不同激光强度经瞳孔温热疗法对色素兔视网膜细胞凋亡的影响

Effectos on apoptosis of retinal cells induced by different laser power transpupillary thermotherapy in pigmented rabbits

目的观察不同激光能量的经瞳孔温热疗法（TTT）对色素兔视网膜的病理损伤及对细胞凋亡的影响。方法将14只有色家兔随机按不同激光能量分为空白组、50、70、90、110、130、150mW组，每组2只兔4只眼行TTT治疗照射后，分别于24、48h后取视网膜组织进行光学显微镜检查及用原位末端转移酶标记（TUNEL）技术和流式细胞仪检测细胞凋亡。结果直接检眼镜下激光反应斑颜色随能量增加由灰变白逐渐变浅，即由灰白——白——浓白，直径逐渐增大。苏木素-伊红（HE）染色：50～70mW组光学显微镜下视网膜各层结构无明显改变；90～130mW组视网膜结构完整，视锥、视杆细胞肿胀，内颗粒层可见少量的固缩核和细胞浆空泡化。以上能量组激光照射后24、48h，光学显微镜下视网膜结构与空白对照组无明显差别。150mW组激光照射后24h视网膜组织各层肿胀、变性，感光细胞内外节部分缺失；而激光照射后48h视网膜组织可见明显坏死，全层均可见细胞缺失。TUNEL检测结果显示，各能量组外颗粒层均可见阳性细胞，随激光能量的增加而增加，并逐渐累及内颗粒层和神经节细胞层。流式细胞仪检测结果显示，激光照射后24h，各能量组均可见细胞凋亡峰。结论兔视网膜在能量为50～70mW的TTT照射后，视网膜组织未见明显改变，但感光细胞凋亡显著增加。随着TTT激光能量增加，视网膜组织出现肿胀、变性甚至坏死。细胞凋亡逐渐累及内颗粒层和神经节细胞层。

Objective To observe the histological changes and apoptosis of retinal cells in pigmented rabbits treated by transpupillary thermotherapy （TTT） with different laser power. Methods Fourteen pigmented rabbits （28 eyes） were divided averagely into seven groups （control group, 50, 70, 90, 110, 130, and 150 mW group）according to different laser power of TTT. Light microscopy was performed to observe the histological changes, and TDT-mediated biotin-dUTP nick- end labeling （TUNEL） technique and flow cytometry （FCM） examination were used to detect the apoptotic cells 24 and 48 hours after photocoagulation, respectively. Results The color of retinal burn speckles changed from offwhite to white and super white with the diameter enlarged gradually as the laser power of TTT increased. The results of light microscopy revealed that compared with the control group, the retinal tissue did not change much in 50-70 mW group; in 90-130 mW group, the retinal structure was integrated, but the cone and rod cells became swollen and condensed nuclei and cytoplasmic vacuolization were seen in the inner nuclear layer. The difference of retinal structure in 50-130 mW group 24 and 48 hours after photocoagulation and control group was not significant. In 150 mW group, tumefaction and degeneration were observed in each layer of retina and the inner and outer segments of photoreceptor cells lost 24 hours after photocoagulation, and obvious necrosis and cell loss of retinal tissues were detected hours after photocoagulation. The results of TUNEL examination indicated that positive cells were found in outer nuclear layer in each photocoagulation group which increased as the laser power of TTT was enhanced; the apoptosis gradually involved the inner nuclear layer and ganglion cell layer. The results of flow cytometry （FCM） examination showed the peak of apoptotic cells in each photocoagulation group 24 hours after photocoagulation. Conclusion Under certain subthreshold photocoagulation （50-70 mW）,retinal tissue of rabbits does not change much but apoptosis of photoreceptor cells increase significantly. As the laser power of TTT increases, the retinal tissues become swollen, degenerated and even necrotic; cellular apoptosis gradually involves the inner nuclear layer and ganglion cell layer.