Morphologic preservation and variability of human donor retina.

PURPOSE: To facilitate studies of human retina and utilization of human retinal tissue for treatment of retinal diseases, we studied morphologic preservation in postmortem human retina. METHODS: Morphology of retinas from thirty-one human eyes was examined using light and electron microscopy. The inner and outer retina, rod and cone photoreceptor cells, and central and peripheral retina were compared with regard to morphologic preservation. Possible factors affecting survival were analyzed. RESULTS: The earliest postmortem change was vacuolation of the nerve fiber layer within a few hours postmortem, followed by vacuolation and cytoplasmic swelling of the inner retina. As compared with the inner retina, outer retinal structure was better preserved, i.e., the photoreceptor cells maintained better morphology. Rod cell morphology was better preserved than cone cell morphology, with good preservation of the rod outer segment disc membranes and the inner segment mitochondrial membranes. Thus, well-preserved rod photoreceptor cells were evident in specimens at least 48-hours postmortem. Peripheral retina was better preserved than the central retina including the fovea and perifovea. Factors affecting anatomical integrity included the total time postmortem and, more importantly, the time between death and enucleation. Other factors, including age and sex, did not appear to affect morphological preservation in the present study. CONCLUSIONS: Human retina postmortem remained morphologically intact for a relatively long period of time, with differential preservation among different geographic areas and cell types. This morphologic evidence is consistent with previous findings of functional preservation (e.g. , photoresponses) in such tissue. This study may shed some light on understanding of human retina and its utilization for retinal transplantation.

Survival of cone responses in postmortem human retina.

A cone-mediated electroretinogram was recordable from human retinas 17-18 hours postmortem, after regeneration of visual pigments by application of 11-cis and 9-cis retinal. The cone was separated from the rod component by stimulation with flickering red light. In addition to these neuronal responses, a P2 and a slow P3 were present.

Species differences in the response of mammalian photoreceptor cyclic GMP and PIII to a reduction in calcium.

Changes in the content of cyclic GMP (cGMP), induced by exposure of isolated, dark-adapted mouse, cat and dog retinas to media depleted of calcium, have been compared with the amplitude of the trans-retinal PIII. Major differences exist in the time-course and magnitude of effects between the species and, in the cat and dog, changes in PIII (potentially a reflection of free cGMP in photoreceptor outer segments) do not correlate with those occurring in total cGMP. The observations imply species variation, not only in the enzymes maintaining cGMP homeostasis in photoreceptors, but also in phototransduction and allied processes.

Cyclic GMP, calcium and photoreceptor sensitivity in mice heterozygous for the rod dysplasia gene designated "rd".

The rise in photoreceptor cGMP, induced by less than 1.0 nM extracellular calcium, is delayed in retinas of mice heterozygous for the rod dysplasia gene (+/rd). The calcium ionophore A23187 reduces the delay, suggesting that +/rd outer segments contain more calcium than normal. In turn, this might explain the increased photosensitivity of the +/rd retina. During the response to low calcium there is no correlation in +/rd retinas between the total concentration of cGMP and the photoresponse amplitude and its time to peak. The observations imply that either free cGMP is abnormally independent of the bound pool in the +/rd photoreceptor outer segment or that factors other than cGMP and its phosphodiesterase are modulating the rising phase of the response. The time-to-peak of PIII in a +/rd retina, incubated in a standard medium and stimulated with dim light, is abnormally delayed. Reduction of extracellular calcium induces an abnormal delay as well in responses to higher light levels. In addition to this, a second delay manifests slowly in both the normal and the +/rd retina. More studies are needed to explain these observations.

Non-competitive NMDA-receptor antagonists and anoxic degeneration of the ERG B-wave in vitro.

Studies have been undertaken to see if the non-competitive NMDA antagonists, ketamine, MK-801 and dextromethorphan would preserve the b-wave of the electroretinogram (ERG) in vitro. The drugs had no effect on the ERG b-wave, nor prolonged its survival postmortem. The present results support previous evidence suggesting that NMDA-receptors are not involved directly in synaptic transmission between photoreceptors and ON-bipolar cells. Further, loss of the b-wave in post-mortem anoxia does not appear to be mediated via NMDA-receptors.

Electrophysiologic characteristics of human and rat retinas in vitro.

To promote studies on the human retina, we investigated the survival of function in postmortem specimens. Visual pigment has been regenerated in normal human retinas, 5 to 58 hours postmortem, by exposure to retinal isomers in the dark. Levels from 0.1 to 0.41 nmol/mg protein were reached. Photoresponses were obtained in 9 of 13 retinas: P III maximum amplitudes ranged from 20-398 microV and thresholds, taking the criterion amplitude as 3 microV, ranged from 8.8-1340 quanta/micros2. In three cases, the b-wave was also seen. The P III amplitude vs. log intensity curves gave values of n between 0.6 and 1.0, and sigma (the stimulus intensity for a half maximal response) between 132-3700 quanta/microns2. Recovery of sensitivity did not always correspond to that of maximum response.

Evidence for reduced binding of cyclic GMP to cyclic GMP phosphodiesterase in photoreceptors of mice heterozygous for the rd gene.

The binding of radiolabelled cGMP to rod outer segment proteins has been investigated in mice, heterozygous for the recessive rd gene that leads to rod dysplasia. Two binding sites were detected, by Scatchard analysis, in a crude cGMP phosphodiesterase fraction, extracted with an EDTA wash from outer segments. Affinities were normal but the capacity of both was reduced 25-35%. Photoaffinity labelling with 3H-cGMP, followed by SDS PAGE and fluorography, suggested that cGMP PDE was the principal binding component in the extracts. If the finding reflects cGMP binding in situ, it might explain the 30-40% lower than normal level of cGMP found in the +/rd retina. Visual pigment has been regenerated in isolated normal and heterozygotic retinas by the application of active isomers of cis-retinal, and the time course of cGMP recovery to 'dark-adapted' levels monitored. The increase in the concentration of cGMP was significantly delayed as compared to that of rhodopsin. No differences in time course or kinetics of recovery were discerned between the two genotypes.

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

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.

Cyclic GMP in the retinas of normal mice and those heterozygous for early-onset photoreceptor dystrophy.

Cyclic GMP metabolism has been investigated in the retinas of mice that are heterozygous for a 'photoreceptor dystrophy' gene and have a lowered concentration of cGMP in their photoreceptor cells. The concentration of rhodopsin, retinal morphology and guanylate cyclase kinetics were normal. Cyclic GMP phosphodiesterase had a lowered affinity for cGMP. In accord with previous observations, chelation of exogenous calcium had no effect on cGMP levels in light-adapted retinas but increased them in dark-adapted tissue. The difference between cGMP concentrations in heterozygous and normal retinas in the dark was then eliminated. It was concluded that a modulator of cGMP phosphodiesterase activity is most likely to be causing the lowered steady-state level of cGMP in heterozygous retinas and that calcium is not involved.

Studies on retinitis pigmentosa in man. II. Erythrocyte osmotic fragility.

The osmotic fragility of erythrocytes from patients with genetically classified forms of retinitis pigmentosa (RP) has been studied. The mean fragility was increased in autosomal dominantly inherited RP, where the dystrophy was expressed regionally in the retina, with both rods and cones affected. In contrast it was normal in patients with the dominantly inherited disease, which leads to a diffusely distributed dystrophy of, predominantly, rod photoreceptor cells. Raised osmotic fragility of erythrocytes has also been observed in female patients with multiplex (recessive) RP and in female carriers of the X-linked form of the disease.

Photochemical damage in the albino rat retina: oral taurine has no effect on DNA and protein loss from severely damaged photoreceptor cells.

Albino Wistar rats were exposed continuously to fluorescent light, causing photoreceptor destruction over a period of 4 days. The rate of DNA and protein loss from the cells was not affected by administration of 5% taurine in the drinking water.

Studies on retinitis pigmentosa in man. I. Taurine and blood platelets.

Platelets from patients with various genetically determined forms of photoreceptor dystrophy and with the clinical manifestations of retinitis pigmentosa (RP) have been studied. Variations in protein content have been observed, with less than normal in multiplex RP (probably autosomal recessive inheritance) and more in platelets from patients with autosomal dominant RP. This may reflect variation in platelet size or in surface adsorption of plasma proteins. Several patients presented with thrombocytopenia, the mean platelet count for X-linked hemizygote patients, as a group, being significantly lower than normal. Accumulation of 3H-taurine has been studied in platelets incubated in Ca2+-free Krebs bicarbonate medium containing 1.0 microM or 60.0 microM taurine, and in autologous plasma. Although, in general, platelets from patients with RP showed normal taurine uptake, the capacity of the higher affinity carrier was increased in patients with X-linked hemizygote and multiplex disease. In contrast, plasma from patients with X-linked hemizygote RP reduced the platelet tissue to medium ratio, established for 3H-taurine uptake, by 20%. More studies are needed to ascertain whether this represents a reduced taurine uptake or is caused by an increased concentration of taurine in the plasma.

Photochemical damage in the albino rat retina: morphological changes and endogenous amino acids.

Endogenous amino acids were measured in retinas of rats exposed for up to 48 h to fluorescent light. Typical light damage was seen in photoreceptor cells after 30 h exposure to a maximum luminance of 1544 scotopic lux; and, from this time, taurine levels were significantly reduced. In contrast, the concentrations of other amino acids increased. After 18 h exposure to light, GABA, glycine, glutamate, and aspartate levels were raised in the photoreceptor cells, and GABA, glutamate, and glutamine levels in the inner retina. When 'exposed' animals were returned to their normal environment for 72 h, photoreceptor degeneration progressed and taurine concentrations were further reduced: the results suggest that the loss was from damaged photoreceptor cells. At this time the concentrations of the other amino acids measured had, in general, returned to normal.