Ultrastructural And Immunocytochemistry Studies Of The Local Tissue Damage Induced By Neuwiedase, A Metalloproteinase Isolated From Bothrops neuwiedi pauloensis Snake Venom

¹Baldo, C; Izidoro, L. F. M.; ²Ferreira, M. J.; ²Ferro, E. V.; ¹Hamaguchi, A ; ¹Homsi-Brandeburgo, M. I and ¹Rodrigues, V. M.

1 Instituto de Genética e Bioquímica, UFU. 2 Instituto de Ciências Biomédicas, UFU, Uberlândia-MG.

The present work reports the role of neuwiedase (22 KDa), a metalloproteinase with a weak hemorrhagic activity isolated by B. neuwiedi pauloensis snake venom, in the development of tissue damage in mouse gastrocnemius muscle. Miotoxicity was measured by determination of plasma creatina kinase (CK) activity after i. m. injection of 200 mg of neuwiedase or 50 ml of PBS, at various time intervals (1, 3, 6 and 24 h). The plasma CK activity showed that the neuwiedase was able to induced a low, but significant increment in plasma CK levels, reaching values of 690 U/L at 3 h, whereas mice controls had CK activity of 350 U/L. The ultrastructural and immunocytochemistry studies were carried out after i. m. injection of 200 mg of neuwiedase or 50 ml of PBS in the gastrocnemius muscle from C57BL/6 mice. After each time intervals (1, 3, 6 and 24 h) the muscle tissue was removed, processed for ultrastructural analysis and observed in a Zeiss 109 electron microscopy. Ultrastructural analysis indicated that neuwiedase induced a degeneration of muscle fibers and swelling of the sarcoplamastic reticulum at the first time after the treatment. The  immunocytochemistry studies were achieved by ligh or electron microscopy analysis after specific time intervals 1, 3, 6, 24 h or 15, 30 min, 1, 3 h respectively. Both were carried out with polyclonal antibody antii-neuwiedase. The immunocytochemistry by light microscopy showed positive reaction for the neuwiedase along the muscle fibers and inside polymorphonuclear cells. Immunogold labeling showed a positive reaction for the neuwiedase on connective tissue, as well as on the specific myofibrils components. In the negative controls the primary antibody was substituted by a irrelevant antibody. The proteolytic activity was carried out on myofibrils extracted by gastrocnemius muscle from normal C57BL/6 mice. Washed myofibrils were incubated with 20 mg of neuwiedase in the presence of 5 mM CaCl2 at 37°C during  5, 15, 30, 60 min, 3, 6 and 24 h. The degradation of miofibrils was evaluated by SDS-PAGE and showed that neuwiedase was able to degrade bands correspondent to actin and miosin according to their molecular weight. Taken together, our results suggests that the tissue damage induced by neuwiedase is due to proteolytic action upon connective tissue and specifics components of muscle fibers.

Support: CNPq and FAPEMIG

Correspondence to: veridiana_avila@hotmail.com