RODENT ORGANOTYPIC BRAIN SLICES AS AN INTEGRATED MODEL TO STUDY THE EFFECTS OF HYPOXIC ISCHEMIA AND REPERFUSION AND TO DISSECT THE SPECIFIC ROLE OF MICROGLIAL CELLS

Stroke is the second leading cause disability in high-income countries and of death worldwide: several pharmacological therapies for acute stroke are currently available, but their efficacy is limited. New potential therapeutical targets have been identified in the neuroinflammatory response after ischemia, where a fundamental role is played by microglial cells, especially during the acute post-traumatic phase. Among other players, ATP and other nucleotides play a pivotal role, being released in high amount in the extracellular spaces, with both beneficial and detrimental effects: their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors and terminated by various ATP metabolizing enzymes; furthermore, they act are key extracellular signalling molecules in many organs and systems, profoundly influencing cell survival under acute or chronic degenerative conditions, including brain ischemia. The first part of my study has been dedicated to set up the best conditions to perform oxygen glucose deprivation (OGD) on an integrated in vitro system consisting of rodent brain organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC). Based on preliminary experiments, we decided to perform our experiments after a 1h OGD followed by 1-6-18-24-48h of reperfusion. The second part of this work has been aimed at evaluating the effects induced by OGD and reperfusion damage on neurons and glial cells in our slice co-culture system, giving particular attention to the effects induced on microglial cells. General cytotoxicity data (LDH release, PI staining) correlated well with specific neuronal markers, highlighting that this population is the most damaged by OGD and reperfusion injury. On the other hand, astrocytes were not impaired by hypoxic injury (we documented reactive gliosis connected to both slice preparation and OGD), while oligodendrocytes underwent light damage. Furthermore, our study showed that OGD- and reperfusion-induced injury triggers the activation of resident microglia, and induces the expression of specific phenotype markers (IB4, CD68) that develop over time with a specific timing and with regional-specificity. Our data suggest that the citotoxicity and neurotoxicity induced by OGD and reperfusion in organotypic slices could be related to a reduction in the microglial phagocytic activity and to the absence of the macrophage contribute to tissue clearance. The last part of this work has been aimed at evaluating the involvement of purinergic transmission in neuroprotection. In order to verify the appropriateness of our model as a tool to assess neuroprotection, we applied the non selective P2 antagonist PPADS, a known neuroprotective agent. We confirmed a reduced amount of damaged neurons. We also showed unchanged amounts of activated microglia, but a reduced phagocytic CD68+ profile, suggesting a putative link between enhanced neuronal survival and switching to a different microglial phenotype, triggered by PPADS exposure. Sponsored by Fondazione Cariplo “Functional regeneration of the mesocorticolimbic dopaminergic system as a model to study novel neuroreparative strategies” (Rif. 2008.2907), bando 2008 “Promuovere progetti internazionali finalizzati al reclutamento di giovani ricercatori” to Enzo Wanke and Maria Pia Abbracchio.