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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.6 n.2 Botucatu  2000

http://dx.doi.org/10.1590/S0104-79302000000200007 

The use of snake venom derived fibrin glue in hysterorrhaphy of ovine caesarean surgery

 

M. CHALHOUB , N. C. PRESTES , M. D. LOPES , N. S. ROCHA , I. A. THOMAZINI-SANTOS , M.J. MENDES-GIANNINI

1 Department of Pathology and Clinics of the School of Veterinary Medicine, Federal University of Bahia, Salvador, Bahia State, Brazil; 2 Department of Animal Reproduction and Veterinary Radiology of the School of Veterinary Medicine and Animal Husbandry (FMVZ) São Paulo State University (UNESP) at Botucatu, São Paulo State, Brazil; 3 Department of Veterinary Clinics the School of Veterinary Medicine and Animal Husbandry (FMVZ) São Paulo State University (UNESP) at Botucatu, São Paulo State Brazil; 4 School of Medicine of Botucatu (FMB) São Paulo State University (UNESP) at Botucatu, São Paulo State Brazil; 5 Department of Clinical Analysis, School of Pharmaceutical Sciences of Araraquara, São Paulo State University (UNESP) at Araraquara, São Paulo State, Brazil; 6 Center for the Study of Venoms and Venomous Animals (CEVAP) São Paulo State University (UNESP) at Botucatu, São Paulo State, Brazil.

 

 

ABSTRACT. Fibrin glue has been used on its own or in conjunction with suturing materials to promote hemostasis, reduce adherence, strengthen the wound site, and improve healing. Snake venom derived fibrin glue was evaluated as an alternative to conventional uterine suturing after ovine caesarean surgery. Twenty-eight pregnant ewes of known mating date were used. The animals submitted to conventional caesarean sections showed a better wound healing process. As expected, all the operated animals had retained placenta, compromising coaptation of wound edges. This had a strong influence in the results observed with the animals in which fibrin glue was used. The animals were divided into four groups GI, GII, GIII, and GIV and sacrificed, respectively, 3, 7, 15, and 30 days after surgery for macro and microscopic examination of the uterus. From each group, six animals underwent surgery using fibrin glue and four animals were submitted to comparative conventional hysterorrhaphy using catgut.
 KEY WORDS: Fibrin tissue adhesive, viper venoms, uterus/sutures, sheep, fibrin glue.

 

 

INTRODUCTION

Ovine caesarean sections on healthy animals are performed routinely with excellent results. The most common reasons for ovine caesarean sections include: relative fetal oversize, most commonly in the primiparous animals with a single fetus; incomplete cervical dilation; fetal abnormalities; vaginal prolapse; and the presence of an emphysematous lamb in the uterus (10). Caesarean sections are usually performed to treat dystocia, to obtain gnotobiotic products for research, and in caprine to obtain arthritis-encephalitis (CAE) free kids (14).

Significant advances in anesthesia and surgical sepsis made laparohysterotomy a usual obstetric procedure in veterinary medicine. A perfect uterine healing following a caesarean section is pivotal for a future pregnancy (13). According to Schlenger et al. (9), the suboptimal healing of uterotomy following caesarean section in women may result in early suture dehiscence or late uterine rupture at the scar site in cases of pregnancy subsequent to vaginal delivery. Despite its clinical importance, studies on the biology of wound healing in uterotomy are rare. Most surgical wounds require a constant and efficient mechanical support to keep the wound edges together during healing, and suture is the method of choice for the synthesis of surgical wounds (11).

In the last 25 years, fibrin glue, a biological adhesive made mainly by mixing fibrinogen and thrombin, has been used more frequently to help sealing of surgical wounds. Fibrin glue is used in combination with suturing to seal needle and suture thread orifices, strengthen the wound site, and improve overall healing (11). Aside from its hemostatic properties, fibrin glue may have a role in promoting wound healing. Fibrin is essential for wound healing because the network in the wound acts as a scaffold for migrating fibroblasts and a hemostatic barrier (3).

Wheaton et al. (15) reported the efficacy of fibrin glue as a hemostatic agent when applied locally in dog liver after biopsy. Hemorrhage control, absence of inflammatory and toxic responses, absence of adherence, and healing stimulation led to the conclusion that this product may be widely used in veterinary surgery.

Schlenger et al. (9) looking for new information about uterine wound, developed a model of uterotomy in rats. Ten mm-incisions were made in the uterus of non-pregnant animals and commercially available fibrin glue (Tissucol DuoÒ) was used. The authors concluded that this proposed model is useful in the investigation of uterine wound and that fibrin associated with tissue necrosis factor a (TNF a) has an important effect only during the initial phase of wound healing.

The only report in literature about the use of snake venom derived fibrin glue in ovine was by Sartori Filho et al. (8) in 1998. They applied this fibrin glue on the surgical wound on scrotal skin following testicular biopsy with excellent results in relation to hemostasis and scar formation.

The objective of this experiment was to evaluate the use of snake venom derived fibrin glue as an alternative to conventional suture in ovine caesarean surgery.

 

MATERIALS AND METHODS

STANDARDIZATION AND PREPARATION OF SNAKE VENOM DERIVED FIBRIN GLUE. The isolation of the thrombin-like enzyme from snake venom and the preparation of the cryoprecipitate containing buffalo fibrinogen are described by Iuan et al. (2) and Thomazini-Santos (12). The glue consists of equal amounts of cryoprecipitate and venom fraction, previously diluted in calcium chloride. The mixture of these components occurs only at the time of glue application to tissue during surgery.

EXPERIMENTAL DESIGN. Twenty-eight pregnant ewes of known mating date were used. Surgery was performed between 143 and 145 days post mating. From each group, six animals underwent surgery using fibrin glue and one animal was submitted to comparative conventional hysterorrhaphy using catgut. The animals were randomly divided into four groups of seven and were sacrificed GI 3 days after, GII 7 days after, GIII 15 days after, and GIV 30 days after surgery for post-mortem macro and microscopic examinations.

SURGICAL PROCEDURE. The animals were submitted to a 12-hour fast and the wool on the left flank was removed by shaving. They were then submitted to lumbosacral epidural anesthesia, using 3 to 4 ml of 2% lidocaine (Astra Química e Farmacêutica Ltda). The animals were positioned in right lateral recumbency and normal surgical procedures were adopted. Ten IU of oxytocin (Hoeschst Roussel Vet.) was administered subcutaneously in an attempt to promote a uniform contraction of the uterus in order to achieve a perfect coaptation of the wound edges, so that fibrin glue could act properly. An incision of approximately 15 cm was made through the left abdominal wall and into the peritoneum cavity. The gravid uterus was exposed and another incision was made along the greater curvature of the pregnant uterine horn between the cotyledons, where the limbs of the fetus projected onto the uterus surface. This incision was longitudinal and of sufficient length to avoid transversal rupture of the uterus wall during fetus extraction. The length of this incision in the uterus required three to four separated stitches, which were made with nonabsorbable cotton suture material. These stitches were alternated equidistantly in relation to the wound edges in order to diminish tension on the surgical wound so that the edges could be satisfactorily bonded with the glue. Snake venom derived fibrin glue (0.3 - 0.4 ml) was applied along the incision (Figure 1). The components were mixed at the time of application to the site. Two syringes were simultaneously used for the application of calcium chloride/snake venom fraction and fibrinogen. The bond sites were manually kept held for three minutes to permit formation of the fibrin network. The other four animals were submitted to conventional hysterorrhaphy using 2-0 chromic catgut (Brasmedica Ind. Brasil S/A). Conventional laparorrhaphy and skin suturing were then performed on all the animals.

 

 

POST-SURGICAL PROCEDURE AND POST-MORTEM. After surgery, the animals received a subcutaneous injection of 30,000 IU/kg of penicillin and streptomycin (Laboratórios Wyeth Ltda) repeated at 48 and 96 hours and 1.1 mg/kg of subcutaneous flunixin meglumine (Shering Plough Veterinária S/A) repeated every 24 hours for three days. Repellent once a day and 2% iodine twice a day were applied to the wound for 10 days. Stitches were removed on the seventh day after surgery. The animals were sacrificed by electrocution 3, 7, 15, and 30 days after surgery and detailed macroscopic examinations were made of the abdominal cavity and uterus. Later, fragments were collected from the surgical wound site for histopathological examination using hematoxylin and eosin (4). The areas of epithelialization, cellular content, granular reaction near the suture and the wound, collagen deposition, and vascularization were histologically evaluated. Macro and microscopic evaluations were documented.

 

RESULTS

ANESTHETIC TECHNIQUE AND DURATION OF SURGERY. Lumbosacral epidural anesthesia and inverted L infiltration produced analgesia adequate for manipulation of the uterus. Duration of surgery using snake venom derived fibrin glue varied from 18 to 38 min, with a mean of 29.3±5.56 min, while duration of conventional caesarean section varied from 20 to 35 min, with a mean of 25.75±6.65 min.

MACROSCOPIC POST-MORTEM. The animals submitted to caesarean section using fibrin glue showed retained placenta (Figure 2), suture dehiscence, uterine adherence, and peritonitis of varied degrees; some animals did not show any of these signs (Figure 3, Table 1). The animals in the conventional caesarean section group did not show these signs, except for animal 4 of GIII, which had mild uterine adherence, and animal 15 of GI, which had retained placenta (Table 2). During the post-surgery period, three animals died, two of GII and one of GIV, all having undergone hysterorrhaphy using fibrin glue. Necropsy revealed the cause of death as endotoxic shock due to dehiscence of hysterorrhaphy with uterine content extravasation and peritonitis. The mortality rate of operated animals in which fibrin glue was used was 12.5, however, no deaths were observed with conventional hysterorrhaphy.

 

 

 

 

 

 

 

 

MICROSCOPIC POST-MORTEM – FIBRIN GLUE HYSTERORRHAPHY.

Group I. The microscopic examination of all uterus layers showed extensive areas of coagulation necrosis alternating with areas of live tissues. In these latter areas, the simple columnar epithelial lining of the lumen was extremely high. The foam cell layer glands showed intense dilation and movement away from the surface with polymorphonuclear cells and fibroblasts observed in between. A slight deposition of collagen fiber bundles and neovascularization were also observed. On the other hand, all cellular components were supported by highly myxoid and hemorrhagic tissue (Figure 4 and Figure 5). In the areas near the remaining suture lines these structures were not seen. Three days after surgery, vascular neoformation accompanying the proliferative connective tissue occurred.

 

 

 

 

Group II. The areas of coagulation necrosis in the uterus layers in GII behaved in the same way as GI, with all the other cell components more evident than in GI.

Group III. The areas of coagulation necrosis in the uterus layers were less evident. The simple columnar epithelial lining of the lumen which was high decreased. The number of foam cell layer glands was much higher than in GI or GII. In addition, the glands were diffuse and mostly arranged in the form of nests. There were a large number of macrophages filled with brownish substances called hemosiderin (Figure 6). In the muscular layer, the remaining suture thread was surrounded by foreign-body type granulation tissue.

 

 

Group IV. Small areas of coagulation necrosis were seen. The lining epithelium showed complete recovery. There was a remarkable presence of glands occupying the entire foam cell layer, arranged in the forms of nests and tubules. The number of macrophages filled with hemosiderin was high (Figure 7).

 

 

MICROSCOPIC POST-MORTEM – CONVENTIONAL HYSTERORRHAPHY. These animals showed the same reactions as those in the fibrin glue group, but they were less intense. In GI and GII, the epithelium was better preserved with smaller areas of coagulation necrosis. In GIII and GIV, near the catgut, a pyogranulomatous inflammatory process was observed surrounded by mononuclear infiltrate and giant cells with fibroblasts covering the catgut, away from the epithelium. This distant catgut did not interfere with the wound epithelialization. The integrity of the chromic catgut in the histological sections, especially in the muscular layer of GI and GII indicated an absence of reabsorption up to seven days after surgery. On the 15 and 30 days after surgery (GIII and GIV), reaction around the suture thread showed some reabsorption.

 

DISCUSSION

The techniques used permitted the performance of caesarean sections in a short time, avoiding undesirable effects on the uterus involution or the animals. Surgery using fibrin glue was approximately 4 minutes longer due to the time needed for the formation of the fibrin network during uterotomy. However, this small time difference did not compromise the use of fibrin glue. Uterine contraction due to the action of exogenous and endogenous oxytocin was not uniform, with problems to achieve perfect coaptation of wound edges. The 12.5% mortality rate of the animals submitted to caesarean section using fibrin glue was considered high, as according to Scott (10) and Majeed et al. (7), conventional surgery in healthy ovine has excellent results. On the other hand, the 2.78% mortality rate of lambs was considered low, with only one dead lamb out of 36. This death was caused by asphyxia due to pulmonary immaturity, even though caesarean section was performed on day 144 of pregnancy.

As expected, all ruminants submitted to caesarean section had retained placenta. This greatly increased pressure on the suture, compromising the healing process. Except for retained placenta, animals undergoing conventional caesarean section did not have these above problems. Dehiscence occurred in some cases. Cotton suture was chosen for hysterorrhaphy using fibrin glue to minimize the tensile force on the sites in which glue was applied. This material was chosen because it is readily available, low cost, and highly resistant, as in tissues submitted to tension, it avoids adherence and promotes healing (6).

The progressive decrease in the lumen of the endometrial glands was shown in this study both in the animals submitted to caesarean section with fibrin glue and those which underwent conventional suturing. This study also demonstrated that large quantities of hemosiderin-laden macrophages were present, and this was probably due to injuries sustained during the caesarean section. In addition, these brownish substances present in the histological sections of GIII and GIV are due to the fact that ovine caruncles are frequently pigmented as the result of the presence of menoblasts, which is in agreement with McEntee’s report (5). Histologically, however, up to the 30 day after surgery, the uterus has not returned to pre-pregnancy conditions.

Wound healing was of good macroscopic appearance 30 days after surgery (GIV) with the application of fibrin glue. However, microscopically, the uterus of the ewes did not return to pre-pregnancy conditions 30 days after experimental caesarean section.

The hemostatic potential of fibrin glue could be seen during surgery, with the formation of small clots and consequent bleeding control. Adhesion was considered relatively firm 3min after glue application.

The animals submitted to conventional caesarean section showed a better wound healing process than those where fibrin glue was used. Retained placenta frequently occurs in ovine submitted to experimental caesarean section. Therefore, these animals are probably not the best for studies on experimental hysterorrhaphy using low-tensile strength materials, such as snake venom derived fibrin glue.

This was the first study in which snake venom derived fibrin glue was used in hysterorrhaphy after caesarean section in ewes. In view of advantages of fibrin glue, including tissue compatibility, biodegradability, and efficacy when applied to humid or highly vascularized surfaces (1), further studies on the use of snake venom derived fibrin glue in hysterorrhaphy after caesarean sections in several animal species are needed.

 

ACKNOWLEDGEMENTS

The authors are very grateful to the Research Support Foundation of São Paulo State – FAPESP (No.97/033980) for financing this study. They also wish to thank professor Eduardo L. T. Moreira of the Federal University of Bahia – UFBA - and the residents working in the areas of animal reproduction and veterinary pathology for their assistance.

 

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Received 03 November 1999
Accepted 15 December 1999

 CORRESPONDENCE TO:
M. CHALHOUB – Departamento de Patologia e Clínicas, Escola de Medicina Veterinária, Universidade Federal da Bahia, CEP 40.000-000, Salvador, Bahia, Brasil.
E-mail:chalhoub@ufba.br

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