MNU诱导视网膜外层退行变性的细胞电生理和超微结构特征

The Electrophysiological Properties and Morphological Ultrastructure of N-nitroso-N-methylurea-induced Retinal Degeneration in Mice

目的：观察N-甲基-N-亚硝基脲（MNU）诱导的小鼠视网膜外层变性损伤的细胞电生理特征和超微结构变化。方法：实验研究。通过腹腔注射PBS和MNU，将C57/BL小鼠60 只随机分为对照组和MNU造模组。运用膜片钳电生理技术，观察位于外丛状层的双极细胞的电生理活动。用透射电子显微镜观察视网膜的超微结构。结果：膜片钳结果显示，给MNU后7 d，ON型双极细胞（ON-BC）对药物氨基（环丙基）（ 4-膦酰苯基）乙酸电生理反应完全消失，而OFF型双极细胞（OFF-BC）对药物α-氨基-3-羟基-5-甲基-4-异恶唑丙酸的刺激仍有电生理反应。给MNU后2 d，位于光感受器细胞外节（OS）的盘膜结构变薄。给MNU后5 d，OS消失，位于光感受器细胞内节（IS）的线粒体等细胞器严重肿胀。给MNU后10 d，IOS消失，光感受器细胞的核层变薄，核呈不同程度的浓染。下游的双极细胞核周间隙增宽，在双极细胞胞浆中有大量的自噬体。结论：MNU对ON型信号通路的损伤，比对OFF型信号通路的损伤要严重一些。MNU导致的视网膜损伤主要位于外侧（包括外核层和外丛状层）。MNU致光感受器细胞损伤的机制是凋亡。

Objective： To study the electrophysiological properties and morphological ultrastructure of MNU-induced retinal degeneration in mice. Methods： In this experimental study, C57/BL mice were randomly divided into two groups： a control group and a MNU group. Patch-clamp recording was used to observe the responses of bipolar cells to several drugs. The ultrastructure of the retina was observed with a transmission electron microscope. Results： The results of the patch-clamp recordings showed more serious damage to ON-bipolar cells （ON-BC） than OFF-bipolar cells （OFF-BC）. Seven days after MNU application, no response was observed from the application of mGlu antagonist （RS）-tt-cyclopropyl-4-phosphonophenylglycine （CPPG） in ON-BC of the MNU group, while a reduced current （pA） of the ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid （AMPA） induced response was observed in OFF-BC. Two days after MNU administration, a decrease inthe thickness of disk membranes was observed in outer segments. Five days after MNU administration, outer segments could not be observed; swollen mitochondria were observed in the inner segment. Ten days after MNU administration, inner segments could not be observed, retinas displayed decreased thickness and concentrated nucleus coloring in the outer nuclear layer. In the retinal bipolar cell remnants, widening of the perinuclear space and autophagosome was detected by electron microscopy. Conclusions： MNU leads to more serious damage in ON-BC than OFF-BC. MNU-induced retinal degeneration is limited to the outer retina, including the photoreceptor and outer plexiform layer. The mechanism of MNU-induced damage is apoptosis.