In this study we have used fluorescence imaging and pharmacological intervention to determine both the final fate of neurons, and the processes occurring during neurodegeneration following OGD, or specific insults, in order to establish the causal and temporal relationships between the factors involved. Neuronal viability was determined with the fluorescent indicator propidium iodide. Statistically significant neuroprotection following OGD was observed due to inhibition of the NMDA receptor (MK-801), intracellular calcium ion scavenging (BAPTA-AM), inhibition of mitochondrial calcium ion uptake (Ru360), free radical scavenging (MnTBAP), nitric oxide (NO) synthase inhibition (L-NAME) and NO scavenging (carboxy-PTIO). However, blockade of the mitochondrial permeability transition pore (Cyclosporin A), or inhibition of calcium induced calcium release (Dantrolene) did not significantly attenuate this neurodegeneration. Calcium ion concentration, superoxide (O₂~) levels and NO production were monitored with the fluorescent indicators fluo-3 AM, dihydroethidium and DAF-FM respectively. These events were found to increase during OGD or NMDA excitotoxicity. Pharmacological intervention while monitoring these events led to several key observations. Scavenging of NO, and blockade of mitochondrial calcium ion uptake, attenuated the observed free radical increase. Neuronal death induced by and NO donor (SNAP) or O₂~ generating agent (duroquinone), in combination with pharmacological intervention, demonstrated the need for the presence of both O₂~ and NO for the neurodegeneration induced by either agent. In conclusion, the mechanism of neurodegeneration following OGD of hippocampal organotypic slice cultures involves a positive feedback, where elevated NO, superoxide and [Ca²⁺]m are required; this feedback is highly dependent on peroxynitrite formation.