实验性光损伤大鼠视网膜光感受器细胞凋亡的研究

目的：观察较强可见光所致大鼠视网膜光感受器细胞凋亡现象，探讨视网膜光化学损伤发病机制。方法：取健康成年SD大鼠50只，随机分成正常对照组（CON）、光损伤后1天（D1）、3天（D3）、5天（D5）、7天（D7）组，每组10只。各组大鼠在12h明12h暗环境中饲养7天，然后暗适应36h。D1、D3、D5、D7组大鼠采用4178Lux照度的可见光进行12h间歇光照射，连续3天，总计36h，然后在正常环境中分别饲养1天、3天、5天、7天。10％水合氯醛麻醉大鼠，摘取眼球，制备视网膜石蜡切片及超薄切片，行HE染色、TUNEL法标记及重金属染色，光镜、透射电镜观察，MIAS-1000图像分析系统检测。结果：正常大鼠视网膜组织结构层次清楚，外核层排列规则，染色质电子密度均匀。TUNEL法染色阴性。光照后1天，外核层开始变薄，其厚度减少11.88％。核染色质开始固缩。可见少量TUNEL法标记的阳性细胞核，染色质向核膜下聚集，呈现典型的凋亡细胞表现。光照后3天，外核层厚度减少26.80％。电镜下核染色质向中心聚集。TUNEL法标记的凋亡细胞增加。光照后5天，外核层厚度减少39.76％。TUNEL法标记的凋亡细胞更。光照后7天，外核层厚度和TUNEL法标记的凋亡细胞数与光照后5天基本相似。视网膜色素上皮细胞、节细胞及内核层均无明显变化。也未了现炎症细胞。结论：实验性光损伤导致大鼠光感受器丧失，光感受器丧失的性质为细胞凋亡。实验结果初步证实光感受器细胞凋亡是实验性大鼠视网膜光损伤的重要机制。

A Study on the Apoptosis of Photoreceptors in Experimental Retinal Photo-injury Rats

Objective To study the apoptosis of photoreceptors of the retinal injury in rats under strong visible light and investigate the pathologic mechanism of retinal photo-injury.Methods 50 healthy adult SD rats were randomly divided into 5 groups (10 rats in each group)by control group(CON)and 1 day(D1),3 days(D3),5 days(D5)and 7 days(D7)groups after the light-injury.The rats in each group were given an intermittent light exposure for 7 days,12 hours light exposure every day and 12 hours dark-adaptation before every exposure.Then they were given 36 hours dark-adaptation.The rats in Group D1,D3,D5 and D7 were given 12 hours intermittent light exposure of 4178 Lux illumination visible light for 3 days successively,36 hours in number,then they were raised in normal enrironment for l day,3 days,5 days and 7 days respectively.Afterwards,all the rats in each group were anesthetized with 10% chloral hydrate and the eyes were extracted to make retinal paraffin sections and ultrathin sections.The former were stained by HE and TUNEL method and the later by heavy metals,observed under light and transmission electronic microscope,and determined by MIAS-1000 Photo Analysis System.Results Normal rats have clear layers of retinal structures.The outer nuclear layer(ONL)arranged orderly and regularly.Nuclear chromatin of photoreceptors had even electronic density.No positive marked cell was found when we distinguished apoptotic cells by TUNEL method.On the 1st day after light-exposure,the outer nuclear layer became thinner by 11 88%,nuclear chromatin began to shrink.A few apoptotic cells could be seen in yellow-brown color.The nuclear chromatin was distributed unevenly,shrinked and gathered under the nuclear membrane,showing a typical symbol of apoptotic cells with a circled nucleus.On the third day after the light-exposure,the ONL became much thinner by 26 80% than that of the normal retinas.Nuclear chromatin gathered toward the center with the island shape examined under electronic microscope.The apoptotic cells recorded by TUNEL method increased in number.On the fifth day after light-exposure,the thickness of the ONL reducde by 39.76%,and much more apoptotic cells were distinguished by TUNEL method.On the 7th day after the light-exposure,the changes are generally similar to the cases on fifth day after light-exposure.No obvious changes could be seen on the retinal pigmental epithelium,the inner nuclear layer and the layer of ganglionic cells.No inflammation cells could be seen in our experimentation.Conclusion It is illustrated that the disappearing of the retinal photoreceptor cells were induced by experimental photo-injury.The nature of photoreceptor dropout is apoptosis.The results of our experiment preliminarily prove that photoreceptor cell apoptosis is the most important pathologic mechanisms which cause the retinal photic injury in expcrimental rats.