Mechanisms of secondary injury to spinal cord axons in vitro: Role of Na+, Na+-K+-ATPase, the Na+-H+ exchanger, and the Na+-Ca2+ exchanger

Abstract

There is evidence that intracellular Na+ entry potentiates hypoxic-ischemic cell death by causing cytotoxic cell edema, intracellular acidosis, and gating of Ca2+ entry by reverse activation of the Na+-Ca2+ exchanger. In this study, we examined the role of Na+ in mediating traumatic injury to spinal cord axons. Dorsal column segments from adult rats (n = 87) were isolated and maintained in an in vitro recording chamber while being superfused with oxygenated Ringer's solution (95% O2/5% CO2, 25°C). Selected experiments (n = 10) also were done at 33°C. Compound action potentials (CAP) were recorded from microelectrodes. Injury was performed by compression of the dorsal column segment for 15 sec with a modified aneurysm clip exerting a closing force of 2 gm. With injury, the CAP decreased to 72.1 ± 9.6% of baseline values. Removal of extracellular Na+ and replacement with the impermeant cation N-methyl-D-glucamine enhanced recovery of the CAP to 98.3 ± 18.3% (p < 0.05) of baseline. The Na+ channel blockers tetrodotoxin and procaine also improved recovery of the CAP to 96.3 ± 23.7% (p < 0.05) and 82.8 ± 4.6% (p < 0.05) of baseline values, respectively. In contrast, increasing Na+ permeability with veratridine resulted in greater attenuation of CAP amplitude after 1 hr of trauma (60.1 ± 8.4%, p < 0.05). Similarly, prevention of extrusion of Na from the intracetlular compartment by inhibiting the Na+-K+-ATPase pump with ouabain resulted in greater attenuation of CAP amplitude at 1 hr after trauma (56.7 ± 3.6%, p < 0.05). The Na+-H+ exchange blockers amiloride (100 μM) and harmaline (100 μM) significantly improved recovery after injury to 89.6 ± 17.0% (p < 0.05) and 85.7 ± 7.2% (p < 0.05) of baseline, respectively. However, administration of the Na+-Ca2+ exchange biockers benzamil (100 or 500 μM) and bepridil (50 μM) was ineffective. In summary, reduction of extracellular Na+ confers neuroprotection after spinal cord injury in vitro. Intracellular sodium rises appear to be mediated by voltage-gated Na+ channels. Blockade of the Na+-H+ exchanger also is neuroprotective, possibly by reducing intracellular acidosis. Furthermore, prevention of extrusion of intracellular Na+ by the Na-K+-ATPase pump exacerbates the effects of compression trauma. However, reverse operation of the Na+-Ca2+ exchanger does not explain the injurious effects of Na+ in traumatically injured CNS white matter.