A microsystem to assay the activity of lysosomal enzymes in a small number of cultured RPE cells is described. The activities of acid phosphatase, a-mannosidase, B-glucuronidase and N-acetyl-B-glucosaminidase were estimated in different human RPE cultures of varying passages. Some biochemical characteristics for each of the enzyme assays were studied including the effect of pH, the saturating concentrations of the appropriate substrates and the relationship between the enzyme activity and the number of cells assayed. The method presented is straightforward, avoids complicated tissue fractionation procedures and is able to estimate enzyme activities in as few as 10(4) cells.
To compare the fluorescence properties of autofluorescent granules generated by retinal pigment epithelial (RPE) cells in vitro with those of the lipofuscin of RPE in vivo.Cultured human RPE cells were maintained in basal medium for as long as 1 year, fed rod outer segments (ROS) daily for as long as 56 days, fed ROS in the presence and absence of leupeptin, or fed liposomes consisting of the major phospholipids in ROS. At different time points, cells were examined for overall fluorescence, and their fluorescence spectra were determined. In addition, chloroform-methanol extracts were examined by thin-layer chromatography and compared with those generated from RPE lipofuscin.Autofluorescent granules accumulated in cultured RPE cells, regardless of the presence of an exogenous substrate or the nature of the substrate. The rate of accumulation of autofluorescent granules was greatest in cells fed ROS. The autofluorescent material generated in cultured RPE cells had some spectral similarities with RPE lipofuscin but differed in solubility and chromatographic mobility of their constituent fluorophores. CONCLUSIONS. The autofluorescent granules generated by cultured RPE, even with different specific substrates, differ from lipofuscin granules in vivo, suggesting that additional properties of RPE cells or of the materials they phagocytose are required to produce autofluorescent materials with the characteristics of lipofuscin.
Protein kinase C (PKC) is involved in both the physiological and pathophysiological processes in the retina and plays an important role in signal transduction. The aim of this study was to determine the PKC isoenzyme profile in three retinal cell types in culture, namely RPE cells, pericytes and retinal microvascular endothelial cells. Confluent cultures were lysed and isoenzyme expression detected by Western blotting. PKC isoenzymes α, β<sub>2</sub> and δ were observed for all three cell types while β<sub>1</sub> was specific for RPE cells. This study has characterised the PKC isoenzyme profile in three retinal cell types and suggests that defining the cell-specific isoenzyme pattern is an important step in understanding their precise physiological role and regulation in the retina.
This randomized, double-masked study compared the effectiveness of two commercially available ocular lubricants containing either 0.3% Carbomer 934 or 0.18% sodium hyaluronate (SH) in treating moderate dry eye.Sixty-five subjects with dry eye were recruited and supplied with eyedrops containing either Carbomer or SH to use for a month. Principle outcome measures were the severity of symptoms of ocular irritation, tear break-up time without (NIBUT) and with (TBUT) fluorescein, and corneal and conjunctival staining with fluorescein and lissamine green, respectively. At the end of the experiment, subjects were also asked, on average, how many times a day they used the treatment and the duration of any post-instillation blur.Both Carbomer and SH reduced the symptom severity and ocular surface staining, but neither had a lasting effect on NIBUT or TBUT. The treatment effects of Carbomer and SH were equivalent for symptoms, NIBUT and TBUT. However, for both corneal and conjunctival staining, SH outperformed Carbomer in improving the integrity of the ocular surface. There was no difference in the average instillation frequency of the two products. Visual disturbance after instillation of either formulation was generally short, but lengthy periods of blur were significantly more common after the use of Carbomer.Both of the eyedrops trialled are suitable for patients with moderate dry eye, but of the two, the SH-containing treatment has marginal benefits in therapeutic efficacy and has less propensity to cause visual disturbance.
Panretinal photocoagulatlon was carried out in mini pigs in an attempt to elucidate the mechanisms involved in laser therapy. The effect of the vitreous from these eyes on the proliferation of retinal microvascular endothelial cells was then studied. Vitreous removed 4 days after panretinal photocoagulation had no effect on the proliferation of bovine retinal microvascular endothelial cells in basal medium but inhibited proliferation in growth medium. Control vitreous was mitogenic for the microvascular cells in basal medium but this effect was not observed in growth medium.
Glycoconjugates are likely to be of fundamental importance in the complex interactions between photoreceptors and the retinal pigment epithelium, but few have been characterized, especially in human tissue. As a preliminary step towards determining their biological functions in health and disease, a lectin-based histochemical study of the glycan expression of human outer retina was performed on glutaraldehyde-fixed, semi-thin, resin-embedded sections. The interphotoreceptor matrix and photoreceptor plasma-lemmata expressed complex bisected and non-bisected biantennary and/or triantennary N-glycans. In addition, both the rod and cone outer segments bound strongly Galanthus nivalis agglutinin (which binds terminal Man alpha 1, 3Man-) and the rod outer segments bound selectively the isolectin II of Bandeiraea simplicifolia (which binds terminal GlcNAc-). The cilia of the rods and cones were labelled selectively with Glycine max agglutinin after sialidase pretreatment. Four putative glycan outer sequences were identified within the interphotoreceptor matrix: (i) sialylated glycans with subterminal GalNAc alpha 1,3GalNAc-sequences; (ii) a sialylated type with subterminal N-acetyl-lactosamine residues; (iii) Gal beta 1,3GalNAc alpha 1- residues which were substituted with sialic acid except in the cone matrix sheath; (iv) GalNAc alpha 1,6Gal beta 1- residues which were substituted in part with sialic acid. The sialic acid expression throughout was predominantly of the 2,3-linked form with lesser amounts of 2,6-linkage, and rod-associated structures (including the surrounding interphotoreceptor matrix) were labelled more strongly with the sialic acid-binding lectins than cone-associated structures (including the cone matrix sheath).
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
