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PW Goletz, B Rohrer, J-X Ma, RK Crouch; Correlation of Regenerable Opsin with Rod ERG Signal in Rpe65-/- Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4543.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:RPE65 has been shown to be an essential component for the production of 11-cis retinal in the visual transduction cascade. Mutations of RPE65 are known to be associated with severe forms of early-onset retinal dystrophy. This project was designed to examine the amount of regenerable opsin in RPE65 knockout (Rpe65-/-) mice at various ages, and to compare it to the amounts of rod electroretinography (ERG) signals in C57BL/6 and Rpe65-/- mice. Methods:All experiments were performed on dark-adapted mice (1, 4, and 13 mo-of-age). Maximally recoverable amounts of rhodopsin after the addition of 11-cis (rhomax) were measured in dodecyl-maltoside solubilized retinal extracts using absorption difference spectrophotometry. A- and b-wave ERG amplitudes were recorded using scotopic single-flash electroretinography. Results: Through 4 mo-of-age, amounts of regenerable opsin in Rpe65-/- mice were comparable to that of both C57BL/6 and BALB/c mice; however, at 13 mo-of-age the level of regenerable opsin in the Rpe65-/- dropped relative to that of the other two strains. No endogenous levels of rhodopsin were measurable in the Rpe65-/- mice (detection limit: 0.225 pmol). Unlike rhomax, ERG parameters deteriorated in young adult Rpe65-/- mice. At 1 mo the ERG consisted of both a- and b-waves, whereas in older mice, only a b-wave could be recorded. Using the b-wave as a readout of rod function, we determined that the b-wave amplitudes dropped continuously from their maximal levels of 112.96 ±15.5 µV (mean ±SEM) at 1 mo, to 60.8 ±10.75 at 4 mo, and then to 49.7 ±15.7 µV at 13 mo. Age-matched wild type mice showed very little change in a- and b-wave amplitudes during aging. Conclusion:In the Rpe65-/- mouse, the largest ERG signal deterioration occurs between 1 and 4 mo-of-age; yet, rhomax remains unchanged. This suggests that the decrease in retinal function is due to a lack of chromophore. The decrease in regenerable opsin levels at 13 mo-of-age in the Rpe65-/- relative to the wild type animals suggests either there is less opsin present, or that the opsin has been structurally modified thereby rendering it nonfunctional.
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