Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy

PURPOSE. Retinal microglia have been implicated in the pathogenesis of various retinal diseases, but their basic function and cellular phenotype remain incompletely understood. Here, the authors used a novel ex vivo retinal imaging preparation to examine the behavioral phenotype of living retinal microglia in intact tissue and in response to injury. METHODS. Fluorescence-labeled microglia in retinal explants from CX3CR1(+/GFP) transgenic mice were observed using time-lapse confocal imaging. High spatial and temporal resolution imaging parameters were used to follow dynamic microglial behavior in real time. RESULTS. Under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes. Process movements are highly random in direction but are balanced to maintain overall cellular symmetry and arbor size. At rest, however, these exuberant process movements do not result in overt cellular migration. After focal laser injury, microglial processes increase significantly in their motility and direct themselves toward the injury site. Microglia rapidly transition their morphologies from symmetric to polarized toward the laser lesion. Microglia also transition from a fixed to a migratory phenotype, translocating through tissue while retaining their ramified morphology. CONCLUSIONS. 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 cellular response to focal laser photocoagulation. Ex vivo live imaging in the retina is an experimental approach well suited to the study of dynamic aspects of microglial physiology.