June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Microglial activation in a mouse model of RP59: Contributor or bystander?
Author Affiliations & Notes
  • Sriganesh Ramachandra Rao
    Ophthalmology & Biochemistry, SUNY-University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
  • Lara Ann Skelton
    Ophthalmology & Biochemistry, SUNY-University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
  • Steven J Pittler
    Optometry and Vision Science, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Steven J. Fliesler
    Ophthalmology & Biochemistry, SUNY-University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Sriganesh Ramachandra Rao, None; Lara Skelton, None; Steven Pittler, None; Steven Fliesler, None
  • Footnotes
    Support  NIH/NEI (1 R01 EY029341; SJP, SJF) and P30 EY003039 (SJP), NIH/NCATS (1UL1 TR001412; SJF); VAWNYHS facilities and resources (SRR, LAS, SJF); UAB Nanostring Laboratory (SJP)
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 709. doi:
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      Sriganesh Ramachandra Rao, Lara Ann Skelton, Steven J Pittler, Steven J. Fliesler; Microglial activation in a mouse model of RP59: Contributor or bystander?. Invest. Ophthalmol. Vis. Sci. 2020;61(7):709.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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