Abstract
Purpose :
To investigate the role of microglial activation in the rapid degeneration of photoreceptors (PRs) observed in a mouse model of RP59, involving rod-specific ablation of the dehydrodolichyl diphosphate synthase (Dhdds) gene.
Methods :
Rod-specific Dhdds knockout (KO) mice were generated (Rao et al., ARVO, 2017), with Dhddsflx/flx iCre+ (KO) and Dhddsflx/flx iCre- (control) mice selected by PCR tail snip analysis for the loxP-modified Dhdds allele and the Rho-iCre transgene. Retinas harvested from PN 4-wk old KO and control mice were subjected to gene expression analysis using NanoString® neurodegeneration/neuroinflammation panels. Immunohistochemistry (IHC) was performed on eyes from PN 5-wk KO and control mice, probing with anti-microglial markers (Iba-1, CD68, F4/80). Retinal whole mounts were probed by IHC to quantify microglia infiltration into the ONL; particle analysis was performed using ImageJ® software. KO mice were maintained on a diet containing PLX3397 (CSF1R inhibitor, 300 mg/kg chow), starting at PN 3 wk, to deplete retinal microglia. ERG analysis was performed on control and KO mice ± PLX3397 at PN 4/5 wk (N=3-4/group). Statistical analysis: Statistical significance: Student’s t-test, P<0.05.
Results :
Retinal gene expression analysis from PN 4-wk old KO and control mice (N=3 ea.) indicated significant changes (fold-change cutoff: 2) in the levels of molecules involved in neuroinflammation and microglial activation (Cxcl-10, TMEM119, C1q, Lif) in KO mice. IHC analysis of KO retinas revealed microglial infiltration into the ONL and subretinal space, with microglia forming phagocytic cups around TUNEL-negative (live) photoreceptors. Quantitative IHC analysis of microglial ONL infiltration demonstrated a significant increase in Iba-1/CD68-positive area in the ONL of KO vs. control retinas (N=3/group, P<0.05). Scotopic ERG analysis at PN 4-5 wk demonstrated rapid rod photoreceptor degeneration in KO mice, regardless of PLX3397 treatment, comparable to that observed in KO mice fed normal chow, at all tested flash intensities.
Conclusions :
Gene expression signatures of retinal microglial activation were upregulated in a rod-specific Dhdds-KO mouse model of RP59, at a time point (PN 4 wk) prior to observable retinal degeneration. Microglial infiltration into the ONL was observed at PN 5 wk, with phagocytosis of live PRs. However, dietary PLX3397 treatment did not rescue the retinal degeneration.
This is a 2020 ARVO Annual Meeting abstract.