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Anne M Hanneken, Thomas Neikirk, Jennifer Johnson, Masahiro Kono; Measurements of 11-cis retinal deficiency in human autopsy eyes with macular degeneration.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5245.
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The clinical findings of delayed dark adaptation in patients with age-related macular degeneration has been proposed to be due to a deficiency in the availability of 11-cis retinal in rod outer segments. To test this hypothesis, we measured the presence of free opsin extracted from rod outer segments in human autopsy eyes from normal and macular degeneration tissues.
Human autopsy eyes were harvested within 4-12 hours postmortem under low light conditions and dark adapted in black canisters. The anterior segments were removed and the retinas were protected from light and dissected from the globes under infrared lighting. The presence, absence and stage of macular degeneration was determined by a careful examination of the RPE layer following the dissection of the retinas. Rod outer segments were extracted using the protocol of Fulton et al, IOVS, 1999, or purified over a discontinuous sucrose gradient according to standard protocols. UV-visible scanning spectroscopy was used to measure the concentration of rhodopsin before and after bleaching with 520 nm orange light. Free opsin was measured by adding an excess of 11-cis retinal to the retina extracts and incubating the sample for 20-30 minutes. Samples were rescanned and the difference in the rhodopsin absorption at 498 nm was calculated before and after the addition of 11-cis retinal.
Human autopsy eyes could be dark adapted following death when the tissue was promptly placed into black canisters following enucleation. The amount of rhodopsin recovered from the human retinas measured 5-6 nmoles under optimal conditions, in good agreement with published reports. Addition of 11-cis retinal did not result in an increase in rhodopsin in normal human retina extracts. Likewise, there was no increase in rhodopsin after the addition of 11-cis retinal in retina extracts from donors with macular degeneration.
While previous assumptions have held that the delayed dark adaptation in macular degeneration patients is likely due to a deficiency of 11-cis retinal, our studies did not reveal a deficiency of the chromophore in autopsy eyes from either normal donors or donors with macular degeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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