Apoptose na patogenia do do loxoscelismo cutâneo experimental em coelho: abordagens morfológica e imunoistoquímica

Abstract

Accidents with Loxosceles spiders represent a relevant medical problem in Brazil. The Loxosceles bite to a dermonecrotic and hemorrhagic injury that can be followed by systemic manifestations. The dermonecrotic injury is preceded by thrombus formation in dermal vessels near to the bite site. It was shown recently that apoptosis of endothelial cell surrounding atherosclerotic plaques predisposes to thrombosis. Apoptosis is a kind of cellular death that is carried out with e nergy expenditure, besides protein synthesis and degradation. Up to now, there are no studies in vivo showing whether the poison of L. similis induces apoptosis of endothelial cells. Additionally, there are no descriptions of the histological lesion induced by the L. similis bite. The aim of this study is: (1) to analyze histologically at first, the induced injury in the skin of rabbits inoculated with the poison and (2) to evaluate the importance of apoptosis in the mechanism of the loxoscelism. Forty male rabbits were divided in 2 groups: (1) control - inoculated intradermically with 50 µl of PBS and (2) treated- inoculated intradermically with 0,5 µg of the L. similis crude venom diluted in 50 µl of PBS. Animals were analyzed 2, 4, 6 and 8 hours after inoculation. Fragments of skin were collected, fixed in 10% buffered formalin, routinely processed and paraffin embedded. Sections of 5 µm were stained with HE. Some sections were submitted to the TUNEL reaction and other sections were submitted to immunohistochemical detection of Bax. Skin samples were also fixed in glutaraldehyde and embedded in methacrylate. Sections 3 µm thick were stained with HE adapted to methacrylate. The crude L. similis venom induces a lesion characterized by oedema, inflammatory infiltrate predominantly heterophilic, fibrinoid necrosis of the arterial and arteriolar walls, thrombosis in all dermal layers and necrosis of the cutaneous muscle whose intensity progressed with the time interval. The lesion intensity increased with the inoculation time becoming into severe heterophilic dermatitis 8 hours post inoculation. A similar lesion is observed in experiments using the crude venom of other Loxosceles species. Additionally, 2 hours post inoculation, shrunken endothelial cells were noted, losing adhesion one to another and to the extracellular matrix (anoykic cells), containing basophilic nuclei, and condensed chromatin, suggesting apoptosis. Some sections were subjected to TUNEL assay and immunohistochemistry for Bax. Present data suggest that the crude L. similis venom induces apoptosis of endothelial cells. On the basis of the present study it can be concluded that the poison of L. similis induces apoptosis of endothelial cells.