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Fibrin sealant in laparoscopic adhesion prevention in the rabbit uterine horn model
Reproductive animal research /' FERTILITY AND STERILITY Copyright
©
Vol. 62, No.2, August 1994
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Fibrin sealant in laparoscopic adhesion prevention in the rabbit uterine horn model
PierAndrea De Iaco, M.D.*t Alberto Costa, M.D.* Guido Mazzoleni, M.D.:j:
Gianandrea Pasquinelli, M.D.§ Leona Bassein, C.Stat.11 Alberto Marabini, M.D.*
S. Orsola Hospital, University of Bologna, Bologna, Italy
Objective: To assess the effects of fibrin sealant on adhesions after laparoscopic surgery. Design: Standardized surgical trauma was induced in 60 female rabbits. The animals were randomized in three groups for different adhesion prevention treatment. Setting: University research laboratory. Interventions: After standardized trauma was induced, group 1 (n = 20) received no treatment, group 2 animals (n = 20) were injected in the abdominal cavity with 60 mL of Ringer's lactate, and human fibrin sealant was applied on the surgical lesions under laparoscopic vision in group 3 (n = 20). Main Outcome Measures: Five weeks after laparoscopy, a laparotomy was performed, and the adhesions were scored. Results: Fourteen of 20 rabbits in the control group (70%) presented postoperative adhesions, 11 of 20 (55%) in the Ringer's group, and 5 of 20 (25%) in the fibrin sealant group. High-score adhesions were seen in 15% of cases in control and Ringer's group and in 5% of cases in the fibrin sealant group. Conclusions: When used during laparoscopic surgery, fibrin sealant has a preventive effect on de novo postsurgical adhesions. To assess the efficacy in reproductive surgery, a trial on recurrent postsurgical adhesions is required. Fertil Steril 1994;62:400-4 Key Words: Fibrin sealant, laparoscopy, surgery, adhesion
Development of postoperative adhesions is a frequent occurrence after laparotomic pelvic surgery. Nezhat et al. (1) have suggested that laparoscopic surgery could reduce this complication because it is more precise, more hemostatic, and less traumatic. For these reasons, laparoscopic surgery is becoming the procedure of choice in fertility-promoting sur-
Received June 3, 1993; revised and accepted February 23, 1994. * II Department of Obstetrics and Gynecology. t Reprints requests: PierAndrea De Iaco, M.D., Ospedale S. Orsola, Universita di Bologna, via Massarenti 13, 40138 Bologna, Italy (FAX: 39-51-349774). :j: Department of Pathology. § Institute of Clinical Electron Microscopy. II Department of Clinical Pharmacology and Therapy. 400
De Iaeo et al.
Fibrin glue in laparoscopic surgery
gery, even though clinical observations and some animal trials demonstrate the presence of adhesion even after endoscopic surgery (2, 3), The prevention of postoperative formation of adhesions remains a major goal for this surgical procedure. A wide variety of substances have been tested to prevent adhesions !lfter laparotomic surgery, but few have been evaluated after laparoscopic surgery (4). Ringer's lactate (Ravizza, Verona, Italy) intraperitoneal instillation is a frequently used adjuvant; it is simple to use, and it has often been administered as a control in comparative trials (5-7), Its efficacy is due to the hydroflotation of intra -abdominal organs. Fibrin glue (Tissucol; Immuno spa, Pisa, Italy) represents an ancillary method because it aids wound healing by producing local hemostasis and Fertility and Sterility
by creating an absorbable barrier (8-11). Its use during laparotomies has demonstrated adhesion prevention properties (11). This study compared fibrin glue with Ringer's lactate in terms oftheir ability to prevent adhesion formation after peritoneal injury inflicted by laparoscopy and assessed the histology of adhesions in the presence of different substances. MATERIALS AND METHODS
Sixty adult female Hibridum Provisal white rabbits (Provisal spa, Bologna, Italy) weighing 2.5 to 3.5 kg and aged 3 to 4 months were used in this study. The rabbits were selected at this age because the female has a completely developed reproductive apparatus. All rabbits were housed in individual stainless steel rabbit cages in the stud farm (Provisal spa). They were fed with commercial rabbit feed and water ad libitum. The rabbits were placed in the supine position; the abdomen was shaved and prepped with Betadine solution and draped in sterile fashion. They were then anesthetized with 3 mg/kg fenotiazine phosphate (Combelen, Bayer, Germany) and 80 mg/kg ketamine hydrocloride (Ketavet 100; Farmaceutici Gallini, Aprilia, Italy). A Verres needle was inserted into the peritoneal cavity, entering the abdominal wall in the midline. Carbon dioxide gas was insufflated by laparoinsufflator (Karl Storz, Tuttlingen, Germany) until the pneumoperitoneum was sufficient for safe introduction of a cystoscope (17-Fr diameter; Karl Storz). The cystoscope was inserted into the peritoneal cavity under visual control with the aid of a Storz 250 W light source. After inspection of the abdominal cavity, laparoscopic forceps and laparoscopic scissors were introduced through short lateral incisions. Standardized surgical trauma was induced in all subjects by the following: [1] denuding a l-cm2 area of the peritoneum of the right uterine mesometrium; [2] crushing the medial right uterine horn for 30 seconds with forceps; and [3] making a l-cm incision in the distal right uterine horn with scissors. The injury was accurately measured using the open jaws of a pair of scissors of 1 cm in width. The rabbits had been randomized into three groups of 20; group 1 received no treatment (control). In group 2, 60 mL of Ringer's lactate were injected in the abdominal cavity. In group 3, human fibrin sealant was applied on the peritoneal and uterine lesions under laparoscopic vision. Vol. 62, No.2, August 1994
Table 1
Adhesion Scoring System*
Grade
Description
o
No adhesions Thin or narrow, easily separable adhesions Thick adhesions, limited to one area Thick and widespread adhesions Thick and widespread adhesions, plus adhesions of viscera to anterior and/or posterior abdominal wall
1 2 3 4
* From Blauer and Collins (6).
Human fibrin sealant (Tissucol; Immuno spa) is a multicomponent adhesive, a freeze-dried protein concentrate of human fibrinogen (120 mg/mL) reconstituted in a solution of aprotinin (3,000 kIU / mL) mixed with a solution of thrombin (500 U /mL) and calcium chloride (40 mmoljL). The two components were preheated at 37°C and drawn up in separate syringes. The syringes were fitted in a specially designed syringe holder (Duploject; Immuno AG, Vienna, Austria) and connected to a three-way catheter, 7-Fr diameter, 15-cm long, with highgrade steel shaping wire (Duploject-Spray Catheter 15; Immuno AG). The catheter was inserted through a lateral abdominal incision. Approximately 0.3 mL of fibrin sealant was used for each rabbit. All rabbits received 62,500 U of penicillin G and 25 mg 1M of streptomycin (Rubrocillina; Istituto Sieroterapico Milanese, Milano, Italy) after laparoscopy. Five weeks later, a laparotomy was performed, and the adhesions to each uterine horn were blindly assessed by the same observer, according to Blauer's scoring system (Table 1) (6). At laparotomy, all major lesions were removed and fixed in 10% formalin, and multiple segments containing areas of adhesions were routinely processed for histologic examination. Sections were stained with hematoxylin and eosin. Serial sections were studied to evaluate the following histologic features: inflammation, fibrosis, fat necrosis, and granulomatosis. The histologic characteristics were graded according to a 0 to 3 sc·ale. In one case for each study group, the injured tissues were removed during postoperative laparotomy, and the specimens were evaluated by electron microscopy (EM). The three groups were compared by means of Ridit analysis (12), followed by the Bonferroni correction for multiple comparisons at the overall 5% level (13). When possible, the reference distribution was derived from the control group; if anomalous results were obtained, the whole sample was De Iaeo et al.
Fibrin glue in laparoscopic surgery
401
Table 2
Adhesion Scores for Each Treatment Group Score
Control Ringer's lactate Fibrin sealant
No. of rabbits
0
1
2
3
4
Ridit*
20 20 20
6 11 15
3 2 1
8 4 3
1 0 0
2 3 1
0.50 0.39 0.28t
* Statistical ridit analysis was used for scoring system arranged in ordered categories. t p < 0.05 versus control group.
used to estimate the reference distribution. Ridit analysis offers a scoring system that is useful to summarize data arranged in ordered categories, in situations in which ranking would lead to too many ties. The average ridit in a particular treatment group represents the probability that a randomly chosen individual in that group will have more severe lesions than a subject chosen at random from the reference group. Significance tests were performed only for the principal question: are the treatments associated with different probability distributions of lesions? The average ridits are presented without testing for the results of the histologic analyses because of difficulties in finding comparable subsamples.
RESULTS
The adhesions score assessed at second-look laparotomy is listed in Table 2. Fourteen of the 20 rabbits (70%) in the control group presented postoperative adhesions, whereas 9 of the 20 rabbits (45%) treated with Ringer's lactate and only 5 of the 20 (25%) of the fibrin sealant group were associated with adhesive disease. First grade lesions were present in three cases of group 1, two cases of group 2, and one case of group 3. High-grade lesions (score 3 or 4) were seen in three rabbits in the control group and in Ringer's group (15%) and in one in the fibrin sealant group (5%). The adhesion ridit for the fibrin sealant group was significantly less (P = 0.008) than that for control rabbits. No significant differences were seen between the control group and Ringer's group (P = 0.218) or between Ringer's group and the fibrin sealant group (P = 0.204). No adhesions were seen in the left uterine horn in any of the groups, where no trauma had been 402
De Iaeo et al.
Fibrin glue in laparoscopic surgery
induced. Also, no notable complications were seen in any group of rabbits. Fibrin sealant application through the abdominal incision was easy to perform and accurate under wide endoscopic magnification. At second-look laparotomy, small yellowish remnants of fibrin sealant were found in most cases. The histologic features of peritoneal scars and of major adhesions showed no differences among the groups with respect to fibrosis, fat necrosis, or granulomatosis (Table 3). Inflammation was considerably more frequent in the fibrin glue group. This finding was related to the presence of macrophages as well as histiocytic-like giant cells at EM in this group. DISCUSSION
The use of fibrin sealant is gaining interest in gynecological surgery because of its hemostatic properties and its favorable effects on wound healing, leading to a good plastic reconstruction of the genitals (8). In 1984 Lindenberg and Lauritsen (11) reported an adhesion prevention effect with a thrombin -containing preparation. This could be explained by a barrier effect obtained by the glue after consolidation (14). Moreover, fibrin sealant enhances the normal wound healing process by increasing the influx of macrophages that produce factors causing angiogenesis, fibroblast proliferation, and collagen production (15-18). Because of fibrin's hemostatic effect, hematoma and peritoneal bleeding are avoided; consequently, the protein exudation from peritoneal injury is markedly reduced, minimizing the fibrin-bridge development
Table 3
Histologic Features of Major Lesions Biopsies Score
Histologic feature
Group (no.)
0
1
2
3
Ridit
Inflammation
Control Ringer Fibrin glue
3 4 0
2 3 0
0 0 1
0 0 5
0.33 0.47 1.00
Fibrosis
Control Ringer Fibrin glue
0 0 2
3 6 3
2 1 0
0 0 1
0.50 0.52 0.38
Fat necrosis
Control Ringer Fibrin glue
0 2 1
3 3 4
2 1 1
0 1 0
0.50 0.54 0.40
Granulomatosis
Control Ringer Fibrin glue
0 2 0
5 4 5
0 1 1
0 0 0
0.50 0.60 0.70
Fertility and Sterility
between adjoining structures (19). The use of fibrin sealant in laparoscopic surgery is a promising ancillary method because postoperative adhesions are still a major problem. Laparoscopy has noticeably reduced the incidence of postoperative adhesions because of the more precise surgical technique, reduced tissue trauma, and avoidance of abdominal organ manipulation (7, 20). Mc Entee (21) reports that simple handling of the bowel with starch-rich gloves in a rat model induced postsurgical adhesion in 78% of the animals. Extensive injury to the peritoneal surfaces and punctuate bleeding at laparoscopic surgery can cause postoperative adhesions. Intraperitoneal blood is therefore a key point because Luciano et al. (22) showed an impressive rise in adhesion formation in surgical procedures complicated by significant bleeding. This study demonstrates that fibrin sealant used during laparoscopic surgery is a potent inhibitor of post-traumatic adhesion formation as shown by the absence of adhesions in 75% of the fibrin glue group. The literature on fibrin glue in laparotomy is inconclusive. de Virgilio et al. (10) concluded his trial on animals with standardized trauma with the statement that fibrin glue inhibits intra-abdominal adhesion formation in rats. Only fibrin glue with a high concentration of fibrinogen was able to prevent adhesions. Other investigators have not reported good results with fibrin sealant as an adhesion prevention method (14, 23) in laparotomic procedures. These authors tested the fibrin sealant in fallopian tubes (23) and colonic anastomosis (14), thus introducing additional uncontrolled variables. This study showed a fairly high within-group variability, as is observed in many animal models (2,10,24), despite attempts to standardize, but the relationship between trauma and adhesion was confirmed by the absence of adhesive disease on the left (untouched) uterine horn. Surprisingly, only 70% of the control animals presented postsurgical adhesions; only 15% of the controls had high-grade (grade 3 or 4) adhesions. This suggests the lesion was a poor stimulus for adhesion formation. A more traumatic technique could have been helpful in giving more precise information on the antiadhesive properties of fibrin glue. Finally, the procedure of fibrin glue application during laparoscopic surgery is easy to perform and accurate, even though a ready-to-use kit is useful. Vol. 62, No.2, August 1994
This study demonstrated a significant reduction in de novo adhesion formation after laparoscopic injection of fibrin glue on the wound surface. The common situation encountered in infertility surgery is the presence of adhesions at first-look operation; the effect of applying fibrin glue to prevent adhesion reformation after adhesiotomy was not tested in this study. Furthermore, clinical trials will be needed to assess whether this procedure is of value in laparoscopic surgery in humans.
Acknowledgments. We thank Zaccanti srI (Bologna, Italy) for useful technical support. The authors acknowledge Mrs. Agnese Vignaga for her excellent assistance with data collection and Mrs. Anne Collins for linguistic assistance.
REFERENCES 1. Nezhat CR, Nezhat FR, Metzger DA, Luciano AA. Adhesion reformation after reproductive surgery by videolaseroscopy. Fertil SterilI990;53:1008-11. 2. Filmar S, Gomel V, McComb PF. Operative laparoscopy versus open abdominal surgery: a comparative study on postoperative adhesion formation in the rat model. Fertil Steril 1987;48:486-9. 3. LundorffP, Hahlin M, Kallfelt B, Thorburn J, Lindblom B. Adhesion formation after laparoscopic surgery in tubal pregnancy: a randomized trial versus laparotomy. Fertil Steril 1991;55:911-5. 4. Diamond MP, Hershlag A. Adhesion formation and re-formation. In: di Zerega GS, Diamond MP, Malinak R, Linsky CB, editors. Treatment of postsurgical adhesions. New York: Wiley-Liss Inc., 1990:23-7. 5. Fayez JA, Schneider PJ. Prevention of pelvic adhesion formation by different modalities of treatment. Am J Obstet GynecoI1987;157:1184-8. 6. Blauer KL, Collins RL. The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits. Fertil Steril 1988;49:144-9. 7. Luciano AA, Maier DB, Koch EI, Nulsen JC, Whitman GF. A comparative study of postoperative adhesions following laser surgery by laparoscopy versus laparotomy in the rabbit model. Obstet GynecoI1989;74:220-4. 8. Orlandi C, Marabini A, Vendra C, Mancini L. The use of fibrin sealant (Tissucol/Tisseel) in gynaecological surgery. In: Schlag G, Redl H, editors. Fibrin sealant in operative medicine. gynaecology and obstetrics-urology. Berlin: Springer-Verlag, 1986:63-5. 9. Oka H, Harrison RC, Burhenne HJ. Effect of biologic glue on the leakage rate of experimental rectal anastomoses. Am J Surg 1982;143:561-4. 10. de Virgilio C, Dubrow T, Sheppard BB, Mac Donald WD, Nelson RJ, Lesavoy MA, et al. Fibrin glue inhibits intra-abdominal adhesion formation. Arch Surg 1990;125:1378-82. 11. Lindenberg S, Lauritsen JG. Prevention of peritoneal adhesion formation by fibrin sealant: an experimental study in rats. Ann Chir GynaecoI1984;73:11-3.
De Iaco et al. Fibrin glue in laparoscopic surgery
403
12. Fleiss JL. Statistical methods for rates and proportions. 2nd ed. New York: Wiley and Sons, 1981. 13. Glantz SA. Primer of biostatistics. 3rd ed. New York: McGraw Hill, 1992:91-2. 14. van der Ham AC, Kort WJ, Weijma 1M, van den Ingh HFGM, Jeekel J. Effect of fibrin sealant on the healing colonic anastomosis in the rat. Br J Surg 1991;78:49-53. 15. Leibovich SJ, Ross R. A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vivo. Am J Pathol 1976;84:501-13. 16. Pohl J, Bruhn HD, Christophers E. Thrombin and fibrininduced growth of fibroblasts: role in wound repair and thrombus organization. Klin Wochenschr 1979;57:273-7. 17. Ross R. The fibroblast and wound repair. Bioi Rev 1968;43:51-96. 18. Beck E, Duckert F, Vogel A, Ernst M. The influence of fibrin stabilizing factor on the growth of fibroblasts in vitro and wound healing. Thromb Diath Haemorrh 1961;6:48591.
404
De Iaco et al.
Fibrin glue in laparoscopic surgery
19. Schlag G, Redl H, Turnher M, Dinges HP. The importance of fibrin in wound repair. In: Schlag G, Redl H, editors. Fibrin sealant in operative medicine. gynaecology and obstetrics-urology. Berlin: Springer-Verlag, 1986;3-12. 20. Hershlag A, Diamond MP, De Cherney AH. Adhesiolysis. Clin Obstet Gynecol 1991;34:395-402. 21. Me Entee GP, Stuart RC, Byrne PJ, Leen E, Hennessy TP. Experimental study of starch-induced intraperitoneal adhesions. Br J Surg 1990;77:1113-4. 22. Luciano AA, Hauser KS, Benda J. Evaluation of commonly used adjuvants in the prevention of postoperative adhesions. Am J Obstet Gynecol 1983;146:88-92. 23. Dargenio R, Ranelletti OF, Cimino C, Ragusa G, Panetta V, Garcea N. Fibrin glue vs. nylon in the anastomosis of rabbit fallopian tubes. J Reprod Med 1986;31:961-5. 24. Steinleitner A, Lambert H, Kazensky C, Danks P, Roy S. Pentoxifylline, a methylxanthine derivative, prevents postsurgical adhesion reformation in rabbits. Obstet Gynecol 1990;75:926-8.
Fertility and Sterility
©
Vol. 62, No.2, August 1994
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Fibrin sealant in laparoscopic adhesion prevention in the rabbit uterine horn model
PierAndrea De Iaco, M.D.*t Alberto Costa, M.D.* Guido Mazzoleni, M.D.:j:
Gianandrea Pasquinelli, M.D.§ Leona Bassein, C.Stat.11 Alberto Marabini, M.D.*
S. Orsola Hospital, University of Bologna, Bologna, Italy
Objective: To assess the effects of fibrin sealant on adhesions after laparoscopic surgery. Design: Standardized surgical trauma was induced in 60 female rabbits. The animals were randomized in three groups for different adhesion prevention treatment. Setting: University research laboratory. Interventions: After standardized trauma was induced, group 1 (n = 20) received no treatment, group 2 animals (n = 20) were injected in the abdominal cavity with 60 mL of Ringer's lactate, and human fibrin sealant was applied on the surgical lesions under laparoscopic vision in group 3 (n = 20). Main Outcome Measures: Five weeks after laparoscopy, a laparotomy was performed, and the adhesions were scored. Results: Fourteen of 20 rabbits in the control group (70%) presented postoperative adhesions, 11 of 20 (55%) in the Ringer's group, and 5 of 20 (25%) in the fibrin sealant group. High-score adhesions were seen in 15% of cases in control and Ringer's group and in 5% of cases in the fibrin sealant group. Conclusions: When used during laparoscopic surgery, fibrin sealant has a preventive effect on de novo postsurgical adhesions. To assess the efficacy in reproductive surgery, a trial on recurrent postsurgical adhesions is required. Fertil Steril 1994;62:400-4 Key Words: Fibrin sealant, laparoscopy, surgery, adhesion
Development of postoperative adhesions is a frequent occurrence after laparotomic pelvic surgery. Nezhat et al. (1) have suggested that laparoscopic surgery could reduce this complication because it is more precise, more hemostatic, and less traumatic. For these reasons, laparoscopic surgery is becoming the procedure of choice in fertility-promoting sur-
Received June 3, 1993; revised and accepted February 23, 1994. * II Department of Obstetrics and Gynecology. t Reprints requests: PierAndrea De Iaco, M.D., Ospedale S. Orsola, Universita di Bologna, via Massarenti 13, 40138 Bologna, Italy (FAX: 39-51-349774). :j: Department of Pathology. § Institute of Clinical Electron Microscopy. II Department of Clinical Pharmacology and Therapy. 400
De Iaeo et al.
Fibrin glue in laparoscopic surgery
gery, even though clinical observations and some animal trials demonstrate the presence of adhesion even after endoscopic surgery (2, 3), The prevention of postoperative formation of adhesions remains a major goal for this surgical procedure. A wide variety of substances have been tested to prevent adhesions !lfter laparotomic surgery, but few have been evaluated after laparoscopic surgery (4). Ringer's lactate (Ravizza, Verona, Italy) intraperitoneal instillation is a frequently used adjuvant; it is simple to use, and it has often been administered as a control in comparative trials (5-7), Its efficacy is due to the hydroflotation of intra -abdominal organs. Fibrin glue (Tissucol; Immuno spa, Pisa, Italy) represents an ancillary method because it aids wound healing by producing local hemostasis and Fertility and Sterility
by creating an absorbable barrier (8-11). Its use during laparotomies has demonstrated adhesion prevention properties (11). This study compared fibrin glue with Ringer's lactate in terms oftheir ability to prevent adhesion formation after peritoneal injury inflicted by laparoscopy and assessed the histology of adhesions in the presence of different substances. MATERIALS AND METHODS
Sixty adult female Hibridum Provisal white rabbits (Provisal spa, Bologna, Italy) weighing 2.5 to 3.5 kg and aged 3 to 4 months were used in this study. The rabbits were selected at this age because the female has a completely developed reproductive apparatus. All rabbits were housed in individual stainless steel rabbit cages in the stud farm (Provisal spa). They were fed with commercial rabbit feed and water ad libitum. The rabbits were placed in the supine position; the abdomen was shaved and prepped with Betadine solution and draped in sterile fashion. They were then anesthetized with 3 mg/kg fenotiazine phosphate (Combelen, Bayer, Germany) and 80 mg/kg ketamine hydrocloride (Ketavet 100; Farmaceutici Gallini, Aprilia, Italy). A Verres needle was inserted into the peritoneal cavity, entering the abdominal wall in the midline. Carbon dioxide gas was insufflated by laparoinsufflator (Karl Storz, Tuttlingen, Germany) until the pneumoperitoneum was sufficient for safe introduction of a cystoscope (17-Fr diameter; Karl Storz). The cystoscope was inserted into the peritoneal cavity under visual control with the aid of a Storz 250 W light source. After inspection of the abdominal cavity, laparoscopic forceps and laparoscopic scissors were introduced through short lateral incisions. Standardized surgical trauma was induced in all subjects by the following: [1] denuding a l-cm2 area of the peritoneum of the right uterine mesometrium; [2] crushing the medial right uterine horn for 30 seconds with forceps; and [3] making a l-cm incision in the distal right uterine horn with scissors. The injury was accurately measured using the open jaws of a pair of scissors of 1 cm in width. The rabbits had been randomized into three groups of 20; group 1 received no treatment (control). In group 2, 60 mL of Ringer's lactate were injected in the abdominal cavity. In group 3, human fibrin sealant was applied on the peritoneal and uterine lesions under laparoscopic vision. Vol. 62, No.2, August 1994
Table 1
Adhesion Scoring System*
Grade
Description
o
No adhesions Thin or narrow, easily separable adhesions Thick adhesions, limited to one area Thick and widespread adhesions Thick and widespread adhesions, plus adhesions of viscera to anterior and/or posterior abdominal wall
1 2 3 4
* From Blauer and Collins (6).
Human fibrin sealant (Tissucol; Immuno spa) is a multicomponent adhesive, a freeze-dried protein concentrate of human fibrinogen (120 mg/mL) reconstituted in a solution of aprotinin (3,000 kIU / mL) mixed with a solution of thrombin (500 U /mL) and calcium chloride (40 mmoljL). The two components were preheated at 37°C and drawn up in separate syringes. The syringes were fitted in a specially designed syringe holder (Duploject; Immuno AG, Vienna, Austria) and connected to a three-way catheter, 7-Fr diameter, 15-cm long, with highgrade steel shaping wire (Duploject-Spray Catheter 15; Immuno AG). The catheter was inserted through a lateral abdominal incision. Approximately 0.3 mL of fibrin sealant was used for each rabbit. All rabbits received 62,500 U of penicillin G and 25 mg 1M of streptomycin (Rubrocillina; Istituto Sieroterapico Milanese, Milano, Italy) after laparoscopy. Five weeks later, a laparotomy was performed, and the adhesions to each uterine horn were blindly assessed by the same observer, according to Blauer's scoring system (Table 1) (6). At laparotomy, all major lesions were removed and fixed in 10% formalin, and multiple segments containing areas of adhesions were routinely processed for histologic examination. Sections were stained with hematoxylin and eosin. Serial sections were studied to evaluate the following histologic features: inflammation, fibrosis, fat necrosis, and granulomatosis. The histologic characteristics were graded according to a 0 to 3 sc·ale. In one case for each study group, the injured tissues were removed during postoperative laparotomy, and the specimens were evaluated by electron microscopy (EM). The three groups were compared by means of Ridit analysis (12), followed by the Bonferroni correction for multiple comparisons at the overall 5% level (13). When possible, the reference distribution was derived from the control group; if anomalous results were obtained, the whole sample was De Iaeo et al.
Fibrin glue in laparoscopic surgery
401
Table 2
Adhesion Scores for Each Treatment Group Score
Control Ringer's lactate Fibrin sealant
No. of rabbits
0
1
2
3
4
Ridit*
20 20 20
6 11 15
3 2 1
8 4 3
1 0 0
2 3 1
0.50 0.39 0.28t
* Statistical ridit analysis was used for scoring system arranged in ordered categories. t p < 0.05 versus control group.
used to estimate the reference distribution. Ridit analysis offers a scoring system that is useful to summarize data arranged in ordered categories, in situations in which ranking would lead to too many ties. The average ridit in a particular treatment group represents the probability that a randomly chosen individual in that group will have more severe lesions than a subject chosen at random from the reference group. Significance tests were performed only for the principal question: are the treatments associated with different probability distributions of lesions? The average ridits are presented without testing for the results of the histologic analyses because of difficulties in finding comparable subsamples.
RESULTS
The adhesions score assessed at second-look laparotomy is listed in Table 2. Fourteen of the 20 rabbits (70%) in the control group presented postoperative adhesions, whereas 9 of the 20 rabbits (45%) treated with Ringer's lactate and only 5 of the 20 (25%) of the fibrin sealant group were associated with adhesive disease. First grade lesions were present in three cases of group 1, two cases of group 2, and one case of group 3. High-grade lesions (score 3 or 4) were seen in three rabbits in the control group and in Ringer's group (15%) and in one in the fibrin sealant group (5%). The adhesion ridit for the fibrin sealant group was significantly less (P = 0.008) than that for control rabbits. No significant differences were seen between the control group and Ringer's group (P = 0.218) or between Ringer's group and the fibrin sealant group (P = 0.204). No adhesions were seen in the left uterine horn in any of the groups, where no trauma had been 402
De Iaeo et al.
Fibrin glue in laparoscopic surgery
induced. Also, no notable complications were seen in any group of rabbits. Fibrin sealant application through the abdominal incision was easy to perform and accurate under wide endoscopic magnification. At second-look laparotomy, small yellowish remnants of fibrin sealant were found in most cases. The histologic features of peritoneal scars and of major adhesions showed no differences among the groups with respect to fibrosis, fat necrosis, or granulomatosis (Table 3). Inflammation was considerably more frequent in the fibrin glue group. This finding was related to the presence of macrophages as well as histiocytic-like giant cells at EM in this group. DISCUSSION
The use of fibrin sealant is gaining interest in gynecological surgery because of its hemostatic properties and its favorable effects on wound healing, leading to a good plastic reconstruction of the genitals (8). In 1984 Lindenberg and Lauritsen (11) reported an adhesion prevention effect with a thrombin -containing preparation. This could be explained by a barrier effect obtained by the glue after consolidation (14). Moreover, fibrin sealant enhances the normal wound healing process by increasing the influx of macrophages that produce factors causing angiogenesis, fibroblast proliferation, and collagen production (15-18). Because of fibrin's hemostatic effect, hematoma and peritoneal bleeding are avoided; consequently, the protein exudation from peritoneal injury is markedly reduced, minimizing the fibrin-bridge development
Table 3
Histologic Features of Major Lesions Biopsies Score
Histologic feature
Group (no.)
0
1
2
3
Ridit
Inflammation
Control Ringer Fibrin glue
3 4 0
2 3 0
0 0 1
0 0 5
0.33 0.47 1.00
Fibrosis
Control Ringer Fibrin glue
0 0 2
3 6 3
2 1 0
0 0 1
0.50 0.52 0.38
Fat necrosis
Control Ringer Fibrin glue
0 2 1
3 3 4
2 1 1
0 1 0
0.50 0.54 0.40
Granulomatosis
Control Ringer Fibrin glue
0 2 0
5 4 5
0 1 1
0 0 0
0.50 0.60 0.70
Fertility and Sterility
between adjoining structures (19). The use of fibrin sealant in laparoscopic surgery is a promising ancillary method because postoperative adhesions are still a major problem. Laparoscopy has noticeably reduced the incidence of postoperative adhesions because of the more precise surgical technique, reduced tissue trauma, and avoidance of abdominal organ manipulation (7, 20). Mc Entee (21) reports that simple handling of the bowel with starch-rich gloves in a rat model induced postsurgical adhesion in 78% of the animals. Extensive injury to the peritoneal surfaces and punctuate bleeding at laparoscopic surgery can cause postoperative adhesions. Intraperitoneal blood is therefore a key point because Luciano et al. (22) showed an impressive rise in adhesion formation in surgical procedures complicated by significant bleeding. This study demonstrates that fibrin sealant used during laparoscopic surgery is a potent inhibitor of post-traumatic adhesion formation as shown by the absence of adhesions in 75% of the fibrin glue group. The literature on fibrin glue in laparotomy is inconclusive. de Virgilio et al. (10) concluded his trial on animals with standardized trauma with the statement that fibrin glue inhibits intra-abdominal adhesion formation in rats. Only fibrin glue with a high concentration of fibrinogen was able to prevent adhesions. Other investigators have not reported good results with fibrin sealant as an adhesion prevention method (14, 23) in laparotomic procedures. These authors tested the fibrin sealant in fallopian tubes (23) and colonic anastomosis (14), thus introducing additional uncontrolled variables. This study showed a fairly high within-group variability, as is observed in many animal models (2,10,24), despite attempts to standardize, but the relationship between trauma and adhesion was confirmed by the absence of adhesive disease on the left (untouched) uterine horn. Surprisingly, only 70% of the control animals presented postsurgical adhesions; only 15% of the controls had high-grade (grade 3 or 4) adhesions. This suggests the lesion was a poor stimulus for adhesion formation. A more traumatic technique could have been helpful in giving more precise information on the antiadhesive properties of fibrin glue. Finally, the procedure of fibrin glue application during laparoscopic surgery is easy to perform and accurate, even though a ready-to-use kit is useful. Vol. 62, No.2, August 1994
This study demonstrated a significant reduction in de novo adhesion formation after laparoscopic injection of fibrin glue on the wound surface. The common situation encountered in infertility surgery is the presence of adhesions at first-look operation; the effect of applying fibrin glue to prevent adhesion reformation after adhesiotomy was not tested in this study. Furthermore, clinical trials will be needed to assess whether this procedure is of value in laparoscopic surgery in humans.
Acknowledgments. We thank Zaccanti srI (Bologna, Italy) for useful technical support. The authors acknowledge Mrs. Agnese Vignaga for her excellent assistance with data collection and Mrs. Anne Collins for linguistic assistance.
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