目的:糖尿病视网膜病变的特征是神经炎症,驱动神经元和血管退行性病变,在许多个体中可导致视网膜缺血和新生血管形成。浸润的巨噬细胞和活化的视网膜驻留小胶质细胞与糖尿病视网膜病变的进展有关。尽管这些免疫细胞的不同作用仍不明确。我们的目的是阐明巨噬细胞/小胶质细胞在增生性缺血性视网膜病变发病机理中的不同作用。
方法:小鼠氧诱导的视网膜病变通常用作缺血诱导的增生性糖尿病视网膜病变(PDR)的模型。我们通过免疫染色评估了巨噬细胞/小胶质细胞的表型,定量实时RT-PCR(qRT-PCR),流式细胞术和scRNA-seq分析。在糖尿病视网膜病变的临床影像学研究中,我们使用光学相干断层扫描(OCT)和OCT血管造影.
结果:免疫染色,qRT-PCR和流式细胞术显示M1样巨噬细胞/小胶质细胞标志物(CD80、CD68和一氧化氮合酶2)和M2样巨噬细胞/小胶质细胞标志物(CD206、CD163和巨噬细胞清道夫受体1)的表达水平在视网膜缺血区域和新血管周围上调,分别。对缺血性视网膜的scRNA-seq分析揭示了表达M1标记和C-C趋化因子受体2的巨噬细胞/小胶质细胞的不同的缺血相关簇。Rho激酶(ROCK)的抑制抑制了缺血区域的CCL2表达并减少了CCR2阳性M1样巨噬细胞/小胶质细胞。此外,不仅通过ROCK抑制剂和单核细胞趋化蛋白-1抗体,而且通过GdCl3抑制血液巨噬细胞浸润,从而减少了视网膜缺血的面积。使用OCT对糖尿病性视网膜病变进行的临床成像研究表明,在灌注减少的区域中,巨噬细胞/小胶质细胞的潜在参与以高反射病灶为代表。
结论:这些结果共同表明,异型巨噬细胞/小胶质细胞在视网膜血管疾病(包括糖尿病性视网膜病变)中不同程度地促进视网膜缺血和新生血管形成。这增加了重要的新信息,可以为更有针对性地提供基础,细胞特异性治疗方法,以防止进展为威胁视力的PDR。
OBJECTIVE: Diabetic retinopathy is characterised by neuroinflammation that drives neuronal and vascular degenerative pathology, which in many individuals can lead to retinal ischaemia and neovascularisation. Infiltrating macrophages and activated retina-resident microglia have been implicated in the progression of diabetic retinopathy, although the distinct roles of these immune cells remain ill-defined. Our aim was to clarify the distinct roles of macrophages/microglia in the pathogenesis of proliferative ischaemic retinopathies.
METHODS: Murine oxygen-induced retinopathy is commonly used as a model of ischaemia-induced proliferative diabetic retinopathy (PDR). We evaluated the phenotype macrophages/microglia by immunostaining, quantitative real-time RT-PCR (qRT-PCR), flow cytometry and scRNA-seq analysis. In clinical imaging studies of diabetic retinopathy, we used optical coherence tomography (OCT) and OCT angiography.
RESULTS: Immunostaining, qRT-PCR and flow cytometry showed expression levels of M1-like macrophages/microglia markers (CD80, CD68 and nitric oxide synthase 2) and M2-like macrophages/microglia markers (CD206, CD163 and macrophage scavenger receptor 1) were upregulated in areas of retinal ischaemia and around neo-vessels, respectively. scRNA-seq analysis of the ischaemic retina revealed distinct ischaemia-related clusters of macrophages/microglia that express M1 markers as well as C-C chemokine receptor 2. Inhibition of Rho-kinase (ROCK) suppressed CCL2 expression and reduced CCR2-positive M1-like macrophages/microglia in areas of ischaemia. Furthermore, the area of retinal ischaemia was reduced by suppressing blood macrophage infiltration not only by ROCK inhibitor and monocyte chemoattractant protein-1 antibody but also by GdCl3. Clinical imaging studies of diabetic retinopathy using OCT indicated potential involvement of macrophages/microglia represented by hyperreflective foci in areas of reduced perfusion.
CONCLUSIONS: These results collectively indicated that heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation in retinal vascular diseases including diabetic retinopathy. This adds important new information that could provide a basis for a more targeted, cell-specific therapeutic approach to prevent progression to sight-threatening PDR.