RESUMO
Phoneutria nigriventer spider venom (PNV) induces, in rats, local edema as result of an increased vascular permeability, as well as causes blood-brain barrier (BBB) breakdown by altering transendothelial transport routes in hippocampal microvessels. In this work we investigated the in vitro effects of PNV on cell viability and cellular transport routes using three cell lines, the ECV304 endothelial-, the C6 glioma- and the MDCK epithelial cells. We showed that PNV (14.6 and 292 microg crude venom/ml culture medium) had no direct cytotoxic effect on both the ECV304 and the MDCK cell lines but slightly reduced the viability of C6 glioma cells (P<0.05) at the highest concentration, as revealed by the cellular neutral red uptake assay. The PNV effects on cell transport were evaluated in MDCK cell line. PNV seems do not cause any disturbance in the paracellular barrier function of the cultured MDCK cells, as shown by the lack of a significant change in the distribution and expression of the junctional proteins, ZO-1, occludin, E-cadherin and the cytoskeletal F-actin. In contrast, PNV-treated MDCK monolayers showed an enhancement in the transepithelial electrical resistance and a tendency towards an increased occludin expression. In addition, the PNV significantly increased the apical endocytosis of HRP, which was not followed by an equivalent exocytosis at the basal side, as revealed by biochemical and ultrastructural methods. We conclude that the venom of P. nigriventer displays a relatively low cytotoxicity in vitro as well as activates directly the endocytic transport pathway in MDCK cells without disrupting the paracellular route.
Assuntos
Venenos de Aranha/toxicidade , Aranhas/química , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Impedância Elétrica , Endocitose/efeitos dos fármacos , Peroxidase do Rábano Silvestre , Proteínas de Membrana/efeitos dos fármacos , Vermelho NeutroRESUMO
The blood-brain barrier (BBB) is responsible for selective flux of substances between blood and brain. The selective permeability of the BBB is crucial for the maintenance of the brain microenvironment homeostasis, and alterations in the barrier may be involved in many pathophysiological processes. Phoneutria nigriventer armed spider venom produces excitatory signals and symptoms in humans, and its recognized neurotoxic action suggests a potential ability to alter BBB permeability. The aim of the present study was to investigate the capacity of P. nigriventer venom (PNV) in promoting BBB breakdown in adult rats. After intravenous injection of 850 micro g/kg of the whole venom, BBB lesions were evaluated after 18 h to 9 days by ultrastructural methods using the extracellular tracer lanthanum nitrate. Clinical signs and symptoms of rats showed acute neurotoxicity, with some of the animals presenting convulsions, but which were clinically resolved by 12 h post-envenoming. The results showed that PNV is able to increase BBB permeability, particularly in the hippocampus. Changes were first detected in arterioles and post-capillary venules 18 h to 5 days after venom inoculation. The increased permeation of the extracellular tracer peaked on day 1, representing about 42% of the examined vessels (P<0.01). This appeared to occur by both transendothelial and intercellular routes, i.e., by pinocytic transport and through interendothelial junctions. Concomitantly, the surrounding tissue showed vasogenic edema and swollen astrocytic processes, without inflammatory infiltrates. The peak of the edema occurrence was observed on day 3, in about 60% of the vessels (P<0.001). Enhanced capillary permeability was observed on day 9, and affected 36% of all capillaries (P<0.05). The affected capillaries were characterized by increased number of pinocytotic vesicles, which, in addition, were filled with the extracellular tracer, but without visible transport through the interendothelial pathway. This study demonstrates that systemic PNV inoculation induces BBB breakdown through trans- and paracellular routes. It is concluded that BBB breakdown is an event not associated with the acute neurotoxicity exhibited by the rats.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Venenos de Aranha/farmacocinética , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Edema Encefálico/induzido quimicamente , Espaço Extracelular , Masculino , Microscopia Eletrônica , Síndromes Neurotóxicas/patologia , Síndromes Neurotóxicas/fisiopatologia , Ratos , Ratos WistarRESUMO
Bothrops moojeni snake venom induces acute renal failure (ARF) as a consequence of morphological and functional alterations in glomerular and tubular cells. It is still unclear whether the ARF results from a direct cytotoxic effect on renal epithelia or from a renal ischemia due to systemic hemodynamic disturbances. This work investigated the in vitro effect of B. moojeni crude venom, using cultured Madin-Darby canine kidney (MDCK) monolayers as a model. The crude venom induced a significant time- and dose-dependent decrease in transepithelial electrical resistance across MDCK monolayers. In addition, the exposure to the venom resulted in cell detachment from the substratum, as revealed by transmission electron microscopy. Immunocytochemical analysis showed no change in the distribution of some junctional proteins, such as occludin, ZO-1, and E-cadherin. Nevertheless, the staining with labeled phalloidin revealed a disarray of the cytoskeleton, specifically of the stress fibers and of the focal adhesion-associated F-actin at the cell-to-matrix contact region. The treatment with B. moojeni venom also increased the cell release of lactate dehydrogenase and decreased cellular uptake of the vital neutral red. In conclusion, B. moojeni crude venom appears to have a direct cytotoxic effect on a renal tubule-derived cell line, also inducing impairment of the cell-matrix interaction.