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。我们使用体外延时共聚焦成像技术对转基因CX3CR1+/GFP小鼠的荧光标记小胶质细胞进行可视化，并实时跟踪完整视网膜移植块中小胶质细胞的动态行为。以前的研究已经证明，大脑皮层的小胶质细胞在其分支过程中表现出结构性的动态，但这种行为是否延伸到大脑以外的区域之前还没有被研究过。我们的成像系统使我们能够跟踪单个小胶质细胞突起结构的详细变化，并对完整的视网膜外植体的过程和迁移速度进行量化。我们还使用与网格激光治疗糖尿病视网膜病变相似的参数，检测了聚焦激光治疗的动态小胶质细胞反应。 使用实时成像，我们发现在正常情况下，静息的视网膜小胶质细胞在结构上并不是静态的，而是在其细胞过程中表现出广泛的结构动态，以每分钟微米的尺度以惊人的速度改变其结构。这些过程向各个方向延伸，似乎以随机的方式对周围的细胞外空间进行采样。尽管有明显的活力，但细胞的总体面积保持相对恒定；突起没有进入邻近的细胞区域，细胞体的位置保持相对固定，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动物和成年动物的视网膜中，是发育中和成熟系统的一种特性。 我们还检查了视网膜小胶质细胞行为如何改变对视网膜局灶性损伤的反应。用Ar激光进行局灶性光凝损伤，记录并分析周围小胶质细胞的行为。损伤后，我们发现小胶质细胞突起移动率较基线显著增加(增加67%)。此外，损伤附近的小胶质细胞优先将突起引向损伤部位，而撤回细胞另一侧的突起，从而采用极化的细胞表型。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了迁移能力，并能够以分叉状态在组织中转移。极化小胶质细胞的迁移速度约为0.5微米/分钟。 综上所述，正常占据未损伤组织的视网膜小胶质细胞表现出一种连续的、动态的行为，提示具有组织监视和细胞间通讯的功能。小胶质细胞的行为受到局灶性组织损伤的高度调节，并立即对其作出反应，并可能构成对局灶性激光光凝的治疗反应。未来的工作将集中在阐明细胞信号，从过程运动和迁移动力学的角度来调控和指导小胶质细胞的动态行为。

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.