Peritoneal Infusion with Cold Saline Decreased Postoperative Intra-Abdominal Adhesion Formation
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Postoperative intra-abdominal adhesion is a common morbidity after laparotomy. We try to determine whether peritoneal infusion with cold saline may decrease postoperative intra-abdominal adhesion formation.
Ninety-six male BALB/c mice weighing 25-30 g were randomized into four groups: (I) adhesion model with infusion of 4°C cold saline, (II) adhesion model with infusion of room temperature saline, (III) adhesion model without infusion of saline, and (IV) sham operation without infusion of saline. Adhesion scores, incidence of adhesion, and serum cytokines were measured at postoperative days 1, 3, 7, and 14.
Group I had lower adhesion scores than groups II and III (P < 0.0001). IL-6, IL-10, and TNF-α were significantly increased in the groups I, II, and III compared to group IV (P < 0.0001). IL-6 in group I was significantly decreased compared to that in group III (P < 0.0004). IL-10 in group I was significantly increased compared to that in groups II (P < 0.0001) and III (P < 0.05). TNF-α in group I was significantly decreased compared to that in groups II (P < 0.0004), and III (P < 0.05).
Peritoneal infusion with cold saline may decrease the degree of postoperative intra-abdominal adhesion formation.
Postoperative intra-abdominal adhesion is a major source of morbidity after laparotomy and is thought to be the most common cause of small-bowel obstruction in adults [1, 2, 3, 4, 5, 6]. Adhesive small-bowel obstruction is a lifetime risk for a patient after abdominal and pelvic surgery, and its treatment, both conservative and operative, is accompanied by substantial morbidity and mortality and has a high socioeconomic impact [7, 8, 9]. The estimated annual cost of lower abdominal-pelvic adhesionolysis was in excess of $US 1,000 million . Although most adhesive small-bowel obstruction can be managed successfully with a conservative method, it is a potentially fatal condition with a mortality rate of approximately 3-30% [1, 9].
Adhesion formation, the adhering of two separate surfaces due to trauma or inflammation, can result in complications following surgical procedures . When the peritoneum is injured, the injured surface becomes re-epithelialized with mesothelial cells within 5-6 days. A subsequent inflammatory response consisting of polymorphonuclear leukocytes, macrophages, fibroblasts, fibrin, and new blood vessels may occur following peritoneal injury. These processes may lead to the development of mature and fibrous adhesions .
Many methods have been used to inhibit intra-abdominal adhesion, most with limited success [13, 14, 15]. Because adhesion formations are formed through a subsequent inflammatory reaction, a recent study reported the reduction of intra-abdominal adhesion in a mice cecal abrasion model of adhesions with nonsteroidal anti-inflammatory drugs . We hypothesized that intra-abdominal adhesion may be reduced if the process of inflammation is depressed. The current study was undertaken to determine whether decreased local inflammatory response using peritoneal infusion of cold saline would effectively decrease the incidence and the degree of postoperative intra-abdominal adhesion formation.
The animal experiments reported in this study adhered to the guidelines established in the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Health Research Institutes (NHRI). All animals used were humanely cared for. BALB/c mice were kept on a 10-h light:14-h dark cycle and allowed free access to drinking water and laboratory chow.
Male BALB/c mice were anesthetized with intraperitoneal anesthetic (pentobarbital 50 mg/kg) and the operation was carried out under semisterile conditions. After the abdominal hair was shaved and the skin was sterilized with chlorhexidine, a low 2-cm midline laparotomy was performed. Ninety-six male BALB/c mice weighing 25-30 g were randomized into four groups as follows: (I) adhesion model with infusion of 4°C cold saline (n = 24), (II) adhesion model with infusion of room temperature saline (n = 24), (III) adhesion model without infusion of saline (n = 24), and (IV) sham operation without infusion of saline (n = 24). The standard adhesion model was performed as previously described . The anterior cecal wall was abraded with 20 strokes of a toothbrush and full-thickness 4-0 silk sutures were placed in the traumatized anterior cecal wall to increase the adhesion formation.
Peritoneal infusion of cold saline
After the adhesion model was completed, the bilateral abdominal wall was lifted 0.5 cm higher than the abdominal viscera with a 3-0 silk suture. Group I received an additional 30 min of cold saline infusion. The infusion of cold saline was made by continuous dripping of 4°C normal saline into the abdominal cavity until the cold saline is ran out. Group II received 30 min of room temperature saline infusion. In groups III and IV without infusion of saline, their abdominal cavity was opened for 30 min. When 30 min of infusion of saline ended, the residual saline in the abdominal cavity was drained out and the abdominal incision was closed with 3-0 absorbable surgical suture. The temperature was measured by inserting a temperature probe into the abdominal cavity beneath the small-bowel loop before and 30 min after the 4°C and room temperature saline infusion.
After 1, 3, 7, and 14 days, groups of six mice were killed by cervical dislocation. A well-documented adhesion-severity scoring system was used to measure the degree of adhesion formation . In brief, a score of 0 denotes no visible adhesion; 1 denotes that only one adhesion is found at one area in the peritoneal cavity; 2 denotes that mild adhesions are found at more than one area in the peritoneal cavity; 3 denotes that moderate adhesions are found at more than one area in the peritoneal cavity; and 4 denotes that severe adhesions are found at more than one area in the peritoneal cavity. Mild adhesions are those that can be lysed with traction, moderate adhesions lysed with blunt dissection, and severe adhesions lysed with sharp dissection.
Blood samples were obtained for interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) at days 1, 3, 7, and 14. IL-6, IL-10, and TNF-α were determined in cell culture supernatants by a commercially available enzyme-linked immunosorbent assay, with specific monoclonal antibodies performed according to the manufacturer’s instructions (Biosource International, Camarillo, CA, USA). The detection limit was 4, 8, and 5 pg/ml of rat TNF-α, rat IL-6, and rat IL-10, respectively. The reliability of this kit has been proven in previous studies [17, 18].
All values are presented as mean ± standard deviation using SPSS-PC statistical package (release 13.0, SPSS Inc., Chicago, IL, USA). Statistical significance was assessed with a two-sided t test by comparing the adhesion scores of the study group with corresponding the scores derived from sham controls. A P value of less than 0.05 was considered significant. Differences of serum level cytokine between and within the three groups of mice were analyzed by repeated analysis of variance (emANOVA) with a grouping factor. The repeated measure was time, and treatment strategy was used as the grouping factor. Post hoc analysis was performed using Tukey’s method.
Presence of adhesions
The incidence of adhesions in group I was 50.0% at day 1, 66.7% at day 3, 66.7% at day 7, and 66.7% at day 14. The incidence of adhesions in group II was 83.3% at day 1, 100% at day 3, 100% at day 7, and 100% at day 14. The incidence of adhesions in group III was 100% from day 1 to day 14. The incidence of adhesions in group IV was 0 from day 1 to day 14. A comparison of the number of adhesions between groups I, II, and III shows that group I animals had decreased the incidence of adhesion formation.
The mean temperature with cold and room temperature saline infusion from time 0 to 30 min in groups I and II is 34.4-21.5°C and 34.2-28.6°C, respectively. The mean temperature without saline infusion from time 0 to 30 min in groups III and IV is 34.2-34.5°C and 34.5-34.1°C, respectively.
At the end of the study, infusion of cold saline resulted in two deaths due to hypothermia. No mortality was found in other groups.
To our knowledge this is the first study to demonstrate that peritoneal infusion of cold saline may decrease postoperative intra-abdominal adhesion formation. Infusion of cold saline not only decreased proinflammatory cytokine production but also decreased adhesion formation. When postoperative inflammation was depressed, the degree of intra-abdominal adhesion formation was decreased.
The process that leads to adhesion formation, caused by surgical trauma, has largely been unexplored. The mechanism of peritoneal repair is now beginning to become clear as being a complex process in which several cell types and molecular mediators act in concert to restore tissue integrity. From a pathophysiologic standpoint, the process of adhesion formation could be separated into four phases: (1) a postinjury inflammatory phase, (2) a fibrin dissolution phase, (3) a fibrous phase, and (4) a remodeling and phagocytotic phase . In the postinjury inflammatory phase, leukocytes are recruited and fibrin is deposited. A characteristic feature of inflammation is the extravasation of plasma proteins, including fibrinogen, and the magnitude of inflammation is clinically likely to have a greater impact on the amount of fibrin deposited.
It has been reported that postoperative fluid concentrations of IL-6 and TNF-α were elevated in peritoneal lavage [20, 21, 22]. IL-6 and TNF-α are proinflammatory cytokines that are derived mainly from activated macrophages and contribute to the immunosuppressive effect after trauma . These proinflammatory cytokines are thought to be partly responsible for the early activation of the acute phase response . We did not check the peritoneal fluid and used cold saline to determine the degree of inflammation and adhesion formation. We also checked the serum IL-6, IL-10, and TNF-α levels and found that they had increased after the adhesion model was completed. However, the level of proinflammatory cytokine IL-6 was significantly decreased after cold saline infusion. On the other hand, the level of anti-inflammatory cytokine IL-10 was significantly increased after cold saline infusion. These cytokine responses confirmed that cold saline might reduce the degree of inflammation thereby decreasing the degree of adhesion formation. Because two deaths due to hypothermia were noted during cold saline infusion, use of cold saline at temperature greater than 4°C is suggested. Besides the two mortalities, no other prominent side effect was noted. The reason why we chose 4°C normal saline is because it was easily obtained from the common refrigerator.
It has been reported that saline could promote intraperitoneal adhesions in rat ; however, a recent report showed that saline was associated with low adhesion formation . Peritoneal lavage with lactated Ringer’s solution also can decrease the frequency of adhesion formation . Our study found that infusion of cold saline could decrease the incidence and degree of postoperative adhesion formation. Peritoneal infusion with cold saline may reduce the incidence and degree of adhesion formation by four probable mechanisms: (1) lowering the degree of inflammation, (2) removal of inflammatory mediators that promote fibrin production, (3) separation of small bowel loops, and (4) removal of fibrin from serosal surfaces and subsequently decrease the adhesion formation.
Many strategies for adhesion prevention using drugs to inhibit inflammatory reaction have been reported. Nonsteroidal anti-inflammatory drugs (NSAIDs), which interfere with prostaglandin synthesis and decrease the initial inflammatory response, have been used to reduce adhesions. The results of studies on NSAIDs have been contradictory in terms of their effectiveness in decreasing adhesions [28, 29, 30]. Guvenal et al.  reported the reduction of adhesions in a rat uterine horn model of adhesions with the selective cyclooxygenase-2 enzyme (COX-2) inhibitor nimesulide. Their proposed mechanism of action in this model was the anti-inflammatory effect. Greene et al.  used a mice cecal abrasion model of adhesions and showed that a selective COX-2 inhibitor, i.e., celecoxib, inhibited intra-abdominal adhesion. Corticosteroid also has been tested to see if it prevents adhesions, but its value has been found to be equivocal or even harmful in some studies [32, 33, 34]. Our study contributes to the literature by showing the anti-inflammatory effect by peritoneal infusion of cold saline which also has the effect of reducing intra-abdominal adhesion formation.
Another common method of preventing adhesion formation is the use of a barrier agent or gel to separate damaged peritoneal surfaces from the bowel loop. The most extensively studied barrier, and the most efficacious, is a hyaluronan-based agent that is available as both a viscous solution and a membrane. Hyaluronan-based agents have the potential to decrease adhesion formation . The bioresorbable membrane consisting of hyaluronan and carboxymethylcellulose is most commonly known as Seprafilm (Genzyme Corporation, Cambridge, MA). Both animal and human studies have found a significant decrease in adhesion with the use of Seprafilm [36, 37, 38]. However, a prospective, randomized, multicenter, multinational, single-blind, controlled study showed that there was no difference between the Seprafilm-treated group and the control group in overall rate of bowel obstruction. Stepwise multivariate analysis indicated that Seprafilm was the only predictive factor for reducing adhesive small-bowel obstruction requiring reoperation .
To date, there is no way to completely prevent postoperative adhesion formation. Compared with other studied barriers, infusion of cold saline is a simple, easy, and cost-effective method to prevent postoperative adhesion formation.
There are several issues that need to be answered in future studies. First, we infused 4°C cold saline in this study and had two deaths due to hypothermia. The most appropriate temperature for cold saline infusion is not known and use cold saline warmer than 4°C is suggested. Whether the lower the temperature the less the adhesion warrants further study. Second, the duration of the infusion of cold saline in this study was 30 min but the ideal duration of infusion needs to be defined. Whether the longer the infusion the less the adhesion warrants more study. Third, we used the cecal wall abrasion model which is different from the use of a silicone patch to generate adhesion . Different adhesion models may produce different results. Fourth, different adhesion models yield different adhesion scores. Our adhesion score is only the grade of cecal adhesion and does not include the tenacity and extent of adhesion associated with the patch. Further investigations to determine the role of different adhesion models and scores are needed.
In conclusion, our study showed that peritoneal infusion of cold saline might decrease postoperative intra-abdominal adhesion formation in a mouse model.
This study was partly supported by the National Science Council Grant NSC 93-2314-B-002-260.
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