We generated a mouse model of primary microglial dysfunction by deleting two negative immune regulatory genes, Cx3cr1 and Socs3 (in LysM+ cells). This study aimed to understand how primary microglial dysfunction impacts retinal neurons during aging. The LysMCre-Socs3fl/flCx3cr1gfp/gfp double knockout (DKO), LysMCre-Socs3fl/fl, Cx3cr1gfp/gfp and Socs3fl/fl mice were maintained up to 12 months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKCα and GABA. Brain microglia from DKO and WT mice were stimulated with LPS + IFN-γ or IL-4. The expression of TNF-α, IL-1β, IL-6, iNOS, IL-12p40, IL-23p19, CCL2, CCL5, CXCL2, IL-10, CD206 and Arg1 were examined by qRT-PCR and protein production was measured by Luminex assay. Retinal explants from C57BL/6 J mice were co-cultured with microglia from DKO or WT mice for 24 h, after which the number of cone arrestin+ cells in retinal flatmount were quantified. In 3–5 month old mice, the number of microglia in retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) were comparable in all strains of mice. The DKO mice had a significantly higher number of microglia in the outer plexiform layer (OPL) but significantly lower numbers of cone arrestin+, secretagogin+ and GABA+ cells compared to Socs3fl/fl and single KO mice. During aging, 57% of the DKO mice died before 12 months old. The 10–12 months old DKO mice had significantly higher numbers of microglia in GCL/IPL and OPL than age-matched Socs3fl/fl and single KO mice. The aged DKO mice developed retinal pigment epithelial (RPE) dysmorphology accompanied by subretinal microglial accumulation. The number of photoreceptors, bipolar cells (Secretagogin+ or PKCα+) and GABA+ amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3fl/fl and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-α, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3fl/fl mice. Co-culture of retinal explants with LPS + IFN-γ or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival. The LysMCre-Socs3fl/flCx3cr1gfp/gfp DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration. Not applicable. The article does not contain any results from human participants.

中文翻译：

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LysM+细胞和Cx3cr1中Socs3的缺失导致视网膜小胶质细胞病的年龄依赖性发展


我们通过删除两个负免疫调节基因 Cx3cr1 和 Socs3（在 LysM+ 细胞中）建立了原发性小胶质细胞功能障碍的小鼠模型。这项研究旨在了解原发性小胶质细胞功能障碍如何在衰老过程中影响视网膜神经元。 LysMCre-Socs3fl/flCx3cr1gfp/gfp 双敲除 (DKO)、LysMCre-Socs3fl/fl、Cx3cr1gfp/gfp 和 Socs3fl/fl 小鼠维持长达 12 个月。收集眼睛并进行 IBA-1、视锥抑制蛋白、促泌素、PKCα 和 GABA 的免疫组织化学分析。用 LPS + IFN-γ 或 IL-4 刺激 DKO 和 WT 小鼠的脑小胶质细胞。通过 qRT-PCR 检查 TNF-α、IL-1β、IL-6、iNOS、IL-12p40、IL-23p19、CCL2、CCL5、CXCL2、IL-10、CD206 和 Arg1 的表达，并通过Luminex 测定。将 C57BL/6 J 小鼠的视网膜外植体与 DKO 或 WT 小鼠的小胶质细胞共培养 24 小时，然后对视网膜平贴片中视锥细胞抑制蛋白 + 细胞的数量进行定量。在 3-5 个月大的小鼠中，所有品系小鼠的视网膜神经节细胞层 (GCL) 和内丛状层 (IPL) 中的小胶质细胞数量相当。与 Socs3fl/fl 和单个 KO 小鼠相比，DKO 小鼠外丛状层 (OPL) 中的小胶质细胞数量显着较高，但视锥细胞抑制蛋白 +、促泌素 + 和 GABA + 细胞数量显着较低。在衰老过程中，57%的DKO小鼠在12个月大之前死亡。 10-12 个月大的 DKO 小鼠 GCL/IPL 和 OPL 中的小胶质细胞数量显着高于年龄匹配的 Socs3fl/fl 和单个 KO 小鼠。老年 DKO 小鼠出现视网膜色素上皮 (RPE) 畸形，并伴有视网膜下小胶质细胞积聚。 与年龄匹配的 Socs3fl/fl 和单个 KO 小鼠相比，老年 DKO 小鼠的光感受器、双极细胞（Secretagogin+ 或 PKCα+）和 GABA+ 无长突细胞的数量显着较低。与 Socs3fl/fl 小鼠的小胶质细胞相比，DKO 小鼠的小胶质细胞表现出明显更高水平的吞噬活性，并产生更高水平的 TNF-α、IL-6、CCL2、CCL5、CXCL2 和 CXCL10。视网膜外植体与 LPS + IFN-γ 或 IL-4 预处理的 DKO 小胶质细胞的共培养显着降低了视锥细胞的存活率。 LysMCre-Socs3fl/flCx3cr1gfp/gfp DKO 小鼠表现出原发性小胶质细胞功能障碍，并出现与年龄相关的视网膜小胶质细胞病，其特征是小胶质细胞活化加剧和多发性视网膜神经元和 RPE 变性。不适用。该文章不包含人类参与者的任何结果。