Huang, Jun Chuan; (1990) A study of structure and function in postmortem human retina. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of the present study was to investigate structure and function in postmortem human retina and to evaluate systems for prolonging survival in vitro. The main factors affecting survival were total time postmortem and the time between death and enucleation of the eye. Limited sample size precluded analysis of potential changes with donor age. Histological studies on 31 human eyes indicated that, in the periphery, photoreceptor and inner retinal structure were reasonably preserved for at least 2 to 3 days postmortem, although some vacuolation was present. In contrast to the periphery, the fovea and perifovea were always poorly preserved. Function of human retinae was assessed under three categories: photochemistry, electrophysiology, and biochemistry. To optimise conditions for restoring phototransduction, pilot experiments were undertaken on rat tissue. Freshly isolated, bleached rat retinae were incubated in the dark at 37°C for 60 minutes in Earle's medium containing 500 uM of a mixture of retinal isomers encapsulated in liposomes. This procedure induced recovery of rhodopsin to 91%, cyclic GMP to 75% and PIII amplitude to 68% of the values obtained for dark-adapted retina. Human retinae 5 to 58 hours postmortem were incubated in fortified Eagle's medium. Dark-adapted values could not be obtained for human retina. Rhodopsin was regenerated from zero to 0.1 - 0.41 nmol/mg protein, and the regeneration rate was about twice as fast as that in the rat. Cyclic GMP increased from 15.5 - 18.8 to 23.5 - 49.2 pmol/mg protein. Photoresponses were obtained in nine out of thirteen retinae: PIII maximum amplitudes ranged from 20 - 398 uV, and thresholds from 8.8 - 1340 quanta/um2. In three cases, b-waves were also seen. Studies on high-affinity uptake of taurine, glycine and GABA by five isolated human retinae 5 to 36 hours postmortem showed that taurine was accumulated predominantly by photoreceptor and possibly bipolar cells, glycine by cells located in the inner nuclear layer, and GABA by Muller cells. Protein synthesis has been investigated in seven human retinae 5 to 48 hours postmortem. Incorporation of 3H-leucine into proteins and specifically into opsin was followed. Incubation in the fortified medium resulted in an increase in opsin and total protein synthesis, and a concurrent reduction of retinal vacuolation. Autoradiography revealed that in the fresh retina general protein synthesis was occurring in both neurones and glia, whereas, at longer postmortem times it was predominantly in Muller cells. The studies have shown that human retinae and, in particular, rod photoreceptor cells survive reasonably well postmortem. Some improvements in structure and metabolism have been obtained in short-term explant culture, and it is likely that more could be achieved.

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