Abstract
Purpose :
Following IR injury, retinal microglia in the plexiform layers migrate to the ganglion cell layer (GCL) and nerve fiber layer (NFL), where neurodegeneration is taking place, and circulating monocytes infiltrate the retina and transform into MDM. We hypothesized that MDM populate the vacated plexiform layers and adopt a microglia-like phenotype, filling the microglial niche.
Methods :
Microglia were lineage traced using mice with a yellow fluorescent protein (EYFP) and tamoxifen (Tam)-inducible Cre bicistronic transgene inserted into the Cx3cr1 allele and a floxed-STOP tdTomato (tdTom) transgene. A 4-week washout after Tam treatment eliminated tdTom+ monocytes in circulation. IR injury was induced by elevation of intraocular pressure for 90 minutes via injection of saline into the anterior chamber. Retinal microglia and MDM were identified at 1, 2, 3 and 6 weeks after injury by immunofluorescence (IF) of EYFP, tdTom and endogenous cell markers in the inner retina and outer plexiform layer (OPL) of central, medial and peripheral regions of retinal flatmounts. Aivia image analysis software was used to compare cell morphologies. Retinas were obtained at 2 weeks after injury, immune cell populations were enriched by fluorescence activated cell sorting with CD45 and CD11b as markers single cell sequencing (scSeq) by the 10X fluidics method. scSeq cell cluster differential gene expression was analyzed using 10X Loupe Browser software.
Results :
EYFP+/tdTomneg MDM densities were significantly increased in IR vs sham-treated retinas at 1-3 weeks post injury in all retinal regions, both in the inner retina and OPL. The mean density of MDM was greatest in the central inner retina at 1 week, with 473 cells/mm2 in IR compared to 11 cells/mm2 in sham (p<0.0001). In all regions MDM increases gradually declined after 1 week and were not different from sham at 6 weeks. In the OPL, invading MDM replaced microglia and took on ramified morphologies resembling microglia, but with reduced numbers of dendrites and branches (p≤0.0001). scSeq identified Sall1 and P2ry12 as the most discriminating microglial markers, and IF confirmed replacement of SALL1+/P2RY12hi microglia with SALL1neg/P2RY12low MDM in C57BL/6J mice.
Conclusions :
After IR injury, MDM infiltrate the retina, replacing and mimicking microglia in the OPL. Microglia almost fully reclaim this niche by 6 weeks after injury.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.