雄激素预处理对实验性变态反应性脑脊髓炎大鼠视神经和视网膜神经节细胞的影响

Effects of testosterone on optic nerve and retinal ganglion cells in experimental autoimmune encephalomyelitis

目的观察雄激素预处理对实验性变态反应性脑脊髓炎（EAE）大鼠视神经形态功能和视网膜神经节细胞（RGC）凋亡的影响。方法将雌性Wistar大鼠41只随机分为正常组10只、未用药对照组15只和雄激素组16只。正常组和未用药对照组每天以1%乙醇灌胃,雄激素组每天以0.25mg/kg甲睾酮灌胃。用药20d后,未用药对照组和雄激素组注射0.4ml完全弗佐剂-豚鼠脊髓匀浆,并辅以注射百日咳疫苗诱导EAE模型。诱导前7d,大鼠上丘和外侧膝状体注射荧光金以逆行标记RGC。继续用药至诱导后14～30d,取正常鼠和出现症状48～72h内的未用药对照组和雄激素组大鼠,应用光学显微镜和闪光视觉诱发电位（FVEP）观察其视神经形态和功能的变化;荧光显微镜下观察视网膜铺片并计数RGC;原位缺口末端标记法（TUNEL）观察RGC的凋亡情况。结果诱导后10d开始,EAE大鼠相继出现尾及后肢无力,麻痹等症状。与未用药对照组相比,雄激素组的发病潜伏期延长（P=0.035）,症状评分降低（P=0.042）。未用药对照组大鼠视神经纤维走行纡曲呈波浪状,弥漫脱髓鞘改变;雄激素组视神经纤维走行较规则,脱髓鞘改变不明显。FVEP显示与未用药对照组相比,雄激素组的N1、P和N2波潜伏期明显缩短（P〈0.05）,N1-P和P-N2振幅提高（P〈0.05）。正常组、未用药对照组和雄激素组RGC数分别为（2284±132）、（934±78）、（1725±95）个/mm^2,雄激素组RGC数较未用药对照组增多（P=0.028）。正常组、未用药对照组和雄激素组TUNEL阳性细胞数分别为（4.02±0.16）、（24.44±2.22）、（9.84±2.36）个/高倍视野,雄激素组TUNEL阳性细胞数较未用药对照组明显减少（P=0.025）。结论雄激素可降低EAE发病率和症状评分,抑制EAE鼠RGC的凋亡,减轻视神经脱髓鞘改变,改善视神经传导功能。

Objective To observe the effects of testosterone on optic nerve and retinal ganglion cells （RGC） in experimental autoimmune encephalomyelitis （EAE）. Methods Fourty-one female Wistar rats were randomly divided into 3 groups： the normal group （10 rats）, the untreated control group （15 rats） and the testosterone group （16 rats）. The rats in the first two groups were fed with 1% ethanol every day, and the rats in the testosterone group were fed with methyltestosterone （0.25 mg/kg） every day. On the 20th day, EAE model was induced in the untreated control group and the testosterone group by injecting guinea pig spinal cord homogenate in complete Freund＇s adjuvant and bordetella pertussis vaccine. RGC were labeled with flurogold （FG） by injecting it in superior colliculus and lateral geniculate body 7 days before establishing EAE model. All rats were fed with drugs continuously, and after 14-30 days, rats in normal group and rats in untreated control and testosterone groups who had symptoms within 48-72 hours were observed by light microscopy and flash visual evoked potential （FVEP） to detect the functional and morphological changes of optic nerve. The number of RGC was counted by fluorescence microscopy, and apoptosis of RGC was observed by terminal deoxynucleotidyl transferase-mediated biotlnylated UTP nick end labeling （TUNEL）. Results EAE rats presented weakness or paralysis of tail and hind limbs 10 days after establishing EAE model. Compared with the rats in the untreated control group, the rats in the testosterone group had longer disease delitescence and lower clinical score （P=0. 042）. Extensive demyelination of optic nerves with the circuitous configuration was found in the untreated control group; while mild demyelination of optic nerves with regular figure was found in the testosterone group. In the testosterone group, the latency of N1,P and N2 wave was shorter while the amplitude of N1-P and P-N2 was higher than that in the untreated control group （P〈0. 05）. The number of RGC was （2284±132）, （934±78, and （1725±95）cells/mm^2 in the normal, untreated control and testosterone groups, respectively; which was higher in testosterone group than that in untreated control group （P = 0. 028）. The number of TUNEL positive cells was （4.02±0.16）, （24.44±2.22）, and （9.84±2.36） cells per high power field （×400） in the 3 groups, respectively; which was less in testosterone group than that in untreated control group （P=0. 025）. Conclusions Testosterone may reduce the incidence and clinical score of EAE, inhibit the apoptosis of RGC, alleviate the demyelination of optic nerves, and improved the conduction function of optic nerves.