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Priscilla Gonzalez-Fernandez, Dongjin Sung; Retinal Degeneration in IRBP(-/-) Mice, and Patterns of Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4768.
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The mechanism of retinal degeneration in transgenic mice lacking interphotoreceptor retinoid-binding protein (IRBP -/-), or in humans with an RP associated IRBP mutation is largely unknown. IRBP, which is the most abundant soluble protein component of the interphotoreceptor matrix, appears to have a complex poorly understood role in the translocation of hydrophobic molecules particularly 11-cis and all-trans retinol, and 11-cis retinal during the visual cycle. Its role in protecting the oxidative state of retinol in this cycle may be a particularly important function of the protein. We hypothesized that lack of IRBP in transgenic mice leads to photoreceptor degeneration by enhancing oxidative stress in the outer retina compared to the inner retina.
IRBP (-/-) mice, and C57BL/6 age matched controls were maintained from birth under cyclic dim light. Eyes were fixed in 4% paraformaldehyde from p5 to p80. The eyes were sectioned at 5 microns in a vertical orientation. Nuclear cell layer counts were made at 6 locations evenly distributed on both sides of the optic nerve. The distribution of 4-hydroxynonenal (HNE) was determined by avidin-biotin immunohistochemistry using mouse monoclonal HNEJ-2 at a 1:20 dilution. Pre-adsorbed controls utilized HNE-ovalbumin. Successful conjugation of HNE to the ovalbumin was confirmed by Western blot analysis. Three month-old albino mice were exposed for 24 hrs to intense green light (1700 lux, 490-580 nm) as positive controls.
Between p5 and p20 the ONL thickness decreased by 30% and 20% for IRBP(-/-) and C57BL/6 mice respectively. In C57BL/6, little further decrease in retinal thickness was seen over the next 2.5 months (5 -10%). In contrast, the ONL thickness in IRBP (-/-) mice decreased by 35% over the same period. The thickness of the INL was unaffected. In p20 IRBP (-/-) mice, staining for HNE was prominent in the outer and inner segments, and lens with little effect on the nuclear or plexiform layers. Age matched C57BL/6 mice showed significantly less staining compared to the IRBP(-/-) animals. In light damaged albino retinas, the distribution of immunospecific HNE staining was associated with the ONL, inner plexiform and ganglion cell layers, and not the lens or the photoreceptor inner and outer segments.
Absence of IRBP primarily affects the ONL with sparing of the INL and ganglion cells. Reduction in ONL thickness was biphasic with an initial reduction in between p5 - p20 in both strains but more so in the IRBP (-/-) mice. A further profound degeneration occurred in the IRBP(-/-) retina over the next 2.5 months. Increased HNE deposition in the inner/outer segments suggests that absence of IRBP increases oxidative stress through a different mechanism(s) than in light toxicity.
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