斜视幼猫发育过程中视皮层神经元NMDA-R1的表达

Expression of NMDA-R1 in developing visual cortex of strabismic kittens

目的 本研究希望了解斜视性弱视猫视皮层神经元N 甲基 D 天门冬氨酸亚基 1 (简称NMDA R1 )的表达减少是在斜视幼猫发育中哪一阶段出现的。方法 幼猫 1 3只 ,其中 6只于 2周龄时做一眼外直肌断腱术产生单眼内斜。分为 4个实验组 :2周龄组 ,2只正常幼猫 ;3周龄组 ,2只正常和 2只斜视幼猫 ;5周龄组 ,2只正常和 2只斜视幼猫 ;成年组 ,1只正常和 2只斜视性弱视猫。动物均在深度麻醉下经心脏 4 %福尔马林灌注后 ,取初级视皮层冷冻切片 ,NMDA -R1单克隆抗体标记后显色 ,焦油紫复染。结果 ①正常幼猫发育过程中初级视皮层神经元NMDA R1的表达主要在视皮层Ⅱ～Ⅲ层 (P <0 0 5) ,3周龄达峰值 ,随后逐渐降低 ,5周龄时接近成年猫水平。②与同龄正常猫比较斜视幼猫视皮层NMDA R1的表达在斜视术后第 1周开始减少 (P <0 0 5) ,以第Ⅳ层较显著。斜视手术后 3周与正常猫比较 ,斜视幼猫视皮层NMDA R1的表达在视皮层各层均降低 (P <0 0 5) ,接近成年斜视性弱视猫的表达水平。结论 斜视性弱视猫视皮层神经元NMDA R1的表达减少发生在斜视手术后 1周。表明NMDA中介了外侧膝状体到视皮层的输入。这与视觉电生理实验中发现 。

Objective To investigate at which period the NMDA-R1 receptor's distribution is changing in young strabismic amblyopic animals after surgery. Methods Thirteen kittens were used for this study. Five squint kittens had been inflicted to be monocularly esotropic (by tenotomy) at 2 weeks of age, and grew up with a pronounced squint measuring from 15 oto 20o. Two normal kittens were sacrificed 2 weeks after born. One pair of normal and strabismic kittens was sacrificed in 3 weeks of ages, which was 1 week after tenotomy. Another pair of normal and strabismic kittens was sacrificed in 5 weeks of ages. One normal and two strabismic amblyopic cats were sacrificed after 6 months of age. Animals were deeply anaesthetized and perfused transcardially with 4% paraformaldehyde. Cryostat sections of frontal central area P5 P0 were cut to 25 μm thickness. The mouse anti NMDA R1 monoclonal antibody (mAb54.1, PharMingen) was used. The positive and the negative controls were used in each test. A computer assisted systematic morphometric counting procedure was used to enumerate immunopositive cells. Results In normal development the percentage density of NMDA R1 labeled cells in layer II to layer III was prominent at 3 weeks of age ( P <0.05). The reduction of NMDA R1 labeled cells in visual cortex of strabismic kitten was started 1 week after surgery, predominantly in layer IV ( P <0.05). After 3 weeks of surgery, the immunopositive neurons of NMDA R1 in visual cortex of strabismic animals were decreased at each layer compared with the normalize though adulthood ( P <0.05,0.01). Conclusion The NMDA R1 receptor distribution in young strabismic amblyopic animal is changed following the visual environment variation quickly, implying that the plasticity associated with NMDA mediated mechanism involve critical period of ocular dominance development