A study of excitotoxicity as pathological process responsible for spinal cord injury: insight from an in vitro rat spinal cord model with a focus on neuroprotection

Excitotoxicity is considered to be a major contributor to pathophysiological mechanisms responsible for spinal cord damage after acute injury. Hyperactivation of poly(ADP-ribose) polymerase (PARP) is the chief effector of neuronal death which leads to cell energy depletion and DNA damage with the manifestation of non-apoptotic cell death termed parthanatos. Glutamate mediated excitotoxicity is also one important component of post-traumatic degeneration following spinal cord injury (SCI). Kainate (1 h), a potent non-degradable analog of glutamate, was used to induce excitotoxic injury in our in vitro model. The neonatal rat isolated spinal cord preparation allowed investigating changes in locomotor network activity after application of this excitotoxic agent with or without potential neuroprotective drugs. Synaptic transmission, cumulative depolarization, fictive locomotion and disinhibited bursting were studied in order to observe the functional properties of locomotor network and related to network hictology. Our results showed that the locomotor network was very sensitive to excitotoxic damage and excitotoxicity grew gradually leaving a time window in which neuroprotection might be attempted to preserve the circuits which are still capable of expressing basic rhythmogenesis. Our result confirmed that PARP-1 overactivity is closely related to neuronal loss after kainate induced excitotoxicity. Application of PJ-34 (60 µM; PARP-1 selective inhibitor) blocked PARP-1 activation and preserved dorsal, central and ventral grey matter with maintained reflex activity. Fictive locomotion was restored in more than half of the preparations when the excitotoxic stimulus was moderate. Histological damage by 50 µM kainate (moderate injury) was widely prevented by PJ-34. At the same time, this drug strongly increased spontaneous network discharges which occured synchronously on ventral roots and persisted for 24 h even after PJ-34 washout. Neurochemical experiments showed that PJ-34 induced up to 33% inhibition of synaptosomal glutamate uptake with no effect on GABA uptake. However, chemically or electrically induced fictive locomotion was present 24 h after PJ-34 application and neurons and glia remain unchanged. The other PARP-1 inhibitor, PHE, failed to recover locomotor network function, while basic network rhythmicity persisted and appeared to be a moderate histological neuroprotector. Riluzole, by inhibiting glutamate release and neuronal excitability, could prevent neuronal loss 24 h later, but this drug per se (applied for 24 h) exerted strong and persistant neurodepressant effects on network synaptic transmission from which recovery was very slow and partial. Although, the number of pyknotic cells in the grey matter was decreased by riluzole application after kainate washout, no fictive locomotion was observed. Three hours application of riluzole was not sufficient enough to contrast the deleterious effect of kainate on locomotor activity. In conclusion, the divergence between histological and functional outcome in case of PARP-1 inhibition, proposes a narrow borderline between loss of fictive locomotion and neuronal preservation and suggests that protecting the motoneurons is not enough to ensure the persistence of fictive locomotion. It is also discovered that PJ-34 plays the role of a partial blocker of glutamate uptake. Despite the increased network discharges induced by PJ-34, there was no neurotoxic consequence due to this drug. In general, in order to protect the damaged spinal locomotor network, the neuroprotective strategy should be directed toward identified biomedical targets which play the most important role in cell death mechanism. On the other hand, neuroprotection should be applied at the early stages after the insult. Survival of premotoneurons in the grey matter is as important as motoneuron protection in order to achieve the goal of locomotor function preservation.