The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. Cellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Student’s t-test and one-way ANOVA. Single-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

中文翻译：

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脂素 B4 对视神经疾病相关小胶质细胞和高眼压症的调节


常驻星形胶质细胞-视网膜神经节细胞 （RGC） 脂质毒素回路在视网膜应激期间受损，包括高眼压症引起的神经病变。稳态星形胶质细胞产生的脂氧素 B4 直接作用于 RGC，以提高高眼压症诱导的神经病变的存活率和功能。视网膜中的 RGC 死亡和视神经中的轴突变性是由小胶质细胞和大胶质细胞之间的复杂相互作用驱动的。LXB4 神经保护作用是否包括调节视网膜和/或视神经中的其他细胞类型是一个重要的知识空白。通过单细胞 RNA 测序定义视网膜中 LXB4 的细胞靶标和信号传导。通过将硅油注射到小鼠眼睛的前房中诱导视网膜神经变性，从而诱导持续稳定的高眼压增高症。通过 MorphOMIC 和伪时间轨迹分析建立视网膜和视神经中小胶质细胞群的形态学特征。使用大量 RNA 测序研究 LXB4 在视神经中的通路和作用机制。转录组学数据通过 qPCR 和免疫组化验证。通过 Student t 检验和单因素方差分析评估实验组之间的差异。单细胞转录组学确定小胶质细胞是健康视网膜中 LXB4 的主要靶标。LXB4 下调驱动小胶质细胞环境感应和反应反应的基因。小胶质细胞功能的分析显示，高眼压症在视网膜中诱导了独特的、时间定义的和动态的表型，出乎意料地在远端有髓鞘视神经中诱导了表型。 CD74 的小胶质细胞表达是大脑中疾病相关小胶质细胞的标志物，仅在独特的视神经小胶质细胞群中被诱导，而在视网膜中未被诱导。脂蛋白形成的基因缺失与正常血压眼中 CD74 视神经小胶质细胞群的存在相关，而高眼压症期间的 LXB4 治疗使视神经小胶质细胞转向稳态形态和非反应状态，并下调 CD74 的表达。此外，我们确定了 CD74 与视神经中磷酸磷酸肌醇 3-激酶 （p-PI3K） 表达水平之间的相关性，LXB4 治疗降低了这种相关性。我们确定了远端视神经中小胶质细胞功能表型、反应性和独特 CD74 小胶质细胞群诱导的早期和动态变化是高眼压症诱导的神经变性的关键特征。我们的研究结果将小胶质细胞调节确定为视网膜和视神经中的新型 LXB4 靶标。LXB4 维持稳态视神经小胶质细胞表型和抑制疾病相关表型是常驻 LXB4 通路的潜在神经保护机制。