Role of ATP and ATP-metabolizing enzymes in neuroreparative processes after hypoxic-ischemic injury in rodent organotypic brain slices

Besides their role as metabolic intermediates and as donors of energy and of phosphate group, ATP and extracellular nucleotides act as key extracellular signalling molecules in many organs and systems, including the central nervous system (CNS), by activating the P2 purinergic receptors (i.e., the 7 ionotropic P2X and 8 G protein-coupled P2Y receptors). Extracellular nucleotides show pro-survival and protective effects on both neurons and glial cells, but they may also contribute to neurodegeneration under deregulated conditions, such as those occurring in acute conditions (stroke or spinal cord damage) or in chronic diseases, like Alzheimer’s or Parkinson’s. Understanding these opposite actions is important to exploit the positive actions of nucleotides and their receptors while limiting the harmful ones. We are particularly interested in verifying if and how the purinergic signalling can contribute to the survival, repair and regeneration of neural cells following ischemic/hypoxic insults. To this purpose, we have focussed our attention on an experimental model that has been set up by Illes and co-workers : organotypic cultures of rodent brain slices from mesencephalic ventral tegmental area/substantia nigra (VTA-SN) and prefrontal cortex (PFC). Freshly dissected slices from these two brain areas are put in contact and maintained in culture up to 3 weeks, to allow studying the influence of purinergic modulators on nerve fibers regeneration and on the reconstruction of neuronal circuitries under control conditions and after induction of hypoxia/ischemia. As a first step, we have evaluated the damage induced by the application of oxygen-glucose deprivation (OGD) to VTA-SN/PFC co-cultures, obtained by maintaining slices in a glucose-free medium and in a controlled humidified atmosphere of 95% N2 in an hypoxia chamber, which mimics in vitro the cytotoxicity induced by ischemia in vivo. Lactate Dehydrogenase (LDH) cytotoxicity assay highlighted a significant increase of LDH enzyme release in the culture medium after 1h OGD (+91% with respect to control cultures). These data correlate with the increased number of PI-positive cells in OGD-treated cultures (+89% with respect to control). Due to the more extended damages induced by longer times of OGD, in particular on the VTA-SN portion that underwent partial or complete detachment from the culture dish and disruption into small pieces, we decided to set up our experiments on 1h OGD, followed by different times of reperfusion. We have then focussed our attention on the effects on neurons, the cellular population most sensitive to OGD, and on the glial population (namely, astroglia, microglia and oligodendroglia), in order to understand the modification of the glial response after injury and its possible role in either damage propagation or recovery. Preliminary data suggest that, in the PFC, the number of astroglial cells is not affected by OGD, while an induction of microglial cells activation (approx. +50%) and an increased number of oligodendroglial cells (approx. +60%) were detected. Since extracellular nucleotide concentrations and functions depend on the activity of nucleotide-hydrolysing enzymes, and some of them (i.e., NTPDases, which hydrolyze ATP to AMP) have been reported to increase upon ischemia , we have set up a lead-phosphate method for the in situ localization of their activity. This method has been initially characterized and validated in dissociated cell cultures from rat CNS (endothelial cells, pericytes and astrocytes). NTPDases activity results in a lead phosphate precipitate that can be visualized by light microscopy as a brown deposit by converting phosphate to sulphide, and subsequently quantified by densitometric analysis. Results highlight that the exposure of these cells to hypoxic/hypoglycaemic conditions results in an increased activity of ATP-hydrolyzing enzymes. We are currently translating this method to the in vitro organotypic VTA-SN/PFC cultures.