Abstract
Purpose :
A CRISPR/Cas9 knock-in murine model of G1528C in Hadha was generated to recapitulate the human LCHADD phenotype. Although the 12-month retinal phenotype was reported, this study aims to further correlate in vivo images with an in-depth analysis of RPE morphology and potential cell death mechanisms.
Methods :
LCHADD and wild type (WT) mice underwent live, in vivo fundus imaging and Optical Coherence Tomography (OCT) imaging at 12-months. Each fundus image was scored (yes/no) for the presence of hypopigmentation indicative of RPE degeneration. Photoreceptor thickness was quantified through manual segmentation of OCT images. Eyes were enucleated and embedded for cryosectioning. Retinal cross-sections underwent Hematoxylin and Eosin (H&E), immunofluorescence (IF), and TUNEL staining. H&E images were used to categorize four types of RPE disruption which were compared between LCHADD and WT mice. To further assess RPE changes, retinal sections were stained with an antibody specific to RPE65. This expression was semi-quantified via confocal microscopy. To elucidate cell death mechanisms involved in the phenotype, IF for CD68 and TUNEL staining were conducted. Macrophages (CD68+) and TUNEL+ cells in the retina of LCHADD and WT mice were counted and compared.
Results :
At 12-months, more LCHADD mice (67%) had hypopigmentation in the RPE compared to WT mice (31%). However, photoreceptor thickness was not significantly different between the strains. H&E scores indicate that a larger percentage of LCHADD retinas exhibit RPE disruption (88%) compared to WT (47%). Furthermore, only LCHADD retinas display the most severe RPE phenotype including rigidity loss (23%) and RPE dropout (12%). RPE65 staining supports the histological findings as its expression is weaker and more intermittent in LCHADD retinas. Macrophage infiltration was doubled in LCHADD retinas compared to WT retinas.
Conclusions :
The increased hypopigmentation on fundus images in LCHADD mice correlated with more severe types of RPE disruption on H&E and lower RPE65 expression on IF. Additionally, macrophages may play a role in eliminating damaged RPE cells. In vivo imaging and retinal cross-section staining support the presence of a distinct retinal phenotype in the LCHADD murine model.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.