Abstract
Purpose :
Models of microglia deficiency are used to probe their functions in the retina. In these microglia-deficient tissues, phagocytosis may be compensated by other glial cell types. We found Müller glia (MG) increase phagocytic load in microglia deficient retinas (Mitchell et al. IOVS 2021). We further use a microglia-deficient genetic zebrafish mutant to investigate MG phagocytic activity and determine downstream effects on MG. We hypothesized increased phagocytic load leads to MG reactivity.
Methods :
Developing whole eyes and retinal cryosections from microglia-deficient and sufficient siblings were analyzed. We used confocal microscopy to visualize apoptotic cells (TUNEL), MG, and lysosomes (LAMP1). We visualized apoptotic cell engulfment in live, transient transgenic zebrafish expressing TP1:mcherry-CAAX stained with acridine orange and 3D image rendering (n=4). To see if phagocytosis induced apoptosis in MG, we correlated phagocytosis to MG cell death using cleaved caspase 3 staining (n=8-20/group). To examine MG reactivity, we measured gfap transcripts in individual pairs of eyes by qPCR (n=10-20/group), and GFAP intensity from stained retinal cryosections (n=3-4). To inhibit MG phagocytosis, we used L-SOP treatment (n=8-20/group). We used a transgenic reporter to quantify reactive MG (gfap:nGFP, Bernardos et al., 2007; n=5-8). To examine possible cell cycle re-entry by post-mitotic MG, we used EdU immersion and quantified EdU+MG (n=8-9/group). We also examined id2a and ascl1a transcripts in eyes using qPCR (n=5-9 & n=9-19/group, respectively).
Results :
MG increase their phagocytic load to engulf dying cells in the absence of microglia, from ~15% in wt to ~50% TUNEL+ cell engulfment in mutants (p<0.0001). LAMP1 staining of MG increased in the absence of microglia. Correlation analysis of engulfment to MG cell death is ongoing. Increased expression of gfap in microglia-deficient eyes (p=0.01) was detected but lost after L-SOP treatment. In the absence of microglia, we found increased gfap:nGFP+ MG compared to wt siblings (p=0.007). We detected no increased incorporation of EdU by MG in the absence of microglia, or differences in expression of id2a or ascl1a.
Conclusions :
MG compensate phagocytic function in the absence of microglia, leading to signs of MG reactivity. However, increased MG phagocytic activity alone does not trigger cell cycle re-entry of differentiated MG in the absence of tissue damage.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.