Tissue Damage and Inflammation Induced by Snake Venoms

Abstract

Some characteristics of the local tissue damage and inflammatory reactions induced by snake venoms were analyzed in a mouse model. Tissue damage was studied by intravital, light, and electron microscopic techniques, and by the use of biochemical markers. Detailed information on the early development and dynamics of local tissue damage was obtained by intravital microscopy. Main alterations were microvascular plasma leakage, hemorrhage, blood flow disturbances, thrombosis, and myonecrosis. A new technique for the quantification of myonecrosis in vivo was established, based on the principle of MTT reduction. The method was tested for its usefulness in the evaluation of antibody-mediated neutralization of myotoxicity. The inflammatory response to venom included early lymphopenia and neutrophilia, thrombocytopenia, with edema and leukocyte extravasation at the site of injection. A rapid plasma peak of IL-6 was induced by venoms, as well as by purified muscle-damaging and hemorrhagic toxins, in response to cellular damage. The effects of a purified hemorrhagic toxin on cultured endothelial cells were studied. The toxin was not directly lytic to these cells even at high concentrations, and caused moderate cell detachment due to its metalloproteinase activity, suggesting that the endothelial cell damage in vivo occurs via an indirect mechanism, probably initiated by proteolytic degradation of the basal lamina of microvessels. Myotoxin II from Bothrops asper venom was shown to lyse a variety of cell types in culture, including myoblasts and endothelial cells. This property was exploited in a cytotoxicity assay for the evaluation of myotoxin neutralization. Heparin was found to be a potent inhibitor of its cytolytic activity, by forming complexes, held at least in part by electrostatic interactions. The ability of heparin to neutralize several related non neurotoxic phospholipase A2 myotoxins present in crotalid venoms, was not dependent on its anticoagulant effect. Thus, nonanticoagulant heparin has a potential as a therapeutic aid, which should be further evaluated. The structural characteristic of the binding interaction of heparin with myotoxin II were analyzed, indicating that a hexasaccharide is the minimal heparin chain size required, and that both N-sulfate and O-sulfate groups participate in the binding.