Eye Research Institute of Oakland University, Rochester, Minnesota, United States of America.
PLoS One. 2011 Mar 31;6(3):e18305. doi: 10.1371/journal.pone.0018305.
Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection.
反应性神经胶质增生是许多视网膜神经退行性疾病的标志，包括青光眼。尽管迄今为止大多数研究都集中在视神经头部的反应性神经胶质增生，但很少有研究开始研究反应性神经胶质增生在视网膜中的作用。我们之前已经表明，反应性胶质细胞合成高水平的蛋白酶，而这些蛋白酶反过来又促进视网膜神经节细胞 (RGC) 的死亡。在这项研究中，我们使用了两种神经胶质毒素来抑制反应性神经胶质增生，并评估了它们对蛋白酶介导的 RGC 死亡的影响。在 C57BL/6 小鼠的玻璃体内注射海人酸 (KA) 以诱导反应性神经胶质增生和 RGC 死亡。C57BL/6 小鼠还接受神经毒素 alpha-氨基己二酸 (AAA) 或神经稳定素 (Neurostatin) 与 KA 联合治疗。通过用胶质纤维酸性蛋白 (GFAP) 和波形蛋白抗体对视网膜切片和视网膜平面进行免疫染色来评估反应性神经胶质增生。通过 TUNEL 测定评估细胞凋亡死亡。通过用 Brn3a 抗体对平面视网膜进行免疫染色来确定 RGC 的损失。通过酶谱测定评估基质金属蛋白酶-9 (MMP-9)、组织纤溶酶原激活物 (tPA) 和尿激酶纤溶酶原激活物 (uPA) 的蛋白水解活性。KA 诱导视网膜星形胶质细胞和 Muller 细胞中的反应性神经胶质增生，GFAP 免疫反应性表明。AAA 单独或与 KA 联合使用可降低 Muller 细胞中的 GFAP 和波形蛋白免疫反应性，但不能降低星形胶质细胞中的 GFAP 和波形蛋白免疫反应性。此外，AAA 未能降低 KA 介导的蛋白酶水平和 RGC 的凋亡死亡。相比之下，Neurostatin 单独或与 KA 联合使用可降低星形胶质细胞和 Muller 细胞中的反应性神经胶质增生。此外，Neurostatin 降低了蛋白酶水平并防止了 RGC 的凋亡死亡。我们的研究结果首次表明，抑制反应性神经胶质增生可降低视网膜中的蛋白酶水平，防止视网膜神经元的凋亡死亡，并提供实质性的神经保护作用。
