Abstract
Purpose :
Immune cell activation and synapse disassembly in the inner retina have been previously demonstrated in ocular hypertension models. Conflicting studies report neuroprotective vs neurotoxic roles of microglia in neurodegeneration. Here we characterize microglia activation and synaptic density changes in an NMDA-induced model of RGC death. We hypothesize that microglia activation will be neurotoxic and contribute to synapse disassembly after NMDA exposure.
Methods :
The eyes of adult Cx3Cr1-GFP mice, in which microglia are fluorescently labeled, were intravitreally injected with PBS (N=4), 0.05mM, 0.1mM, or 1mM NMDA (N=5/group). Three days post-injection, retinas were fixed and RGCs and presynaptic components were visualized by immunolabeling with antibodies to RBPMS and CtBP2, respectively. Image stacks of the inner plexiform layer (IPL) were acquired with confocal microscopy. Cell densities were computed by counting cells in a maximum image projection in ImageJ and synaptic densities computed using ObjectFinder (https://lucadellasantina.github.io/ObjectFinder/). The Mann-Whitney U test was used to calculate differences between groups.
Results :
RGC density was significantly reduced in 1mM NMDA-injected mice (4.18±0.32cells/1000μm2) compared to control (1.93±0.66cells/1000μm2; p=0.015). Interestingly, the 0.05 and 0.1mM groups showed no decrease, indicating a critical concentration for RGC death between 0.1 and 1mM NMDA. Microglia density was elevated in the 1mM NMDA condition (0.75±0.21cells/1000μm2) compared to control (0.52±0.19cells/1000μm2); however, the elevation did not reach statistical significance (p=0.11). Finally, despite RGC death, there was no difference in the density of presynaptic ribbons in the 1mM group (0.15±0.03puncta/μm3) compared to control (0.17±0.04puncta/μm3).
Conclusions :
Our results demonstrate greater RGC death and microglia activation as NMDA concentration increases. However, presynaptic ribbons appear to persist despite RGC loss, suggesting that NMDA is not toxic to bipolar cells. Analysis of bipolar cell density, postsynaptic component density, and the colocalization of microglial processes with these synaptic proteins will further elucidate the role of microglia activation in the synapse disassembly of the bipolar to ganglion cell synapse.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.