The propagation of Ca 2ϩ waves in a network of microglial cells, after its initiation by glutamate, is mediated by purinergic transmission. In this study, we investigated the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The 4-fold increase in ATP release from microglia in response to glutamate (0.5 mM) was blocked by ␣-aminohydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione and specific AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) but not by N-methyl-D-aspartic acid or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evoked an ATP release that was blocked by antagonizing the rise in intracellular Ca 2ϩ as a result of its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release was significantly antagonized by the cystic fibrosis transmembrane conductance regulator (CFTR) blockers flufenamic acid and glibenclamide. A role for the CFTR was further confirmed using microglia from CFTR knockout mice, which released significantly less ATP than microglia from control wildtype mice in response to glutamate. Use of 6-methoxy-1-(3-sulfopropyl)quinolinium fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acted on microglial AMPA receptors to stimulate release of Ca 2ϩ from intracellular stores as well as a Ca 2ϩ -dependent isoform of protein kinase C, which then acts to trigger release of ATP with the CFTR acting as a regulator of the ATP release process, perhaps through another channel or transporter.

Sprouting of peptidergic nociceptive and descending supraspinal projections to the dorsal horn following spinal cord injury (SCI) has been proposed as a mechanism of neuropathic pain. To identify structural changes that could initiate or maintain SCI pain, we used a complete transection model in rats to examine how structural remodeling in the dorsal horn rostral to the lesion relates to distance from injury, laminar region, and duration of injury. The major classes of C-fiber primary afferents differed greatly in their susceptibility to structural and chemical changes and their ability to undergo plasticity. Peptidergic primary afferents showed a widespread loss throughout the dorsal horn of segments approaching the injury site. Some of this loss may have been due to decreased neuropeptide expression. The reduction in peptidergic fibers was transient, indicating compensatory sprouting and perhaps also increased neuropeptide expression within the cord. Nonpeptidergic afferents expressing GFRalpha1 were largely unaffected by SCI. In contrast, in GFRalpha2-expressing nonpeptidergic afferents SCI caused a permanent loss of dorsal horn innervation. Unexpectedly, GFRalpha2 was transiently induced throughout deeper laminae but this was not due to upregulation of GFRalpha2 in dorsal root ganglia. We also observed permanent sprouting of catecholamine terminals of supraspinal origin. This was restricted to the superficial laminae. Our results show that SCI caused a loss of sensory input as well as structural remodeling such that the balance of nociceptive inputs and descending modulation was permanently altered. These changes may contribute to mechanisms rostral to the site of SCI that trigger and maintain neuropathic pain.

Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C-fibers, and descending catecholamine and serotonergic projections, were examined in segments T9-T12 at 2 and 12 weeks after a T13 clip-compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene-related peptide (CGRP) or GDNF-family receptors (GFRalpha1, GFRalpha2, GFRalpha3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRalpha1, GFRalpha2 and GFRalpha3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain.

No abstract