Effects of bromopride on the healing of left colonic anastomoses in rats with induced abdominal sepsis 1

PuRPOSE: To evaluate the effects of bromopride on the healing of left colonic anastomoses in rats with induced abdominal sepsis. METHOdS: Forty rats were divided into two groups to receive either bromopride (experimental groupE) or saline (control groupC). Each group was divided into subgroups of ten animals each to be euthanized on third (E3 and C3) or seventh day (E7 and C7) after surgery. Sepsis was induced by cecal ligation and puncture. The rats underwent segmental left colon resection and end-to-end anastomosis. Adhesion formation, tensile strength and hydroxyproline concentration were assessed. Histomorphometry of collagen and histopathological analysis were also performed. RESuLTS: On postoperative third day, anastomoses in bromopride-treated animals showed lower tensile strength (p=0.02) and greater reduction in hydroxyproline concentration (p=0.04) than in control animals. There was no statistical difference in these parameters on seventh day, and the remaining parameters were similar across subgroups. Collagen content was also similar across subgroups. CONCLuSION: In the presence of abdominal sepsis, the administration of bromopride was associated with decreased tensile strength and hydroxyproline concentration in left colonic anastomoses in rats three days after surgery.


Introduction
Several factors may affect the healing of intestinal anastomoses, including intraperitoneal infection and drug use [1][2][3] .
One of the most frequently used models to study the pathophysiology of abdominal sepsis and its systemic consequences is the cecal ligation and puncture (CLP) model.This model is associated with polymicrobial sepsis and bacteremia and has the advantage of combining tissue injury with the ability to modulate the degree of bacterial contamination and mortality 4 .In a previous study, we found that administration of bromopride in the absence of sepsis was associated with decreased tensile strength of left colonic anastomoses in rats three days after surgery 5 .A search of the literature returned no studies specifically related to the influence of bromopride on the healing of intestinal anastomoses in the presence of infection.
This study aimed to evaluate the effects of bromopride on the healing of left colonic anastomoses in rats with induced abdominal sepsis.

Methods
The study was performed at the Laboratory of The rats were randomly divided into two groups of 20 animals each to receive either bromopride (experimental group-E) or saline (control group-C) postoperatively.Each group was then divided into subgroups of 10 animals each to be euthanized on third (E3 and C3) or seventh day (E7 and C7) after surgery (Figure 1).Laparotomy consisted of a 4.0-cm midline incision of the abdominal wall, starting 1.0 cm above the external genitalia of animals.The distal colon was exposed, and 0.5 cm of the left colon was resected 2.5 to 3.0 cm above the peritoneal reflection, followed by end-to-end anastomosis of the segment, in a single transmural layer, using running 6-0 polypropylene suture and a blunted needle.CLP-induced abdominal sepsis was performed as described by Rittirsch et al. 6 .After anastomosis, 50% of exposed cecum was ligated with 3-0 silk suture (Shalon ® ).Then, 10 random perforations were made at the end of the cecum distal to the ligation site using a 40 x 13 mm venipuncture needle, and a small amount of feces was extruded.The abdominal wall was closed in two layers with running 3-0 silk suture.
After the procedure, bromopride (total daily dose of 1 mg/100 mg of body weight) was administered subcutaneously to animals in the experimental subgroups every 12h until the day of euthanasia.Control animals received identical volumes of saline in a similar regimen.
In the postoperative period, clinical patterns of apathy, bristling hair, diarrhea, abdominal distension, and wound complications, such as hematoma and signs of surgical site infection, were assessed.
A second operation was performed on the day previously scheduled for each subgroup.After exposure of the abdominal cavity, signs of peritonitis, abscess, obstruction or anastomotic dehiscence were investigated.Intra-abdominal adhesions were quantitatively scored according to the adhesion scoring system defined by Nair et al.The right lateral segment underwent histopathological analysis and analysis of collagen content.For histopathological analysis, the specimens were fixed in formalin and stained with hematoxylin and eosin.An experienced pathologist, blind to which subgroup the specimens belonged, examined the slides by light microscopy.The following parameters were assessed: congestion, edema, hemorrhage, ulceration, necrosis, mononuclear and polymorphonuclear infiltration, neovascularization, granulation, fibrosis, and fibroblasts (extent, arrangement, and maturity).Each of these parameters was graded as absent (0), mild (1), moderate (2), marked (3), or intense (4).
Quantitative analysis of collagen was performed only in animals euthanized on postoperative seventh day.The slides that more significantly represented all subgroups were selected.
In picrosirius-stained sections, the perianastomotic area was analyzed by polarized light microscopy 9 .The total field area was examined using a microscope with a 10x objective, and the material was analyzed by an observer blind to which group the specimens belonged.Images were acquired using a 5.

Results
There were no deaths among animals euthanized on postoperative third day.There was one death in each subgroup of animals scheduled for euthanasia seven days after surgery, none of them due to complications related to anastomosis.The animals' clinical course was similar across subgroups.
On third day, an anastomotic dehiscence blocked by omentum and adjacent organs occurred in the bromopride-treated subgroup, and free dehiscence, with presence of feces in the abdominal cavity, occurred in the control subgroup.On seventh day, each subgroup showed one case of blocked anastomotic dehiscence.
Bromopride-treated animals had adhesion scores similar to those of controls both on third (p=0.77) and seventh days (p=1.00)postoperatively (Table 1).* Adhesion scoring system defined by Nair et al. 7 .

Effects of bromopride on the healing of left colonic anastomoses in rats with induced abdominal sepsis
Acta Cirúrgica Brasileira -Vol.27 ( 6) 2012 -373 The percentage of anastomosis covered with adhesions was also similar across groups both on third (p=0.58) and seventh days (p=0.77)postoperatively (Table 2).Tensile strength of anastomoses was lower in animals in group E3 than in group C3, with a statistically significant difference (p=0.02)(Figure 2).There was no statistical difference in tensile strength between groups on seventh day (p=0.79)(Figure 3).On third day, bromopride-treated animals showed a greater reduction in hydroxyproline concentration than controls (p=0.04)(Figure 4).There was no difference between groups on seventh day (p=0.73)(Figure 5).Collagen content on seventh day was also similar across subgroups (p=0.41).On postoperative third day, control animals showed a higher number of bacterial colonies in the anastomotic region than bromopride-treated animals (p=0.02).On seventh day, bromopridetreated rats showed a higher abscess rate in the anastomotic region than controls (p=0.01).There was no difference between subgroups in the remaining histopathological parameters.

discussion
The colon is the region of the gastrointestinal tract most vulnerable to anastomotic dehiscence 12 .The occurrence of dehiscence is particularly high when suture is performed in the distal colon due to associated fecal loading and technical difficulty 13 .High mortality and severe morbidity associated with dehiscence support the need for further evaluation of methods aiming to reduce its incidence 14 and the deleterious effects of drugs on wound healing.
Bromopride is a prokinetic agent used in the treatment of postoperative gastroesophageal reflux, nausea, vomiting, and gastrointestinal motility disorders.This is a substituted benzamide drug, such as metoclopramide, and its main action involves the blockade of D2 dopamine receptors in the central nervous system (CNS) and gastrointestinal tract.
In previous studies, we evaluated separately the effects of abdominal sepsis 15 and administration of bromopride 5 on the healing of left colonic anastomoses in rats.CLP-induced abdominal sepsis increased the number of intra-abdominal adhesions on postoperative third and seventh days, caused a reduction in tensile strength of anastomoses on third day, with subsequent increase on seventh day, and produced no changes in hydroxyproline concentration or collagen content in the anastomotic region 15 .
Treatment with bromopride in the absence of abdominal sepsis produced no changes in adhesion formation within the abdominal cavity or in the anastomosis and caused a reduction in tensile strength of anastomoses on third day, but not on seventh day.In addition, there was no difference in the variation of hydroxyproline concentration in the two experimental periods 5 .
In the present study, we analyzed the joint action of these two factors and confirmed the deleterious effect of bromopride on colonic anastomosis healing, reducing not only tensile strength, but also hydroxyproline concentration three days after surgery.Again, there was no difference between subgroups on postoperative seventh day.
There is no consensus in the literature regarding the mechanism of action of prokinetic agents on the healing of anastomoses or whether such action is due to changes in adhesion formation.García-Olmo et al. 16 evaluated the effects of pharmacological manipulation of gastrointestinal motility on the resistance of colonic anastomoses and concluded that treatment with metoclopramide was associated with fewer adhesions to the anastomosis and a significant decrease in anastomotic resistance four days after surgery.In another study 17 , metoclopramidetreated animals showed adhesion formation within the abdominal cavity and in the anastomosis similar to that of animals receiving saline three and seven days after surgery.Likewise, tensile strength of anastomoses was similar across subgroups, but hydroxyproline concentration on postoperative third day was higher in metoclopramide-treated animals than in controls.Springall and Spitz 18 investigated the action of cisapride, another prokinetic agent, on the healing of anastomoses in rats seven days after surgery.Drug administration was associated with a reduced number of adhesions, but with no effects on anastomotic resistance.
In this study, treatment with bromopride in the presence of CLP-induced abdominal sepsis was detrimental in the early stages of healing, with no changes in adhesion formation.Thus, we can infer that the effect of prokinetic drugs on the healing of left colonic anastomoses in rats cannot be attributed to changes in adhesion formation in the anastomosis, suggesting that other mechanisms may be involved.
No differences were found between subgroups regarding collagen content in the tissue.Collagen is the main extracellular matrix protein and a major component of the final scar.In the gastrointestinal tract, collagen is found mainly in the submucosal layer 19 .Collagen is important at all stages of wound healing and is critical to the overall strength and integrity of the tissue.However, the final anastomotic strength is not always related to the absolute concentration of collagen, the structure and arrangement of the collagen matrix may be more important 20 .

Conclusion
In the presence of abdominal sepsis, the administration of bromopride was associated with decreased tensile strength and hydroxyproline concentration in left colonic anastomoses in rats three days after surgery.

Bromopride ( 4 -
amino-5-bromo-N-[2-(diethylamino) ethyl]-2-methoxybenzamide) is a prokinetic agent that has antiemetic action and the ability to stimulate gastrointestinal motility.This drug is often used in the treatment of paralytic ileus and control of postoperative nausea and vomiting.

FIGuRE 1 -
FIGuRE 1 -Allocation of animals into groups.

7 : 0 - 1 - 2 - 3 - 4 -
Complete absence of adhesions; Single band of adhesion, between viscera, or from one viscus to abdominal wall; Two bands, either between viscera or from viscera to abdominal wall; More than two bands, between viscera, or viscera to abdominal wall, or whole of intestines forming a mass without being adherent to abdominal wall; Viscera directly adherent to abdominal wall, irrespective of number and extent of adhesive bands.A 4-cm long colon segment, with the anastomosis located in its central part, was removed along with the structures adhering to it.Subsequently, the type of structures adhering to the anastomosis was analyzed.The extent of adhesions present was determined as the percentage of anastomosis covered with adhesions.The specimen was cut open on the antimesenteric border and divided into three longitudinal segments.The central segment was subjected to tensile strength testing using a VersaTest test stand (Mecmesin VersaTest, United Kingdom) fitted with a digital force gauge (Mecmesin AFG 2500N, United Kingdom).A rectangular tissue sample was fixed at its two ends to the test apparatus by the upper fixing clamp of the force gauge and the lower clamp of the VersaTest, with the anastomotic region equidistant and parallel to the clamps.The tensile test was performed at a speed of 30 mm/ min 8 .Tensile strength was measured in Newton (N).
0 mega pixel Moticam 2500 digital camera, coupled to an Olympus CX41 light microscope.All images were analyzed using ImageJ (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA).The left lateral segment was used to measure hydroxyproline concentration according to the technique described by Stegemann and Stalder 10 and modified by Medugorac 11 .Data were analyzed using the Statistical Package for the Social Sciences (SPSS ® ) version 18.0.The Mann-Whitney test was used to analyze tensile strength, hydroxyproline concentration, and collagen content.Fisher's exact test was used to evaluate clinical parameters, extent of adhesions, and histological data.The level of significance was set at p<0.05.

FIGuRE 2 -
FIGuRE 2 -Median and range values of tensile strength, on postoperative third day, in animals treated with either bromopride (E3) or saline (C3).

FIGuRE 3 -
FIGuRE 3 -Median and range values of tensile strength, on postoperative seventh day, in animals treated with either bromopride (E7) or saline (C7).

FIGuRE 4 -
FIGuRE 4 -Median and range values of variation in hydroxyproline concentration, on postoperative third day, in animals treated with either bromopride (E3) or saline (C3).

FIGuRE 5 -
FIGuRE 5 -Median and range values of variation in hydroxyproline concentration, on postoperative seventh day, in animals treated with either bromopride (E7) or saline (C7).

TABLE 2 -
Percentage of anastomosis covered withadhesions in animals treated with either bromopride or saline.