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.
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