Valerie Montgomery Rice, M.D. Aida Shanti, M.D. Kamran S. Moghissi, M.D. Richard E. Leach, M.D.

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1 FERTILITY AND STERILITY Copyright 1993 The American Fertility Society Vol. 59, No.4, April 1993 Printed on acid-free paper in U.S.A. A comparative evaluation of Poloxamer 407* and oxidized regenerated cellulose (lnterceed [TC7]*) to reduce postoperative adhesion formation in the rat uterine horn modelt* Valerie Montgomery Rice, M.D. Aida Shanti, M.D. Kamran S. Moghissi, M.D. Richard E. Leach, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Hutzel Hospital, Wayne State University, Detroit, Michigan Objectives: To determine if Poloxamer 407 (poloxamer, Pluronic F -127; BASF Wyandotte Corp., Parsippany, NJ) is as effective as Interceed(TC7) (Ethicon, a Johnson and Johnson company, Sommerville, NJ) in preventing postoperative adhesion formation using the rat uterine horn model and to determine if the presence of blood or lactated Ringer's solution affects the effectiveness of Poloxamer 407. Design, Setting, Participants: Sprague-Dawley white rats, weighing 225 to 250 g in a conventionallaboratory setting. The left or right sidewall was randomly assigned to receive no treatment (control), Interceed(TC7), or poloxamer. Interventions: Each uterine horn and ipsilateral sidewall was subjected to a standardized lesion of denudation. To evaluate the barrier agents in the presence of blood, a sidewall vessel was ligated and a thrombus allowed to form. To evaluate the effectiveness of lactated Ringer's solution, 10 ml was injected intraperitoneally after abdominal closure. Main Outcome Measures: Degree of adhesion formation was evaluated 14 days after surgery. Results: The adhesion score for the poloxamer-treated animals was significantly lower than its control. Interceed(TC7) did not reduce adhesion formation as compared with its control. In animals that received both poloxamer and Interceed(TC7) on either side, the poloxamer-treated sides had a significantly lower adhesion score than Interceed(TC7)-treated sides. The presence of blood and lactated Ringer's reduced the adhesion-reducing properties of poloxamer. Conclusion: In this model, poloxamer is more effective than Interceed(TC7) in the prevention of postoperative adhesion formation. These findings also suggest that the presence of blood compromises the effectiveness of poloxamer to prevent postoperative adhesion formation, therefore requiring complete hemostasis before poloxamer's application. Lactated Ringer's instillation was ineffective in reducing adhesion formation on the control or poloxamer-treated sidewall. Fertil Steril 1993;59:901-6 Key Words: Adhesion prevention, oxidized regenerated cellulose (Interceed[TC7]), Poloxamer 407 Received September 14, 1992; revised and accepted December 10,1992. * Poloxamer 407: Pluronic F-127, BASF Wynadotte Corp, Parsippany, New Jersey; Interceed(TC7): Ethicon, a Johnson and Johnson Company, Sommerville, New Jersey. t Supported in part by the American College of Obstetrics and Gynecology/Ethicon Research Award for Innovations in Gynecologic Surgery and Mediventures, Detroit, Michigan. :j: Presented in part at the 39th Annual Meeting of The Society for Gynecologic Investigations, San Antonio, Texas, March 18 to 21,1992. The use of barrier surgical adjuvants has been shown to effectively reduce primary adhesion formation and, in some series, adhesion reformation in both animal and human studies (1-8). The presumed Reprint requests: Richard E. Leach, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Hutzel Hospital, 4707 Saint Antoine Boulevard, Detroit, Michigan Vol. 59, No.4, April 1993 Montgomery Rice et al. Evaluation of poloxamer and Interceed(TC7) 901

2 mechanism of action is the separation of opposing injured peritoneum until the mesothelium is regenerated 5 to 7 days later. Two reports have documented that the barrier separation of Interceed(TC7) (Ethicon, a Johnson and Johnson Company, Sommerville, NJ) and Poloxamer 407 (Pluronic F-127; BASF Wyandotte Corp., Parsippany, NJ) are in fact absent by 72 hours, suggesting that some other factor or factors are involved in adhesion reduction in addition to tissue separation (1,5). Alternatively, separation for periods less than those required for complete reperitonealization may be sufficient to impact favorably on adhesion formation. Each adjuvant has its unique advantages that provide for specific surgical application. Interceed(TC7) allows for the separation of large areas of injured parietal peritoneum with relative ease of application. Further, Interceed(TC7) has been documented to be efficacious in women with endometriosis (6). Poloxamer 407 is a copolymer that is in the liquid phase at room temperature. When the solution is raised to body temperature, viscosity is increased and the solution forms a firm gel. This adjuvant is effective in reducing adhesion formation in several animal models (1, 2, 7). The advantages of poloxamer is that it can be applied to areas not accessible by Interceed(TC7) and ease of laparoscopic placement. The purpose of this investigation was to compare directly the adhesion reducing properties of these adjuvants and to evaluate the effects of the addition of blood and lactated Ringer's solution to the efficacy of poloxamer. Animals MATERIALS AND METHODS Adult female Sprague-Dawley rats weighing 200 to 300 g were obtained from Charles River Breeding Laboratories (Port Huron, MI) and housed one to three animals per cage in a 14-hour light, lo-hour dark cycle. Animals were fed standard rat chow and water ad libitum. After surgery, animals were housed separately in an insulated cage. The study was approved by the Animal Investigation Committee, Wayne State University, Detroit, Michigan. Barrier Materials Poloxamer 407 is a 28% aqueous solution of an osmotically balanced polyol and other additives which is a proprietary formulation prepared by Mediventures Inc., Detroit, Michigan. Poloxamer 407 is metabolized in the liver and excreted in bile and urine (1). During the operative procedure, poloxamer is maintained in an icebath at 4 DC and is applied to the defect with an applicating needle that is also maintained at 4 DC. Interceed(TC7) is a woven fabric of oxidized-regenerated cellulose that is biodegradable. Surgical Technique Anesthesia was induced with a combination of ketamine (80 mg/kg) and xylazine (4 mg/kg) intraperitoneally through the left subcostal region of the ventral wall. The abdominal wall was shaved and prepped with betadine. Laparotomy was performed / under clean but not sterile techniques through a 5-cm midline abdominal vertical incision exposing the uterus and parietal sidewalls. A bilateral l-cm parietal area on the lateral side at the level of uterine horn was excised, including part of the underlying muscle. Hemostasis was maintained with pressure followed by cauterization as needed. A l-cm segment of each uterine horn at the level of the sidewall defect was stripped of its serosa and approximated to within 0.5 cm of the sidewall defect using a single 3.0 Vicrylligature (Fig. 1). In the first experiment, the rats were randomly assigned to three groups (Table 1). Within each group, randomization ofthe respective agent to the left or right side was also performed. In the Interceed(TC7) group the sheet was sized to adequately cover the denuded area on the side wall. Poloxamer held at 4 DC was applied to the sidewall defect with the cooled applicating needle and smoothed in place. Approximately 1.0 ml of the poloxamer solution was needed to cover the sidewall defect. Care was taken that both Interceed(TC7) and poloxamer remained in place at the completion of the operation. The control sides had both injuries induced and approximated to the sidewall without treatment. The abdominal wall was closed using a single layer of continuous 3.0 Vicryl suture, followed by staples for closure of the skin. In the second experiment (group 4), the above described injury was induced with each side randomized to receive poloxamer or nothing. After closure of the abdominal wall, 10 ml of lactated Ringer's solution mixed with methylene blue was injected into the abdominal cavity. The skin was closed with staples and the animal placed on a white towel to assess possible leakage ofthe lactated Ringer's from the abdominal cavity. In the third experiment, to explore the effect of the presence of blood in combination with poloxamer 902 Montgomery Rice et al. Evaluation of poloxamer and Interceed(TC7) Fertility and Sterility

3 or Interceed(TC7), the above surgical technique with the exception of controlling for hemostasis was performed. Each side was randomized to receive poloxamer or Interceed(TC7) to the left or right side. During the step of excising the parietal peritoneum, a ventral sidewall vessel was incised ensuring that a thrombus was present over the injured area before applying the barrier agent (group 5). The remainder of the surgical technique was the same. Fourteen days after surgery, the animals were lightly anesthetized using carbon dioxide and killed via cervical dislocation. At laparotomy the presence and degree of adhesions were graded independently by two investigators who were blinded to the therapy used. The adhesions on each side were graded using the following modified Linsky Grading System (3): 0, no adhesions; 1, adhesions on 1% to 25% of the traumatized area; 2, adhesions on 26% to 50% of the traumatized area; and 3, 51% to 100% of the Table 1 Group * 5t Treatment Groups No. of animals * Lactated Ringer's instillation. t In the presence of blood. Treatment Poloxamer versus control Interceed(TC7) versus control Poloxamer versus Interceed(TC7) Poloxamer versus control Poloxamer versus Interceed(TC7) traumatized area. The character of the adhesions was noted but not included in the grading system because the majority of the adhesions observed required minimum force for separation. Therefore the adhesion scores were graded on a scale of 0 to 3. Statistical Methods The differences seen in the total adhesion scores between the Interceed(TC7), poloxamer, and control A. ~_,!ft_, \ '\...""f B c Figure 1 The rat uterine horn model employed to induce injury to the uterine horn and sidewall. (A), A 1- cm parietal area on the sidewall at the level of the uterine horn is excised, including part of the underlying muscle and a 1-cm area on the ipsilateral uterine horn is stripped of its serosa. (B), The barrier agent is applied. (C), The two defects are approximated to within 0.5 cm of each other. (Reprinted by permission of Mayo Foundation, Rochester, Minnesota.) Vol. 59, No.4, April 1993 Montgomery Rice et a1. Evaluation of poloxamer and Interceed(TC7) 903

4 Control Treatment Pol o Treatment Te7 With LR Without LR Poloxamer TCl Pol Te7 Barrier agent Figure 2 Mean (±SE) adhesion score 14 days after surgery. Each treatment group is as depicted in Table 1. *P s 0.01 (X2). Poloxamer (Pol), Interceed(TC7). sides were analyzed using X2 test, with significance at P < All results are expressed as means ± SEM. RESULTS On the control sides, adhesions were produced in 46 of 59 (78%) of the animals. The total adhesion score of the untreated control side in groups 1 (n = 20) and 2 (n = 20) was 1.55 ± 0.27 and 1.20 ± 0.24 (mean ± SE), respectively, thereby demonstrating the ability of the model to promote adhesion formation. As shown in Figure 2, there was a significant difference in the total adhesion score between the poloxamer-treated side and its paired control untreated side (P < 0.01). In the Interceed(TC7) treated group (group 2), the total score of the adhesion formation was similar on the treated side when compared with the untreated side. When comparing the total adhesion score for the animals treated with poloxamer and Interceed(TC7) on opposite sides, the poloxamer-treated sides showed a significant reduction in adhesion formation as compared with the Interceed(TC7)-treated sides. This difference was found to be significant at a level of P = In the presence of lactated Ringer's (group 4; Fig. 3), the poloxamer-treated side had a total adhesion score similar to its control side. With poloxamer treatment, the addition of lactated Ringer's resulted in a significantly higher score (0.95 ± 0.18 to 1.53 ± 0.22, P < 0.05). On the control side, in the presence of lactated Ringer's the total adhesion score decreased but not significantly (1.55 ± 0.27 to 1.42 ± 0.23, P = 0.48). There was no leakage of lactated Ringer's solution observed postoperatively. Poloxamer Barrier Agent Control Figure 3 Mean (±SE) adhesion score 14 days after surgery. Group 1 (Table 1) represents the treatment without the presence of lactated Ringer's group 4 (Table 1) represents treatment in the presence of lactated Ringer's. * P s 0.05 (X2). The addition of blood to the injured site appeared to attenuate the adhesion-reducing properties of both poloxamer and Interceed(TC7). As shown in Figure 4, the mean adhesion score for the nonhemostatic poloxamer and Interceed(TC7)-treated sides (group 5) was increased as compared with its hemostatic-treated side (group 3). The difference seen between the hemostatic and nonhemostatic sides treated with poloxamer was significantly different at P < 0.05 but not for the Interceed(TC7) sides, P = There was no statistical difference observed in any group when the barrier agent was placed on the right or left side. DISCUSSION The rat uterine horn model used in this investigation has been previously reported in the rabbit Poloxamer Barrier Agent TCl Hemostatic II Non-hemostatic Figure 4 Mean (±SE) adhesion score 14 days after surgery. Group 3 (Table 1) represents the hemostatic-treated sides. Group 5 (Table 1) represents the non-hemostatic-treated sides. *P s 0.01 (X2). Interceed(TC7). 904 Montgomery Rice et al. Evaluation af palaxamer and Interceed(TC7) Fertility and Sterility

5 (8). The model has been generally reliable in inducing adhesion formation ranging from 74% to 100% (1,3,8). We have previously reported a 98% adhesion formation rate using the rat uterine horn model (1). The adhesions formed were dense and vascular in the control untreated sites with a mean score of 3.6 out of a total of 4 from a scale proposed by Linsky et al. (3). In this experiment the control untreated sides generated adhesions in 78% of the animals. However, the adhesions present were generally filmy and avascular, precluding the assignment of a character score. For this reason, only the area of the injured area covered by adhesion was analyzed. One reason for the difference in the grade of adhesions from these two series includes the difference in the time that the adhesions were graded postoperatively, 14 days in this study compared with 21 days in the previous study (1). Second, seasonal differences for biological responses have been described and may be a variable not controlled for because the former study was performed in the spring and the current study in the fall and winter. This possible seasonal propensity to generate adhesions of differing grade is currently under investigation. The area of adhesion generated remained a statistically valid measure of comparing the various treatment groups with control sides and with each other. As has been previously described (1, 2, 7), poloxamer in our study was found to be efficacious in reducing adhesion formation when compared with untreated sides. On the other hand, no difference in the adhesion score was observed between the Interceed(TC7)-treated side and the untreated side. Three other studies evaluating Interceed(TC7) failed to identify a significant reduction in adhesion formation in various animal models (9-11). Recently, one group of investigators has documented evidence of macroscopic and microscopic inflammatory reactions after simple placement of Interceed(TC7) into the abdomen of mice (12). The inability of Interceed(TC7) to reduce adhesions and its propensity to incite an inflammatory reaction cited above are in sharp contrast to several previous reports (3-6, 8, 13). Using similar animal models, these studies showed Interceed(TC7) was effective in reducing adhesions (3, 5, 8,13). Further, Interceed(TC7) was found to be effective in two large multicenter clinical trials (4, 6). These contradictory findings among various investigators may be explained by the differences in the species used, the injury introduced and its location, the interval between primary injury and adhesion scoring, the presence of blood and suture, and adhesion grading scale. The establishment of a uniform animal model would eliminate the need to contend with these variables when attempting to compare the findings from different reports. Ultimately, the model selected should attempt to reflect biological similarities with adhesion formation in human. The failure of previous studies to document the efficacy of Interceed(TC7) could be due to inability to achieve strict hemostasis. In this series meticulous hemostasis was achieved by needle point electrocautery before the application ofinterceed(tc7) and poloxamer. The higher score for the Interceed(TC7) treated sides exposed to blood in group 5 compared with the hemostatic Interceed(TC7) side in group 3, although not significant, suggests that the presence of blood was not a confounding variable in the inability to document adhesion reduction in group 3. Poloxamer and Interceed(TC7) have also been shown to reduce adhesion reformation in the rabbit model, although different animals were used with each agent to make comparisons (7). An issue to consider in this study is the possibility of poloxamer spilling over onto the Interceed(TC7)-treated side and attenuating its effect. This appears to be unlikely because placement of both barriers was confirmed before closure ofthe abdominal wall. Further, poloxamer is a gel at body temperature and has little osmotic effect that would lead to an increase of free water in the peritoneum, which would attenuate Interceed(TC7)'s efficacy (1). To achieve beneficial adhesion-reducing properties, barrier adjuvant placement should ensure that separation of injured tissue be maintained. It is recommended that Interceed(TC7) be placed in a dry operating field after the establishment of complete hemostasis. Repositioning a patient to a level attitude from a Trendelenburg position can introduce irrigating fluid collected in the upper abdomen into a previously dry field, thus displacing the Interceed(TC7). Poloxamer's efficacy is also attenuated in the presence of lactated Ringer's and blood, prompting the recommendation that it also be applied into a pelvis free of irrigating fluid and blood. The reports on the efficacy of lactated Ringer's to reduce adhesion formation have been conflicting (14-16). Our findings did not demonstrate any beneficial effect of lactated Ringer's alone or in combination with poloxamer. In conclusion, we have confirmed the ability of poloxamer to reduce primary adhesion formation in a rat uterine horn model when compared with an untreated side. The inability of Interceed(TC7) to Vol. 59, No.4, April 1993 Montgomery Rice et al. Evaluation of poloxamer and Interceed(TC7) 905

6 reduce adhesion formation as compared with its control side needs further evaluation. Poloxamer was more efficacious than Interceed(TC7) in reducing adhesions when applied in the same animal. The addition of blood and lactated Ringer's to poloxamer-treated tissue reduced its adhesion-reducing properties. Acknowledgment. We thank Kenneth A. Ginsburg, M.D., Wayne State University School of Medicine, Detroit, Michigan, for his critical review of this manuscript and Michael Kruger, M.A., Wayne State University School of Medicine, Detroit, Michigan, for his review of the statistical evaluation of the data. REFERENCES 1. Leach RE, Henry RL. Reduction of postoperative adhesions in the rat uterine horn model with poloxamer 407. Am J Obstet GynecoI1990;162: Steinleitner A, Lambert H, Kazensky C, Cantor B. Poloxamer 407 as an intraperitoneal barrier amterial for the prevention of postsurgical adhesion formation and reformation in rodent models for reproductive surgery. Obstet GynecoI1991;77: Linsky CB, Diamond MP, Cunningham T, Constantine B, Decherney AH, dizerega G. Adhesion reduction in a rabbit uterine horn model using TC-7. J Reprod Med 1987;32: Interceed(TC7) Adhesion Barrier Study Group. Prevention of postsurgical adhesions by Interceed(TC-7), an absorbable adhesion barrier: a prospective, randomized multicenter clinical study. Fertil Steril 1989;51: Diamond MP, Cunningham T, Linsky CB, Kamp L, McDonnell RF, Gracy RW. Interceed(TC7) as a adjuvant for adhesion reduction: animal studies. In: dizerega GS, editor. Treatment of postsurgical adhesions. New York: Wiley-Liss Inc., 1990: Sekiba K. Obstetrics and Gynecology Adhesion Prevention Committee. Use of Interceed(TC7) absorbable adhesion barrier to reduce postoperative adhesion reformation in infertility and endometriosis surgery. Obstet Gynecol 1992;79: Steinleitner A, Lopez G, Suarez M, Lambert H. An evaluation of Flowgel as an intraperitoneal barrier for the prevention of postsurgical adhesion reformation. Fertil Steril 1992;57: Diamond MP, Linsky CB, Cunningham T, Constantine B, dizerega GS, DeCherney AH. Development of a model for sidewall adhesions in the rabbit and their reduction by an absorbable barrier. Microsurgery 1987;8: Pagidas K, Tulandi T. Effect of Ringer's lactate, Interceed(TC7), and Gore-Tex surgical membrane on postsurgical adhesion formation. Fertil Steril 1992;57: Best CL, Rittenhouse D, Sueldo CE. A comparison of TC7 and 32% dextran 70 for prevention of postoperative adhesions in hamsters. Obstet Gynecol 1991;78: Maxson WS, Herbert CM, Oldfield EL, Hill GA. Efficacy of a modified oxidized cellulose fabric in the prevention of adhesion formation. Gynecol Obstet Invest 1988;26: Haney AF, Doty E. Murine peritoneal injury and de novo adhesion formation caused by oxidized-regenerated cellulose (Interceed TC7) but not expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane). Fertil Steril1992;57: Linsky CB, Diamond MP, Cunningham T, Constantine B, Decherney AH, dizerega G. Effect of blood on the efficacy of barrier adhesion reduction in the rabbit uterine horn model. Infertility 1988;11: Yaacobi Y, GoldbergEP, Habal MB. Effect of Ringer's lactate irrigation in the formation of postoperative abdominal adhesions. J Invest Surg 1991;4: Fayez JA, Schneider PJ. Prevention of pelvic adhesion formation by different modalities of treatment. Am J Obstet Gynecol 1987;157: Caballero J, Tulandi T. The effect of Ringer's lactate and fibrin glue in postsurgical adhesions. J Reprod Med 1992;37: Montgomery Rice et al. Evaluation of poloxamer and Interceed(TC7) Fertility and Sterility