In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma
In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma
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DOI：
10.3791/52731
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发表时间：
2015-05-01
影响因子：
1.2
通讯作者：
Vetter, Monica L.
中科院分区：
文献类型：
--
作者：
Bosco, Alejandra;Romero, Cesar O.;Vetter, Monica L.
小胶质细胞是中枢神经系统内的神经免疫细胞，在中枢神经系统损伤时改变其形态和大小，转换为具有不同功能和基因表达谱的激活状态。小胶质细胞的激活在健康、损伤和疾病中的作用尚不完全清楚，因为它们对微环境变化的响应是动态和复杂的。因此，在完整的生物体中，非侵入性监测和分析小胶质细胞激活随时间的变化是至关重要的。由于在不改变中枢神经系统环境的情况下追踪小胶质细胞行为的技术限制，小胶质细胞活化的体内研究被推迟。这在慢性神经退行性疾病中尤其具有挑战性，因为必须追踪长期变化。视网膜是一种中枢神经系统器官，适合于非侵入性实时成像，它提供了一个强大的系统，可以可视化和表征慢性疾病期间小胶质细胞激活的动态。本方案概述了视网膜小胶质细胞的长期体内成像方法，使用共聚焦眼镜检查（cSLO）和CX3CR1（GFP/+）报告小鼠，以细胞分辨率观察小胶质细胞。此外，我们还描述了量化大细胞亚群（每个视网膜200-300个细胞）中细胞激活和密度的月度变化的方法。我们确认使用染色体面积作为一个有用的指标，实时跟踪视网膜中的小胶质细胞激活，通过应用体内图像的自动阈值为基础的形态计量学分析。我们使用这些实时图像采集和分析策略来监测慢性青光眼小鼠模型视网膜神经变性早期小胶质细胞激活和小胶质细胞增生的动态变化。这种方法应该有助于研究影响视网膜和视神经的慢性中枢神经系统疾病中小胶质细胞对神经元和轴突下降的贡献。
Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1(GFP/+) reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.