Volume 25, Issue 1 , Pages 39-43, January 2011
Effect of bovine amniotic fluid on intra-abdominal adhesion in diabetic male rats
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results
- 4. Discussion
- Acknowledgments
- References
- Copyright
Abstract
Background
Postsurgical adhesion formation is a significant clinical problem within every surgical specialty. In type I diabetic patients, the problem is more severe and wound healing is slow. A wide variety of treatments have been proposed to deal with the problems that adhesion causes. One of the modalities that have not been studied extensively yet is the use of amniotic fluid. The purpose of the present study was to evaluate the clinical value of bovine amniotic fluid (BAF) efficacy in the treatment of postsurgical adhesion formation in diabetic male rats.
Materials and Methods
Fifty male Wistar rats in five groups were used for our study, with animal identification being facilitated by a microchip implant system. Diabetes was induced in all groups except for the control group by intraperitoneal alloxan injection (120 mg/kg). Based upon blood glucose concentration, rats received either one third of the required insulin (two groups) or all the required insulin (remaining groups). After 2 weeks, a laparotomy was performed on each rat and adhesions were scaled. Bovine amniotic fluid was then applied to two groups, and, as a control, sterilized water was applied to the other groups. After 2 weeks, a laparotomy was again performed on each rat and adhesion was rescored.
Results and Conclusion
Significant reductions (P<.05) in adhesions were seen with BAF only in those diabetic rats that had received the required insulin. The results of our study suggest that BAF could be effective in the treatment of adhesion formation during diabetes.
Keywords: Adhesions, Treatment, Diabetic rat, Bovine amniotic fluid
1. Introduction
Diabetes is a hereditary, chronic metabolic disease characterized by hyperglycemia and eventual glycosuria (Aguwa, 1998). It is caused by the inability of tissues to carry out normal metabolism of carbohydrates, fats, and proteins due to a deficiency in or absolute lack of insulin. It occurs in two main forms. The first is classified as type I, insulin-dependent diabetes mellitus or juvenile-onset diabetes. In this type of diabetes, there is an absolute lack of insulin and although it usually begins in late childhood (8–12 years), it can occur at any age. Type I diabetes is managed by diet and insulin. The second type is classified as type II, non–insulin-dependent diabetes mellitus or maturity-onset diabetes. One of the major pathophysiological features of type II diabetes is insulin resistance in peripheral tissues, i.e., a lack of insulin effects despite hyperinsulinaemia. A majority of patients in this class are obese. Type II diabetes may be controlled by diet and oral hypoglycaemic agents.
Postoperative adhesion formation is a significant clinical problem within every surgical specialty. Specifically, following almost every intra-abdominal surgical procedure, adhesions are frequently an ensuing complication. Bowel obstruction and/or chronic pelvic pain is a common complication, which results from a formation of adhesions. Furthermore, several studies have reported the occurrence of postsurgical intra-abdominal adhesions in the majority of women who had undergone gynecological surgery (Diamond, 1995).
A number of compounds and/or techniques for the purpose of prevention or treatment of the formation and reformation of adhesions have been proposed, but, unfortunately, an effective solution has not yet been achieved (Ellis, 1971, Ellis, 1982, Ellis, 2004, Ellis, 2005, Ellis, 2007). Studies on most of these treatments have produced inconsistent results. The application of various fluids to the peritoneum is one of the most frequent methods have been used. In an attempt to reduce the inflammatory reaction at the site of the peritoneal trauma, corticosteroids have been used and, subsequently, the antiadhesive properties of calcium channel blockers and antihistamines were studied. More recent studies deal with the use of nonsteroidal anti-inflammatory drugs, tissue plasminogen activators, and selective immunosuppressors (i.e., tumor necrosis factor and interleukin-1 antibodies). Varying results with all of these treatments have been reported (Bakkum and Trimbos, 1996, diZerega, 1994, diZerega and Tulandi, 2008, Pijlman et al., 1994, Risberg, 1997).
One of the modalities that have not been studied extensively yet is the use of amniotic fluid. Since bovine has a well-developed allantoic cavity and a large amount of amniotic fluid, bovine could be a useful source of amniotic fluid for use in various aspects of human diseases (Durmus and Han, 2006, Golan and Stolik, 1991, Gonenci and Altug, 2009, Ozgenel and Samli, 2001).
Bovine amniotic fluid (BAF) consists of proteins, minerals, and cells, the relative amounts of which may change during pregnancy (Thomsen, 1975).
The purpose of the present study was to evaluate the efficacy of amniotic fluid in the treatment of postsurgical adhesion formation in diabetic male rats.
2. Materials and methods
2.1. Animals
All experiments were performed on 50 almost 1-year-old Wistar male rats, weighing 200–250 g, in accordance with the guidelines of the Animal Ethics Committee of the University of Isfahan. All groups of rats were allowed to acclimate to the animal houses under standard conditions at the Isfahan Medical University for 1 week before use. The 50 rats were assigned to five different groups randomly before the diabetic induction procedure was initiated: a nondiabetic control group and four diabetic groups (each consisting of 10 rats). A microchip implant system was used for identification of individual rats.
2.2. Induction of diabetes
Diabetes was induced in four groups of rats using alloxan. Rats were fasted for 18 h with free access to water. A dose equivalent to 120 mg alloxan/kg body weight was dissolved in normal saline and administered intraperitoneally to the rats under light anesthesia (with ether). After 24 h, the extent of diabetic induction was monitored based on blood sugar levels. At regular time intervals, blood samples were withdrawn from the eye medial cantus using micropipettes after anesthesia with isofluran. The blood glucose was measured using a glucometer machine (Biomine, USA). Blood glucose levels of at least 280 mg/dl were accepted as the basal level for diabetes. Diabetes in two groups of rats was controlled by injection of insulin, whereas the other two groups received only one third of the required insulin.
2.3. Surgical techniques
The rats were anaesthetized with a combination of isofluran and nitrous oxide inhalation, after which the abdomen was shaved and swabbed with povidine iodine in preparation for surgery. The surgical procedures were performed under aseptic, but not sterile, conditions. Surgical gloves were washed extensively with saline before the start of the operative procedure in order to remove any particles of powder. The same researcher performed all surgical procedures.
This model involves excision of a defect of 3 cm in the white line. Then, three 1- to 2-cm abrasions were made on one side of the abdominal wall and 1–2 cm on another side was cut off. Manipulation of other tissues was minimized and care was taken to avoid gross bleeding from the injured sites. At the end of the procedure, the abdomen was closed in double layer in a continuous method (muscle and peritoneum with 3-0 catgut suture, skin with 3-0 silk suture). All rats were housed under controlled temperature (25±5°C) and 12/12 light/dark cycles with food and water.
2.4. Adhesions scoring and BAF application
The adhesion model that we used in this study is a highly reproducible and semiquantitative rat model for the evaluation of therapeutic modalities used for the treatment of postoperative adhesion formation and was developed specifically for this purpose (Canbaz et al., 1999). Adhesions were scored from 0 to 4 (Fig. 1) based on Canbaz's scoring method (Table 1). At 2 weeks after surgery, adhesions were scored and documented macroscopically according to their extent (Table 1). The same researcher scored all adhesions.
Fig. 1.
Grades 1 to 3 (A, B, and C, respectively) of intra-abdominal adhesion in rat.
Table 1. Canbaz adhesion severity scoring method
| Grade | Description |
|---|---|
| 0 | No adhesion |
| 1 | One adhesion band, no vessel, easily separated |
| 2 | Two thin adhesion bands, no vessel, easily separated |
| 3 | Three thin adhesion bands, no vessel, easily separated |
| 4 | More than three thin adhesion bands, easily separated with no vessel or defused adhesion bands with vessels |
The uteri of cows that carried only female fetus (5 months) were selected at the slaughterhouse after governmental inspection of each animal. Three milliliters of BAF from those cows was applied to the defect in each rat in one group of rats treated with insulin and in one group treated with one third of required insulin. For all other groups, 3 ml of sterilized water was applied. The abdomen was closed as before during the induction of adhesion, and after 2 weeks the adhesions were rescored.
2.5. Statistical analysis
A two-tailed Student's t test was used to analyze the results. All tests were done at the 5% level of significance. Calculations were done using either Microsoft Excel or GraphPad Prism. All data were expressed as mean±S.E., and statistical significance was defined as P<.05.
3. Results
An increase in the serum glucose level from 40 mg/dl in control to at least 280 mg/dl (474.8±66.7) was observed after alloxan administration. In some rats, the serum glucose level was higher than 500 mg/dl, which is the maximum serum glucose level that the machine can detect. The high level of blood glucose is indicative of type I diabetes in the groups to which alloxan was applied (Dunn & Letchie, 1943).
The scoring of adhesions in each group is shown in Table 2. Both the number of each adhesion grade and mean adhesions are reported. It should be noted that there were no surgical or postoperative mortalities, and in all animals studied, adhesion formation was confined to the peritoneal defects. Table 2 shows that more animals were scored as Grades 2 and 3 compared to the other grades. In this study, no Grade 4 was recorded. Adhesion formation had occurred in almost all animals following surgery. However, the peritoneal defects of the control animals had fewer adhesions than those of the alloxan-treated animals (Table 2). This result is consistent with the observation that diabetes can cause significantly more adhesion formation. During postoperative evaluation, nondiabetic control animals that were operated on without applying BAF showed an average adhesion score of 1.7±0.3 per peritoneal defect in the first scoring (fs) and 1.5±0.3 in the second scoring (ss). Thus, no significant differences in the mean adhesion scores between the first and second postoperative evaluations were observed in control animals (Fig. 2). In diabetic animals to which BAF was applied (Groups 2 and 3, Table 2), mean adhesion scores were 2.5±0.2 (Group 2; one third insulin) and 2.3±0.2 (Group 3; required insulin) in the first evaluation and 1.9±0.4 and 1.5±0.4 (respectively) in the second evaluation. A significant difference (P<.05) was observed only in those diabetic animals that had received the required insulin (Fig. 2). In diabetic animals to which BAF was not applied (Groups 4 and 5, Table 2), adhesion scoring was 2.6±0.2 and 2.2±0.2 in the first scoring and 2.3±0.2 and 1.8±0.3 in the second scoring, respectively. No significant difference was observed between the first scoring and second scoring in these animals (Fig. 2). An important reduction in adhesion was observed with all five groups, but only in Group 3 was it significant (P<.05).
Table 2. Grade of adhesions according to the Canbaz classification
| Groups | Grade | Mean±S.E. | |||||
|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | |||
| 1 (Control) | C-fs | 1 | 3 | 4 | 2 | 0 | 1.7±0.3 |
| C-ss | 1 | 5 | 2 | 2 | 0 | 1.5±0.3 | |
| 2 | D1/3i-fs | 0 | 1 | 3 | 6 | 0 | 2.5±0.2 |
| D1/3i-ss | 2 | 1 | 3 | 4 | 0 | 1.9±0.4 | |
| 3 | Dri-fs | 0 | 1 | 5 | 4 | 0 | 2.3±0.2 |
| Dri-ss | 3 | 2 | 2 | 3 | 0 | 1.5±0.4 | |
| 4 | D1/3i-fs | 0 | 1 | 2 | 7 | 0 | 2.6±0.2 |
| D1/3i-ss | 0 | 1 | 5 | 4 | 0 | 2.3±0.2 | |
| 5 | Dri-fs | 0 | 2 | 4 | 4 | 0 | 2.2±0.2 |
| Dri-ss | 1 | 3 | 3 | 3 | 0 | 1.8±0.3 | |
Fig. 2.
Comparison of mean scores between the first and second evaluation. Only in diabetic rats treated with BAF and that received the required insulin was a significant reduction in adhesion observed in the first scoring compared to the second scoring. C-fs: Control group, first scoring; C-ss: control group, second scoring; D1/3i-fs: diabetic group received one third insulin, first scoring; D1/3i-ss: diabetic group received one third insulin, second scoring; Dri-fs: diabetic group received the required insulin, first scoring; Dri-ss: diabetic group received the required insulin, second scoring.
4. Discussion
In the present study, bovine amniotic fluid (BAF) was studied for its efficacy in treating postoperative adhesion formation in a rat diabetic adhesion formation model. The rat models used were fasted. This shows that the amount of glucose in the gastrointestinal tract would have reduced to the barest minimum. During the period of fasting, the rats were allowed free access to water. With the very dilution of the abdominal contents, there could be glucose resumption (Franz, 1989) back to the intestine, which could act as a continual reservoir of glucose for further reabsorption into the blood when the blood glucose decreases drastically.
To prevent and to treat postoperation peritoneal adhesion formation, various substances, such as cow peritoneal and amniotic membrane, hyaluronic acid derivatives, oxidized celluloses, olive oil, soybean oil, liquid made from glucose, starch, glycerol, and many other materials, have been used with different mechanisms (Alponat and Lakshminarasappa, 1997, Avital and Bollinger, 2004, Dunn and Lyman, 2001, Ellis, 1982, Tayyar and Turan, 1993). One substance that is not considered widely used yet is BAF. The aim of this study was to determine the efficacy of BAF in treating peritoneal adhesion formation in diabetic rats.
In the present study, adhesion formation was more severe in diabetic rats than in the control group, but it was not significant. This enhancement in adhesion formation in diabetic rats could be due to the effect of diabetes in wound healing, which is very slow (Diamond, 1995).
The results demonstrate that application of distilled water has no effect on the treatment of adhesion formation. We chose distilled water as a control because no report has been found which shows its benefits in preventing or treating adhesion formation. Other animal studies reported a benefit from Ringer's lactate in preventing adhesions (Caballero and Tulandi, 1992, Tulandi, 1993). Because of these results, it was not considered as a control in this project. However, clinical data on the use of Ringer's lactate show no beneficial effect. In two clinical studies, the effect of dextran solution on postoperative adhesion formation was compared with that of Ringer's lactate solution (Jansen, 1985, Rosenburg and Board, 1984). In both studies, no beneficial effect of Ringer's lactate was found, and many of the patients treated with Ringer's lactate had more adhesions at the time of second-look laparoscopy than before the initial procedure.
The use of BAF has not been considered widely yet. In the present study, the peritoneal adhesions treated with BAF showed a significant decrease in the second laparotomy compared with the first one only in diabetic rats that received the required insulin, but no significant difference was found when compared with the control group. The results obtained with diabetic rats that did not receive the required insulin were somewhat disappointing. Although there were no significant differences between the first scoring and second scoring, the data showed a decrease in adhesion formation in these groups. On the other hand, on second-look laparotomy, in all groups a reduction in adhesion formation was found.
The mechanisms of substances that have been used to prevent or treat adhesion formation are various and for some they are unknown. One mechanism of these materials is the formation of a mechanical barrier between peritoneal surfaces. In the case of BAF, this could be true, but other parts such as BAF's cells, proteins, and minerals may play a role which should be studied in another project.
In summary, we have described the effect of BAF on a diabetic rat model. The adhesion model we used in the present study was very consistent in producing adhesions. Furthermore, no beneficial effect of sterilized water on adhesion formation was seen. Significant reductions in adhesion were seen with BAF, but only in diabetic animals that received the required insulin. It clearly shows the importance of controlling and treating diabetes. In conclusion, the results of this study suggest that BAF could be used in treating adhesion formation because it is inexpensive, readily available, and has minimal side effects. It is not clear which part of BAF is effective in preventing or treating adhesion formation. It is obvious that more investigations are needed on this issue.
Acknowledgments
We profoundly thank Dr. Tim Webb for his advice.
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PII: S1056-8727(09)00094-4
doi:10.1016/j.jdiacomp.2009.09.003
© 2011 Elsevier Inc. All rights reserved.
Volume 25, Issue 1 , Pages 39-43, January 2011
