Mechanisms involved in the blood–brain barrier increased permeability induced by Phoneutria nigriventer spider venom in rats

Abstract

We have recently demonstrated by electron microscopy, using lanthanum nitrate as an extracellular tracer, that the intravenous injection of Phoneutria nigriventer spider venom (PNV) induces blood–brain barrier (BBB) breakdown in rat hippocampus. One and nine days after PNV injection, tracer was found in pinocytic vesicles crossing the endothelium and in the interendothelial cleft, suggesting that BBB breakdown had occurred through enhanced transendothelial transport and/or tight-junction opening. In the present work, we investigated the mechanisms by which PNV (850 μg/kg, i.v.) increased the hippocampal microvascular permeability in rats 24 h after the endovenous administration. The expression and phosphorylation of some tight- and adherens junctions-associated proteins in hippocampal homogenate and hippocampal microvessel homogenate were assessed by Western blotting and immunoprecipitation. The microtubule-dependent transcellular transport was also evaluated by quantitative ultrastructural methods in pretreated rats with colchicine (0.5 mg/kg, i.p.), prior to PNV injection. Western blots showed no significant increase in the expression of the tight junction-associated proteins ZO-1 and occludin or in the adherens junction-associated β-catenin after 24 h of PNV administration. Morphological study showed no alterations of the immunolabeling for occludin and ZO-1 in rat brain cryosection following PNV. In addition, no changes were observed in phosphotyrosine content of occludin and β-catenin in PNV-treated rats compared with control animals. However, the disruption of microtubule-dependent transcellular transport by colchicine completely prevented ( p<0.001) PNV-induced leakage of the BBB tracer. These findings indicate that the increased BBB permeability evoked by PNV in rats probably resulted from enhanced microtubule-dependent transendothelial vesicular transport, with no substantial involvement of the paracellular barrier in the time interval studied.