大鼠视神经损伤后Toll样受体4在视网膜中的表达

背景Toll样受体4（TLR4）是一种重要的免疫相关受体，在多种疾病的发生中起致炎作用。研究发现，视神经损伤后继发的炎症反应可进一步引起视网膜损伤，因此视神经损伤后TLR4的表达及其效应值得研究。目的研究大鼠视神经损伤后视网膜TLR4的表达情况。方法选取成年健康SPF级SD大鼠24只，按随机数字表法随机分为视神经损伤3d组和视神经损伤7d组。取大鼠右眼用视神经钳夹法制备视神经损伤模型，左眼不予处理为对照组。分别于视神经损伤后3d和7d用过量麻醉法处死大鼠并分离视网膜，采用免疫荧光法检测各组大鼠视网膜中TLR4的表达；分别采用逆转录PCR法（RT—PCR）和Westernblot法检测大鼠视网膜中TLR4mRNA及其蛋白的表达；采用TUNEL染色法观察各组大鼠视网膜神经节细胞（RGCs）的凋亡情况。结果视网膜免疫荧光法检测结果显示，TLR4在大鼠视网膜中呈绿色荧光，视神经损伤3d组和视神经损伤7d组造模眼视网膜中的荧光强度较对照组左眼均明显增强，绿色荧光主要分布在视网膜内层。RT—PCR法检测表明，模型眼视网膜损伤后3d和7d视网膜中TLR4mRNA相对表达量分别为2．92±0．06和3．92±0．12，对照眼TLR4mRNA的相对表达量分别为2．87±0．12和3．44±0．17，大鼠模型眼TLR4mRNA表达的灰度值较对照眼明显增加，差异均有统计学意义（t3d=-12．888，P〈0．001；t7d=-4．669，P=0．010）。Westernblot法检测显示，大鼠模型眼视网膜损伤3d和7d视网膜中TLR4蛋白的相对表达量分别为1．14±0．05和1．49±0．03，对照眼TLR4蛋白的相对表达量分别为0．99±0．09和1．38±0．07，模型眼视网膜中TLR4蛋白表达量明显高于对照眼，差异均有统计学意义（t3d=-11．324，P〈0．001；t7d=-5．638，P=0．005）。TUNEL染色显示，模型眼RGCs凋亡数较对照眼增多。结论TLR4在视神经损伤大鼠视网膜内层的表达明显上调，提示TLR4通路可能参与RGCs的损伤。

Expression of Toll-like receptor 4 in retina following optic nerve crush in rat

Background Toll-like receptor 4 （TLR4） is an immune related receptor. It plays an important role in inducing inflammation response. The inflammatory response secondary to optic nerve crush will results in serious retinal damage. It is worthy of studying the expression and effect of TLR4 in retina after optic nerve crush. Objective This experimental study was to explore the role of TLR4 in the loss of retinal ganglion cells（RGCs） after optic nerve crush. Methods Twenty-four SPF adult health Sprague-Dawley （SD） rats were used in the study and radomized into two groups based to the experimental time. The optical nerve crush models were established by crushing the optical nerve in the right eyes of the rats, and the left normal eyes served as controls. The rats were sacrificed by over anesthesia and retinas were isolated 3 days and 7 days after operation. Expression of TLR4 in the retinas was detected using immunofluorescence method. Reverse trancription PCR （RT-PCR） and Western blot were applied respectively for the detection of TLR4 mRNA and protein in the retinas. The apoptosis of RGCs was evaluated using TUNEL staining. The use and care of experimental animals followed the ＂Guide for the Care and Use of Laboratory Animals＂ of NIH. This study was approved by the Institutional Animal Care and Use Committee at the Zhongshan Ophthalmic Center. Results The expression of TLR4 in rat retinas presented with green fluorescence mainly in the inner layer of retinas. The fluorescence was enhanced in the model 3 days group and the model 7 days group compared with the corresponding control groups. The relative expressing values of TLR4 mRNA in the retinas were 2.92±0.06 and 3.92±0. 12 in the model 3 days and 7 days groups,respectively,which were significantly higher than 2.87±0. 12 and 3.44±0. 17 in the control 3 days and 7 days group （t3d =-12. 888,P〈0. 001 ;t7a =-4. 669,P=0. 010）. In the model 3 days group and model 7 days group,the grey values of TLR4 protein were 1. 14±0.05 and 1.49±0.03 ,and those in the control 3 days and 7 days groups were 0.99 ± 0.09 and 1.38 ± 0.07, showing significant differences between them（t3d =-11. 324,P〈0. 001 ;t7d =-5. 638,P=0. 005）. Apoptotic RGCs were obviously increased in the optic nerve damage group in comparison with the control group. Conclusions The TLR4 is over-expressed in the inner layer of retina after optic nerve crush,which suggestes that TLR4 is probably involved in the loss of RGCs after optic nerve crush.