Role of axon resealing in retrograde neuronal death and regeneration after spinal cord injury

Abstract

Spinal cord injury leads to persistent behavioral deficits because mammalian central nervous system axons fail to regenerate. A neuron's response to axon injury results from a complex interplay of neuron-intrinsic and environmental factors. The contribution of axotomy to the death of neurons in spinal cord injury is controversial because very remote axotomy is unlikely to result in neuronal death, whereas death of neurons near an injury may reflect environmental factors such as ischemia and inflammation. In lampreys, axotomy due to spinal cord injury results in delayed apoptosis of spinal-projecting neurons in the brain, beyond the extent of these environmental factors. This retrograde apoptosis correlates with delayed resealing of the axon, and can be reversed by inducing rapid membrane resealing with polyethylene glycol. Studies in mammals also suggest that polyethylene glycol may be neuroprotective, although the mechanism(s) remain unclear. This review examines the early, mechanical, responses to axon injury in both mammals and lampreys, and the potential of polyethylene glycol to reduce injury-induced pathology. Identifying the mechanisms underlying a neuron's response to axotomy will potentially reveal new therapeutic targets to enhance regeneration and functional recovery in humans with spinal cord injury.

Keywords: PEG; axon resealing; calcium signaling; mitochondrial dysfunction; regeneration; retrograde neuronal death; sea lamprey; spinal cord injury.

Figure 1

Polyethylene glycol (PEG)-induced axon sealing reduces post-complete spinal cord transection (TX) caspase activation. (A, C) At 24 hours after spinal cord TX and application of control Ringer solution (A) or PEG (C) to the cut ends, neurons with unsealed axons were labeled retrogradely with dextran-tetramethylrhodamine (DTMR) applied to the lesion. (B, D) Two weeks later, the brains were dissected live and labeled by fluorochrome-labeled inhibitors of caspases (FLICA) to identify neurons that contained activated caspases. Neurons with delayed sealing were more likely to be FLICA⁺. (E) Hypothesis to explain results. Delayed resealing raises cytosolic calcium levels and injures mitochondria, which releases accumulated calcium along with low molecular weight mitochondrial molecules including cytochrome c, which propagates the intrinsic caspase activation pathway, leading to cell death. PEG rapidly reseals the axolemma independently of the calcium-dependent endogenous pathway. Extracellular calcium chelation with ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) reduces calcium influx but degeneration is not inhibited, either because of the entry of other toxic substances, or because sodium influx promotes calcium release from intracellular stores.