Cell death in pure-neuronal and neuron-astrocyte mixed primary culture subjected to oxygen-glucose deprivation: The contribution of poly(ADP-ribose) polymerases and caspases

Primary cortical neurons subjected oxygen-glucose deprivation (OGD) is an . in vitro model that mimics fundamental aspects of neonatal hypoxic-ischemic encephalopathy (HIE) and is widely used to test neuroprotective treatments. However, controversial results characterize the existing literature on the OGD model. To shed some light on the initial cell death triggers in OGD, we first investigated the contribution of glucose- or oxygen-deprivation, alone or in combination, to cell viability/death in two cell systems, i.e. pure neuronal: 98% neurons; 2% astrocytes- vs. mixed neuron/astrocytes: 50% neurons; 50% astrocytes- culture. Cell viability was evaluated biochemically (MTT, and LDH release) and morphologically by high-content screening. We first found that neuronal death triggered by OGD (3. h OGD. +. 24. h re-oxygenation) was mainly driven by glucose rather than oxygen deprivation. Astrocytes survival was not substantially affected. Caspase-3 activation was found both in neuronal and mixed neuron/astrocytes cultures, whereas PARP activation was evident only in pure neuronal cultures. To pharmacologically dissect the contribution of these pathways, we measured the effect of TIQ-A (PARP 1 inhibitor) and ZVAD-fmk (pan-caspase inhibitor), individually or in combination, on culture viability after 3. h OGD. We found that only the combination treatment exerts a significant neuroprotective effect particularly evident in pure neuronal cultures. In sum, glucose deprivation is the major cell death trigger in OGD and neurons are more sensitive to OGD than astrocytes. Both PARP and caspases are concurrently activated in pure neuronal cultures and both contribute to neuronal cell death suggesting that neuroprotective strategies may require the simultaneous inhibition of multiple death pathways to be effective.