Agar Ashish, Li Shaojuan, Agarwal Neeraj, Coroneo Minas T, Hill Mark A
Cell Biology Laboratory, School of Anatomy, University of New South Wales, Sydney, NSW, Australia.
Brain Res. 2006 May 1;1086(1):191-200. doi: 10.1016/j.brainres.2006.02.061. Epub 2006 Apr 25.
Cellular responses to changes in pressure are implicated in numerous disease processes. In glaucoma apoptosis of retinal ganglion cells (RGCs) is associated with elevated intra-ocular pressure, however, the exact cellular mechanisms remain unclear. We have previously shown that pressure can induce apoptosis in B35 and PC12 neuronal cell lines, using an in vitro model for pressure elevation. A novel RGC line allows us to study the effects of pressure on retinal neurons. 'RGC-5' cultures were subjected to elevated ambient hydrostatic pressure conditions in our model. Experimental pressure conditions were 100 mm Hg and 30 mm Hg, representing acute (high) and chronic (lower-pressure) glaucoma, and 15 mm Hg for normal intra-ocular pressure, set above atmospheric pressure for 2 h. Negative controls were treated identically except for the application of pressure, while positive controls were generated by treatment with a known apoptotic stimulus. Apoptosis was determined by a combination of cell morphology and specific TUNEL and Annexin V fluorescent markers. These were assessed simultaneously by laser scanning cytometry (LSC), which also enabled quantitative marker analysis. RGC-5 neurons showed a significantly increased proportion of apoptotic cells compared with controls; maximal at 100 mm Hg, moderate at 30 mm Hg and not statistically significant at 15 mm Hg. This graded response, proportionate to the level of pressure elevation, is representative of the severity of analogous clinical settings (acute, chronic glaucoma and normal). These results complement earlier findings of pressure-induced apoptosis in other neuronal cultures. They suggest the possibility of novel mechanisms of pressure-related mechanotransduction and cell death, relevant to the pathogenesis of diseases such as glaucoma.
细胞对压力变化的反应与众多疾病过程相关。在青光眼中，视网膜神经节细胞（RGCs）的凋亡与眼内压升高有关，然而，确切的细胞机制仍不清楚。我们之前利用压力升高的体外模型表明，压力可诱导B35和PC12神经元细胞系发生凋亡。一种新型的RGC细胞系使我们能够研究压力对视网膜神经元的影响。在我们的模型中，对“RGC-5”培养物施加升高的环境静水压力条件。实验压力条件为100毫米汞柱和30毫米汞柱，分别代表急性（高）和慢性（低压力）青光眼，正常眼内压为15毫米汞柱，设定为高于大气压并持续2小时。阴性对照除不施加压力外，处理方式相同，而阳性对照通过用已知的凋亡刺激物处理产生。通过细胞形态以及特异性TUNEL和膜联蛋白V荧光标记物的组合来确定凋亡。通过激光扫描细胞术（LSC）同时评估这些指标，该技术还能够进行定量标记分析。与对照相比，RGC-5神经元显示凋亡细胞比例显著增加；在100毫米汞柱时最大，在30毫米汞柱时中等，在15毫米汞柱时无统计学意义。这种与压力升高水平成比例的分级反应代表了类似临床情况（急性、慢性青光眼和正常）的严重程度。这些结果补充了早期在其他神经元培养物中关于压力诱导凋亡的发现。它们提示了与压力相关的机械转导和细胞死亡新机制的可能性，这与青光眼等疾病的发病机制相关。
