The microglia response to electrical overstimulation of the retina imaged under a transparent stimulus electrode

Objective.We investigated using the morphological response of retinal microglia as indicators of tissue damage from electrical overstimulation by imaging them through an optically transparent stimulus electrode.Approach.To track the microglia, we used a transgenic mouse where the microglia expressed a water soluble green fluorescent protein. The clear stimulus electrode was placed epiretinally on the inner limiting membrane and the microglia layers were imaged using time-lapse confocal microscopy. We examined how the microglia responded both temporally and spatially to local overstimulation of the retinal tissue. Using confocal microscope vertical image stacks, the microglia under the electrode were imaged at 2.5 min intervals. The retina was overstimulated for a 5 min period using 1 ms 749μC cm^-2ph^-1biphasic current pulses and changes in the microglia morphology were followed for 1 h post stimulation. After the imaging period, a label for cellular damage was applied to the retina.Main results.The microglia response to overstimulation depended on their spatial location relative to the electrode lumen and could result in three different morphological responses. Some microglia were severely injured and became a series of immotile ball-like fluorescent processes. Other microglia survived, and reacted rapidly to the injury by extending filopodia oriented toward the damage zone. This response was seen in inner retinal microglia outside the stimulus electrode edge. A third effect, seen with the deeper outer microglia under the electrode, was a fading of their fluorescent image which appeared to be due to optical scatter caused by overstimulation-induced retinal edema.Significance.The microglial morphological responses to electrical overstimulation injury occur rapidly and can show both direct and indirect effects of the stimulus electrode injury. The microglia injury pattern closely follows models of the electric field distribution under thinly insulated disc electrodes.