丰富环境对成年弱视大鼠视皮层17区N-甲基-D-天冬氨酸受体亚基的影响

Effects of enriched environment on the subunits N-mehyl-D-aspartate receptors at the 17th area of the visual cortex in adult amblyopia rats

目的研究丰富环境(EE)对关键期后的单眼形觉剥夺性弱视大鼠视皮层17区N-甲基-D-天冬氨酸(NMDA)受体亚基2A(NMDA-NR2A)和NMDA-NR2B蛋白水平的影响,探讨EE对弱视视皮层突触可塑性的影响。方法选取出生后2周龄的Wistar大鼠80只,按照随机数字表法分为正常对照组和实验组。实验组在整个关键期内行右侧眼睑缝合,制作单眼形觉剥夺(MD)弱视模型。45日龄时按照随机数字表法将实验组大鼠分为弱视组、标准环境组(SE组)和EE组,其中SE组和EE组于46日龄时打开右侧眼睑。60、75和105日龄时各组取3只大鼠,全身麻醉后行多聚甲醛心脏灌注,剪刀于颈部端头,取左侧大脑视皮层17区。采用苏木精-伊红染色观察大脑视皮层17区的分层,并采用免疫组织化学法检测在关键期后不同时期每组大鼠NMDA-NR2A和NMDA-NR2B的蛋白在大脑视皮层中的分布及表达。应用Image-Proplus6.0分析软件对实验产生的阳性反应产物进行累计吸光度(A)值检测。结果光学显微镜下视皮层17区均可见NMDA-NR2A和NMDA-NR2B染色阳性细胞,多呈圆形或椭圆形,主要位于细胞膜。60、75和105日龄各组大鼠视皮层17区NMDA-NR2A表达总体比较,差异均有统计学意义(均P<0.05)。各时间点弱视组阳性细胞较正常对照组少;EE组阳性细胞较正常对照组少,较弱视组多。EE组60、75和105日龄NMDA-NR2A表达较SE组增强,但并未达到正常对照组水平,差异有统计学意义(均P<0.05)。60、75和105日龄各组大鼠视皮层17区NMDA-NR2B表达总体比较,差异均有统计学意义(均P<0.05)。各时间点弱视组阳性细胞较正常对照组多;60日龄EE组阳性细胞较正常对照组多,75日龄和105日龄和正常对照组接近。EE组60日龄NMDA-NR2B在视皮层的表达较SE组减弱,但并未达到正常对照组水平,差异均有统计学意义(均P<0.05)。EE组75日龄和105日龄时NMDA-NA2B表达量较SE组减弱,与正常对照组比较,差异均无统计学意义(均P>0.05)。结论视觉系统的可塑性不但存在于视觉发育的关键期以前,且在关键期之后视觉系统的可塑性仍然存在。作为一种非侵入性的干预方法,EE能通过改变NMDA-NR2A和NMDA-NR2B在视皮层中的表达使成年弱视大鼠的可塑性得以改善和恢复。

Objective To explore the effect of enriched environment on the level of NR2A and NR2B subunits of N-mehyl-D-aspartate (NMDA) receptors which belong to glutamate receptors with excitability at the 17th area of the visual cortex in amblyopia rats after the critical period,and to understand the possible mechanism of synaptic plasticity of the visual cortex in adult amblyopia rats. Methods Eighty Wistar rats were divided into normal group and experimental group by random number table.Right eyelids of all rats were sutured through the whole critical period in order to establish monocular deprivation (MD) amblyopia model.The rats in experimental group were divided into the amblyopia group,standard environment (SE) group and environmental enrichment (EE) group on P45 in random.The sutured right eyelids were opened on P46 in the SE group and EE group.All rats were sacrificed to get the 17th area of the left visual cortex on P60,P75 and P105.Three rats were used at different time points from each group.The Ⅰ-Ⅵ layers of the visual cortex area 17 were observed by using hematoxylin-eosin staining.The expression of NMDA-NR2A and NMDA-NR2B was detected by immunohistochemistry.Integrated optical density of NMDA-NR2A and NMDA-NR2B was detected by using special image analysis software (Image-Pro Plus 6.0 ).The use of animals complied with Regulation on the Managenment Experimental Ainimals from Shandong Eye Institute and Association for Research in Vision and Ophthalmology (ARVO). Results The positive expression of NMDA-NR2A and NMDA-NR2B were observed in the visual cortex.The positive cells were mostly round or elliptical and mainly expressed in cell membrane.The expression of NMDA-NR2A in P60,P75 and P105 from four groups had statistical differences (all at P <0.05).There were less positive cells in amblyopia group and EE group than normal control group on P60,P75 and P105,while there were more positive cells in EE group than amblyopia group.Amblyopia can lead to reduced NMDA-NR2A expression in the visual cortex.The expression of NMDA-NR2A was stronger than that in the amblyopia group by intervention 15 days,30 days,and 60 days with the rich environments,but did not reach the normal level (all at P <0.05).The expression of NMDA-NR2B in P60,P75 and P105 from four groups also had statistical difference (all at P <0.05).There were more positive cells in amblyopia group than those in normal control group on P60,P75 and P105.There were more positive cells in EE group than normal control group on P60,while there were equal positive cells in EE group and normal control group on P75 and P105.Amblyopia can lead to increase NMDA-NR2B expression in the visual cortex.The expression of NMDA-NR2B was weaker than that in the amblyopia group by intervention 15 days with the rich environments,but did not reach the normal level (all at P <0.05).The expression of NMDA-NR2B after intervention 30 days and 60 days reached the normal levels (all at P >0.05). Conclusions The plasticity of visual cortex exists not only in the critical period but also after the critical period of visual development.EE,as a non-invasion method,can improve and recover the synaptic plasticity in visual cortex of adult rats by the expression of NMDA-NR2A and NMDA-NR2B.