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观察谷氨酸转运体抑制剂苏-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、500μmol/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氨酶(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。
To investigate the effect of glutamate transporter (THA) on the organotypic spinal cord slices and to explore the role of glutamate in motor neuron injury. Twenty-eight days after birth, the lumbar spinal cord tissue sections of neonatal rats were used to do spinal cord organotypic culture. Different concentrations of THA (50μmol / L, 100μmol / L, 500μmol / L) were added into the culture medium. SMI-32, a non-phosphorylated neurofilament marker, was used to identify α motor neurons in the ventral horn of the spinal cord. The calftinin antibody was used to count the dorsal horn interneurons. The medium Lactate deaminase (LDH) content, and compared with the control group. The results showed that the number of α motor neurons in the control group was constant, THA could induce the increase of LDH content and the decrease of the number of α motor neurons in the dose-dependent medium, while the damage of the interneurons in the dorsal horn of the spinal cord was relatively mild. THA100μmol / L After 4 weeks of in vitro culture, pathological changes similar to amyotrophic lateral sclerosis (ALS) occurred: the number of α motor neurons decreased significantly compared with that of the control group, while there was no significant change in the number of interneurons in spinal dorsal horn. Increased extracellular glutamate is mainly caused by motor neuron damage, spinal motor neurons than sensory neurons glutamate excited more sensitive role.