Inibição dos efeitos tóxicos da Crotoxina pelo Fator Neutralizante de Crotalus (CNF) em preparações neuromusculares de camundongos

Abstract

Crotalus Neutralizing Factor (CNF) is a glycoprotein present in the blood plasma of C.d. terrificus, capable to inhibit the in vivo toxic effects of CTX, the main component found in the venom of this snake. However, there are no in vitro studies demonstrating the neutralizing action of CNF on the CTX toxic actions at the neuromuscular junction, the main site of action of this toxin. In this sense, the objective of this study was to evaluate the ability of CNF to inhibit neuromuscular blockade and myotoxicity induced by CTX and its CB subunit in mice phrenic nerve-diaphragm muscle preparations. The paralyzing activity was evaluated through the recording of muscle twitches evoked indirectly and directly. At the end of the myographic experiments, the muscles were fixed, and the blades subjected to hematoxylin and eosin staining, the myotoxicity was assessed by optical microscopy. The results showed that the control and CNF groups did not induce alterations in the amplitude of indirectly or directly evoked twitches over time. CTX 5 μg/mL alone induced total blockade of indirectly evoked twitches in 60 minutes, however, in the direct contractions, the blockade was partial. The CNF 50 μg/mL was able to inhibit neuromuscular block induced by CTX 5 μg/mL in indirectly evoked preparations, whereas in the direct twitches the inhibition was partial. Concerning the CB 20 μg/mL subunit, the proposed concentrations of CNF were not sufficient to promote the inhibition of the toxic effect of this PLA2 alone. The morphological study showed that the pre-incubation of CTX 5 μg/mL with CNF 50 μg/mL reduced the number of injured fibers compared to the muscles exposed to CTX alone. Therefore, the results revealed for the first time that CNF is able to inhibit the neurotoxic and myotoxic activities of CTX. Although inhibition of myotoxicity was partial in myographic experiments, muscle injury was completely inhibit, as observed in the morphological study. Thus, CNF can be an important tool for the study of the CTX mechanisms of action, since it is able to neutralize its toxic effects, as well as a substance with clinical potential to assist in the treatment of individuals poisoned by snakes of the Crotalus durissus terrificus species.