We recently discovered the cyclosporin A (CsA), a commonly used immunosuppressant, is neuroprotective in a rat model of spinal cord injury (SCI), CsA inhibitors calcineurin, a key intracellular phosphatase in neurons and lymphocyts. Recent studies have shown that calcineurin induces and regulates immune and inflammatory responses of cells, as well as calcium channels, neurotransmitter receptors, and secretion. We consequently propose to study CsA and other calcineurin inhibitors further. Our first aim is to determine whether calcineurin inhibition protects the spinal cord. We will compare the effects of methylprednisolone (MP, the current standard therapy for human SCI), CsA, CsA+MP, FK506, rapamuycin, and FK506+rapamycin. FK506 is a macrolide antibiotic which inhibits calcineurin but by binding to a different protein. Rapamycin is a macrolide that does not inhibit calcineurin and antagonizes FK506 inhibition of calcineurin. If Cs
and FK506 are neuroprotective, rapamycin is not and antagonizes FK506's neuroprotective effect, the data would argue strongly that calcineurin plays a major role in SCI. Our second aim is to establish that GsA and FK506 improves motor recovery and saves axons in the spinal cord. We will assess locomotor recovery, count spinal axons, and assess myelination in the rats at 6 weeks after injury and CsA, MP, CsA+MP, FK506, and FK506+ rapamycin treatment. Our third aim is to study the effects of calcineurin inhibitors on blood flow in injured spinal cords. Calcineurin stimulates the nitric oxide synthase which produces nitric oxide, a potent vasodilating agent. We propose the nitric oxide causes an initial increase in flow and delays onset of blood flow loss in SCI. Our fourth aim is to determine whether serotonin (5-HT) and gamma-amino- butyrate acid (GABA) contribute to SCI. Both neurotransmitters are released in injured spinal cords. We recently found that spinal axons have 5-HT and GABA receptors. Calcineurin prevents desensitization of 5-HT gated calcium channels and CsA may protect spinal axons by turning off 5-HT receptors. Mianserin, a 5-HT receptor blocker, has been reported to be neuroprotective in SCI. We will therefore assess the effects of calcineurin on 5-HT and GABA-induced excitability changes in vitro and in vivo. Our fifth aim is to assess the role of glutamate in spinal cord injury. Glutamate is known to injure neurons and glutamate receptor blockers have been reported to be neuroprotective in spinal cord injury. Recent studies, however, report that calcineurin inactivates glutamate channels and also enhances glutamate release. Calcineurin inhibitors therefore may paradoxically enhance glutamate-induced neurotoxicity in SCI. To resolve this issue, we will examine glutamate sensitivity in spinal cords and determine the effects of calcincurin inhibitors on glutamate-mediated axonal changes, in vitro and in vivo.