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International Journal of Surgery and Research (IJSR)
ISSN 2379-156X
Colorectal Anastomotic Leakage: A new, Validated Rat Model
Research Article
Kevin WY van Barneveld1, Konstantinos A Vakalopoulos2, Joanna WAM Bosmans1, Ruben RM Vogels1, Geesien SA Boersema2, Marion JJ Gijbels3,4,5,
Jack PM Cleutjens4, Johannes Jeekel2, Johan F Lange2, Nicole D Bouvy1*
1
Department of
Department of
3
Department of
4
Department of
5
Department of
2
Surgery, Research Institute NUTRIM, Maastricht University Medical Centre, AZ, Maastricht, The Netherlands.
Surgery, Erasmus University Medical Centre Rotterdam, CA, Rotterdam, the Netherlands.
Molecular Genetics, Research Institute CARIM, Maastricht University Medical Centre, AZ, Maastricht, The Netherlands.
Pathology, Research Institute CARIM, Maastricht University Medical Centre, AZ, Maastricht, The Netherlands.
Medical Biochemistry, Academic Medical Centre, Meibergdreef AZ, Amsterdam, The Netherlands.
Abstract
Background: Anastomotic leakage (AL) remains an important complication after colorectal surgery. Experimental research aims to ind a solution to overcome this serious complication; however, no validated AL model exists in the rat. This
study was designed to develop a feasible new, reproducible AL model for use in colorectal anastomotic research.
Methods: Forty-four male Wistar rats, randomly divided into 4 groups, underwent a midline laparotomy and transection
of the proximal colon. An anastomosis was created with 3, 4, 5, or 12 (control) sutures. After 7 days, the rats were euthanized and scored for the presence of AL. The secondary outcomes were anastomotic bursting pressure (ABP), histological
evaluation, and maturation of collagen. After the initial experiment, the AL model was reproduced in an external academic
center to ensure reproducibility.
Results: AL occurred in 68.8% of rats treated with an insuficient anastomosis, compared to 10% of the control group
(P=0.005). AL rates were highest (81.8%) in the 3-suture group and lowest (50%) when 5 sutures were used. The experiment with 4 sutures was reproduced at an external academic center and showed similar AL rates as in our center (72.7%
vs. 63.6%).
Conclusions: Our rat model for inducing AL by creating an anastomosis using an insuficient number of sutures is feasible
and reproducible. When combining all outcome measures, the AL model for colorectal leakage using 4 sutures is the most
suitable for use in future research.
Keywords: Anastomosis; Animal Model; Bowel; Colonic Anastomotic Leakage; Experimental; Rodent.
Introduction
In colorectal surgery, anastomotic leakage (AL) is known to be a
frequent and life-threatening complication. The literature reports
incidences of 5 to 17% of cases [1-3]. AL is associated with
morbidity and mortality: AL patients undergo more radiological
diagnostics, need prolonged Intensive Care Unit (ICU) admission,
require more re-operations, require the construction of stomas,
and, although debated, might even suffer from higher local and
distant oncological recurrence rates and decreased long-term
survival [4-9].
During the past decades, much research has been carried out to
overcome the problem of AL. Part of this research is focused
on the identiication of risk factors, prediction, and diagnostics
for anastomotic leakage [3, 10, 11] whereas other studies focus
on developing new techniques to improve anastomotic strength
or healing [12-14]. This latter research often includes the use of
*Corresponding Author:
Nicole D Bouvy MD, PhD,
Professor, Department of Surgery, Research Institute NUTRIM, Maastricht University Medical Centre, PO Box 5800, 6202 AZ, Maastricht, the Netherlands.
Tel: +31 43 3875492
Fax: +31 43 3875473
E-mail: n.bouvy@mumc.nl
Received: February 12, 2016
Accepted: December 22, 2016
Published: December 27, 2016
Citation: Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat
Model. Int J Surg Res. 3(6), 61-67. doi: http://dx.doi.org/10.19070/2379-156X-1600013
Copyright: Nicole D Bouvy© 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat Model. Int
J Surg Res. 3(6), 61-67.
61
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animal models for AL. Unfortunately, a wide variety of animal
models are in use, mainly for the testing of tissue adhesives in the
bowel [15-18], internal devices [19-21], or systemic therapies [22].
This large variety of AL models seriously impairs the possibility
of adequately comparing outcomes between studies, making it
dificult for new therapies to progress into the clinical testing
phase [23]. Furthermore, most studies use primary outcomes
like anastomotic bursting pressure (ABP), breaking strength or
histological outcomes to deine anastomotic healing; outcomes
which cannot be measured in leaking anastomoses and do not
necessarily predict the onset of AL [24-27].
Only a few studies have been designed speciically to develop a
standardized animal model for use in research regarding AL [25,
28-30]. Komen and colleagues introduced a new experimental
mouse model and provided information about its reproducibility
[25]. However, most present-day AL research is performed in the
rat, as the mouse has been found to be too small to adequately test
anastomotic healing/sealing devices such as external applications
to enhance wound healing, or tissue adhesives to reinforce
anastomotic strength [18-31]. For the purpose of anastomotic
testing, there is a need for a low-cost, easy to use, and reproducible
animal model.
Therefore, in this study, we aim to evaluate the feasibility and
reproducibility of a new experimental model of AL induced by
standardized technically insuficient anastomoses in rats.
Materials and Methods
Male Wistar rats weighing 250-350g were housed and cared
for at the Central Animal Facility of the Maastricht University
Medical Centre, the Netherlands. Standard rat chow and water
were supplied ad libitum, according to local standards. Similar to
our local human pre-operative procedures, no bowel preparation
or irrigation was performed. The experimental protocol complied
with the Dutch Animal Experimentation Act and was approved
by the local Animal Experimental Committee.
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the abdominal muscle layers with a running suture of polyglactine
4/0 (Vicryl, Ethicon, Johnson & Johnson, USA). The skin was
closed intracutaneously with a running suture of polyglecaprone
4/0 (Monocryl, Ethicon, Johnson & Johnson, USA). After the
operation, moist rat chow was available to enhance postoperative
feeding and to reduce animal discomfort. Daily check-ups of all
animals were carried out to evaluate animal welfare, and in cases
of severe distress/illness, additional analgesics (buprenorphine)
were administered to diminish discomfort. In the experimental
protocol, discomfort endpoints were pre-deined, partly adapted
from Roughan & Flecknell [32]. Once these endpoints were
reached, the rats would have to be euthanized preliminarily.
Study Design
This study consisted of 3 intervention groups in which a
technically insuficient end-to-end colonic anastomosis was
created using 3, 4 or 5 sutures. The control group received 12
equidistant sutures, which is considered a suficient anastomosis
and technically feasible without compromising reproducibility. A
leakage rate of 12% was expected in the control group (based on
prior research) and based on an expected difference of 48% in
an ideal group, an alpha of 0.05 and power of 0.8, a total of 10
rats per group were required. Because previous research showed
a dropout rate of 10%, the group size was estimated at 11 rats
per group. Therefore, atotal of 44 rats were divided randomly
into these 4 groups (Table 1). The goal of this experiment was to
identify the amount of sutures needed to create an anastomosis
leading to a leakage rate of 70%, considered the best suitable rate
for use in models in comparison studies. This high percentage
reduces the number of animals needed to reach signiicance,
without compromising the level of animal discomfort, dropouts,
or inancial means.
The primary outcome of this study was the percentage of AL; the
secondary outcome measures were anastomotic bursting pressure,
histological scoring, and the maturation of collagen.
Macroscopic Evaluation
Anesthesia and Surgical Procedures
Anesthesia was induced by the inhalation of isolurane 5.0 vol%
(Forene, Abbott Laboratories, USA), followed by a maintenance
dose of 2.5 vol%. The animals received buprenorphine 0.1mg/kg
(Temgesic, Schering-Plough, USA) pre-operatively for analgesia.
During the surgical procedure, the animals were placed on a
temperature-controlled heating pad. The abdominal skin was
shaved, disinfected with iodine 1% and covered with sterile
drapes. Experienced researchers, certiied for animal research,
performed all surgical procedures.
The abdominal cavity was accessed through a 5cm midline
incision. The cecum was identiied and placed on sterile gauzes
hydrated with sterile saline solution to prevent dehydration. The
colon was transected 2 cm distally to the cecum, taking care
not to damage the mesenteric vessels. An end-to-end inverting
anastomosis was created using evenly distributed polypropylene
6/0 sutures (Prolene, Ethicon, Johnson & Johnson, USA), after
which the intestines were repositioned into the abdominal cavity.
For postoperative hydration, a bolus of 5 mL sterile saline solution
(37°C) was injected into the abdominal cavity prior to the closure of
Because the majority of AL occurs in the irst week following
surgery and gastrointestinal healing measured by bursting
pressure will be at its maximum after 7 days [33], this period was
chosen for follow-up. After 7 days, the rats were euthanized by an
overdose of inhaled carbon dioxide. Thereafter, a re-laparotomy
was performed and signs of anastomotic leakage were scored
intra-abdominally. The Hinchey classiication was used to score
anastomotic leakage, ranging from Hinchey I (the presence of
localized abscess para-colonic), Hinchey II (the presence of
pelvic abscess), Hinchey III (purulent peritonitis: the presence of
pus in the abdominal cavity) to Hinchey IV (feculent peritonitis:
the presence of pus and feces in the abdominal cavity). AL was
deined as the manifestation of fecal or purulent peritonitis
(Hinchey III-IV) or the presence of one or more abscesses in the
abdominal cavity (Hinchey I-II) [34].
Next, signs of bowel obstruction were scored since this was a
frequently observed side effect in previous anastomotic leakage
research [13, 15]. Furthermore, adhesion formation to the
anastomotic region was scored using the Zühlke scoring system
[35].
Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat Model. Int
J Surg Res. 3(6), 61-67.
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Subsequently, anastomotic bursting pressure (ABP) was measured:
the colon distal to the anastomosis was clamped, a plastic tube
was inserted into the proximal end, and ligated with a single
polyglactine 4/0 suture. Thereafter, the abdomen was illed with
sterile saline solution and the pressure was gradually increased
using a pressure device (IDEE, Maastricht, the Netherlands).
ABP was monitored using a digital manometer until there were
clear signs of air leakage: i.e. the presence of air bubbles in the
saline solution [15, 19]. The anastomotic segment was resected
and prepared for histological evaluation.
Microscopic Evaluation
Specimens were ixated in formaldehyde 4%, after which they
were dehydrated using increasing concentrations of ethanol
prior to being embedded in parafin. After parafin embedding,
tissue sections 4μm thick were prepared and these tissue sections
were stained with hematoxylin and eosin, according to standard
histological protocols. Semiquantitative scoring was used to
evaluate the inlammatory response: not present, slightly present,
moderately present or abundantly present [34].
To evaluate collagen formation, tissue sections were stained with
Picrosirius red [36]. When Picrosirius red staining is combined
with digital imaging, different types of collagen can be identiied
and quantiied. A red color indicates thick and mature collagen
type 1 ibers and green indicates thin immature collagen type 3
ibers. Sections were exposed to a 0.1% solution of Sirius red
in saturated aqueous picric acid for 90 minutes followed by 2
minutes of washing in 0.01N HCl, dehydration and mounting
with entellan. Images of the (peri) anastomotic region were
taken at a magniication of 200x using cross-polarization light
microscopy (Leica DM5000B, Leica Microsystems, Switzerland).
The percentages of collagen in the anastomotic tissue were
calculated. The maturity level of the collagen was estimated
by calculating the red versus green area ratio using the Qwin
morphometry-system (Leica QWin V3.5.1, Leica Microsystems)
[15, 19]. Three randomly chosen images per tissue sample were
analyzed per group. An experienced animal pathologist, who was
blinded to the identity of the groups, microscopically evaluated all
samples in a randomized matter.
The External Test for Reproducibility
In the surgical research facilities of the Erasmus Medical Center
in Rotterdam, we performed an additional experiment with
11 rats, using the exact same operative procedure. During the
operation, performed by a researcher not present during the
earlier experiments (GB), the construction of an anastomosis
was performed using 4 sutures, which was the best performing
interventional group. After 7 days of follow up, the same
experimental protocol as above was used to evaluate the primary
outcomes.
Statistical Analysis
All data are expressed as the means with the standard error of the
mean (SEM). Differences were analyzed using one-way ANOVA
and t-tests. A Bonferroni corrected P-value < 0.05 was considered
statistically signiicant. All analyses were performed using IBM
SPSS Statistics, version 21.0 for Mac (IBM SPSS, USA). All
graphs were composed using GraphPad Prism, version 5.0 a for
Mac (GraphPad Software, USA).
Results
At baseline, the rats weighed 308 ± 3.0g, and no signiicant
differences between groups were observed (P = 0.292; Table
1). Perioperatively, a total of 2 animals died, 1 in the control
group and 1 in the 5-suture group, most likely due to respiratory
depression following anesthesia. During follow up, no discomfort
endpoints were reached. However, 1 animal in the 4-suture group
died, and the postmortem examination showed bowel obstruction
and AL. All other animals had normal postoperative recovery.
After 7 days of follow up, the average weight was 285 ± 3.1g, an
average weight loss of 6.7 ± 0.79 %. No signiicant differences
between groups were observed.
Clinical Outcomes
After the 7-day follow-up period, signiicantly higher AL
percentages were observed in the interventional groups compared
to the control group: 68.8% vs. 10.0%, P = 0.005 (Figure 1). No
signiicant differences were found between the 3 interventional
groups: 81.8%, 72.7%, and 50.0%, for the 3-, 4-, and 5-suture
groups, respectively.
In contrast to animals with AL, animals without the presence
of AL showed no adhesions to the anastomotic region (Zühlke
score 0). Animals suffering from AL showed only mild to
moderate adhesions to the anastomosis (Zühlke score 2-3),
except for 1 animal with a total dehiscence of the anastomosis,
in which massive adhesion formation (Zühlke score 4) and fecal
peritonitis were observed (Table 2). Furthermore, comparable
rates of postoperative bowel obstruction were observed in the
interventional groups versus the control group: 36.3% vs. 10.0%,
P = 0.183 (Table 2). Between the individual interventional groups,
Table 1. Baseline and Postoperative Weight Characteristics of Experimental Groups.
Number of animals (n)
Number of sutures
Baseline weight (g)
Endpoint weight (g)
Weight loss (%)
Control
11
12
298 ± 4.8
284 ± 4.7
4.0 ± 0.7
3 sutures 4 sutures
11
11
3
4
313 ± 5.9 312 ± 5.2
292 ± 6.7 289 ± 7.1
6.6 ± 1.4 6.9 ± 1.5
5 sutures
11
5
308 ± 7.6
276 ± 5.7
9.5 ± 2.2
P - value
0.292
0.280
0.117
All measurements in this table are presented as mean (± SEM)
Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat Model. Int
J Surg Res. 3(6), 61-67.
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Figure 1. Anastomotic leakage (AL) rates per group after 7 days of follow up. Dark grey bars represent cases in which signs
of AL were present at the time of euthanasia. After 7 days, signiicantly higher AL rates were found in the interventional
groups compared to the control group. No signiicant differences were observed between the interventional groups. AL is
deined by Hinchey I-IV scores.
P = 0.005
n.s
100
Precentage (%)
80
60
40
20
0
ol
ntr
Co
res
utu
3S
res
utu
4S
res
utu
5S
Table 2. Synopsis of Clinical Outcomes.
Number of animals
Fecal peritonitis*
Mechanical ileus*
Adhesions total (mean)
Median Zühlke score
Control
10*
0
1
0.2
3
3 sutures
11
1 (severe)
5
1.4
3
4 sutures
10***
0
3
0.5
3
5 sutures
10*
1 (severe)
3
0.5
3
* One rat in this group died following anesthesia-related complications
** One rat died during follow-up due to AL
*** Number of affected animals.
no signiicant differences were observed in the rates of bowel
obstruction: 54.5%, 30%, and 30% for the 3-, 4-, and 5-suture
groups, respectively. All observed bowel obstructions were
classiied as mild, meaning a mild distention of the bowel segment.
Anastomotic Bursting Pressure
The bursting pressure analysis after 7 days showed a signiicant
difference between the 3-suture group (232.3 ± 20.9 mmHg)
and the control group (324.8 ± 24.4, P = 0.024, Figure 2). Also,
the 5- suture group (206.11 ± 12.8) differed signiicantly from
the control group (P = 0.004). The difference between the
4-suture group (241.7 ± 22.1) and the control group showed a
trend towards signiicance (P = 0.074). No signiicant differences
between the 3 interventional groups were observed.
Histology
The analysis of tissue sections stained for hematoxylin and
eosin showed a well-organized anastomotic area in which clear
differences were found between the extent of the inlammatory
response in the control group compared to the 4-suture group
(Figure 3). In the anastomotic region, no clear differences were
observed between the different experimental groups (3, 4, or
5 sutures) in terms of the amount of collagen, macrophages,
granulocytes, and neoangiogenesis (data not shown). However,
the control group had less inlammation and collagen deposition
compared to the 4-suture experimental group. Picrosirius
red staining of the anastomotic region showed comparable
percentages of collagen in all groups (P = 0.339; Table 3). When
focusing on collagen maturity, similar red/green ratios were
observed for all groups, with the experimental groups having
slightly more maturated collagen (P = 0.240).
External Test for Reproducibility
The 4-suture group was selected as the most feasible candidate
for further reproducibility testing, with a leakage rate closest to
the desired rate of 70%. Furthermore, it showed comparable
results in terms of secondary outcome measures. During the
reproducibility testing at the Erasmus Medical Centre Rotterdam,
an anastomotic leakage rate of 63.6% was observed. This rate
is comparable with the leakage rate found in the 4-suture group
during the earlier experiment (72.7%). No animals died during
the follow-up period. The postoperative recovery and weight loss
were comparable to those observed in the initial experiment.
Discussion
In the present study we introduce a new, feasible, and reproducible
Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat Model. Int
J Surg Res. 3(6), 61-67.
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Figure 2. Anastomotic bursting pressure (ABP) measured in situ after 7 days of follow up. The ABP measured in 2 of the
interventional groups (3- and 5 sutures) was signiicantly lower compared to that of the control group. No signiicant differences were observed between the interventional groups.
400
P = 0.024
P = 0.004
n.s
Bursting pressure (mmHg)
300
200
100
0
ol
ntr
Co
res
utu
3S
res
utu
4S
res
utu
5S
Figure 3. Semi-quantitative scoring of important aspects of wound healing, with more inlammation and granulocyte
iniltration in the experimental groups (A), a representative image of the anastomotic site in the control group (B) and a
representative anastomosis in the experimental group with 4 sutures (C). *P = <0.05.
Wound healing (day 7)
Mean + SE
4
Inlammation
Granulocytes iniltration
Collagen deposition
3
2
1
0
ol
ntr
A
Co
s
ure
ut
4s
B
C
Table 3. Total Collagen and Mature/Immature Collagen Ratios.
Collagen percentage (%)
Red/green ratio
Control
3 sutures
29.6 ± 3.5 22.4 ± 2.1
5.56 ± 0.72 8.72 ± 1.80
4 sutures
22.7 ± 2.1
6.40 ± 1.30
5 sutures P-value
25.4 ± 4.1
0.339
9.91 ± 2.17 0.240
The relative collagen area was quantiied as the percentage of total tissue surface. Maturity of collagen was estimated by calculating the
red/green ratio. Red indicates thick, mature collagen. Green indicates thin, immature collagen. In terms of collagen percentage and
red/green ratio, no signiicant differences were found between groups. All measurements in this table are presented as mean (± SEM).
rat model for use in colorectal AL research. The model uses an
insuficient number of sutures to create a leaking anastomosis.
This model is easy to perform, achieves high percentages of AL,
and was reproducible in an external academic center.
Although leaks in adults may occur due to a number of causes, many
of which are patient-speciic factors (i.e., gender, comorbidities,
and medications), procedural factors (i.e., hypotension), or other
technical issues (i.e., anastomotic ischemia) [37, 38], most AL
models described in the literature are based on iatrogenic leakage,
in which a partial defect or ischemia is created in the anastomosis
and are considered models of colonic perforation [20, 21, 29, 30].
With these types of models, leakage rates of up to 100% have
been described. Only a few models use a technically insuficient
Kevin WY van Barneveld, Konstantinos A Vakalopoulos, Joanna WAM Bosmans, Ruben RM Vogels, et al., (2016) Colorectal Anastomotic Leakage: A new, Validated Rat Model. Int
J Surg Res. 3(6), 61-67.
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anastomosis with an insuficient number of sutures; however, such
models have not been developed in rats [15, 18, 19, 25]. Clearly,
in clinical situations a complete anastomosis is constructed;
therefore, it was suggested that a model that develops dehiscence
over time would be clinically more relevant [39]. Nonetheless,
we believe that this model has its strengths in being artiicial and
standardized, making it more suitable for reproducing and for
comparisons between different research centers.
In this study, we considered an AL rate of 70% most suitable for
future colorectal AL research. With this percentage, fewer animals
are needed to reach signiicant outcomes, which is ideal for
studies regarding new leakage prevention strategies. Higher rates
of AL, however, could be associated with a higher level of animal
discomfort and possible death and are therefore not appropriate.
Several important characteristics are needed for a successful
colorectal AL model. First of all, the anastomosis needs to be
large enough to be suitable for anastomotic device testing, such
as intraluminal stents or external sealing. Secondly, the colorectal
tract should be comparable with that of humans, in terms of fecal
consistency and gross anatomy. Furthermore, the costs should be
low; larger animals are very costly, and a large numbers of animals
are needed to achieve suficient statistical power. The procedures
should be easy to perform to minimize technical variability and to
increase reproducibility. Finally, an optimal colorectal AL model
should cause high percentages of AL to achieve a maximum
effect per animal and to reduce the number of needed animals.
To create an optimal rat colorectal AL model, we adapted parts
of animal models used in earlier experiments from our research
groups [15, 19, 25]. To overcome the problem observed by Van
der Ham, who reported low levels of AL in an AL rat model
with a left-sided anastomosis, we used a model with a right-sided
anastomosis to obtain fecal consistency comparable to the human
situation [16].
This experimental colorectal AL model clearly shows that the use
of an insuficient number of sutures signiicantly increases the
leakage rate from 10% (control) up to 50 to 82%, depending on
the number of sutures used. The lowest number of sutures (i.e.,
3 sutures) showed the highest leakage rate, whereas the 4-and
5-suture groups showed similar AL rates. The leakage rate from
the control group is comparable with the clinical situation, where
AL rates are reported to be as high as 5 to 17% [1, 3]. As discussed
above, for an experimental model to be used in colorectal AL
research, higher leakage rates around 70% are desirable, as a lower
number of animals per group are needed to reach signiicance.
The group with the highest number of sutures (the 5-suture group)
experience the highest amount of weight loss (9.5%), whereas the
3- and 4-suture groups did not show any differences (6.6% and
6.9%) compared to the control group. In terms of postoperative
bowel obstruction, the 3-suture group had the highest rates (54%),
whereas the 4- and 5-suture groups did not show any differences.
No differences between the interventional groups and control
group were observed based on the histological examination.
Furthermore, all interventional groups had signiicantly lower
ABPs compared to the control group. Interestingly, the correlation
analysis revealed no correlation between the ABP and AL rates,
indicating that this measurement does not provide information
on the onset of AL as observed by others [24-26].
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Microscopically, clear differences were found between the control
group (12 sutures) and the experimental groups. Leakage resulted
in an evident larger inlammatory process that was evaluated
histologically. No clear differences were found between the
experimental groups, which is understandable because the semiquantitative scoring in animals that developed leakage is similar,
probably due to the same amount of leakage. This might also
explain why there were no differences in the collagen content
and mature/immature collagen ratios between the experimental
groups. However, due to the relatively high leakage rates, collagen
content could have been used to discriminate between rats with
and without AL, as recently described by Shogan and colleagues
[40]. A big difference between their study and this work is that
in this model no devascularization of mesenterial arteries was
performed. Because it is known that adequate oxygen is critical
for optimal collagen-crosslinking and synthesis [41], this could
explain why no clear differences were found. In addition, the
method of determining collagen ratios with Picrosirius red
staining is perhaps not suficiently sensitive enough to test subtle
pathological changes that may have occurred in this model [42].
Repeating the experiment in an external academic center tested
the reproducibility of this new colorectal AL model. This
demonstrated the good reproducibility of the model, with
comparable AL rates and postoperative recovery, indicating that
the model may be valuable for use in various experimental centers.
This study does have potential drawbacks. There is ongoing
discussion about the comparability of physiological processes in
rats and humans. For example, several exogenous factors differ
between the human and experimental situation, which can limit
the extrapolation of results to the human context. Because nonabsorbable polypropylene sutures have been shown to cause
less tissue reaction as compared to absorbable sutures, we used
non-absorbable sutures in our model, whereas in the majority
of human cases, absorbable PDS sutures would have been used
[43]. Although physiological processes in rats may not be a
perfect relection of the human situation, this validated model
can be used to obtain results that provide a good indication of
future outcomes in human subjects, especially regarding newly
developed preventive measures such as sealants for AL.
In the present study, we introduce a new animal model for
research regarding colorectal anastomotic leakage. This model,
consisting of an end-to-end colonic anastomosis with 4 sutures,
ensures a high onset of anastomotic leakage and has been proven
to be reproducible. Therefore, this AL model may be useful in
future experiments, in particular for testing novel colonic sealing
devices to prevent AL.
Acknowledgements and Declarations
The authors would like to thank Mrs. L. Platenkamp, BSc for her
contribution to data processing. The authors declare no conlicts
of interest. Financial disclosure: None reported.
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J Surg Res. 3(6), 61-67.
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