视神经夹持损伤模型大鼠视网膜和视神经小胶质细胞的活化

Activation of retinal and optic nerve microglia following optic nerve crush in rats

背景外伤性视神经病变（TON）是继发于外力创伤下的急性视神经损伤，预后较差。小胶质细胞作为中枢神经系统中重要的免疫细胞，参与了中枢神经系统疾病与多种眼科疾病的病理生理过程。然而，小胶质细胞在TON的病理发展及损伤修复过程中的作用尚不明确。目的比较大鼠视神经夹持损伤后视神经与视网膜中小胶质细胞的形态变化、激活数量、分布情况及活化水平的差异。方法将35只SPF级健康雌性成年Sprague-Dawley（sD）大鼠按照随机数字表法分为正常对照组，造模后6h、3d、7d、14d、30d组和假手术组，每组5只大鼠。造模后各时间点组用夹持钳以50g的夹持力在大鼠眼球后约2mm处钳夹视神经10S，建立大鼠视神经夹持模型，假手术组大鼠行相同的手术操作但不夹持视神经，正常对照组不做任何处理。分别于上述时间点制备大鼠视神经和视网膜冰冻切片，采用lectin-FITC荧光标记抗体检测各组大鼠视神经和视网膜中的小胶质细胞数量和激活的小胶质细胞数量。结果正常对照组和假手术组大鼠视网膜中小胶质细胞主要位于内丛状层（IPL），少部分位于内核层（INL）和神经节细胞层（GCL），外核层（ONL）和外丛状层（OPL）未见小胶质细胞分布。正常对照组大鼠视网膜小胶质细胞的细胞体较小，以分支状为主，突触细长，可见二级分支。各模型组大鼠视网膜中小胶质细胞主要位于GCL和IPL，小胶质细胞在GCL的数量明显多于假手术组，小胶质细胞多为阿米巴状，部分呈半激活态，少见分支静息态。正常对照组、假手术组及造模后6h、3d、7d、14d和30d组大鼠视网膜中小胶质细胞数分别为6．40±1．52、7．20＋2．05、12．00±3．54、14．00±4．06、18．00±4．36、18．40±3．13和10．80＋1．92，造模后各时间点大鼠视网膜中小胶质细胞数量均明显多于正常对照组，造模后30d小胶质细胞数量明显少于造模后7d和14d组，差异均有统计学意义（均P〈0．05）；造模后3、7和14d组大鼠视网膜中激活小胶质细胞数量明显多于假手术组，差异均有统计学意义（P=0．024、0．009、0．023）。正常对照组和假手术组大鼠视神经中小胶质细胞较小，呈棒状或分枝状，分布均匀且稀疏。造模后各时间点组小胶质细胞较假手术组细胞体积增大，呈阿米巴状并分布在近视神经夹持部位。造模后6h、3d、7d、14d大鼠视神经中小胶质细胞数量明显多于正常对照组，差异均有统计学意义（P=0．007、0．001、0．003、0．014）。造模后30d大鼠视神经中小胶质细胞数量明显少于造模后3d、7d和14d组，差异均有统计学意义（均P〈0．05）。造模后6h、3d和7d组大鼠视神经中活化小胶质细胞数量明显多于假手术组，差异有统计学意义（P=0．005、0．004、0．030），造模后14d、30d大鼠视神经中活化的小胶质细胞数量较造模后3d组明显减少，差异均有统计学意义（P=0．021、0．004），造模后6h组视神经中激活态小胶质细胞增加并持续到造模后14d。结论大鼠视神经夹持损伤后一定时间内视网膜及视神经中小胶质细胞增加并活化，视神经中小胶质细胞的活化及其衰减均早于视网膜，视神经中小胶质细胞活化程度更明显。

Background Traumatic optic neuropathy （TON） is an acute injury of the optic nerve secondary to trauma. As an important immune cell, mieroglia is involved in a variety of pathologic changes in central nervous system diseases and eye diseases. However, the role of mieroglia in the lesion development and neural restoration in.TON is still unclear. Objective This study was to compare the differences of microglial activation between optic nerve axon and retina after optic nerve crush injury,including the morphological changes, number, and distribution of microglia. Methods Thirty-five SPF female Sprague-Dawley （SD） rats were numbered from 1 to 35 and randomized into normal control group, sham operation group and modeling 6-hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group according to the random number table. Optic nerve injury models were established by crushing optic nerve at retrobulbar 2 mm for 10 seconds. The same operation was carried out but not clamping optic nerve in the sham operation group, and normal control group without any treatment. The morphological changes, number and distribution of microglia in retina and optic nerve axon were assayed and compared by immunofluorescence staining with lectin-FITC fluorescence labeled antibody detection. The use and care of the experimental animals complied with ARVO. Results Microglia were displayed mainly in inner plexiform layer （IPL） ,and a few of microglia were in inner nuclear layer （INL） and ganglion cell layer （GCL） ,not any microglia were seen in outer nuclear layer （ONL） and outer plexiform layer （OPL） in the normal control group and sham operation group. Thining synapsis and secondary branches were seen with the dendroid in shape in normal control group. In the model groups,the microglia were more in GCL compared with sham operation group and showed amoeboid microglial cells. The number of microglia in retinas was 6.40 ＋- 1.52,7.20 -＋ 2.05,12.00 -＋ 3.54,14.00 -＋ 4.06,18.00＋4.36,18.40 ＋ 3.13 and 10. 80-＋ 1.92 in the normal control group, sham operation group, modeling 6- hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group, respectively,and the microglia number were significantly increased in modeling 6-hour group, modeling 3-day group, modeling 7-day group, modeling 14-day group and modeling 30-day group in comparison with the normal control group, and in the modeling 30-day group, the microglial number in retinas was significantly reduced in comparison with the modeling 7-day group and modeling 14-day group （ all at P〈0.05 ）. The activited microglia in the retinas were Much more in the modeling 3-day group, modeling 7-day group and modeling 14-day group than that in the sham operation group （P=0. 024,0. 009,0. 023 ）. In the optic nerve, the microglial cells were small in size and sparse in arrangement in the normal control group and sham operation group. The cells were enlarged with the amoeboid-like in shape in the model groups and distributed in injury area. The cell number was significantly higher in the modeling 6- hour group,modeling 3-day group, modeling 7-day group and modeling 14-day group than that in the normal control group （P=0. 007,0. 001,0. 003,0. 014）. The cell number in modeling 30-day group was significnatly reduced in comparison with the modeling 3-day group,modeling 7-day group and modeling 14-day group （ all at P〈0.05 ）. The activated microglial number were significantly elevated in the modeling 6-hour group,modeling 3-day group,modeling 7-day group compared with the sham operation group （P=0. 005,0. 004,0. 030） ,and the cell number in the modeling 30-day group was evidently lower than that in the modeling 3-day group （P = 0. 021,0. 004）. The ceils of activated state in the optic nerve were significantly increased in modeling 6-hour group and modeling 14-day group. Conclusions Microglia are activated and keep increasing number in retina and optic nerve at a certain period after optic nerve iniurv.and these changes are earlier and more distinct in oDtic nerve axon than those in retina.