connected with the processes of other reticular cells to
form a cellular reticulum (or cellular network). This cellular network (reticular
fibres are scant in the thymus) provides support for other cells of the thymus.

Reticular cells sheathe the cortical capillaries; they form an epitheloid layer
which delimits the cortical tissue from the connective tissue and secrete
substances (hormones and other factors) important for thymic function. Thereby
they create and maintain the microenvironment necessary for the development
of T-lymphocytes in the cortex. Their functions thus go beyond those of
"typical" reticular cells and, to reflect this, they are also referred to as thymic
epitheliocytes.

Macrophages

occur in both cortex and medulla. They are difficult to distinguish from the
reticular cells in H&E stained sections.

Lymphocytes

are present in both cortex and medulla, but are more numerous (denser) in the
cortex. Their sizes are variable (5 - 15 µm) in the cortex but generally small in
the medulla. The vast majority of them will be developing T-lymphocytes. They
are also called thymic lymphocytes or thymocytes.
 176

Thymus, foetal human - H&E

Identify the connective tissue capsule
and septa, a lobule and its cortex and
medulla at low magnification. Identify
lymphocytes and thymic corpuscles.
They look pretty much like a sliced
(very, very small) onion. Take a close
look at the medulla and try to find
some cells which contain large and
light nuclei. They will be either
macrophages or reticular cells.
Sketch part of the thymus at low
magnification. Identify medulla and
cortex. Draw a segment of a lobule at
high magnification and identify
lymphocytes and nuclei of reticular
cells/macrophages and, if possible a
Hassall corpuscle.
 177

Function of the Thymus

The thymus is necessary for the development of the recirculating pool of small, long-
lived (in humans many years) lymphocytes, the T-lymphocytes. These cells are mainly
responsible for the cell-mediated part of an immune response. Stem cells invade the
cortical regions of the thymus, where they divide to form lymphocytes. Only a small
fraction (estimates range from 10-30%) of the cells generated in the cortex leave the
thymus. They migrate via the medulla into the blood stream to populate the T-
lymphocyte areas of other lymphoid tissues and organs. Cells which do not express the
necessary receptors to recognize antigens presented to them or which react incorrectly
towards "self-antigens" die and are removed by cortical macrophages.
Since the function of the thymus is to produce lymphocytes for the other lymphoid
tissues it is a primary lymphoid organ.

Involution of the thymus

After puberty much of the parenchyma of the thymus, in particular cortical lymphoid
tissue, is replaced by adipose tissue. The process, which is called involution, initially
proceeds rapidly but slows down in adulthood. Involution is under the control of steroid
hormones (both sexual hormones and stress hormones). Although most pronounced in
the thymus, involution is a common feature of all lymphoid tissues.

Another age-related phenomenon is the increase in size of the thymic (or Hassall's)
corpuscles. Thymic corpuscles are rounded eosinophilic structures, which consist of
concentrically arranged, flattened cells. Thymic corpuscles are likely to be formed by
reticular cells. Similar structures occur also in the tonsils. The size of these structures
varies from 20 µm to more than 100 µm in diameter. Thymic corpuscles may calcify,
and their core may "dissolve" leading to the formation of a cyst.
 178

Thymus, adult human - H&E

Draw a part of the tissue, which
illustrates the presence of adipose
tissue, and the decrease in the amount
of cortical and medullary thymic
tissue. Include thymic corpuscles in
your drawing if you can find them.

Lymph Nodes

Lymph nodes are small, flattened, oval or bean shaped organs, which are situated in the
course of the collecting lymph vessels. Their size is variable (from a few mm to more
than 2 cm). The capsule and trabeculae of lymph nodes are formed by connective
tissue. Afferent lymph vessels penetrate the capsule and empty into the subcapsular
space. The lymph continues thereafter through cortical and medullary sinuses towards
the efferent lymph vessels, which emerge from the hilus of the lymph node. The walls
of the sinuses can be traversed freely by all components of the lymph, which allows
lymphocytes to enter/leave the lymphoid tissue (as part of their constant circulation) or
to get in contact with antigens/antigen-presenting cells that may arrive with the lymph.

In lymph nodes we find B- and T-lymphocytes, macrophages and reticular cells.

 179

Reticular cells

(and reticular fibres) form a

delicate network between the
capsule and trabeculae. Only
their large and light nuclei are
easily visible in the
microscope. The cytoplasm of
reticular cells is only weakly
eosinophilic. Lymphocytes and
macrophages are housed in the
network of reticular cells and
the reticular fibres formed by
them. The processes of reticular
cells and reticular fibres extend
into and criss-cross within the
sinuses.
Lymphocytes

which are located in the outer cortex of the lymph node are likely to represent
B-lymphocytes. They are organised into spherical masses - lymphoid nodules or
follicles. Sites within the cortex at which B-lymphocytes have been stimulated
to proliferate (by contact with an antigen) appear lighter than the surrounding
tissue and allow you to identify the centres of lymphoid nodules. The lighter
stained parts of the nodules are called germinal centres. Mature B-lymphocytes
(plasma cells) are located in cord-like extensions of the lymphoid tissue into the
medulla, the medullary cords. T-lymphocytes are located in the more diffuse
tissue between the nodules and in the paracortex, i.e. the deep part of the cortex.

Macrophages

are found scattered within the lymphoid tissue. In many preparations they are
difficult to distinguish from the reticular cells, but if an H&E stain turns out
nice, macrophages can be distinguished from the reticular cells in the sinus
system of the lymph node.

Blood Vessels

Blood vessels enter the lymph nodes through the hilus and travel initially in the
connective tissue trabeculae that extend from the hilus into the parenchyma of the
lymph node. They continue in the medullary cords towards the cortex and give off
capillaries to the surrounding tissue as they do so. High-endothelial venules (or
postcapillary venules) in the deep cortex have a characteristic low cuboidal epithelium -
quite unlike the squamous epithelium that we usually would expect to see.
Lymphocytes, which reach the lymph node via the blood stream, may migrate through
this epithelium as part of their recirculation. Larger venules accompany the arteriolar
 180

branches as they leave the lymph nodes.

Lymph node, rabbit - H&E

The subcapsular and cortical sinus system was hardly (if at all) visible in the slides I
looked at. Identify the connective tissue capsule and trabeculae, cortex and medulla of
the lymph node, lymph nodules with germinal centres, medullary cords and
postcapillary venules.
Draw a section of the lymph node in which you can see the capsule and a nodule.
Include, if possible, a postcapillary venule at high magnification.
 181

Lymph node - H&E, carbon injected

This slide illustrates the distribution of
macrophages in lymph nodes - take a
quick look at them. Note that most of
them are located in the paracortex and
medulla.
 182

page content and construction: Lutz Slomianka

last updated: 5/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology-Integumentary System

INTEGUMENTARY SYSTEM

The skin or cutis covers the entire outer surface of the body. Structurally, the skin
consists of two layers which differ in function, histological
appearance and their embryological origin. The outer layer or
epidermis is formed by an epithelium and is of ectodermal origin . The
underlying thicker layer, the dermis, consists of connective tissue and
develops from the mesoderm. Beneath the two layers we find a
subcutaneous layer of loose connective tissue, the hypodermis or
subcutis, which binds the skin to underlying structures. Hair, nails
and sweat and sebaceous glands are of epithelial origin and
collectively called the appendages of the skin.

The skin and its appendages together are called the integumentary system.

Suitable Slides
sections of skin - H&E, trichrome or van Gieson
 183

Skin, thick - H&E,

trichrome
A good starting point is
to identify the main
layers (epidermis,
dermis and
hypodermis) of the skin
at low magnification.
The three layers
forming the skin can be
identified in all skin
sections. The
epithelium forming the
surface layer, the
epidermis, is usually
the darkest layer
visible. Sublayers are
visible in the
epidermis. Their
staining varies - not
just between stains but
also between different
H&E stained
preparations (possibly
depending on tissue
preservation and how
fresh the staining
solutions were). At the
transition from the
epidermis to the
dermis, staining will
become lighter. The
lighter stained layer,
the dermis, consists of
dense irregular
connective tissue. The
dermis is much thicker
than the epidermis. In
thick skin, dermal
papillae create a very
irregular border
between epidermis and
dermis. The
hypodermis is the
lightest layer visible
and consists mainly of
 184

adipose tissue. Dense

connective tissue
strands may extend
from the dermis deep
into the hypodermis
and anchor the skin to
underlying structures.

Epidermis

The epidermis is a keratinised stratified squamous epithelium. The

main function of the epidermis is to protect the body from harmful
influences from the environment and against fluid loss. Five
structurally different layers can be identified:

1. The stratum basale

is the deepest layer of the epidermis (closest to the dermis). It consists of a
single layer of columnar or cuboidal cells which rest on the basement
membrane. Basal cells are the stem cells of the epidermis. Their mitotic activity
replenishes the cells in more superficial layers as these are eventually shed from
the epidermis. The renewal of the human epidermis takes about 3 to 4 weeks.

2. In the
stratum
spinosum,
the cells
become
irregularly
polygonal. The
cells are often
separated by
narrow,
translucent
clefts. These
clefts are
spanned by
spine-like
cytoplasmatic
extensions of
 185

the cells (hence

the name of the
layer and of its
cells: spinous
cells), which
interconnect the
cells of this
layer. Spines of
cells meet end-
to-end or side-
to-side and are
attached to each
other by
desmosomes. In
addition to the
usual organelles
of cells, EM
shows
membrane-
bound lamellar
granules in the
cytoplasm of
the spinous
cells.

3. The stratum granulosum

consists, in thick skin, of a few layers of flattened cells. Only one layer may be
visible in thin skin. The cytoplasm of the cells contains numerous fine grains,
keratohyalin granules. The keratohyalin is not located in membrane-bound
organelles but forms "free" accumulations in the cytoplasm of the cells. The cells
begin to release the contents of the lamellar granules. The lipids contained in the
granules come to fill the entire interstitial space, which is important for the
function of the epidermis as a barrier towards the external environment.

4. The stratum lucidum

consists of several layers of flattened dead cells. Nuclei already begin to
degenerate in the outer part of the stratum granulosum. In the stratum lucidum,
faint nuclear outlines are visible in only a few of the cells. The stratum lucidum
can usually not be identified in thin skin.

5. In the stratum corneum,

cells are completely filled with keratin filaments (horny cells) which are
embedded in a dense matrix of proteins. Individual cells are difficult to observe
because (1) nuclei can no longer be identified, (2) the cells are
 186

very flat and (3) the space between the cells has been filled with
lipids, which cement the cells together into a continuous
membrane. In the EM, the cell membranes appear thickened and interdigitate
with those of neighbouring cells. Closest to the surface of the epidermis, the
stratum corneum has a somewhat looser appearance. Horny cells are constantly
shed from this part of the stratum corneum.
The protection of the body by the epidermis is essentially due to
the functional features of the stratum corneum.

Variations in the thickness of the epidermis (~0.1 mm in thin skin, 1 mm or more in

thick skin) are mainly the result of variations in the thickness of the stratum corneum,
although the other layers also vary in thickness. Cells of the epidermis of the skin will
at some time of their life keratinise and are collectively also called keratinocytes.

Keratinisation should not be used as a synonym for the formation of the stratum
corneum: other stratified squamous epithelia may become keratinised but may not form
a stratum corneum in which cells join to form a horny cell membrane.

Suitable Slides
sections of skin - H&E, trichrome or van Gieson

Skin, thin - H&E and Skin, thick, trichrome

The most superficial part of the epidermis is formed by the stratum corneum. Nuclei are
not visible in this layer. Cell outlines may be visible at high magnification or, in the
form of artefacts, as cracks or clefts in the stratum corneum. The stratum granulosum is
formed by a single layer of very dark and flattened cells in thin skin. Several layers of
cells containing keratohyalin granules are visible in thick skin. Polyhedral cells with
clear outlines form the stratum spinosum. The stratum basale is formed by a single layer
of cuboidal or columnar cells and delimits the epidermis from the dermis.
At high magnification, the basal cytoplasm of the basal cells seem to interdigitate with
the underlying dermis. Similar to the dermal papilla, this irregular border at the cellular
level, the dermal-epidermal junction, anchors individual basal cells firmly to the
underlying dermis.
Identify and draw the epithelium in thick and thin skin. Identify in your drawing as
many of the layers of the epidermis as possible.
 187

Other Cells of the Epidermis

The red and yellow hues of the skin are due to haemoglobin in the red
blood cells, which pass through the capillaries beneath the epidermis,
and carotene, which accumulates in fat cells found in the dermis and
hypodermis.

Melanocytes

The brown colour component is due to melanin, which is produced in the skin
itself in cells called melanocytes (typically 1000-2000 / sqr. mm). These cells are
located in the epidermis and send fine processes between the other cells. In the
melanocytes, the melanin is located in membrane-bound organelles called
melanosomes. The cell bodies of melanocytes are difficult to distinguish in
ordinary LM preparations, because the melanosomes are located mainly in the
 188

processes of the cells.

Melanocytes
can transfer
melanin to
keratinocytes -
mainly to the
basal cells. The
fine processes
of melanocytes
may invade
keratinocytes
and bud-off part
of the
melanocyte
cytoplasm,
including the
melanosomes,
within the
keratinocytes.
Melanin
protects the
chromosomes
of mitotically
active basal
cells against
light-induced
damage.

Pigmentation is
not just under
the control of
light. Hormones
produced by the
pituitary and the
adrenal glands
also affect
pigmentation.
Diseases of
these two
endocrine
organs often
result in
changes of
pigmentation of
 189

the skin.

Although
melanocytes are
also
ectodermal
in origin, they
are derived
exclusively
from the neural
crest of the
embryo, from
where they
migrate to all
other parts of
the body.

Langerhans Cells

are another cell type found within the epidermis. Morphologically they are not
unlike melanocytes, but functionally they are more closely related
to macrophages. They are important in immune reactions of the epidermis.
Their fine processes form a network between the cells of the epidermis and
phagocytose antigens which have entered the epidermis. Langerhans cells
may only be temporary residents of the skin. If they have come into contact with
an antigen, they can migrate to regional lymph nodes, where they initiate an
immune response.

T-lymphocytes

are, like Langerhans cells, a group of cells functioning in the immune

system. Some of them will be present in the epidermis. Together with
Langerhans cells they are sometimes referred to as SALT, i.e. skin-associated
lymphoid tissue.

Dermis

The dermis is the thick layer of connective tissue to which the epidermis is attached. Its
deepest part continues into the subcutaneous tissue without a sharply defined boundary.
Its thickness is for this reason difficult to determine but 1-2 mm is a good
 190

guestimate for "average" skin. The dermis may be divided into two
sublayers (again without a sharp boundary):

1. The papillary layer consists of loose, comparatively cell-rich

connective tissue, which fills the hollows at the deep surface (dermal
papillae) of the epidermis. Capillaries are frequent. Collagen fibres
appear finer than in the reticular layer.
2. The reticular layer appears denser and contains fewer cells. Thick
collagen fibres (5-10 µm) often aggregate into bundles (up to 100 µm
thick). The fibres form an interlacing network, although their
predominant direction is parallel to the surface of the skin. A preferred
orientation of the collagen fibres is not visible in the sections, but the
main orientation of the fibres differs in skin from different parts of the
body. Usually, their main orientation will follow the "lines of greatest
tension" in the skin (Kraissl lines). This is of some surgical
importance since incisions parallel to these lines will heal
faster and with less formation of scar tissue.

Kraissl lines have been defined in living humans. They not always coincide with
the cleavage lines, which Langer defined (Langer's cleavage lines) about a
century before Kraissl in cadavers.

Elastic fibres are found in both the papillary (fine fibres) and reticular
(coarse fibres) layers.
They can not be identified in H&E stained sections.

Suitable Slides
sections of skin - H&E, van Gieson
Van Gieson stained sections are particularly nice if the van Gieson stain has
been combined with an elastin stain.

Skin, thin - H&E and Skin, thick - van Gieson & elastin
How easy it is to differentiate between the papillary and reticular layer of the dermis
depends on the preparation - you may have to look at several preparations. Immediately
beneath the epidermis you should see a layer which at low magnification appears rather
evenly stained. At high magnification the stain should resolve into a fine network of
collagen fibres, which blend with equally fine elastic fibres. Cells are more numerous in
the papillary layer and you should see more nuclei in this area than in the deeper
reticular layer. Also, the papillary layer contains the capillary network which supplies
the epidermis, The reticular layer contains coarse collagen and elastic fibres and the
larger vessels which feed into the capillary network of the papillary layer..
Draw part of the epidermis and the underlying dermis. Label the layers of the dermis
 191

and structures contained within them.

Appendages of the Skin

Hair

A characteristic feature of the human skin is the apparent lack of hair

(pili) on most of the body surface. This is actually not quite true. Most of the skin is
haired although the hair in most areas is short, fine and only lightly pigmented. This
type of hair is called vellus hair.

Truly hairless are only the palms of hands and soles of feet, the distal
phalanges and sides of fingers and toes and parts of the external
genitalia.
 192

In those parts of the skin which we perceive as "hairy" we find terminal hairs. The
free part of each hair is called the shaft. The root of each hair is anchored in a tubular
invagination of the epidermis, the hair follicle, which extends down into the
dermis and, usually, a short distance into the hypodermis. The deepest end of the
hair follicle forms an enlargement, the bulb. Cells in the bulb are mitotically active.
Their progeny differentiates into the cell types which form the hair and the cells that
surround its root, the root sheath. Hair cells keratinise within the lower one-third of the
hair follicle. Above this level it is not possible to identify individual cells within the
hair. Each hair follicle has an associated bundle of smooth muscle, the
arrector pili muscle. This muscle inserts with one end to the papillary layer of the
dermis and with the other end to the dermal sheath of the hair follicle.

Hair growth is discontinuous. Hairs are lost and replaced by new ones. The hair follicle
goes through different stages that reflect the discontinuous hair growth.
Anagen is the phase of growth. The resting stage is called telogen. The length of
the anagen is variable in different regions of the body - lasting only a
few months for hair of the eyebrows and eyelashes but 2 to 5 years for
hair of the scalp. Hair growth is controlled by a number of hormonal
and hereditary factors and their interactions.

Suitable Slides
Sections of hairy skin or scalp - H&E
With a few exceptions (thick skin and skin covering parts of the external
genitalia), all skin sections should contain a few hair follicles.

Skin, hairy - H&E

Hair follicles of terminal hair span the entire dermis and usually extend deep into the
hypodermis. Most of them will be cut at odd angles and only a few good longitudinally
or transversely cut profiles are visible. The hair may have been lost during the
preparation of the specimen and not all hair follicles will contain hairs. Although it is
often possible to see the attachment of the arrector pili muscle into the hair follicle or
the papillary layer of the dermis, both attachments are hardly ever visible in the same
section.
Draw a hair follicle at low magnification. Try to draw a composite from several hair
follicles and associated structures, which captures their appearance from the bulb to the
epidermis.
 193

Sebaceous Glands

Sebaceous glands empty their secretory product into the upper parts of the hair follicles.
They are therefore found in parts of the skin where hair is present. The hair follicle
and its associated sebaceous gland form a pilosebaceous unit.

Sebaceous glands are also found in some of the areas where no hair
is present, for example, lips, oral surfaces of the cheeks and external
genitalia.

Sebaceous glands are as a rule simple and branched (Remember the nomenclature of
glands!). The secretory portion consists of alveoli. Basal cells in the outermost layer of
the alveolus are flattened. Basal cells are mitotically active. Some of the new cells will
replenish the pool of basal cells, while the remaining cells are displaced towards the
 194

centre of the alveolus as more cells are generated by the basal cells. The secretory cells
will gradullay accumulate lipids and grow in size. Finally their nuclei disintegrate, and
the cells rupture. The resulting secretory product of lipids and the constituents of the
disintegrating cell is a holocrine secretion.

The lipid secretion of the sebaceous glands has no softening effect

on the skin, and it has only very limited antibacterial and antifungoid
activity. Its importance in humans is unclear. Clinically the
sebaceous glands are important in that they are liable to infections
(e.g. with the development of acne).

Suitable Slides
slides of hairy skin or thin skin - H&E, trichrome, van Gieson

Skin, hairy - trichrome, H&E

Sebaceous glands will be present in all types of skin other than thick skin. Their
numbers should correlate with the number of hair follicles. If your section does not
contain hair follicles you are unlikely to see a good sebaceous gland. Sebaceous glands
are usually embedded in the dermis. Although they empty into the hair canal of the hair
follicle, this point will only be visible for a few of them because of the thinness of the
sections. It should however be possible to follow the fate of the secretory cells. Deep in
the sebaceous glands cells are smaller with intact nuclei. Cell size increases with the
accumulation of sebum as the cells are gradually displaced towards the opening of the
gland into the hair follicle. The nuclei condense, become darker and irregularly shaped.
Draw a sebaceous gland. Emphasise the appearance of the secretory cells in different
parts of the gland. If possible include part of the associated hair follicle.
195
 196

Sweat Glands

Two types of sweat glands are present in

humans. They are distinguished by their
secretory mechanism into merocrine
(~eccrine) sweat glands and apocrine
sweat glands. In addition, they differ in their
detailed histological appearance and in the
composition of the sweat they secrete.

Merocrine sweat glands are the only glands of the

skin with a clearly defined biological
function. They are of critical importance
for the regulation of body temperature.
The skin contains ~3,000,000 sweat
gland which are found all over the body - with the
exception of, once again, parts of the external
genitalia.

1. Sweat glands are simple

tubular glands. The secretory
tubulus and the initial part of the
excretory duct are coiled into a
roughly spherical ball at the border
between the dermis and hypodermis.
2. The secretory epithelium is
cuboidal or low columnar.
Two types of cells may be
distinguished: a light type,
which secretes the watery
eccrine sweat, and a dark
type, which may produce a unusually thick, haematoxylin
mucin-like secretion. The cells stained section of the skin
have slightly different shapes and, as
a result of the different shapes, the
epithelium may appear
pseudostratified.
3. A layer of myoepithelial cells is
found between the secretory cells of
the epithelium and the basement
membrane.
 197

4. The excretory duct has a

stratified cuboidal epithelium
(two layers of cells).

The excretory ducts of merocrine sweat

glands empty directly onto the surface of
the skin.

Apocrine sweat glands occur in, for example, the axilla. They are
stimulated by sexual hormones and are not fully developed or
functional before puberty. Apocrine sweat is a milky, proteinaceous
and odourless secretion. The odour is a result of bacterial
decomposition and is, at least in mammals other than humans, of
importance for sexual attraction.

The histological structure of apocrine sweat glands is similar to that of merocrine sweat
glands, but the lumen of the secretory tubulus is much larger and the secretory
epithelium consists of only one major cell type, which looks cuboidal or
low columnar. Apocrine sweat glands as such are also much larger
than merocrine sweat glands.

The excretory duct of apocrine sweat glands does not open directly onto the surface of
the skin. Instead, the excretory duct empties the sweat into the upper part of the hair
follicle. Apocrine sweat glands are therefore part of the pilosebaceous unit.

Some texts argue that the apocrine sweat glands use a merocrine or a combined
merocrine / apocrine secretory mechanism.

Suitable Slides
merocrine sweat glands: sections of thick skin or thin skin - H&E
apocrine sweat glands: sections of skin from the areolae (pigmented skin
surrounding the nipples), the axilla (arm pit) or skin covering the external
genitalia - H&E

Skin, thick - H&E

Scan along the border between dermis and hypodermis and locate a sweat gland. The
secretory tubulus and the initial segment of the duct usually form a cluster of round or
irregularly shaped profiles, which stain darker than the surrounding connective tissue.
The structural preservation of the sweat glands may vary quite a bit in the different
 198

preparations. The different cell types in the secretory epithelium of merocrine sweat
glands are only visible in well preserved glands. The red rim around the secretory
tubulus is formed by the cytoplasm of myoepithelial cells. Their small, dark nuclei may
be visible close to the periphery of the tubulus.
Draw a small schematic illustrating the relative position of the sweat gland in the skin.
Identify and draw the secretory tubulus and excretory duct. Label as many features as
can be identified.
 199

Nipple - H&E
Like merocrine sweat
gland, the secretory
tubulus of apocrine
sweat glands will coil
close to the border
between the dermis and
the hypodermis. Only
one type of secretory
cell is present, and the
lining of the secretory
tubulus looks more
uniform than that of
merocrine sweat
glands. The key feature
though is the very wide
lumen of the secretory
tubulus. The secretory
cells are surrounded by
a layer of myoepithelial
cells. Their cytoplasm
forms the slightly
darker outline of the
secretory tubulus.
Draw the secretory
tubulus of an apocrine
seat gland - preferably
next to your drawing of
the merocrine sweat
gland. Label the
structures included in
your drawing.

<HRpage content and construction: Lutz Slomianka

last updated: 6/08/09

School of Anatomy and Human Biology - The

University of Western Australia
 200

Blue Histology - Oral Cavity and Oesophagus

ORAL CAVITY
The oral cavity is formed by a bewildering array of tissues which function in or are
associated with the processes that are performed with what we typically refer to as
our mouth. Lecture and lab focus on the one organ found within the oral cavity, the
tongue, and the glands which empty their secretory products into the oral cavity, the
salivary glands. In the lab you will also have the opportunity the examine one other
specialized epithelial area, the lip. The oesophagus is the first part of the alimentary
canal. Its organisation is also typical for all parts of the gastrointestinal tract (GIT).

The oral cavity is divided in a vestibule, the area "outside" the teeth, and an oral cavity
proper. The entire oral cavity is lined by a stratified squamous epithelium. The
epithelial lining is divided into two broad types:

1. Masticatory epithelium covers the surfaces involved in the processing of food (tongue,
gingivae and hard palate). The epithelium is keratinized to different degrees
depending on the extent of physical forces exerted on it.
2. Lining epithelium, i.e. non-keratinised stratified squamous epithelium, covers the
remaining surfaces of the oral cavity.

The Tongue

The dorsal surface of the tongue is divided by the sulcus terminalis into an oral part, the
anterior two-thirds, and a pharyngeal part, the posterior one-third. The dorsal surface of
the oral part has a characteristic appearance due to the presence of a large number of
small projections, the lingual papillae. The epithelium of the pharyngeal part forms a
somewhat irregular surface which covers the lingual tonsils.

The lingual papillae consist of a connective tissue core

covered with a stratified squamous epithelium. On the basis of
their appearance four types of papillae can be distinguished -
filiform, fungiform, circumvallate and foliate papillae.

Filiform papillae
 201

are the smallest and most numerous papillae. By providing the tongue with a
rough surface they aid in the manipulation and processing of foods.
Prof. Oxnard brought another function to my attention, i.e. the cleaning of the
surfaces of the mouth, in particular the teeth.

Fungiform papillae

occur singly and are fairly evenly spaced between the filiform
papillae. Their connective tissue core is richly vascularised.
The epithelium is slightly thinner than on the remaining
surface of the tongue.

Circumvallate papillae

are the largest and least numerous papillae - in humans there are between 8 and
12 of them. They occur in depressions of the surface of the tongue and are
surrounded with a trench formed by the infolding of the epithelium. Taste buds
are particularly numerous on the lateral surfaces of these papillae. The excretory
ducts of serous glands open into the trenches surrounding the papillae ("rinsing
glands" or glands of von Ebner).

Foliate papilla

are not well developed in humans and may be absent in aged individuals. If
present, they form lamellae along the posterior and lateral border of the
 202

tongue.

The epithelium of the dorsal surface of the tongue rests on a fairly dense layer of
connective tissue, which connects the epithelium firmly with the underlying muscular
and connective tissues.

The muscles of the tongue (skeletal muscle) are organized into strands oriented more or
less perpendicular to each other. Their actions provide the tongue with the necessary
motility to participate in the formation of speech and to aid in the initial processing of
foods. Control of the movement of the tongue muscles and the collection of sensory
information necessitate a profuse innervation of the tongue in which a number of the
cranial nerves participate (V, trigeminal nerve - sensory - anterior two-thirds; VII, facial
nerve - taste; IX, glossopharyngeal nerve - sensory/taste - posterior one-third; XII,
hypoglossal nerve - motor).

Suitable Slides

Tongue, human - H&E

This section illustrates the general
organisation of the tongue. Taste buds
will be rare, if present at all, in this
section. Examine the tissue and have
a close look at the small salivary
glands located in the connective
tissue of the tongue. You should be
able to identify mucous acini and
serous demilunes (see below)
attached to the acini.
Draw a part of the section in which
papillae on the surface of the
tongue, muscular tissue and possible
some of the glands embedded
between the muscular tissue of the
tongue are visible.

Taste Buds
 203

Taste buds are most numerous in the fungiform, circumvallate and foliate papillae. In
addition, taste buds are found in the palate, palatoglossal and palatopharyngeal arches
and in the pharynx and larynx.

In histological sections they appear as ovoid lightly stained bodies, which extend
perpendicular from the basement membrane to a little opening formed in the
epithelium, the taste pore. The elongated cells that form the taste bud can functionally
be divided into three groups: sensory cells, supporting (or sustentacular) cells, and basal
cells. Sensory cells extend microvilli into the taste pore. These microvilli contain the
receptors for the different basic taste modalities (sweet, salty, bitter and acid). Basal
cells regenerate the two other cell types.

Cell turnover is quite high, and it is thought that the cells of the taste buds are
replaced (on average) every 10th day.

Suitable Slides

Circumvallate Papilla and Taste Buds, sheep - Alcian blue & van Gieson, H&E
Find and inspect the taste buds embedded in the epithelium of the lateral walls of the
circumvallate papillae. The taste pore may not always be visible (outside the plane of
section). Now look at the bottom of the trench surrounding the circumvallate papillae.
Sometimes it is possible to find a duct opening into the trench. If the actual opening is
outside the plane of section it is usually possible to find a section of the duct in the
underlying connective tissue. Slightly deeper in the connective tissue you may be able
to identify the serous glands, which rinse the trenches surrounding the circumvallate
papillae.
Draw a part of the tissue in which these structures (as many as possible) are visible.
 204

Salivary Glands

Saliva is a mixed secretion, which is derived from numerous large and small salivary
glands that all open into the oral cavity. Small salivary glands are situated in the
connective tissue beneath the epithelia lining the oral cavity, and, in the case of the
tongue, they may also be found between the muscular tissue. Depending on the
localisation they are grouped into lingual, labial, buccal, molar and palatine glands.

The large salivary glands form three paired groups:

1. the sublingual glands, which are positioned beneath the tongue and embedded deeply
in the connective tissue of the oral cavity,
2. the submandibular glands and
3. the parotid glands, which lie outside the oral cavity.

All of these glands are tubuloacinar glands, i.e. they have secretory acini but the first
part of the duct system originating from the acini also participates in the secretory
process. The salivary glands are divided by connective tissue septa into lobes, which are
further subdivided into lobules.

Functionally the secretory acini can be divided into two groups: those that secrete a
 205

rather liquid product - serous acini, and those that secrete a very viscous product -
mucous acini. This functional differentiation is reflected in the appearance of these
acini in histological sections.

 The cells forming the serous acini contain a round or slightly ovoid nucleus which is
placed basally in the cell. In an H&E stain, the apical cytoplasm may appear
pinkish/red or, in well-preserved tissue, contain reddish granules. The granules
represent the vesicles which contain the secretory products of the cell. The digestive
enzyme α-amylase is also secreted by the acinar cells.
 The cells forming the mucous acini usually contain flattened nuclei which appear to be
"pressed" against the basal surface of the cell. Secretory vesicles fill much of the apical
cytoplasm. The secretory product has either been dissolved during the staining
process or remains unstained. Small amounts of cytoplasm which remain between the
vesicles gives the apical part of the cell a distinct "spongy" appearance.

Occasionally, and in particular in glands

located relatively close to the oral cavity,
serous cells and mucous cells may form
compound or mixed acini. The serous cells
form in these cases small half-moon or
crescent-shaped structures, which attach to
mucus producing acini and empty their
secretory product into interstices between
the mucus-producing cell. Following their
appearance they are called serous
demilunes.

Both serous and mucous acini and parts of

the secretory duct system are surrounded
by myoepithelial cells which by their
contraction participate in the secretory
process. They are usually difficult to
distinguish in histological sections.

Glands located close to the oral cavity have mainly mucous secretions, whereas glands
located further away from the oral cavity have mainly serous secretions. Following this
general rule, the parotid glands contain almost exclusively serous acini, the
submandibular glands contain both serous and mucous acini, and the sublingual glands
contain mainly mucous acini or mucous acini with serous demilunes.

Ducts of the Salivary Glands

 206

The ducts of the salivary glands can, according to their position in relation to the lobes
and lobules of the glands, be divided into two parts. Interlobular and interlobar ducts
are embedded in the connective tissue surrounding the lobes and lobules of the
glands. Intralobular ducts are located in between the secretory acini within the lobules
and, consequently, only surrounded by scant, if any visible connective tissue.

Interlobar and interlobular ducts function mainly in the conduit of the saliva and are
formed by a stratified cuboidal or stratified columnar epithelium. The epithelium is
replaced by the stratified squamous epithelium as they approach the opening into the
oral cavity.

The product of serous glands is extensively modified by the initial part of the duct
system. Intralobular ducts can on the basis of their function be divided into intercalated
ducts and striated ducts. The secretory acini empty into intercalated ducts which merge
into the striated ducts.

 Cells forming the intercalated ducts add bicarbonate ions to the saliva (buffering
function) and absorb chloride from the saliva. They are typically formed by cuboidal
epithelium.
 Striated ducts are formed by columnar cells. In contrast to many other columnar
epithelia, the nucleus of these cells is located approximately midways between the
apical and basal cell surfaces. The striations of the striated duct are found in the basal
part of the cytoplasm of the cells where numerous mitochondria are found between
infoldings of the basal cell membrane. This specialisation provides the cell with the
necessary energy and surface area to perform its task in the modification of the saliva
- the secretion of potassium and the absorption of sodium. Cells of the striated ducts
also take up a secretable form of antibodies and release them into the saliva.

Intercalated ducts are difficult to

identify in mucous glands and striated
ducts are absent in purely mucous
glands. Following the main secretory
product of the major salivary glands,
well-differentiated intercalated and
striated ducts are a prominent feature
of the parotid glands, rare in the
submandibular glands and absent in
the sublingual gland. An additional
feature that may aid in the
identification of the parotid gland are
fairly large amounts of adipose tissue
which is found between the secretory
tissue of the lobules.
 207

Suitable Slides

Parotid Gland - H&E,

Submandibular Gland - H&E and
Sublingual Gland - H&E
Inspect these slides. Try to identify
serous and mucous acini, intercalated
ducts and striated ducts and (if
present) secretory ducts in the
surrounding connective tissue.
High magnification images of
secretory acini and ducts of the
parotid and sublingual gland are
available on the "Epithelia and
Glands" page. The slides of one or
maybe two of the glands do not
always completely conform to
textbook descriptions. Which ones,
and why?.
Draw part of the tissues of the
glands in which the structures
characteristic for each major
salivary gland are illustrated.
 208

The Oesophagus

Throughout the remainder of the digestive system, the histological composition of the
alimentary canal can be described by the following blue-print:

 The lumen is lined by an epithelium, which rests on a vascular connective tissue, the
lamina propria. The lamina propria is in turn surrounded by a narrow band of smooth
muscle (muscularis mucosae). These three tissues are collectively referred to as the
mucosa of the alimentary canal.

 Beneath the mucosa we find a

wider zone of loose connective
tissue, the submucosa, which in
addition to vessels contains a
nerve plexus (submucosal
plexus or Meissner's plexus),
which is one of the two plexi
innervating the alimentary
 209

canal.

 The submucosa is surrounded by

smooth muscle, which is
typically divided into two
differently oriented layers - an
inner circular and an outer
longitudinal layer. These layers
of muscle are together referred
to as the muscularis externa.
Between two muscle layers we
find the second nerve plexus
innervating the alimentary canal
(myenteric plexus or
Auerbach's plexus).

 The alimentary canal is delimited from other tissues by a layer of loose connective
tissue, the adventitia. In the case of the intraperitoneal parts of the alimentary canal,
i.e. those parts which are suspended in the peritoneal cavity, a simple squamous
epithelium, the serosa, delimits the adventitia from the peritoneal cavity.

Glands may be present in some parts of the wall of the alimentary canal canal. These
glands are called mucosal glands if they are located luminal (or superficial) to the
muscularis mucosae. If the glands extend into the submucosa they are called
submucosal glands.

In the oesophagus the mucosa is formed by a stratified squamous epithelium (non-

keratinised) and a well-defined lamina propria and muscularis mucosae.

Oesophageal glands are located in the submucosa. These submucosal glands produce a
mucous secretion, which lubricates the epithelium and aids the passage of food. In the
part of the oesophagus closest to the stomach there may be mucosal mucus-producing
glands, which resemble the glands in the adjacent mucosa of the stomach.
The mucous glands in the part of the oesophagus closest to the stomach protect the
oesophageal mucosa from acidic reflux from the stomach. Mucous glands in the
adjacent mucosa of the stomach are called cardiac glands, and this name is also used
for submucosal mucosal glands in the the part of the oesophagus closest to the stomach.

The muscularis externa is somewhat unusual in that it contains striated muscle in its
upper one third, a mixture of striated muscle and smooth muscle in its middle one-third
and smooth muscle in its lower one-third.
 210

The adventitia consists only of a layer of loose connective tissue. Only the lowest part
of the oesophagus (approx. the lowest 2 cm) enters the peritoneal cavity. A serosa
forms the outermost part of the adventitia of this short intraperitoneal segment of the
oesophagus.

Suitable Slides

Oesophagus, human - H&E

Identify the different tissues that form
the wall of the alimentary canal. The
two slides differ in the types of
muscle present in the muscularis
externa and in the organisation of the
muscularis mucosae. It may be
difficult to identify parts of the nerve
plexus innervating the oesophagus,
and oesophageal glands are not
present in all sections. Look for these
structures, but don't get too upset if
you do not find them.
Draw a section of the wall of the
esophagus. Identify in your drawing
the main parts of the wall of the
alimentary canal (mucosa,
submucosa, muscularis externa and
adventitia).

The Lips

When we think of lips we usually only think of a small part, the vermilion border (or
prolabium), of the "anatomical" lips, which comprise the entire fleshy fold surrounding
the oral orifice. The outside and inside of the lips are lined by skin and oral mucosa
respectively. Between the two, we find labial vessels, nerves, the orbicularis oris
muscle (striated), which shapes the lips, and labial salivary glands.

The vermilion border is the area of transition from the skin to the oral mucosa. The
epithelium is somewhat thicker than in other parts of the facial skin. Connective tissue
papilla extend deep into the epithelium and are heavily vascularized. It is the proximity
 211

of these vessels to the surface of the epithelium which gives the prolabium it's red
appearance.

Suitable Slides

Lip, human - H&E

Hairs and glands (aside from a few
sebaceous glands) are usually not
present beneath the epithelium of the
prolabium. Find the point of
transition to the stratified squamous
epithelium of the oral mucosa. In the
connective tissue beneath the oral
mucosa you may be able to find the
small labial salivary glands.
If you can not find them in the
section, try to find them in your
mouth by palpating the oral surface of
the lips with the tips of your tongue -
the glands are the small nodules that
you feel. The sebaceous glands
present in the section are very pretty -
have a look - it should become
clear(er) why they are alveolar
glands. The thin skin present in the
section is also quite stunning. The
spines of the spiny cells in the stratum
spinosum are actually very clearly
visible.
Draw a survey of the lip and identify
the prolabial and oral sides, the
orbicularis oris muscle and, if
present, glands embedded in the
connective tissue beneath the two
sides of the lip.

page content and construction: Lutz Slomianka

last updated: 1/10/06
 212

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Gastrointestinal Tract

GASTROINTESTINAL TRACT
The gastrointestinal tract (GIT) comprises the stomach,
duodenum, jejunum, ileum, colon, rectum and anal canal. The
GIT and oesophagus form the alimentary canal. The basic
structure of the walls of the alimentary canal has been described
on the "Oral Cavity and Oesophagus" page.

The Stomach

The stomach functions both as a reservoir and as a digestive

organ. It empties its contents in small portions (suitable for
continued digestion) into the small intestine.

Anatomically, the stomach is divided into

 a cardiac part,
 fundus,
 body or corpus, and
 a pyloric part (pyloric antrum and pyloric canal)
Histologically, most of the layers of the wall of the stomach
appear similar in its different parts. Regional differences are
mainly restricted to the appearance of the gastric mucosa.
 213

The Mucosa (epithelium, lamina propria, muscularis mucosae)

The mucosa is thrown into longitudinal folds (gastric folds or
rugae), which disappear when the stomach is fully distended. A
network of shallow grooves divides the mucosa into gastric areas
(1-5 mm). On the mucosal surface we see small, funnel-shaped
depressions (gastric pits). Almost the entire mucosa is occupied
by simple, tubular gastric glands which open into the bottom of
the gastric pits.

The structure and cellular composition of the surface epithelium

(simple, tall columnar) does not change throughout the stomach.
It contains mucus-producing cells, surface mucous cells, which
form a secretory sheath (glandular epithelium). The mucus is
alkaline and adheres to the epithelium. The mucus forms an ~ 1
mm thick layer, which protects the mucosa from the acidic
contents of the stomach. The surface epithelium is renewed
approximately every third day. The source of the new cells is the
isthmus, i.e. the upper part of the neck, of the gastric glands,
where cells divide and then migrate towards the surface
epithelium and differentiate into mature epithelial cells.

In contrast to the surface epithelium, cellular composition and

function of the gastric glands are specialized in the different parts
of the stomach.

Cardiac glands
 214

Cardiac glands are heavily

branched tubular glands
(similar to the cardiac
glands of the esophagus),
which contain mainly
mucus-producing cells. A
few of the secretory cells
characteristic for the
principal glands (see
below) may be present.

Principal (or corpus-

fundic) glands
Each glandular tubule
(oriented more or less
perpendicular to the
surface of the epithelium)
consists of three parts: a
deep body, an intermediary
neck and an upper isthmus.
In principal glands we find
four cell types: chief cells, parietal
cells, mucous neck cells and
endocrine cells.
Chief cells (or zymogenic cells)
are the most numerous of the four types. They occur
primarily in the body of the glands. They produce
pepsinogen, which is a precursor of the proteolytic enzyme
pepsin.
The pH optimum of of pepsin is about 2. This enzyme is
able to break down collagen.

Parietal cells (or oxyntic cells)

 215

occur most frequently in the neck of the glands, where they

reach the lumen of the gland. They are situated deeper,
between and below chief cells, in lower parts of the gland.
Parietal cells secrete the hydrochloric acid of the gastric
juice. Aside from activating the pepsinogen the
hydrochloric acid also effectively sterilizes the contents of
the stomach.

Note that so far only one type of bacteria has found which
can live happily in the stomach - Helicobacter pylori.
Unfortunately these bacteria are involved in the
pathogenesis of gastritis and gastric ulcers.

Parietal cell also secrete intrinsic factor, which is necessary

for the resorption of vitamin B12.

Vitamin B12 is a cofactor of

enzymes which synthesise
tetrahydrofolic acid, which,
in turn, is needed for the
synthesis of DNA .
components. An impairment
of DNA synthesis will affect
rapidly dividing cell
populations, among them the
haematopoietic cells of the
bone marrow, which may
result in pernicious anemia.
This condition may result
from a destruction of the
gastric mucosa by e.g.
autoimmune gastritis or the
resection of large parts of the
lower ileum, which is the
 216

main site of vitamin B12

absorption, or of the
stomach.

Mucous neck cells

are found between the
parietal cells in the neck of
the gland.
They are difficult to
distinguish from chief cells
in plain H&E stained section.

Endocrine cells
Endocrine cells are scattered, usually solitary, throughout
the epithelium of the gastrointestinal tract. They are part of
the gastro-entero-pancreatic (GEP) endocrine system. The
best characterized endocrine cells in the gastric mucosa are
gastrin-producing cells (G cells) and somatostatin-
producing cells (D cells). G cells are most frequent in the
middle third of the glands. They stimulate the secretion of
acid and pepsinogen. G cell function is stimulated by
nervous input, the distension of the stomach or
secretagogues. D cells are found mainly in glands of the
pyloric antrum. They inhibit G cells and thereby acid
production. D cell function is stimulated by acid in the
lumen of the stomach and duodenum.

Other types of endocrine cells encompass VIP-producing

cells (or D1 cells; vasoactive intestinal peptide) and
serotonin-containing cells (enterochromaffin cells).
Endocrine cells in the gastrointestinal tract are alternatively
named APUD-cells: amine precursor uptake and
 217

decarboxylation cells.

Pyloric glands
Pyloric glands are more coiled than principal glands, and they
may be branched. Endocrine cells, in particular gastrin-producing
cells, are more frequent than in principal glands. A few parietal
cells may be present but chief cells are usually absent.

The lamina propria is formed by a very cell-rich loose connective

tissue (fibroblasts, lymphocytes, plasma cells, macrophages,
eosinophilic leukocytes and mast cells). The muscularis mucosae
of the stomach contains both circular and longitudinal layers of
muscle cells. Its organization is somewhat variable depending on
the location in the stomach.

Large blood vessels, lymph vessels and nerves are located in the
submucosa which consists of loose connective tissue.

Note that the muscularis externa consists of three layers of

muscles: an inner oblique layer, a middle circular layer and an
outer longitudinal layer.

Suitable Slides
slides of stomach - H&E (with PAS), van Gieson (with
Alcian Blue)

Stomach, cat - H&E

It is helpful to find a spot of the gastric mucosa where the glands
have been cut along their long axis.
Draw part of the stomach wall, and identify in your drawing the
mucus-producing cells of the secretory sheath, the light,
undifferentiated looking mucous neck cells, the slightly pink,
fairly large parietal cells and the somewhat darker bluish-violet
chief cells.
 218

Small Intestine
The small intestine is divided into duodenum (25-30 cm),
jejunum (about first two-fifths of the rest) and ileum. The three
segments merge imperceptibly and have the same basic
histological organization.

The Mucosa
The mucosa of the small intestine has various structural features
which considerably increase the luminal surface area and
consequently support the main function of the small intestine -
the absorption of the degraded components of the food.

Plicae circulares (of Kerkering) are macroscopically visible,

 219

crescent-shaped folds of the mucosa and submucosa. Plicae

circulares extend around one-half to two-thirds of the
circumference of the lumen of the small intestine.

Plicae circulares

 are permanent structures, i.e. their presence does not

depend on the state of distension of the small intestine.
 are absent from the first few centimetres of the duodenum
and the distal part of the ileum.
 Are particularly well developed in the jejunum.
 increase the surface area of the mucosa by a factor of ~
three.
The entire intestinal mucosa forms intestinal villi (about one mm
long), which increase the surface area by a factor of ~ ten. The
surface of the villi is formed by a simple columnar epithelium.
Each absorptive cell or enterocyte of the epithelium forms
numerous microvilli (1 µm long and about 0.1 µm wide).
Microvilli increase the surface area by a factor of ~ 20.

Between the intestinal villi we see the openings of simple tubular

glands, the crypts of Lieberkühn. They extend through the lamina
propria down to the muscularis mucosae. Undifferentiated cells
close to the bottom of the crypts regenerate the epithelium
(epithelial cell turnover time is less than one week). Other
 220

epithelial cells in the crypts correspond largely to those in the

epithelium of the intestinal villi. One exception are Paneth cells
which are located at the bottom of the crypts. They release a
number of antibacterial substances, among them lysozyme, and
are thought to be involved in the control of infections.

One function of the crypts of Lieberkühn is the secretion of

"intestinal juice" (about 2 liter/day), which in its composition
closely resembles extracellular fluid and which is rapidly
reabsorbed. The only enzymes which can be demonstrated in the
intestinal juice are enteropeptidase (or enterokinase), which
activates the pancreatic enzyme trypsin, and small amounts of
amylase. In addition to enterocytes, the epithelium is composed
of mucus-secreting goblet cells and endocrine cells.

In addition to gastrin- and somatostatin-producing cells, we also

find endocrine cells secreting cholecystokinin and secretin.
Cholecystokinin stimulates the secretion of digestive enzymes in
the pancreas and the contraction of the gall bladder. Secretin
stimulates the pancreas to release "pancreatic juice", which is rich
in bicarbonate ions. Secretin also amplifies the effects of
cholecystokinin.

The lamina propria is, similar to the lamina propria of the

stomach, unusually cell rich. Lymphocytes often invade the
epithelium or form solitary lymphoid nodules in the lamina
propria. Lymph nodules may form longitudinal aggregations of
30-50 nodules in the lamina propria of the ileum. These large
aggregations are called Peyer's patches.

The muscularis mucosae has two layers and extends into the
intestinal villi, where the smooth muscle cells form a longitudinal
bundle in the centre of the villi.

The Submucosa
 221

The submucosa contains glands only in the duodenum.

Submucosal glands of the duodenum are also called Brunner's
glands. Their secretion is mucous and slightly alkaline due to
bicarbonate ions (pH 7-8). The amount of bicarbonate is however
too low to neutralize the acidic contents of the duodenal lumen.
Instead, the secretion of Brunner's glands protects the duodenal
mucosa - similar to the mucus which protects the gastric mucosa.

Suitable Slides
slides of the duodenum - H&E, van Gieson, trichrome

Duodenum - H&E
Take a close look at the epithelium lining the villi and crypts of
the duodenum, and note the absence of plicae circulares. Where
in the duodenum are we? You will see this tall columnar
epithelium composed of enterocytes, goblet cells and endocrine
cells throughout the remainder of the GIT. Now identify the
lamina propria and muscularis mucosae and the "packages" of
glandular tissue (Brunner's glands) in the connective tissue
between the muscularis mucosae and muscularis externa, i.e. in
the submucosa. Occasionally you will see ducts of Brunner's
gland which penetrate the muscularis mucosae and ascend
through the lamina propria. Note that goblet cells are absent from
these ducts.
You will not be able to identify endocrine cells in the H&E
stained sections.
Draw a section of the duodenal wall in which these structures are
visible, and identify them in your drawing.
 222

Suitable Slides
slides of jejunum or ileum - H&E, van Gieson

Jejunum - H&E
Look at the slide without the microscope and see if you can
identify plicae circulares, muscularis externa and villi. Next
identify surface epithelium (simple columnar with goblet cells),
crypts, muscularis mucosae, submucosa and muscularis externa.
Crypts will probably be smaller than you expect them from
schematic drawings. They are fairly short and narrow. The
connection of the lumen of the crypt with the lumen of the
intestine will not always be visible in the plane of the section.
Accumulations of lymphocytes are common in the mucosa of the
GIT, and they occur frequently in the small intestine. They can
form very large aggregates in particular in the ileum, where they
may be covered by a specialised form of epithelium which
facilitates their function in the immune-defence of the body
 223

against possible pathogens in the lumen of the intestine. These

specialised parts of the small intestine are called Peyer's patches,
which are described in more detail on the "Lymphoid Tissues II"
page.
Draw part of the wall of the ileum in which a patch of
lymphocytes is visible. A schematic presentation of other features
should be sufficient.

Large Intestine
The large intestine constitutes the terminal part of the digestive
system. It is divided into three main sections: cecum including the
appendix, colon, and rectum with the anal canal. The primary
function of the large intestine is the reabsorption of water and
inorganic salts. The only secretion of any importance is mucus,
 224

which acts as a lubricant during the transport of the intestinal

contents.

The surface of the mucosa is relatively smooth as there are no

plicae circulares or intestinal villi. Crypts of Lieberkühn are
present and usually longer and straighter than those of the small
intestine. Goblet cells account for more of the epithelial cells than
in the small intestine.

There is only little lamina propria squeezed between the glands.

The muscularis mucosae again forms two layers.

Considerable amounts of fat may be found in the submucosa.

The appearance of the muscularis externa is different from that of

the small intestine. The inner circular layer of muscle forms the
usual sheath around the large intestine, but the outer longitudinal
muscle layer forms three flattened strands, the taenia coli. Only a
thin layer of longitudinal muscle surrounds the inner circular
muscle layer between the taenia coli .

The adventitia forms small pouches (appendices epiploicae) filled

with fatty tissue along the large intestine.

Suitable Slides
slides of the colon - H&E, van Gieson, trichrome

Colon - H&E
Again look at the slide without the aid of the microscope.
Bundles of longitudinal muscle should be clearly visible on the
outside of the colon. Plicae circulares are absent from the luminal
side of the colon.
Draw a small schematic figure of these features.
Now have a closer look at the components of the wall of the
colon. Villi are absent and the crypts appear deeper than the ones
 225

you observed in the small intestine. Goblet cells are numerous.

The lamina propria and muscularis mucosae may be difficult to
distinguish. Note also that a thin layer of longitudinal muscle is
found between the taenia coli on the outside of the inner circular
muscle layer.
Draw a small segment of the wall of the colon and label
specialized features of the colon in your drawing.

Specialized Sections of the Large Intestine

The vermiform appendix

is a small blind-ending diverticulum from the cecum. The
most important features of the appendix is the thickening of
its wall, which is mainly due to large accumulations of
 226

lymphoid tissue in the lamina propria and submucosa.

Intestinal villi are usually absent, and crypts do not occur as
frequently as in the colon. There is often fatty tissue in the
submucosa. The muscularis externa is thinner than in the
remainder of the large intestine and, the outer, longitudinal
smooth muscle layer of the muscularis externa does not
aggregate into taenia coli.

An extreme proliferation of lymphocytes (lymphoid

hyperplasia) as a consequence of bacterial or viral
stimulation may lead to the obstruction of the lumen of the
appendix and thereby cause appendicitis, but this is only
one of many possible causes.

The anal canal

is the 2.5-4 cm long terminal part of the digestive tract. The
mucosa has a characteristic surface relief of 5-10
longitudinal folds, the anal columns.
Each column contains a terminal branch of the superior
rectal artery and vein.

Small mucosal folds between the anal columns (anal valves)

form the pectinate line. This line defines sections of the anal
canal with different arterial and nerve supplies, different
venous and lymphatic drainages and different
embryological origins. Crypts disappear below the pectinate
line and the epithelium changes from the tall, columnar type
seen in other parts of the large intestine to a stratified
squamous epithelium. The muscularis externa gradually
becomes thicker and forms the involuntary internal anal
sphincter.

Suitable Slides
 227

slides of the appendix - H&E, van Gieson, trichrome

Appendix, human - H&E
Note the even thickness of the muscularis externa and the smooth
outline of the lumen of the appendix. Lymphoid tissue is present
beneath the epithelium around almost the entire circumference of
the appendix and does (try to identify the muscularis mucosae)
extend into the submucosa.
Occasionally you may see crypts extending deep into the
lymphoid tissue. Compare the epithelium embedded in lymphoid
tissue with that closer to the lumen of the appendix. Goblet cells
are rare or absent and the epithelium appears low columnar.
Small, darkly stained nuclei seem to be embedded in the
epithelium - they represent T-lymphocytes and other immune
cells in intraepithelial pockets.
Draw a spot where you can see "normal" and specialised
epithelium and lymphoid tissue.
 228

Junctions
Junctions between the major parts of the alimentary canal share a
rapid transition from tissues characteristic of one part to those
characteristic for the next part, e.g. the transition from the
stratified squamous epithelium of the oesophagus to the glandular
epithelium of the stomach. Many junctions are in addition
accompanied by morphological specializations, e.g. the pylorus at
the gastro-duodenal junctions or the ileo-caecal valve at the ileo-
caecal junction.

Compared with these rapid changes, "junctions" between parts of

the small intestine are very gradual morphological transitions.

Suitable Slides
sections containing the gastro-oesophageal, gastro-
duodenal, ileo-caecal or ano-rectal junction - van Gieson,
H&E, trichrome
 229

Ano-rectal junction, human -

van Gieson
Identify in this or another
section which contains a
junction of two parts of the
alimentary canal the
histological features
characteristic for joining
parts.
If a section of the gastro-
oesophageal junction is
available, have a look at the
cardiac glands of the
stomach. These glands are
typically not visible in
"regular" stomach sections.
Note that the tubules of the
glands branch and that they
are lined by an almost
homogenous population of
mucus-producing cells.

page content and construction: Lutz Slomianka

last updated: 5/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Accessory Digestive Glands

 230

The Pancreas

 is positioned retroperitoneally on the posterior wall of the abdominal

cavity at the level of the second and third lumbar vertebrae.
 has no distinct capsule, but is covered by a thin layer of loose
connective tissue.
 is both an exocrine and endocrine gland. The exocrine part produces
about 1.5 l of pancreatic juice every day. The endocrine part, which
accounts for ~1% of the pancreas, consists of the cells of the islands
of Langerhans. These cells produce insulin, glucagon and a number
of other hormones.

Components of the exocrine pancreas

The exocrine pancreas consists of tubuloacinar glands. A single layer of

pyramidal shaped cells forms the secretory acini. The apical cytoplasm
(towards the lumen of the acini) is filled with secretory vesicles containing
the precursors of digestive enzymes. The first portion of the duct system
extends into the centre of the acini, which is lined by small centroacinar
cells. These cells form the first part of intercalated ducts. Intercalated ducts
are lined by low columnar or cuboidal epithelium. They empty into
interlobular ducts, which are lined by a columnar epithelium. Interlobular
ducts in turn empty into the main pancreatic duct (of Wirsung), which is
lined by a tall columnar epithelium.

The main pancreatic duct opens into the summit of the major duodenal
papilla, usually in common with the bile duct. A duct draining the lower
parts of the head of the pancreas, the accessory pancreatic duct (of
Santorini), is very variable. If present, it may open into the minor duodenal
papilla ~2 cm above the major papilla in the duodenum.

Pancreatic juice is a clear alkaline fluid which contains the precursors of

enzymes of all classes necessary to break down the main components of
the diet:

 trypsin, chymotrypsin and carboxypeptidase hydrolyse proteins into

smaller peptides or amino acids;
 ribonuclease and deoxyribonuclease split the corresponding nucleic
 231

acids;
 pancreatic amylase hydrolyses starch and glycogen to glucose and
small saccharides;
 pancreatic lipase hydrolyses triglycerides into fatty acids and
monoglycerides;
 cholesterol esterase breaks down cholesterol esters into cholesterol
and a fatty acid.

Proteolytic enzymes are secreted as zymogens - inactive precursors of the

enzymes. They are activated in the lumen of the digestive canal. The
enzyme enteropeptidase is associated with the brush border of enterocytes.
It catalyses the conversion of trysinogen into trypsin. Trypsin can activate
a number of the other pancreatic zymogens.

While the enzymes are secreted by the secretory cells of the pancreatic
acini, the bulk of fluid and bicarbonate ions of the pancreatic juice are
secreted by the cells which form the intercalated ducts of the pancreas.
Bicarbonate ions in the pancreatic juice neutralize the acidic contents
which the stomach empties into the duodenum.

Pancreas, human - H&E

Look at the slide at low magnification and note the subdivision of the
pancreas into numerous lobes and lobules. Identify the connective tissue
between the lobes and lobules and try to find interlobar or interlobular
excretory ducts. Their outline is often irregular and their lumen is lined by
a tall columnar epithelium. If you find a large duct you may see a number
of smaller ducts streaming towards the larger duct and, occasionally,
connecting with it.
The overall appearance of the pancreas is that of a serous gland. At a first
glance it may be possible to confuse the pancreas with other serous glands,
e.g. the parotid gland. Note that the pancreas does not contain structures
resembling the large intralobular ducts of the salivary glands and that the
interlobular and interlobar secretory ducts of large salivary glands are lined
by ... which type of epithelium?
Draw the excretory duct.
 232

Now have a closer look at the secretory tissue within the lobules. At low
magnification most of the tissue appears to be composed of small reddish
packages, the secretory acini. Intercalated ducts are difficult to find and so
are the initial segments of the (non-secretory) intralobular ducts (cuboidal
epithelium). You may try to find them and include them in your drawing,
but don't get upset if you or the demonstrators have difficulties locating
them.
Draw at the largest magnification a number of exocrine secretory acini. If
possible, include in your drawing some centroacinar cells.
 233

Components of the Endocrine

Pancreas

Islands of Langerhans, usually

containing several hundred
endocrine cells, are scattered
throughout the exocrine tissue of
the pancreas. The
vascularization, composed of
many fenestrated capillaries, is
more extensive than that of the
exocrine tissue.

Although the quantitative cellular composition of the islands is quite

variable, we find typically:

 75% beta-cells which secrete insulin. Insulin stimulates the

synthesis of glycogen, protein and fatty acids. It also facilitates the
uptake of glucose into cells and activates glucokinase in liver cells.
Beta-cells are fairly easy to recognize in EM pictures because the
contents of their secretory vesicles typically form one or two
crystals.
 20% alpha-cells which secrete glucagon. The effects of glucagon
are generally opposite to those of insulin. The contents of the
secretory vesicles of alpha-cells are fairly electron dense, and they
are typically smaller than those of delta-cells (average of about 300
nm).
 5% delta-cells which secrete somatostatin, a locally acting hormone
which inhibits other endocrine cells.
 other endocrine cells of the islands secrete
o pancreatic polypeptide, which stimulates chief cell in gastric
glands and inhibits bile and bicarbonate secretion,
o vasoactive intestinal peptide, which has effects similar to
glucagon, but also stimulates the exocrine function of the
pancreas,
o secretin, which stimulates the exocrine pancreas, and
o motilin, which increases GIT motility.
 234

Pancreas, human - H&E and Pancreas, rat - ICC

If you scan over the secretory tissue at low or medium magnification, you
may be able to identify areas of tissue with a slightly different hue and
texture. These areas are likely to represent the islands of Langerhans.
Draw at low or medium magnification a part of the pancreas in which you
see an island of Langerhans. Make sure that the difference in texture and/or
hue between the endo- and exocrine pancreas is visible (at least to you).

The Liver

 is the largest gland of the body (about 2% of the body weight in an

adult).
 receives both venous blood, through the portal vein (~75% of the
blood supply), and arterial blood, through the hepatic artery (~25%
of the blood supply).
 is surrounded by a well defined but thin capsule of connective tissue.
The connective tissue extends into the liver parenchyma and divides
it into the basic structural units of the liver, the "classical" liver
 235

lobules.
 functions as an exocrine gland because it secretes bile.

The portal vein, hepatic artery and bile duct enter the liver through the
porta hepatis. These three vessels travel together through the liver
parenchyma. If one of these vessels gives off a branch it is usually
accompanied by branches of the other two vessels. Terminal branches of
one of the vessels will consequently be accompanied by terminal branches
of the other two vessels.

These groups of three tubes - a branch of the portal vein, a branch of the
hepatic artery and a branch of the bile duct - are called portal triads. Portal
triads are a key feature of the organization of the liver. Portal triads are
embedded in interlobular connective tissue.

The Liver Lobule

An idealized representation of
the "classical" liver lobule is a
six-sided prism about 2 mm long
and 1 mm in diameter. It is
delimited by interlobular
connective tissue (only little, if
any, visible in humans; plentiful
in e.g. pigs). In its corners we
find the portal triads. In cross
sections, the lobule is filled by
cords of hepatic parenchymal
cells, hepatocytes, which radiate
from the central vein and are
separated by vascular sinusoids.

There are other ways of dividing the parenchyma of the liver into units.
Two common ways are divisions into portal lobules and liver acini. Portal
lobules emphasize the afferent blood supply and bile drainage by the
vessels of the portal triads. The secretory function of the liver is
emphasized by the term liver acinus. Acini are smaller units than portal or
"classical" liver lobules and relate structural units to terminal branches
formed by the vessels of the portal triad but not the portal triad itself.
Representations of portal lobules and liver acini vary in different
 236

textbooks.

Hepatocytes are separated from the bloodstream by a thin discontinuous

simple squamous epithelium, which lines the sinusoids. Between the
hepatocytes and the epithelial cells is a narrow perisinusoidal space (of
Disse). Contents of the blood plasma can freely enter the perisinusoidal
space through the openings in the epithelium lining the sinusoids. Fixed
macrophages, Kupffer cells, are attached to the epithelium .

The liver lobule is drained by the central vein, which open into the
intercalated or sublobular veins of the liver. These in turn coalesce to form
the hepatic veins. They run alone through the tissue, are usually covered by
connective tissue and eventually empty into the inferior vena cava.

Adjoining liver cells form the walls of the bile canaliculi , which form
a three dimensional network within the sheets of hepatocytes. Bile
canaliculi connect via very short canals (of Hering; formed by both
hepatocytes and cells similar to those in the epithelium of bile ducts) to
terminal bile ducts (cholangioles) which empty into the interlobular bile
ducts found in the portal triads.

Liver - H&E and Liver, rabbit - trichrome & carbon

The Kupffer cell section does not show the detailed organization of the
liver very well. It is however the best section to identify liver lobules. Scan
over the tissue at low magnification and identify lobules. Once you have
recognized them, change to the H&E stained section. It is difficult, if not
impossible, to clearly identify liver lobules in the H&E stained section. The
best indication of a liver lobule are the large central veins and the
strands/sheets of hepatocytes, which seem to radiate out from the central
veins. Change to a higher magnification in the region of a central vein and
try to identify the epithelial cells forming the walls of the liver sinusoids.
Draw a central vein and adjacent sheets of liver cells and sinusoids. Try to
find and draw a portal triad. Identify in your drawing the branches of the
portal vein, hepatic artery and bile duct. Their size will very much depend
on how close you are to the terminal branches of these structures. Try to
avoid the largest portal triads you see.
 237

Liver, rabbit - trichrome &

carbon and Liver - reticulin
The first slide will allow you to
identify the macrophages which
adhere to the wall of the liver
sinusoids. They are represented
by the accumulations of small
brown/black dots, the carbon
particles ingested by the
macrophages.
Draw a spot in which you
observe some macrophages.
Have a quick look at the
remaining tissue, and try to
identify structures you have seen
in the H&E stained section. Now
take a look at the reticulin stained
section, and again identify the
structures you saw in the H&E
 238

stained section.
Draw a small part of sheets of
liver cells and their supporting
reticular connective tissue.
Include in your drawing some of
the hepatocyte nuclei.

Hepatocytes

 make up about 80% of the cells in the liver.

 are typically large polyhedral cells, with large round centrally
located nuclei. Hepatocytes are frequently polyploid.
 function in the storage of glucose in the form of glycogen (SER),
vitamin A (possibly in specialized adipocytes), vitamin B12, folic
acid and iron.
 participate in the turnover and transport of lipids. The synthesis of
plasmalipoproteins takes place almost exclusively in the liver
(RER/SER).
 synthesize some of the plasma proteins (albumin, α and β globulins,
prothrombin, fibrinogen; RER).
 metabolize/detoxify fat soluble compounds (drugs, insecticides;
SER).
 participate in the turnover of steroid hormones.
 secrete bile (up to 1 liter per day).

Biliary System

Bile contains both organic components (e.g. lecithin, cholesterol and

bilirubin - the latter is a breakdown product of haemoglobin and
accumulates in the blood in jaundice) and inorganic components (bile
salts). The bile salts facilitate the digestion and absorption of fat in the
small intestine.

Terminal bile ducts merge to form interlobular, intrahepatic bile ducts,

which eventually coalesce to form first the left and right hepatic ducts and
then the common hepatic duct, which connects to the cystic duct and the
bile duct (ductus choledochus). The bile duct carries the bile to the
 239

duodenum. The cystic duct leads to the gall bladder.

Terminal bile ducts are lined by a cuboidal epithelium. All other parts of
the bilary system are lined by a tall columnar epithelium. In the gall
bladder the epithelium is often folded and "caved". The gall bladder
functions in the storage and concentration of bile. Microvilli on the apical
surface of the epithelial cells facilitate the resorption of water from the bile.

The epithelium lining the biliary system does not contain mucus-producing
cells and a muscularis mucosae is absent. These features allow you to
distinguish the gall bladder from other parts of the gastrointestinal tract.

Many of the components of bile are not secretory products of the

hepatocytes in a strict sense. They are reabsorbed in the gut and return to
the liver through the portal vein. Here they are taken up by the hepatocytes
and excreted again - a phenomenon called enterohepatic circulation.

Gall Bladder, human - H&E

Have a look at the slide at low magnification. Note the irregular outlines of
the epithelium, the relatively dense irregular connective tissue beneath it,
and the irregular appearance of the muscular layer of the gall bladder. Now
take a close look at the epithelium.
Draw the epithelium and part of the underlying connective and muscular
tissues.
 240

page content and construction: Lutz Slomianka

last updated: 5/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Respiratory

System

RESPIRATORY SYSTEM
 241

The complex of organs and tissue which are necessary to exchange blood
carbon dioxide (CO2) with air oxygen (O2) is called the respiratory system. It
consists of

1. structures, which function as ducts, and which together are called the
conductive portion of the respiratory system
2. structures which form the respiratory portion of the respiratory system, in
which the exchange of CO2 and O2 is occurring and
3. the parts of the thoracic musculo-skeletal apparatus and specialisations
of the lung which allow the movement of air through the respiratory
system - the ventilating mechanism.

Nasal Cavity

The Nasal cavity is divided into three structurally and functionally different parts.

1. The vestibules (the first ~1.5 cm of the conductive portion following the
nostrils) are lined with a keratinised stratified squamous epithelium.
Hairs, which filter large particulate matter out of the airstream, and
sebaceous glands are also present.

2. At the transition from the vestibule to the respiratory region of the nasal
cavity the epithelium becomes first stratified squamous and
then pseudostratified columnar and ciliated. This type of epithelium is
characteristic for all conductive passages dedicated to the respiratory
system and therefore also called respiratory epithelium. Mucus
producing goblet cells are present in the epithelium.
The surface of the lateral parts of the nasal cavity is thrown into folds by
bony projections called conchae. These folds increase the surface area
of the nasal cavity and create turbulence in the stream of passing air,
both of which facilitate the conditioning (warming, cooling and filtration)
of the air. Mucous and serous glands in the connective tissue underlying
the epithelium, the lamina propria, supplement the secretion of the goblet
cells. Veins in the lamina propria form thin-walled, cavernous sinusoids,
 242

also called cavernous bodies.

3. Tissues on the superior concha and the nasal septum form the olfactory
region of the nasal cavity. Cilia in the epithelium of the olfactory region
arise from olfactory cells. Although their internal structure resembles
largely that of normal cilia they do not move, because they lack dynein
arms which are necessary for cilial motility. The cell membrane covering
the surface of the cilia contains olfactory receptors which respond to
odour-producing substances, odorants, dissolved in the serous covering
the epithelium. The axons of the olfactory cells collect into bundles in the
lamina propria. The olfactory cells and their processes receive
mechanical and metabolic support from supporting cells (or
sustentacular cells). Basal cells can divide and differentiate into either
olfactory or supporting cells.

The supporting cells and the secretion of the serous glands contain lipofuscin
granules, which give a yellow-brown colour to the surface of the olfactory
region.

Pharynx

The pharynx connects the nasal cavity with the larynx. Depending on the
extent of abrasive forces on the epithelium, the pharynx is either lined
with respiratory epithelium (nasopharynx or epipharynx) or with a
stratified squamous epithelium (oropharynx or meso- and hypopharynx),
which also covers the surfaces of the oral cavity and the oesophagus.
Lymphocytes frequently accumulate beneath the epithelium of the
pharynx.

Accumulations of lymphoid tissues surrounding the openings of the

digestive and respiratory passages form the tonsils.

The nasal cavity and pharynx form the upper respiratory passages.

Suitable Slides
 243

sections of the respiratory region of the nasal cavity - H&E, van Gieson

Inferior Concha, human - Alcian blue & van Gieson

Thin structures which consist of tissues with very different physical properties - like
loose connective tissue and bone - may easily get damaged during tissue preparation.
Find a spot where the layers from epithelium to bone appear intact. Identify goblet
cells, basal cells and ciliated cells in the epithelium. Now have a look at the lamina
propria. Depending on the exact location you may only see connective tissue between
the epithelium and bone or large spaces (either empty or filled with red blood cells)
which represent the cavernous sinusoids, or glandular tissue (mucous glands appear
green and muco-serous glands appear brownish-green in this preparation), or
combinations of these features. Lamellae and osteocytes in lacunae may be visible in
the bone, while Haversian systems are rare or absent. Which type of bone is it?
Draw a survey of the tissue which includes bone, the lamina propria and structures
within it, and epithelium.

Suitable Slides
sections of the olfactory region of the nasal cavity - H&E, van Gieson

Nasal Cavity, Olfactory Region, rat - Alcian blue & van Gieson
In humans, olfactory epithelium lines the superior concha and parts of the nasal septum.
The bony structures beneath the epithelium form an irregular surface, which increases
 244

turbulence in the air passing them and thereby the chances that odorants come into
contact with the olfactory epithelium. In macrosmatic animals, like the rat, the olfactory
epithelium also covers the middle conchae and the surface is considerably more
irregular than in humans.
The olfactory epithelium is formed by olfactory cells, sustentacular cells and basal
cells. Basal cells can be identified by their location. Sustentacular cells are
preferentially located in the superficial cell tier of the epithelium but are difficult to
distinguish from olfactory cells in this preparation. Cilia are not visible and goblet cells
are absent from the olfactory epithelium. Lightly stained rounded areas in the lamina
propria represent bundles of olfactory axons in the lamina propria. Small mucous
glands, olfactory glands or Bowman's glands, in the lamina propria moisturise the
epithelium.
Draw the olfactory epithelium and underlying lamina propria at high magnification.
Label the features included in your drawing.

Larynx, trachea, bronchi and bronchioles form the lower respiratory passages.

Larynx

The larynx connects the pharynx and trachea. The vocal folds of the larynx
 245

control airflow and allow the production of sound. The vocal folds are lined by
stratified squamous epithelium and contain the muscle (striated, skeletal) and
ligaments needed to control the tension of the vocal folds. The larynx is
supported by a set of complexly shaped cartilages.

Trachea

The trachea is a fairly short tube (10-12 cm) with a diameter of ~2 cm.

Epithelium, Mucosa and Submucosa

The trachea is lined by respiratory epithelium. The number of goblet cells

is variable and depends on physical or chemical irritation of the
epithelium which increase goblet cell number. Prolonged intense
irritation of the epithelium may lead to its transformation to a stratified
squamous epithelium (squamous metaplasia).
In addition to the staple of basal cells, ciliated cells and goblet
cells , the respiratory epithelium also contains brush cells,
endocrine cells (or small granule cells, function not clear),
surfactant-producing cells (or Clara cells), and serous cells.

Epithelium and underlying lamina propria are called the mucosa. The
lamina propria consists of loose connective tissue with many elastic
fibres, which condense at the deep border of the lamina propria to form
an elastic membrane. This elastic membrane forms the border between
the mucosa and the connective tissue below it, which is called the
submucosa. Muco-serous glands in the submucosa (submucosal glands)
supplement the secretions of cells in the epithelium. The submucosa
ends with the perichondrium of the tracheal cartilages.

Tracheal cartilages

The trachea is stabilised by 16-20 C-shaped cartilages (hyaline

cartilage). The free dorsal ends of the cartilages are connected by
bands of smooth muscle (trachealis muscle) and connective tissue
fibres. Longitudinal collagenous and elastic connective tissue
fibres (annular ligaments) link the individual cartilages and allow
both the lengthening and shortening of the trachea for example
during swallowing or movements of the neck . They are
inseparable from the fibres of the perichondrium. The tracheal
cartilages may ossify with age.

Cartilages, annular ligaments and the trachealis muscle form the

"skeleton" of the trachea which sometimes is referred to as tunica
fibromusculocartilaginea. If you want to impress someone with this term
 246

make sure that you can pronounce and/or spell it.

The trachea bifurcates to give rise to the main bronchi. Their histological
structure corresponds largely to that of the trachea.

Suitable Slides
sections of the trachea - H&E, van Gieson
Trachea, human - H&E
In the trachea you should be able to
identify the following structures:
respiratory epithelium, basement
membrane, submucosal glands (both
serous and mucous parts),
perichondrium, tracheal cartilage and
trachealis muscle (smooth muscle).
One can perceive different appearances
of the connective tissue immediately
below the epithelium and the
connective tissue surrounding the
submucosal glands, but the elastic
lamina forming the border between the
mucosa and submucosa is not visible
in H&E stained slides. Accumulations
of very dark small dots represent
lymphocytes (not illustrated). If
present, you are likely to see them
close to the glandular tissue.
Draw a composite, i.e. assemble all the
main features of the trachea in one
drawing, of a segment of the trachea.
Label the main features.

Conductive Structures in the Lung

Bronchi

In the lungs we find the last segments of the conductive portion of the
respiratory system. The main bronchi divide into lobar bronchi which in
turn give rise to segmental bronchi. The latter supply the bronchopulmonary
segments of the lungs. Bronchial branches are accompanied by branches of
the pulmonary artery, nerves and lymph vessels. These structures usually
 247

travel in intersegmental and interlobar sheets of connective tissue. Conductive

structures of a size down to ~1 mm are termed bronchi. Smaller ones are called
bronchioles. Aside from their different sizes, bronchi are characterized by the
presence of glands and supporting cartilage. The cartilage supporting the
bronchi is typically found in several small pieces.

The histological structure of the epithelium and the underlying

connective tissue of the bronchi corresponds largely to that of the
trachea and the main bronchi. In addition, bronchi are surrounded by a
layer of smooth muscle, which is located between the cartilage and
epithelium.

Bronchioles

Bronchioles are the terminal segments of the conductive portion. At the

transition from bronchi to bronchioles the epithelium changes to a
ciliated columnar epithelium, but most of the cell types found in the
epithelium of other parts of the conductive portion are still present. Glands and
cartilage are absent. The layer of smooth muscle is relatively thicker than
in the bronchi.

Respiratory Structures in the Lung

Bronchioles divide into respiratory bronchioles, which are the first structures
that belong to the respiratory portion of the respiratory system. Small out
pouchings of the walls of the respiratory bronchioles form alveoli, the site of gas
exchange. The number of alveoli increases as the respiratory bronchioles
continue to divide. They terminate in alveolar ducts. The "walls" of alveolar
ducts consists of entirely of alveoli.

Histological Structure of Alveoli

The wall of the alveoli is formed by a thin sheet (~2µm) of tissue

separating two neighbouring alveoli. This sheet is formed by epithelial
cells and intervening connective tissue. Collagenous (few and fine),
reticular and elastic fibres are present. Between the connective tissue
fibres we find a dense, anastomosing network of pulmonary capillaries.
The wall of the capillaries are in direct contact with the epithelial lining of
the alveoli. The basal laminae of the epi- and endothelium may actually
fuse. Neighbouring alveoli may be connected to each other by small
alveolar pores.

The epithelium of the alveoli is formed by two cell types:

 248

1. Alveolar type I cells (small alveolar cells or type I pneumocytes) are

extremely flattened (the cell may be as thin as 0.05 µm) and form the
bulk (95%) of the surface of the alveolar walls. The shape of the cells is
very complex, and they may actually form part of the epithelium on both
faces of the alveolar wall.

2. Alveolar type II cells (large alveolar cells or type II pneumocytes) are

irregularly (sometimes cuboidal) shaped. They form small bulges on the
alveolar walls. Type II alveolar cells contain are large number of granules
called cytosomes (or multilamellar bodies), which consist of precursors
to pulmonary surfactant (the mixture of phospholipids which keep
surface tension in the alveoli low) . There are just about as many type
II cells as type I cells. Their small contribution to alveolar area is
explained by their shape.

Cilia are absent from the alveolar epithelium and cannot help to remove
particulate matter which continuously enters the alveoli with the inspired air.
Alveolar macrophages take care of this job.

They migrate freely over the alveolar epithelium and ingest particulate matter.
Towards the end of their life span, they migrate either towards the bronchioles,
where they enter the mucus lining the epithelium to be finally discharged into
the pharynx, or they enter the connective tissue septa of the lung.

Suitable Slides
sections of lung - H&E, elastin, reticulin
Sections may not contain bronchi.
 249

Lung, human - H&E, elastin

You should be able to find at least
bronchioles, alveolar ducts and alveoli
in the section. Respiratory bronchioles
should be present but may require
some time to find. Note that
bronchioles are usually accompanied
by a vessel, a branch of the pulmonary
artery. Interlobular connective tissue is
also present and may be identified by
holding the slide against the light. It is
represented by continuous and fairly
straight streaks of tissue visible
without a microscope. Have a look at
the bronchioles and alveoli at high
magnification. The bronchiolar
epithelium is usually not well
preserved, but the smooth muscle is
clearly visible. Note the absence of
cartilage and glands from the
bronchioli. You should be able to
identify both type I and II alveolar
cells and capillaries in the alveolar
walls. Note that red blood cells usually
appear thicker than the entire wall of
the alveoli.
Sketch part of the tissue at low
magnification. Include an alveolar duct
and, if possible, a bronchiole and/or
respiratory bronchiole in your drawing.
 250

Lung, cat - reticulin

Reticular and elastic fibres form the
bulk of the connective tissue present
in the walls of the alveoli. You have
seen both types of fibres previously.
Note that if you mentally
superimpose the elastin and reticulin
stains there is not much space for
anything other than capillaries.
Collagenous fibres are sparse and
fine in the alveolar walls. Note also
that the tissue stained for reticular
fibres looks much denser than the
other sections. This lung collapsed
prior to fixation because of the
recoil of the elastic fibres. Because
of this artifact it may be a little
easier to recognize alveolar ducts
than in the other sections.
 251

Development of the Lungs

The formation of the lower respiratory passages begins in the fourth foetal
week. An outpouching of the foregut gives rise to the laryngotracheal tube. The
lining of this tube will eventually give rise to the epithelia covering the surfaces
of the larynx, trachea, bronchi, bronchioles and alveoli. Most of the other
tissues of the lower respiratory passages are derived from splanchnic
mesoderm. The laryngotracheal tube divides distally to form two lung buds.

Dependent of the state of maturity of the lung, development is divided

into three periods:

1. The bronchi grow and branch during the glandular period, which last until
approx. the 17th foetal week. Alveoli are not present at this time.
2. Bronchi and bronchioles expand and branch during the canalicular
period. The lung tissue is vascularised during the canalicular period.
Bronchi and bronchioli begin to form terminal sacs (developing primitive
alveoli), in which cuboidal and squamous cells become associated with
vessels. Respiration becomes possible towards the end of this period
around the 25th foetal week.
3. The number of terminal sacs increases during the intial part of the
alveolar period (sometimes also considered a separate period of lung
development and called terminal sac period). The capillary network is
developing between the terminal sacs. The late alveolar period is
marked by the development of mature alveoli from the terminal sacs.
The period begins shortly before birth, but the first mature alveoli appear
only after birth. Alveolar sacs continue to be formed during early
childhood (up to year 8) and mature into alveoli. Alveolar maturation and
growth continue for another decade, but their numbers do not increase
further.

Suitable Slides
Sections of developing lung - H&E

Foetal lung, human - H&E

The lung tissue on the slides available in the tray should according to the above scheme
come from the late canalicular period. Mucous connective tissue fills fairly wide spaces
between the terminal sacs. Both slides contain developing bronchi and cartilage. Have a
quick look at the developing cartilage and note that histologically it somewhat
resembles the intramembranous formation of bone.
 252

Draw a small section of developing lung including terminal sacs and connective tissue
at high magnification.

page content and construction: Lutz Slomianka

last updated: 6/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Urinary System

URINARY SYSTEM
 253

The kidneys, ureters, urinary bladder and urethra are the main components
of the urinary system. A function of the urinary system that immediately
comes to mind is the excretion of waste products from the body. This is
only one of many functions of the system. Others are

 elimination of foreign substances

 regulation of the amount of water in the body
 control of the concentration of most compounds in the extracellular
fluid

Most of these tasks are performed in the kidneys. Functionally the

processes can be divided into two steps, each of which have their
anatomical correlate:

 filtration - glomeruli of the kidney

 selective resorption and excretion - tubular system of the kidney

In addition, the kidney also functions as an endocrine organ. Fibrocytes in

the cortex release the hormone erythropoietin, which stimulates the
formation of red blood cells. Modified fibrocytes of the medulla secrete
prostaglandins which are able to decrease blood pressure.

Kidney

Glomeruli and the tubular system are both part of the basic functional unit
of the kidney, the nephron.

The Glomerulus (or renal corpuscle)

The glomerulus is the round (~0.2 mm in diameter) blind beginning of the

nephron. It is invaginated by a tuft of capillaries at the vascular pole of the
glomerulus. The tuft of capillaries and other cells in contact with them
form the anatomical glomerulus. Substances which leave the capillaries
enter the renal tubule at the urinary pole of the glomerulus.

The anatomical glomerulus is enclosed by two layers of epithelium,

Bowman's capsule. Cells of the outer or parietal layer of Bowman's capsule
form a simple squamous epithelium. Cells of the inner layer, podocytes in
 254

the visceral layer, are extremely complex in shape. Small foot-like

processes, pedicles, of their cytoplasm form a fenestrated epithelium
around the fenestrated capillaries of the glomerulus. The openings between
the pedicles are called filtration slits. They are spanned by a thin
membrane, the filtration slit membrane. Between the podocytes and the
endothelial cells of the capillaries we find a comparatively thick basal
lamina, which can be subdivided into an outer lamina rara externa, a
middle lamina densa and an inner lamina rara interna. The basal lamina
and the slit membranes form the glomerular filtration barrier, which
prevents some large molecules from entering the capsular space between
the outer and inner epithelial layers of Bowman's capsule.

Mesangial cells in the glomerulus form the connective tissue that gives
structural support to podocytes and vessels.

Blood pressure is the driving force in the formation of about 125 ml of

glomerular filtrate per minute. About 124 ml of the glomerular filtrate is
reabsorbed in the tubules of the nephron.

Kidney - H&E
Locate the cortex of the kidney and scan over the tissue at low
magnification. Note the presence of numerous glomeruli and the apparent
absence of any preferred orientation of the tubules visible between the
glomeruli (convoluted parts of proximal and distal tubuli). You should be
able to identify the vascular pole of a good glomerulus by the attachment
of the capillary tuft to the wall of the glomerulus. What would make your
glomerulus VERY good would be the presence of a tubulus which contains
a dense row of nuclei in the part of its wall closest to the vascular pole of
the glomerulus, the macula densa. The nuclei are located side by side or
may even overlap. It should be possible to find this structure in all slides. It
is also very likely that it may take you a few minutes of carefully scanning
the tissue at high magnification before you will find it. Proximal tubules
are characterised by their eosinophilic (pink) low, columnar cells and by
large amounts of fuzzy material, which may fill the entire lumen of the
tubulus. This fuzzy material represents the remains of the brush border of
the cells of the proximal tubules, which is difficult to preserve during the
preparation of the tissue.
Draw a glomerulus and label its components: the anatomical glomerulus,
 255

the parietal blade of Bowman's capsule (squamous cells), podocytes (fairly

large and light nuclei ), endothelial cells (smaller and darker nuclei),
vascular pole. Include a proximal tubulus and a distal tubulus in your
drawing.

Tubules of the Nephron

The tubular system can be divided into proximal and distal tubules, which
in turn have convoluted and straight portions. Intermediate tubules connect
the proximal and distal tubules. Running from the cortex of the kidney
towards the medulla (descending), then turning and running back towards
the cortex (ascending), the tubules form the loop of Henle.
 256

The proximal tubule

is the longest section
of the nephron
(about 14 mm). The
convoluted part of
the proximal tubules
coils close to the
glomerulus in the
cortex. The diameter
of proximal tubules
is ~65 µm. Their
walls are formed by
a low columnar
epithelium. The
eosinophilic cells of
the epithelium have
a wide brush border
(long microvilli -
What is their
function?) and are
active in
endocytosis. They
almost completely
resorb substances of
nutritional value
from the glomerular
filtrate (glucose,
amino acids, protein,
vitamins etc. -
Which organelles
would you expect to
be numerous in
proximal tubule
cells?). In the
proximal tubules the
volume of the
glomerular filtrate is
reduced by about
75%. Sodium ions
 257

are actively resorbed

from the glomerular
filtrate. They are
followed by
passively diffusing
chloride ions and the
osmotic absorption
of water. The
straight portion of
the proximal tubule
descends towards the
medulla.

The straight portion

of the proximal
tubule merges with
the intermediate
tubule (thin segment
of the loop of
Henle). A flattened,
only ~1-2 µm high
epithelium forms the
intermediate tubule,
which is only ~15
µm wide.
Descending parts of
the straight proximal
and intermediate
tubules are
permeable to water
but not to solutes.

The thin segment of Henle's loop leads into the straight part of the
distal tubule, which is formed by low cuboidal cells without a
brush border. A few short microvilli are present, but they are
difficult to see in the light microscope. The diameter of the tubule
expands to ~35 µm. Epithelial cells in the ascending parts of the
intermediate and straight distal tubules cells transport chloride
 258

(active) and sodium ions (passive) out of the tubular lumen into
the surrounding peritubular space. The epithelium can not be
penetrated by water. Consequently, the transport of ions over the
epithelium sets up a gradient in osmotic pressure, which serves as
driving force in the further concentration of the urine.

The straight portion of the distal tubule contacts the glomerulus

forming the macula densa. Thereafter, the distal tubule forms its
convoluted portion (about 5 mm long). Cells in the distal tubulus
are sensitive to the hormone aldosterone, which is produced in the
zona glomerulosa of the adrenal glands. Aldosterone stimulates
the active resorption of sodium ions and the excretion of
potassium ions.

The convoluted distal tubule merges, via connecting tubules, with

the collecting ducts. In the presence of antidiuretic hormone
(ADH), the epithelia of the collecting ducts are permeable to
water but not to sodium ions. Osmotic forces move water out of
the lumen of the tubules as they pass through the medulla, where
cells of the ascending intermediate and straight distal tubules of
the loop of Henle have established high concentrations of sodium
in the extracellular space.

Collecting ducts merge to form papillary ducts (of Bellini), which

terminate on the tips of the renal papilla and empty into a
distended, funnel shaped part (minor calyx) of the ureter.

Kidney, human - H&E

Find a good spot in the medulla of the kidney. What is good?
Preferably a spot in which you are able to identify a collecting
ducts (cuboidal to columnar cells, well-defined boundaries
between cells, cytoplasm only weakly stained or unstained, large
ducts) and an intermediate (very flat epithelium, nuclei bulge into
 259

the lumen of the tubulus, diameter of the duct is small) and distal
tubule (cuboidal epithelium, cells stain weakly pink). Both
transversely or longitudinally cut tubules are suitable. Note that it
will be difficult to identify ALL tubules that are visible.
In most of our sections only little medulla is present - try to scan
along the margins of the tissue and see if you can find some
medulla. If that should not be possible take a look at medullary
rays instead, although they will contain few, if any, good thin
tubules.
Draw the tubules you could identify and label them. Also include
some tubules which could not be identified in your drawing,
which will give you a better impression of how the medulla of the
kidney looks than just three circles on a white background.

The Juxtaglomerular Apparatus

 260

As mentioned above, the distal tubule contacts the glomerulus

forming a specialized section of tubular epithelium, the macula
densa. At the point of contact with the glomerulus, the distal
tubule is always in close contact with the efferent and afferent
arterioles of the glomerulus.

Other parts of the juxtaglomerular apparatus are extraglomerular

mesangial cells and the juxtaglomerular cells surrounding the
afferent arteriole (modified smooth muscle cells), which produce
and secrete renin. Renin activates angiotensinogen, a precursor
found in the bloodstream, leading to the formation of angiotensin
I, which is converted to angiotensin II. Angiotensin II is the most
potent vasoconstrictor known. It also stimulates the secretion of
aldosterone.

Different theories exist that try to explain the interactions

between the cells that eventually lead to the release of renin. One
of them, the baroreceptor theory, assumes that the
juxtaglomerular cells function as stretch receptors (high blood
pressure would inhibit the release of renin). Another theory, the
macula densa theory, claims that the secretion of renin is
regulated by the composition of the fluid in the distal tubule
and/or the afferent arteriole (low sodium would increase in the
release of renin).

Excretory Passages

The minor calyces merge to form major calyces within the

kidney, which in turn merge to form the renal pelvis (still within
the kidney). The urine flows through these structures to the ureter
and is channelled to the bladder.

The basic structure of all these components is the same. The

mucosa is lined with a transitional epithelium , which occurs
exclusively in the urinary system. The epithelium is virtually
 261

impenetrable to any components of the urine , which

consequently does not change in composition as it passes through
the excretory passages. The lamina propria consists mainly of
dense connective tissue, with many bundles of coarse collagenous
fibres. The muscularis usually consists of an inner longitudinal
and outer circular layer of smooth muscle cells . In lower parts of
the ureter and the bladder an additional outer longitudinal layer of
muscles is added to the first two.

The bladder is finally emptied through the urethra. Initially, the

urethra is lined by a transitional epithelium in males and females.
In males, it is replaced by a pseudostratified or stratified
columnar epithelium below the openings of the ejaculatory ducts
into the urethra. The distal parts of the female urethra and the
distal end of the male urethra are lined by a stratified squamous
epithelium. The lamina propria contains loose connective tissue.
Smooth muscle cells in the muscularis are mainly oriented
longitudinally. They are surrounded, in the middle part of the
urethra (below the prostate in males), by striated muscle cells of
the sphincter urethrae.
 262

Ureter, primate - H&E and

Bladder, human - H&E
You should focus your
attention first on the
epithelium and, second, on
the general appearance of the
musculature in the walls of
the ureter and bladder. Find
the place in your tissue
(either bladder or ureter) in
which the transitional
epithelium has the most
textbook-like appearance.
Although the precise
orientation of the muscles in
the wall of the bladder
depends on where the tissue
block was taken and at which
angle it was sectioned, it
should be possible to identify
three tiers of muscle bundles
at least in some parts of the
bladder wall. Note also that
the epithelium is smooth,
without appreciable crypts or
folds.
Draw these features of the
muscular wall and
epithelium. You may want to
include a similar low-
magnification drawing of the
ureter.
 263

page content and construction: Lutz Slomianka

last updated: 6/08/09

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Endocrines

ENDOCRINE GLANDS
 264

Endocrine (or internally secreting) glands are also named ductless glands, since they
lack excretory ducts. Instead, the secretory cells release their products, hormones, into
the extracellular space. From the extracellular space, the hormones may enter the
blood stream, by which they reach their target organs. Alternatively, the hormones may
affect nearby cells (paracrine acting hormones).

The major endocrine glands are the pituitary gland, the pineal body, the thyroid gland,
the parathyroid gland, the pancreas, the adrenal glands, the ovaries and the testes. In
some of these glands/organs the endocrine tissue constitutes only part of the
parenchyma of the organ (Which ones?). Small groups or individual endocrine cells are
also found in a variety of other organs, e.g. the GIT and the kidneys.

Pituitary Gland

The pituitary gland (or hypophysis) is attached to the inferior surface of the
brain by an extension of the nervous tissue of the tuber cinereum /eminentia
mediana of the hypothalamus, the infundibulum. The infundibulum and small
amounts of non-neural secretory tissue surrounding it form the hypophyseal
stalk. The pituitary gland is located in the sella turica, the hypophyseal fossa of
the sphenoid bone. The pituitary gland is surrounded by a thin connective
tissue capsule. The loose connective tissue between the capsule and the
periosteum of the sphenoid bone contains a dense plexus of thin-walled veins,
which surround the entire pituitary gland.

Macroscopically, the pituitary gland

can be divided into neurohypophysis,
which includes all neuroectodemal
hypophyseal derivatives, and
adenohypophysis, which includes all
ectodermal hypophyseal derivatives.
Adenohypophyseal tissue extending
dorsally along the anterior and lateral
surfaces of the hypophyseal stalk is
also called pars tuberalis of the
hypophysis. The remainder of the
adenohypophysis can be divided into
a pars intermedia and a pars distalis.

It becomes a little easier to

understand the structural divisions of
the pituitary gland if you understand
 265

the pattern of the development of this

gland.

Any pituitary slide which contains both the adenohypophysis and neurohypophysis is
suitable to look at the general organization of the pituitary. Try to identify the portal
venules. Several of them can usually be seen in the pars tuberalis, where they descend
towards the pars distalis of the adenohypophysis.
Draw the pituitary at low magnification and identify its divisions (those visible in the
slide) and portal venules in your drawing.

Cells and secretory products of the hypophysis :

Adenohypophysis

The pars distalis of the adenohypophysis accounts for about 75% of the hypophyseal
tissue. The glandular cells are arranged in irregular clumps or cords between a network
of capillaries with large and irregular lumina. Connective tissue, which supports the
glandular cells, is scant. Traditionally, glandular cells are subdivided into chromophobe
cells and acidophil and basophil (chromophil) cells. This division into three cell types
is based on their differential staining with H&E. Cocktails of other dyes, some of which
are mentioned below, also allow a differentiation between these cell types.

All known hormones of the adenohypophysis are proteins or glycoproteins.

The contents of the secretory vesicles are responsible for the staining characteristics of
the chromophil cells.

Acidophil cells (or acidophils)

Acidophils are rounded cells and typically smaller than basophil cells. Acidophils
account for roughly 65% of the cells in the adenohypophysis.

 The most frequent subtype of acidophils are the somatotrophs

(which can be stained with the dye orange G). Somatotrophs
produce growth hormone (GH or somatotropin), which e.g.
stimulates liver cells to produce polypeptide growth factors which
stimulate growth (e.g. somatomedin which stimulates epiphyseal
cartilage - overproduction of this hormone may result in (gigantism
or acromegaly).
 266

 Mammotrophs (or lactotrophs), the second group of acidophils,

secrete prolactin. Their number increases significantly in late
pregnancy and the early months of lactation.

Basophil cells (or basophils)

Based on their hormone products basophils are divided into three subtypes.

 Thyrotrophs produce thyroid stimulating hormone (TSH or

thyrotropin).
 Gonadotrophs produce follicle stimulating hormone (FSH), which
stimulates the seminiferous epithelium in males in addition to early
follicular growth in females. Gonadotrophs also produce
luteinizing hormone (LH), which stimulates production of
testosterone by Leydig cells in males in addition to late follicular
maturation, oestrogen secretion and formation of corpus luteum in
females.
 Corticotrophs (or adrenocorticolipotrophs) secrete
adrenocorticotropic hormone (ACTH or corticotropin) and
lipotropin (LPH), no known function in humans). Corticotropes are
the most frequent cell type in the pars intermedia. In the pars
intermedia, the precursor of ACTH and LPH undergoes further
hydrolysis into melanocyte stimulating hormone (MSH), increased
pigmentation in patients with Addison's disease) and a number of
other peptides (among them endogenous opioids).

When stained with the PAS reaction all three types of basophils appear reddish.
Morphological criteria may be used, aside from the secretory products, to distinguish
between the cells, but differences are so subtle that it is hopeless to try to tell them
apart in the available preparations.

Chromophobe cells

Chromophobe cells are unstained or weakly stained cells. Most

chromophobe cells can be assigned to the different classes of
chromophils if EM and immunocytochemistry are used. They are now
thought to represent acidophil and basophilic cells in a dormant or
recently degranulated stage (degranulation = release of most of the
secretory vesicles), but may also include stem cells of the secretory
cells.
 267

Pituitary, sheep - PAS/ORANGE G or Pituitary, sheep - H&E

The best slide to identify the different cell types of the adenohypophysis is the
PAS/Orange G stained one. Identify acidophils, basophils and chromophobes. Survey
the tissue, and verify that the relative frequencies of the cells are different in different
parts of the adenohypophysis.
In the H&E stained sections acidophils are dark pink and basophils look light
pink/blue.
Draw part(s) of the adenohypophysis which contains, if possible, all three cell types at
high magnification.

The blood supply to the pituitary gland is extraordinary complex. At this stage it is
important to know that the primary capillary network in the neural part of the
hypophyseal stalk drains into 20 or more portal venules, which form a secondary
capillary network in the pars distalis of the pituitary gland.

The release of hormones from the adenohypophysis is under the control of hormones
which are produced by nerve cells in the hypothalamus.

For each hormone released by the adenohypophysis there exist release-inhibitory

factors and releasing-factors. These factors are also hormones. The axons of
hypothalamic nerve cells terminate within the neural stalk and release these regulatory
 268

factors into the extracellular space associated with the primary capillary plexus. They
are transported towards the adenohypophysis within the portal venules and reach their
target cells via the secondary capillary plexus.

Neurohypophysis

neurohypophysis consists of

 unmyelinated nerve fibres derived from neurosecretory cells of the

supraoptic and paraventricular hypothalamic nuclei and
 pituicytes.

Usually only the oval or round nuclei of the pituicytes are visible. Hypothalamic nerve
fibres typically terminate close to capillaries. Scattered, large, and bluish-violet (in
PAAB/PAS/Orange G stained sections) masses represent dilations of these nerve fibres.
The dilations are named Herring bodies. They are filled by small vesicles which contain
the neurosecretory products of the hypothalamic cells.

The neurophypophysis expands posterior to the adenohypophysis, where it

forms the posterior lobe of the pituitary.

Release-inhibiting and releasing factors, which regulate the activity of the

adenohypophysis, are not the only hormones secreted in the neurohypophysis.
Two additional hormones are oxytocin, which stimulates the contraction of
smooth muscle cell in the uterus and participates in the milk ejection reflex, and
antidiuretic hormone (ADH or vasopressin), which facilitates the concentration
of urine in the kidneys and, thereby, the retention of water.
 269

Pituitary, sheep -
PAAB/PAS/Orange G
Identify nuclei of pituicytes and
Herring bodies. Herring bodies are
not quite as well-defined structures
as e.g. Hassall bodies, but rather
areas of darker staining in the tissue.
You may also want to take a quick
look at neurones of the
hypothalamus, part of which should
be visible on the slides.
Staining in adenohypophysis of the
PAAB/PAS/Orange G section
corresponds largely to that in the
other PAS/ORANGE G section, but
the colours are somewhat subdued
because of the additional PAAB
stain.
Sketch the appearance of the tissue
of the neurohypophysis at high
magnification and label the
structures you can identify.

PINEAL BODY

The pineal body is a flattened, cone-shaped organ attached to the roof of the third
ventricle, where it occupies a depression between the superior colliculi.
The pineal body is surrounded by pia mater, which functions as its capsule and which
sends connective tissue septa into the pineal body, subdividing it into lobules.

In the pineal we find two cell types: pinealocytes (about 95% of the cells;
large, light and round nuclei) and astrocytes (glial cells; dark, elongated
nuclei).

Aside from the cells the pineal gland also contains ..... sand - well - brain sand (or
acervuli cerebri or - just for good measure - corpora arenacea). These are calcium-
containing concretions in the pineal parenchyma, which increase in size and number
with age. These concretions are radioopaque, and, since the pineal is located in the
midline of the brain, they provide a good midline-marker. They have no other known
function.

The most prominent secretory product of the pineal body is melatonin. The
cocktail of substances released by the pinealocytes can have several functions:
 270

they may decrease secretory activity in most other endocrine glands (in part
indirectly, by way of influencing hypothalamic neurones), and they may "delay"
puberty through antigonadotrophic effects.

Secretory activity in the pineal gland is stimulated by darkness and inhibited by

light. Via the effects of pineal hormones on the adenohypophysis and sex
hormones it is likely that the pineal body is involved in phenomena associated
with the circadian rhythm and seasonal phenomena (e.g. seasonal affective
disorder, SAD). The pineal body is innervated by postganglionic sympathetic
fibres derived from the superior cervical ganglion.

Pineal gland, sheep - H&E

The parenchyma of the pineal gland
looks rather homogeneous at low
magnification. A few blood vessels
are visible criss-crossing through the
gland. At higher magnification three
types of nuclei can be distinguished.
Small dark nuclei belong to the
astrocytes found in the pineal gland.
Pinealocytes have larger, lighter and
round nuclei, which are surrounded
by a broad rim of light cytoplasm.
Most nuclei present are the nuclei of
pinealocytes. Endothelial cell nuclei
are found in association with the
vessels and capillaries traversing the
tissue. Both pinealocytes and
astrocytes have long processes which
give the tissue between the nuclei its
"stringy" appearance.
Brain sand is not visible in this section.
Draw a small part of the parenchyma
of the pineal gland at high
magnification. Label pinealocytes and
astrocytes.

THYROID GLAND

The thyroid gland is situated on the lateral sides of the lower part of the larynx and
upper part of the trachea. The two lateral lobes of the thyroid are connected by a narrow
isthmus in front of the trachea. The size is quite variable but typically ranges around
20g (slightly larger in females than in males).
 271

The thyroid gland consists almost entirely of rounded cysts, follicles, which are
separated by scant interfollicular connective tissue. The follicle is the structural and
functional building block of the thyroid gland. It consists of a simple cuboidal
epithelium (variable - depending on the functional state) which surrounds a lumen
filled with a viscous substance, colloid. The size of the follicles is variable ranging from
about 50 µm to about 1 mm.

The colloid is the secretory product of the follicular cell (extracellular storage!). Its
main component, thyroglobulin, consists of triiodothyronine and tetraiodothyronine
(or thyroxine).

C cells (or parafollicular cells) are part of the follicles. There are only few of them, and
they are typically situated basally in the epithelium, without direct contact with the
follicular lumen. They are always situated within the basement membrane, which
surrounds the entire follicle.

Arterial supply is abundant with a dense network of capillaries between the follicles.
Sympathetic fibres (from the superior, middle and inferior cervical ganglia) are mainly
vasomotor (there is some evidence that sympathetic input may have a stimulatory effect
on secretory activity).

Hormones of the thyroid gland

The main secretory products of the thyroid gland are thyroxine and
triiodothyronine. TSH stimulates the endocytosis of thyroglobulin from the
follicular lumen and the subsequent release of its components into the blood stream.
TSH also stimulates their synthesis and release into the follicular lumen.

These thyroid hormones increase metabolic activity in almost all tissues

and organs. Many of the other effects of the hormones are secondary to
an increased oxygen consumption of the affected cells and the energy
that, as a consequence, becomes available for cellular processes
(exceptions to this rule are e.g. effects on lipid metabolism and
increases in carbohydrate absorption in the small intestine).

C cells produce the hormone calcitonin, which decreases blood calcium concentration
by inhibiting the resorption of bone (primarily by inhibiting osteoclast activity).

Thyroid gland, human - H&E

Identify the follicles of the thyroid gland. Have a look at the height of the epithelium
and make an educated guess at the functional activity in the thyroid (well, when it was
still alive!). Notice the capillaries in the interstices between the thyroid follicles. C cells
are very difficult to identify.
 272

Pick a nice follicle and draw it.

PARATHYROID GLANDS

The parathyroid glands are four small oval bodies located at the posterior surface of
the thyroid gland (close to the middle and inferior ends of the lateral thyroid lobes - but
a bit variable; the inferior pair may actually be located in the mediastinum).

These glands are small (average total weight is about 130 mg - that's 130
milligrams) but essential for life.

Each parathyroid gland is surrounded by a thin connective tissue capsule. Parenchymal

cells are arranged in anastomosing chords surrounded by delicate connective tissue
septa. Capillaries are abundant. A considerable number of fat cells infiltrate the gland
(beginning around puberty) and may account for about half the weight of the
parathyroid glands in adults.

Two cell types can be distinguished in the parathyroid glands:

1. Chief cells are the most numerous type. They are rather small, a round, light and
centrally placed nucleus and a very weakly acidophilic cytoplasm. They
 273

synthesise parathyroid hormone (PTH or parathormone) which is of pivotal

importance for normal calcium concentrations in the fluids and tissues of the
body. The effect is mediated by a stimulation of osteoclastic bone resorption,
intestinal calcium uptake and calcium resorption in the kidneys. If the
parathyroid glands are removed completely, calcium concentrations decrease
rapidly, leading to tetany within 2-3 days and eventually death if left untreated.
2. Oxyphilic cells are less frequent (entirely lacking in small children; occurring
first in children six to seven years old and afterwards increasing in number with
age - funny enough they have so far only been demonstrated in Rhesus
monkey, the ox and, of course, humans). Their cytoplasm is strongly
acidophilic, the nucleus is small and uniformly intense basophilic. They contain
large amounts of mitochondria.

There are plenty of transitional cells, i.e. cells that morphologically represent
transitions between chief cells and oxyphilic cells.

Both the release of calcitonin by C cells in the thyroid gland and the release of
parathyroid hormone are regulated by negative feedback from blood calcium
concentrations.

Parathyroid gland, human - H&E

Your first task, which may not be that easy, is to find the parathyroid glands. The
glands are small and usually occupy only a small fraction of the tissue on the slide.
Give it a good try before you call for help. Identify chief cells and oxyphilic cells.
Draw a part of the tissue in which both cell types are both visible. Include if possible
some of the fat cells which may occupy a large part of the parenchyma of the
parathyroid.
 274

ADRENAL GLANDS

The adrenal (or suprarenal) glands consist of an outer cortex (the main part of
the adrenal glands) and an inner medulla (which accounts for about 10% of the
adrenal glands). The gland is surrounded by a thick connective tissue capsule.
Vessels and nerves reach the medulla by way of connective tissue trabeculae
which extend from the capsule towards the medulla. Cortex and medulla are
two distinct endocrine organs(in lower vertebrates they may actually form two
entirely separate organs).

Cortex

The cortex is divided into three concentric zones which, from the surface
inwards, are termed the zona glomerulosa (accounting for about 15% of the
cortical thickness), the zona fasciculata (about 75%) and the zona
reticularis(about 10%). Transitions are usually gradual.

 Cells of the zona glomerulosa are organised into small rounded

groups or curved columns. Cells are smaller than in the two other zones,
their nuclei are dark and round, and the cytoplasm is light basophilic.
The zona glomerulosa is not influenced by ACTH.
 275

 The zona fasciculata consists of radially arranged cell cords separated

by fenestrated sinusoid capillaries. The nucleus is light and typically
located centrally. The cytoplasm is also light and often has a
characteristic foamy or spongy appearance (lipid droplets in the
cytoplasm extracted during tissue processing) - they are for this reason
also called spongiocytes.
 Anastomosing cell chords separated by sinusoid spaces form the
zona reticularis. Cells are typically smaller than in the zona fasciculata.
Their cytoplasm is eosinophilic and less spongy than that of other cells in
the cortex. The nucleus is rather light and large. Lipofucsin, a pigment,
accumulates in the cells with age. These accumulations have an orange
tinge in H&E stained preparations.

Both the zona fasciculata and zona reticularis depend on ACTH to sustain their
function and survival.

Adrenal gland, monkey - H&E

Try to find a transect through the
adrenal gland where you can see all
three zones of the cortex and, if
possible, also a bit of the medulla.
Note that the relative thickness of
the cortical zones may vary. It is not
always possible to identify the
adrenal medulla beneath the cortex.
In addition to chromaffin cells you
may find ganglion cells, sometimes
in small clusters, in the medulla.
They can be recognized by the
"typical" ganglion cell nucleus -
LARGE, light and with a distinct
nucleolus.
Draw and label a transect of the
adrenal gland which contains the
three zones and the medulla.
 276

Hormones produced in the cortex are all steroids. Consequently, cortical cells contain
large amounts of smooth endoplasmatic reticulum and lipid droplets. Since the
hormones are synthesised in the cortex they are more precisely termed corticosteroids.
Corticosteroids are further subdivided into mineralocorticoids and glucocorticoids .
The most important mineralocorticoid is aldosterone, which regulates the resorption of
sodium and excretion of potassium in the tubules of the kidney. The most important
glucocorticoids is cortisol, which has a wide range of effects on most cells of the body.
Cortisol effects protein catabolism in almost all cells aside from liver
cells, gluconeogenesis, glycogen storage, mobilisation of fat from
adipocytes, anti-inflammatory effects, inhibition of allergic reactions) .

Small amounts of androgens, oestrogens and progesterone are also

produced.The morphological zonation of the cortex reflects a functional
zonation in that

 mineralocorticoids are produced in the zona glomerulosa,

 glucocorticoids are produced in the zona fascicularis and
reticularis, and
 sex hormones are produced in the zona reticularis.
 277

Medulla

The medulla is not sharply delimited from the cortex. Cells are arranged in strands or
small clusters. Capillaries and venules in the intervening spaces. The cytoplasm of the
cells is weakly basophilic. They are called chromaffin cells because the granules of
these cells can be stained with potassium bichromate. Chromaffin cells correspond to
the adrenaline- (80%) and noradrenaline-producing cells of the medulla. The two
groups cannot be distinguished using routine histology. Chromaffin cells are innervated
by preganglionic sympathetic fibres and correspond functionally to postganglionic
neurones. The correspondence is not only functional - chromaffin cells are, like
ganglion cells of the PNS, derived from neural crest cells.

The adrenal medulla is not essential for life.

<hr size=2 width="100%" align=center>

page content and construction: Lutz Slomianka

last updated: 1/10/06

School of Anatomy and Human Biology - The University of Western Australia

Blue Histology - Male Reproductive System

MALE REPRODUCTIVE SYSTEM

The internal male genitalia consist of the testes with the adjoining epididymis, the vas
deferens and the accessory sex glands, namely the seminal vesicles, the prostrate and
 278

the bulbourethral glands (the latter sometimes are included in the external genitalia).

Testes

The testes have, like the ovaries, two functions: they produce the male gametes or
spermatozoa, and they produce male sexual hormone, testosterone, which stimulates the
accessory male sexual organs and causes the development of the masculine extragenital
sex characteristics.

The testis is surrounded by a thick capsule, the tunica albuginea, from which a conical
mass of connective tissue, the mediastinum testis, projects into the testis. The tunica
albuginea is covered externally by a serosa.

From the mediastinum, delicate fibrous septa radiate towards the tunica albuginea and
divide the parenchyma of the testis into about 300 lobuli testis, which communicate
peripherally. Each lobule contains 1-4 convoluted seminiferous tubules (about 150-300 µm
in diameter, 30-80 cm long).

Interstitial tissue between the convoluted tubules is continuous with a layer of loose
vascular connective tissue, the tunica vasculosa testis, which is found beneath the tunica
albuginea.

Each seminiferous tubule continues near the mediastinum into a straight tubule, a
tubulus rectus. The straight tubules continue into the rete testis, a labyrinthine system of
cavities in the mediastinum.

Suitable Slides

-
 279

Testis, young and

mature - H&E
Use the lowest
magnification
available. Identify the
capsule and the
connective tissue
septa extending from
it. Identify lobules,
convoluted
seminiferous tubules
and clusters of
interstitial cells. The
mediastinum testis
and rete testis are not
visible in all sections.
Draw a survey picture
of the testis.

The Convoluted Seminiferous Tubules

These tubules are enclosed by a thick basal lamina and surrounded by 3-4 layers of
smooth muscle cells (or myoid cells). The insides of the tubules are lined with
seminiferous epithelium, which consists of two general types of cells: spermatogenic
cells and Sertoli cells.

Spermatogenic cells:
Spermatogonia

are the first cells of spermatogenesis. They originate in the 4th week of foetal
development in the endodermal walls of the yolk sac and migrate to the
primordium of the testis, where they differentiate into spermatogonia.
Spermatogonia remain dormant until puberty. They are always in contact with
the basal lamina of the tubule.
 280

Two types of spermatogonia can be distinguished in the human seminiferous

epithelium:
Type A spermatogonia have a rounded nucleus with very fine chromatin grains
and one or two nucleoli. They are stem cells which divide to form new
generations of both type A and type B spermatogonia.
Type B spermatogonia have rounded nuclei with chromatin granules of variable
size, which often attach to the nuclear membrane, and one nucleolus. Although
type B spermatogonia may divide repeatedly, they do not function as stem cells
and their final mitosis always results in the formation of

Primary spermatocytes

which lie in the cell layer luminal to the spermatogonia. They appear larger than
spermatogonia. They immediately enter the prophase of the first meiotic
division, which is extremely prolonged (about 22 days!). A large number of primary
spermatocytes is always visible in cross-sections through seminiferous tubules.
Cell divisions, from the formation of primary spermatocytes and onwards, to the
production of the spermatocytes, are incomplete. The cells remain connected by
bridges of cytoplasm. The completion of the first meiotic division results in the
formation of

Secondary spermatocytes,

which are smaller than primary spermatocytes. They rapidly enter and complete
the second meiotic division and are therefore seldom seen in histological
preparations. Their division results in the formation of

Spermatids,

which lie in the luminal part of the seminiferous epithelium. They are small
(about 10 µm in diameter) with an initially very light (often eccentric) nucleus. The
chromatin condenses during the maturation of the spermatids into spermatozoa,
and the nucleus becomes smaller and stains darker.

The terminal phase of spermatogenesis is called spermiogenesis and consists of the

differentiation of the newly formed spermatids into

Spermatozoa

The mature human spermatozoon is about 60 µm long and actively motile. It is

divided into head, neck and tail.
 281

The head (flattened, about

5 µm long and 3 µm wide)
chiefly consists of the
nucleus (greatly condensed
chromatin!). The anterior
2/3 of the nucleus is
covered by the
acrosome, which
contains enzymes
important in the
process of
fertilisation. The
posterior parts of the
nuclear membrane
forms the so-called
basal plate.

The neck is short (about

1 µm) and attached to
the basal plate. A
transversely oriented
centriole is located
immediately behind
the basal plate. The
neck also contains
nine segmented
columns of fibrous
material, which
continue as the outer
dense fibres into the
tail.

The tail is further

divided into a middle
piece, a principal
piece and an end
piece. The axonema
(the generic name for the
arrangement of microtubules
in all cilia) begins in the
middle piece. It is
surrounded by nine
outer dense fibres,
which are not found
in other cilia. In the
middle piece (about 5
 282

µm long), the axonema

and dense fibres are
surrounded by a
sheath of
mitochondria. The
middle piece is
terminated by a dense
ring, the annulus. The
principal piece is
about 45 µm long. It
contains a fibrous
sheath, which consists
of dorsal and ventral
longitudinal columns
interconnected by
regularly spaced
circumferential hoops.
The fibrous sheath
and the dense fibres
do not extend to the
tip of the tail. Along
the last part (5 µm) of
the tail, called the end
piece, the axonema is
only surrounded by a
small amount of
cytoplasm and the
plasma membrane.

It takes about 48 days from the time cells enter meiosis until morphologically mature
spermatozoa are formed. Depending on the length of reproduction of spermatogonia
(which is not precisely determined) it takes approximately 64 days to complete
spermatogenesis.

Spermatogenesis is regulated by follicle stimulating hormone (FSH), which in males

stimulates the spermatogenic epithelium, and luteinizing-hormone (LH), which in males
stimulates testosterone production by Leydig cells in the interstitial tissue.

Sertoli cells

are far less numerous than the spermatogenic cells and are evenly distributed
between them. Their shape is highly irregular - columnar is the best
approximation. Sertoli cells extend from the basement membrane to the luminal
surface of the seminiferous epithelium. Processes of the Sertoli cells extend in
between the spermatogenic cells (cell limits are therefore not clearly visible in
 283

the LM). The nucleus of Sertoli cells is ovoid or angular, large and lightly
stained and often contains a large nucleolus. The long axis of the nucleus is
oriented perpendicular to wall of the tubule. A fold in the nuclear membrane is
characteristic for Sertoli cells but not always visible in the LM (well ... actually
... it's not that difficult to find, but not that easy either ....).
Lateral processes of Sertoli cells are interconnected by tight junctions, which are
likely to be the structural basis for the blood-testis barrier. Spermatogonia and
primary spermatocytes are located in the basal compartment, other cellular
stages of spermatogenesis are located in the adluminal compartment. Tight
junctions may temporarily open to permit the passage of spermatogenic cells
from the basal into the adluminal compartment. Sertoli cells provide mechanical
and nutritive support for the spermatogenic cells. Sertoli cells also secrete two
hormones - inhibin and activin - which provide positive and negative feedback
on FSH secretion from the pituitary.

Interstitial tissue

Leydig cells (15-20 µm), located in the interstitial tissue between the convoluted
seminiferous tubules, constitute the endocrine component of the testis. They synthesise
and secrete testosterone. Ledig cells occur in clusters , which are variable in size and
richly supplied by capillaries. The cytoplasm is strongly acidophilic and finely granular.
The nucleus is large, round and often located eccentric in the cell.

Suitable Slides

-
 284

Testis, young and mature - H&E

Find a nice seminiferous tubule and
identify smooth muscle cells that surround
the tubule, spermatogonia (it is difficult to
distinguish between A and B spermatogonia),
primary spermatocytes and spermatids.
Look at different tubules to see different
stages of spermio- and spermatogenesis.
Identify Sertoli cells and Leydig cells.
Draw a composite picture of your
observations.

Have a quick look at the slide TESTIS

H&E RABBIT. It illustrates nicely the
interspecific variation of the testis
(compare e.g. the amount of Leydig cells
with the other testis slides). In this slide it
somewhat easier to distinguish A and B
spermatogonia.
 285

Ducts of the Testis

Spermatozoa pass via the tubuli recti (low columnar epithelium) and the rete testis
(flattened or cuboidal epithelium) into numerous ductuli efferentes, which are lined by a
columnar epithelium, which consists of both absorptive and ciliated cells. The height of
the two cells types which form the epithelium of the ductuli efferentes is variable which
gives the lumen a characteristic wavy outline.

The ductuli efferentes leave the testis and open into a common duct, the ductus
epididymidis (about 6 m long!). It is lined by a very tall pseudostratified columnar
epithelium. Most cells of the epithelium, also called principal cells, have long
stereocilia. Stereocilia are non-motile structures, which in the EM resemble large
microvilli. Towards the basal lamina we see a number of small nuclei, which belong to
the basal cells of the ductus epididymidis. These cells regenerate the epithelium.

Peristaltic contractions of smooth muscle cells surrounding the ductus epididymidis

 286

move the spermatozoa towards the middle segment of the duct, which is the site of final
functional maturation of the spermatozoa - now they are motile. The terminal segment
of the ductus epididymidis is the site of storage of the mature spermatozoa. Smooth
muscle fibres of the terminal part of the ductus epididymidis do not contract
spontaneously. They contract during sexual stimulation concurrently with the
contraction of the musculature of the duct into which it opens, the vas deferens.

Suitable Slides

sections of the epididymis or testes sections which include the epididymis - H&E, van Gieson
Note that ductuli efferentes are located mainly in the head of the epididymis, whereas the ductus
epididymidis forms the body and tail of the epididymis. Sections of epididymis may therefore not
contain both types of ducts.

Epididymis, monkey - van Gieson

It is usually easy to see at low magnification if two types of ducts are present in your
sections. Find a spot where the two types are located close together, and identify the
ductus epididymidis and ductuli efferentes. Identify the cell types found in the ductus
epididymidis, stereocilia and the thin layer of smooth muscle which surrounds both
types of ducts.
? Try not to confuse the ducts with blood vessels, which are also abundant in parts of the epididymis. A quick look at
the epithelium lining the lumen of the visible tubules should tell you if you a looking at a blood vessel (simple
squamous epithelium) or the ducts of the epididymis.
Draw a nice profile of the ductus epididymidis and of the ductuli efferentes side by
side. Label the cell types / features visible in your drawing.
 287

The Vas deferens (or ductus deferens)

The mucosa of the vas deferens forms low longitudinal folds. It is lined by a
pseudostratified columnar epithelium. Similar to the epididymis, cells have long
stereocilia. The lamina propria is unusually rich in elastic fibres. The muscularis is well
developed (up to 1.5 mm thick) and consists of a thick circular layer of smooth muscle
between thinner inner and outer longitudinal layers. The muscularis is the structure
which makes the vas deferens palpable in the spermatic cord. The vas deferens is
surrounded by an adventitia, which is slightly denser than usual.

Suitable Slides

Vas deferens, human - H&E

Find the vas deferens, and identify the epithelium and the surrounding muscle layers.
The epithelium appears much more folded than illustrated in any of the textbooks I have consulted. Slide TESTIS
MATURE H&E - Although the epithelium of the duct was badly damaged in my slide it was possible to find the vas
deferens - give it a try in your section!
Draw the ductus deferens and label the layers.
 288

Male Accessory Reproductive Glands

The accessory (or secondary) male sex glands consist of the seminal vesicles, the
prostrate and the bulbourethral glands.

Prostate

The prostate is the largest accessory sex gland in men (about 2 × 3 × 4 cm). It contains
30 - 50 tubuloalveolar glands, which empty into 15 - 25 independent excretory ducts.
These ducts open into the urethra. The glands are embedded into a fibromuscular
stroma, which mainly consists of smooth muscle separated by strands of connective
tissue rich in collagenous and elastic fibres. The muscle forms a dense mass around the
urethra and beneath the fairly thin capsule of the prostrate.

The secretory alveoli of the prostate are very irregularly shaped because of papillary
projections of the mucosa into the lumen of the gland. The epithelium is cuboidal or
columnar. Basal cells are again present, and the epithelium may look pseudostratified
where they are found. The secretory cells are slightly acidophilic and secretory granules
may be visible in the cytoplasm. Small extensions of the apical cytoplasm into the
lumen of the alveoli may represent cells which release their secretory products
(secretion is apocrine/merocine). The secretion of the prostate contains citric acid, the
 289

enzyme fibrinolysin (liquefies the semen), acid phosphatase, a number of other

enzymes and lipids. The secretion of the prostate is the first fraction of the ejaculate.

The secretory ducts of the prostate are lined by a simple columnar epithelium, which
changes to a transitional epithelium near the openings of the ducts into the urethra.

A characteristic feature of the prostate is the appearance of corpora amylacea in the

secretory alveoli. They are rounded eosinophilic bodies. Their average diameter is
about 0.25 mm (up to 2 mm). They appear already in the seventh month of foetal
development. Their number increases with age - in particular past 50. They may undergo
calcification. Corpora amylacea may appear in semen.

Macroscopically the prostrate can be divided into lobes, but they are inconspicuous in
histological sections. In good histological sections it is possible to distinguish three
concentric zones, which surround the prostatic part of the urethra.

 The peripheral zone contains large, so-called main glands, whose ducts run
posteriorly to open into the urethra.
 The internal zone consists of the so-called submucosal glands, whereas
 the innermost zone contains mucosal glands.

This subdivision of the prostate is of clinical importance. With age the prostate
becomes enlarged due to benign nodular hyperplasia. The onset age of these
hyperplastic changes is 45. About 3/4 of the males above 60 are affected of which half
will be symptomatic. This condition affects the mucosal glands. Cancer of the prostate,
which is the second most common malignant tumor in western males, involves the
peripheral zone.

Suitable Slides

-
 290

Prostate, human - H&E

Have a look at the epithelium and the
interstitial tissue. Try to convince
yourself that the interstitial tissue is
quite cellular (smooth muscle). It may
help to compare it with the dense connective tissue
visible in Slide NONLAC BREAST H&E. Find
corpora amylacea and the urethra.
You may also be able to identify
submucosal glands.
Draw a part of the tissue where you
can see the secretory epithelium,
corpora amylacea and part of the
interstitial tissue.
 291

Seminal Vesicles

The seminal vesicles develop from the vas deferens. Their histological organisation
resembles to some extent that of the vas deferens. They are elongated sacs (about 4 cm long
and 2 cm wide), which taper where they unite with the vas deferens. Each seminal vesicle
consists of one coiling tube (about 15cm long). All the lumina visible in sections of the
seminal vesicle are in continuity in the intact organ.

The mucosa shows thin, branched, anastomosing folds. The structure of the epithelium
is variable appearing columnar or pseudostratified columnar (columnar cells and basal
cells). The lamina propria of the mucosa is fairly thin and loose. The muscularis
consists of inner circular and outer longitudinal layers of smooth muscle.

Seminal vesicles were thought to store semen - hence there name. This turned out to be
wrong. They are glands, whose secretion constitutes 60-70 % of the ejaculate. The
secretory product of the columnar cell, which may be seen in the lumen of the seminal
vesicles, is strongly acidophilic. It contains large amounts of fructose which the
spermatozoa utilise as a source of energy. Furthermore, the secretion contains
prostaglandins, flavins (yellow fluorescing pigment - of use in forensic medicine to detect
semen stains) and several other proteins and enzymes.

The cocktail of compounds which is released by the seminal vesicles in addition to

 292

fructose has three main functions:

1. the formation of the sperm coagulum,

2. the regulation of sperm motility and
3. the suppression of immune function in the female genital tract.

The secretion of the seminal vesicles is the third fraction of the ejaculate (the
spermatozoa are released with the second fraction - the contents of the vas deferens).

Suitable Slides

Seminal Vesicle - H&E

I hope your slide is as nice as mine - quite colourful. Identify the epithelium. Secretory
vesicles may be visible in the apical cytoplasm of the columnar cells. The lumen of the
seminal vesicles is often filled with the their acidophilic secretion. Try to understand
the appearance of the epithelium by looking at spots where it is cut parallel or
perpendicular to its surface. Sections passing tangentially through the anastomosing
epithelial folds of the mucosa will often show a honeycomb-like structure.
Draw a survey picture of the seminal vesicle and draw part of the epithelium at high
magnification.
 293

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page content and construction: Lutz Slomianka

last updated: 1/10/06

School of Anatomy and Human Biology - The University of Western Australia

 294

Blue Histology - Female Reproductive System

Female Reproductive System

This section focusses on the internal female reproductive organs: the ovaries, oviducts,
uterus and vagina. We will also have a look at the mammary gland, an accessory
reproductive gland. The external female genitalia include the labia minora and majora,
clitoris and vestibule.

The Ovaries

The ovaries have two functions - "production" and ovulation of oocytes and the
production and secretion of hormones. The ovary is attached to the broad ligament by a
short fold of peritoneum, called the mesovarium (or ligament of the ovary), through
which vessels and nerves pass to the ovary and enter it at the hilus of the ovary.

The surface of the ovary is covered by a single layer of cuboidal epithelium, also called
germinal epithelium. It is continuous with the peritoneal mesothelium. Fibrous
connective tissue forms a thin capsule, the tunica albuginea, immediately beneath the
epithelium.

Like so many other organs the ovary is divided into an outer cortex and an inner
medulla. The cortex consists of a very cellular connective tissue stroma in which the
ovarian follicles are embedded. The medulla is composed of loose connective tissue,
which contains blood vessels and nerves.

Ovarian Follicles

Ovarian follicles consist of one oocyte and surrounding follicular cells. Follicular
development can be divided into a number of stages.

Development represents a morphological continuum, and it may not be possible to

assign all follicles to a specific stage. This said, it's pretty easy most of the time.

Primordial follicles

are located in the cortex just beneath tunica albuginea. One layer of flattened
follicular cells surround the oocyte (about 30 µm in diameter). The nucleus of
the oocyte is positioned eccentric in the cell. It appears very light and contains a
prominent nucleolus.
Most organelles of the oocyte aggregate in the centre of the cell, where they
form the vitelline body (probably not visible in any of the available
 295

preparations).

The primary follicle

is the first morphological stage that marks the onset of follicular maturation
(Which hormone stimulates follicular maturation and where is this hormone
produced?). The previously flattened cell surrounding the oocyte now form a
cuboidal or columnar epithelium surrounding the oocyte. Their cytoplasm may
have a granular appearance, and they are for this reason also called granulosa
cells. The continued proliferation of these cells will result in the formation of a
stratified epithelium (with a distinct basement membrane) surrounding the
oocyte. The zona pellucida (glycoproteins between interdigitating processes of
oocyte and granulosa cells) becomes visible. Parenchymal cells of the ovary
surrounding the growing follicle become organised in concentric sheaths, the
theca folliculi.

Secondary follicle

Small fluid-filled spaces become visible between the granulosa cells as the
follicle reaches a diameter of about 400 µm. These spaces enlarge and fuse to
form the follicular antrum, which is the defining feature of the secondary
follicle. The oocyte is now located eccentric in the follicle in the cumulus
oophorus, where it is surrounded by granulosa cells. The theca folliculi
differentiates with the continued growth of the follicle into a theca interna and a
theca externa. Vascularization of the theca interna improves, and the spindle-
shaped or polyhedral cells in this layer start to produce oestrogens. The theca
externa retains the characteristics of a highly cellular connective tissue with
smooth muscle cells. The oocyte of the secondary follicle reaches a diameter of
about 125 µm. The follicle itself reaches a diameter of about 10-15 mm.

The mature or tertiary or preovulatory or Graafian follicle

increases further in size (in particular in the last 12h before ovulation). The
Graafian follicle forms a small "bump" on the surface of the ovary, the stigma
(or macula pellucida). The stigma is characterised by a thinning of the capsule
and a progressive restriction of the blood flow to it. Prior to ovulation the
cumulus oophorus separates from the follicular wall. The oocyte is now floating
freely in the follicular antrum. It is still surrounded by granulosa cells which
form the corona radiata. The follicle finally ruptures at the stigma and the
oocyte is released from the ovary.

Ovary, macaque - H&E

Identify cortex and medulla at low magnification and verify the presence of large
numbers of blood vessels in the medulla. Now have a look at the cortex at medium/high
 296

magnification. Identify the cuboidal epithelium covering the ovary and the underlying
tunica albuginea. Find a part of the cortex where you can observe primordial, primary
and secondary follicles.
Draw this section of the cortex with its follicles, the surrounding theca (if present),
connective tissue stroma, tunica albuginea and epithelium.

Atresia

Atresia is the name for the degenerative process by which oocytes (and follicles) perish
without having been expelled by ovulation. Only about 400 oocytes ovulate - about
99.9 % of the oocytes that where present at the time of puberty undergo atresia. Atresia
may effect oocytes at all stages of their "life" - both prenatally and postnatally. By the
sixth month of gestation about 7 million oocytes and oogonia are present in the ovaries.
By the time of birth this number is reduced to about 2 million. Of these only about
400.000 survive until puberty.

Atresia is also the mode of destruction of follicles whose maturation is initiated during
the cyclus (10-15) but which do not ovulate. Atresia is operating before puberty to
remove follicles which begin to mature during this period (none of which are ovulated).
Given that atresia affects follicles at various stages of their development it is obvious
that the process may take on quite a variety of histological appearances.
 297

The Corpus luteum

The corpus luteum is formed by both granulosa cells and thecal cells after ovulation has
occurred. The wall of the follicle collapses into a folded structure, which is
characteristic for the corpus luteum. Vascularization increases and a connective tissue
network is formed. Theca interna cells and granulosa cells triple in size and start
accumulating lutein (Which hormone stimulates this process? Where is this hormone
produced?) within a few hours after ovulation. They are now called granulosa lutein
cells and theca lutein cells and produce progesterone and oestrogens.

Hormone secretion in the corpus luteum ceases within 14 days after ovulation if the
oocyte is not fertilised. In this case, the corpus luteum degenerates into a corpus
albicans - whitish scar tissue within the ovaries.

Hormone secretion continues for 2-3 month after ovulation if fertilisation occurs.

Corpus luteum - H&E

Hold the slide against the light and try to identify the corpus luteum. It appears as a
large (5mm-1cm) rounded but somewhat irregularly shaped structure in the periphery of
the ovary. It stains homogeneously bright red except from a reddish irregular structure
at its core. Now have a look using the low magnification and verify the "folded"
appearance of the tissue forming the corpus luteum. You may be able to find spots in
the periphery of the corpus luteum in which a fairly thin layer of slightly darker cells
surround the otherwise light red cell forming most of the corpus luteum. The dark cell
represent theca lutein cell the lighter ones are granulosa lutein cells.
Sketch the corpus luteum and ovary at low magnification and make sure that the
relative size of the corpus luteum becomes apparent in your sketch. Draw, if possible, a
spot where you can differentiate between theca and granulosa lutein cells.
 298

The Oviduct

The oviduct functions as a conduit for the oocyte, from the ovaries to the uterus.
Histologically, the oviduct consists of a mucosa and a muscularis. The peritoneal
surface of the oviduct is lined by a serosa and subjacent connective tissue.

The mucosa

is formed by a ciliated and secretory epithelium resting on a very cellular lamina

propria. The number of ciliated cells and secretory cells varies along the oviduct
(see below). Secretory activity varies during the menstrual cycle, and resting
secretory cells are also referred to as peg-cells. Some of the secreted substances
are thought to nourish the oocyte and the very early embryo.

The muscularis

consists of an inner circular muscle layer and an outer longitudinal layer. An

inner longitudinal layer is present in the isthmus and the intramural part (see
below) of the oviduct. Peristaltic muscle action seems to be more important for
the transport of sperm and oocyte than the action of the cilia.

Texts usually refer to four subdivisions of the oviduct.

 299

1. The infundibulum is the funnel-shaped (up to 10 mm in diameter) end of the

oviduct. Finger-like extensions of its margins, the fimbriae, are closely applied
to the ovary. Ciliated cells are frequent. Their cilia beat in the direction of
2. the ampulla of the oviduct. Mucosal folds, or plicae, and secondary folds which
arise from the plicae divide the lumen of the ampulla into a very complex shape.
Fertilization usually takes place in the ampulla.
3. The isthmus is the narrowest portion (2-3 mm in diameter) of the parts of the
oviduct located in the peritoneal cavity. Mucosal folds are less complex and the
muscularis is thick. An inner, longitudinal layer of muscle is present in the
isthmus and the
4. last, intramural part of the oviduct, which penetrates the wall of the uterus. The
term "intramural" should be familiar to you ..... The mucosa is smooth, and the
inner diameter of the duct is very small.

Oviduct is a nice descriptive term, but (sigh) not the only one commonly used for these
structures - you will also find the terms Fallopian tubes or uterine tubes. The term
salpinx (Greek, trumpet) seems to have passed its "use-by-date" in many histology text
but (sigh) not in pathology, where salpingitis refers to chronic or acute inflammation of
the oviduct. Let's see how "tubal inflammation" will fare in the future.

Obstruction of the oviduct as a consequence of salpingitis is one possible cause of

infertility, and alterations of luminal structure by inflammatory processes are a risk
factor for tubal pregnancies.

Oviduct - H&E and Ovary, macaque - H&E

Unfortunately, we do not have many oviduct slides, but some sections of the macaque
ovary and the uterus slide contains segments of the oviduct. In the former you should be
able to see both the muscularis and the folded mucosa. Ciliated cells and peg-cells are
present. In the intramural part of the oviduct (uterus slide) the mucosa is smooth and
ciliated cells are rare or absent. The intramural part of the uterus should remind you of a
structure in the male reproductive system - Which one?
Draw part of the wall of the oviduct, identify the segment and, if possible, ciliated and
peg cells.
 300

The Uterus

The uterus is divided into body (upper two-thirds) and cervix. The walls of the uterus
are composed of a mucosal layer, the endometrium, and a fibromuscular layer, the
myometrium. The peritoneal surface of the uterus is covered by a serosa.

Myometrium

The muscle fibres of the uterus form layers with preferred orientations of fibres
(actually 4), but this is very difficult to see in most preparations. The muscular
tissue hypertrophies during pregnancy, and GAP-junctions between cells
become more frequent.

Endometrium

The endometrium consists of a simple columnar epithelium (ciliated cells and

secretory cells) and an underlying thick connective tissue stroma. The mucosa is
invaginated to form many simple tubular uterine glands. The glands extend
through the entire thickness of the stroma. The stromal cells of the endometrium
are embedded in a network of reticular fibres. The endometrium is subject to
cyclic changes that result in menstruation. Only the mucosa of the body of the
uterus takes part in the menstrual cycle.
 301

The endometrium can be divided into two zones based on their involvement in the
changes during the menstrual cycle: the basalis and the functionalis.

 The basalis is not sloughed off during menstruation but functions as a

regenerative zone for the functionalis after its rejection.
 The functionalis is the luminal part of the endometrium. It is sloughed off during
every menstruation and it is the site of cyclic changes in the endometrium.
These cyclic changes are divided into a number of phases: proliferative (or
follicular), secretory (or luteal), and menstrual.

Uterus, proliferative phase - H&E

Identify the muscular wall of the uterus and the endometrium lining the lumen of the
uterus. Identify uterine glands embedded in the stroma of the endometrium. Finally try
to find a spiral artery. These are arteries ascend through the endometrium and form a
coil/spring like structure while they do so. How would you expect this structure to look
like in a section?
Sketch a small section of the endometrium.

Vagina
 302

The vagina is a fibromuscular tube with a wall consisting of three layers: the mucosa,
muscularis and adventitia of the vagina

Mucosa

The stratified squamous epithelium (deep stratum basalis, intermediate stratum

spinosum, superficial layers of flat eosinophilic cells which do contain keratin
but which do not normally form a true horny layer) rests on a very cellular
lamina propria (many leukocytes). Towards the muscularis some vascular
cavernous spaces may be seen (typical erectile tissue).

Muscularis

Inner circular and outer longitudinal layers of smooth muscle are present.
Inferiorly, the striated, voluntary bulbospongiosus muscle forms a sphincter
around the vagina.

Adventitia

The part of the adventitia bordering the muscularis is fairly dense and contains
many elastic fibres. Loose connective tissue with a prominent venous plexus
forms the outer part of the adventitia.

Vagina, human - H&E

Identify the layers of the vagina. Note
that the organization of the wall of the
vagina corresponds in many respects to
the organization of the wall of the
oesophagus. It should not be necessary
to do a drawing.
Compare your observations in the
vagina with those in the oesophagus.
Try to define how the layers differ
although they are composed of similar
tissue types.
 303

Female Accessory Reproductive Glands - Mammary Glands

The mammary glands are modified glands of the skin. Their development resembles
that of sweat glands. They are compound branched alveolar glands, which consist of
15-25 lobes separated by dense interlobar connective tissue and fat. Each lobe contains
an individual gland. The excretory duct of each lobe, also called lactiferous duct, has its
own opening on the nipple.

The lactiferous duct has a two layered epithelium - basal cells are cuboidal whereas the
superficial cells are columnar. Beneath the nipple, the dilated lactiferous duct forms a
lactiferous sinus , which functions as a reservoir for the milk. Branches of the
lactiferous duct are lined with a simple cuboidal epithelium. The secretory units are
alveoli, which are lined by a cuboidal or columnar epithelium. A layer of myoepithelial
cells is always present between the epithelium and the basement membrane of the
branches of the lactiferous duct and the alveoli.

The above description corresponds basically to the appearance of the resting mammary
gland. Pregnancy induces a considerable growth of the epithelial parenchyma leading to
the formation of new terminal branches of ducts and of alveoli in the first half of
pregnancy. Growth is initiated by the elevated levels of oestrogen and progesterone
produced in the ovaries and placenta. Concurrently, a reduction in the amount of intra-
and interlobular connective tissue takes place. The continued growth of the mammary
glands during the second half of pregnancy is due to increases in the height of epithelial
cells and an expansion of the lumen of the alveoli. They contain a protein-rich (large
amounts of immunoglobulins) eosinophilic secretion - the colostrum or foremilk).

Secretion of milk proteins proceeds by exocytosis (merocrine secretion), whereas lipids

are secreted by apocrine secretion. Secretion is stimulated by prolactin. Prolactin
secretion in turn is stimulated by sensory stimulation of the nipple, which also initiates
the so-called milk ejection reflex via the secretion of oxytocin from the
neurohypophysis. Milk is ejected from the glandular tissue into the lactiferous sinuses -
now it's up to the baby to get things out.

The glandular tissue of the mammary gland is frequently subject to pathological

changes - the most serious being mammary cancer, which is the most frequent
malignancy in women (about 6.5% of all women develop the disease).

Non-lactating breast - H&E and Lactating breast, human - H&E

Identify lactiferous ducts in the section of the non-lactating breast. See if you can
identify resting alveoli and lactiferous sinuses.
Draw a nice lactiferous duct and, if possible, a few resting alveoli.
Identify the secretory alveoli and interlobular ducts in the slide of the lactating
 304

mammary gland. Do all parts of the secretory tissue look similar? Why/why not?
Draw and label part of the secretory tissue.

<HRpage content and construction: Lutz Slomianka

last updated: 5/08//09

School of Anatomy and Human Biology - The University of Western

Australia
Blue Histology - The Eye

The Eye

The eye is formed by three layers, or tunics. Each of these three layers contributes with
parts that have structural / nutritive functions and parts that form the optic and
 305

photoreceptive apparatus of the eye. From the outside to the inside of the eyeball the
three tunics are the

1. fibrous tunic, which forms a capsule enclosing and protecting the other
components of the eye. It is subdivided into the sclera, with primarily structural
functions, and the cornea, which is part of the optic apparatus.
2. vascular tunic, which forms the choroid, ciliary body and iris. This tunic is also
called the uveal tract. The choroid has primarily nutritive functions. The ciliary
body generates the aqueous humor of the eye, but the ciliary muscle also
functions in the optic apparatus. The iris is part of the optic apparatus in which it
functions a contractile diaphragm, i.e. the aperture of the eye.
3. neural tunic consists of the retina. The retina proper forms the photoreceptive
layer of the eye. As a double-layered epithelium, the retina also covers the
ciliary process and the posterior surface of the iris, where it has both nutritive
and structural functions.
The ciliary and iridial parts of the retina are described together with the ciliary
process and iris.

The Fibrous Tunic: Cornea and Sclera

Cornea

The cornea forms the anterior surface of the eye in an area largely corresponding to the
pigmented iris, which is visible behind the cornea. The diameter of the cornea is ~11
mm; the thickness ranges from ~0.5 mm centrally to ~1mm along the margins of the
cornea. The cornea is formed by three cellular layers, which are separated from each
other by two thin, acellular layers. Blood vessels are not normally found in the cornea,
and the cells are not pigmented.

The anterior surface of the cornea is lined by a stratified squamous epithelium. The
basement membrane of this anterior corneal epithelium rests on the first acellular layer,
the anterior limiting lamina or Bowman's membrane. It separates the epithelium from
the corneal stroma and consists of densely packed collagen fibrils embedded in ground
substance.

The corneal stroma consists of 200 - 250 layers of regularly organized collagen fibrils
(mainly tropcollagen type I, but also types III, V and VI). Collagen fibres within each
layer will run parrallel to each other but at large angles to collagen fibres in the next
layer. Flattened fibrocytes, referred to as keratocytes, are located between the layers of
collagen fibres. The regular arrangement of the collagen fibres and their small diameter
(20 - 60 nm) acount for the transparency of the cornea.
 306

The posterior surface of the cornea is lined by an endothelium, the posterior

endothelium. The posterior endothelium and the corneal stroma are separated from each
other by the posterior limiting lamina or Descemet's membrane, which corresponds to
the basement membrane of the posterior endothelium.

The lateral margins of the cornea are continuous with the conjunctiva (anterior corneal
epithelium) and sclera (corneal stroma).

Sclera

The sclera is a tough layer of dense connective tissue consisting of collagenous fibres
and networks of elastic fibres. Melanocytes are present in deep parts of the sclera in
addition to the usual complement of connective tissue cells. Distended by the
intraocular pressure, the sclera maintains the shape of the eyeball. It is also the site of
attachment of the ocular muscles.

Anteriorly, the sclera forms a slight protrusion into the eyebal before it merges with the
cornea - the scleral spur, which provides a point of insertion for part of the ciliary
musle. The sclerocorneal junction houses the canal of Schlemm, through which the
aqueous humor is drained into ciliary veins.

Suitable Slides
sections of the eye - H&E, van Gieson
It is difficult to prepare good sections of the eye. The sclera is quite tough, while
the hyaline body is very soft and contains a high proportion of water.
Differential shrinkage and hardness typically give rise to a number of artefacts.
Detachment of the outer retina from the pigment epithelium and distortions of
the lens and cornea are the most common ones. The preparation of only parts of
the eye is one way to overcome at least some of the problems. You may have
access to sections of just 'retina' or 'anterior eye'.
 307

Cornea, rat - H&E and Cornea,

monkey - van Gieson
Identified the layers of the cornea.
Keratocytes are easiest to identify in
H&E stained sections. The posterior
endothelium may not be well
preserved. If that is the case in your
section, scan along the posterior
boundary of the cornea and see if there
is a spot with at least a few remaining
endothelial cells. An irregular, "wavy"
surface of the anterior corneal
epithelium is a preparation artefact.
Draw a small segment of the cornea at
high magnification and label the layers
that can be identified.

The Vascular Tunic: Choroid, Ciliary Body and Iris

Choroid

The coroid consists of loose connective tissue, which houses a dense network of blood
vessels. Connective tissue cells and melanocytes are numerous. The latter give the
choroid its dark colour. Small blood vessels are especially frequent in the innermost
part of the choroid, which is called the choriocapillary layer. This layer supplies the
retina with nutrients. Bruch's membrane is located between the choroid and the retina. It
consists of two layers of collagen fibres and a network of elastic fibres between them.

Ciliary body

The ciliary body is an inward extension of the choroidea at the level of the lens. Ciliary
processes are short extensions of the ciliary body towards the lens. A small amount of
loose connective tissue similar to that of the choroid is located between smooth muscle
cells which form the bulk of the ciliary body. They form three bundles, the ciliary
muscle.

The inner surface of the ciliary body and its processes are lined by two layers of
 308

columnar cells which belong to the retina - the ciliary epithelium formed by the pars
ciliaris of the retina. The outer cell layer is pigmented, whereas the inner cell layer, i.e.
the layer that faces the posterior chamber of the eye, is nonpigmented.

The ciliary processes contain a dense network of capillaries. The cells of the inner layer
of the ciliary epithelium generate the aqueous humor of the eye. , i.e. they transport the
plasma filtrate generated by the capillaries in the ciliary processes into the posterior
chamber of the eye. Thight junctions between the cells form the blood - aqueous humor
barrier.

Fibers, which consist of fibrillin, extend from the ciliary processes towards the lens and
form the suspensory ligament of the lens. These fibres are also called zonule fibres.
Two of the bundles of the ciliary muscles attach to the sclera and strech the ciliary body
when they contract, thereby regulating the tension of the zonule fibres. The reduced
tension will result in a thickening of the lens which focusses the lens on close objects -
a process called accomodation.

Iris

The posterior surface of the iris is covered by the retina. The inner layer of the retina,
i.e. the layer facing the posterior chamber, is called the posterior epithelium of the iris.
Both layers of the retina are pigmented, but pigmentation is heavier in the inner layer.
In the region of the central opening of the iris, the pupil, the retina extends for a very
short distance onto the anterior surface of the iris. The iridial stroma consists of a
vascularized loose connective tissue rich in melanocytes in addition to macrophages
and fibrocytes, which are all surrounded by a loose meshwork of fine collagen fibers.
The anterior surface of the iris is not covered by an epithelium - instead of we find a
condensation of fibrocytes and melanocytes, the anterior border layer of the iris.

The iris forms the aperture of the eye. Myoepithelial cells in the outer (or anterior) layer
of the retina, i.e. the layer adjacent to the stroma of the iris, have radially oriented
muscular extensions. These extensions form a flat sheet immediately beneath the
anterior layer of the retina, the dilator pupillae muscle. Embedded in the central portion
of the iridial stroma are smooth muscle cells which form the annular sphincter pupillae
muscle. In humans, this muscle surrounds the pupil as a less than 1 mm wide and only
0.2 mm thick band. The two muscles regulate the size of the pupil.
Pupillary constriction, which is mediated by the sphincter pupillae muscle, is clinically
refered to as miosis - dilation, mediated by the dilator pupillae muscle, as mydriasis.

The pigmentation of cells in the stroma and anterior border layer of the iris determines
to color of the eyes. If cells are heavily pigmented the eyes appear brown. If
pigmentation is low the eyes appear blue. Intermediate levels create shades of green and
grey.

Suitable Slides
 309

sections of the

Eye, monkey, van Gieson

The iris, ciliary body, sclera and cornea meet at the iridocorneal angle, where you also
can see the trabecular meshwork and the canal of Schlemm.
Draw the structures joining at the iridocorneal angle at low magnification and
suplement your drawing with high magnification inserts of part of a ciliary process and
the iris.

Lens

The lens consists of a lens capsule, the subcapsular epithelium and lens fibres. It does
not contain blood vessels or nerves.

The lens capsule is generated by the cells of the subcapsular epithelium and
corresponds to a thick, elastic basal lamina. The zonule fibres insert into the lens
capsule.

Cells of the subcapsular epithelium (or anterior lens cells) are mitotically active. In
adult individuals they only cover the anterior "hemisphere" of the lens. As they divide,
 310

cells gradually move towards the equator of the lens where they tranform into lens
fibres. The apical part of the gradually elongating cell extends between the subcapsular
epithelium and adjacent lens fibres towards the anterior pole of the lens. The basal part
extends towards the posterior pole. The nucleus remains close to the equatorial plane of
the lens.

The mature lens fibres, i.e. very long

(up to 12 mm), hexagonal cells, form
the body of the lens. They are located
immediately deep to the cells of the
subcapsular epithelium. Lens fibres are
nucleated in the soft, outer cortex of
the lens. As new lens fibres are added
to the periphery of the cortex, lens
fibres located deeper in the cortex
loose their nuclei and become part of
the somewhat harder nucleus of the
lens. In the intact lens, lens fibres are
tightly connected to each other. Few
organelles are scattered in a cytoplasm
filled with cystallin proteins. These
proteins are responsible for the
transparency and refractive properties
of the lens and account for up to 60%
of the mass of lens fibres.

The optical properties of the lens change from periphery to central parts because of
differences in the amounts of crystallins contained in lens fibres. These difference
correct for distortions of colours and shapes (called spherical and chromatic
aberrations) which commonly occur at the margins of glass lenses. These aberrations
are easy to observe when you look through a loupe - or even in not-so-good
microscopes at the margins of the field of view, where they are easy to detect when to
slide is moved

Suitable Slides
sections of eye - H&E, van Gieson

Eye, rat, H&E

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The Neural Tunic: Retina

Similar to the retinal lining of the iris and ciliary body, the outer layer of the light
sensitive retina forms a single layer of cuboidal cells - the pigment epithelium. The
inner layer of the retina contains the photoreceptors, the first neurones which process
the sensory information, and the neurones which convey the pre-processed sensory
information to the central nervous system. Receptors, neurones, supporting cells and
their processes are segregated into nine layers:

1. The layer of rods and cones contains the outer, rod- or cone-shaped light
sensitive segements of the photoreceptive cells. The lights sensitive part and the
perikayon of the rods and cones are connected by a narrowed bridge of
cytoplasm. At the level of this connection the rods and cones are surrounded by
the processes of a specialised type of glial cells, Müller cells, which form the
2. outer limiting membrane.
3. The outer nuclear layer contains the nuclei and perikarya of the rods and cones.
Their processes form part of the
4. outer plexiform layer, where they form synapses with the processes of neurones
whose cell bodies are located in the
5. inner nuclear layer. The cells of the inner nuclear layer are concerned with the
 312

initial processing of the sensory input. The three major neurone types are
horizontal, bipolar and amacrine cells. The inner nuclear layer also houses the
perikarya of the Müller cells.
6. The inner plexiform layer contains the processes of the inner nuclear layer
neurones which convey the sensory input to the
7. ganglion cell layer. Ganglion cells are not evenly distributed. There are few of
them towards the periphery of the retina. Close to the fovea, ganglion cells form
a densely packed layer. Both ganglion cells and the cell bodies located in the
inner nuclear layer which contact the rods and cones of the fovea are displaced
towards the margins of the fovea.
8. Layer of optic nerve fibres. The axons of the ganglion cells travel in this layer
towards the optic disc. Towards the optic disc, the thickness of this layer
increases as more and more axons are added to it.
9. The inner limiting membrane corresponds to a basal lamina formed by the
Müller cells.

Suitable Slides
The eyes of most mammals are suitable to look at the general organization of
the retina. However, the retina of some mammals does not contain cones, and,
as mentioned before, cell layers which would be pigmented in normal humans
will not be so if the eye has been collected from an albino (many laboratory
strains of small ammals are albinoid).

Retina, monkey, methylene blue and Retina, albino rat, H&E

You will probably not be able to identify the inner and outer limiting membranes. The
other layers should not be a problem. Ganglion cell density is quite variable and,
although some ganglion cells will always be visible, they may not form a continuous
layer.
Draw and label the layers of the retina.
 313

Optic Nerves

Because of the origin of the retinae and optic nerves from the developing forebrain, the
optic nerves (cranial nerves II) corresponds to fibre tracts connecting parts of the CNS -
in this case the ganglion cells of the retina with neurones in the lateral geniculate
nucleus of the thalamus and neurones in the superior colliculus and pretectum of the
midbrain.

Ganglions cell axons run towards the optic disc where they turn towards the sclera.
Numerous bundles (or fascicles) of axons pass through the choroid and openings in the
sclera, the lamina cribosa. The axons become myelinated in this region. Collectively,
the bundles form the optic nerve. Like other parts of the CNS, the optic nerve is
surrounded by the three meninges - the outer dura mater, the middle archnoid and the
inner pia mater, which are separated from each other by subdural and subarachnoid
spaces. At the eyeball, the dura fuses with the sclera while the arachnoid and pia mater
merge with the choroid. Connective tissue septa, which arise from the pia mater,
separate the fibre bundles in the optic nerve. The axons in the optic nerve are supported
by astrocytes and oligodendrocytes. Microglia is also present.
 314

Suitable Slides
sections of the optic nerve - H&E, trichrome
Optic nerve, monkey, van Gieson
The organisation of the optivc nerve
corresponds at a first glance to that of
peripheral nerves. Differences relate to
the connective tissue surrounding the
nerve and the fascicles of nerve fibres.
In the optic nerve, the dura mater,
arachnoid and pia mater take the roles
of the epi-, peri- and endoneurium of
peripheral nerves. Narrow cleft-like
spaces are found between the
meninges, forming the subdural space
(between dura and arachnoid) and
subarachmoid space (between
arachnoid and pia).
These spaces may expand during tissue
preparation due to differential
shrinkage of the tissues and appear
much wider than they are in the living
organism. This applies also to the optic
nerve illustrated.
Draw the optic nerve at low
magnification and label its
components.

Eyelid

The posterior (facing the eyeball) and anterior (facing the world) surfaces of the eyelid
are also called conjunctival and cutaneous parts. The cutaneous part is covered by skin
and contains sweat glands, sebaceous glands and, along the margins of the lid, 3-4 rows
of hairs - the eyelashes. Modified apokrine sweat glands, the glands of Moll, empty into
the follicles of the eyelashes. The eyelashes lack arrector pili muscles.

The inner, conjunctival part of the lids is lined by conjunctiva. Beneath the conjunctiva,
large sebaceous glands are embedded in a plate of dense connective tisssue containing
many elastic fibres. The plate and the sebaceous glands within it are called the tarsal
plate the tarsal glands (also Meibomian glands). Extensive skeletal muscle bundles
between the tarsal plate and the skin belong to the orbicularis oculi muscle.

Conjunctiva
 315

The margins of the cornea merge with the conjunctiva. The conjunctiva extends over
the the 'white of the eye', which corresponds to the anterior part of the sclera, folds back
and continues over the posterior part of the eyelid. At the opening formed by the
eyelids, the conjunctive merges with the skin which covers the anterior surface of the
eyelids.

The epithelium of the conjunctiva varies from stratified squamous (most of it) to
stratified columnar (at the reflection from the sclera to the eyelid). It contains goblet
cells. The conjunctival epithelium rest on the loose connective tissue of its lamina
propria.

page content and construction: Lutz Slomianka

last updated: 5/08/09

School Of Anatomy And Human Biology-The University Of Western Australia

Blue Histology- EAR.

Ear
 316

 R
e
g
i
o
n
s
o
f
t
h
e

e
a
r
.

 Overview of inner ear.

o Bony labyrinth and membranous labyrinth
o Hair cells
o Otolith organs (saccule and utricle)
o Semicircular canals
o Cochlea
o Organ of Corti
o Endolymph and perilymph

 Images
o Cochlea (overview)
o Cochlea (low magnification)
o Organ of Corti (high magnification)
o Spiral ganglion (high magnification)
o Saccule and ampulla (low magnification)
o Macula of saccule (high magnification)
o Crista of ampulla (high magnification)
 317

REGIONS of the ear

The ear has three distinct regions -- outer ear, middle ear, and
inner ear.

 The outer ear includes the pinna (the visible ear, consisting
mostly of skin and cartilage) and the ear canal. The latter
is lined by keratinized stratified squamous epithelium. This
lining differs from skin by the presence of specialized
ceruminous (ear-wax) glands.

 The middle ear is basically a space, communicating via the

eustacian tube with the oropharynx. It is lined by a very
thin non-keratinized stratified squamous epithelium.
Spanning the space of the middle ear are the three middle
ear bones, the malleus (hammer), incus (anvil), and stapes
(stirrup).

 The eardrum is a thin membrane separating the outer ear

from the middle ear. It is sandwich of tissues, with
keratinized stratified squamous epithelium facing the outer
ear, non-keratinized stratified squamous epithelium facing
the middle ear, and a very thin layer of connective tissue in
between.

 The inner ear is the portion of the ear which contains

sensory receptors. The remainder of this study guide
describes the inner ear.

OVERVIEW of the inner ear

The inner ear has a complex structure. The following basic
 318

concepts should help organize that complexity.

 The
inner
ear,
locat
ed
withi
n the
bony
labyr
inth,
cont
ains Image courtesy Alec Salt, Cochlear Fluids
sens
Laboratory, Washington University; used with
e
orga permission.
ns
serving both balance and hearing.

o Head position (i.e., gravity; also linear acceleration)

is sensed by the otolith organs of the saccule and
utricle.
o Head rotation (i.e., angular acceleration) is sensed by
the cristae ampularis of the semicircular canals.
o Hearing is sensed by the organ of Corti within the
scala media of the cochlea.

 All of these several senses of the inner ear utilize the same
 319

mechanoreceptor cell type, epithelial hair cells.

 Hair cells are housed within an elaborately-shaped

chamber called the membranous labyrinth.

 The membranous labyrinth is filled with a unique fluid

called endolymph, secreted by cells of the stria vascularis.
Endolymph differs substantially from all other fluids of the
body and provides a special fluid environment for the hair
cells

 The membranous labyrinth includes interconnection

among the cochlea, saccule, utricle, and semicircular
canals.

 The membranous labyrinth is housed within the bony

labyrinth.

 Perilymph fills the space of the bony labyrinth around the

membranous labyrinth.

BONY LABYRINTH and MEMBRANOUS LABYRINTH

 320

The inner ear

resides within a
space called the
bony
labyrinth.

 The oval
window
forms a
potential
opening
from the
middle ear Image courtesy Alec Salt, Cochlear Fluids
into the Laboratory, Washington University; used with
bony permission.
labyrinth.
o The
stapes of the middle ear plugs this opening; but . . .
o The stapes is flexibly attached and can vibrate to
transmit pressure waves to the fluid that fills the
bony labyrinth. (Sound is carried from the eardrum
across the middle ear by the three middle ear
ossicles, ending with the stapes at the oval window.)

Suspended within the bony labyrinth, and approximating its

shape, is an interconnected set of membrane-lined chambers and
passageways called the membranous labyrinth.

 In the diagram, the color orange occupies the space of the

bony labyrinth, while the membranous labyrinth is blue.

 The name labyrinth suggests the complex shape of these

chambers and passageways.
o The vestibule is (logically enough) the "entry room"
 321

into the deeper passageways.

 The vestibule of the bony labyrinth contains the
saccule and utricle of the membranous
labyrinth;
o Three semicircular canals comprise looping tubules
which leave and return to the vestibule.
 Within each semicircular canal of the bony
labyrinth is a semicircular canal of the
membranous labyrinth.
o The cochlea is shaped like a snail-shell which spirals
away from the vestibule.
 A single coiled tunnel of the bony labyrinth is
subdivided into three levels ("scalae") by
membranes of the membranous labyrinth.
 The portion of the membranous labyrinth within
the cochlea is called the scala media.

 Inside the membranous labyrinth is a unique fluid called

endolymph. Surrounding the membranous labyrinth (i.e.,
filling the remaining space of the bony labyrinth) is a fluid
called perilymph.

HAIR CELLS
Hair cells, the specialized mechanoreceptor cells of the auditory
and vestibular systems, are found in several positions along the
chambers and passageways of the membranous labyrinth.

 Hair cells are basically columnar epithelial cells.

o At the apical end of each hair cell is a set of "hairs"
(cytoplasmic projections, kinocilium and stereocilia)
embedded in a mass of extracellular jelly.
 322

o At the basal end of each hair cell are synapses onto

sensory axons.

 Hair cells work similarly throughout the inner ear.

o A hair cell responds when movement of the
extracellular jelly displaces its kinocilium and
stereocilia. Displacement is excitatory in one
direction (toward the kinocilium) and inhibitory in the
opposite direction.
o Displacement of the kinocilium and stereocilia alters
conductance of ion channels, in turn affecting release
of neurotransmitter onto the associated sensory
axon. (These axons project along the auditory and
vestibular nerves, cranial nerve VIII).
o Hair cells function within a fluid environment, the
endolymph, with a unique ionic composition.
o The process of sensory transduction in hair cells (i.e.,
the conversion of an external stimulus, in this case
small movement, into neural activity) has been
intensively investigated. For detailed information,
consult your print resources (e.g., Kandel at al.)

 The mechanical disposition of the jelly in relation to the

spaces of the membranous labyrinth determines how hair
cells respond.
o Hair cells in the semicircular canals respond to
angular acceleration (rotation).
o Hair cells in the in the otolith organs respond to linear
acceleration.
o Hair cells in the organ of Corti of the cochlea respond
to sound.
 323

OTOLITH ORGANS (SACCULE and UTRICLE)

The saccule and utricle contain patches of hair cells
called maculae ("macula" means "spot" or "patch").

 A small mass of jelly rests on top of the hair cells of the

macula.
 In this jelly are numerous tiny mineral concretions, called
otoliths ("earstones" or "earsand").
o Clinical Note: If any otoliths break loose (e.g., due to
head trauma), they may come to rest in an
inappropriate place, stimulating the hair cells in a
semicircular canal) and cause disturbance in balance
(see benign positional vertigo).

 Hair cells of the macula are deflected by the weight or

inertia of the otoliths. Together the two pairs of otolith
organs (one of each in each ear) can sense head
orientation (gravity) or linear acceleration in any direction.

Image courtesy Alec Salt, Cochlear Fluids

 324

Laboratory, Washington University; used with

permission.

SEMICIRCULAR CANALS

Each semicircular canal of the bony labyrinth is a

hollow passageway looping out from and back to the
vestibule. Within each of these passageways is a
semicircular canal of the membranous labyrinth.

 At one end of each membranous semicircular canal is a

small enlargement called the ampulla.
 Within each ampulla is a ridge or "crest" called
the crista.
 The crista is covered with hair cells.
 A small mass of jelly, called the cupola ("cap") rests on top
of the hair cells of the crista.

Hair cells of the ampullae respond to angular acceleration (i.e.,

rotation of the head).

 There are three semicircular canals in each ear, oriented in

three mutually-perpendicular planes.
 Rotation of the head in any direction will cause inertial
fluid movement in one or more of the semicircular canals.
 Fluid motion in a semicircular canal pushes the the cupola
like a swinging door.
 Movement of the cupola in turn deflects the projections of
the hair cells.
o Clinical Note: Should loose otoliths enter a
semicircular canal, they may stimulate the hair cells
inappropriately and cause disturbance in balance (see
 325

benign positional vertigo).

The planes of orientation of the semicircircular canals correspond

to the planes of action of the extraocular muscles, allowing
simple reflexes to coordinate eye movement with head rotation.

Image courtesy Alec Salt, Cochlear Fluids

Laboratory, Washington University; used with
permission.
 326

COCHLEA
The cochlea houses an elaborate
configuration of membranous
labyrinth and hair cells, called the
organ of Corti, designed for
auditory reception.

The basic shape of the cochlea is

that of a snail-shell, or tapering
helix.

The human cochlea is short and

broad; micrographs at this website
(and in many other references) show
the cochlea of a laboratory rodent which is proportionately taller
and narrower.

The spiraling tunnel (blue, in image at right) that

forms the cochlea of the bony labyrinth is divided
into three distinct channels by portions of the
membranous labyrinth attached to bony ridges. Each of these
channels is called a "scala", meaning "ramp" or "incline" (think
of a musical "scale").

 The scala vestibuli ascends from the vestibule (hence

vestibuli in the name) to the tip of the cochlea.
o The scala vestibuli contains perilymph.

 The scala tympani descends from the tip of the cochlea to

the round window. (There is an elastic energy-dissipating
membrane covering the round window (hence tympani in
the name).
o The scala tympani, like the scala vestibuli, contains
perilymph.
 327

o At the tip of the cochlea, the scala vestibula and the

scala tympani are connected through the
helicotrema.

 The
scala
medi
a,
also
calle
d the
cochl
ear
duct,
lies
alon Image courtesy Alec Salt, Cochlear Fluids
g the
Laboratory, Washington University; used with
lengt
h of permission.
spiral
cochlea, in a "medial" position between the scala vestibuli
and scala tympani.
o The scala media contains endolymph.
o The organ of Corti lies within the scala media.
o The scala media is separated from the scala vestibuli
by the very thin Reissner's membrane.
o The scala media and the scala tympani are separated
by the basilar membrane.

 Clinical note: A cochlear implant is inserted into

the scala tympani, where it lies close to the
 328

organ of Corti and can artificially stimulate

axons of the auditory nerve.

The central column (the modiolus) of the helical

cochlea contains axons serving the organ of Corti
on their way to the auditory nerve.

A bony ridge, the spiral lamina, extends out from

the modiolus and provides support for the organ
of Corti. A tubular cavity within the spiral lamina
contains the cell bodies of the axons of the
auditory nerve. Because this collection of nerve
cell bodies has a helical shape paralleling the
cochlear scalae, it is called the spiral ganglion.

ORGAN OF CORTI

The organ of Corti is an elaborate structure with more named

parts than the rest of inner ear. Several key features are listed
below. For additional detail, including the basic physiology of
hearing, consult a textbook or click here, Anatomy and pathology
of organ of Corti (Bohne laboratory, Washington University).

 The organ of Corti is contained within the scala

media.

o The organ of Corti is a long strip of tissue that extends

the length of the scala media, from the base of the
cochlea to its apex.
 329

o The organ of Corti is usually illustrated in cross-

section. Tissue sections of the cochlea typically
contain several appearances of the organ of Corti, as
the organ is sliced in each turn of the helix.

 The fluid environment for the organ of Corti is endolymph,

which fills the scala media. (Endolymph is secreted by cells
of the stria vascularis.)

 Within the complex strip of tissue that comprises the organ

of Corti are specialized sensory hair cells.

o The entire complex (the whole organ of Corti) rests

on the basilar membrane.

o This basilar membrane supports the basal ends of the

hair cells in the organ of Corti.

o The apical ends of hair cells touch the tectorial

membrane, a "shelf" of jelly that is supported
immovably on the spiral lamina.

o When the basilar membrane flexes in

respond to sound waves (i.e., pressure
waves delivered to inner-ear fluid by the
middle-ear ossicles), the organ of Corti, including its
hair cells, also moves.

o Thus, when the basilar membrane is moved by

pressure waves (i.e., sound), the hair cells move
relative to the tectorial membrane, causing
 330

stimulatory deflection of the apical ends of the hair

cells.

Clinical note: A cochlear implant is inserted into the scala

tympani, where it lies close to the organ of Corti and can
artificially stimulate axons of the auditory nerve. (more)

The organ of Corti is considerably more complex than this simple

account implies, with, among other things, two functionally
distinct classes of hair cells (inner and outer). Synapses from the
inner hair cells apparently supply most of the sensory information
that goes to the brain, while the outer hair cells (the ones which
are most readily recognized by light microscopy) have a curious
mechanical function (for more, see for example How the Ear
Works).

Image courtesy Alec Salt,

 331

ENDOLYMPH and Cochlear Fluids Laboratory,

PERILYMPH
Washington University; used
The membranous labyrinth is with permission.
filled with endolymph and
surrounded by perilymph.

Endolymph (blue, in image at right) is a unique fluid, with high

K+ concentration and very low Na+ concentration. This
endolymph provides the proper ionic environment for hair cell
function.

Endolymph is secreted by cells of the stria vascularis, along the

scala media of the cochlea. The stria vascularis resembles a
stratified cuboidal epithelium, but unlike any proper epithelium
(and as the name vascularis indicates) this tissue contains
capillaries among the cuboidal cells.

For additional detail, see Cochlear Fluids Lab at Wash. U.

Perilymph (orange, in image at right) is similar to ordinary

interstitial fluid. Perilymph fills the spaces of the bony labyrinth
surrounding the membranous labyrinth.

In the vestibular system (surrounding the saccule, utricle, and

semicircular canals), perilymph simply provides a cushioning
support for the membranous labyrinth.

In the cochlea, perilymph of the ascending scala vestibuli and the

descending scala tympani conveys pressure waves (sound) across
the scala media. Pressure waves flex the basilar membrane and
thereby stimulate hair cells of the organ of Corti.
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