calcimycin and Spinal-Cord-Injuries

Both glibenclamide and riluzole reduce necrosis and improve outcome in rat models of spinal cord injury (SCI). In SCI, gene suppression experiments show that newly upregulated sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channels in microvascular endothelial cells are responsible for "persistent sodium currents" that cause capillary fragmentation and "progressive hemorrhagic necrosis". Glibenclamide is a potent blocker of Sur1-regulated NC(Ca-ATP) channels (IC(50), 6-48 nM). Riluzole is a pleotropic drug that blocks "persistent sodium currents" in neurons, but in SCI, its molecular mechanism of action is uncertain. We hypothesized that riluzole might block the putative pore-forming subunits of Sur1-regulated NC(Ca-ATP) channels, Trpm4. In patch clamp experiments, riluzole blocked Sur1-regulated NC(Ca-ATP) channels in endothelial cells and heterologously expressed Trpm4 (IC(50), 31 μM). Using a rat model of cervical SCI associated with high mortality, we compared the effects of glibenclamide and riluzole administered beginning at 3h and continuing for 7 days after impact. During the acute phase, both drugs reduced capillary fragmentation and progressive hemorrhagic necrosis, and both prevented death. At 6 weeks, modified (unilateral) Basso, Beattie, Bresnahan locomotor scores were similar, but measures of complex function (grip strength, rearing, accelerating rotarod) and tissue sparing were significantly better with glibenclamide than with riluzole. We conclude that both drugs act similarly, glibenclamide on the regulatory subunit, and riluzole on the putative pore-forming subunit of the Sur1-regulated NC(Ca-ATP) channel. Differences in specificity, dose-limiting potency, or in spectrum of action may account for the apparent superiority of glibenclamide over riluzole in this model of severe SCI.

Topics: Action Potentials; Analysis of Variance; Animals; Calcimycin; Calcium; Calcium Ionophores; Capillaries; Cell Count; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Glyburide; Green Fluorescent Proteins; Hand Strength; Hypoglycemic Agents; Motor Activity; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Rats; Riluzole; Spinal Cord Injuries; Transfection; Treatment Outcome; TRPM Cation Channels

Periaxonal glia play an important role in maintaining axonal function in white matter. However, little is known about the changes that occur in glial cells in situ immediately after traumatic injury. We used fluo-3 and confocal microscopy to examine the effects of localized (<0.5 mm) mechanical trauma on intracellular calcium (Ca(i)(2+)) levels in glial cells in a mature rat spinal cord white matter preparation in vitro. At the injury site, the glial Ca(i)(2+) signal increased by 300-400% within 5 min and then irreversibly declined indicating cell lysis and death. In glial cells at sites adjacent to the injury (1.5-2 mm from epicenter), Ca(i)(2+) levels peaked at 10-15 min, and thereafter declined but remained significantly above rest levels. At distal sites (6-9 mm), Ca(i)(2+) levels rose and declined even slower, peaking at 80-90 min. Injury in zero calcium dampened Ca(i)(2+) responses, indicating a role for calcium influx in the generation and propagation of the injury-induced Ca(i)(2+) signal. By 50-80 min post-injury, surviving glial cells demonstrated an enhanced ability to withstand supraphysiological Ca(i)(2+) loads induced by the calcium ionophore A-23187. Glial fibrillary acidic protein (GFAP) and CNPase immunolabeling determined that the glial cells imaged with fluo-3 included both astrocytes and oligodendrocytes. These data provide the first direct evidence that the effects of localized mechanical trauma include a glial calcium signal that can spread along white matter tracts for up to 9 mm within less than 3 h. The results further show that trauma can enhance calcium regulation in surviving glial cells in the acute post-injury period.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Aniline Compounds; Animals; Astrocytes; Calcimycin; Calcium; Calcium Signaling; Cell Death; Cell Survival; Female; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Homeostasis; Immunohistochemistry; Ionophores; Microscopy, Confocal; Neuroglia; Oligodendroglia; Potassium; Rats; Rats, Wistar; Spinal Cord Injuries; Xanthenes

Activation of arachidonic acid occurs after spinal cord injury. Leukotriene B4 is a lipoxygenase metabolite of arachidonic acid. In a rat model of experimental spinal cord injury, we found that the leukotriene B4 content was less than the sensitivity of our assay (8 pg/mg of protein) in non-traumatized spinal cord. Leukotriene B4 was detectable in traumatized cord (mean +/- SE, 25 +/- 5 pg/mg of protein; n = 3). Release of leukotriene B4 from spinal cord slices into the incubation medium was also noted after trauma (9 +/- 1 pg/mg of protein; n = 12) and was enhanced by exposure of traumatized spinal cord slices to the calcium ionophore A23187 (375 +/- 43 pg/mg of protein; n = 12). The amount of leukotriene B4 released corresponded to the extent of post-traumatic polymorphonuclear cell infiltration determined by a myeloperoxidase assay. Results from this study suggest that the source of leukotriene B4 in spinal cord injury is infiltrating polymorphonuclear cells.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Leukocyte Count; Leukotriene B4; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries