Zeng Hui-Yang, Zhu Xiu-An, Zhang Cheng, Yang Li-Ping, Wu Le-Meng, Tso Mark O M
Peking University Eye Centre, Peking University Third Hospital, Beijing, China.
Invest Ophthalmol Vis Sci. 2005 Aug;46(8):2992-9. doi: 10.1167/iovs.05-0118.
To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration.
Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), interferon-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-alpha in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-alpha was determined by double immunolabeling.
Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-alpha was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-alpha was predominantly expressed in the activated microglial cells in the ONL.
Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-alpha), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-alpha, produced by the activated microglia, may accentuate the photoreceptor cell death.
通过识别视网膜变性过程中与小胶质细胞激活、迁移和细胞毒性相关的一系列事件及因素，阐明活化的小胶质细胞在rd小鼠光感受器凋亡中的作用。
采用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测出生后第8、10、12、14、16和18天rd小鼠的光感受器凋亡情况。用CD11b抗体识别视网膜小胶质细胞。通过逆转录-聚合酶链反应（RT-PCR）检测视网膜中趋化因子mRNA的表达，包括单核细胞趋化蛋白（MCP）-1、MCP-3、巨噬细胞炎性蛋白（MIP）-1α、MIP-1β、正常T细胞激活后表达和分泌的调节因子（RANTES）、干扰素-γ诱导的10 kDa蛋白（IP-10）和fractalkine。通过酶联免疫吸附测定（ELISA）和免疫组织化学分析研究营养不良视网膜中肿瘤坏死因子（TNF）-α的产生。通过双重免疫标记确定小胶质细胞中TNF-α的表达。
rd小鼠的光感受器凋亡在出生后第10天开始，在第16天达到高峰，而小胶质细胞的激活和迁移在出生后第10天观察到，并在第14天达到高峰。MCP-1、MCP-3、MIP-1α、MIP-1β和RANTES转录本在出生后第8天被检测到，并在第12天达到高峰。rd小鼠的外核层（ONL）在出生后第8天检测到TNF-α的产生，并在第12天达到高峰。在小胶质细胞活性高峰时，TNF-α主要在ONL中活化的小胶质细胞中表达。
小胶质细胞的激活及其信号分子（趋化因子）和小胶质细胞衍生的毒性因子（TNF-α）的表达与光感受器凋亡的发生同时或先于其发生，提示活化的小胶质细胞在rd小鼠视网膜变性中起主要作用。趋化因子MCP-1、MCP-3、MIP-1α、MIP-1β和RANTES参与小胶质细胞向退化的光感受器细胞层的激活和募集。活化的小胶质细胞产生的TNF-α可能会加剧光感受器细胞的死亡。
