Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat
Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat
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DOI：
10.1016/j.exer.2005.06.002
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发表时间：
2006-01-01
影响因子：
3.4
通讯作者：
Ball, AK
中科院分区：
文献类型：
--
作者：
Lau, J;Dang, M;Ball, AK
血管收缩肽，内皮素-1（ET-1）已被发现在青光眼眼内水平升高。在这项研究中，为了表征体内视网膜神经节细胞（RGC）特异性细胞死亡模型，将5 μ l的ET-1单次眼内注射到大鼠眼中，使用5、50和500 μ m浓度的ET-1确定注射后2周诱导RGC损失的ET-1的最有效浓度。通过计数荧光金标记的RGC来确定存活的RGC的密度。当与PBS注射的对照相比时，仅使用500 μ π ι浓度观察到RGC的显著损失（25%）。GFAP免疫组化显示，在苗勒细胞终足GFAP表达增加，以及GFAP表达的总增加（80%），ET-1治疗后。通过测量视网膜厚度和TUNEL标记的变化来确定ET-1介导的RGCS细胞死亡的特异性。使用共聚焦和光学显微镜定量视网膜厚度。在共聚焦测量中，使用Yo Pro-1对核层进行染色，并从重建中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。当通过常规透射光显微镜观察时，在半薄切片中观察到相同的观察结果。外核、外丛状层或内核层中缺乏显著的厚度变化表明，除了RGC层之外，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唯一共定位为RGC特异性死亡提供了额外的证据，其最可能通过凋亡机制发生。与注射PBS的对照组相比，在注射ET-1后1、2、3和4周观察到RGC损失25、25、36和44%。在注射ET-1后3周，治疗眼的总ON轴突损失了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治疗导致瞳孔速度减慢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ET-1注射可产生RGC特异性细胞死亡和许多类似于青光眼的细胞变化。这种潜在的青光眼模型保留了完整的视神经，并可用于后续实验，这些实验涉及增加RGC存活和功能恢复。(C)2005爱思唯尔有限公司保留所有权利。
The vasoconstrictive peptide,Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5 mu l 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-I at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500 mu m 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 500 pm concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Muller 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 1.85 sec (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. (C) 2005 Elsevier Ltd. All rights reserved.