Cannabinoid-induced neuroprotection in vitro and in a middle cerebral artery occlusion model.

CALTANA LAURA; HEIMRICH BERND; BRUSCO ALICIA

Washington

Congreso; Neuroscience Annual Meeting; 2011

Society for Neuroscience

Ischemic insult of the brain is known to lead to neuronal cell death. Excessive glutamate release, intracellular Ca2+ rise, recruitment of pro-inflammatory cells and increase in reactive oxygen species preceed this fatal outcome. Cannabinoid receptor 1 (CB1) activation has been show to exert neuroprotective potential counteracting these processes. Here we used mouse hippocampal slices cultures as a model for evaluation of neuroprotective impact of cannabinoid agonists and antagonists. The CB1 agonist, ACEA, and its antagonist, AM251, were administered to the cultures at different concentrations. At different time points (6, 9 and 12 days days in vitro) we analyzed glial reponse, morphological changes of neurons and expression patterns of the propapototic marker capase-3. Treatment at low concentration of ACEA (0,5 µM) did not affect cytoarchitecture of the culture neither neuronal morphology nor glial response. In contrast, at 10µM ACEA many neurons exhibit strong caspase-3 immunoreactivity. After treatment with AM251 we observed an increase in caspase-3 immunoreactivity and a downregulation of CB1 expression. Our data indicate that long term hippocampal slice cultures are a sensitive tool to evaluate cellular alterations induced by CB1 activation and inactivation. In a second set of experiments C57Bl/6 mice were subjected to focal brain ischemia (MCAO). 3, 24 and 48 hours after MCAO, animals received ACEA 1mg/kg, AM251 1mg/kg or vehicle. To assess motor activity, neural deficit score and motor tests (cylinder test, corner test and hanging wire) were performed 1 day before and 3 and 7 after MCAO. At 7 days post-lesion, animals were sacrificed and their brains processed for immunohistochemistry to detect glial response (GFAP, vimentin, S100β), MAP-2 as a neuronal marker and nuclear Hoechst staining. ACEA treatment reduced neuronal death, dendrite loss, astroglial hypertrophy and hyperplasia compared to control. In contrast, examination of these parameters after AM251 treatment indicates a strong increase in damage of brain tissue. Motor test data analyzed showed that there is a progressive deterioration to D7 in ischemic animals and the CB1 agonist treatment produces an improvement in motor activity. Non-ischemic animals treated with different drugs, behaved similarly to the group of untreated non-ischemic animals, except the group treated with CB1 antagonists, which showed a significant decrease in motor activity in both ischemic and non-ischemic groups. Our results suggest that cannabinoids agonists may be involved in neuronal survival and the regulation of neuroprotection during focal cerebral ischemia in mice.