The

Journal of Pathology
Vol. 117 No. 1

T H E PATHOLOGY OF ELASTASE-INDUCED PANACINAR

EMPHYSEMA IN HAMSTERS

J. A. HAYES,AGNESKORTHY L. SNIDER
AND GORDON
Mallory Institute of Pathology, Boston City Hospital, Boston, Mass. 02118
Department of Pathology and Medicine, Boston University School of Medicine,
Boston, Mass. 02118
Veterans Administration Hospital, Boston, Mass. 02130

PLATES
I-VI

HOMOZYGOUS alpha I antitrypsin deficiency is known to be associated with

the premature onset of severe panacinar-type emphysema which is widespread
throughout the lungs (Eriksson, 1965; Orell and Mazodier, 1972), although
probably more severe in the lower lobes (Greenberg et al., 1973). Gross et al.
(1964, 1965) developed an experimental model of lung damage simulating
emphysema by the intratracheal instillation of papain, a finding subsequently
confirmed by others (Goldring, Greenberg and Ratner, 1968; Johanson, Pierce
and Reynolds, 1971). Using different commercial types of papain we have
found that there is a marked variation in the severity of emphysema induced by
the different products, the severity being proportional to the ability of each type
of papain to induce dissolution of elastin from bovine ligamentum nuchae
(Snider et al., 1974). This finding suggested that emphysema might be produced
experimentally by exposing the lung to porcine pancreatic elastase, an enzyme of
well-defined chemical composition (Shotten and Hartley, 1973), which acts
predominantly, although not exclusively, on elastin (Mandl, 1961). Emphy-
sematous change has recently been demonstrated in hamsters by intratracheal
instillation of pancreatic elastase (Kaplan, Kuhn and Pierce, 1973). These
workers found that the emphysema-producing effect of elastase was abolished by
administering elastase mixed with normal serum (PiMM), but not when mixed
with serum deficient in alpha1 antitrypsin (PiZZ).
The present paper confirms that the intratracheal injection of elastase
produces a lesion with a striking resemblance to panacinar type emphysema
Received 17 Sept. 1974; Accepted 11 Oct. 1974
Correspondence to J. A. Hayes, Mallory Institute of Pathology, Boston City
Hospital, Boston, Massachusetts 021 18.
J. PATH.-VOL. 117 (1975) 1 A
2 J. A. HAYES, AGNES KORTHY AND GORDON L. SNIDER

(Ciba Symposium, 1959) and describes the pathology of the lesion, its relation to
the amount of elastase administered, its reproducibility and evolution to a
stable “ repaired ” state.
MATERIALSAND METHODS
All experiments were carried out on male golden hamsters (Cricetufusgriseus) weighing
between 90 and 130 g. Transoral intratracheal cannulation was performed using a fine metal
cannula under light anaesthesia produced by intraperitoneal injection with sodium metho-
hexital solution (Brevital SodiumR, Eli Lillie, Indianapolis), dosage 0.1 mg per 100 g body
weight.
Experiment I . Relation of elastase dosage to severity of emphysema and mortality
Hamsters were given intratracheal injections of crystalline, porcine pancreatic elastase
(pancreatico-peptidase (E.C.3.4.4.7) Whatman) with a specific activity of 26.8 units per mg.

TABLE I
Deaths occurring after injection of different concentrations of elastase

Elastase dosage-mg/lOO g body weight

Total
0.05 0.1 0.2 0.3 0.4 0.5
-----___-

I
Number of animals 10 16 120 5 7
injected
Deaths 0 0 2 1 4 27 34
56 214*
Percentage of 57 16
deaths
0 0 2 20
48 j
* The numbers include animals injected in other studies and not reported in this paper.
The dose of elastase varied between 0.05 mg to 0.5 mg per 100 g body weight and was admini-
stered as a solution in physiological saline at a volume of 0.5 ml per 100 g body weight. Two
control groups were used. The &st was given a comparable volume of intratracheal saline;
the second group was anaesthetised and the trachea cannulated, but no intratracheal injection
was made. All the animals were killed 16 days after elastase injection by an overdose of
pentobarbitol administered intraperitoneally.
The chest was opened and the aorta and venae cavae ligated, divided and the lungs
removed. The trachea was intubated and the lungs distended with 10 per cent. neutral,
phosphate-buffered formalin or paraformaldehyde-gluteraldehyde (Karnovsky, 1967) at a
pressure of 25 cm for 24 h. Sagittal sections from parahilar and lateral regions of both lungs
were embedded in paraffin. Histological sections were prepared at 5 pm, 10 pm and 40 p m
thickness and stained with hematoxylin and eosin, Foot’s reticulin, Verhoeff‘s elastic with
van Gieson’s stain, and the Prussian-blue reaction for hemosiderin. All sections were
examined for parenchymal abnormalities, particularly for emphysema, which was considered
to be present when there was a significant increase in alveolar diameter, usually accompanied
by alveolar wall thinning, diminished prominence of alveolar septa and abnormality of the
elastic fibers on Verhoeff’s stain.
These subjective judgments were substantiated by measurement of the mean linear
intercept (Dunnill, 1962), internal surface area (Weibel, 1962), and mean alveolar density
(Weibel), assessed in 20 randomly selected fields on each section. Measurements were made
on a representative number of animals from each group (table I). In each animal measure-
ments were made on two slides, one from the lateral, the other from the medial (parahilar)
 ELASTASE-IND UCED EMPHYSEMA 3

aspect of the left lung, and were made without knowledge of the prior subjective microscopical
assessment. The mean value and standard error was calculated for each group, and the
values compared with those from the controls.

Experiment 2. Early stages of elastase damage and repair

Hamsters were injected with 0.2 mg elastase per 100 g body weight, which was enough to
produce a lesion of moderate severity with minimal mortality, as indicated by the results
from Experiment 1. The exposed hamsters were killed at 4 and 8 hr, 1, 2,4, 8, and 16 days
after injection. The methods of anaesthesia, injection, preparation and morphometry were
identical with those described above.

Experiment 3. Late evolution of elastase damage

Hamsters were injected intratracheally with 0.2 mg elastase per 100 g body weight and
were killed 16, 45 and 90 days later. Preparation and morphometry were carried out as
described previously. In all experiments unexposed and saline-exposed hamsters were used as
controls.

Elastase dose, mg/lOOg body weight

FIG.1.-Mortality related to dose of elastase administered.

RESULTS
Experiment I . Relation of elastase dosage to mortality and lesion severity
There was an overall mortality of 15.4 per cent. irrespective of elastase
dosage. Mortality was clearly related to dosage because below 0.3 mg elastase
per 100 g body weight there were only two deaths, whereas at the 0.5 mg dose
4 J. A . HAYES, AGNES KORTHY AND GORDON L. SNIDER

mortality approached 50 per cent. (fig. 1). What this does not show is that as
our experience with intratracheal injection increased, the mortality fell, even
with higher doses. In particular, the points plotted for the 0.3 mg and 0.4 mg
doses represent only small numbers of animals in an early experiment (table I) in
which the mortality for the 0.5 mg level was approximately 80 per cent. With
repeated use of the 0.5 mg dose, however, the mortality dropped to about 50 per
cent., so the LD,, is probably a dose greater than 0.5 mg per 100 g body
TABLE I1
Mean linear intercept values for control and elastase-exposed hamsters

Mean linear intercept

Dose of elastase/ Number of “t” test Significance(P)
100 g body wt. animals
Group Standard
mean (cm) ~ error

Unexposed control
Saline control
6
6
0.00594
0.00591
04002
O~OoO1
} 0.1439* NS

Combined controls 12 040593 0.0003 ... ...

Elastase 0.05 mg 10 040730 04003 3.231t 0.01>P>OOol

0.10 mg 6 0.00996 O.Oo08 4.729 P< om1
0.20 mg 6 0.01386 0.0006 11.80 P<0.001
0.30 mg 4 0.01273 00094 13.61 P<0.001
0.40 mg 3 0.01340 0.0014 5.23 P < 0.001
0.50 mg 15 0.01447 0.0009 9.01 P<O.OOl

* Unexposed controls compared with saline-injected controls.

t Elastase-treatedcompared with combined control group.

weight, using Whatman purfied elastase. No further attempt was made to

define the mortality for the 0.3 mg and 0.4mg levels because the results show
that a dosage of 0.2 mg produces negligible mortality, yet yields a consistent
widespread and easily discernible lesion.
No subjective nor objective difference could be detected between the un-
exposed and saline-exposed animals (table I]), so that the measurements from
these two groups were combined as a single control group for statistical analysis.
Strikingly, on opening the pleural cavity, the majority of the lungs in elastase-
treated animals showed increased volume and failed to collapse as does the
normal hamster lung. This was mostly seen with the high dose of elastase.
Subpleural bullae were often visible to the naked eye. Microscopically there
was alveolar enlargement with thinning of the wall and increased shallowness of
individual alevoli. An intriguing feature was the widespread uniformity of the
 ELASTASE-IND UCED EMPHYSEMA 5

alveolar change which was invariably present at the 0.5 mg level, but was also
usually seen with the 0.2 mg/100 g dose level (fig. 2).

' 0 4 -2 *3 *4 .5
Elastase dose, mg/lOOg body weight

FIG.3.-Alveolar density in relation to dose of injected elastase. Each dot represents observations
on a single hamster. Mean values for different dose levels are joined by the continuous line.
f
o Mean value with standard error for control animals.
A

U
n
-0100
I
*
.-
L

c
e
a0050

1 0.05 1

01 0.2 0.3 04 05
Elastase dose, mg/l Wg body weight
FIG.4.-Mean linear intercept at differing elastase doses represented by dark line drawn through
points. Interrupted line represents mean control value.

These changes correlated closely with the measurements of mean linear

intercept (M.L.I.) which showed larger values with increasing dosage (table 11)
and all elastase dose levels showed a highly significant increase in M.L.I. when
compared with the control values. Coincident with increased alveolar size,
there was a prominent diminution of the mean alveolar count per unit area
(fig. 3). Plotting the values for M.L.I. at different dose levels gave a curve
6 J. A . HAYES, AGNES KORTHY AND GORDON L. SNIDER

showing that a dose of 0.2 mg produced major damage, larger doses producing
only marginally more severe damage (fig. 4). The internal surface area was
calculated using the mean linear intercept (Tomkieff, 1945) and the volume of
air in the lungs at a transpulmonary hydrostatic pressure of 25 cm as a measure
of total lung capacity. The mean internal surface area with standard error in 15
untreated hamsters was 0.34 (k0.017 m2) compared with a value of 0.25
(&0.008 m2) in eight hamsters treated with 0.2 mg elastase per 100 g body
weight ( t = 4.39, P<O.OOl). This represents about a 25 per cent. reduction in
internal surface area consequent to elastase exposure.

Experiment 2. Early stages of acute damage and repair

The pathology reported in this section is based on animals dying spontan-
eously, or after killing by barbiturate overdosage, the time intervals and numbers
TABLE 111
Deaths at early stages

Mode of
death
4 hr ' 8 hr 1 day
1 2 days 4 days
_ _ _ ~ _ _ _ _ _ _ _ _ _
6 days 8 days 16 days 1 Total

Spontaneous 25 3 ... 2 ... ...

Barbiturate
overdosage ... 5 5 30

1
Total 8 5 5 63

being shown in table 111. In hamsters dying spontaneously, the majority of

deaths occurred within 3 days of injection, most being within 24 hr. The lungs
of these animals were invariably heavy and red due to diffuse haemorrhage.
Microscopically there was patchy intra-alveolar haemorrhage which was
confluent in many instances. Often haemorrhage was seen in the adventitial
tissues of pulmonary vessels and bronchi. Perivascular oedema was invariably
present even in areas where haemorrhage was not conspicuous (fig. 5), but
intra-alveolar oedema was only seen focally. This haemorrhagic phase with
increased lung weight was transitory and disappeared by the 4th day after
injection, as shown in fig. 6. Lungs from hamsters dying spontaneously showed
more severe changes than those killed, but the process was essentially the same
in both groups.
By 4 hr the alveolar walls showed a mild infiltration of polymorphonuclear
leucocytes (PML) which increased to a maximum by 1 day. In hamsters dying
spontaneously there were focal areas showing alveolar wall necrosis and dense
PML infiltrate (fig. 7). The PML infiltrate decreased rapidly in the lungs of the
surviving animals and had disappeared by the 2nd day. However, it must be
emphasised that a PML infiltrate was a focal lesion and was not a major feature
 ELASTASE-IND UCED EMPHYSEMA 7

of the early changes. At 8 hr the lungs showed increasing numbers of macro-

phages, both about the bronchi and arteries and in alveolar walls, and these
reached a maximum by 24 hr. The interstitial infiltrate had virtually cleared by
the 4th day. However, intra-alveolar collections of macrophages persisted up to
the 16th day. The cytoplasm of alveolar macrophages gave a strongly positive
reaction for haemosiderin. Macrophages aggregated in groups in bronchioles
and some alveoli, although most alveoli were cell free (fig. 8) and resembled the
focal aggregations seen when blood is injected into normal animal lungs
(Margarey, 1951). Saline-injected animals did not show this phenonemon.
In two hamsters given the 0.5 mg dose of elastase and dying within 24 hr of
injection some alveoli showed structures resembling hyaline membranes.

4 8 24 4 8 16
hours days
Time after elastase exposure

FIG.6.-Mean lung weight at intervals following injection (elastase injection 0-0, saline in-
jection 0-0). Transitory weight increase affects only elastase-treatedhamsters.

The bronchial and bronchiolar walls showed similar infiltrates of PML in

which few or no eosinophils could be identified. This infiltrate cleared in the
same time-course as did the alveolar wall infiltrate. In the hamsters killed at
4 hr, and in the spontaneous deaths within 24 hr, the bronchial and bronchiolar
epithelium generally remained intact, although focal ulceration did occur
and was quite extensive in some early spontaneous deaths.
Animals dying spontaneously within the 1st day after injection showed
clear evidence of intra-arterial thrombosis in the main pulmonary artery and
its larger branches, none being seen in the smaller arteries or arterioles. The
thrombi showed a lamellar pattern of fibrin deposition and a smooth luminal
surface, without obvious endothelialisation. The deep portion of the thrombus
had a broad mural attachment (fig. 9). Elastic stains showed segmental necrosis
of the arterial wall underlying the thrombus with destruction of both inner and
outer elastic membranes and the muscle coat (fig. 10). In some instances only
the inner elastic lamina was destroyed. There was a variable amount of PML
infiltration at these sites, the infiltrate at times being minimal, at others marked.
Mural thrombosis was generally restricted to the area of underlying necrosis,
although several areas of segmental necrosis occurred without overlying
8 J. A . HAYES, AGNES KORTHY AND GORDON L. SNIDER

thrombi. Careful search revealed no evidence of pulmonary venous lesions of a

similar nature. Half of the 28 hamsters dying in the first 24 hr after injection
showed mural thrombosis. In hamsters surviving a 0.5 mg elastase dose,
60 per cent. showed thrombi in pulmonary arteries in the first 24 hr, but none was
seen after 4 days. A single focal arterial wall lesion was the only evidence
which suggested a late, healed lesion. This consisted of focal reduplication
and thickening of the inner elastic membrane in a hamster exposed to 0-5 mg
elastase and killed 16 days after injection (fig. 11).

Experiment 3. Late evolution of elastase damage

All animals exposed to a dose of 0 3 mg elastase showed widespread,
uniform emphysematous change of the panacinar type with little subjective

0 20 40 60 80 100
Days after exposure
FIG.12.-Mean linear intercept (fstandard error) in control hamsters and elastase-treated hamsters
at differing time intervals after injection ( $ control, $ elastase 0.5 mg/100 g body weight).

difference between lungs examined 16 and 90 days after injection of elastase.

The values for mean linear intercept in each group confirmed this interpretation
(fig. 12), but as the numbers are small these should be looked on as preliminary
results. The measurements showed a highly significant increase over the
control values (table IV), but the difference between the mean linear intercept at
16 and 90 days was not significant ( t = 1.338, P not significant).
Elastic stains on the lungs of elastase-exposed animals showed striking
alterations when compared with those of controls. In control animals alveolar
walls show moderate numbers of fine, crossed elastic fibrils while the pleura
shows a cascade-like pattern of fine, interwoven fibrils (fig. 13). In contrast,
elastic fibres in the alveoli of elastase-exposed hamsters are coarse, nodular
and few in number, while some appear to be ruptured, although this interpreta-
tion is difficult to substantiate. The changes are more readily appreciated in
the pleura where the cascade of fine fibrils is replaced by scanty coarse fibres
with a clumped and fragmented appearance (fig. 14).
HAYES,KORTHYAND SNIDER PLATEI
ELASTASE-INDUCED
EMPHYSEMA

Fig. 20.-Control hamster injected with saline alone. Haematoxylin and eosin (HE). x 12.

FIG.2b.-Hamster injected with 0.2 mg elastase per 100 g body weight. HE. x 12.
HAYES,KORTHYA N D SNIDER PLATEI1
ELASTASE-INDUCED
EMPHYSEMA

FIG.2c.-Control at higher magnification. HE. x 35.

FIG.2d.-Elastase-damaged magnified. HE. x 35.

HAYES,KORTHY
AN D SNIDER PLATE111

ELASTASt-INDUCED tMPHYSEh4A

FIG.5.-Lung of hamster dying spontaneously 24 h after 0.5 mg/100 g elastase injection showing
prominent perivascular oedema and alveolar haemorrhage. Only small numbers of PML are
present. HE. x100.

FIG.7.-Lung of hamsters dying 24 h after elastase injection showing alveolar haemorrhage and focal
necrosis of alveolar wall associated with P ML aggregation. HE. x 240.
HAYES,KORTHYAND SNIDER PLATE1V
ELASTASE-INDUCED
EMPHYSEMA

FIG.8.-Lung 16 days after 0.05 ing/lOO g elastase injection showing focal aggregations of haemo-
siderin containing macrophages. Prussian-blue reaction. x 180.

FIG.9.-Spontaneous death at 24 h. Thrombus in pulmonary artery. Note adjacent PML infiltrate

in wall. HE. x110.
HAYES, KORTHYAND SNIDER PLATEV
ELASTASE-INDUCED EMPHYSEMA

FIG.10.--Comparable lesion showing thrombus overlying zone i n arterial wall with focal absence of
inner and outer elastic laminae. Elastic and van Cieson. x 200.

FIG.11.-Sixteen days after elastase injection. Pulmonary artery shows segmental reduplication of
elastic lamina resulting in intimal thickening (small arrows). Underlying there is breaching of
both outer and inner elastic laminae (large arrow). EVC. Y 200.
HAYES,KORTHYAND SNIDER PLATEVI

ELASTASE-INDUCED
EMPHYSEMA

F I G.13. --Control hamster lung showing fine elastic fibres in pleura and alveolar walls. EVG. x 400.

FIG.14.-Elastase-cxposed hamster lung showing coarse granular pattern of elastic fibres in pleura
and adjacent alveolar wall. EVG. x 400.
 ELASTASE-IND UCED EMPHYSEMA 9

DISCUSSION
These experiments demonstrate a striking difference between the lungs of
elastase-exposed animals and control groups, a conclusion supported both by
the illustrations and by the measured indices. The widespread structural
change is accompanied by physiological abnormalities in the anaesthetised
living animal, similar to those seen in human emphysema (Koo et al., 1973,
1974). Our studies showed that the quasi-static deflation volume-pressure
curve is shifted upward and to the left with an increase in compliance measured
just above the total lung capacity. The functional residual capacity and lung
volume at 25 cm water transpulmonary pressure (analogous to total lung
capacity in humans) are markedly increased (fig. 15). These physiological and
structural changes fulfil criteria which have been suggested for the identifica-
tion of emphysema in experimental animals (Wright, 1968).

TABLE
IV
Mean linear intercept at later stages of elastase damage

M.L.I. (cmx 105)

Time of Number of 6Gt,, test Comparison with
killing animals controls (P)
Mean SE

16 days 5 0.01335 04008 8.983 <0.001

45 days 4 0.01458 04006 12.91 <0~001
90 days 4 0.01575 04016 6.082 < 0.001
Controls 12 0.00593 04003 ... ...

The evolution of the lesion is relatively rapid, the stable, " healed " state
being established within a few days, thus making identification of the initial
site and nature of the injury difficult. There is an initial, transitory increase in
lung weight, pulmonary hemorrhage and mild polymorphonuclear infiltration
in alveolar walls which contrasts markedly with the prominent PML infiltrate
seen in papain-induced emphysema (Goldring et al., 1972). The finding
suggests that there is some underlying tissue destruction. However, focal
destruction was only seen in animals dying spontaneously from large doses of
elastase. It is not clear whether the initial haemorrhagic changes represent
tissue injury associated with cell death, or whether they simply result from a
transitory phase of increased vascular permeability not associated with cell
death. So far, our data do not help resolve this question.
An important aspect of the mechanism of elastase damage lies in the
possibility that human emphysema may result from the liberation of enzymes
from damaged cells within alveoli, as in patients with homozygous alpha I
antitrypsin deficiency. Proteolytic enzymes are present within PML (Janoff and
Scherer, 1968) and alveolar macrophages (Janoff, 1972), and it has been shown
10 J. A . HAYES, AGNES KORTHY AND GORDON L. SNIDER
that lung destruction resembling human emphysema can be produced in dogs by
aerosols of enzymes extracted from dog and human PML and from dog
alveolar macrophages (Mass et al., 1972). Recently, the same group of workers
has demonstrated that washings from normal lungs have antiprotease activity
which can be neutralised by excess enzyme (Weinbaum et aE., 1974a). Conse-
quently the minor PML exudate seen both in the observations reported here
and in reports by others (Weinbaum et al., 1974b) does not seem to adequately
account for the severe, widespread changes produced. These considerations
suggest that the lesion is directly due to the introduction of the elastase solution
into the lung rather than from proteases liberated from cells evoked as part of
an inflammatory reaction to the injection.
Lung volumes and volume-pressure diagrams in 15 untreated
and 8 elastase treated hamsters

PL (cm H@)
FIG.15.-Quasi-static deflation volume-pressure curves from control and elastase-exposed hamsters.
Elastase damage shifts the curve upward and to the left. This is associated with a marked in-
crease in functional residual capacity and lung volume at 25 cm water transpulmonary pressure.

The damage produced in the rat lung by elastase in vitro appears to affect
mainly the elastic framework (Johanson and Pierce, 1972). Morphologically,
it is difficult to determine how and where elastic fibres are damaged because
alveolar shape and size are so markedly altered. However, damage to elastic
fibres in the pleura probably occurs because the interweaving cascades of fine
fibrils are replaced by thick, lumpy fibres often with knobs at their ends. It
is reasonable to infer that elastic fibres are similarly damaged throughout the
lungs, a conclusion supported by the physiological abnormalities mentioned
above (fig. 15). The changes in elastic fibres have not yet been studied by
electron microscopy, but in papain-induced emphysema, which has some
 ELASTASE-IND UCED EMPHYSEMA 11

features of the elastase lesion, damage was thought to be restricted to elastic

fibres (Johanson et aZ., 1973). Johanson considered that the elastic fibres were
disrupted but not removed. This interpretation agrees with the observations
reported here and also with the minimal alterations detected in the total elastin
content, and elastin-collagen ratio of aging and emphysematous human lungs
(Pierce et aZ., 1961; Pierce and Ebert, 1965). In other words, the elastic fibres
could have undergone marked physiological impairment without a significant
loss in total lung content of chemically identifiable elastin.
It is extremely difficult to produce lung damage by intravenous administra-
tion of elastase (Weinbaum et al., 1974). These workers describe no pulmonary
arterial lesions similar to those noted in this paper, even after pulmonary
arterial infusion of elastase. The only record of vascular damage in enzyme-
induced emphysema is a fleeting reference to disrupted and beaded " elastic
fibres in the walls of arterioles, venules and bronchioles " within a few hours of
exposure to papain aerosol (Johanson et al., 1973). Presumably this is due to
the relatively large amounts of antiprotease activity present in the circulating
blood.
The mechanism of the arterial damage describe here is puzzling. If elastase
is directly absorbed across the alveolar walls into the capillaries, lesions might be
expected in pulmonary veins, but these were not seen. Moreover, no micro-
scopic evidence of acute capillary destruction was seen in the first 24 hr, the
lesions being essentially arterial in location. Absorption of elastase followed by
systemic circulation seems unlikely in view of the findings by Weinbaum and
his colleagues mentioned above. Transmural absorption with direct damage to
the arterial wall seems most unlikely because the arteries affected have thick
media covered by an appreciable layer of adventitia and interstitial connective
tissue which would present a distinct anatomic barrier. Furthermore, in some
lesions the artery showed breaching only of the internal elastic lamina which
suggests that injury is originating from within the arterial lumen. This, of
course, assumes that the internal elastic lamina and external elastic lamina are
equally susceptible to elastase digestion. Lymphatic transportation of elastase
to produce injury is unlikely because absorption would be predominantly
perivascular and so damage might be expected to affect the external rather than
the internal elastic lamina. Theoretically it might be explained by distribution
via vasa vasorum except these do not appear to be present in the medium-sized
pulmonary arteries of the hamster. Arterial thrombosis including pulmonary
arterial involvement has been reported in dogs and rabbits after intravenous
injection of dilute trypsin solutions (Tagnon, 1945; Taylor and Wright, 1954).
It is thought that the trypsin promotes the conversion of prothrombin to
thrombin with subsequent thrombus formation. Neither paper mentioned
damage to the arterial wall. A recent publication has noted that in man
pulmonary thromboemboli (Salyer et al., 1974) produce damage of the under-
lying arterial wall which includes damage to and even rupture of the elastic
lamina in almost 20 per cent. of the cases studied. In theory, systemic circula-
tion of elastase could produce systemic venous thrombosis and subsequent
embolism leading to arterial damage. We think this is also an unlikely
12 J . A . HAYES, AGNES KORTHY AND GORDON L. SNIDER

mechanism. The presence of the single " healed " arterial lesion at 16 days
after injection is intriguing. Morphologically it resembles changes seen in
elderly human lungs, particularly in persons suffering from chronic obstructive
lung disease (Mclean, 1958; Hayes, 1968). Acute pulmonary arterial lesions
with thrombosis have not been described in human emphysema, although they
may occur in septicemic processes (Rabin et al., 1961) or polyarteritis nodosa
(Symmers, 1952). The relationship of these arterial lesions to human emphy-
sema is obscure.
Two additional points deserve mention. Firstly, the lesion with the 0.5 mg
dose in our hands is invariably uniform throughout the lung, whereas papain
damage tends to involve entire segments served by a separate bronchi, the
intervening lung being uninvolved. This may in part be due to the dose
administered because the 0.2 mg dose level shows many examples of a segmental
lesion. However, this is not the entire explanation because papain rarely
produces a diffuse lesion even when the LD50 is approached (Snider et al., 1974).
Secondly, our data does not clearly establish whether or not the panacinar
lesion becomes more severe as the interval following the initial injury increases.
Although there is no statistically significant difference in the M.L.l. between
16 days and 90 days after damage (table IV), the points in the diagram (fig. 14)
do suggest that the alveoli of the damaged lung become bigger with increasing
age. However, as the numbers are small, further study is necessary to clarify
this point.
It is very difficult to assess the presence of parenchymal destruction histo-
logically, particularly in lungs showing increased alveolar size. It has been
suggested that the loss of " spurs " at the mouth of alveoli, and increase in
alvoelar diameter indicate tissue destruction (Gross et al., 1964). We have
found it difficult to interpret such changes. However, the present experiment
shows that there is a decrease in internal surface area which is consistent with
alveolar loss.
The study reported here shows that the intratracheal injection of elastase
provides an animal model with some of the characteristic anatomical and
physiological features of the uncomplicatedpanacinar type of emphysema seen in
man (Ciba Symposium, 1959). The model is useful for studying the possible
structural changes and pathogenesis of human emphysema and for assessing
the impact of such factors as infection, smoking amd inhalation of toxic matter
of various kinds.
SUMMARY
A single dose of crystalline, porcine pancreatic elastase injected intra-
tracheally into hamsters induces widespread alveolar enlargement with sub-
pleural bullae. A uniformly severe lesion is consistently induced by 0.2 mg
elastase per 100 g body weight and with negligible mortality. Compared with
controls, which showed no lesion, elastase-damaged lungs show a highly signifi-
cant (P ~0.001)increase in alveolar size and a decrease in internal surface area.
Taken with the associated physiological abnormalities, these findings closely
simulate human emphysema of the panlobular (panacinar) type. Histologically
 ELASTASE-IND UCED EMPHYSEMA 13

it appears that elastase converts the fine elastic fibres in alveolar walls and pleura
into thickened, nodular fibres which may also be broken along their length.
With higher doses of elastase, i.e., 0.5 mg/100 g body weight, many pulmonary
arteries showed segmental loss of inner and outer elastic laminae, usually with
thrombosis on the overlying endothelium. The mechanism of this thrombosis
is unclear.
These experiments suggest that damage to elastic fibres may be an important
element in the development of human panacinar emphysema, and that the
damage could be one pathogenetic mechanism which produces damage of
elastic fibres.
We wish to express our thanks to Olva Delmano, Bradford Milne, Eveline Gillette and
Frank Harris for their technical assistance in preparing this paper, and Claire Costello for
secreterial assistance.
The study was supported by Grants HL15563 (NIH) and MRIS 8019 (VA).

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