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Administracao topica per-operatoria da mitomicina C, em diferentes concentracoes, sobre a cicatrizacao de mioplastias do reto dorsal de coelhos.

Intraoperative topical administration of mitomycin C, in different concentrations, on the cicatrization of mioplasties of the dorsal rectus of rabbits

INTRODUCTION

Myoplasties of extra ocular muscle may cause adhesions between the operated muscle and the adjacent tissues, allowing the occurrence of oculomotor dysfunctions and cicatricial strabismus (CRUZ, 1996). To prevent them physical barriers have been tested such as gelatinous sponge of animal skins, synthetic polypeptides, polyglactin 910, viscoelastic substances, capsules of Tenon and silicone (SILVA, 1983; ELSAS et al., 1992; HWANG & CHANG, 1996). Furthermore anticicatricial agents were also used such as daunorubicin, bleomycin, mitramycin, 5-fluoracil, cyanoacrylate and mitomycin C (LEE et al., 1990; CRUZ, 1996; MAMEDE et al., 2004).

Antifibrotic effects of mitomycin C have been reported in in vitro and in vivo studies (KUNITOMO & MORI, 1969; YAMAMOTO et al., 1990). There have been several communications regarding the use of antifibrotics effects in the correction of strabismus in concentration varying from 0.02% to 0.05% (BROOKS et al., 1996; CRUZ, 1996; OH et al., 1999; MINGUINI et al., 2000; MAHINDRAKAR et al., 2001; MAMEDE et al., 2004). Those studies evaluated the effects of mitomycin C after its utilization on myoplasties, by contact between the substances and the tissues in time periods varying from 1 to 5 minutes (BROOKS et al., 1996; CRUZ, 1996; OH et al., 1999; MINGUINI et al., 2000; MAMEDE et al., 2004).

Despite the good results confirmed by many researchers, there have been some controversies about the efficacy of mitomycin C as an antifibrotic agent (BROOKS et al., 1996; MINGUINI et al., 2000). Side effects have been reported such as discomfort, persistent photophobia, conjunctival hyperemia and chemosis. Conjunctival granulomas, punctiform keratitis, melting and perforation of the cornea, areas of scleral calcifications, secondary glaucoma and cataract have also been reported. (RUBINFELD et al., 1992; ANDUZE & BURNETT, 1996; DOUGHERTY et al., 1996; MAMEDE et al., 2004).

The present study aimed at investigating the antifibrotic action of mitomycin C, applied during the myoplastic of the dorsal rectus, in different concentrations, based on the clinical, histological and immunohistochemical studies.

MATERIAL AND METHODS

Prior to its performance the research project was evaluated and approved by the 'Chamber of Ethics in Animal Research' of the College of Agricultural and Veterinarian Sciences, Sao Paulo University--UNESP--Jaboticabal, Sao Paulo, Brazil. The rules of Association for Research in Vision and Ophthalmology --ARVO (National Institutes of Health Publications No. 85-23: Revised 1985) and the Nuremberg Code (GOLDIM, 1995) were also complied with.

Fifty-six rabbits (Oryctolagus cuniculus, LILLJEBORG, 1874) of the New Zealand race, adults, males and females, weighing above 2kg were utilized. They were divided in five categories: Negative Control Group (NCG), Positive Control Group (PCG) and groups subjected to treatment with mitomycin C in the concentrations 0.008% (GT-1) 0.02% (GT-2) and 0.04% (GT-3).

To minimize the effects of individual variations and to increase the amount of samples, both eyes of each animal were utilized excepting the animals of the control group, NCG, which had only one of the eyes operated. The dorsal rectus muscle was recessed 5 to 8 mm. In the NCG, 0.9% physiological solution was instilled in the myoscleral suture for 5 minutes. And in the PCG, after the recession, a 0.9% physiological solution was instilled in the myoscleral suture for five minutes, and in the contra-lateral eye the effects of mitomycin C were evaluated. This group was study considering the possibility of the mitomycin C affecting the untreated eye by absorption and systemic dissemination.

In the groups GT-1, GT-2 and GT-3 after the recession of the dorsal rectus muscle, mitomycin C in the concentration of 0.008%, 0.02% and 0.04% was instilled on the myoscleral suture for five minutes.

To perform the surgical techniques the rabbits were anesthetized with xilazina (5mg kg-1, IV) (Virbaxyl 2%-Cloridrato de xilazina--Virbac-Sao Paulo, SP, Brasil) and ketamina (25mg [kg.sup.-1] IV) (Vetaset--Cloridrato de Quetamina--Fort Dodge--Campinas, SP, Brasil) and maintained in the anesthetic plan with halothane (Fluothane--Halotano--AstraZeneca--Cotia, SP, Brasil) in a semi open circuit (KOHN et al., 1997). The employed surgical technique for the muscle recession and myoplasties was the divulsion of the bulbar conjunctiva from the corneosclera limbus between 11 and 14 hours of a clock, identification and isolation of the dorsal rectus muscle, resection close to its scleral insertion and myoscleral suture with Vicryl 6-0 (Vicryl 6-0 (J544G)- Poliglactina 910-Ethicon--Sao Jose dos Campos, SP, Brasil) (Figure 1) (PARKS, 1983). Afterwards physiological solution or mitomycin C in the different concentrations were applied on the myoplastic areas. After the surgical procedures the area was washed with physiological solution and the suture of the capsule of Tenon and bulbar conjunctiva was performed using Vicryl 6-0.

During the postoperative period the ocular surface was submitted to daily rinsing and prophylactic instillation of eye drops composed of neomycin, polymyxin B and dexamethasone (Maxitrol--Sulfato de Neomicina, Sulfato de Polimixina B e Dexametasona --Alcon do Brasil--Sao Paulo, SP, Brasil) every six hours for seven consecutive days. For pain control buprenorphine (Temgesic 0.3mg [ml.sup.-1]--Cloridrato de Buprenorfina--Schering-Plough - Rio de Janeiro, RJ, Brasil) was administered subcutaneously in the dosage of 0.01 mg [kg.sup.-1] every 12 hours for five consecutive days.

[FIGURE 1 OMITTED]

The parameters concerning chemosis, blepharospasm, conjunctival hyperemia, hemorrhage, secretion and strabismus were evaluated for up to 120 days. Qualitative and quantitative criteria were followed: absent (nihil), discrete (+), moderate (++) and intense (+++).

Macroscopical, histological and immunohistochemical evaluation of the myoplastic areas were conducted for the postoperative periods of 15, 30, 60 and 120 days. For this purpose the animals were again subjected to anesthesia following the adopted protocol for the surgical procedures.

For adhesions evaluations as a quantity criterion, nihil represents absence of adhesions, (+) few adhesions, (++) moderate adhesions, (+++) many adhesions. To evaluate its intensity the adhesions were qualified from 0 to 4, where 0- absence of adhesions; 1--limited adhesions easily separated by dissection. 2--mild to moderate adhesions; 3--moderate to dense adhesions with difficult dissection and 4--strong nondissectible adhesions.

For histopathological purposes, tissues containing sclera and muscular portions were extracted from the adjacent surgical areas. They were fixed for 24 hours in 10% buffered formalin and sectioned in paraffin. Sections of 5 [micro]m were prepared and the laminas were stained with hematoxylineosin and Masson's Trichrome. Thereafter, for scientific documentation purposes, a photomicroscope was used to evaluate the cells and events related to fibrosis. In addition, immunohistochemical was used to demonstrate FGF-2.

The quantified events were analysis statistically using Kruskal-Wallis (one-way analysis of variance for nonparametric data) with the level of significance set at 5% (P=0.05).

RESULTS

The clinical evaluation of the operated eyes showed chemosis only in the first days of the postoperative follow up. Moderate levels of blepharospasm were assessed on the first day of evaluation only in the NCG animals. In the other groups blepharospasm was absent or present in minimal levels. Intense conjunctival hyperemia was observed up to the fifth day of evaluation in the majority of the animals of every group, tending to diminish in the subsequent periods. Ocular hemorrhage was not observed during the evaluation process. Secretion, when present, was of the mucous type and appeared moderately in the NCG animals. In the other groups the secretion, when present, was even discrete. Statistical differences were not observed (Figure 2).

Discrete to moderate hypotropia was observed. At every moment of the evaluation the eyes treated with mitomycin C, regardless its concentration, showed adhesions in lesser significant quantities and levels when compared to the control group of myoplasties. However statistical difference was found only between PCG and GT-3, at the 15th postoperative day (P<0.01) (Figure 2).

In the early stages of the follow up, the histological evaluation showed intense granulomatous and lymphocytic inflammatory reaction at the mioplastic areas. The granulomatous reaction occurred similarly between the groups on the 15th, 30th and 60th Postoperative. On the 120th day, half the animals of the GT-2 group showed a delayed involution. Regarding the fibrosis, on the 15th day a smaller incidence was observed in the control groups when compared to treated groups. However after 15 days it was observed a tendency to an increase of fibrosis in the control groups and a decrease in the groups treated with mitomycin C. At the end of the evaluation (120 days) the groups showed moderate and with no significant differences (Figure 1).

Immunohistochemical evaluation showed expression of the FGF-2 in fibroblasts and macrophages in different grades and variables, according to the periods of the evaluation. At 15 day it was observed moderate to intense expressions in the animals of NCG groups. In the other groups they varied from discrete to moderate. At 30 day it was observed a great increase in the FGF-2 expressions in all groups. In the NCG group it evolved to an intense grade while in the other groups it was maintained in moderate. After 60 days it was observed a great decrease in the FGF-2 marking in every group, evolving to a discrete on day 120th (Figure 1).

DISCUSSION

The clinical evaluation indicates that most animals showed, at some moment of the postoperative, signs of chemosis, blefarospasm, conjunctival hyperemia and mucous ocular secretion. Chemosis was present in the acute phases of the postoperative and in a discrete form in every animal. Due to its pattern and rapid development we believe it was caused by surgical trauma. The conjunctival hyperemia, present especially in the early days of evaluation, is also due to the surgical procedures. The hyperemia was more intense in the area around the suture as reported by DADEYA & KAMLESH (2001). In general the qualified and quantified clinical signs showed no differences between the groups, allowing us to assume that mitomycin C as applied in the intraoperative did not induce exudation in the eyes surface, in opposition to the findings of MAMEDE et al. (2004) when it was utilized in the postoperative for 14 days.

Adhesions were present in all of myoplasties. When we compared the NCG and PCG groups no differences were seen. Those findings lead us to assume that mitomycin C, according to the utilized protocol, was not absorbed by systemic via in enough quantity to produce any effect in the contra-lateral eyes. The findings confirm those of MATTAR et al. (1995), who inform that mitomycin C acts only in the applied area.

When comparing the NCG and PCG groups with the groups treated with mitomycin C (GT-1, GT-2, GT-3) it was found in the controls a greater quantity and grades of adhesion in all evaluated periods. Those findings corroborate CRUZ & MATKOVICH (1995), CRUZ (1996) OH et al. (1999) and MAHINDRAKAR et al. (2001). However, despite the confirmation of these facts, statistically there was no difference, except when comparing the PCG and GT-3 on 15th postoperative. Adhesions were present in a similar form in the groups treated with mitomycin C in different concentrations. Therefore it is assumed that the use of smaller concentration has the same antifibrotic effect.

[FIGURE 2 OMITTED]

Inflammatory reaction of the granulamatous and lymphocytic type, as seen in the early stages of the postoperative, was similar to the findings of INGRAM (1965), who studied in rabbits the effects caused by recess of the extrinsic muscle of the eyes without the interference of any drugs. Therefore it is fair to assume that the surgical procedure and the suture material have caused this inflammatory reaction.

The granuloma that was formed in the myoplastic areas occurred in the same way between the groups, in the evaluation periods of 15, 30 and 60 days. On the 120th day there was a delayed involution of the granuloma in 50% of the animals treated with mitomycin C at 0.02% (GT-2). Fibrosis occurred more intensively in the treated groups until 60 days. Afterwards it was equivalent to the mitomycin C control groups. Therefore we assume that mitomycin C may have slowed the occurrence of fibrosis on the applied area, which corroborate the results found by RIBEIRO et al. (2003), who study the effects of mitomycin C in rat's derma.

Regarding the immunohistochemical, the FGF-2 expressed for fibroblasts as well as for macrophages confirms the findings of FUNATO et al. (1999) and WANG et al. (2001). In the present research the intensity of the expression of FGF-2 occurred in a very similar way between the studied groups. Those results oppose the findings of OH et al. (1999), which reported a smaller expression in animals that received mitomycin C. Since FGF-2 is produced by macrophages, fibroblasts, mast cells, lymphocytes T and by endothelial cells (KUMAR et al., 2005) and if mitomycin C has effect only over fibroblastic proliferation (YAMAMOTO et al., 1990), we admit that it has little interference on the production of FGF-2.

CONCLUSIONS

Based on our investigation, it is possible to admit that mitomycin C allows clinical occurrence of smaller adhesions in the myoplastic areas, although the result was not statistically significant. Under light microscope study mitomycin C delayed the occurrence of fibrosis formation in the myoplastic areas, especially in the 0.02% concentrations. According to immunohistochemical analyses, the FGF-2 suffered no interference from mitomycin C effect in any moment of the evaluation. The clinical evaluation showed no side effects by using the mitomycin C.

ACKNOWLEDGEMENT

The authors are grateful to FAPESP (Proc. nO 03/ 09527-0), CAPES (Capes-Proap--Demanda social) and FUNDUNESP (Proc. no 00743/03-DFP) for the financial support.

Received 09.18.06 Approved 05.09.07

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Fabricio Villela Mamede (I) Bianca da Costa Martins (I) Ana Leticia Groszewicz Souza (I) Adriana Torrecilhas Jorge Brunelli (II) Karina Pontin (III) Edson Garcia Soares (IV) Sergio de Albuquerque (V) Jose Luiz Laus (I) *

(I) Faculdade de Ciencias Agrarias e Veterinarias (FCAV), Universidade Estadual Paulista (UNESP), Campus de Jaboticabal, SP, Brasil. E-mail: jllaus@fcav.unesp.br. * Autor para correspondencia.

(II) Autonoma.

(III) Instituto de Biologia, Universidade de Campinas (UNICAMP), Campinas, SP, Brasil.

(IV) Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo (USP), Ribeirao Preto, SP, Brasil.

(V) Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, USP, Ribeirao Preto, SP, Brasil.
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Author:Villela Mamede, Fabricio; da Costa Martins, Bianca; Groszewicz Souza, Ana Leticia; Torrecilhas Jorge
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