Hohnholt Michaela C, Blumrich Eva M, Dringen Ralf
Centre for Biomolecular Interactions Bremen and Centre for Environmental Research and Sustainable Technology, University of Bremen, Bremen, Germany.
J Neurosci Res. 2015 Jul;93(7):1127-37. doi: 10.1002/jnr.23502. Epub 2014 Oct 30.
To clarify discrepancies in the literature on the adverse effects of hydrogen peroxide on neurons, this study investigated the application of this peroxide to cultured cerebellar granule neurons with six assays frequently used to test for viability. Cultured neurons efficiently cleared exogenous H2O2. Although viability was not affected by exposure to 10 µM hydrogen peroxide, an exposure to the peroxide in higher concentrations rapidly lowered, within 15 min, the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) reduction capacity to 53% ± 1% (100 µM) and 31% ± 1% (1,000 µM) and the 3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride (neutral red; NR) uptake to 84% ± 6% (100 µM) and 33% ± 1% (1,000 µM) of control cells. The release of glycolytically generated lactate was stopped within 30 min in neurons treated with 1,000 µM peroxide. In contrast, even hours after peroxide application, the cell morphology, the number of propidium iodide-positive cells, and the extracellular activity of the cytosolic enzyme lactate dehydrogenase (LDH) were not significantly altered. The rapid loss in MTT reduction and NR uptake after exposure of neurons to H2O2 for 5 or 15 min correlated well with a strongly compromised MTT reduction and a very high extracellular LDH activity observed after further incubation in peroxide-free medium for a total incubation period of 24 hr. These data demonstrate that cultured neurons do not recover from damage that is inflicted by a short exposure to H2O2 and that the rapid losses in the capacities of neurons for MTT reduction and NR uptake are good predictors of delayed cell damage.
为了澄清文献中关于过氧化氢对神经元不良影响的差异，本研究使用六种常用于测试活力的检测方法，研究了这种过氧化物在培养的小脑颗粒神经元中的应用情况。培养的神经元能够有效清除外源性过氧化氢。虽然暴露于10μM过氧化氢对活力没有影响，但暴露于更高浓度的过氧化物会在15分钟内迅速降低细胞的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）还原能力，降至对照组细胞的53%±1%（100μM）和31%±1%（1000μM），同时3-氨基-7-二甲基氨基-2-甲基吩嗪盐酸盐（中性红；NR）摄取量降至对照组细胞的84%±6%（100μM）和33%±1%（1000μM）。在用1000μM过氧化物处理的神经元中，糖酵解产生的乳酸释放会在30分钟内停止。相比之下，即使在过氧化物处理数小时后，细胞形态、碘化丙啶阳性细胞数量以及胞质酶乳酸脱氢酶（LDH）的细胞外活性也没有明显改变。神经元暴露于过氧化氢5或15分钟后MTT还原和NR摄取的迅速下降，与在无过氧化物培养基中进一步孵育24小时后观察到的MTT还原能力严重受损和细胞外LDH活性非常高密切相关。这些数据表明，培养的神经元无法从短时间暴露于过氧化氢所造成的损伤中恢复，并且神经元MTT还原和NR摄取能力的迅速丧失是延迟细胞损伤的良好预测指标。
