The John Curtin School of Medical Research, College of Medicine, Biology and Environment, The Australian National University, Building 131, Garran Rd, Canberra, ACT 0200, Australia.
J Neuroinflammation. 2012 Sep 19;9:221. doi: 10.1186/1742-2094-9-221.
The recruitment and activation of inflammatory cells is thought to exacerbate photoreceptor death in retinal degenerative conditions such as age-related macular degeneration (AMD). We investigated the role of Müller cell-derived chemokine (C-C motif) ligand (Ccl)2 expression on monocyte/microglia infiltration and photoreceptor death in light-mediated retinal degeneration, using targeted small interfering (si)RNA.
Adult Sprague-Dawley rats were injected intravitreally with 1 μg of either Ccl2 siRNA or scrambled siRNA, and were then exposed to 1000 lux of light for a period of 24 hours. The mice were given an overdose of barbiturate, and the retinas harvested and evaluated for the effects of bright-light exposure. Ccl2 expression was assessed by quantitative PCR, immunohistochemistry, and in situ hybridization. Monocytes/microglia were counted on retinal cryostat sections immunolabeled with the markers ED1 and ionized calcium binding adaptor (IBA)1, and photoreceptor apoptosis was assessed using terminal dUTP nick end labeling.
Intravitreal injection of Ccl2 siRNA significantly reduced the expression of Ccl2 following light damage to 29% compared with controls. In retinas injected with Ccl2 siRNA, in situ hybridization and immunohistochemistry on retinal cryostat sections showed a substantial decrease in Ccl2 within Müller cells. Cell counts showed significantly fewer ED1-positive and IBA1-positive cells in the retinal vasculature and outer nuclear layer of Ccl2 siRNA-injected retinas, compared with controls. Moreover, there was significantly less photoreceptor apoptosis in Ccl2 siRNA-injected retinas compared with controls.
Our data indicate that Ccl2 expression by Müller cells promotes the infiltration of monocytes/microglia, thereby contributing to the neuroinflammatory response and photoreceptor death following retinal injury. Modulation of exaggerated chemokine responses using siRNA may have value in reducing inflammation-mediated cell death in retinal degenerative disease such as AMD.
炎症细胞的募集和激活被认为会加剧视网膜变性疾病（如年龄相关性黄斑变性[AMD]）中的光感受器死亡。我们使用靶向小干扰（si）RNA 研究了 Müller 细胞衍生趋化因子（C-C 基序）配体（Ccl）2 表达在光介导的视网膜变性中对单核细胞/小胶质细胞浸润和光感受器死亡的作用。
成年 Sprague-Dawley 大鼠通过玻璃体内注射 1μg 的 Ccl2 siRNA 或乱序 siRNA，然后暴露于 1000 lux 的光下 24 小时。给小鼠过量注射巴比妥酸盐，然后收获视网膜并评估强光暴露的影响。通过定量 PCR、免疫组织化学和原位杂交评估 Ccl2 表达。在视网膜冷冻切片上用 ED1 和离子钙结合衔接蛋白（IBA）1 标记的免疫标记物对单核细胞/小胶质细胞进行计数，并使用末端 dUTP 缺口末端标记法评估光感受器凋亡。
与对照组相比，光损伤后玻璃体内注射 Ccl2 siRNA 可将 Ccl2 的表达显著降低至 29%。在注射 Ccl2 siRNA 的视网膜中，视网膜冷冻切片的原位杂交和免疫组织化学显示 Müller 细胞内 Ccl2 大量减少。细胞计数显示，与对照组相比，Ccl2 siRNA 注射的视网膜中血管和外核层的 ED1 阳性和 IBA1 阳性细胞明显减少。此外，与对照组相比，Ccl2 siRNA 注射的视网膜中的光感受器凋亡明显减少。
我们的数据表明，Müller 细胞表达的 Ccl2 促进单核细胞/小胶质细胞的浸润，从而促进视网膜损伤后的神经炎症反应和光感受器死亡。使用 siRNA 调节过度的趋化因子反应可能有助于减少 AMD 等视网膜退行性疾病中炎症介导的细胞死亡。
