Surgery Today

https://doi.org/10.1007/s00595-021-02267-9

ORIGINAL ARTICLE

Intestinal mucosa staple line integrity and anastomotic leak pressure

after healing in a porcine model
Daishi Naoi1 · Hisanaga Horie1 · Koji Koinuma1 · Yuko Kumagai1 · Gaku Ota1 · Mineyuki Tojo1 · Yuji Kaneda1 ·
Shuji Hishikawa1 · Ai Sadatomo1 · Yoshiyuki Inoue1 · Noriyoshi Fukushima2 · Alan Kawarai Lefor1 · Naohiro Sata1

Received: 25 December 2020 / Accepted: 7 February 2021

© Springer Nature Singapore Pte Ltd. 2021

Abstract
Purpose  The aim of this study was to evaluate both the intestinal mucosa staple line integrity and anastomotic leak pressure
after healing in a porcine survival model.
Methods  We used two suture models using two different size staples (incomplete mucosal closure model: group G [staple
height 0.75 mm], complete mucosal closure model: group B [staple height 1.5 mm]) in the porcine ileum. Five staple lines
were created in each group made in the ileum for each model, and the staple sites harvested on days 0, 2, and 7. The leak
pressure at the staple site was measured at each time point.
Results  On day 0, the leak pressure for group G (79.5 mmHg) was significantly lower than that for group B (182.3 mmHg)
(p < 0.01). On days 2 and 7, there was no significant difference between groups G and B (171 mmHg and 175.5 mmHg on
day 2, 175.5 mmHg and 175.5 mmHg on day 7, p > 0.05). The histological findings in both groups showed similar healing
at postoperative days 2 and 7.
Conclusion  The integrity of the mucosal staple lines was associated with the postoperative leak pressure on day 0. However,
there was no association with the leak pressure at two days or more postoperatively in a porcine model.

Keywords  Stapler · Anastomosis · Leak pressure · Mucosal suture · Porcine model

Purpose
Intestinal anastomosis is an essential technique in gastroin-
testinal surgery. Gastrointestinal anastomoses made using
stapling device are commonly performed instead of hand-
sewn anastomoses because of safety, the ability to shorten
the operation time, and the ease of use in an area where
hand-sewn anastomosis is technically difficult. In the era
of laparoscopic surgery [1, 2], stapling devices have thus
become especially essential. With stapling device improve-
ments, the rate of anastomotic leakage has been reduced
[3, 4]. However, anastomotic leakage still happens with a
certain frequency and it remains one of the complications
* Daishi Naoi
daishi.naoi@jichi.ac.jp
1
Department of Surgery, Division of Gastroenterological,
General and Transplant Surgery, Jichi Medical University,
3311‑1 Yakushiji, Shimotsuke, Tochigi 329‑0498, Japan
2
Department of Pathology, Jichi Medical University, 3311‑1
Yakushiji, Shimotsuke , Tochigi 329‑0498, Japan
that affects patients most. The risk factors for anastomotic
leakage include an impaired blood flow to the anastomo-
sis, tension, and increased intestinal pressure [5–11]. The
integrity of the staple line is also important when staplers
are used to prevent anastomotic dehiscence.
The integrity of the staple line is usually evaluated on the
serosal surface. The integrity of the mucosal surface is not
evaluated in most gastrointestinal operations, and it is less
understood as to what impact mucosal integrity has on the
completed anastomosis. Thompson et al. [12] reported that
anastomoses created with different staplers lead to differ-
ent levels of mucosal capture, and more complete mucosal
capture is associated with a higher initial leak pressure after
suturing in a porcine ileum model. This suggests that incom-
plete mucosal capture may increase the risk of anastomotic
leakage after gastrointestinal anastomosis. However, their
study did not evaluate the leak pressure or condition of the
staple line throughout the postoperative period.
In this study, we devised two models (incomplete mucosal
closure model, complete mucosal closure model) using two
different size staples in a porcine in vivo experiment. We

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verified whether incomplete mucosal closure affects the leak
pressure of the anastomosis throughout the postoperative
period in survival porcine models. We hypothesized that the
integrity of mucosal closure was associated with the leak
pressure at the staple lines throughout the postoperative
period.
Methods
All animal experiments were approved by the Use and Care
of Experimental Animals Committee of Jichi Medical Uni-
versity Guide for Laboratory Animals (Approval No. 18029-
05) and all procedures were carried out in accordance with
the Jichi Medical University guidelines. All animals were
young female domestic pigs, and their weight ranged from
30 to 40 kg. The animals were kept under standard labo-
ratory conditions before the operations. The animals were
administered anesthesia after 24 h of fasting. At the time
of premedication, we injected medetomidine (0.06 mg/kg),
midazolam (0.3 mg/kg), and atropine (0.02 mg/kg) intra-
muscularly. After Vecuronium bromide (0.1 mg/kg) was
injected intravenously, intubation was performed. Sevoflu-
rane (1–4%) was used for induction and to maintain general
anesthesia, and buprenorphine was administered for anal-
gesia. Subsequently, venous access in the internal jugular
vein was secured by cutdown. The animals were monitored
with standard cardiopulmonary monitoring. During surgery,
lactated Ringer’s solution was administered at 2–10 mL/kg/h
according to vital signs.
In‑vivo suture model
After the induction of general anesthesia, we performed a
laparotomy through a midline incision. In this study, we
used the Powered Echelon Flex GST System (Ethicon, Inc.,
Cincinnati, OH) to make staple lines. Based on a previous
report [12], we estimated the porcine ileum thickness to be
1–2 mm. We selected two staple cartridges (0.75 mm/gray
cartridge; group G, 1.5 mm/blue cartridge; group B) to make
two models. The porcine ileum was stapled in vivo with a
Fig. 1  Creating the staple line
The staple lines were oriented
on the ileum cross to the anti-
mesenteric side through this
study
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Surgery Today
gray cartridge which is less than 1 mm thick, and with blue
cartridges, 10 times each alternately starting at 30 cm from
the ileocecal junction. The staple lines were oriented on the
bowel cross to the anti-mesenteric side (Fig. 1). This stapling
method was used to prevent intestinal obstruction due to
intestinal stenosis during the survival model portion of the
experiment described below. Before suturing, we performed
a 15 s precompression. Then, the ileum was harvested, then
each part of the ileum’s thickness and mucosal integrity was
evaluated immediately. To measure the thickness in each
part of porcine ileum, we used a device that applied a pres-
sure of 8 g/mm2 to the tissue via a 9.5 mm diameter circular
probe for 15 s prior to the measurement of thickness as in
the previous report [12].
The thickness of the sutured ileum was 1.1 ± 0.1 mm in
both groups. All staple lines in group G had some mucosal
defects (Fig. 2a), while all of those in group B had com-
plete mucosal closure (Fig. 2b). The serosal integrity was
maintained in both groups. Therefore, we defined group G
as an incomplete mucosal closure model, and group B as a
complete mucosal closure model.
Postoperative leak pressure test protocol
We devised three animal models to evaluate postopera-
tive leak pressure at the staple line created by the stapling
device. After the induction of general anesthesia, we per-
formed laparotomy through a 5 cm midline incision in
the lower abdomen. The ileum was stapled five times in
each group alternately at approximately 15 cm intervals
starting 30 cm from the ileocecal junction. Staple lines
were created in the same way as in the method for the
in vivo suture model. The ileum of the day 0 experiment
was immediately harvested after surgery while continuing
general anesthesia. For the other two animals, the incision
was closed after an antiadhesion agent was placed under
the incision and then it was recovered after surgery. Fenta-
nyl patch ­(Durotep® MT Patch 16.8 mg, Janssen Pharma-
ceutical K.K., Japan) was applied for postoperative anal-
gesia until postoperative day 2. In the day 2 experiment,
the animal was not fed for 2 days after the surgery and it
Surgery Today

Fig. 2  Findings at the staple line

a all staple lines in Group G had
mucosal defects. b All staple
lines in Group B had complete
mucosal closure

was maintained by fluid infusion (2 mL/kg/h, ­SOLITA®-T Statistical analysis

No.3, AY Pharmaceutical Co., Ltd., Japan) using the cen-
tral venous catheter inserted at surgery. Consecutively,
after the induction of general anesthesia postoperatively
on day 2, the ileums were harvested. In the day 7 experi-
ment, the animal was fed enteral nutrition (RACOL-NF
liquid for Enteral U­ se®, Otsuka Pharmaceutical Co., Ltd.,
Japan, day 3, 4; 400 mL/day, day 5, 6; 800 mL/day) and
water from day 3 after surgery. The ileums were harvested
under general anesthesia postoperatively on day 7. When
removing the ileum, a partial excision of the ileum was
performed over a length of 15 cm centered at the staple
line (Fig.  3a). To determine the leak pressure at each
staple line, the intraluminal pressure needed to cause a
visually apparent leak by putting air into the ileum under
water was measured (Fig. 3b). As a control of intraluminal
bursting pressure, the contiguous proximal ileum was par-
tially resected at five locations about 15 cm each, and the
intraluminal pressure at which the ileum serosa was torn
was measured in the respective experiments (group C).
All efforts were made to minimize animal discomfort and
suffering. After completing these experiments, the animals
were euthanized. After the postoperative leak pressure test,
the tissue samples were fixed in 10% phosphate-buffered
formalin and embedded in resin. The tissue sections were
stained with hematoxylin and eosin (HE), and then the
histological findings at the staple line were observed.
Continuous variables are expressed as the median, the differ-
ences in the leak pressure among the two groups and control
were analyzed by the Mann–Whitney U test. All p value
less than 0.05 were considered to be statistically significant.
All statistical analyses were performed with EZR (Saitama
Medical Center, Jichi Medical University, Saitama, Japan),
which is a graphical user interface for R (The R Foundation
for Statistical Computing, Vienna, Austria). More precisely,
it is a modified version of R commander designed to add
statistical functions frequently used in biostatistics [13].
Results
Postoperative leak pressure test
In the postoperative leak pressure test protocol, all animals
had a good post-operative course and were able to undergo
the leak pressure test. Adhesions around the ileum at reop-
eration were mild and they did not interfere with the evalu-
ation of the anastomoses.
At evaluation for postoperative leak pressure postopera-
tively on day 0, the leak pressure for group G (79.5 mmHg)
was significantly lower than that for group B (182.3 mmHg)
(p < 0.01) (Fig. 4a). There were no significant differences

Fig. 3  Postoperative leak pres-

sure test a a partial excision of
the ileum was performed over a
length of 15 cm centered at the
staple line. b The intraluminal
pressure that is required to
cause a visually apparent leak
by putting air into the ileum
under water was measured

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Fig. 4  Postoperative leak pressure G: group G, B: group B, C: control, *p < 0.01 a leak pressure of postoperative day 0, b leak pressure of post-
operative day 2, c leak pressure of postoperative day 7
between groups B and C (210 mmHg). There was also no
significant difference in the leak pressure for groups G, B
and C (171 mmHg, 175.5 mmHg, and 180 mmHg on day
2, p > 0.05, 175.5 mmHg, 175.5 mmHg, and 174 mmHg on
day 7, p > 0.05) postoperatively on days 2 and 7 (Fig. 4b, c).
Histological examination
The mucosa in Group G postoperatively on day 0 was inter-
rupted and was not caught in the staple line (Fig. 5a). The
mucosa of ileums in Group B was caught completely in the
staple line (Fig. 5c, d). The muscular layer and subserosa in
group G appeared to be slightly compressed compared to
those in group B, but their continuity was maintained, and
no tissue degeneration was seen (Fig. 5b). On postoperative
day 2, similar wound healing was observed in both groups
with inflammatory cells observed infiltrating around the
staples (Fig. 5f, h), the staple line was completely covered
with a fibrous membrane (Fig. 5e, g). Similar wound healing
was also observed in both groups postoperatively on day 7.
Fibroblast proliferation and angiogenesis were seen around
the staples, the subserosa was thickened, and the serosa com-
pletely covered the suture line (Fig. 5i–l).
Discussion
We evaluated the postoperative leak pressure in two models
(incomplete mucosal closure model, complete mucosal clo-
sure model) using two different staple sizes, and determined
whether an incomplete mucosal closure affects the leak pres-
sure at the site of anastomosis in the survival models. We
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Surgery Today
hypothesized that the complete closure model is endowed
with a higher leak pressure even after the healing process
throughout the postoperative period. At day 0, the complete
mucosal closure model showed a higher leak pressure than
the incomplete mucosal closure model, which was the same
result as described in a previous report [12]. Therefore,
the incomplete mucosal closure might be associated with
a lower initial leak pressure. However, the results on days
2 and 7 were somewhat unexpected. There was no signifi-
cant difference in the postoperative leak pressure of the two
models on days 2 and 7. As a result, the staple lines of the
incomplete mucosal closure model have the same leak pres-
sure as the complete mucosal closure model throughout the
postoperative period.
Anastomotic leakage is one of the most serious complica-
tions, with high morbidity, mortality, local recurrences, and
poor anorectal function [14–16]. Therefore, some risk fac-
tors in the patient background, such as advanced age, male
gender, diabetes, impaired blood flow to the anastomosis,
tension, and increased intraluminal pressure, were reported
[5–11, 17]. Additionally, because the stapling device is com-
monly used to create anastomoses in gastrointestinal sur-
gery, the number of the cartridge and cartridge type (staple
height, attached bioabsorbable polyglycolic acid felt) used
for anastomosis were also evaluated to evaluate their effec-
tiveness and impact on anastomotic leakage in some studies
[12, 18–22]. The integrity of the staple lines created by the
stapling device is important to prevent anastomotic leak-
age. Chekan et al. [19] reviewed surgical stapling devices
and tissue interactions in bariatric, thoracic, and colorectal
procedures. They emphasized that the surgeon should take
into consideration adequate tissue compression, appropriate
Surgery Today
Fig. 5  Histological examination of each staple line (H&E) group
G, day 0 experiment (a ×40), (b ×100), group B day 0 experiment
(c ×40), (d ×100) group G day 2 experiment (e ×40), (f ×400) group B
day 2 experiment (g ×40), (h ×400) group G day 7 experiment (i ×40),
(j ×200) group B day 7 experiment (k ×40), (l ×200) The mucosal
layer was interrupted in group G (a). Complete mucosal closure (*)
was observed in group B (c). The muscular layer and subserosa in
staple height for tissue thickness, tissue compressibility, and
the tissue type to be able to maximize the integrity of the
staple line.
However, precisely what impact the integrity of the staple
line has including complete mucosal closure, on the anas-
tomosis is still less well understood. Thompson et al. [12]
reported that more complete mucosal closure was associ-
ated with a higher initial leak pressure after suturing in a
porcine ileum model. A similar trend in which a complete
mucosal closure model showed a higher leak pressure than
an incomplete mucosal closure model was also observed
in these results. The integrity of the staple line including
complete mucosal closure is probably related to the initial
leak pressure. Baker et al. [23] reported that the majority of
group G appeared to be slightly compressed compared to those in
group B, but their continuity was maintained, and no tissue degen-
eration was seen (b, d). Inflammatory cells were observed to infil-
trate around the staples (f, h), the staple line was completely covered
with a fibrous membrane postoperatively day 2 in both groups (e, g).
The subserosa was thickened, and the serosa completely covered the
suture line in both groups (i–l)
leaks in bariatric operations occurred in the first two post-
operative days and they were caused by mechanical/tissue
issues. In this in vivo suture model experiment, we observed
mucosal dehiscence in all staple lines created with a lower
staple height (0.75 mm) for the thickness of porcine ileum
(1.1 ± 0.1 mm). We therefore should select an appropriate
staple height based on the intestinal thickness to ensure
integrity of the staple line, including complete mucosal clo-
sure, to prevent the occurrence of anastomotic leakage in the
early postoperative period.
The muscular layer and subserosa in group G appeared
to be slightly compressed on histologic evaluation of the
day 0 experiment compared to those in group B, but the
continuity was maintained, and no tissue degeneration was
13

observed. Though the compressed tissue may be an indicator
of mechanical damage in the staple line to a degree, our find-
ings suggest that incomplete mucosal closure had a greater
influence on the initial leak pressure than the compression
of other parts of the bowel wall in this study.
Another notable finding in these results was that the leak
pressure of the staple line comparing groups G and B on
postoperative days 2 and 7 was equivalent. Furthermore, it
was comparable to group C which was measured while the
ileal serosa was torn. Penninckx et al. [24] reported that
the bursting pressure of the anastomosis creating by circu-
lar stapling devices gradually increased from approximately
60 mmHg at postoperatively day 1 to above 250 mmHg at
day 7 in their comparative study using single- and double-
row staple circular anastomosis for canine colons. Their
histological findings showed that serosal continuity was
partly restored within 24 h after surgery, and was continu-
ous and smooth at postoperative day 4 in contrast to incom-
plete mucosal healing. Graffner et al. [25, 26] also reported
that the mechanical strength of the anastomosis created by
circular stapling devices at one week postoperatively was
higher than that of the intestine itself in porcine experiments.
There are no reports evaluating either the leak pressure or
histological findings of staple lines postoperatively with
or without complete mucosal closure. In the present study,
the histological findings showed that the staple lines were
already buried in the subserosa at postoperative day 2, and
the subserosa became thicker by postoperative day 7. This
healing process on the serosal side of the staple line might
affect the equivalent leak pressure between groups G and B
during the postoperative periods.
We evaluated the leak pressure at the staple lines post-
operatively in a porcine survival model, and postoperative
leak pressure tests were performed in a non-physiological
maneuver. The overall intraluminal pressure in the gas-
trointestinal tract of human is unknown, but some studies
have investigated the rectal intraluminal pressure. Adame
et al. [27] reported the mean rectal resting pressure lying
in the left position to be 11 (8–14) mmHg, while the rectal
pressure bearing down was 41 (37–45) mmHg. Lee et al.
[28] reported that rectal pressure bearing down on com-
mode is higher than that on the bed (median [interquartile];
105.5 [155.4] vs. 42.3 [32.2] mmHg). Therefore, it could be
expected that a higher intraluminal pressure is applied to a
staple line of an anastomosis as patients leave their bed after
surgery, and it might become one of the risks inducing anas-
tomotic leakage without the integrity of staple line includ-
ing complete mucosal closure. The utility of a trans-anal
decompression tube to prevent anastomotic leakage after
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Surgery Today
rectal surgery has been reported [29]. This clinical perspec-
tive supports the concept that an initial higher intraluminal
pressure may be associated with anastomotic leakage in the
early postoperative period.
The intra-operative selection of the appropriate staple
height for intestinal anastomoses in patients is difficult.
The normal uncompressed thickness of the small intestine
wall is 1–2 mm, while the colorectal wall may be up to
3 mm. The bowel wall can be further thickened by edema,
inflammation, or tumors, as measured by ultrasonography,
ultrasonographic colonoscopy, and computed tomography
[30–32]. When the bowel wall is intraoperatively found to
be edematous, such as due to inflammation by preoperative
radiation or being adjacent to an advanced tumor, a higher
staple height should therefore probably be chosen.
This study is associated with some limitations. First, we
made two anastomosis models using a linear stapler to evalu-
ate the leak pressure and observe the histopathological find-
ings. However, most instances of postoperative anastomotic
leakage are caused by anastomoses created using the double
stapling technique, which are usually performed with a lin-
ear stapler and a circular stapler. Complete mucosal closure
may lead to integrity at the intersection between the linear
and circular staple lines. Further investigations are necessary
to elucidate the responsible mechanisms. Second, wound
healing at the staple line using a linear stapler in a porcine
model was already observed histologically by postoperative
day 2 in this study. However, the healing process and dura-
tion at the staple line both vary depending on the anasto-
mosis method and individuals [24–26], and these phenom-
ena may be different in humans. Therefore, the appropriate
staple height for the intestinal thickness should be selected
to ensure the integrity of the staple line to prevent t anasto-
motic leakage in the early postoperative period.
In conclusion, the integrity of mucosal closure was asso-
ciated with an initial postoperative leak pressure. However,
it was not associated with the leak pressure after healing
in surviving porcine models. To avoid anastomotic leakage
during the early postoperative period, the selection of an
appropriate staple height for the intestinal thickness is there-
fore important.
Acknowledgements  We thank Takuya Ito and Kahori Igarashi for their
expert assistance in animal care, and Dr. Yuko Homma and Dr. Toshiki
Mimura for providing invaluable suggestions.
Declarations 
Conflict of interest  The authors have no financial conflicts of interest
to declare.
Surgery Today

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