Abstract
Purpose :
Microglia activation is a complex process characterized by changes in morphology and gene expression. The activation of microglia results in neuroinflammation. Although needed to reestablish tissue homeostasis, unchecked neuroinflammation has been linked to retinal degeneration. We have previously reported that AAV-mediated inhibition of RelA/p65 (an NF-kB family member) blocks microglial activation and increases retinal expression of LIF, IL10, and IL9 in a mouse model of geographic atrophy. These cytokines activate STAT3 through their receptors. Furthermore, AAV-mediated retinal expression of LIF has neuroprotective effects. In this study we test the hypothesis that RelA and STAT3 are two transcription factors that modulate microglial activation through regulation of gene expression.
Methods :
C57BL/6J mice were injected intravitreally with either LIF (250 ng), TNFα (250 ng), or both (250 ng LIF and 250 ng TNF). Control mice were either injected with an equal volume of normal saline, or not injected at all to account for the impact of our procedure. Eyes from each treatment were harvested 24 hours after the injection and fixed with 4% paraformaldehyde. Both retina whole mounts and RPE flatmounts were prepared. Microglial morphology in whole mounts was studied by immunofluorescence staining for CD11b and Tmem119. Similarly, interaction of microglia with the RPE was evaluated by staining for ZO-1 (RPE marker) and CD11b.
Results :
LIF-treated retinas had similar density and morphology of microglia when compared to either naive or saline-injected retinas. These microglial cells had a predominantly ramified structure, indicative of a homeostatic phenotype. Retinas treated with TNF (a RelA activator) showed a mixture of ameboid and ramified microglia. We observed the highest number of ameboid microglia in the LIF+TNF treated retinas.
Conclusions :
Activation of TNF signaling causes morphological changes in microglia from homeostatic/ramified towards activated/ameboid. Co-activation of TNF and LIF signaling induces a greater number of active microglia, however, with a prominent number of homeostatic microglia. These results suggest that downstream signaling mechanism, such as RelA and STAT3 activation, can modify microglia morphology and potentially activation status. Future studies will correlate these morphological changes in microglia with gene expression profiles.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.