Kitano S, Morgan J, Caprioli J
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06510, USA.
Exp Eye Res. 1996 Jul;63(1):105-12. doi: 10.1006/exer.1996.0096.
The cytotoxic effects of hypoxia and excitatory amino acids on cultured retinal ganglion cells were studied. The influence of coculture with retinal Müller glia and cortical astrocytes on cell survival was evaluated, as were the effects of the N-methyl-DL-aspartate inhibitor, MK-801. Dissociated retinal neurons from 7-day-old Sprague-Dawley rats were plated on a laminin substrate, neocortex-derived glial monolayers, or Müller cell monolayers. Ganglion cells were labeled by injection of DiI into the superior colliculus 2 days prior to dissociation. Exposure of cultured ganglion cells to glutamate and N-methyl-DL-aspartate showed a time- and concentration-dependent survival rate. Exposure of cells to hypoxia demonstrated a survival rate that was dependent on time and O2 concentration. Excitotoxic and hypoxic damage was entirely blocked by the specific non-competitive inhibitor of N-methyl-DL-aspartate, MK-801. Retinal ganglion cells cultured on cortical astrocytes and retina-derived Müller glia showed significantly better survival rates (P < 0.001) than cells cultured on laminin-coated dishes under control conditions, in hypoxia (9% to 15% O2), and after exposure to 200 microM glutamate. Retinal ganglion cells cultured on Müller glia showed significantly better survival rates (P < 0.01) than those cultured on cortical astrocytes under conditions of hypoxia (9% to 15% O2) and exposure to 200 microM glutamate. The results demonstrate that excitotoxic and hypoxic damage to cultured retinal ganglion cells is moderated by NMDA receptor blockade and by the presence of glial cells, especially retinal Müller cells. This system may provide a useful model for studying the pathophysiology of excitotoxicity and hypoxia on cultured retinal ganglion cells, and may be used to help identify potentially clinically useful therapeutic agents.
研究了缺氧和兴奋性氨基酸对培养的视网膜神经节细胞的细胞毒性作用。评估了与视网膜Müller胶质细胞和皮质星形胶质细胞共培养对细胞存活的影响，以及N-甲基-DL-天冬氨酸抑制剂MK-801的作用。将7日龄Sprague-Dawley大鼠的视网膜神经元解离后接种在层粘连蛋白底物、新皮质来源的胶质细胞单层或Müller细胞单层上。在解离前2天将DiI注入上丘，标记神经节细胞。将培养的神经节细胞暴露于谷氨酸和N-甲基-DL-天冬氨酸后，其存活率呈现出时间和浓度依赖性。将细胞暴露于缺氧环境中，其存活率取决于时间和氧气浓度。N-甲基-DL-天冬氨酸的特异性非竞争性抑制剂MK-801可完全阻断兴奋毒性和缺氧损伤。在对照条件下、缺氧（氧气浓度为9%至15%）以及暴露于200微摩尔谷氨酸后，在皮质星形胶质细胞和视网膜来源的Müller胶质细胞上培养的视网膜神经节细胞的存活率显著高于在层粘连蛋白包被培养皿上培养的细胞（P<0.001）。在缺氧（氧气浓度为9%至15%）和暴露于200微摩尔谷氨酸的条件下，在Müller胶质细胞上培养的视网膜神经节细胞的存活率显著高于在皮质星形胶质细胞上培养的细胞（P<0.01）。结果表明，NMDA受体阻断和胶质细胞尤其是视网膜Müller细胞的存在可减轻培养的视网膜神经节细胞的兴奋毒性和缺氧损伤。该系统可能为研究兴奋毒性和缺氧对培养的视网膜神经节细胞的病理生理学提供一个有用的模型，并可用于帮助识别潜在的临床有用治疗药物。
