Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model*

Transcription

1 FERTILITY AND STERILITY Copyright 1991 The American Fertility Society Vol. 56, No.3, September 1991 Printed on acid-free paper in U.S.A. Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model* Biilent Urman, M.D.t Victor Gomel, M.D.H Nazma Jetha, M.D. University of British Columbia and University Hospital, Vancouver, British Columbia, Canada Objective: The aim of this study was to determine the effectiveness of hyaluronic acid solution in preventing intraperitoneal (IP) adhesions. Design and Setting: The study design was prospective, randomized and blinded and involved 83 rats. Interventions: Measured serosal injury was inflicted using a C0 2 laser on the right uterine horn of the rat. Animals randomized to groups 1 and 2 received either 0.4% hyaluronic acid or its diluent phosphate-buffered saline (PBS) intraperitoneally before and after the injury. In groups 3 and 4, the same solutions were used only after the injury. Postoperative adhesions were assessed at secondlook laparotomy. Histologic assessment of the fresh laser injury was carried out on uteri pretreated with hyaluronic acid, PBS, or nothing. Main Outcome: Pretreatment with hyaluronic acid was associated with a significant reduction in postoperative adhesions and a significantly decreased crater depth. Conclusions: Hyaluronic acid appears to reduce postoperative IP adhesion formation by coating the serosal surfaces and decreasing the extent of initial tissue injury. Fertil Steril 56:563, 1991 Prevention of postoperative adhesions is an issue that continues to elude the abdominal and reproductive surgeon. Numerous adjuvants have been used to reduce postoperative adhesions. 1 Crystalloid solutions, dextran, 2-4 corticosteroids, 5 6 heparin, 7 nonsteroidal anti-inflammatory agents, 8 calcium channel blockers, 9 progesterone, 10 tissue plasminogen activator, 11 and several barrier methods such as amniotic membrane grafts, 12 polytetrafl.uoroethylene surgical membrane, 13 and oxidized regenerated cellulose 14 are some of the many that can be men- Received January 4, 1991; revised and accepted April23, * Supported in part by research grant no. 81 (87-1) from the British Columbia Health Care Research Foundation, Vancouver, British Columbia, Canada, and partly by the Genzyme Corporation, Boston, Massachusetts. t Department of Obstetrics and Gynecology. :j: Reprint requests: Victor Gomel, M.D., University of British Columbia, Department of Obstetrics and Gynecology, Room 2H Oak Street, Vancouver, British Columbia V6H 3V5, Canada. Department of Pathology. tioned. There has been a lack of corroboration of the studies reporting beneficial results. Because of the limited success obtained with the numerous substances that have been tried, the search for an effective adjuvant to prevent postoperative adhesions continues. The aim of this study was to determine the effectiveness of hyaluronic acid solution in preventing adhesion formation after a measured laser injury on the rat uterine horn. MATERIALS AND METHODS Eighty-three female Wistar rats weighing 200 to 220 g were used as the animal model. They were housed in the animal unit, which is artificially illuminated from 7:00A.M. to 7:00P.M. and had free access to water and food. Operations were performed under general inhalation anesthesia using Halothane (Fluothane; Ayerst Laboratories, Montreal, Canada). In the initial phase of the study, 40 rats were randomized into two groups (groups 1 and 2). In both Vol. 56, No.3, September 1991 Urman et al. Effect of hyaluronic acid on adhesion formation in the rat model 563

2 groups, the abdominal cavity was entered via a 3-cm vertical midline incision. Immediately on entering the cavity, 2 ml of 0.4% hyaluronic acid ( Genzyme Corporation, Boston, MA) in group 1 or its dilution vehicle phosphate-buffered saline (PBS) in group 2 (control group) was instilled into the abdomen. The right uterine horn was isolated atraumatically, and an additional! ml of hyaluronic acid in group 1 or PBS in group 2 was applied directly onto the horn before any further manipulation. Then a measured injury was inflicted on this horn using a defocused C0 2 laser beam. A Sharplan 1050 C0 2 laser (Sharplan Lasers Inc., Tel-Aviv, Israel; Ingram and Bell, Toronto, Ontario, Canada) with an articulated arm attached to a hand piece was employed for this purpose. The distance from the hand piece of the laser to the surface of the serosa was kept constant (10 mm) with an appropriate attachment to the tip of the hand piece, thus keeping the defocused spot size the same at all times. The power output was 3 watts and the exposure time 0.1 seconds. The laser was fired 30 times, creating two rows of 15 burns each on the antimesouterine surface of the right uterine horn over a distance of 3 cm. 15 The procedure was performed under magnification with the aid of an OPMI 1 operating microscope (Carl Zeiss, Inc., Thornwood, NY) with fiberoptic illumination. The left uterine horn was neither exposed nor handled and served as control for each animal with regard to postoperative adhesion formation. At the completion of the procedure, an additional! ml of hyaluronic acid in group 1 or PBS in group 2 was instilled into the peritoneal cavity. The abdomen was closed in two layers using 4-0 vicryl (Ethicon, Inc., Somerville, NJ) for the peritoneum, muscle, and fascia, and staples for the skin. Fourteen days after the initial procedure, a second-look laparotomy was performed to assess the extent of adhesions. The investigators were blinded to the prior treatment modality. Division of adhesions was performed under magnification using electrosurgery. An insulated microelectrode adapted to the handle of a Valley Lab SSE2L (Valley Lab Inc., Boulder, CO) solid-state electrosurgical unit was employed. The electrode was activated with blend current using settings of 4 watts and 12 watts for coagulation and cutting, respectively. Once lysis of adhesions was complete, the surface area involved in the adhesive process was measured by placing a milimetric acetate on the surface of the uterine horn and tracing the affected areas with a thin tipped marker as previously reported from our center. 15 The total area involved in adhesions was expressed in mm 2 The animals were killed after the completion of this procedure. In the second phase of the study, an additional 40 rats were randomized into two groups (groups 3 and 4). In this phase of the study, injuries were inflicted without prior intraperitoneal (IP) instillation and subsequent direct application of hyaluronic acid or PBS onto the uterine horn. However, 4 ml of either solution (hyaluronic acid in group 3 and PBS in group 4) was instilled into the peritoneal cavity before closure of the abdomen. Adhesions were assessed 14 days later in the manner previously described. Three additional rats were used to assess histologically the extent of laser injury. In the first rat, 2 ml of hyaluronic acid solution was instilled into the peritoneal cavity immediately on entering the abdomen. Both uterine horns were submitted to the previously described injury after pretreating each horn with 1 ml of the same solution. In the second animal submitted to the same injury of both uterine horns, PBS was used in a similar manner. In the third, no solution was used. The uterine horns were removed after the completion of the procedure and the animals killed. The removed uteri were fixed in 10% formalin, and multiple segments containing areas of injury from each of the three groups were routinely processed for histologic examination. Serial sections of the lesions were obtained to visualize the area of maximum damage and to measure the depth and the diameter of tissue injury. Sections were stained with hematoxylin and eosin. For statistical analysis of the results one-way ANOV A and Student's t-test were applied. RESULTS The surface area involved in adhesions (mean ± SD) as assessed during second-look laparotomy is shown in Figure 1. When hyaluronic acid was used before laser injury (group 1), the surface area (mean ± SD) involved in adhesions (2.35 ± 2.7 mm 2 ) was significantly less (P < 0.05) than when PBS (group 2) (6.9 ± 5.4 mm 2 ) was used instead. Adhesions were observed in 30% of the animals in the hyaluronic acid group compared with 75% of the animals in the PBS group. When no solution was instilled before infliction of the laser injury (groups 3 and 4), the extent of adhesions observed during second-look, laparotomy did not differ (P > 0.05) between hyaluronic acid (8.65 ± 5.35 mm 2 ) and PBS (9.21 ± 5.45 mm 2 ) groups. Adhesions were observed in 85% of the animals in both groups. No adhesions were found 564 Urman et al. Effect of hyaluronic acid on adhesion formation in the rat model Fertility and Sterility

3 15 1 DISCUSSION Postoperative adhesions are a frequent result of IP procedures, including reproductive operations, and remain an important issue in surgery. An inflammatory response to tissue injury initiates the production of a serofibrinous exudate and reduction in plasminogen activator activity. The latter results in decreased fibrinolysis and enhanced fibroblast proliferation. These represent the basic cascade of events leading to adhesion formation. 1 Formation 10 ~ c 0 ;;; " ~ 5 HA-B/A PBS-B/A HA-A PBS-A Figure 1 Surface area of the uterine horn involved in adhesions. HA-B/A, hyaluronic acid before and after (group treated with hyaluronic acid applied before and after laser injury); PBS-B/A, PBS before and after (group treated with PBS applied before and after laser injury); HA-A, hyaluronic acid after (group treated with hyaluronic acid applied after laser injury); PBS-A, PBS after (group treated with PBS applied after laser injury). in association with the left uterine horn in any of the groups. A significant reduction in postoperative adhesion formation was achieved only in group 1 in which the laser injury was created after the IP instillation and direct application of hyaluronic acid onto the uterine horn. The depth and the diameter of the lesion crater was measured histologically in the three animals in which the laser injury was inflicted on hyaluronic acid-pretreated, PBS-pretreated, and nontreated uteri. Twenty lesions were assessed in each uterus, and maximal depth and diameter of the lesions were measured. The mean ± SD lesion depth was significantly less (P < 0.001) in the hyaluronic acid-pretreated uteri (0.15 ± mm) when compared with PBS-pretreated (0.265 ± mm) and nontreated (0.29 ± mm) uteri. The difference in lesion depth was not significant when PBS-pretreated and nontreated uteri were compared (P > 0.05). The depth of the lesions damaged the circular but did not affect the longitudinal muscle layer in the hyaluronic acid-pretreated group (Fig. 2A); however, the circular as well as the longitudinal muscular layers were involved in PBS-pretreated and nontreated lesions (Fig; 2B). The diameters of the lesions were the same irrespective of the prior treatment applied (P > 0.05). Therefore, pretreatment of the uterine horn with hyaluronic acid was associated with a decreased penetration of the laser energy, resulting in an injury with a significantly decreased crater depth while its diameter was unchanged. Vol. 56, No.3, September 1991 Urman et al. Figure 2 Extent of laser injury. (A), Hyaluronic acid pretreated uterine horn. (B), Nontreated uterine horn (c, circular muscular layer; l, longitudinal muscular layer; e, endometrium). Effect of hyaluronic acid on adhesion formation in the rat model 565

4 of adhesions is dependent on the extent of pathology, the procedure, the surgical technique employed, and the skill of the surgeon. Minimization of peritoneal injury forms the basis of modern surgical methods and is the single most important component in the prevention or reduction of postoperative adhesions. 1 However, despite strict adherence to this principle, serosal trauma inevitably occurs because of handling and instrumentation of tissues and desiccation, which results after prolonged exposure of the surgical field to room atmosphere and high-intensity surgical lights. Substances that would aid in minimizing microscopic, unintentional, or often unavoidable tissue injury may potentially decrease the extent of postoperative adhesions. Numerous adjuvants have been used in an attempt to either decrease the initial inflammatory response or keep the denuded peritoneal surfaces separated. However, the reported outcomes have been variable and beneficial results frequently marginap 6 17 The disappointing results associated with current adjunctive regimens have instigated a whole field of research directed at new and more effective substances to reduce postoperative adhesions. The present study confirms prior observations 15 of variation in adhesive response in the same species, despite standardization of the injury. Of the control animals, 25% demonstrated no adhesions. A liquid form of 0.4% hyaluronic acid diluted in PBS was found to be effective in reducing the extent of adhesions when it was instilled into the peritoneal cavity and also directly applied to the uterine horn of the rat before the causation of a measured injury using the C0 2 laser. When hyaluronic acid was instilled into the peritoneal cavity after the tissue injury was induced, the extent of postoperative adhesions was not significantly different from the control animals who received the diluent vehicle PBS. Histologic assessment of the lesions demonstrated a significantly shallower crater depth in the hyaluronic acid pretreated uteri compared with PBS-pretreated or nontreated uteri. These results indicate that pretreatment with hyaluronic acid is associated with a decrease in the extent of tissue injury. This fact and the observation that there was no difference in the extent of postoperative adhesions between groups 3 and 4, in which hyaluronic acid or PBS was administered after the creation of injury, suggests that the noted decrease in adhesion formation in group 1 is the result of decreased tissue damage associated with hyaluronic acid pretreatment. The ideal way of preventing or reducing postoperative adhesion formation is minimizing tissue damage. Aside from the use of proper surgical technique, an adjuvant that prevents both desiccation and unavoidable injury by coating the tissues exposed and/or handled during the operative procedure would be beneficial in this respect. Tissue damage because of desiccation may be prevented by constant irrigation of serosal surfaces; however, the efficacy of this approach has not been shown conclusively. In an experimental study on rats, adhesion formation after standardized uterine and cecal injuries was not influenced by irrigation or drying but seemed solely associated with the extent of serosal trauma. 18 Hyaluronic acid is a naturally occurring glycosaminoglycan that is present in the soft tissues of all vertebrates. 19 It also is a component of skin, synovial fluid, subcutaneous and interstitial connective tissue, and the umbilical cord. Hyaluronic acid is believed to play a role in a number of.physiological functions including protection and lubrication of cells, maintenance of the structural integrity of tissues, transport of molecules and cells, and fluid retention and regulation Under aqueous physiological conditions, hyaluronic acid forms a highly viscous solution that appears to coat serosal surfaces. This property appears to provide a certain degree of protection against serosal desiccation and other types of tissue injury. Hyaluronic acid was shown to reduce the extent of postoperative adhesions when instilled into the abdominal cavity before a controlled cecal trauma in the rat. 23 Furthermore, in the dog model, a significant reduction in pericardia! adhesions was observed when hyaluronic acid was used to coat the pericardia! sac before induction of a controlled tissue injury. 24 In the experimental model used for this study, pretreatment of the uterine serosa with hyaluronic acid before induction of a measured injury decreased postoperative adhesions as well as the extent of injury as noted histologically. Further studies are necessary with the model presented herein and other models using different concentrations of hyaluronic acid and more extensive serosal injury. In addition, studies must be carried out to ascertain whether or not hyaluronic acid affects adhesion reformation after lysis. Acknowledgments. We gratefully acknowledge Ms. Stephanie Janssen, AHT, Animal Health Technologist, for her able assistance with the experiments and the care of the animals. Hyaluronic acid solution was provided by the Genzyme Corporation, Boston, Massachusetts. 566 Urman et al. Effect of hyaluronic acid on adhesion formation in the rat model Fertility and Sterility

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