Effects of the Na-K ATPase blocker digoxin on microglia structure and function in real time in the mouse retina.

Abstract

Purpose : Microglia are sensitive indicators of retinal damage, however, it is unclear whether they can identify retinotoxins. The mouse retina contains 2 layers of microglia. We examined how the α2/α3-subunit selective Na-K ATPase blocker digoxin altered microglia morphology and retinal structure, in real time using a new eyecup preparation and confocal microscopy.

Methods : We used a CX3CR1 GFP (green fluorescent protein) transgenic mouse to track microglia in the retina in real time using confocal microscope Z-stack images. In some cases, histology was also performed to validate the changes observed in the microglial population with changes in the retinal structure. Mouse retina microglia were imaged using a miniature eyecup chamber, and superfused with oxygenated Ames Ringer at 35°C. To examine for cell death, the nuclear binding dye 7-amino-actinomycin D (7AAD) was used to label dead cells.

Results : To examine the microglial response, digoxin was bath applied (3-10μM) to the retina for 10 minutes, followed by a 30min washout period and nuclear dye labeling for cell death. In each case examined, the first effect of digoxin (3μM) was a 57±11% (mean±std. dev.) decline of fluorescence in the processes of the outer retinal microglia from their pre drug levels, while the inner microglia fluorescence declined only 14 ±12% (n=7 retinas) (P<0.001). Histological data suggested this inner/outer fluorescence difference may be due to increased optical scatter by the swollen processes of inner retinal cells in digoxin. Digoxin had a curiously delayed action on the mouse retina, with the peak response some 20 minutes after the drug washout. By 30min, there was a retraction of the fine processes of inner retinal microglial indicative of activation. After digoxin, 7AAD labeled small nuclei in the ganglion cell layer and the microglia (as previously reported). Histological analysis of digoxin-applied retinae showed damage to the middle of the inner nuclear layer and swelling in the IPL. Preliminary data using microscopic OCT showed a similar pattern.

Conclusions : Microglia are sensitive indicators of retinal injury by toxins. The loss of fluorescence by the outer microglia in digoxin is suggestive of inner retinal tissue swelling causing optical scatter in confocal images, and suggest microglia morphological analysis may be useful in neurotoxin assays of whole retinal tissue.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.