Neuropathological characterization of spinal motor neuron degeneration processes induced by acute and chronic excitotoxic stimulus in vivo

• Acute perfusion of AMPA in the spinal cord induces excitotoxic rapid motor neuron degeneration mainly by a necrotic process.
• Chronic AMPA infusion induces motor neuron degeneration along several days by an apoptosis process followed by necrosis.
• Necrosis, rather than the caspase 3-mediated apoptosis, is responsible for the motor neuron loss causing paralysis.
• The excitotoxic processes characterized may be relevant for understanding the pathophysiology of motor neuron diseases.

Motor neuron (MN) diseases are characterized by progressive cell degeneration, and excitotoxicity has been postulated as a causal factor. Using two experimental procedures for inducing excitotoxic spinal MN degeneration in vivo, by acute and chronic overactivation of α-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) receptors, we characterized the time course of the neuropathological changes. Electron transmission microscopy showed that acute AMPA perfusion by microdialysis caused MN swelling 1.5 h after surgery and lysis with membrane rupture as early as 3 h; no cleaved caspase 3 was detected by immunochemistry. Chronic AMPA infusion by osmotic minipumps induced a slow degeneration process along 5 days, characterized by progressive changes: endoplasmic reticulum swelling, vacuolization of cytoplasm, vacuole fusion and cell membrane rupture. Quantification of these ultrastructural alterations showed that the increase of vacuolated area was at the expense of the nuclear area. Caspase 3 cleavage was observed since the first day of AMPA infusion. We conclude that acute AMPA-induced excitotoxicity induces MN loss by necrosis, while the progress of degeneration induced by chronic infusion is slow, starting with an early apoptotic process followed by necrosis. In both the acute and chronic procedures a correlation could be established between the loss of MN by necrosis, but not by caspase 3-linked apoptosis, and severe motor deficits and hindlimb paralysis. Our findings are relevant for understanding the mechanisms of neuron death in degenerative diseases and thus for the design of pharmacological therapeutic strategies.

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