Evaluation of the effects of carbon dioxide pneumoperitoneum on abdominal wall wound healing in rats undergoing segmental resection and anastomosis of the left colon 1

PURPOSE: To evaluate the influence of carbon dioxide pneumoperitoneum on abdominal wall wound healing in rats. METHODS: Eighty rats underwent laparotomy, segmental left colon resection, and anastomosis. The animals were divided into three experimental groups and one control group: EI = pneumoperitoneum for 30 minutes before laparotomy (n=20); EII = pneumoperitoneum for 30 minutes after abdominal closure (n=20); EIII = pneumoperitoneum for 30 minutes before laparotomy and 30 minutes after abdominal closure (n=20); C = control group, without pneumoperitoneum (n=20). In each group, 10 animals were killed 7 days and 10 animals 14 days postoperatively. A segment of the abdominal wall was resected and subjected to tensile strength testing. Another segment of abdominal muscle was used for histopathological analysis; the specimens were fixed in formalin and stained with hematoxylin and eosin. RESULTS: There were no differences in histopathology and tensile strength values among animals in the experimental and control groups 7 or 14 days after surgery. CONCLUSION: Under the present experimental conditions, carbon dioxide pneumoperitoneum did not interfere with abdominal wall wound healing.


Introduction
Wound dehiscence and incisional herniation of the abdominal wall are potential complications associated with surgical procedures that require access to the abdominal cavity via open surgery or converted laparoscopic surgery.These are also recurrent complications of poorly healing wounds.
Laparoscopic surgery is a minimally invasive procedure that requires the use of pneumoperitoneum to provide adequate surgical exposure and to enable good visualization and manipulation of the target organ.Carbon dioxide (CO 2 ) has been widely used in this procedure, being insufflated into the abdominal cavity to create a pneumoperitoneum, because it is easily excreted, does not support combustion, and is inexpensive.In addition, CO 2 is highly soluble in blood and diffuses into the tissues, thus decreasing the risk of gas embolism 3 .
The laparoscopic method allows the performance of procedures with reduced operating time, reduced pain, and early recovery after surgery.Furthermore, laparoscopy is associated with fewer postoperative complications, compared to traditional open surgery, and reduced inflammatory response, thus allowing patients to resume their professional and routine daily activities sooner if no surgical complications occur [1][2][3][4][5][6] .In addition to these advantages, pneumoperitoneum has been associated with less tumor growth due to surgical stress, in gastric cancer, in relation to open surgery. 4Despite all the benefits, laparoscopic surgery is still a more expensive alternative. 6though CO 2 pneumoperitoneum induces pathophysiological changes in the kidneys, spleen, and endocrine, cardiovascular, hemodynamic, and pulmonary systems, most changes have a transient effect and no clinical significance in the great majority of cases.The effects produced by pneumoperitoneum depend on the amount of CO 2 absorbed, intra-abdominal pressure, circulatory volume status, and prior medical conditions of each patient 3,5,6 .
The magnitude of the pathophysiological effects of pneumoperitoneum on different organs is highly influenced by intra-abdominal pressure, and the maintenance of pressure levels ≤ 12 mmHg in humans induces minimal effects 3,5 .However, pressures above 15 mmHg increase CO 2 absorption and the risk of gas embolism 5 .An intra-abdominal pressure of 5 mmHg in Wistar rats has been used to experimentally reproduce pathophysiological changes that occur in humans at a pressure of 12 mmHg 7 .
The objective of this study was to evaluate the influence of CO 2 pneumoperitoneum on abdominal wall wound healing in rats undergoing segmental resection and anastomosis of the left colon.

Methods
This study was conducted at the Laboratory of Pneumoperitoneum was induced using a laparoscopic insufflator.After abdominal puncture using a Veress needle, CO 2 was insufflated at a flow rate of 0.5 to 1.0 liter per minute, without preheating, to establish pressure at 5 mmHg.
Before surgical intervention, animals were weighed, anesthetized, and then fixed in supine position at their thoracic and abdominal limbs on a surgical board.Trichotomy of the anterior abdominal wall was performed, and polyvinylpyrrolidone iodine was used for skin antisepsis.
The surgical procedure consisted of a midline incision of the abdominal wall of about 5 cm.In all animals 1 cm of the left colon was resected approximately 2.5 to 3.5 cm proximal to the peritoneal reflection, followed by coloanal reconstruction with end-to-end anastomosis, using running 6-0 polypropylene sutures, whole plane.
The abdominal wall was closed with running 5-0 polypropylene sutures, with approximation of the aponeurosis and muscles.Skin incisions were closed using running 5-0 polypropylene sutures.All procedures were performed by one surgeon.
As previously scheduled, seven and 14 days after surgery, the animals were anesthetized and two samples were obtained from the anterior abdominal wall containing the surgical wound in the central portion: one specimen for histopathological analysis and the other for tensile strength testing.Finally, the inferior vena cava was punctured and the animals were killed with an overdose of thiopental.

Tensile strength
Tensile strength was measured in newton (N) using a VersaTest test stand (Mecmesin VersaTest, United Kingdom) fitted with a digital force gauge with force capacity of 2500 N (Mecmesin AFG 2500N, United Kingdom).A rectangular, skinless sample of the abdominal wall, about 1.0 cm wide, was fixed to the force gauge and subjected to tension at a speed of 25 mm/minute (Figures 1 and 2).
During tensile strength testing, the specimens were carefully placed and fixed in the test apparatus, with standardization of the distance between the surgical scar and the fixing clamps.

Histopathology
For histopathological analysis, the specimens were fixed in formalin, stained with hematoxylin and eosin, and examined under an optical microscope.The amount of collagen, fibroblasts, polymorphonuclear and mononuclear infiltration and neovascularization was analyzed and graded as 0, 1, or 2; with 0 indicating absence of the analyzed parameter, 1 indicating an average amount, and 2 for the samples with the highest amount of the analyzed factor.The presence or absence of abscess, bacterial colony, or foreign body was also analyzed.
The histopathologist examining the slides was blind to which group or subgroup the specimens belonged.

Weighing
Body weight variations between the day of surgery and reoperation were also analyzed in this study.Body mass was measured using a precision scale (0.01 grams resolution). Morais

Results
Four animals died during the study: one animal in the C7 group and three animals scheduled for euthanasia 14 days after surgery (two in EI14 group and one in EIII14 group).Therefore, the sample for final evaluation included 76 animals.
Body weight variation of animals, according to the day of euthanasia, is shown in Table 1.
The mean preoperative body weight of all animals was The main data on tensile strength (minimum and maximum values, median, mean and standard deviation) are shown in Table 2.The mean tensile strength of the abdominal wall of animals killed 7 and 14 days postoperatively was 6.38 and 8.17 N, respectively, with statistically significant difference (p = 0.001) (Table 2 and Figure 3).TS at day 7 < TS at day 14 (p = 0.001).There was no statistically significant difference in tensile strength between experimental groups (EI, EII, EIII) and the control group (C) 7 days after surgery (p = 0.712).Likewise, there was no statistically significant difference in tensile strength among the four groups 14 days after surgery (p = 0.580) (Table 3 and Figure 4).4).
*Tables 4 and 5 on next page.

Discussion
The present study investigated possible effects of CO 2 pneumoperitoneum by experimentally using pressures equivalent to those used in laparoscopic surgery in humans.Four conditions were established in rats in order to simulate those occurring in Postoperative pneumoperitoneum mimics a situation in which, after open surgery, during the postoperative period, there is a need to approach the peritoneal cavity and exploratory laparoscopy is the option adopted.
Pre and postoperative pneumoperitoneum is consistent with a common situation observed in colorectal surgical practice in which laparoscopic colectomy, in most cases, is performed using a hybrid approach or there is a need to open the abdominal wall to remove the surgical specimen.In this situation, laparoscopic procedures often require a small laparotomy, thus leaving a surgical wound in the abdominal wall.
In our study, the weight of the animals decreased significantly within 7 days of surgery.However, the weight of animals analyzed 14 days postoperatively shows a complete reversal of this catabolic process based on the significant weight gain observed on day 14.These data replicate those reported in the relevant literature describing that weight loss occurring in the postoperative period is recovered after the 7th postoperative day 8,9 .
The most commonly used parameters to evaluate the healing process include clinical assessment of animals, tensile strength, analysis of anti-inflammatory activity, collagenous tissue expressed by hydroxyproline concentration or morphometric analysis, and histopathological analysis using optical microscopy [10][11][12] .Among these parameters, measurement of tensile strength is particularly relevant in cases of intervention through the abdominal wall, because tensile strength testing provides accurate and prompt results and reflects the reality of clinical practice, in which a pulling force is exerted on the surgical wound whenever patients contract their abdomen 9,11,13 .reflect interference in some stage of the wound healing process.

Histopathological and tensile strength abnormalities
The first stage of healing involves hemostasis and inflammation and may be affected mainly by anti-inflammatory agents, among others, which may reduce the tensile strength of the abdominal wall due to reduced collagen formation.As a result, higher rates of wound dehiscence, incisional hernia, and weight loss may be observed 14,15 .
CO 2 pneumoperitoneum has been reported to induce a decrease in the amount of type I and II collagen 10 days postoperatively in a dorsal wound.However, in the same period, the use of helium to create the pneumoperitoneum has been shown to increase collagen synthesis 10,12 .
It remains uncertain how CO 2 pneumoperitoneum may affect the healing process in the abdominal wall.It has been suggested that CO 2 pneumoperitoneum may affect tensile strength, because a decrease in pH is observed in the subcutaneous, intraarterial, and intra-abdominal tissue after the creation of a CO 2 pneumoperitoneum, a fact that leads to suppression of cellular differentiation of lineages derived from monocytes (which have a role in healing), thus altering collagen formation locally and systemically 10,12,17 .
However, in the present study, histopathological and biomechanical data evaluated 7 and 14 days postoperatively substantiate the hypothesis that CO 2 pneumoperitoneum at a pressure corresponding to that used in laparoscopic surgical procedures in humans does not affect abdominal wall wound healing.This result is in agreement with previous studies reporting that CO 2 pneumoperitoneum and laparotomy had no systemic influence on wound healing, since no influence was observed in dorsal wound repair in rats 18,19 .
Potential effects of chemical and cellular magnitude, such as those observed in metalloproteinases or peritoneal macrophage concentration, can be regarded as minimal, thus failing to promote changes in the criteria analyzed and, consequently, failing to present important clinical significance 4,8,10,12,17 .
Similarly, CO 2 pneumoperitoneum at a pressure of 5 mmHg may induce only transient effects on the healing process in the abdomen, as observed in other organs, and such effects may therefore not be detectable after 7 or 14 days 3,5 .

Conclusion
Under the present experimental conditions, CO 2 pneumoperitoneum did not interfere with abdominal wall wound healing.

FIGURE 1 -
FIGURE 1 -Sample of the abdominal wall used for measurement of tensile strength, with suture in the central portion.

FIGURE 2 -
FIGURE 2 -Sample of the abdominal wall subjected to tension for measurement of tensile strength.

TABLE 1 -
350.48 g.Animals killed 7 days after surgery showed a significant weight loss.The mean body weight of animals killed 7 days postoperatively was 355.97 g before surgery and 331.54 g at day 7 (p < 0.001).Animals killed 14 days after surgery gained weight in relation to the day of surgery, with mean body weight of 344.70 g before surgery and 354.57g at day 14 (p = 0.001).Body weight in grams (g) at surgery and 7 and 14 days postoperatively (minimum and maximum values, median, mean and standard deviation).

FIGURE 3 -
FIGURE 3 -Tensile strength (TS) of the abdominal wall in Newton (N) of animals killed 7 and 14 days after surgery (p = 0.001).

FIGURE 4 -
FIGURE 4 -Tensile strength (TS) of the abdominal wall in Newton (N) for the four study groups (EI, EII, EIII, C) 7 and 14 days after surgery.
surgical practice: preoperative pneumoperitoneum; postoperative pneumoperitoneum; pre and postoperative pneumoperitoneum; and laparotomy without pneumoperitoneum (control).Preoperative pneumoperitoneum aimed to simulate a situation in which surgery begins with laparoscopy, but requires a switch to laparotomy.According to reports on laparoscopic ventral hernia repair, 7.69% of the procedures required a switch to open surgery due to complications 2 .

TABLE 2 -
Tensile strength (TS) of the abdominal wall in Newton (N) of animals killed 7 and 14 days after surgery (minimum and maximum values, median, mean and standard deviation).

TABLE 4 -
Values of histopathological analysis in groups of animals killed 7 days after surgery.EI = pneumoperitoneum for 30 minutes before laparotomy; EII = pneumoperitoneum for 30 minutes after abdominal closure; EIII = pneumoperitoneum for 30 minutes before laparotomy and 30 minutes after abdominal closure; C = control group, without pneumoperitoneum.

TABLE 5 -
Values of histopathological analysis in groups of animals killed 14 days after surgery.EI = pneumoperitoneum for 30 minutes before laparotomy; EII = pneumoperitoneum for 30 minutes after abdominal closure; EIII = pneumoperitoneum for 30 minutes before laparotomy and 30 minutes after abdominal closure; C = control group, without pneumoperitoneum.Evaluation of the effects of carbon dioxide pneumoperitoneum on abdominal wall wound healing in rats undergoing segmental resection and anastomosis of the left colon Acta Cirúrgica Brasileira -Vol.27(1)2012-69