Roles of NF-kB in spinal cord injury pathophysiology
The pathophysiology of spinal cord injury (SCI) is characterized by multiple locomotor and sensory deficits, as well as chronic pain arising partly from significant cell loss at and around the injury site. Implicated in multiple biochemical pathways affected after SCI is the transcription factor NF-kB. We used two-month-old male rats and subjected them to a contusion spinal cord injury to elucidate the differential changes in NF-kB subunits and NF-kB-dependent changes after SCI. We observed an increase in cell death 12 hours after SCI, with significant decreases in the neuronal cell marker, NeuN, at 2, 12 and 24 hours. We show significant and sustained protein level decreases in IkB family members, IkB-a and Bcl-3, suggesting early and prolonged activation of NF-kB up to 28 days after SCI. Validation of NF-kB activation was performed by immunohistochemical analyses of spinal cord cross sections and Western blot assay analyses. Immunohistochemical co-staining with NeuN and c-Rel antibodies demonstrated decreased neuronal c-Rel up to 24 hours after SCI. We also observed significant decreases in the protein levels NF-kB c-Rel up to 12 hours after SCI, signifying its inability to activate genes involved in anti-apoptotic mechanisms. NeuN co-localization with NF-kB p65 and p50 revealed neuronal increases up to 24 hours, especially in the nucleus of cells, supporting activation of NF-kB p50/p65 and p50/p65-induced apoptotic gene transcription after SCI. Western blots of p65 and p50 showed no changes in their protein levels, but whole tissue blots may dilute the immunohistochemical observations. Significant changes in NF-kB p52 and RelB were not observed, suggesting that they do not play a role in response to SCI. Genes dependent on NF-kB were also up-regulated after SCI, including Cox-2 and iNOS, important molecules in the pathophysiology of injury. We show increased protein expression of Cox-2 at 2 hours and of iNOS at 12 hours, the earliest time in which these proteins have been shown to be up-regulated after SCI. Inhibition of NF-kB p50/p65 was performed using decoy deoxyoligonucleotides containing the NF-kB consensus binding site to the Cox-2 promoter. The decoy treatment was successful in abrogating SCI-induced Cox-2 and iNOS protein increases. The decoys did not appear to significantly affect non-NF-kB-dependent gene transcription. Functional outcomes after SCI were also measured after decoy treatment. Improvements in locomotion and decreased mechanical hypersensitivity were observed. In summary, the following series of experiments were designed to test the hypothesis that NF-kB plays a central role in the pathophysiology of spinal cord injury.