Carolyn A. Converse

Pathology of Hereditary Retinal Degeneration Associated with Hypobetalipoproteinemia

BACKGROUND The clinical features and previously unreported ocular pathology in a case of heterozygous hypobetalipoproteinemia (HBL) associated with a pigment epitheliopathy are documented. Night blindness developed in a white woman with familial heterozygous HBL (cholesterol and low-density lipoprotein levels < 5% of normal) at 51 years of age. Ophthalmoscopy showed bilateral symmetric depigmentation at the posterior pole with pigment clumping and pavingstone configuration in the periphery. By the time the patient died, at 75 years of age, vision had deteriorated to hand motions. METHODS One eye was removed 2 hours postmortem for light and electron microscopic study. RESULTS The photoreceptors were absent, and the outer nuclear layer was replaced by glial cells throughout most of the retina, but there was some focal photoreceptor preservation in isolated regions. The outstanding feature was a massive deposition of basal linear deposit which was calcified in segments and which contained macrophages and the processes of glial cells: trilaminar bodies and melanin granules were identified in the macrophages. The remaining retinal pigment epithelial cells contained melanin but very little lipofuscin: intraretinal migration was minimal. CONCLUSIONS The authors postulate that the pigment epitheliopathy associated with HBL is an abiotrophy in which photoreceptor discs are unable to regenerate due to locally disordered metabolism resulting from or acting in concert with the pigment epitheliopathy.

Stabilization of All-trans-retinol by Cyclodextrins: A Comparative Study Using HPLC and Fluorescence Spectroscopy

Purpose: To formulate preparations incorporating cyclodextrins (CDs) which could be used for direct delivery to the retina of vitamin A (all-trans-retinol), while also protecting it from degradation in the aqueous environment. Vitamin A supplementation is being considered for treatment of several ophthalmic diseases characterised by progressive photoreceptor degeneration. Methods: The complexation between vitamin A and ten cyclodextrins, Captisol®(sulfobutyl ether-7-β-cyclodextrin), hydroxypropyl-β-CD, 2-hydroxypropyl-β-CD, α-CD, hydroxypropyl-γ-CD, hydroxypropyl-α-CD, β-CD, methyl-β-CD,Heptakis-(2,6-di-O-methyl)-β-CD andHeptakis-(2,3,6-tri-O-methyl)-β-CD,was investigated using bothhigh sensitivity fluorescence spectrometry and HPLC (high pressure liquid chromatography). Samples of retinol-CD complexes in phosphate buffer, pH 7.4 were analysed for up to 72 hours. Optimum conditions for formation of the Captisol-retinol complexes were investigated. Results: Using spectroscopic measurements and HPLC, the complexes formed between ten cyclodextrins and all-trans-retinol were evaluated. The results indicate that all cyclodextrins tested were able to form inclusion complexes as shown by the fluorescence signals which are considerably larger than those obtained in the absence of cyclodextrin. Only minimal degradation of retinol over 48 hours was observed with three of these cyclodextrins. Captisol was able to stabilise all-trans-retinol for up to 72 hours, as shown by HPLC, and the optimum ratio of Captisol to retinol was determined to be 50 to 1. Addition of glutathione and decrease in pH did not improve stability of the complex. Conclusions: This survey suggests that Captisol and other cyclodextrins could be used to stabilise and solubilise vitamin A in aqueous media and this establishes the basis for an ocular Captisol-retinol drug delivery system now under development in our laboratory.

Comparison between oleic acid and docosahexaenoic acid binding to interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP), which binds retinoids and fatty acids, is the major soluble protein in the interphotoreceptor matrix (IPM) but its role remains ambiguous. Using competitive fluorescence and tryptophan-quenching assays we found oleic acid and other cis-monounsaturated fatty acids bind much more strongly than does docosahexaenoic acid to bovine IRBP. IPMs fatty acid composition was determined: it was richer in oleic acid than either the retinal pigment epithelium or rod outer segments. This may imply oleic acid has a key role in the balance and transport of retinoids and fatty acids in the retina.

Photosensitized light-induced damage of IRBP (interphotoreceptor retinoid-binding protein): Effects on binding properties

Purpose. To determine if IRBP (interphotoreceptor retinoid-binding protein) is damaged following irradiation by visible light in the presence of bound all- trans retinal. Methods. Following irradiation of the IRBP-all- trans retinal complex, the retinal was removed and damage to IRBP measured as loss of titratable thiol groups, loss of tryptophan fluorescence, and changes in retinol-binding-induced fluorescence. Results. IRBP irradiated by itself showed only minimal loss of tryptophan fluorescence; this loss was substantially in-creased by irradiation in the presence of all- trans retinal. Thiol groups and retinol-binding activity were also shown to be reduced. The damage to IRBP seemed to involve photosen-sitization by the all- trans retinal, which was in turn protected from bleaching by the IRBP. The binding affinity was shown to be reduced ten-fold following irradiation. Conclusion. In the eye, IRBP can stabilise vitamin A and debatably may be responsible for transport of different forms of vitamin A between the photoreceptor cells and pigment epithelium. If this is the case, it would play a key role in rhodopsin regeneration after bleaching. IRBP also appears to be necessary to sustain photoreceptor cells. Light was shown to cause photosensitized damage to IRBP, and thus might impair the regeneration process and photoreceptor viability.

The large intrinsic membrane protein in rod outer segments: in vitro synthesis in cattle, and comparison in humans and rabbits.

Abstract Throughout life, vertebrate photoreceptor discs are continually being renewed at the base, and discarded from the tip, of the outer segment. In the frog, in vivo studies indicate this renewal process includes both rhodopsin and a large intrinsic membrane protein, which is found on the margins and incisures of discs. In this paper, I examine the same events in the cattle retina in vitro, and show there is a similar large intrinsic membrane protein in cattle, which is rapidly synthesized and incorporated into rod outer segment preparations. Human and rabbit photoreceptor outer segments are also shown to possess a large intrinsic membrane protein.

Effect of glycoconjugates on rod outer segment phagocytosis by retinal pigment epithelial explants in vitro assessed by a specific double radioimmunoassay procedure

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

An increased incidence of apolipoprotein E2/E2 and E4/E4 in retinitis pigmentosa

Previous studies from our laboratory have shown that retinitis pigmentosa (RP), a family of hereditary retinal degenerations, is often accompanied by abnormal levels of cholesterol or polyunsaturated fatty acids. The requirement of the retina for n−3 fatty acids is well known, and a defect in the supply of these lipids (e.g., by apolipoproteins) could affect the course of the disease. The present study confirms and extends a report on apolipoprotein E (apo E) isoforms in German RP patients [Jahn, Oette, Esser, Bergmann, and Leiss, (1987)Ophthalmic Res. 19, 285–288] which showed a tenfold increased frequency of the E2/E2 phenotype compared to the average German population. In our study, apo E phenotypes were determined in the probands of 100 Scottish RP families. The findings revealed a 4-fold increase in the incidence of E2/E2 and an 8-fold increase in E4/E4 compared to a Scottish control population. These increases were statistically significant at theP<0.05 andP<0.01 levels, respectively. To investigate the possibility that some of these apparent E2/E2 or E4/E4 phenotypes might actually be new apo E mutations, we examined the behavior of the apo E on sodium dodecyl sulfate-polyacrylamide gels (E2 migrates anomalously) and on isoelectric focusing gels following cysteamine modification of cysteines. These studies showed that two RP patients possibly had new apo E mutations, though amino-terminal sequence analysis revealed no changes in the sequence of the first 19 residues; further sequence analysis is obviously warranted.

Short-term organ culture of the retinal pigment epithelium in microtitration plates: ultrastructural studies

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2–3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.

Specific radioimmunoassay to investigate rod outer segment phagocytosis by retinal pigment epithelium in vitro.

A sensitive radioimmunoassay has been developed which allows rapid quantitation of rod outer segment (ROS) phagocytosis by retinal pigment epithelial (RPE) explants in vitro. It involves the use of an antiopsin antiserum, in conjunction with 125I-protein A as a second antibody, and utilizes permeabilization with ethanol to distinguish between the binding and ingestion phases of phagocytosis. This procedure will be used in the future to investigate potential regulatory factors of ROS phagocytosis by retinal pigment epithelium and to evaluate animal models of retinal degeneration.

D-[3H]-galactose incorporation in the bovine retina: specific uptake and transport of the radiolabel in cones.

When bovine neural retinas are incubated in Krebs-Henseleit buffer with D-[3H]-galactose, autoradiography reveals that there is a rapid uptake of the tritium label into the inner segments of cones, but not of rods. Pulse--chase studies show that the label is first associated with the Golgi apparatus in the cones, then appears to travel around the nucleus and along the cone fibre (homologous to an axon) to the synaptic pedicle. The cone-specific label travels along the fibre at a rate of about 0.5-1.0 mm per day. Label is also found in endothelial cells and Müller cells, but does not persist in the Müller cells as long as in the cones. The striking difference between rod and cone labelling may reflect fundamental differences in the neurochemistry of these two photoreceptor cell types.