Effect of streptokinase in the prevention of intra-abdominal adhesions in the rat

Efeito da estreptoquinase na prevenção de aderências intra-abdominais no rato

Abstracts

O objetivo deste estudo foi determinar o efeito da estreptoquinase, derivada do estreptococo com ação fibrinolítica, na prevenção de aderências intra peritoneais em ratos. Oitenta animais forma distribuídos em 4 grupos de 20 animais cada. O grupo A (controle) recebeu solução salina pela via intraperitoneal; o grupo B recebeu estreptoquinase pela via intraperitoneal na dose de 60.000 unidades por quilograma de peso diluído em 2 ml de solução salina; o grupo C recebeu 60.000 unidades de estreptoquinase diluído em 1mililitro (ml) de solução salina pela via intravenosa e o grupo D recebeu 30.000 unidades de estreptoquinase em 2 ml de solução salina pela via intraperitoneal e 30.000 unidades de estreptoquinase diluídas em 1 ml de solução salina pela via intravenosa. Os animais foram submetidos, aleatoriamente, a laparotomia mediana para a colocação de fios de suturas que induziriam a formação de aderências do tipo isquêmica de acordo com o modelo de FERRAZ-NETO e col. (1991) modificado, e sacrificados com uma dose letal de éter sulfúrico no 3º e no 7º dia de pós operatório. A estreptoquinase mostrou ser efetiva na prevençãode aderências quando utilizadas pela via intraperitoneal (p=0,0349) ou quando ministradas simultaneamente pela via intraperitoneal e pela via intravenosa (p=0,0073). A comparação dos resultados no 3º e do 7º dia de pós operatório, em um mesmo grupo, mostrou que não há diferença estatisticamente significante, sugerindo que a droga age no início do processo de cicatrização. Conclui-se que a estreptoquinase é efetiva na prevenção de aderências quando utilizadas pela via intraperitoneal ou quando se combina a via intraperitoneal com a via intravenosa nas doses utilizadas, em ratos.

Aderências; Cicatrização; Estreptoquinase

5 - ORIGINAL ARTICLE

EFFECT OF STREPTOKINASE IN THE PREVENTION OF INTRA-ABDOMINAL ADHESIONS IN THE RAT.¹

Benjamin Smaniotto²

Maria de Lourdes Pessole Biondo-Simões³

Giocondo Villanova Artigas³

Antonio de Padua Gomes da Silva⁴

Luiz Martins Collaço⁴

Gustavo Verner Ramasco⁵

SMANIOTTO, B.; BIONDO-SIMÕES, M.L.P.; ARTIGAS, G.V.; SILVA, A P.G.; COLLAÇO, L.M.; RAMASCO, G.V. - Effect of streptokinase in the prevention of intra-abdominal adhesions in the rat. Acta Cir. Bras., 12(4):235-9, 1997.

SUMMARY: The objective of the present study was to assess the action of streptokinase, a streptococcal derivative with fibrinolytic capacity, in the prevention of intraperitoneal adhesions in rats. Eighty animals were divided into four groups of 20 each; group A (control) received isotonic saline by the intraperitoneal route, group B received streptokinase by the intraperitoneal route at the dose of 60.000 units/kg body weight diluted in 2 ml isotonic saline, group C received 60.000 streptokinase units diluted in 1 ml isotonic saline by the intravenous route, and group D received 30.000 streptokinase units in 2 ml isotonic saline by the intraperitoneal route and 30.000 streptokinase units diluted in 1 ml isotonic saline by the intravenous route. The animals were submitted at random to median laparotomy for the preparation of stitches inducing adhesions of the ischemic type according to the model of FERRAZ-NETO et al. (1991) modified, and sacrified with a lethal dose of sulfuric ether on the 3rd and 7th postoperative day. Streptokinase was found to be effective in the prevention of adhesions when used by the intraperitoneal route (p=0,0349) or when administered both intraperitoneal and intravenously (p=0,0073). Comparison of the 3rd and 7th posteperative day within the same group showed no significant difference, suggesting that the drug acts during the early period of the healing process. We conclude that streptokinase is effective in the prevention of adhesions formation when injected by the intraperitoneal route or by the combined intraperitoneal and intravenous routes at the dose employed in rats.

SUBJECT HEADINGS: Adhesions, Healing, Streptokinase

INTRODUCTION

Peritoneal adhesions are frequent findings in surgical interventions, either as a consequence of previous surgeries or as a consequence of pelvic infections. Cases of acute occlusive abdomen caused by adhesion are not uncommon in emergency units.

RYAN, GROBÉTY e MAJNO (1971), SCHADE e WILLIAMSON (1968), ELLIS (1978) e NISELL e LARSSON (1978) stated that fibrin deposition occurs as a consequence of peritoneal trauma; fibrin may then become fibrous, giving origin to a permanent adhesion. Several substances have been used in an attempt to prevent the formation of these adhesions, particularly interesting among them being those that interfere with the coagulation system.

The objective of the present study was to determine the effects of streptokinase, a substance with a fibrinolytic action, on the formation of postoperative intra-abdominal adhesions in rats.

METHOD

The experiment was conducted on 80 male Wistar-TECPAR rats (Rattus norvegicus albinus, Rodentia, mammalia) ranging in age from 160 to 193 days (mean: 175.9 days) and weighing 236.8 to 400.2 g (mean: 322.4 g), supplied by the Technological Institute of Paraná

The rats were anesthetized with sulfuric ether and submitted to a 4.0-cm laparotomy. Four ischemic saliences were prepared on the internal surface of the abdominal wall on the right according to the model of FERRAZ NETO, HUNGARO NETO, ANTUNES, AFONSO, RIBAS, PARRA and SAAD (1991), modified. All animals received 2.0 ml isotonic saline intraperitoneally and the abdominal wall was closed. The rats were divided at random into four groups of 20 animals each. Group A rats were used as controls. Group B received intraperitoneally 60,000 U streptokinase added to the isotonic saline, group C was injected intravenously through the dorsal vein of the penis with 60,000 U streptokinase diluted in 1 ml isotonic saline, and group D received 30,000 U streptokinase intravenously and 30,000 U intraperitoneally. Thus, all animals received an equal volume of intraperitoneal saline and all experimental groups received an equal dose of streptokinase.

After recovery from anesthesia, the rats were identified and placed in their cages where they had free access to water and ration until the time for the experimental determinations.Ten animals picked at random from each group were sacrified with excess ether on the 3rd and 7th postoperative days.

The abdominal cavity was opened through a left paramedian incision which continued superiorly contouring the costal margin and inferiorly contouring the upper margin of the pubis. The abdominal wall was retracted to the right and the cavities were examined for the presence of adhesions, number of inducing stitches that gave origin to them, number of adhesions, and macroscopic aspect of the viscera involved. When adhesions were present their width and thickness were measured with a Digimatic electronic pachymeter (Mitutoyo Corporation ^® )of one-thousandth sensitivity. The adhesions were then resected, placed on filter paper, fixed in 10% formalin and processed for histology. Blocks were cut into 5-µm sections which were stained with hematoxylin-eosin, Mallory trichrome and picrosirius. Inflammatory cells such as lymphocytes, neutrophils, plasmocytes, histiocytes and foreign body giant cells were identified. Adhesions were scored as + when few, ++ when present in moderate amounts, and +++ when present in large amounts. Collagen fibers were visualized and classified as dense or sparse according to their quantity, and as mature or immature on the basis of their staining yellow to red and green to the picrosirius technique, respectively. Vascularization was also checked.

Data were analyzed statistically by the nonparametric chi-square test and exact Fisher test, both for independent samples. The level of significance was set at 5.0%.

RESULTS

Adhesions were present in all groups at the 2 times studied and were more numerous in group A (Tables I and ^II). On the 3rd day, the number of stitches that gave origin to adhesions was larger in group A. When the intensity of such stitches was determined, groups B and D were found to have no more than 1 stitch giving origin to adhesions, a significant difference compared to control (Table III). Figures 1, 2, 3 and 4 show the adhesions in response to the inducing stitches in animals from the four groups. On the 7th day, the number of stitches giving origin to adhesions was found to be significantly smaller in all experimental groups compared to control (Table IV). With respect to the transverse dimension, narrow adhesions, i.e., adhesion up to 5 mm wide, were observed in all animals from groups B and D. In group C, 66.7% of the adhesions were 6 to 10 mm wide and were denoted intermediate, and in group A 18.2% of them were wide, i.e. more than 10 mm wide, [(chi-square test = tabulated value 3.84 (0.05) and 5.41 (0.02)] (A x C = 6.40* and A x D = 4.45*). On the 7th day, narrow, intermediate and wide adhesions were observed only in the control group. In group B and C there were narrow and wide adhesions, whereas in group D there were 1 narrow adhesion and 2 intermediate ones, with no significant differences between groups.

Fig. 1
- Behavior of the adhesions. Two adhesions can be observed: one on the right, formed by the greater omentum in which the central vessel on the parietal peritoneal surface extends from one inducing stitch to another, a situation in which there is no bifurcation of the adhesion, and the other on the left, formed by the spermatic cord in which central vessel extends from one stitch to the other on the parietal peritoneum, with the absence of bifurcation.

With respect to thickness, fine adhesions, i.e., adhesions measuring up to 1 mm in thickness, were observed in all groups on the 3rd day. In group A, 5 adhesions measured 1.1 to 2 mm and were denoted intermediate, and 1 each in group A and group C measured more than 2 mm and were denoted thick. The differences between groups were nonsignificant. On the 7th day there was a larger number of adhesions classified as intermediate and thick in group A, whereas in groups B and C there were fine and intermediate adhesions and in group D only fine adhesions. These differences were nonsignificant.

Fig. 2
- Adhesion detected in group B on the 3rd postoperative day, formed by the spermatic cord.

Fig. 3
- Adhesion observed in group C on the 3rd postoperative day, formed by the greater omentum.

Fig. 4
- Detail of the abdominal cavity of a rat in group D on the 7th postoperative day. *Note the absence of adhesions.

The structures that adhered to the inducing stitches were the grater omentum and the fat of the spermatic cord. In only one case, in group A, did we observe adhesion of the liver to the inducing stitch.

In all adhesions, whether they involved the omentum or the spermatic cord, there was the presence of a central vessel that ran towards the inducing stitch. When the adhesion bifurcated to reach two points, the behavior of the vessel was the same.

We observed adhesion of these structures to one or more stitches extending over the parietal peritoneum.

Microscopic examination revealed the presence of polymorphonuclear cells, lymphocytes, plasmocytes and histiocytes scored ++ (moderate) in the adhesions of all groups and at the two times studied. A moderate amount of collagen fibers forming a tissue of weak density was observed.

DISCUSSION

Alterations in vascular permeability and tissue invasion by inflammatory cells and proteins occur during the first 48 hours after trauma. Once the extent of the inflammatory process during this phase is determined, resolution or reorganization of this exudate starts, culminating with tissue repair on the 7th day (ROBBINS et al., 1991).

In reality, tissue repair is an inflammatory reaction that progresses with changes in vascular permeability and consequently extravasation of substances and cells into the interstitial medium, leading to fibrin deposition. The use of streptokinase by the intraperitoneal route may determine the lysis of the fibrin clot by direct contact with the injured area. When administered intravenously, the drug may act systemically reaching the site of tissue damage simultaneously with fibrin deposition. When the two routes, intraperitoneal and intravenous, are combined, there may be a summation of the topic effect with the systemic effect.

The rats were examined on the 3rd and 7th postoperative days to determine the formation of adhesions and their relationship with the healing process. The animals that had received streptokinase by the intraperitoneal route or by the intraperitoneal and intravenous routes, when examined on the 3rd postoperative day presented a significant reduction in the formation of adhesions in terms of number and thickness and organs involved when compared to controls injected with isotonic saline alone. Thus a significant difference in number of adhesions detected on the 3rd day was observed between group A and group B, which was injected with intraperitoneal streptokinase. The same occurred when group A was compared with group D, which received streptokinase intraperitoneally and intravenously. In contrast, on the 7th day only the combination of intraperitoneal and intravenous administration led to a larger number of adhesions compared to control. When streptokinase was injected only intravenously, no significant difference in number of adhesions was observed at either time studied.

Analysis of the number of ischemic saliences that gave origin to adhesions on the 3rd day revealed that more than one did so in group A, whereas this did not occur in groups B and D. On the 7th day, the animals injected with streptokinase by either route showed the same number of ischemic saliences giving origin to adhesions.

Comparison of the number of adhesions within the same group on the 3rd and 7th days showed no significant differences, suggesting that adhesions must be formed at the beginning of the healing process. These findings agree with those reported by WRIGHT, SMITH, ROTHMAN, QUASH and METZGER (1950 e 1951), who observed a smaller number of adhesions in rabbits when they used streptokinase and streptodornase. Similar data were reported by LUTTWAK, FELMAN and NEUMAN (1954) in a study of the prevention of adhesions induced in rats by the presence of talcum in the peritoneal cavity.

The adhesions developed towards the ischemic area of tissue both on the abdominal wall and on the intestinal wall (ELLIS, 1962). In the present experimental study we observed the presence of a central vessel at all sites of adhesion which proceeded towards the adhesion site, supporting the hypothesis of ELLIS.

Analysis of the dimensions of the adhesions showed that they were predominantly wide in group A, although they did not differ significantly from the other groups either on the 3rd or on the 7th day. As to thickness, adhesions were preferentially thick in group A on the 3rd and 7th days, with a significant difference from the remaining groups.

In the pathogenesis of post-traumatic peritoneal adhesions, the initial reaction is a typical inflammatory response (MILLIGAN and RAFFERTY, 1974), a fact that was confirmed in the present experiment.

CONCLUSIONS

Streptokinase at the dose of 60,000 U/kg body weight significantly reduced the formation of intraperitoneal adhesions compared to control when administered intraperitoneally (p = 0.0349) and by the combined intraperitoneal and intravenous routes (p = 0.0073). Streptokinase administered exclusively by the intravenous route did not reduce the number of adhesions at the two times studied.

SMANIOTTO, B.; BIONDO-SIMÕES, M.L.P.; ARTIGAS, G.V.; SILVA, A P.G.; COLLAÇO, L.M.; RAMASCO, G.V. - Efeito da estreptoquinase na prevenção de aderências intra-abdominais no rato. Acta Cir. Bras., 12(4):235-9, 1997.

RESUMO: O objetivo deste estudo foi determinar o efeito da estreptoquinase, derivada do estreptococo com ação fibrinolítica, na prevenção de aderências intra peritoneais em ratos. Oitenta animais forma distribuídos em 4 grupos de 20 animais cada. O grupo A (controle) recebeu solução salina pela via intraperitoneal; o grupo B recebeu estreptoquinase pela via intraperitoneal na dose de 60.000 unidades por quilograma de peso diluído em 2 ml de solução salina; o grupo C recebeu 60.000 unidades de estreptoquinase diluído em 1mililitro (ml) de solução salina pela via intravenosa e o grupo D recebeu 30.000 unidades de estreptoquinase em 2 ml de solução salina pela via intraperitoneal e 30.000 unidades de estreptoquinase diluídas em 1 ml de solução salina pela via intravenosa. Os animais foram submetidos, aleatoriamente, a laparotomia mediana para a colocação de fios de suturas que induziriam a formação de aderências do tipo isquêmica de acordo com o modelo de FERRAZ-NETO e col. (1991) modificado, e sacrificados com uma dose letal de éter sulfúrico no 3^o e no 7^o dia de pós operatório. A estreptoquinase mostrou ser efetiva na prevençãode aderências quando utilizadas pela via intraperitoneal (p=0,0349) ou quando ministradas simultaneamente pela via intraperitoneal e pela via intravenosa (p=0,0073). A comparação dos resultados no 3^o e do 7^o dia de pós operatório, em um mesmo grupo, mostrou que não há diferença estatisticamente significante, sugerindo que a droga age no início do processo de cicatrização. Conclui-se que a estreptoquinase é efetiva na prevenção de aderências quando utilizadas pela via intraperitoneal ou quando se combina a via intraperitoneal com a via intravenosa nas doses utilizadas, em ratos.

DESCRITORES: Aderências, Cicatrização, Estreptoquinase.

Address for correspondence:

Prof^.a Maria de Lourdes Pessole Biondo Simões

Rua Ari José Valle, 1987

CEP 82030-000 - Santa Felicidade - CURITIBA - PR

Accepted for publication on june, 1997

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• 2. ELLIS, H. - Wound repair reaction of the peritonium to injury. Ann.R.Coll., 60:219-21, 1978.
• 3. FERRAZ NETO, J.B.H.; HUNGARO NETO, P.; ANTUNES, M.C.; AFONSO, R.C.; EIRAS,A.M.; PARRA, O.M.; SAAD, W.A. - Aderęncias peritoneais: estudo experimental. Acta Cir.Bras., 6:72-6, 1991.
• 4. LUTTWAK, E.M.; FELDMAN, J.; NEUMAN, Z. - Effect of streptokinase-streptodornase on peritoneal talc adhesions and granulomas: experimental study. Arch.Surg., 68:69-75, 1954
• 5. MILLIGAN, D.W. & RAFTERY, A.T. - Observations on the pathogenesis of peritoneal adhesions: a light and electron microscopical study. Am. J. Surg., 61:274-80, 1974.
• 6. NISELL, H. & LARSSON, B. - Role of blood and fibrinogen in development of intraperitoneal adhesions in rats. Fertil.Steril., 30:470-3, 1978.
• 7. ROBBINS, S.L.; CONTRAN, R.S.; KUMAR, V. - Robbins patologia estrutural e funcional. 4.ed. Rio de Janeiro, Guanabara Koogan, 1991. p.33-72.
• 8. RYAN, G.B.; GROBÉTY, J.; MAJNO, G. - Posteoperative peritoneal adhesions. Am.J. Pathol., 65:117-40, 1971.
• 9. SCHADE, D.S. & WILLIAMSON, J.R. - The pathogenesis of peritoneal adhesions: na ultrastructural study. Ann. Surg., 167:500-10, 1968.
• 10. WRIGHT, L.T.; SMITH, D.H.; ROTHMAN, M.; QUASH, E.T.; METZGER, W.I. - Prevention of postoperative adhesions in rabbits with streptococcal metabolities. J.Int. Coll.Surg., 14:602-4, 1950.
• 11. WRIGHT, L.T.; SMITH, D.H.; ROTHMAN, M.; METZGER, W.I.; QUASH, E.T. - Use of streptokinase-dornase in certain surgical conditions. J. Int.Coll.Surg., 15:286-98, 1951.

1

Part of Master's thesis presented to the Postgraduate Course in Trauma Surgery, Pontificia Universidade Católica do Paraná - PUC-PR

2 Master of "Pontificia Universidade Católica do Paraná"

3 M.D., PhD.

4 M.D.

5 Medical student

Publication Dates

• Publication in this collection
  31 May 2001
• Date of issue
  Dec 1997

History

• Accepted
  June 1997