Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai 200031, People's Republic of China.
Exp Neurol. 2012 Dec;238(2):192-208. doi: 10.1016/j.expneurol.2012.08.029. Epub 2012 Sep 10.
Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3 ms, 20 Hz, 300-1600 μA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1β and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).
许多类型的电刺激（ES）已被证明可促进退化神经元的存活，例如内侧隔核束横断大鼠中的多巴胺能神经元、缺血性皮质神经元和退化视网膜的内核层和外核层细胞。我们实验室使用大鼠光损伤模型先前证明了经角膜电刺激（TCES）对凋亡光感受器细胞的神经保护作用。为了阐明可能在此过程中起作用的机制，本体外研究调查了 ES 对光损伤光感受器变性诱导的小胶质细胞和 Müller 细胞激活的影响。我们的数据显示，ES（3 ms，20 Hz，300-1600 μA）通过 LDH 和 TUNEL 测定分析，增加了与小胶质细胞或 Müller 细胞共培养的光培养的锥体衍生细胞（661W）的存活率。发现挽救程度取决于 ES 电流的不同强度。免疫细胞化学显示，ES 可显著减少与光损伤的 661W 细胞共培养时具有阿米巴样形状的活化小胶质细胞的数量，并增加具有较大胞体的反应性胶质状 Müller 细胞的数量。实时 RT-PCR 和 Western blot 分析表明，ES 对不同共培养物和 661W 细胞条件培养基（661WCM）处理的神经胶质细胞培养物中小胶质细胞中白细胞介素（IL）-1β和肿瘤坏死因子（TNF）-α的分泌具有显著的抑制作用，对 Müller 细胞中脑源性神经营养因子（BDNF）和睫状神经营养因子（CNTF）的产生具有积极的调节作用。用抗白细胞介素（IL）-1β和 TNF-α的中和抗体添加到与小胶质细胞共培养的光暴露的 661W 细胞中，可显著降低小胶质细胞的死亡率。另一方面，当在存在抗 BDNF 的中和抗体的情况下孵育与 Müller 细胞共培养时，光暴露的 661W 细胞的死亡率明显增加，而抗 CNTF 中和抗体不会导致这些 661W 细胞的细胞死亡进一步恶化。这些发现表明，使用 ES 为光损伤的光感受器细胞创造一个培育环境是可行的。这种环境的特征是小胶质细胞活化减弱和强化 Müller 细胞反应性胶质形成，这可能在改善光感受器损伤方面具有很大的潜力。通过这种方式，ES 被确定为治疗色素性视网膜炎（RP）患者中光感受器变性进展的一种新的、有效的治疗选择。
