Abstract
Purpose :
Reactive oxygen species (ROS) are produced by cell metabolism and upon tissue/cellular damage. ROS are known to be dangerous when uncontrolled yet are potentially important factors in development and regeneration. We probed the antioxidant response of microglia (MiG) and the NRF2 transcription factor in a zebrafish system of rod photoreceptor ablation and regeneration.
Methods :
We employed the transgenic line rho:nfsB-eGFP^nt19 (Montgomery et al., 2010) to induce rod cell death in larvae via metronidazole (MTZ) treatment. Larvae were treated at 3.5 days post-fertilization (dpf) with MTZ or DMSO (vehicle) for 48 hours and then fixed at 48-, 72-, 96-, and 120-hours post treatment (hpt), n=6-10 eyes per timepoint. TUNEL and PCNA staining were performed to measure cell death and proliferation, respectively. Fluorescence in-situ hybridization (FISH) was used to visualize induction of NRF2-response genes. Gain-of-function (GOF) and loss-of function (LOF) mutants of nfe2l2a (nrf2) were used to test the effect of nrf2-mediated antioxidant response in regeneration, as well as MiG-deficient irf8 mutants to weigh the contribution of microglia to the oxidative stress response and regeneration of rods.
Results :
We found ample rod ablation by 48hpt and regeneration-induced proliferation rises at 96 and 120 hpt relative to controls (2, 3.8 fold, respectively). In this larval system, outer nuclear progenitor cells constituted a majority (~66-95%) of PCNA+ proliferating cells in regenerating retinas across timepoints. FISH revealed MiG expression of select NRF2-induced genes following rod ablation at 48 hpt. Nrf2-GOF mutants remained susceptible to rod ablation. The nrf2-GOF mutant had increased PCNA+ cell counts compared to wildtype at 96 hpt (p=0.0394), but no difference at 48 and 120 hpt (p>0.05). Crosses to analyze nrf2-LOF mutants are in progress. We did not find evidence of macrophage infiltration following rod ablation in MiG-deficient mutants.
Conclusions :
Damage of rods in larvae induces some inner retinal proliferation but more extensively results in proliferation of progenitors in the outer retina. Expression of NRF2-regulated genes are observed in MiG following the ablation of rods. Interestingly, the post-damage proliferative response in nrf2-GOF mutants was transiently increased, suggesting that the NRF2 response is involved in regulating the proliferative response following rod damage.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.