Agrawal S K, Nashmi R, Fehlings M G
Division of Cell and Molecular Biology, Toronto Western Research Institute, Ontario, Toronto, Canada.
Neuroscience. 2000;99(1):179-88. doi: 10.1016/s0306-4522(00)00165-2.
Recent work has suggested a potential role for voltage-gated Ca(2+) channels in the pathophysiology of anoxic central nervous system white matter injury. To examine the relevance of these findings to neurotrauma, we conducted electrophysiological studies with inorganic Ca(2+) channels blockers and L- and N-subtype-specific calcium channel antagonists in an in vitro model of spinal cord injury. Confocal immunohistochemistry was used to examine for localization of L- and N-type calcium channels in spinal cord white matter tracts. A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber and injured with a modified clip (2g closing force) for 15s. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential at two points with glass microelectrodes. The compound action potential decreased to 71.4+/-2.0% of control (P<0. 05) after spinal cord injury. Removal of extracellular Ca(2+) promoted significantly greater recovery of compound action potential amplitude (86.3+/-7.6% of control; P< 0.05) after injury. Partial blockade of voltage-gated Ca(2+) channels with cobalt (20 microM) or cadmium (200 microM) conferred improvement in compound action potential amplitude. Application of the L-type Ca(2+) channel blockers diltiazem (50 microM) or verapamil (90 microM), and the N-type antagonist omega-conotoxin GVIA (1 microM), significantly enhanced the recovery of compound action potential amplitude postinjury. Co-application of the L-type antagonist diltiazem with the N-type blocker omega-conotoxin GVIA showed significantly greater (P<0.05) improvement in compound action potential amplitude than application of either drug alone. Confocal immunohistochemistry with double labelling for glial fibrillary acidic protein, GalC and NF200 demonstrated L- and N-type Ca(2+) channels on astrocytes and oligodendrocytes, but not axons, in spinal cord white matter. In conclusion, the injurious effects of Ca(2+) in traumatic central nervous system white matter injury appear to be partially mediated by voltage-gated Ca(2+) channels. The presence of L- and N-type Ca(2+) channels on periaxonal astrocytes and oligodendrocytes suggests a role for these cells in post-traumatic axonal conduction failure.
近期研究表明，电压门控性Ca(2+)通道在缺氧性中枢神经系统白质损伤的病理生理过程中可能发挥作用。为探究这些发现与神经创伤的相关性，我们在脊髓损伤体外模型中，使用无机Ca(2+)通道阻滞剂以及L型和N型特异性钙通道拮抗剂进行了电生理研究。采用共聚焦免疫组织化学方法检测L型和N型钙通道在脊髓白质束中的定位。从成年大鼠脊髓中分离出一段30毫米长的背柱，固定于体外记录室，用改良夹（2克闭合力）损伤15秒。通过用玻璃微电极在两点定量测量复合动作电位，电生理监测背柱的功能完整性。脊髓损伤后，复合动作电位降至对照值的71.4+/-2.0%（P<0.05）。去除细胞外Ca(2+)可显著促进损伤后复合动作电位幅度的更大恢复（达对照值的86.3+/-7.6%；P<0.05）。用钴（20微摩尔）或镉（200微摩尔）部分阻断电压门控性Ca(2+)通道可使复合动作电位幅度得到改善。应用L型钙通道阻滞剂地尔硫䓬（50微摩尔）或维拉帕米（90微摩尔）以及N型拮抗剂ω-芋螺毒素GVIA（1微摩尔），可显著增强损伤后复合动作电位幅度的恢复。联合应用L型拮抗剂地尔硫䓬与N型阻滞剂ω-芋螺毒素GVIA，复合动作电位幅度的改善程度显著大于单独应用任一药物（P<0.05）。对胶质纤维酸性蛋白、半乳糖脑苷脂和神经丝蛋白200进行双重标记的共聚焦免疫组织化学显示，脊髓白质中星形胶质细胞和少突胶质细胞上存在L型和N型Ca(2+)通道，但轴突上没有。总之，Ca(2+)在创伤性中枢神经系统白质损伤中的有害作用似乎部分由电压门控性Ca(2+)通道介导。轴周星形胶质细胞和少突胶质细胞上L型和N型Ca(2+)通道的存在表明这些细胞在创伤后轴突传导障碍中发挥作用。
