缺血损伤对神经元膜表面AMPA受体构成变化的影响

背景α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体的过度激活在继发性脑损害的发生过程中起重要作用,研究表明,AMPA受体第二亚单位(GluR2)的减少是引起Ca2+快速内流主要原因,但缺血损伤后神经元膜表面AMPA受体数量及结构(亚单位组成比例)变化特征尚不清楚.目的探讨缺血后神经元膜表面AMPA受体亚单位(GluRs)含量组成变化及其对Ca2+内流和细胞死亡的影响,为脑缺血治疗的特异性干预途径提供理论依据.设计随机对照的实验研究.单位解放军第三军医大学大坪医院野战外科研究所.材料孕18d SD大鼠8只(解放军第三军医大学野战外科研究所动物所提供),进行胚胎海马神经元分离和培养.干预缺血损伤组更换无糖细胞外液后置专用缺氧箱内缺氧30min,然后重新添加维持培养基继续培养,对照组在相同时刻更换正常培养基.采用PI染色、双重免疫荧光标记和细胞比色分析技术定量观察神经元死亡和膜表面GluRs含量变化,采用Fura-2法测定胞内Ca2+含量.主要观察指标①伤后神经元死亡数目.②突触内和膜表面GluRs含量.③胞内Ca2+含量.结果缺氧损伤后24h GluR2阳性突触数目构成比为0.42±0.16,与对照组(0.58±0.15)相比显著降低,差异有显著性意义(t=2.348,P=0.023),膜表面GluR2含量也显著减少,差异有显著性意义(t=2.427,P=0.019);而GluR3阳性突触数和膜表面含量则显著升高,伤后24h膜表面GluR3含量与伤前的比值为1.23±0.23,相差显著(t=2.114,P=0.040);膜表面GluR1伤后也呈增多趋势;缺血损伤组胞内Ca2+含量是对照组的1.36倍,差异有显著性意义(t=2.571,P=0.017),其神经元死亡数量也显著高于对照组,差异有显著性意义(t=4.803,P=0.0001).结论缺血损伤可致神经元膜表面AMPA受体组成结构变化,GluR2含量减少,GluR3,GluR1含量增加,介导了Ca2+快速内流和神经元死亡,在继发性脑损害中发生过程中起重要作用.

Effect of ischemicinjury on constitutional change of AMPA receptors of neuron membrane

BACKGROUND:The over activation of α-amino-3-hydroxy -5methylisoxazole-4-propionic acid(AMPA) receptor plays an important role in the occurrence of brain secondary damage. Recent studies showed that the decrease of the second unit of AMPA receptor(GluR2) mainly induced Ca2 +fast influx.However,how the membrane AMPA quantity and constitution (constructiveratio of subunits) changes after ischemic injury were not clarified.OBJECTIVE:To probe into the changes of the subunits(GluRs) content,constitution of AMPA receptor on neuron membrane involving in ischemia,and furthermore to elucidatethe contribution of these changes to the Ca2 +influx and the death of neuron so as to provide the theoretical basis for the intervention way of highly specific drugs for ischemia.DESIGN: A randomized and controlled experiment was conducted.SETTING: Research institute of field surgery, daping hospital, third military medical university MATERIALS: Eighteen SD rats at gestation for 18 days were provided by animal center of research institute of surgery, and the hippocampal cells were separated from embryos and cultured.INTERVENTIONS: Neuron in the ischemic injury group was put into a specific box for anoxic challenge 30 minutes after replacement of culture medium with glucose free extracellular solution, and then re-cultured after adding culture medium. Neuron in the control group underwent the same medium replacement of normal medium at the same time. The quantities of GluRs on the neuron membrane were quantitatively measured with colorimetric assay and double immuno-fluorescence labeling on neuron anoxic model. The death of neuron was quantitated with PI staining. Intracellular Ca2 + content was determined with Fura-2 method.MAIN OUTCOME MEASURES: ① The number of the dead cells after injury. ② The quantities of GluRs on the neuron membrane and synapse. ③ lntracellular Ca2 + concentration. RESULTS: The constitutional ratio of GluR2 positive staining synapses at 24hours after ischemic injury was 0. 42 ±0. 16, which was significantly lower than that of the control group(0. 58 ± 0.15,t = 2.348,P=0.023).The membrane GluR2 also marked and decreased compared with the control group( t = 2.427, P = 0.019).The GluR3 positive synapses and membrane content, however, increased significantly. The ratio of membrane GluR3 at 24hours to preinjury was 1.23 ± 0. 23, which was statistically different from that in the control group( t = 2.114,P = 0.040).The membrane GluR1 also showed an increasing trend. Intracellular Ca2 + concentration in the injury group was 1.36 times higher than that in the control group(t = 2. 571, P =0.017).The amount of dead neuron in the ischemic injury group was significantly higher that in the control group ( t = 4. 083, P = 0.000 1 ).CONCLUSION: Ischemic injury will induce the constitution changes of AMPA receptors on neuron membrane. The reduction of membrane GluR2and the increase in GluR3 and GluR1 will accelerate Ca2+ influx and the death of neuron,which plays an important role in the occurrence of secondary cerebral damage.