Abstract
Purpose:
We previously showed that the retinal pigment epithelium (RPE) in the AY9944-induced rat model of Smith-Lemli-Opitz syndrome (SLOS) exhibits marked accumulation of phagosomes and other lipid/membrane inclusions, compared to age-matched control rats. We hypothesized that the RPE in this model would contain substantially elevated amounts of A2E and other bis-retinoids, relative to controls, which might contribute to the observed pathology. We tested this hypothesis in the present study.
Methods:
Sprague-Dawley rats were treated with AY9944 to generate the SLOS rat model, as previously described (Fliesler et al., Arch Ophthalmol. 2004); age-matched rats without AY9944 treatment served as controls. At ca. 10-11 wks postnatal, rat were euthanized and eyes (N=4 per group/treatment) were harvested: for biochemical analysis, eyes were flash frozen in liquid nitrogen and stored at -80oC, while for histological analysis eyes were formalin-fixed and stored at 4oC. Analysis of A2E and related bis-retinoids as well as all-trans retinal was performed by HPLC as previously described (Sparrow et al., Methods Molec Biol., 2010). Formalin-fixed, OCT-embedded eyes were cryosectioned, and frozen sections were mounted/coverslipped without staining; RPE autofluorescence was assessed by confocal scanning laser fluorescence microscopy, using 488 nm excitation and 500-600 nm emission.
Results:
Compared to controls, SLOS rat eyes exhibited the following fold-change increases (p<0.05): A2E, 1.52; isoA2E, 2.20; total A2Es, 1.71; all-trans retinal, 1.57. RPE cells in SLOS rat eyes also contained increased numbers of punctate, hyperfluorescent inclusions, compared to age-matched controls, consistent in size and distribution with phagosomes derived from ingested rod outer segment tips.
Conclusions:
RPE cells in the SLOS rat model contain elevated levels of A2E and related bis-retinoids, consistent with the observed increase in their phagosome content, compared to untreated controls. These changes may contribute to the retinal dysfunction and degeneration observed in the AY9944-induced SLOS rat model.
Keywords: 701 retinal pigment epithelium •
705 retinoids/retinoid binding proteins •
636 pathobiology