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Lowell G. Sheflin, Joyce E. Young, Bruce A. Pfeffer, Steven J. Fliesler; Association of the Complement Pathway with Retinal Degeneration in the AY9944-Induced Rat Model of Smith-Lemli-Opitz Syndrome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1007.
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Microarray analysis has demonstrated marked alteration of the retinal transcriptome during progressive retinal degeneration in the AY9944-induced rat model of Smith-Lemli-Opitz syndrome (SLOS), a disease involving defective cholesterol biosynthesis. One of the up-regulated genes was complement component C3, a key player in both the "classical" and "alternative" immune system pathways. Here, we evaluated retinal C3 expression in SLOS rats fed a cholesterol-free vs. high (2%)-cholesterol diet, and also localized C3 in normal and SLOS rat retinas.
The SLOS model was generated by treating Sprague-Dawley rats with AY9944, a cholesterol pathway inhibitor, as previously described (Arch. Ophthalmol. 122:1190, 2004); untreated age-matched rats served as controls. Retinas were harvested at 2-3 months; total RNA and protein were isolated by TRIzol® extraction. Rat-specific C3 primers were used for qRT-PCR. Western blot analysis was performed on equal amounts of retinal protein, probing with anti-rat C3 polyclonal antibody; immunoreactive bands were quantified using a StormTM Imager. Indirect immunofluorescence localization of C3, as well as Iba1 (microglia/macrophage marker), was performed on sections from treated and control retinas.
qRT-PCR and Western analysis validated the up-regulation of C3 mRNA and protein expression in SLOS rat retinas, vs. controls. Feeding SLOS rats a high-cholesterol diet significantly decreased the levels of C3 transcript and protein in retinas, vs. those on a normal diet. C3 immunoreactivity was localized to RPE/choroid and retinal endothelial cells; while no differences in C3 immunoreactivity were noted, Iba1-positive cells were found in the photoreceptor layer of treated, but not control, retinas.
Retinal degeneration in the SLOS rat model appears to involve recruitment of the immune system, via up-regulation of complement C3 and microglia/macrophage migration. Feeding a high-cholesterol diet (the standard of care for SLOS management) suppresses C3 up-regulation. Thus, immunomodulation may be an important factor in the pathophysiology and clinical management of SLOS.
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