Abstract
Purpose :
Challenges persist in determining the molecular features of intact retina tissue. IR spectroscopy measures the appearance of vibration and absorption for atomic bonds, and advances in MAIR allow IR to be applied to nontransparent samples. We sought to identify signature peaks in the complex retina spectra and assign them to anatomic and biochemical processes.
Methods :
We obtained retina and RPE-choroid from adult albino Sprague Dawley rats for four experiments. Dissections were done in 10 min of bright light or under dim red light only. In two experiments, rod outer segments were isolated by titration and centrifugation at 15,000 xG for 5 min at 4 °C. The supernatant corresponded to the soluble interphotoreceptor matrix (IPM). Retinas, RPE-choroid, 50 ul of rod outer segment suspensions, purified outer segments and IPM supernatant were transferred to separate polyethene (PE) membranes. Sample tissue, extract and fluid were air dried on membranes to avoid a strongly absorbing H2O IR peak. We used a PE membrane negative control to distinguish its peaks from sample peaks. The samples on membranes were analyzed by MAIR-IR spectroscopy.
Results :
Washed and unwashed retina had complex spectra including peaks at 1737, 1652, 1584 and 1540 cm-1. The RPE also showed these peaks, except the peak at 1737 cm-1 was distinctly absent. Peaks at 1737 and 1550 cm-1 were associated with the pellet (rod outer segments). Peak 1584 cm-1 was associated with the supernatant (soluble IPM extract). The fluid loosely associated with the retina did not match to purified hyaluronan and its spectra was interpreted to be protein-rich vitreal or aqueous material. Dissections in light and dark revealed changes in the 3000 – 2,800 cm-1 region, which may represent released visual cycle retinoids (all-trans retinol and retinal). The 1737 cm-1 peak was not affected by light. This peak matched purified docosahexaenoic acid.
Conclusions :
Although the retina generates complex MAIR-IR spectra, assignments can be made. The 1737 and 1150 cm-1 peaks represent the rod outer segments. In contrast, the 1600 cm-1 peak represents the soluble IPM proteins. Changes in the 3000 – 2,800 cm-1 region may represent visual cycle retinoids. MAIR is a powerful approach to characterizing the retina in situ.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.