谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate，THA)对器官型培养的脊髓片的影响，探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养，在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L)，用神经元的特异性免疫组化染色剂SMI-32，非磷酸化神经丝标记物，对脊髓腹角α运动神经元进行鉴定，用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数，测定培养液中乳酸脱氢酶(LDH)的含量，并与对照组比较。结果显示对照组α运动神经元数目恒定，THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少，而脊髓背角的中间神经元损伤相对较轻，其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少，而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤，脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。

Effects of glutamate transport inhibitor on organotypic cultured spinal cord slices]

This study was aimed at investigating the effect of glutamate on motor neurons in organotypic cultured spinal cord slices treated by threohydroxyaspartate (THA), an inhibitor of glutamate transporter. The spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentration of THA(50 micromol/L,100 micromol/L,500 micromol/L) was added into the culture medium respectively. Ventral alpha-motor neurons survival was evaluated by immunohistochemistry staining monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, and interneurons in dorsal horn were identified by monoclonal anti-calretinin antibody staining. Lactate dehydrogenase (LDH) level in the culture medium was also measured. The spinal cord slices in the control group could maintain excellent organotypic cellular organization and a stable population of ventral alpha-motor neurons. THA caused a slow dose-dependent loss of alpha-motor neurons and an increase in LDH enzyme activity in the culture medium while dorsal interneurons were less damaged. 100 micromol/L THA resulted in a significant decrease in (alpha-motor neurons after cultured for 4 weeks. On the contrary, the interneurons in the dorsal horn were less affected. It was also observed in patients with amyotrophic lateral sclerosis (ALS). Excellular Glu mainly caused selective alpha-motor neuron death, and motoneurons were more sensitive to glutamate excitotoxicity than sensory neurons in the spinal cord.