We report here that activation of the caspase-3 apoptotic cascade in spinal cord injury is regulated, in part, by calcineurin-mediated BAD dephosphorylation. BAD, a proapoptotic member of the bcl-2 gene family, is rapidly dephosphorylated after injury, dissociates from 14-3-3 in the cytosol, and translocates to the mitochondria of neurons where it binds to Bcl-x(L). Pretreatment of animals with FK506, a potent inhibitor of calcineurin activity, or MK801, an NMDA glutamate receptor antagonist, blocked BAD dephosphorylation and abolished activation of the caspase-3 apoptotic cascade. These findings extend previous in vitro observations and are the first to implicate the involvement of glutamate-mediated calcineurin activation and BAD dephosphorylation as upstream, premitochondrial signaling events leading to caspase-3 activation in traumatic spinal cord injury.

The purpose of this study was to investigate the potential neuroprotective efficacy of the mitochondrial uncoupler 2,4-dinitrophenol (DNP) in rats following a mild to moderate spinal cord contusion injury. Animals received intraperitoneal injections of vehicle (DMSO) or 5 mg/mL of DNP prior to injury. Twenty-four hours following surgery, mitochondrial function was assessed in mitochondria isolated from spinal cord synaptosomes. In addition, synaptosomes were used to measure indicators of reactive oxygen species formation, lipid peroxidation, and protein oxidation. Relative to vehicle-treated animals, pretreatment with DNP maintained mitochondrial bioenergetics and significantly decreased reactive oxygen species levels, lipid peroxidation, and protein carbonyl content following spinal cord injury. Furthermore, pretreatment with DNP significantly increased the amount of remaining white matter at the injury epicenter 6 weeks after injury. These results indicate that treatment with mitochondrial uncoupling agents may provide a novel approach for the treatment of secondary injury following spinal cord contusion.

We examined intracellular pH (pH(i)) regulation in the retrotrapezoid nucleus (RTN), a CO(2)-sensitive site, and the hypoglossal nucleus, a nonchemosensitive site, during development (postnatal days 2-18) in rats. Respiratory acidosis [10% CO(2), extracellular pH (pH(o)) 7.18] caused acidification without pH(i) recovery in the RTN at all ages. In the hypoglossal nucleus, pH(i) recovered in young animals, but as animal age increased, the slope of pH(i) recovery diminished. In animals older than postnatal day 11, the pH(i) responses to hypercapnia were identical in the hypoglossal nucleus and the RTN, but hypoglossal nucleus and RTN neurons could regulate pH(i) during intracellular acidification at constant pH(o) at all ages. Recovery of pH(i) from acidification in the RTN depended on extracellular Na+ and was inhibited by amiloride but was unaffected by DIDS, suggesting a role for Na+/H+ exchange. Hence, pH(i) regulation during acidosis is more effective in the hypoglossal nucleus in younger animals, possibly as a requirement of development, but in older juvenile animals (older than postnatal day 11), pH(i) regulation is relatively poor in chemosensitive (RTN) and nonchemosensitive nuclei (hypoglossal nucleus).

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