Dynamic Imaging of Retinal Microglia

视网膜中的免疫细胞表现出显著的动态运动，报告其生理和功能。我们研究了眼免疫细胞的解剖和行为，包括视网膜中的小胶质细胞和脉络膜中的巨噬细胞。我们的工作采用了体外延时共聚焦成像技术，显示了转基因CX3CR1+/GFP小鼠的荧光标记小胶质细胞，并实时跟踪了完整视网膜和脉络膜外植体中小胶质细胞的动态行为。 脉络膜的免疫和血管改变与老年性黄斑变性(AMD)有关。由于脉络膜免疫细胞的生理学和与脉络膜血管的相互作用尚不完全清楚，我们研究了年轻和老年小鼠脉络膜髓细胞和血管成分之间的关系。 白化CX3CR1(GFP/+)转基因小鼠的脉络膜髓系细胞具有绿色荧光，用活性染料DiI进行铝内灌注以标记脉络膜血管。使用延时实时共聚焦成像和免疫组织化学分析来检测常驻髓系细胞的分布、形态、行为和血管相关性。 树突状髓样细胞是脉络膜免疫细胞的主要组成部分，广泛分布于脉络膜，并与脉络膜血管密切相关，脉络膜血管在脉络膜树中的位置不同。值得注意的是，与脉络膜动脉和小动脉相关的髓样细胞以细长的细胞形式出现在血管的长轴两侧，而与脉络膜毛细血管相关的髓样细胞则以一层星状细胞分布在巩膜表面，而不是玻璃体脉络膜毛细血管表面。树突状髓样细胞处于静止状态时，表现出快速的过程动力，非常适合对血管周围空间进行全面的免疫监测。髓系细胞的密度也随着年龄的增长而增加，局部与脉络膜血管的衰减程度更大相关。 常驻髓系细胞表现出与脉络膜血管密切但动态的物理相互作用，这表明正常脉络膜中存在结构性的免疫-血管相互作用。这些相互作用可能会随着年龄的增长而逐渐改变，这为理解AMD背后的年龄相关性脉络膜功能障碍提供了基础。

Immune cells in the retina demonstrate significant dynamic motility that report on their physiology and function. We have examined the anatomy and behavior of ocular immune cells including 1. microglia in the retina, and 2. macrophages in the choroid. Our work employed ex vivo time-lapse confocal imaging techniques to visualize fluorescence-labeled microglia from transgenic CX3CR1+/GFP mice and follow dynamic microglia behavior in intact retinal and choroidal explants in real time. Immune and vascular alterations in the choroid are implicated in age-related macular degeneration (AMD). As choroidal immune cells are incompletely understood with regard to their physiology and interactions with choroidal vessels, we examined the associations between myeloid and vascular components of the choroid in young and aged mice. Albino CX3CR1(GFP/+) transgenic mice, whose choroidal myeloid cells possess green fluorescence, were perfused intraluminally with the vital dye DiI to label choroidal vessels. The distribution, morphology, behavior, and vascular associations of resident myeloid cells were examined using time-lapse live confocal imaging and immunohistochemical analysis. Dendritiform myeloid cells, comprising most of the resident immune cell population in the choroid, were widely distributed across the choroid and demonstrated close associations with choroidal vessels that varied with their position in the vascular tree. Notably, myeloid cells associated with choroidal arteries and arterioles appeared as elongated cells flanking the long axes of vessels, whereas those associated with the choriocapillaris were distributed as a layer of stellate cells on the scleral but not vitreal choriocapillaris surface. While stationary in position, dendritiform myeloid cells demonstrated the rapid process dynamism well suited to comprehensive immunosurveillance of the perivascular space. Myeloid cells also increased in density as a function of aging, correlating locally with greater choroidal vascular attenuation. Resident myeloid cells demonstrated close but dynamic physical interactions with choroidal vessels, indicative of constitutive immune-vascular interactions in the normal choroid. These interactions may alter progressively with aging, providing a basis for understanding age-related choroidal dysfunction underlying AMD.