| Sažetak (engleski) | Background and objectives: Although great progress in colorectal surgery was
achieved during the last few decades, the problem of colorectal anastomosis dehiscence,
with resulting morbidity and mortality, remains to this day. Colonic anastomosis
creation is an everyday procedure in abdominal surgery and basically does not differ
from any other anastomosis creation. A particular type of colonic anastomosis is a
colorectal-coloanal anastomosis, special due to rectum vascularization, absence of the
serosa in the subperitoneal part of the rectum, and difficulty with the surgical procedure
due to narrow space in the true pelvis. Thus, literature that focuses on colonic
anastomoses usually refers to the colorectal anastomosis. Intestinal anastomosis healing
is a complicated process that depends on the patient's general state, intestine state,
technical conditions, and surgeon's knowledge and experience. Among many factors
that affect the process of anastomosis healing, one of the most important is adequate
arterial and venous circulation of the intestines. Colonic congestion is defined as colonic
venous circulation stasis that causes intestinal wall edema and reduction of arterial
irrigation, which is often observed in patients that suffer from colonic obstruction ileus
caused by malignant tumors. Although ischemia and congestion both lead to tissue
hypoxia, intestinal congestion is considered a more serious and complicated condition.
By comparing ischemic and venous congestion intestinal injury, destruction of the
intestinal mucosa induced by venous congestion arises earlier and is slower to
normalize after etiological factor removal, which makes intestinal congestionreperfusion injury much more severe than ischemic-reperfusion injury. Colonic
congestion is usually found in patients suffering from ileus caused by malignant tumors
located in the colon, and in those cases, a colostomy is preferred over anastomosis
creation. In case of venous congestion, meaning compromised venous circulation,
primary anastomosis is usually not performed because congested intestinal anastomosishealing is risky and burdened with a high possibility of anastomosis dehiscence with
accompanying peritonitis or intraabdominal abscess. Thus, colostomy is usually
performed in those cases.
Pentadecapeptide body protective compound 157 (BPC 157) is a terminal part of the
BPC protein crucial for its activity. Pentadecapeptide BPC was isolated from human
gastric juice and is found to need no additional carriers for absorption and to be stable in
human gastric juice. The synthetic form of BPC 157 has been examined in great detail
and scope via experimental research that has shown its positive effects on burn healing,
gastric and duodenal ulcer healing, different fistula of the gastrointestinal tract, and
intestinal anastomosis healing. The effectiveness of pentadecapeptide BPC 157 in
healing promotion has been investigated in different animal models, proving its effect
on connective tissue and blood vessels. In a burn animal model, it was shown that BPC
157 attenuates the inflammatory response, lessens the edema and necrosis of the burnt
skin, and enhances collagen organization, angiogenesis, and tissue epithelization. In an
animal model of esophagogastric anastomosis and colon-colonic anastomosis in
cysteamine colitis caused by rectal cysteamine enema, greater mortality and
anastomosis dehiscence was found in control groups, in contrast to treated groups.
Pentadecapeptide BPC 157 helps the healing process by stimulating granulation tissue
creation and collagen organization, as well as reducing the edema and stimulating
angiogenesis. Recent studies have shown that pentadecapeptide BPC 157 activates
anatomically present collateral circulation in arterial or venous obstruction conditions
and thus lessens the negative impact of vascular obstruction.The exact mechanism of
action of pentadecapeptide BPC 157 is still not clarified in full, but its effects on
different tissues and organs are explained through many different interacting systems,
the most important one being the NO system. It has been proven that pentadecapeptide
BPC 157 induces NO formation and interferes with agonists (L-arginine, NO substrate)
and antagonists (L-NAME) of the NO system. By inducing NO synthesis,
pentadecapeptide BPC 157 stimulates vasodilatation in the early stages of the healing
process. Additionaly, in early stages of the healing process, pentadecapeptide BPC 157
stimulates the production of EGR1, crucial for activation of genes that code for various
cytokines and growth factors, thus stimulating collagen production and organization. It
has been established that pentadecapeptide BPC 157 stimulates expression of EGR-1mRNA 15 minutes after application, and 30 minutes after application, BPC 157
stimulates NAB2 mRNA expression, which is a corepressor of EGR-1. Hsieh et al. have
demonstrated that pentadecapeptide BPC 157 affects aortic tone via NO synthesis
stimulation through the Src-Cav-1-eNOS signal pathway. The angiogenesis-modulating
potential of pentadecapeptide BPC 157 manifests through stimulation of VEGF and
VEGFR2 expression, which was examined in an animal model of limb ischemia.
Pentadecapeptide BPC 157 increased VEGFR2 expression in blood vessel endothelium
and thus enabled quick reestablishment of the circulation through the ischemic
extremity. Angiomodulatory effects of pentadecapeptide BPC 157 were also examined
in an animal model of duodenal lesions caused by ligation of superior anterior
pancreaticoduodenal vein that revealed that pentadecapeptide BPC 157 causes collateral
circulation activation through the NO system, and thus attenuates the congestion and
duodenal mucosal lesions development. To this day, pentadecapeptide BPC 157 was
administered in various different doses (µg/kg, ng/kg, or pg/kg) and applied in various
different modes of drug delivery (locally: baths, cremes, eyedrops; systemically:
intraperitoneally, intragastrically, perorally). Additionally, to this day no serious toxicity
of pentadecapeptide BPC 157 has been noted. Based on described effects of
pentadecapeptide BPC 157, we hypothesized that pentadecapeptide BPC 157 may have
positive effects on colon-colonic anastomosis healing under venous congestion
conditions. The aim of this study is to examine the effect of pentadecapeptide BPC 157
on colon-colonic anastomosis healing under venous congestion conditions caused by
ligation of the caudal mesenteric vein in rats.
Material and methods: The experiment was performed using 105 male albino Wistar
rats, bodyweight 200-220 g, aged 8-10 weeks, divided into 15 groups and 5 time
intervals (3, 5, 7, 15, or 30 days). Each group included 7 animals. For each time
interval, there was one control group, one treated group that received pentadecapeptide
BPC 157 dose of 10µg/ kg, and the second treated group that pentadecapeptide BPC
157 dose of 10 ng/kg. Two different doses of BPC 157 were used to examine the dosedependent effect of BPC 157. Animals were anesthetized via intraperitoneal injection
containing diazepam 5 mg/kg (Apaurin, Krka, Slovenia), thiopental 50 mg/kg
(Thiopental Injection BP, Rotexmedica, Germany), and buprenorphine 1 mg/kg
(Bupredine Multidose, Dechra, Great Britain).In deeply anesthetized rats, median laparotomy was performed and caudal mesenteric
vein (CMV) was ligated to achieve venous congestion in the descending colon. After
venous congestion was achieved, transection of the colon descendens and adjacent
mesocolon was done, after which terminoterminal colon-colonic anastomosis was
created. After anastomosis creation, treated animals received pentadecapeptide BPC 157
intraperitoneally in doses 10 µg/ kg or 10 ng/kg and control animals were given 1 mL
saline, also intraperitoneally. At each time interval (3, 5, 7, 15 or 30 days
postoperatively) animals were again deeply anesthetized and relaparatomised to
examine macroscopic changes: anastomosis dehiscence, adhesions, anastomosis
passability, and venous arcade (vasa recta) presentation on the ventral and dorsal side of
the colon, orally and aborally from the anastomosis. The anastomosis was tested as
follows: intestinal clamps were used to occlude the descending colon 2.5 cm orally and
2.5 cm aborally from the anastomosis site and saline wasapplied intraluminally until
volume that caused anastomosis leakage was reached. The colonic segment containing
the anastomosis was explanted from euthanized animals (2 cm orally and 2 cm aborally
from the anastomosis site). Surrounding mesocolon was removed from the resected part
of the colon, the resected part was then rinsed in saline, placed over a styrofoam panel,
and submerged into 4% formaldehyde solution for histopathological analysis.
Mentioned analysis was performed by two independent pathologists who had no
previous knowledge of tissue specimens. 5 fields of view were analyzed using 200x
magnification. The morphological analysis included epithelisation, necrosis, edema, and
granulation tissue formation evaluation. Histochemical coloring methods included
hemalaun-eosine, Gommori for reticulin fibers and Masson for collagen fibers, and
immunohistochemical coloring SMA for new smooth muscle cells visualization. All
stages of the experiment were recorded using a Veho Discovery VMS-004 Deluxe USB
camera with a microscope (Veho, Dayton, OH, SAD). Recordings took place before
CMV ligation, after CMV ligation, after anastomosis creation and pentadecapeptide
BPC 157 application, and at 3, 5, 7, 15 and 30 days postoperatively. Thus obtained
films images were then saved as digital photographs to enable comparison between
treated and control animals. Using digital photographs, two venous arcades (vasa recta)
were analyzed on the ventral and dorsal side of the colon, orally and aborally from the
anastomosis, as well as communicating branches between mentioned venous arcades.Program STATISTICA 12.1. (StatSoft, Inc, Tulsa, USA) was used for statistical
processing. For the data distribution assessment, the Kolmogorov-Smirnoff test was
used.
Results are expressed as mean +/- standard deviation (SD) and as
minimum/median/maximum. For parametric data, one way ANOVA test with NewmanKeuls post hoc test was used, and for nonparametric data Kruskal-Wallis test with
Mann-Whitney post hoc test was used. Qualitative data in control and treated groups
were analyzed using the Fisher test. p<0,05 was considered statistically significant.
Results: Anastomosis dehiscence was evidenced in 11 of 35 control animals (31.42%)
and 2 of 70 treated animals (2.85%). There was no evidence of diffuse peritonitis or
intraabdominal abscess. Adhesions and intestinal obstruction of the anastomosis were
significantly mitigated in treated groups at all time intervals. Better presentation of the
blood vessels, meaning higher volume of venous arcades orally and aborally from the
anastomosis and greater number of communicating branches between mentioned venous
arcades, was found in treated animals. Anastomosis testing revealed better anastomosis
solidity in treated animals. Histopathological analysis showed less edema and necrosis
in treated animals. Granulation tissue was present at an earlier time interval in treated
animals, with the visible organization of collagen and reticulin fibers. In control
animals, no healing organization was found. Immunohistochemical staining showed
smooth muscle cell bundles organization in treated animals, which was absent in control
animals. Additionally, epithelization developed earlier in treated animals. At the end of
the experiment, treated animals exhibited 80%nanastomosis epithelization, in contrast,
control animals exhibited only 10% anastomosis epithelization. There was no
statistically significant difference in any of the observed parameters between groups that
received the pentadecapeptide BPC 157 dose of 10 µg /kg and 10 ng/kg.
Conclusions: Pentadecapeptide BPC 157 enhances colon-colonic anastomosis healing
under venous congestion conditions, which is observable at both macroscopic and
microscopic levels. The enhancing effect of pentadecapeptide BPC 157 on coloncolonic anastomosis healing under venous congestion conditions was present in 10
µg/kg and 10 ng/kg doses without statistically significant differences between the doses.
This experiment confirmes already established role of pentadecapeptide 157 in healingpromotion and opens the possibility of pentadecapeptide BPC 157 use in abdominal
surgery. |
