Functional study of Anorectal Malformations

Shilpa Pandey e t a l

Research article
A FUNCTIONAL STUDY OF NEONATAL RECTUM IN ANORECTAL
MALFORMATIONS
Shilpa Pandey1, M.B. Mandal2, A.N. Gangopadhyay3
1.
2.
3.

Resident, Department of Physiology,Institute of Medical Sciences,Banaras Hindu University,Varanasi, India.

Professor, Department of Physiology,Institute of Medical Sciences,Banaras Hindu University,Varanasi, India
Professor, Department of Pediatric Surgery,Institute of Medical Sciences,Banaras Hindu University,Varanasi,
India

ABSTRACT
Objectives: The present study was undertaken to assess the contractile status of proximal & distal segments of high
type of anorectal malformations (ARM) from neonates, for better understanding of colorectal motility problems in
these patients. Methods: Circular muscle strips were prepared from freshly excised specimens of high ARM (n= 17)
obtained directly from paediatric surgery operation theatre. The in vitro contractile activity of the tissue was assessed
initially without any chemical interventions and thereafter with application of acetylcholine, histamine and their
antagonists as well as hexamethonium (ganglion blocker) in an organ bath filled with physiological solution. The
isometric contractile activity was recorded with the help of force transducer and computerised data acquisition
system. Result: Spontaneous contractions were recorded only in the proximal segment of 18 % of ARM cases.
Histamine and acetylcholine (0.1-100 M) evoked contractions were significantly (p < 0.05) greater in proximal
segment as compare to distal segment. As compared to acetylcholine, histamine produced stronger contractions.
Responses of histamine were significantly (p < 0.05) blocked by pre-application of H 1 blocker pheniramine (90%)
and hexamethonium (50%).

Pre-application of atropine significantly (p < 0.05) blocked (85%) acetylcholine

responses where as hexamethonium enhanced the response. Conclusion: Largely absence of spontaneous activity
in ARM cases indicated that the tissue is functionally abnormal. Further, as compared to the proximal segment, the
distal segment of ARM was badly affected as evidenced by severely impaired contractility in this segment. Thus, the
result of this study may help formulation of better surgical management strategies.
Keywords: Anorectal Malformation, Contractile responses, Invitro study, Neonate; Rectum

Int j clin surg adv 2014; 2(2):9-18

Functional study of Anorectal Malformations

Shilpa Pandey e t a l

INTRODUCTION

aimed to explore the functional aspect by recording the

Anorectal malformation (ARM) is common congenital

spontaneous and chemically evoked contractions of

problem in neonates in which the rectum ends either in

muscle strips from proximal and distal segment of

a blind pouch that does not connect with the anus or

ARM using in vitro preparations. Thus, this study may

may open in the urethra, bladder, base of the penis

help understanding the functional alterations in

scrotum in male and in vagina in girls by forming

anorectal malformation and the mechanisms involved,

fistula 1.

and thereby may provide a guideline for formulating

Management of this condition is surgical. Despite the

better surgical management strategies.

development of several surgical techniques aimed for

MATERIALS & METHODS

improved outcome, the result remains to be fully

Collection of Samples

satisfactory due to appearance of motility related

The study was carried out on the excised specimen

disorders like constipation; incontinence and soiling as

from 17 neonatal patients of anorectal malformation.

common sequelae after surgical repair of ARM it still2.

The specimens were collected immediately after

Histological

anorectal

excision from the Department of Pediatric Surgery

malformation showed the immaturity of the enteric

operation theater B.H.U., Varanasi in a wide mouth

nervous system

studies

available

on

and absence or reduced number of

bottle containing pre- oxygenated ice-cold (4C-6C)

interstitial cells of Cajal 4. It also has been seen that the

Krebs-Ringer

pelvic floor and the smooth muscle of the terminal

composition (in mmol/L): NaCl, 119; KCl, 4.7;

solution

having

the

following

rectum in ARM remained maldeveloped . Further,

CaCl2.2H2O, 2.5; KH2PO4, 1.2; MgSO4.7H2O,

studies on fetal rats showed that there were abnormal

1.2;NaHCO3, 5; and glucose, 11 . They were quickly

innervations of neural plexus in anorectum in ARM 6.

transferred to the laboratory in the Department of

Although these histological had thrown some light

Physiology for contractile studies.

on pathophysiology of ARM, the functional status of

All the experiments were conducted as per the

the condition remains to be determined. Earlier, in this

guidelines laid down by the ethical committee of the

laboratory, the functional studies on pouch colon also

Institute

demonstrated alteration in contractility of colonic

University.

of

medical

sciences,

Banaras

Hindu

smooth muscle 7. Therefore, the present study was

Int j clin surg adv 2014; 2(2):9-18

10

Functional study of Anorectal Malformations

Shilpa Pandey e t a l

Dissection and Preparation of muscle strips

glass tube ends). The parts of tissue which were lying

The excised specimens were transferred to a petri dish

beyond the point of attachment were not involved in

containing ice-cold (4C-6C) Krebs-Ringer solution

contractions and therefore, were removed. The rest of

continuously bubbled with 100% O 2 . Each specimen

the muscle strip was then lightly soaked on a bloating

was thoroughly cleaned with freshly prepared cold

paper to remove extra water and the weight of the strip

Ringer solution. After removing the adventitious layer,

was recorded to express the response in g/g of wet

2 to 3 mm wide and 15 to 20 mm long, circular

tissue.

muscular strips were prepared from proximal and

Drugs and solution

distal end of samples of ARM.

Aqueous solutions of Acetylcholine Chloride (Ach),
Mounting and recording of contractile responses

Histamine

(Hist),

Atropine

sulphate

(Atrp),

The muscle strips were mounted in Krebs-Ringer filled

Hexamethonium (Hxm; Sd-Fine Chemicals, Mumbai

organ bath (15ml) at 30 2 C and continuously

india) and Pheniramine maleate (Phn; Aventis pharma

bubbled with 100 % O 2. One end of the strip was tied

limited) were used for contractile study. The stock

by thread with curved end of the oxygen tube and the

solutions of these chemicals were prepared with

other end with fine force transducer (MLT 0210, AD

distilled water in the strength of 10 mmol/L. Final

Instrument, Australia). The tissue was left for 30-45

strength (0.1-100 M) were made by diluting with

min for equilibration after application of Initial tension

Krebs Ringer solution just before the experimentation.

of 0.5 gm on muscle strip. Recording of Isometric

Statistical analysis

muscle contractions were made through Bridge

amplifier and displayed onto personal computer with
the help of Power lab data acquisition system and
software CHART-5 for windows (AD Instruments,
Australia).

The amplitude of contractions were converted to

tension (gram) with the help of the Chart-5 software
and then the tension so developed was expressed as
tension per unit mass (g/g wet tissue) using the tissue
weight determined at the end of the experiments. The

Once the preparation was stabilzed, recordings were

values were then pooled to calculate mean SEM.

made to observe the spontaneous and chemically

The statistical significance of differences between

evoked contractions. After the recording, the strips

Mean values was determined by using paired or

were removed from their ends (force transducer and

Int j clin surg adv 2014; 2(2):9-18

11

Functional study of Anorectal Malformations

Shilpa Pandey e t a l

unpaired t-test and two-way ANOVA as applicable. P

value < 0.05 was considered significant.

Characteristics of Histamine induced contractions

Contractions induced by Hist at different conc. (0.1-

OBSERVATION AND RESULTS

100 M) were studied in proximal and distal segments

Contractile study was carried out with a total of 50

of ARM (n=6). Proximal and distal segments of all

rectal muscle strips (Hist=12, Ach=8, Hxm=30)

samples showed responses to different conc. of Hist.

obtained from 17 ARM cases to evaluate spontaneous

There was dose dependent increase in response to Hist

as well as chemically evoked contractions.

in proximal and distal segments.

Responses of

different conc. of Hist on proximal segment were

Spontaneous contractions in ARM Cases

higher as compare to distal segment. The difference of

Weak spontaneous contractions (without application of
responses of Hist was statistically significant (*P <
chemical) were observed only in proximal segment of
0.05, two way ANOVA; Fig 2).
2 cases of ARM. None of the distal segment showed
any spontaneous contractions.
spontaneous

contractions

were

In five

samples

induced

after

Fig 2: Representative recordings of (A) proximal (B)

distal segments of ARM before and after applying
different concentrations (0.1, 1, 10 & 100 M) of

application of different concentrations (0.1-100 M)

Hist. Arrows indicate the point of application of

of Hist. The required conc. of Hist for such drug

drug. Vertical & horizontal calibrations represent

induced spontaneous contractions was higher in distal

the tension (g) and time (min) respectively. Note the

presence of spontaneous contractions appearing in

segment. Another four samples showed Hist evoked

the proximal segment after application of Hist

spontaneous contractions only in proximal segment.

(Histamine). (C) Comparison of dose-response

(Fig 1)

curve of proximal and distal segments of ARM.

Data points represent Mean SEM values of Hist
evoked

contractions

(g/g)

with

the

different

concentrations of Hist. Responses were significantly

Fig 1: Recording of feeble spontaneous contractions

different (*P < 0.05, two way ANOVA, n= 6).

seen in proximal segment of ARM in one of the

sample.

Int j clin surg adv 2014; 2(2):9-18

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Functional study of Anorectal Malformations

Characteristics

of

response

produced

Shilpa Pandey e t a l

by

Acetylcholine

Responses induced by Ach at different conc. (0.1-100

M) were studied in Proximal and distal segment of
ARM (n=4).There was dose dependent increase in
response to Ach in proximal and distal segments.
Responses of different conc. of Ach on proximal
segment were higher as compare to distal segments.
Comparison of responses produced by
The difference of responses of Ach was statistically
Histamine and Acetylcholine
significant (*P < 0.05, two way ANOVA; Fig 3).
Responses produced by Hist were significantly (*P <
Fig 3: Representative recordings of (A) proximal
segment (B) distal segments of ARM before and
after applying different concentrations (0.1, 1, 10 &
100 M) of Ach (Acetylcholine). Arrows indicate the

0.05, two way ANOVA) greater than responses

produced by equimolar conc. of Ach in both proximal
and distal segments of ARM. EC-50 for Hist in both

point of application of drug. Vertical & horizontal

calibrations represent the tension (g) and time
(min.) respectively. (C) Dose response curve of
proximal and distal segments of ARM. Mean SEM

proximal (6.31M) and distal (17.44 M) segment was

lower

than

the

EC-50

for

Ach

(proximal

segments=28.73 M, distal segment=31.04 M).

values of Ach induced contractions (g/g) with the

different concentrations of Ach. Responses were
significantly different (*P<0.05 twoway ANOVA, n=
4).

Int j clin surg adv 2014; 2(2):9-18

Histamine-induced contractions were blocked by

pheniramine maleate (H 1 receptor blocker)

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Functional study of Anorectal Malformations

Shilpa Pandey e t a l

Responses of Hist (100 M) were significantly (P <

0.05, paired t test, n=6) blocked after application of
Phn (100 M) in both proximal and distal segments.
The blockade of responses in proximal and distal
segments was 90 % and 85 % respectively after
application of Phn (Fig 4).

Fig 4: Original recordings of effect of Phn

(Pheniramine)

on

Hist

(Histamine)

Acetylcholine induced contractions were blocked by

induced

contractions in (A) Proximal (B) distal segments of

atropine

ARM. Arrows indicate the point of application of

drug. Vertical & horizontal calibrations represent

Responses of Ach (100 M) were significantly (P <

the tension (g) and time (min.) respectively. (C)

0.05, paired t test, n=4) blocked after application of

Histogram showing Mean SEM values of % of

the initial responses to Hist 100 M before and

Atrp (100 M) in proximal as well as in the distal

after applications of Phn. Responses of Hist after

segments. In proximal segments, Ach produced 12 %

Phn were significantly decreased (*P < 0.05, paired

of initial responses (88 % blockade) after application

t test , n=7).
of Atrp. Where as in the distal segment 28% of initial
responses (72 % blockade) were produced (Fig 5).

Hexamethonium (ganglion blocker) pre-treatment

blocked histamine induced contractions

Hxm blocked nearly 55 % of Hist induced responses in

proximal & distal segments and was statistically
significantly (P <0.05, paired t test, n=7; Fig 6). On the
contrary Ach induced responses were enhanced by 49
% & 20% in proximal and distal segments respectively
but was not statistically significant (P 0.05, paired t
test, n=6; Fig 7).

Int j clin surg adv 2014; 2(2):9-18

14

Functional study of Anorectal Malformations

Fig 5: Representative recordings to show the effect

of Atrp (Atropine) on Ach induced contractions in

Shilpa Pandey e t a l

Fig 6: Original recordings of effect of Hxm

(Hexamethonium) on Hist induced contractions in

(A) Proximal (B) distal segments of ARM. Arrows

indicate the point of application of drug. Vertical &
horizontal calibrations represent the tension (g)
and time (min.) respectively. (C) Histogram

(A) Proximal (B) distal segments of ARM. Arrows

indicate the point of application of drug. Vertical &
horizontal calibrations represent the tension (g)

showing Mean SEM values of % of the initial

responses to Ach 100 M before and after
application of Atrp in proximal and distal segments
of ARM. Responses of Ach after Atrp were

and time (min.) respectively.(C) Histogram showing

Mean SEM values of % of the initial response to
Hist 100 M before and after application of Hxm.

significantly decreased (*P < 0.05, paired t test,

n=4).

Responses of Hist after Hxm were significantly

decreased (*P < 0.05, paired t test, n=7).

Int j clin surg adv 2014; 2(2):9-18

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Functional study of Anorectal Malformations

Shilpa Pandey e t a l

Fig 7: Histogram showing Mean SEM values of

% of the initial responses to Ach 100 M in

(devoid

of

demonstrated

spontaneous
spontaneous

contractions)
contractions

samples
after

proximal and distal segments before and after

applications of Hxm (Hexamethonium). Responses

application of Hist and not with Ach. This indicated

of Ach after Hxm were insignificantly increased (P

that the ICC may get activated only by an agonist like

0.05, paired t test).

Hist suggesting a differential effect of Hist and Ach on

ICC. Dose of Hist required to stimulate ICC were
higher in distal segment as compared that of proximal
segment.

On examination of Hist and Ach induced contractile

strength of proximal and distal segment, it was noted
that there was dose dependent increase in the response
.These agonistic effect is known to be mediated by
DISCUSSION

their actions on muscarinic and H 1 receptors for Ach

In the present investigation the contractility

and Hist respectively. In this study also it was

of rectal smooth muscle was evaluated by recording

observed that Atrp (muscarinic blocker) and Phn (H 1

spontaneous as well as chemically (Ach and Hist)

blocker) could largely block the response.

evoked

contractions

from

proximal

and

distal

segments of rectal tissue of high type of ARM to

Rectal tissue of high type of ARM

understand the severity and extent of contractile

demonstrated higher contractility in proximal segment

impairment.

for both Hist as well as Ach. Since ICC was only

In the proximal segment, only 2 out of 17 cases

(nearly 18%) of ARM and none of the distal segments
showed spontaneous contractions. The origin of
spontaneous contraction is believed to be related to
activity of interstitial cells of Cajal (ICC) 8. Thus the
absence of spontaneous contractions in these tissues
indicated abnormal functioning of ICC. Five silent

Int j clin surg adv 2014; 2(2):9-18

activated by an agonist like Hist. therefore it is

possible that the higher response seen in proximal
segment may not be due to the stimulant effect of Hist
on ICC. Abnormal functioning of receptor or
dysfunction of signalling molecules at intracellular
level cannot be ruled out for decrease contractility in
distal segment. For example, in Irritable Bowel

16

Functional study of Anorectal Malformations

Shilpa Pandey e t a l

Syndrome (IBS) with diarrhea, serotonin signalling

of Ach in the tissue pretreated with Hxm. This

path was reported to be altered 9. The Hist induced

phenomenon suggested role of inhibitory transmitter that

contractions were greater than that of Ach in

might be relasing through ganglion.

equimolar conc. which may be due to the stimulant

Thus, the present investigation demonstrated that high

action

suggesting

type of ARM the rectal tissue is excitable by

contribution of spontaneous contractions also in some

Histamine and Acetylcholine. However, the tissue

samples. The higher potency of histamine to induce

appeared to be functionally abnormal on account of

greater contractions is also evidenced by the EC-50 in

absence of spontaneous contractions in most of the

proximal (Hist = 6.31 M, Ach = 28.73 M) and distal

cases, with severe impairment of contractility in the

segments of ARM (Hist = 17.44 M, Ach = 31.04

distal

M). Such higher responses to Hist were also

implications in surgical management of ARM problem

observed in neonatal colonic tissue in earlier studies 10.

in neonate, pertaining to the most controversial issue

Thus, it is possible that large gut is more responsive to

of preserving or excising the distal most part of the

Hist. as compared to Ach in neonate. However, such

rectal tissue of high type of ARM.

of

Hist

on

ICC,

therefore,

segment.

These

observations

may

have

differential contractile response to Hist and Ach were

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CORRESPONDENCE
Dr.Shilpa Pandey
Junior Resident,Department Of Physiology
Institute Of Medical Sciences,Banaras Hindu
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Int j clin surg adv 2014; 2(2):9-18

acetylcholine

and

18