McElnea E M, Quill B, Docherty N G, Irnaten M, Siah W F, Clark A F, O'Brien C J, Wallace D M
1Institute of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.
Mol Vis. 2011;17:1182-91. Epub 2011 May 5.
Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH.
Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium (Ca(2+)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively.
Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). Ca(2+) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR.
Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or Ca(2+).
氧化应激与青光眼相关的病理变化密切相关。本研究的目的是比较从5只正常（NLC）和4只青光眼（GLC）人类供体眼的视神经乳头（ONH）区域获得的胶质纤维酸性蛋白阴性（GFAP）筛板（LC）细胞中的氧化应激水平，并研究ONH该区域的线粒体功能和钙稳态。
通过硫代巴比妥酸反应物质（TBARS）测定法检测细胞内活性氧（ROS）的产生，该方法测量丙二醛（MDA），脂质过氧化的天然产物，用作氧化应激的指标。分别使用JC-1（5,5',6,6'-四氯-1,1',3,3'-四苯并咪唑羰花青碘化物）和fluo-4/AM探针通过流式细胞术评估线粒体膜电位（MMP）和细胞内钙（Ca(2+)）水平。分别使用基因特异性引物/探针组通过实时聚合酶链反应（RT-PCR）和蛋白质免疫印迹法测定抗氧化剂和Ca(2+)转运系统基因及蛋白质表达。
与NLC相比，GLC中细胞内ROS产生增加（27.19±7.05μM MDA对14.59±0.82μM MDA，p<0.05）。与NLC对照相比，GLC中抗氧化剂醛糖酮还原酶家族1成员C1（AKR1C1）和谷氨酸半胱氨酸连接酶催化亚基（GCLC）的表达显著降低（p = 0.02）。与NLC（41.8±5.3%）相比，GLC中的MMP较低（57.5±6.8%）。发现GLC细胞中的Ca(2+)水平高于NLC（p<0.001）。与NLC相比，GLC中质膜Ca(2+)/ATP酶（PMCA）和钠钙（NCX）交换器的表达较低，而细胞内肌浆网Ca(2+)/ATP酶3（SERCA）的表达显著较高。通过RT-PCR测定，将NLC细胞置于氧化应激（200μM H(2)0(2)）下会降低钠/钙交换器1（NCX 1）、质膜Ca2+ ATP酶1（PMCA 1）和PMCA 4的表达。
我们的数据发现，与NLC细胞相比，GLC细胞存在氧化应激、线粒体功能障碍和钙外排受损的证据，并表明它们在青光眼ONH发生的病理变化中的重要性。未来的治疗可能针对降低氧化应激和/或Ca(2+)。
