Survival of structure and function in postmortem rat and human retinas: rhodopsin regeneration, cGMP and the ERG

Abstract

Procedures for regenerating visual pigment and restoring phototransduction have been established with freshly isolated, bleached rat retinas. Phosphatidyl choline liposomes containing a 500 microM mixture of retinal isomers, including the 9-cis and 11-cis forms, were employed and the results compared with dark-adapted retinas, incubated similarly but without retinal. The following were recovered in a 60 min incubation, rhodopsin (plus isorhodopsin) to 91% of the original rhodopsin concentration, 87% of cGMP and 89% of PIII amplitude at saturation. PIII amplitude vs. log intensity curves gave values of n between 0.6 and 1/2.0 and sigma between 85 and 439 quanta/micron 2. Human retinas, ranging from 18 to 58 hours postmortem and treated as above, also produced photoresponses. Of the 7 retinas studied so far, rhodopsin has been regenerated to 0.1-0.35 nmol/mg protein, cGMP to 23.5-49.2 pmol/mg protein, and PIII to 20-50 microV: in some cases a b-wave was also seen. Values of n varied between 0.6 and 1.0, and sigma between 132 and 3700 quanta/micron 2. PIII responses were also seen after retinas, approximately 30 hours postmortem, were incubated for a further 24 hours in fortified medium. After incubation, retinal vacuolation was reduced.

Electrophysiologic characteristics of human and rat retinas in vitro

“…In line with previous observations in retinas 18-43 hours postmortem [2], Table 2), and its PIII amplitude vs. log intensity curve. The retina was incubated with a 500/~M photoisomerate of retinal for 60 minutes (see text).…”

“…In these studies, the choice of a 60-minute exposure to retinal to regenerate visual pigment in the human retina was based on preliminary findings with mouse and rat tissue [1,2]. However, further experiments have now shown that the maximum level of regenerated rhodopsin is more likely to be attained after about 30 minutes, compared with about 60 minutes for the rat (Fig.…”

“…The media, containing 100#M l l-cis retinal or 500#M retinal photoisomerate, were gassed throughout and gently shaken. In preliminary studies [2], we found that 9-cis plus 11-cis retinal constituted 27 _ 2% of a photoisomerate, as estimated by HPLC. Also, the efficiency of this preparation in inducing an increase in the concentration of cGMP and the photoresponse in the rat retina paralleled its content of active isomers and, on this basis, appeared approximately equivalent to that of pure 11-cis retinal [2].…”

“…In the present report we summarize our electrophysiologic findings. Previous reports have appeared [1,2]. Pilot studies also have been undertaken on the effects of medium composition on the survival of the rat electroretinogram postmortem.…”

Dietary 20:4n-6 and 22:6n-3 Modulates the Profile of Long- and Very-Long-Chain Fatty Acids, Rhodopsin Content, and Kinetics in Developing Photoreceptor Cells

“…The value for rhodopsin at 6 wk of age lies within the range of values for whole eye rhodopsin level reported in the literature for similar body weights (44). The level of rhodopsin at 9 wk of age is between 1.5 and 1.7 nmol/mg protein (submitted for publication), implying that the retinas used in this experiment are still developing.…”