Dynamic Imaging of Retinal Microglia

我们的工作集中在使用实时成像技术来检查活组织中视网膜小胶质细胞的行为。 我们采用离体时间推移共聚焦成像技术，可视化荧光标记的小胶质细胞从转基因CX 3CR 1 +/GFP小鼠，并遵循动态小胶质细胞的行为在完整的视网膜外植体在真实的时间。 先前的研究已经证明，大脑皮层中的小胶质细胞在其分支过程中表现出结构动力学，但这种行为是否延伸到大脑以外的区域尚未得到研究。 我们的成像系统使我们能够跟踪单个小胶质细胞过程结构的详细变化，并定量完整视网膜外植体中的过程和迁移速度。我们还研究了动态小胶质细胞的反应，局部激光治疗使用的参数类似于网格激光治疗糖尿病视网膜病变。 使用实时成像，我们发现在正常条件下，静息视网膜小胶质细胞的结构不是静态的，而是在其细胞过程中表现出广泛的结构动态性，以微米/分钟的速度快速改变其结构。 这些过程向各个方向延伸，并且似乎以随机的方式对周围的细胞外空间进行采样。 尽管显着的动态，细胞的整体面积保持相对恒定;过程没有交叉到相邻的细胞领土，和细胞体的位置保持相对固定，并没有显示任何明显的细胞迁移。 我们发现这种现象存在于新生和成年动物的视网膜中，并且是发育和成熟系统的特性。 我们还研究了视网膜小胶质细胞的行为如何改变，以响应局灶性视网膜损伤。使用氩激光进行局灶性光凝损伤，记录并分析附近的小胶质细胞行为。 损伤后，我们发现小胶质细胞突起运动的速率比基线速率显著增加（增加67%）。 此外，损伤附近的小胶质细胞优先向损伤部位定向突起，而撤回细胞另一侧的突起，因此采用极化细胞表型。 此外，虽然静止的小胶质细胞具有固定的细胞体位置，但激光损伤后的小胶质细胞获得迁移能力，并且能够以分枝状态通过组织移位。 极化小胶质细胞的迁移速度在0.5微米/分钟的范围内。 总之，视网膜小胶质细胞通常占据未受伤的组织显示出连续的，动态的行为，这表明组织监视和细胞间通讯的功能。 小胶质细胞的行为是高度调节，并立即响应，局灶性组织损伤，并可能构成一个治疗反应局灶性激光光凝。 未来的工作将集中在阐明细胞信号调节和指导的动态行为的小胶质细胞的过程运动和迁移动力学。

Our work focused on the use of live imaging techniques to examine the behavior of retinal microglia in living tissue. We 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 explants in real time. Previous studies have demonstrated that microglia in the cerebral cortex demonstrate structural dynamism in their ramified processes, but whether this behavior extends to areas outside the brain has not been previously examined. Our imaging system enables us to follow detailed changes in the structure of individual microglial processes and to quantitate process and migration velocities in intact retinal explants. We have also examined dynamic microglial responses to focal laser treatment using parameters similar to those used in the grid laser treatment of diabetic retinopathy. Using live imaging, we found that under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes, changing their structure at remarkably rapid rates on the scale of micrometers per minute. These processes extended in all directions, and appeared to sample the surrounding extracellular space in a random fashion. Despite marked dynamism, the overall area of the cell remained relatively constant; processes did not cross into a neighboring cells territory, and the position of cell bodies remain relatively fixed, and did not display any overt cellular migration. We found that this phenomenon was present in the retina both neonatal and adult animals and is a property of both developing and mature systems. We also examined how retinal microglia behavior changes in response to focal retinal injury. Focal photocoagulative injury using an argon laser was applied, and microglial behavior in the vicinity was recorded and analyzed. After injury, we found that the rate of microglia process movement increased significantly (67% increase) over baseline rates. Also, microglia in the injury vicinity directed processes preferentially toward the injury site, while withdrawing processes on the other side of the cell, hence adopting a polarized cellular phenotype. In addition, while resting microglia have stationary cell body positions, microglia post-laser injury acquire a migratory capacity, and are capable of translocating through tissue in a ramified state. The migratory speed of polarized microglia was on the scale of 0.5 microns/minute. In summary, retinal microglia normally occupying uninjured tissue display a continuous, dynamic behavior that suggests functions of tissue surveillance and intercellular communication. Microglial behavior is highly regulated by, and immediately responsive to, focal tissue injury and may constitute a therapeutic response to focal laser photocoagulation. Future work will focus on elucidating the cellular signals modulating and directing the dynamic behavior of microglia in terms of process movement and migratory dynamics.