International Journal of Surgery 8 (2010) 639e642

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International Journal of Surgery

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Effect of bovine amniotic uid on intra-abdominal adhesion in male rats

Abolghasem Esmaeili a, *, Behnam Abbasian b, Hamidreza Kazemini b, Shahriyar Adibi c
a
Cell, Molecular & Developmental Division, Department of Biology, Faculty of Sciences, University of Isfahan, Hezar Jarib, Isfahan 8174673441, Iran
b
Graduated from Faculty of Veterinary Medicine, Shahrekord Azad University, Shahrekord, Iran
c
Torabinejad Dental Research Centre, Dental Faculty, The University of Isfahan Medical Sciences, Isfahan, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Background: A wide variety of treatments have been proposed in order to deal with prevention of
Received 4 April 2010 postoperative adhesions formation, but, no denitive results have been achieved. In the present study,
Received in revised form bovine amniotic uid (BAF) has been investigated as a possible option.
8 July 2010
Materials and methods: 84 male Wistar rats were undergone a laparotomy. After 2 weeks, adhesions were
Accepted 26 July 2010
Available online 14 August 2010
scaled grossly. Bovine amniotic uid (in whole combination, without cells or without cells and proteins)
extracted from cows carried either male or female calves was then applied to treated groups 2 weeks
later during the second laparotomy. Adhesions were rescored 2 weeks later during a third laparotomy.
Keywords:
Adhesions
Results and conclusion: A signicant reduction (P < 0.05) in adhesions formation was observed only in rats
Bovine amniotic uid treated with male bovine amniotic uid without cells and proteins. Therefore, BAF could be used in
Rat treatment of adhesion formation because it is inexpensive, readily available, and has minimal side effects.
2010 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

1. Introduction are killed in slaughterhouses. Therefore, bovine amniotic uid

Postoperative adhesion formation is an important clinical problem
in every surgical speciality. In particular, following almost every intra-
abdominal surgical procedure, adhesions are often formed. Bowel
obstruction and/or chronic pelvic pain are common complications,
which result from a formation of adhesions. Furthermore, several
studies have reported the occurrence of postsurgical intra-abdominal
adhesions in the majority of women undergoing gynaecological
surgery.1,2
Various compounds and/or techniques for adhesion prevention
have been proposed, but unfortunately an efcient solution has not
yet been achieved.3 Studies on most of these treatments have
produced inconsistent results. The application of various uids to
the peritoneum is one of the most frequent methods to have been
used. In an attempt to reduce the inammatory reaction at the site of
the peritoneal trauma, corticosteroids have been used and subse-
quently, the antiadhesive properties of calcium channel blockers
and antihistamines were studied. More recent studies deal with the
use of nonsteroidal anti-inammatory drugs (NSAIDs), tissue plas-
minogen activator (tPA) and selective immunosuppressors. Varying
results with all of these treatments have been reported.4e6
One of the modalities that have not been studied widely is the
use of amniotic uid. Each year many unrecognized pregnant cows
* Corresponding author. Tel.: 98 3117932490; fax: 98 3117932456.
E-mail addresses: abesmaeili@yahoo.com, aesmaeili@biol.ui.ac.ir (A. Esmaeili).
(BAF) could be inexpensive, readily available material in this issue.
Moreover, cows have a well-developed allantoises cavity and
a large amount of amniotic uid, so they could be a useful source of
amniotic uid for use in various aspects of human diseases.
Meanwhile, the characteristics of amniotic uid environment may
have some roles in the scar-free healing of the foetus.7 It could be
due to amniotic uid factors such as hyaluronic acid and growth
factors.8 Therefore, BAF may have positive effect on prevention of
intra-abdominal adhesion.9
We have previously reported that BAF could be effective in the
treatment of adhesion formation during diabetes.10 Bovine amni-
otic uid constitutes of proteins, minerals and cells, the relative
amounts of which may change during pregnancy.11 The purpose of
the present study was to evaluate the efcacy of bovine amniotic
uid with and without proteins, minerals and cells in the treatment
of postsurgical adhesion formation in male rats.
2. Methods
2.1. Animals
All experiments were performed on 84 almost one-year-old Wistar male rats,
weighing 200e250 g, in accordance with the Animal Ethics Committee for the
Isfahan Medical University. All groups of rats were allowed to equilibrate in standard
conditioned animal houses at the Isfahan Medical University for one week before
use.10 The 84 rats were assigned to 7 different groups randomly before the procedure
was initiated; a control group and six treated groups (each consisting of 12 rats).
Microchips implant system was used for identication of individual rats.

1743-9191/$ e see front matter 2010 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijsu.2010.07.301
640 A. Esmaeili et al. / International Journal of Surgery 8 (2010) 639e642

2.2. Preparing BAF
Bovine Amniotic Fluid (BAF) was obtained under aseptic conditions from healthy
pregnant cows carried out either male or female calves brought to a slaughterhouse
postmortem at ve month of gestational age after governmental inspection of each
animal. These animals were healthy and not detected pregnant due to the lack of
recognition of pregnancy. To prepare BAF without cells, the uid was centrifuged at
3000 rpm for 15 min. BAF was heated in a water bath for 30 min at 70  C to denature
proteins and centrifuged at 3000 rpm for 15 min to prepared BAF without cells and
proteins. The supernatant then was preserved at 20  C. To be sure there were no
cells and proteins in samples, smears were stained by a standard Giemsa method
and electrophoresis carried out respectively. The samples were kept frozen until
immediately before application and then stored at 4  C during the study in an effort
to minimize potential bacterial proliferation in the samples.
2.3. Surgical techniques
The rats were anaesthetized with a combination of isouran and nitrous oxide
inhalation, after which the abdomen was shaved and swabbed with povidine iodine
in preparation for surgery; the surgical procedures were performed under aseptic,
but not sterile, conditions.10 All surgical procedures were blind and performed by
the same surgeon using the same techniques.
This model consisted of excision of a defect of 3 cm in white line. Then, three
2 cm abrasions were made on one side of anterior abdominal wall and 2 cm of
another side was cut off. Manipulation of other tissues was minimized and care was
taken to avoid gross bleeding from the injured sites. At the end of the procedure, the
abdomen was closed in double layer in a continuous method (muscle and perito-
neum with 3e0 catgut suture, and skin with 3e0 silk suture). All rats were housed
under controlled temperature (25  5  C) and 12/12 light/dark cycles with food and
water.
2.4. Adhesions scoring and bovine amniotic uid application
The adhesion model used in this study is a highly reproducible and semi
quantitative rat model for the evaluation of therapeutic modalities used for the
treatment of postoperative adhesion formation, and was developed specically for
this purpose.10,12
A surgical injury was performed during rst laparotomy. After 2 weeks, a second
laparotomy was performed on each rat and adhesions were graded and documented
macroscopically on a scale of 0e4 (rst scoring) based on Canbazs scoring method
(Table 1). All adhesions were scored in a blinded manner by the same researcher.
Three ml bovine amniotic uid (BAF) was then applied to the injury in each rat. Two
groups of rats were treated with male and female BAF, another two groups with
male and female BAF without cells and two groups treated with male and female
BAF without cells and proteins. For control group 3 ml distilled water was used.
Third laparotomy after two weeks carried out again and the adhesions rescored
(second scoring).
2.5. Statistical analysis
A two tail Students t-test was used to analyze the results. All tests were done at
the 5% level of signicance. Calculations were done using either Microsoft Excel or
GraphPad Prism 5. All data were expressed as mean  SE, and statistical signicance
was dened as P < 0.05.
almost all animals following surgery. However, the peritoneal
defects of the animals treated with male BAF without cells had
fewer adhesions than the peritoneal defects of other animals
(Table 2). During postoperative evaluation, control animals that
were operated on without appliance of BAF showed an average
adhesion score of 1.83  0.27 per peritoneal defect in the rst
scoring (fs) and 1.66  0.28 second scoring (ss). Thus, no signicant
differences in the mean adhesions scoring between the rst and
second postoperative evaluations were observed in control animals
(Figs. 1 and 2; P 0.34). In animals to which whole male and female
BAF were applied (groups 2 and 3; Table 2) mean adhesion scores
were 1.58  0.23 (group 2; whole male BAF) and 1.75  0.28 (group
3; whole female BAF) in the rst evaluation and 1.25  0.25 and
2.16  1.34 (respectively) in the second evaluation. There were not
signicant differences in the rst scoring and the second in both
whole male and female BAF (P 0.39 and P 0.32 respectively). In
animals to which male and female BAF without cells were applied
(groups 4 and 5, Table 2), adhesion scoring was 1.25  0.13 and
1.67  0.26 in rst scoring and 2.00  0.30 and 1.50  0.29 in the
second scoring, respectively. A signicant difference (P 0.012)
was only observed in those animals that treated with male BAF
without cells (Fig. 1). No signicant difference was observed
between the rst scoring and second scoring in animals treated
with female BAF without cells (Fig. 2). In animals to which male and
female BAF without cells and proteins were applied (groups 6 and 7,
Table 2), adhesion scoring was 2.50  0.19 and 2.17  0.21 in rst
scoring and 1.42  0.29 and 1.17  0.34 in the second scoring,
respectively. Signicant difference was observed between the rst
scoring and second scoring in animals treated with male BAF
without cell and proteins (Fig. 1, P 0.04) whereas, no signicant
difference was observed between the rst scoring and second
scoring in animals treated with female BAF without cell and
proteins (Fig. 2, P 0.08).
4. Discussion
In the present study, bovine amniotic uid (BAF) was studied for
its efcacy in treatment of postoperative adhesion formation in a rat
adhesion formation model. Various substances, such as cow peri-
toneal, amnion membrane, hyaluronic acid derivatives, oxidized
celluloses, olive oil, soybean oil, liquid made from glucose, starch,
Table 2
Grade of adhesions according to the Canbaz classication.

Groups Grade Mean  Std.

3. Results error
0 1 2 3 4
1 Control fs 1 3 5 3 0 1.83  0.27
The scoring of adhesions in each group is shown in Table 2. Both ss 1 5 3 3 0 1.66  0.28
the number of each adhesion grade and mean adhesions are
2 Treated with whole male BAF fs 0 7 3 2 0 1.58  0.23
reported. It should be noted that adhesion formation was conned
ss 3 3 6 0 0 1.25  0.25
to the peritoneal defects. Table 2 demonstrates that more animals
were scored as grade 2 and 3 in rst scoring (fs) and 1 and 3 in 3 Treated with whole female BAF fs 1 4 4 3 0 1.75  0.28
ss 3 0 1 8 0 2.16  0.39
second scoring (ss) than were scored as other grades. In this study,
no grade 4 was recorded. Adhesion formation had occurred in 4 Treated with male BAF without cells fs 1 8 2 1 0 1.25  0.13
ss 2 3 4 3 0 2.00  0.30

5 Treated with female BAF without cells fs 1 4 5 2 0 1.67  0.26

Table 1 ss 1 7 1 3 0 1.50  0.29
Canbaz adhesion severity scoring method.
6 Treated with male BAF without cells fs 0 1 4 7 0 2.50  0.19
Grade Description and proteins ss 2 5 3 2 0 1.42  0.29
0 No adhesion
7 Treated with female BAF without cells fs 0 2 6 4 0 2.17  0.21
1 One adhesion band, no vessel, easily separated
and proteins ss 5 2 3 2 0 1.17  0.34
2 Two thin adhesion bands, no vessel, easily separated
3 Three thin adhesion bands, no vessel, easily separated Sum fs 3 21 26 21 0
4 More than three thin adhesion bands, easily separated ss 15 22 17 18 0
with no vessel or defused adhesion bands with vessels
fs: rst scoring. ss: second scoring.
 A. Esmaeili et al. / International Journal of Surgery 8 (2010) 639e642 641

200 factors.8 In a rat model it was shown that there is no direct effect of
First scoring * the spilled amniotic uid that inhibits adhesion formation after the
Second scoring performance of caesarean sections.22 However, it is reported that
150
Adhesion (% )

BAF had positive effect on prevention of intra-abdominal adhesion

* in a rat model.9 Topical application of human amniotic uid (HAF)
100 immediately after tenorrhaphy is signicantly effective in pre-
venting peritendinous adhesion formation without impairment of
tendon healing in a rabbit model.23 BAF may also have positive
50 effects in severe burns.24
The results of this study demonstrate that application of
distilled water had no effect on treatment of adhesion formation.
0
We chose distilled water as control because, no report was found to
l

ns
ls
show its benets in preventing or treatment of adhesion formation.
o

el
tr

ei
B
on

tC

Other animal studies reported a benet from Ringers lactate in

ot
le
C

Pr
ou
ho

preventing adhesions.25,26 Because of these results, it was not

ith

d
W

an
considered as a control in this project. However, clinical data on the
W

ls
F

el

use of Ringers lactate show no benecial effect. In two clinical

A
B

tC

studies, the effect of Dextran solution on postoperative adhesion

ou

formation was compared with that of Ringers lactate solution.27,28

ith
W

In both studies, no benecial effect of the Ringers lactate was

F
A
B

Fig. 1. Comparison of mean percentage adhesion between the rst and the second
evaluation in male rats treated with male BAF. Only in rats treated with BAF without
cells and proteins a signicant reduction in adhesion was observed when the rst
scoring compared with second scoring.
glycerol, bioresorbable barriers, icodextrina solution, honey, sodium
hyaluronate-carboxymethylcellulose, bioresorbable barriers and
many other materials to prevent postoperation peritoneal adhesion
formation have been used with different mechanisms.13e21
One substance that is not considered widely yet is BAF. The
foetus is surrounded by amniotic uid, known to be rich in nutri-
ents, growth factors, and other substances important in foetal
development. The characteristics of this uid environment may
play some roles in the scar-free healing of the foetus.7 Amniotic
uid contains many factors such as hyaluronic acid and growth
First scoring
150 Second scoring
Adhesion (% )
found, and many of the patients treated with Ringers lactate had
more adhesions at the time of second look laparoscopy than before
the initial procedure.
In the present study, the peritoneal adhesions treated with
bovine amniotic uid showed a signicant decrease in third lapa-
rotomy (second scoring) compared with the second (rst scoring)
only in rats treated with male BAF without cells and proteins. The
results obtained with rats treated with female BAF were somewhat
disappointing. Although there were no signicant differences
between rst scoring and second scoring, data showed a decrease
in adhesion formation in these groups. On the other hand, at second
look laparotomy, in most groups a reduction in adhesion formation
was found.
The mechanisms of substances have been used to prevent or
treatment of adhesion formation are various and for some are
unknown. One mechanism of these materials is forming
a mechanical barrier between peritoneal surfaces. In the case of BAF
this could be true. However, other mechanisms such as BAFs cells,
proteins and minerals may play a role. Our results showed BAF
containing cells and proteins had no positive effect in reduction of
adhesion formation.
BAF proteins and cells may interact with many types of cell and
proteins at sites of injury in which cells can proliferate, organize, and
carry out specialized functions. Interaction or binding of BAF
proteins to brinogen and brin or proteins of intra-abdominal

100 wound is possible. Proteins such as plasminogen may bind to

brinogen and brin that are involved in angiogenesis as brinolytic
system; these are important interactions in wound healing.29,30 This
50 may increase adhesion formation. Also it is possible some BAF
proteins interact to some intra-abdominal wound cells and result in
enhancement of adhesion formation. Since the best results of this
study obtained using BAF without cells and proteins, then differ-
0
ences in cells and proteins could not be the case. To the best of our
F

ns

knowledge there is no information about gender differences in BAF

l

ls
ro

el

ei
t

B
on

tC

composition. It seems if there is any difference it may be in

ot
le
C

Pr
ou
ho

hormones or enzymes. On the other hand, differences in organic and

i th

d
W

an

minerals components between male and female BAF may inuence

W

ls
F

adhesion formation. However, the reasons for that are not clear yet
el
A
B

tC

and need to be studied.

ou
i th

In summary, we have described effect of BAF on rat model as an

W

experimental adhesion model. The adhesion model we used in the

F
A

present study was very consistent in producing adhesions.

B

Fig. 2. Comparison of mean percentage adhesion between the rst and the second
Furthermore, no benecial effect of distilled water on adhesion
evaluation in male rats treated with female BAF. No signicant reduction in adhesion formation was seen. Signicant reductions in adhesion were seen in
was observed when the rst scoring compared with second scoring. rats treated with male BAF without cells and proteins. The results of
642 A. Esmaeili et al. / International Journal of Surgery 8 (2010) 639e642
this study suggest that bovine amniotic uid could be used in
treatment of adhesion formation because it is inexpensive, readily
available, and has minimal side effects. However, active ingredient
of BAF needs to be elucidated.
Conict of interest
There are not any conicts of interest in this manuscript.
Funding
We funded for this research from our grants.
Ethical approval
The Animal Ethics Committee for the Isfahan Medical University
allowed us working on animals according to 185265 reference
number.
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