Abstract
Purpose :
Inflammation has emerged as a key driver of the Müller glial (MG) response to retinal damage in several species. Inflammatory signals may impact formation/proliferation of MG-derived progenitors as well as production of regenerated neurons. To date, experimental manipulations of inflammation have predominantly involved broad-acting immunosuppressants, whole cell depletions, or exogenously supplied compounds/factors. Cellular sources of inflammatory signals presumably include the microglia (MiG), though pathways involved in stimulating such signals from MiG are unknown. We focus on the regulation of MYD88 signaling in MiG to understand downstream effects of this microglial pathway’s activation on the MG regenerative response in the zebrafish retina. We hypothesize that forcing inflammatory signals from MiG via sustained MYD88 signaling will alter the outcome of MG-mediated regenerative responses.
Methods :
We examined published RNA-seq datasets (Mitchell et al., 2019, Oosterhof et al., 2017). We generated transgenic zebrafish in which forced MYD88 signaling occurs cell-selectively in MiG/macrophage. We stimulated MG-mediated regenerative response using the neurotoxin ouabain to induce death of inner retinal neurons in transgenic zebrafish. At selected timepoints, we analyzed tissue damage, leukocyte response, proliferation of MG-derived progenitors, selected gene expression, and early regeneration of inner retinal neurons using TUNEL/immunostaining, microscopy, image analysis, and RT-qPCR (n=4-6/group).
Results :
Transcriptome data indicated downregulation of myd88, and upregulation of inhibitors of this pathway, in zebrafish MiG during retinal damage and regeneration. In transgenic lines with forced MYD88 signaling in MiG, initial levels of cell death and early leukocyte responses were not significantly altered. However, at 6 days post injury numbers of PCNA+ cells were increased (p<0.03), as was expression of gfap (p=<0.01) and GFAP staining, compared to controls. Analysis of progenitor markers and regenerated neurons is in progress.
Conclusions :
Our results provide evidence that inflammatory signals produced by MiG/macrophage via MYD88-pathway activation affect MG reactivity and proliferation of MG-derived progenitors. We consider that temporal regulation of inflammatory signals from MiG drive the proper transition from inflammatory/proliferative to regenerative responses.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.