大鼠视神经损伤后视网膜小胶质细胞表达的初步观察

摘要

目的 研究大鼠视神经损伤后视网膜中小胶质细胞的表达情况.方法 实验研究.选取30只成年雌性健康SD大鼠,按照随机数字表法分为实验组和对照组各15只,分别用于细胞计数、免疫组织化学及免疫印迹实验.实验组在眼球后约1.5 ～2.0 mm处行右眼视神经鞘内切断术,术后5d于视神经断端处用荧光金逆行标记视网膜节细胞,手术后7d处死取材.对照组小鼠右眼行视神经切断术并标记,2d后处死取材.视网膜做铺片用于计数.用免疫组织化学法于视网膜切片上行小胶质细胞的表面标记物Iba-1染色,观察小胶质细胞的形态及数量,同时应用免疫印迹法检测视网膜内Iba-1蛋白含量的变化.两组间比较采用非配对student t-检验进行统计学分析.结果 对照组视网膜中有少量小胶质(Iba-1阳性)细胞表达,并呈非活化状态.视神经切断7d后小胶质细胞明显增多且呈半活化状态,免疫印迹结果显示损伤后Iba-1蛋白表达量明显增加到对照组的2.3倍(t=7.669,P=0.001).视视神经切断7d后节细胞数量为(1182±64)个/mm2,明显减少至对照组的51％(t=23.850,P＜0.01).结论 大鼠视神经损伤后小胶质细胞表达增多且呈部分激活状态,可能是视网膜受损后自我保护的表现之一.%Objective To investigate the expression of microglia after optic nerve transection in rats.Method Thirty healthy adult female SD rats were randomly grouped to experimental and control groups according to the random number table.The experimental group received an optic nerve transection (ONT) on their right eyes.ONT was performed 1.5 mm from the eye ball.The control group received a surgery on day 5. Rats were killed 7 days after the first operation. Retinal ganglion cells (RGC) were retrograde labeled by placing fluorogold dye on the stump of the optic nerve. Microglia was immunohistochemically stained in the retinas of normal and 7-day injury groups using ionized calcium adaptive molecular 1 ( Iba-1 ) as the marker.The protein levels of Iba-1 in the retinas were also examined using Western blotting.The number of RGCs was quantified using flatmount of retinas in the two groups.Results There were some resting microglia (Iba-1 positive cells) in the nerve fiber layer and inner plexiform layer in the normal control retina.However,microglia showed a moderately activated morphology with increased Iba-1 immunoreactivity in the stroma and processes up to outer plexiform layer. Western blotting showed that the levels of Iba-1 was lower in the normal retina and increased to 2.3-fold 7 days after injury (t =7.669,P =0.001 ).The number of RGCs after injury was ( 1182 ± 64 ) per mm2,decreased to 51％ in the normal groups (t =23.85,P ＜ 0.0001 ).Conclusions The results implicated that microglia was increased and partially activated after the optic nerve transection.This may be a manifestation of the self-protection mechanism of RGCs against the injury insult.