皮层神经元机械性损伤后代谢型谷氨酸受体表达及其拮抗剂的保护作用

Expression of metabotropic glutamate receptor la and neuroprotective effects of its antagonist 1-aminoindan-1, 5-dicarboxylic acid in mechanically injured cortical neuron of mouse in vitro

目的研究体外培养小鼠脑皮层神经元机械性损伤后代谢型谷氨酸受体1a(metat-ropicglutamatereceptor1a,mGluR1a)的表达规律及其选择性拮抗剂1-氨基茚-1,5-二羧酸(1-aminoindan-1,5-dicarboxylicacid,AIDA)的保护作用。方法孕15-16d昆明种小鼠胚胎脑皮层神经元体外培养7d,以微量移液器塑料滴头在培养孔内划割,造成机械性损伤,伤后不同时间点(10,30min、1,3,6,12,24,72h)行神经元免疫组化染色,并留取培养液上清,检测乳酸脱氢酶(lactatedehydrogenase,LDH)活性;对照组除不划伤神经元,其他处理同损伤组。AIDA处理组在损伤前30min,每孔加入AIDA使其终浓度为20μmol/L,其余步骤同前,激光扫描共聚焦显微镜检测AIDA处理前后神经元细胞内Ca2+含量变化。结果mGluR1a在正常神经元中表达呈弱阳性,染色颗粒分布于胞浆、胞膜及突起;机械性损伤后10min起,mGluR1a表达明显增强,胞核周围胞浆内染色明显加深,持续至伤后24h,神经元突起中也有明显表达,伤后72h,存活神经元数量明显减少,mGluR1a染色呈弱阳性。对照组LDH活性为(80.5±21.9)U/L,损伤后30min,LDH活性较对照组明显增加(P<0.05),3,6h时与对照组相比,差异无统计学意义(P>0.05),伤后12-72h,LDH活性较对照组明显增强(P<0.05)。伤后12-72h,AIDA处理组LDH活性较单纯损伤?

Objective To study the expression of mGluR1a ( metabotropic glutamate receptor 1a) and the effects of AIDA ( 1-aminoindan-1 , 5-dicarboxylic acid) in mechanically injured cortical neuron in vitro. Methods The cortical neurons of mouse fetus ( 15-16 days old) were cultured in vitro for seven days. Mechanical injury was caused by plastic tip piercing the cortical neuronal culture in the injury group, while the control group underwent all the same managements except for the mechanical injury. Immunohistochemical examination was made on these neurons at 10, 30 minutes,1 ,3,6, 12, 24 and 72 hours after injury. At the same time, the data were collected to detect the activity of lactate dehydro-genase ( LDH). A1DA was added to the wells in the AIDA treated group 30 minutes before injury so that the end concentration of AIDA reached 20 μmol/L Ca2+ level in neurons was detected with laser scanning confocal microscopy. Results There was weak expression of mGluR1a in neurons in the control group, with the positive particles distributing in the neuronal cytoplasm, cyto-membrane and dendrites. While in the injury group, there was widespread expression of mGluR1a positive neurons from 10 minutes to 24 hours after injury and distribution of the positive particles mainly in the cytoplasm around the nucleus 10 minutes after injury. Neurons were rarely alive 72 hours after injury, with weak expression of mGluR1a. The concentration of LDH reached (80.5±21.9) U/L in the control group but that in the injury group increased significantly 30 minutes after injury (P < 0.05). However, the concentration of LDH at the 3rd and 6th hours in the injury group showed insignificant statistical difference compared with control group (P>0. 05). The concentration of LDH decreased significantly 12-72 hours after injury in the AIDA treated group compared with that of the injury group alone (P < 0. 05 ). Ca2+ level was decreased significantly in AIDA treated group than that in control group, with statistically significant difference (P < 0.05). Conclusions Expression of mGluR1a increases significantly in mechanically injured cortical neurons of mouse in vitro, and its selective mGluR1a antagonist AIDA has obvious neuropro-tective effects after neuronal trauma.