Damage to the spinal cord, whether caused by injury or disease, cannot currently be repaired. The molecular mechanisms underlying the early pathophysiological stages of spinal cord injury remain largely unknown. After the initial insult, the tissue damage considerably spreads (secondary damage) because of further destruction of neuronal and glial cells caused by metabolic dysfunction (including ischemia), excitotoxicity, reactive oxygen radicals and neuroinflammatory reactions. We wished to explore the temporal evolution of such processes using, as a model, the thoracic-lumbar spinal cord of the neonatal rat maintained in vitro for up to 24 h. We have recently observed that excitotoxicity and severe metabolic perturbation differentially contribute to the dysfunction of locomotor networks, spinal reflexes and intrinsic network rhythmicity (Taccola et al., Neuroscience 2008, 155:538-555). Kainate evoked excitotoxicity suppresses fictive locomotion irreversibly, while a pathological medium (containing free radicals and hypoxic/aglycemic conditions) slows down fictive locomotion. Histological analysis shows significant cellular damage around the central canal after kainate treatment, while the pathological treatment induces preferential damage to white matter.

To clarify the molecular mechanisms underlying these distinct types of acute spinal cord dysfunction, we analyzed mRNA and protein content of the neonatal rat spinal cord at various times after applying either kainate or pathological medium (1 h application). Gene expression levels of different cell type markers (GFAP for astroglia, Mac 1 for microglia), the neuronal injury marker ATF-3, and various genes involved in neuroinflammation (interleukin 1b, IL-1b; serpine-1), and cell proliferation (egr-1) were studied using Real-Time PCR or large-scale Superarrays. Our data indicate early (4 h) upregulation of ATF-3, egr-1, IL-1b and serpine-1 expression after kainate treatment. Conversely, following pathological medium, there was significant downregulation of GFAP and MAC1 expression starting already at 4 h after treatment. Western blots and immunohistochemistry (anti-NeuN, anti-GFAP and anti-SMI-32) strongly suggested large neuronal damage by kainate treatment, whereas predominant glial damage by the pathological medium.

Our results validate distinct molecular consequences of excitotoxicity and metabolic dysfunction after experimental spinal cord injury, indicating different cellular sensitivity to acute damage. Combination of different strategies might therefore be necessary to treat the various components contributing to early spinal cord damage.

Supported by PRIN and FVG grants.