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A longitudinal study of the effects of retinal ablation on the organization of the retinal target lamina of the optic tectum in the teleost, Rutilus rutilus.

The optic tecta of 55 Rutilus rutilus were examined at intervals varying from 2 days to 4 years after unilateral retinal ablation. Qualitative ultrastructural examination of the retinal target lamina of the optic tectum (stratum fibrosum et griseum superficiale, SFGS) revealed that an initial period of degeneration and glial reaction, each of which could take one of a variety of forms and which lasted for 1-3 months after ablation, was followed by the temporary formation of heterologous synapses which persisted for a further 1-12 months. This in turn was followed by the degeneration of these synapses during the second year after ablation. Quantitative analysis at the level of the light microscope revealed a shrinkage of the SFGS throughout the level of the light microscope revealed a shrinkage of the SFGS throughout the first 14 postoperative months with no further reduction taking place thereafter. Analysis at the ultrastructural level revealed that this shrinkage was due to the disappearance, and not to the reduction in size, of pre- and postsynaptic profiles accompanied by glial reaction. No signs of collateral sprouting were seen throughout the survival period. Thus, partial deafferentation of the SFGS leads in the long run to a marked impoverishment of its neuronal network, without any apparent compensation.

Animals↗

Possible involvement of cholinergic and glycinergic amacrine cells in the inhibition exerted by the ON retinal channel on the OFF retinal channel.

In the frog retina, the inhibition exerted by the ON channel on the OFF channel was evidenced by the increase in transient ganglion cell OFF responses, when the ON channel was blocked by 2-amino-4-phosphonobutyrate (APB). Intraocular administration of the neurotoxic choline analog ethylcholine mustard arizidinium ion (ECMA) also provoked an increase in the number of spikes of transient ganglion cell OFF responses, without suppressing the ON responses. APB, when administrated after ECMA, abolished the ON responses, but did not modify the OFF responses already increased by ECMA. Neurons located in the inner part of the inner nuclear layer were histologically altered by the toxin, and choline acetyltransferase activity was significantly depressed in ECMA-treated retinas. A double immunostaining experiment showed that amacrine cells containing glycine bear muscarinic binding sites. These results confirm the participation of cholinergic neurons in the inhibition exerted by the ON retinal channel on the OFF retinal channel, and suggest the involvement of a cholinergic/glycinergic loop of amacrine cells in this mechanism.

Acetylcholine↗

Antibodies against a retinal guanine nucleotide-binding protein cross-react with a single plasma membrane protein in non-retinal tissues.

Antisera (AS/1-AS/6) to purified bovine retinal transducin, a guanine nucleotide-binding protein, were produced in 6 rabbits. Immunoblots showed that the antisera varied in their reactivity with the subunits of transducin; AS/1 reacted strongly with all 3 subunits, while the others reacted with only the beta and/or gamma subunits. Only AS/1 specifically immunoprecipitated the alpha subunit radiolabeled with non-covalently bound guanine nucleotides. Immunostaining of plasma membrane proteins from non-retinal tissues with AS-1 revealed a single protein (approx. 35 kDa), most likely representing the beta subunit of the guanine nucleotide-binding proteins (Gs and Gi) associated with adenylate cyclase. Cerebral cortex showed the highest content of this protein. Antisera against transducin provide a highly specific and sensitive probe for quantitation of the beta subunit of Gs and Gi.

Animals↗

Effect of sodium fluorescein on neurite outgrowth from the retinal explant culture: an in vitro model for retinal toxicity.

Effect of sodium fluorescein was examined on neurite outgrowth from the retinal explants of chick embryos cultured with chicken gizzard extract, containing macromolecules which promote neurite outgrowth. A dose more than 0.5 mg/ml of fluorescein completely prevented the neurite outgrowth from the retinal explants. The minimum dose of toxic effect of fluorescein on the neurite outgrowth was about 0.2 mg/ml. The fluorescein also caused lysis of pre-existing neurites which had been fully extended by gizzard extract.

Animals↗

Plasmalogens in the retina: in situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT)--the first enzyme involved in their biosynthesis--and comparative study of retinal and retinal pigment epithelial lipid composition.

Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond in the sn-1 position of the glycerol backbone. The physiological role of Pls is still enigmatic, especially within the eye where their deficiency leads to developmental abnormalities. In order to learn more about the functions of Pls in the posterior eye, we evaluated retinal Pl content as well as the expression of the first enzyme involved in Pls biosynthesis, dihydroxyacetone phosphate acyltransferase (DHAP-AT) in the retina. In situ hybridization of DHAP-AT mRNA was performed on rat eye sections. The Pl contents of calf retina and retinal pigment epithelium (RPE) samples were determined by high-performance liquid chromatography, thin-layer chromatography, and gas chromatography. DHAP-AT was highly expressed in the inner segment of photoreceptors and in the RPE, suggesting two distinct sites for Pl biosynthesis. Plasmenyl-ethanolamine was the prominent class of Pls in both neural retina and RPE (28-29% of the total phospho-ethanolamine-glycerides). According to the nature of the alkenyl residue linked to the sn-1 position of Pls, the most striking finding was the greater proportion of octadecanal-aldehyde in the sn-1 position of plasmenyl-ethanolamine of the neural retina compared to all the other classes of Pls in the neural retina and the RPE. These findings might be relevant to the biological functions of Pls against oxidative stress and in the formation of lipid rafts.

Acyltransferases↗

9-cis-retinoic acid in combination with retinal pigment epithelium induces apoptosis in cultured retinal explants only during early postnatal development.

Retinoic acid is one of the active metabolites of vitamin A and has profound effects on the development of the CNS including retina. Previously, we have shown that rod-specific apoptosis is induced in retinal explants from neonatal mice by exposure to 9-cis-retinoic acid (9CRA) when the retinal pigment epithelium (RPE) is present. In explants lacking RPE, it instead has a differentiation-promoting effect seen as an accelerated opsin expression on postnatal day 3. To investigate the long-term effect of 9CRA exposure, we have explanted retinas from neonatal C3H mice with or without RPE attached and placed in organ culture. After 19 or 48 h in culture or 7, 8 or 13 days in culture, the explants were either fixed for histochemical examination or frozen for assay of DEVDase activity. We found that long-term exposure to 9CRA caused a decrease in the number of cell layers in the outer nuclear layer (ONL) only in explants with the RPE attached. When explants with RPE attached were exposed to 9CRA only during the second postnatal week, neither an increase in DEVDase activity, TUNEL-positive cells, nor a decrease in cell layers of the ONL could be demonstrated, indicating that the retina was insensitive to the apoptosis-inducing effect of 9CRA after the first postnatal week. The absence of RPE in control explants resulted in a higher number of rosettes and the extrusion of cells into the subretinal space.

Aging↗

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells.

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Animals↗

Immunization with the cross-reactive antigens Ov39 from Onchocerca volvulus and hr44 from human retinal tissue induces ocular pathology and activates retinal microglia.

Antigen Ov39, derived from Onchocerca volvulus, cross-reacts on both the T and B cell level with a nonhomologous human retinal antigen, hr44. Lewis rats were immunized to investigate the potential of these antigens to induce eye disease. Histologic and immunohistologic examination of ocular tissues revealed pathologic changes as early as day 12, which included induction or up-regulation of class II and CD68-like antigen on perivascular cells, ramified retinal microglia, dendritiform cells of the iris epithelium, and ciliary epithelium and significant breakdown of anterior and posterior blood-ocular barriers. Extravascular immunoglobulin and staining for CD68-like antigen was detected in the optic nerve after immunization with Ov39. Unrelated structural abnormalities of retina and lens seen in 8% of eyes examined significantly predisposed eyes to the development of Ov39- or hr44-induced pathology. These findings suggest a role for cross-reactive immune responses in the development of ocular onchocerciasis.

Animals↗

Retinal and post-retinal components of visual illusions.

A technique is presented permitting control of input to each hemiretina and each hemisphere, of the various elements of a visual illusion. When the retinal effects and post-retinal effects are added, the result is greater than the total illusory effect. This is believed due to the mixing between hemispheres via the corpus callosum, and suggests a possible means of assessing the role of the corpus callosum.

Corpus Callosum↗

Retinal thickness measurements with optical coherence tomography and the scanning retinal thickness analyzer.

PURPOSE: To assess the reproducibility of retinal thickness measurements in normal subjects and to compare foveal thickness using optical coherence tomography (OCT) and the scanning retinal thickness analyzer (RTA). METHODS: Two examiners performed foveal thickness measurements in 24 eyes of 12 healthy subjects using both OCT and the scanning RTA. Intraclass correlation coefficients (ICC) (intra-examiner and inter-examiner) were calculated for the paired foveal thickness measurements obtained with each instrument. RESULTS: The average foveal thicknesses measured with OCT and the scanning RTA were 155.1 +/- 14.9 microm and 107.8 +/- 18.6 microm, respectively. The intra-examiner ICCs from the two sessions using the OCT and the scanning RTA were 0.99 and 0.78 and 0.89 and 0.99, respectively. The inter-examiner ICCs of the OCT and the scanning RTA were 0.99 and 0.99, respectively. There was a significant correlation between the foveal thickness measurements with these two instruments (R2 = 0.629, P < 0.0001). CONCLUSION: The foveal thicknesses measured using OCT and the scanning RTA in healthy subjects agreed with the previously reported data on foveal thickness. Both instruments can reproducibly quantitate foveal thickness.

Adult↗

Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular IP3-sensitive Ca2+ pools.

A comparative study of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca2+]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP3 in a dose-dependent manner. However, when stimulated by various concentrations of IP3 (1-10 microM), the increase in [Ca2+]i of BRCP was always two- to threefold higher than that in BRPE. Subcellular-fractionation studies showed that a single population of IP3 binding site with a high affinity and high specificity of IP3 mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [32P]-IP3 binding sites (Kd 1.9-2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP-dependent Ca2+ uptake and IP3-dependent Ca2+ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca2+ whereas BRPE only released 0.07 nmol Ca2+ with the same dose of IP3 (5 microM). The selectively higher density of IP3 binding sites in coupling to the larger Ca(2+)-release in the membrane of BRCP suggests that the quantity of Ca2+ mobilized is determined by the spatially preferential distribution of membrane-associated IP3 binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP3-induced DNA replication.

Binding Sites↗

Culture of retinal pigment epithelium from chorio-retinal biopsies.

The recently devised methods for surgical sampling of chorio-retinal tissue permits morphological evaluation of retina in various diseases. In the present report it is demonstrated, that the retinal pigment epithelium in such samples can be isolated as a viable cell population, and that the cell mass can be increased sufficiently in culture to permit biochemical as well as morphological studies. This procedure thereby expands the diagnostic potential of the sample tissue.

Animals↗

Early diagnosis of retinal changes in diabetes: a comparison between electroretinography and retinal biomicroscopy.

The present study was undertaken in order to find out whether electroretinographic examinations could reveal signs of functional abnormalities before morphological changes are detected in the diabetic retina. Pattern-reversal and flash electroretinograms (ERG) and oscillatory potentials (OP) were recorded in 24 diabetics and 10 age-matched normal controls. The diabetic group consisted of 11 patients without retinopathy and 13 patients with background retinopathy. No significant changes in pattern-reversal or flash ERG or OP amplitudes were observed in the diabetic group with normal fundus or with background retinopathy. The findings imply that ERG examination with the described techniques does not reveal retinal dysfunction in diabetics before retinopathy can be detected by means of retinal biomicroscopy.

Adult↗

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation.

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.

Animals↗

Partial dehydration of the retinal binding pocket and proof for photochemical deprotonation of the retinal Schiff base in bicelle bacteriorhodopsin crystals.

In bicelle bacteriorhodopsin (bcbR) crystals, the protein has a different structure from both native bacteriorhodopsin (bR) and in-cubo bR (cbR) crystals. Recently, we studied the ability of bcbR crystals to undergo the photocycle upon laser excitation, characterized by the appearance of the M intermediate by single crystal resonance Raman spectroscopy. Calculation of the M lifetime by flash photolysis experiments demonstrated that in our bcbR crystals, the M rise time is much faster than in the native or cbR crystals, with a decay time that is much slower than these other two forms. Although it is now known that the bcbR crystals are capable of photochemical deprotonation, it is not known whether photochemical deprotonation is the only way to create the deprotonated Schiff base in the bcbR crystals. We measured both the visible and Raman spectra of crystals dried under ambient lighting and dried in the dark in order to determine whether the retinal Schiff base is able to thermally deprotonate in the dark. In addition, changes in the visible spectrum of single bcbR crystals under varying degrees of hydration and light exposure were examined to better understand the retinal binding environment.

Bacteriorhodopsins↗

Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium.

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Animals↗

Polarity of 11-cis retinal release from cultured retinal pigment epithelium.

PURPOSE: Fetal bovine retinal pigment epithelium (RPE) was grown on porous supports to investigate the polarity of 11-cis retinal (RAL) release from these cells and the influence that the interphotoreceptor retinoid-binding protein (IRBP) has on this process. METHODS: [3H]all-trans retinol (ROL) was delivered to the basal surface of the cultured RPE by serum retinol-binding protein (RBP). Apo IRBP was added to either the apical or basal medium, or was absent from the incubation entirely. RESULTS: The greatest level of [3H]11-cis RAL was detected in the apical medium but only when apo IRBP was present there. When apo IRBP was present only in the basal medium, or was absent from the incubation entirely, low levels of [3H]11-cis RAL were released apically and basally. CONCLUSIONS: If 11-cis RAL release were constitutive, one would expect to find elevated levels of this retinoid in the apical and basal media in the absence of apo IRBP. Instead, the enhancement of [3H]11-cis RAL release into the apical, but not the basal, medium in the presence of apo IRBP suggests that [3H]11-cis RAL release is polarized and dependent on the presence of apo IRBP. It is postulated, therefore, that a mechanism such as an IRBP membrane receptor in the apical plasma membrane may be responsible for this polarity.

Animals↗

Slowly progressive changes of the retina and retinal pigment epithelium in Briard dogs with hereditary retinal dystrophy. A morphological study.

Seven eyes from 2 generations of Briard dogs (5 weeks--7 years old) with congenital night blindness and (in the second generation) impairment of day vision to varying degrees, were examined by light and electron microscopy. Specimens from 4 locations were studied: the central area, the midperiphery of the tapetal area, the upper periphery and the lower periphery. Disorientation of rod outer segment disc membranes was seen in the 5-week-old dog. Large electron-lucent inclusions were found in the RPE at 3.5 months of age. These inclusions occurred most frequently in the central and midperipheral-tapetal areas and seemed to increase in numbers and spread towards the periphery with increasing age. The content of these inclusions is not elucidated. Rod photoreceptor degeneration was apparent from 7 months of age and was most prominent in the peripheral areas. The cones were better preserved. The 7-year-old dog showed reduction of photoreceptors in the central and midperipheral-tapetal areas and almost complete photoreceptor degeneration in the periphery. This dog also showed severe changes of the inner retina in the peripheral fundus. It appears that these Briard dogs suffer from a very slowly progressive retinal degeneration, in which the photoreceptor degenerative changes do not correlate anatomically to the changes in the RPE cells. The disease seems to be different from the retinopathy described in the English Briards. It is not clear yet whether the lipid type of retinopathy found in American Briards is identical to the present disease.

Animals↗