Lau Jonathan, Dang Matthew, Hockmann Karlo, Ball Alexander K
Department of Pathology and Molecular Medicine, HSC Rm 1R1, McMaster University, 1200 Main St. West, Hamilton, Ont., Canada.
Exp Eye Res. 2006 Jan;82(1):132-45. doi: 10.1016/j.exer.2005.06.002. Epub 2005 Jul 19.
The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5mul intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500mum concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500mum concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.
血管收缩肽内皮素 -1（ET-1）在青光眼患者眼中的水平升高。在本研究中，向大鼠眼内单次注射5微升ET-1，以建立体内视网膜神经节细胞（RGC）特异性细胞死亡模型。使用5、50和500微摩尔浓度的ET-1确定注射后2周诱导RGC损失的最有效浓度。通过计数荧光金标记的RGC来确定存活RGC的密度。与注射PBS的对照组相比，仅使用500微摩尔浓度时观察到RGC显著损失（25%）。GFAP免疫组织化学显示，ET-1处理后，Müller细胞终足中GFAP表达增加，GFAP表达总量增加（80%）。GFAP表达的这些变化表明神经胶质细胞对应激的反应过度活跃。通过测量视网膜厚度和TUNEL标记的变化来确定ET-1介导的RGC细胞死亡的特异性。使用共聚焦显微镜和光学显微镜对视网膜厚度进行定量。在共聚焦测量中，用Yo Pro-1对核层进行染色，并根据重建确定视网膜层的厚度。在任何视网膜层中均未观察到厚度的显著损失。当通过传统透射光显微镜观察半薄切片时，也得到了相同的结果。外核层、外网状层或内核层缺乏显著的厚度变化表明，除了RGC层外，视网膜中没有显著的细胞损失。TUNEL标记的细胞核与荧光金标记的细胞质的排他性共定位为RGC特异性死亡提供了额外证据，这种死亡很可能通过凋亡机制发生。进行细胞死亡时间进程以确定随时间的RGC损失。与注射PBS的对照组相比，在ET-1注射后1、2、3和4周观察到RGC损失分别为25%、25%、36%和44%。通过将每单位面积的视神经（ON）轴突数量乘以横截面积来确定剩余RGC轴突的总数。注射后3周，ET-1处理的眼中ON轴突总数损失31%。通过观察瞳孔对光反射的变化来确定视觉系统的功能完整性。ET-1处理导致瞳孔速度减慢31%，收缩持续时间平均增加1.85秒（增加32%）。这些实验提供了证据，表明急性注射ET-1可导致RGC特异性细胞死亡以及许多与青光眼相似的细胞变化。这种潜在的青光眼模型使视神经保持完整，可用于后续涉及提高RGC存活率和功能恢复的实验。
