Objective

Microglia/macrophage activation is previously reported to be involved in various ocular diseases. However, the separate role of M1/M2 phenotype microglia/macrophage in the pathological process of oxygen-induced retinopathy (OIR) remains unknown. In this research, we explored the role and regulatory mechanism of M1/M2 microglia/macrophage in OIR in C57BL/6J mice. Furthermore, we demonstrated the time phase of M1/M2 shifting of microglia/macrophage during the natural process of OIR, which is very essential for further investigations.

Materials and methods

C57BL/6j pups were exposed to hyperoxia environment from postnatal 7(P7) to P12 then returned to normoxia. The mice were then euthanized, and the eyes were harvested at a series of time points for further investigation. The M1/M2 phenotype microglia/macrophage activity was presented by immunofluorescent staining and real-time quantitative polymerase chain reaction (qPCR). The NF-κb-STAT3 signaling and IL-4-STAT6-PPAR-γ signaling pathway activity was examined by western blot analysis.

Results

The microglia/macrophage were activated when the OIR model was set up after P12. The M1 microglia/macrophage activation was found in neovascularization (NV) tufts in both central and peripheral retina, which started from P12 when the mice were returned to normoxia environment and peaked at P17. During this period of time, the NF-κb-STAT3 signaling pathway was activated, resulting in the upregulated M1 phenotype microglia/macrophage polarization, along with the enhanced inflammatory cytokine expression including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β. Consequently, the NV tufts were observed from P12 and the volume continued to increase until P17. However, the M2 phenotype microglia/macrophage activity took over during the late phase of OIR started from P17. The IL-4-STAT6-PPAR-γ signaling activity was upregulated from P17 and peaked at P20, inducing M2 phenotype microglia polarization, which consequently led to the inhibition of inflammatory cytokines and spontaneous regression of NV tufts.

Conclusions

Microglia/macrophage participate actively in the natural process of OIR in mice, and two phenotypes exert different functions. Treatment modulating microglia/macrophage polarize toward M2 phenotype might be a novel and promising method for ocular neovascular diseases such as retinopathy of prematurity (ROP), wet age-related macular degeneration (wAMD), and diabetic retinopathy (DR).

中文翻译：

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小鼠氧诱导视网膜病变后小胶质细胞/巨噬细胞M1/M2表型的相变

 客观的

先前报道小胶质细胞/巨噬细胞激活与多种眼部疾病有关。然而，M1/M2表型小胶质细胞/巨噬细胞在氧诱导性视网膜病变（OIR）病理过程中的单独作用仍不清楚。在本研究中，我们探讨了M1/M2小胶质细胞/巨噬细胞在C57BL/6J小鼠OIR中的作用和调节机制。此外，我们还证明了 OIR 自然过程中小胶质细胞/巨噬细胞 M1/M2 转变的时间阶段，这对于进一步研究非常重要。

 材料和方法

C57BL/6j幼仔出生后7(P7)至P12暴露于高氧环境，然后恢复常氧。然后对小鼠实施安乐死，并在一系列时间点摘取眼睛以进行进一步研究。通过免疫荧光染色和实时定量聚合酶链反应 (qPCR) 呈现 M1/M2 表型小胶质细胞/巨噬细胞活性。通过蛋白质印迹分析检查 NF-κb-STAT3 信号传导和 IL-4-STAT6-PPAR-γ 信号传导通路活性。

 结果

P12后建立OIR模型时小胶质细胞/巨噬细胞被激活。在中央和周边视网膜的新生血管（NV）簇中发现了M1小胶质细胞/巨噬细胞的激活，该激活从小鼠返回常氧环境的P12开始，并在P17达到峰值。在此期间，NF-κb-STAT3信号通路被激活，导致M1表型小胶质细胞/巨噬细胞极化上调，同时炎症细胞因子表达增强，包括肿瘤坏死因子-α（TNF-α）、白介素-6 (IL-6) 和 IL-1β。因此，从 P12 开始观察到 NV 簇绒，并且体积持续增加，直到 P17。然而，M2 表型小胶质细胞/巨噬细胞活性在从 P17 开始的 OIR 晚期阶段接管。 IL-4-STAT6-PPAR-γ信号活性从P17开始上调，并在P20时达到峰值，诱导M2表型小胶质细胞极化，从而导致炎症细胞因子的抑制和NV簇的自发消退。

 结论

小胶质细胞/巨噬细胞积极参与小鼠 OIR 的自然过程，两种表型发挥不同的功能。调节小胶质细胞/巨噬细胞极化至 M2 表型的治疗可能是治疗早产儿视网膜病变 (ROP)、湿性年龄相关性黄斑变性 (wAMD) 和糖尿病视网膜病变 (DR) 等眼部新生血管疾病的一种新颖且有前景的方法。