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Steven J Fliesler, Christopher C Goulah, Bruce A Pfeffer, Keiko Ueda, Janet R Sparrow; Elevated ocular A2E and bis-retinoid levels in a rat model of Smith-Lemli-Opitz syndrome. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1707.
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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.
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.
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.
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.
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