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At least 289 records · Page 16Linked to original sources
PubMed1999

Retina-derived fetuin (RDF): analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot.

PURPOSE: This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. METHODS: Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. RESULTS: Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. CONCLUSIONS: Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

Animals↗
PubMed2002

FGFR1 expression and FGFR1-FGF-2 colocalisation in rat retina: sites of FGF-2 action on rat photoreceptors.

AIM: To identify sites of FGF-2 action on photoreceptors of the rat retina, by localizing FGFR1 in the intact retina, and to assess the colocalisation of FGF-2 with FGFR1. METHODS: Immunohistochemistry and confocal microscopy were used to localise FGF-2 and FGFR1 in cryosections of the rat retina, both normal retina and retina stressed by exposure to bright continuous light (1000 lux, 24h). Antibodies to synaptophysin (SY), cytochrome oxidase (CO) and opsin were used to relate FGFR1-labelling to synaptic vesicles, mitochondria and the photoreceptor cell membrane. Electron microscopy was used to demonstrate the location of synapses within the outer plexiform layer (OPL). RESULTS: FGFR1 was most prominent in the outer nuclear layer (ONL), as diffuse labelling of photoreceptor cytoplasm and as granules between photoreceptor somas. FGFR1 labelling was also observed in the outer synapse-rich sublayer of the OPL where it colocalised with SY, but not with CO-labelled mitochondria. In stressed retina, both at the edge of normal retina and after light stress, FGFR1 expression was upregulated in both the ONL and the OPL. Colocalisation of FGFR1 with FGF-2 could not be demonstrated in unstressed retina, but was demonstrable in stressed retina, in both the ONL and OPL. CONCLUSIONS: FGFR1 is prominent in the cytoplasm of photoreceptors, and in their axon terminals, where it is closely associated with synaptic vesicles. Colocalisation of FGFR1 and FGF-2 could be demonstrated in stressed retina, in the cytoplasm and the axon terminals of photoreceptors. The known protective action of FGF-2 may be exerted at the photoreceptor soma. The action of FGF-2 in inhibiting the ERG b-wave may be exerted at the axon terminal.

Animals↗
PubMed2003

Low oxygen consumption in the inner retina of the visual streak of the rabbit.

The oxygen requirements of different retinal layers are of interest in understanding the vulnerability of the retina to hypoxic damage in retinal diseases with an ischemic component. Here, we report the first measurements of retinal oxygen consumption in the visual streak of the rabbit retina, the region with the highest density of retinal neurons, and compare it with that in the less-specialized region of the retina underlying the vascularized portion of the rabbit retina. Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of retinal depth in anesthetized animals. Measurements were performed in the region of the retina containing overlying retinal vessels and in the center of the visual streak. Established mathematical analyses of the intraretinal oxygen distribution were used to quantify the rate of oxygen consumption in the inner and outer retina and the relative oxygen contributions from the choroidal and vitreal sides. Outer retinal oxygen consumption was higher in the visual streak than in the vascularized area (means +/- SE, 284 +/- 20 vs. 210 +/- 23 nl O2.min(-1) x cm(-2), P = 0.026, n = 10). However, inner retinal oxygen consumption in the visual streak was significantly lower than in the vascular area (57 +/- 4.3 vs. 146 +/- 12 nl O2 x min(-1) x cm(-2), P < 0.001). We conclude that despite the higher processing requirements of the inner retina in the visual streak, it has a significantly lower oxygen consumption rate than the inner retina underlying the retinal vasculature. This suggests that the oxygen uptake of the inner retina is regulated to a large degree by the available oxygen supply rather than the processing requirements of the inner retina alone.

Animals↗
PubMed1998

Modulation of sustained and transient lateral inhibitory mechanisms in the mudpuppy retina during light adaptation.

Two functionally and anatomically distinct types of lateral inhibition contribute to the receptive field organization of ganglion cells in the vertebrate retina: sustained lateral inhibition (SLI), which is present during steady illumination and transient lateral inhibition (TLI), evoked by changes in illumination. We studied adaptive changes in these two lateral inhibitory mechanisms in the mudpuppy retina by measuring the responses of ON-OFF ganglion cells to spots of light in the receptive field center, in the absence and presence of a concentric broken annulus (windmill) pattern, which was either stationary or rotating. SLI was measured as the percent suppression of the centered spot response by the stationary windmill and TLI was measured as the additional suppression produced when the windmill was rotating. In dark-adapted retinas SLI was elicited by windmills of 600 or 1,200 micron ID, but TLI could not be elicited by windmills of any size, over a wide range of windmill intensities and rotation rates. Exposure of dark-adapted retinas to diffuse adapting light caused an immediate decrease in the response to the spot alone, followed by slowly developing changes in both SLI and TLI: SLI produced by 1,200 micron ID windmills became weaker, whereas SLI produced by 600 micron ID windmills became stronger. After several minutes strong TLI could be elicited by both 600 and 1,200 micron ID windmills. The changes in SLI and TLI were usually complete within 5 and 15 min, respectively, and recovered to dark-adapted levels slightly more slowly after the adapting light was turned off. However the changes in sensitivity of the spot response were complete within one minute after onset and termination of the adapting light. The adaptive changes in SLI and TLI did not depend on the presence of the adapting light; after a brief (1 min) exposure to the adapting light, the changes in SLI and TLI slowly developed and then decayed back to the dark-adapted level. The effects of the adapting light on SLI were mimicked by dopamine and blocked by D1 dopamine receptor antagonists. However dopamine did not enable TLI in dark-adapted retinas and dopamine antagonists did not prevent enablement of TLI when dark-adapted retinas were exposed to light or disable TLI when applied to light-adapted retinas. The results suggest that light-adaptive changes in SLI are mediated by dopamine and are consistent with a reduction in electrical coupling between neurons that conduct the SLI signal laterally in the retina. In contrast, TLI appears to be switched off or suppressed in the dark-adapted retina and enabled in light-adapted retinas, by a relatively slow modulatory mechanism that does not involve dopamine.

Adaptation, Ocular↗
PubMedDate not supplied

Expression of nitric oxide synthase during the development of RCS rat retinas.

Nitric oxide (NO) has been reported to be both neurodestructive and neuroprotective in the central nervous system and could possibly play an important role in neurodegenerative disorders. On the assumption that NO synthesis may influence degenerative processes in the retina, we have examined the development and distribution of nitric-oxide-synthase(NOS)-immunoreactive cells in developing Royal College of Surgeons (RCS) rat retinas, which is an animal model for retinal degeneration. An antibody against constitutive neuronal NOS was used for immunocytochemistry on RCS rat retinas from postnatal (PN) days 3, 7, 10, 14, 35, 70 and 281 and compared with that in the normal rats of PN days 3, 7, 10, 14, 54 and adults. Immunoreactive cells were not seen in PN 3 retinas but were distinctly seen in the PN 7 retina along with a plexus in the inner plexiform layer. In both groups (normal and RCS rats) a distinct sublayering of the plexus in the inner plexiform layer could be seen at PN 10, which became more distinct at PN 14. The immunoreactive cells were detected also in the oldest retina examined, which was PN 281 in the case of RCS rats. In both groups, certain amacrine cells, certain bipolar cells and certain horizontal cells were found to be immunoreactive. In conclusion, the developmental timetable of the NOS immunoreactivity was identical in the normal and the RCS rat retinas. The NOS-immunoreactive cells persisted in the RCS retinas even when the retina had degenerated extensively. Abnormalities with the inducible isoforms of NOS cannot be ruled out from this study. We conclude that the chronological and qualitative development of the constitutive neuronal NOS immunoreactivity is normal in RCS rat retinas.

Animals↗
PubMed2003

Expression of beta-carotene 15,15' monooxygenase in retina and RPE-choroid.

PURPOSE: Beta-carotene 15,15' monooxygenase (beta-CM) catalyzes the central cleavage of beta-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of beta-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE. METHODS: RT-PCR was used to detect expression of beta-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize beta-CM in mouse and monkey retina with an anti-peptide antibody specific for beta-CM. RESULTS: By RT-PCR, beta-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, beta-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed beta-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for beta-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. beta-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of beta-CM immunoreactivity in monkey RPE. CONCLUSIONS: Beta-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of beta-CM in the retina and RPE, it can be concluded that beta-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.

Animals↗
PubMed2003

Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD.

PURPOSE: To demonstrate structural and immunocytochemical changes associated with light-induced degeneration in albino rat retinas and human AMD retinas. METHODS: Retinas from Wistar rats aged 3, 6, or 10 months were examined by immunocytochemistry, with antibodies to neuronal and glial markers. Results were compared with human nonneovascular AMD retinas. RESULTS: In aging rat retinas, many photoreceptors were lost in response to normal ambient light exposure. Photoreceptor loss was preceded by loss of RPE cells. Müller cells extended processes through gaps in Bruch's membrane, into the choroid. immunolabeling for gamma-aminobutyric acid (GABA), the glycine transporter Glyt-1, and the rod bipolar cell marker PKC revealed the presence of numerous neuronal somata and processes that appeared to have migrated into the choroidal region. Processes of presumptive ganglion cells remodeled and stratified in the choroid, where strong labeling for synaptic vesicle antigens was present. Myelination of retinal ganglion cell axons was also observed, especially in the peripheral retina. In AMD retinas, glial rearrangement and displacement of neurons suggestive of their migration were also observed. CONCLUSIONS: In response to loss of RPE and photoreceptor cells, adult retinal neurons migrate out of the retina along remodeled processes of Müller cells. The presence of synaptic vesicle antigens suggests the formation of new synapses between migrating neurons. The myelination is probably due to the ingress of Schwann cells from the sclera. The presence of some similar changes in human AMD retinas suggests that these findings are of broad significance for determining the likely events in transplantation of neurons in the human retina and elsewhere.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Oxygen distribution in the mouse retina.

PURPOSE: To make the first measurements of intraretinal oxygen distribution in the mouse, an animal model of increasing importance in ophthalmic research. METHODS: Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of depth through the retina and choroid in anesthetized mice (n = 8). All measurements were performed under light-adapted conditions, with the animals spontaneously inspiring room air. The oxygen distribution in the avascular portion of the outer retina was analyzed by an established three-layer mathematical model that determines outer retinal oxygen consumption. RESULTS: The intraretinal oxygen distribution in the inner retina in individual profiles was often characterized by sharp peaks associated with elements of the retinal microvasculature, but, in the outer retina, the oxygen distribution was much more predictable and reflected the high oxygen uptake of the photoreceptors. Average choroidal oxygen tension was 42.0 +/- 1.2 mm Hg (mean +/- SE), and P(O2) at the surface of the retina was 21.7 +/- 0.8 mm Hg. The average minimum oxygen tension in the outer retina was 4.2 +/- 0.5 mm Hg. Average outer retinal oxygen consumption in the mouse was 193.3 +/- 10.6 nL O2/min per square centimeter, which is similar to that previously reported in the outer retina in vascularized areas of retina in the rat and monkey using similar techniques. CONCLUSIONS: The intraretinal oxygen distribution in the mouse is qualitatively and quantitatively similar to that in other species with vascularized retinas. The rate of oxygen consumption in the outer retina is also similar. These baseline data can now be used in studies employing mouse models of retinal disease.

Animals↗
PubMed2006

Oxygen distribution and consumption in the developing rat retina.

PURPOSE: To determine the intraretinal oxygen distribution and oxygen consumption in the rat before eye opening and maturation of the retina. METHODS: Oxygen-sensitive microelectrodes were used to measure the oxygen tension as a function of depth through the retina in anesthetized Sprague-Dawley rats at postnatal day (P)15. Measurements were made under air-breathing conditions and at increasing levels of systemic oxygenation (20%, 40%, 60%, 80%, and 100% oxygen) under light-adapted conditions. Oxygen consumption in the outer retina and in the predominantly avascular region of the inner retina was assessed by fitting the oxygen profiles to a mathematical model of oxygen consumption. The retinas were processed for histology and compared with retinas from mature animals. RESULTS: Under normal conditions, the intraretinal oxygen distribution at P15 was significantly different in some respects from that in mature animals. Oxygen consumption analysis indicated an average outer retinal oxygen consumption rate of 103 +/- 15 nL/min per cm2 and an inner retinal oxygen consumption rate of 42.4 +/- 11.7 nL/min per cm2. Inner retinal oxygen consumption was significantly (P < 0.001) lower than that previously measured in mature rats, but outer retinal oxygen consumption was similar. Systemic hyperoxia increased the oxygen level throughout the retina, but choroidal PO2 in particular remained significantly lower than in adult rats (P < 0.001). At P15 there were marked differences in the relative thickness of some retinal layers when compared with adult rats. In particular the inner and outer nuclear layers were much thicker at P15, the outer segments of the photoreceptors and the inner and outer plexiform layers were not fully developed. CONCLUSIONS: At P15, before eye opening, the oxygen consumption of the inner retina is lower than in mature retinas, presumably reflecting the immaturity of the retina in such young animals so soon after their first exposure to light stimuli.

Animals↗
PubMed2006

Slit proteins regulate distinct aspects of retinal ganglion cell axon guidance within dorsal and ventral retina.

An early step in the formation of the optic pathway is the directed extension of retinal ganglion cell (RGC) axons into the optic fiber layer (OFL) of the retina in which they project toward the optic disc. Using analysis of knock-out mice and in vitro assays, we found that, in the mammalian retina, Slit1 and Slit2, known chemorepellents for RGC axons, regulate distinct aspects of intraretinal pathfinding in different regions of the retina. In ventral and, to a much lesser extent, dorsal retina, Slits help restrict RGC axons to the OFL. Additionally, within dorsal retina exclusively, Slit2 also regulates the initial polarity of outgrowth from recently differentiated RGCs located in the retinal periphery. This regional specificity occurs despite the fact that Slits are expressed throughout the retina, and both dorsal and ventral RGCs are responsive to Slits. The gross morphology and layering of the retina of the slit-deficient retinas is normal, demonstrating that these distinct guidance defects are not the result of changes in the organization of the tissue. Although displaced or disorganized, the aberrant axons within both dorsal and ventral retina exit the eye. We also have found that the lens, which because of its peripheral location within the developing eye is ideally located to influence the initial direction of RGC axon outgrowth, secretes Slit2, suggesting this is the source of Slit regulating OFL development. These data demonstrate clearly that multiple mechanisms exist in the retina for axon guidance of which Slits are an important component.

Adaptation, Physiological↗
PubMed2000

Heat shock cognate-70 gene expression declines during normal aging of the primate retina.

PURPOSE: Despite documented age-related changes in retinal function and histology, little is known about the pattern of gene expression during normal aging of the vertebrate retina. This study was undertaken to definitively characterize gene expression in the primate retina during aging. METHODS: Human retina cDNA library clones were arrayed at high density on nylon membranes and screened with mixed cDNA probes generated from young (4-year-old) and old (80-year-old) human retinae. Clones showing a more than twofold difference in intensity were rescreened by dot blot analysis with the same probes and with mixed cDNA probes generated from young (2-3 years) and old (27-35 years) rhesus monkeys. One clone identified by its differential (age-putative) signal, and age-related differential expression was used for analysis of Northern blot analysis of total retinal RNA from human donors (35 weeks to 94 years of age) and two rhesus monkeys (2 and 27 years of age). The identified clone was sequenced and compared with entries in the GenBank/EMBL databases. Western blot analysis was performed on protein isolated from the retina of human donors aged 4 to 64 years and rhesus monkeys aged 18 months and 35 years. RESULTS: Approximately 1.6% of the 55,368 retina-expressed sequences examined show age-related changes between tissues from young and old donors. The mRNA level one clone, identical with heat shock cognate (HSC)70, was altered during normal retinal aging in primates. Regression analysis of Northern blot analysis signals from 23 human donors suggested that there may be a two- to threefold decrease in HSC70 mRNA levels in the human retina by the eighth decade of life. Western blot analysis also showed lower levels of the 70-kDa HSC protein in older tissues of both primates. CONCLUSIONS: HSC70 mRNA levels apparently decline during normal aging of the primate retina. Because the heat shock 70 protein family may play important roles in ocular development and protection from various biologic and environmental stresses, decreased HSC70 levels in the retina during aging may contribute to the apparent increased susceptibility of the retina to age-acquired retinal disease.

Adolescent↗
PubMed1983

The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific for intermediate filament proteins.

Rat retina structure was studied between embryonic day 14 and adult with antibodies specific for vimentin, glial fibrillary acidic protein (GFA) and the proteins of the neurofilament triplet. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond, in the developing retina, to ventriculocytes, and in the mature retina to Müller cells. They could not normally be stained with any of the other intermediate filament antibodies employed here. We did find, however, that some older albino rats possessed GFA staining in addition to vimentin in these processes. Since we never saw such staining in the retinae of mature non-albino rats, and the retinae of older albino rats often showed signs of degeneration, we concluded that such GFA expression was most likely pathological. Neurofilament protein-positive processes were first detectable at embryonic day 15 1/2 in the inner regions of the retina, and corresponded to the axons of retinal ganglion cells. Such processes were equivalently displayed with antibodies to 68 K and 145 K protein, but were negative with 200 K protein. Some 68 K and 145 K positive fibers could also be decorated with vimentin antibody at this stage, though at later stages this was not the case. At later development stages more 68 K and 145 K neurofilament positive processes appeared, and after the first post-natal week progressively more of such processes became in addition 200 K positive, so that almost all neurofilament positive fibers in the adult stained for all three proteins. Such fibers, in the mature retina corresponded to 68 K and 145 K positive optic nerve fibers, and the processes of neurones in the inner plexiform layer. All fibers in the mature optic nerve fiber layer, but not all of those in the inner plexiform layer were stainable with 200 K antibodies. At 4 days post-natal we were able to detect 68 K and 145 K protein positive profiles in the outer regions of the developing retina, the prospective outer plexiform layer. Such profiles were always in addition vimentin positive, but negative for 200 K protein. During further development such profiles became ordered into a well defined layer and from about post-natal day 13 all of them began to acquire 200 K protein. They could be identified as the processes of horizontal cells. They continued to express vimentin in addition to the three triplet proteins in the adult, a so far unprecedented situation. We were able to detect neurofilament staining in the mature retina only in the above described regions, the inner and outer nuclear layer and the photoreceptor processes being completely free of staining. GFA was first detected in short processes adjacent to the inner limiting membrane which penetrated the optic nerve fiber layer. Such profiles were first detectable in the eye of the newborn animal, and were invariably identically stainable with vimentin at this age. These profiles could be stained with both vimentin and GFA at all later stages examined, although GFA staining became very much stronger than vimentin staining in some profiles in the adult. The results presented here are discussed in terms of development of the different retinal cell types.

Age Factors↗
PubMed1996

Macrophages in the retina of normal Lewis rats and their dynamics after injection of lipopolysaccharide.

PURPOSE: To investigate the density, distribution, and morphology of macrophages (bone marrow-derived microglia) and major histocompatibility complex (MHC) class II-positive cells in the retina of Lewis rats and the dynamics of these cells after systemic lipopolysaccharide (LPS) injection. METHODS: Immunohistochemistry was carried out using monoclonal antibodies specific to monocytes and macrophages (ED1, ED2) and MHC class II-positive cells (OX-6) on whole-mounts of the retina obtained from Lewis rats before and at different time points after footpad injection of 200 micrograms of LPS. RESULTS: The inner layers of the normal retina contained a network of macrophages, whereby ED1 and ED2 staining revealed similar results. Macrophages were either dendritiform or pleiomorphic in morphology, with the former predominant. The density of positive cells was higher at the peripheral part and the periequatorial part (271 +/- 10 cells/mm2 and 267 +/- 9 cells/mm2, respectively) than at the posterior part (196 +/- 11 cells/mm2; P < 0.0001 in both cases). Lipopolysaccharide injection induced an early adherence of monocytes to retinal blood vessels, followed by a massive influx of the macrophages into the retina. The ED1-ED2 positive cells showed a variety of morphologic appearances: large round cells, pleiomorphic cells, and dendritiform cells. Pleiomorphic cells were striking at 48 hours, whereas dendritiform cells were predominant in the whole retina at 72 hours and thereafter. On day 14, the dendritiform cell numbers returned to approximately preinjection levels. Major histocompatibility class II-positive cells could not be found in the normal retina, nor after LPS injection. CONCLUSIONS: The network of MHC class II-negative microglia in the retina were studied. These cells may play an important role in immunoregulation and stability of the immunologic microenvironment within the retina. Systemic LPS injection was followed by a massive influx of macrophages into the retina. The absence of MHC class II-positive cells in the retina after LPS challenge may be an important protective mechanism against possible autoimmune damage.

Animals↗
PubMed1996

Cloning and mRNA expression of vascular endothelial growth factor in ischemic retinas of Macaca fascicularis.

PURPOSE: To identify and isolate cDNAs for the alternatively spliced vascular endothelial growth factor (VEGF) mRNAs present in retina and to compare the relative levels of the splice variants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas. METHODS: Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the main branch retinal veins. Reverse transcription-polymerase chain reaction was used to amplify the VEGF coding region of RNA from ischemic and control retinas, and amplification products were analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing. Analysis of VEGF mRNA expression was accomplished by in situ hybridization. RESULTS: Control and ischemic retinas produce mRNAs that correspond to the 121 and 165 amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively spliced VEGF transcripts between control and ischemic retinas. Cloning and sequencing revealed that simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteins identical to their respective human homologues. In situ hybridization of nonischemic retinas revealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer. VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion, when there was a small but reproducible increase in signal. The expression peaked at approximately 13 days, coincident with maximal iris neovascularization, and was significantly reduced by 28 days, when the iris vessels largely regressed. CONCLUSIONS: The elevation of simVEGF121 and VEGF165 in ischemic retinas is consistent with a role for diffusible, retina-derived angiogenic factors in the development of ocular neovascularization.

Alternative Splicing↗
PubMed1997

Glucose dependence of glycolysis, hexose monophosphate shunt activity, energy status, and the polyol pathway in retinas isolated from normal (nondiabetic) rats.

PURPOSE: To measure glucose-dependent metabolic activities and selected parameters of the polyol pathway in retinas isolated from normal rats to test the hypothesis recently proposed by Van den Enden et al that incubation of whole retinas for 2 hours with elevated concentrations of glucose results in activation of the polyol pathway, which is the cause of a redox imbalance, as measured by an increase in the retinal cytosolic lactate-pyruvate ratio and a diabetic-like state. METHODS: Retinas obtained from nondiabetic rats and separated from other ocular tissues were incubated for several hours in incubation medium containing glucose at concentrations ranging from 5 to 30 mM. Measurements were made under aerobic and anaerobic conditions of lactic acid production, retinal adenosine triphosphate (ATP), lactic acid content, the hexose monophosphate shunt pathway, aldose reductase activity, and levels of sorbitol and galactitol. Morphology was examined by light microscopy at the end of the incubations. RESULTS: Incubation of isolated rat retinas with 20 mM glucose increased lactic acid production by approximately 25% in comparison to the rate observed in 5mM glucose under aerobic and anaerobic conditions. The content of ATP and lactate in the retinas after a 2-hour incubation in the presence of oxygen and 20 mM glucose was equal to the amounts found in fresh tissues, whereas these metabolites declined, respectively, by 25% and 45% when 5 mM glucose was used. The activity of the hexose monophosphate shunt pathway in isolated rat retinas was not increased increased significantly when the concentration of glucose was raised from 5 to 30 mM. Aldose reductase activity and polyols were below our limits of detection, 0.5 nmol/minute.mg protein and 3.5 nmol/retina, respectively, under all conditions tested. The morphologic appearance of the retina was similar in the presence of normal and high concentrations of glucose. CONCLUSIONS: These results show that incubation of isolated rat retinas, obtained from nondiabetic rats, with elevated concentrations of glucose for 2 hours leads to increases in glycolysis and a higher tissue content of lactic acid and ATP in comparison to values obtained with 5 mM glucose. However, the magnitude of the glucose-dependent increase in the retinal level of lactate in the current study and in that of Van den Enden et al is six to seven times greater than the calculated flux of glucose through the polyol pathway. These results, therefore, do not support the hypothesis of Van den Enden et al. Rather, it is suggested that supranormal concentrations of glucose yield more lactate and ATP in a whole retina because they optimize the supply of this essential nutrient to cells throughout the tissue by overcoming diffusional limitations that result when the retina is separated from its normal choroidal and intraretinal blood supplies.

Adenosine Triphosphate↗
PubMed1998

The target of the antiproliferative antibody (TAPA) in the normal and injured rat retina.

PURPOSE: The target of the antiproliferative antibody (TAPA, CD81) is a member of the tetramembrane spanning superfamily of proteins and appears to be involved in the regulation of mitotic activity and the stabilization of cellular contacts [J Neurosci 1996; 16:5478-5487]. The present study examines the distribution of this protein in the normal rat retina and its role in reactive gliosis occurring after retinal injury. METHODS: An immunoblot was used to define the relative level of TAPA in the normal rat retina. The distribution of the protein was examined using indirect immunohistochemical methods. Both of these methods were used to define the upregulation of TAPA in the rat retina injured with a needle scrape. RESULTS: The immunohistochemical analysis of the retina shows that TAPA is found in all layers of the normal retina with a distinct lack of labeling in the inner and outer segments of the photoreceptors. After retinal injury, a dramatic upregulation of TAPA was observed. The immunohistochemistry also revealed a pattern of expression similar to that observed in the normal retina with two notable exceptions: (1) small finger-like projections extending down into the outer segments are immunopositive, and (2) the elevated levels of TAPA can be seen outlining cell bodies in the outer nuclear layer and the ganglion cell layer. CONCLUSIONS: TAPA is found in the normal rat retina and there is a dramatic upregulation of this protein following injury. The distribution of the protein within the retina is consistent with its expression in retinal glia, the Muller cells which span the thickness of the retina, and astrocytes found in the ganglion cell layer. These data suggest that TAPA may play a role in the proliferative response of non-neuronal cells that occurs following a mechanical injury to the retina.

Animals↗
PubMed1998

Estrogen receptor expression in bovine and rat retinas.

PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.

Animals↗
PubMed1996

Comparison of topographical patterns of ganglion and photoreceptor cell differentiation in the retina of the zebrafish, Danio rerio.

Earlier studies suggested retinal differentiation in the zebrafish commences ventrally rather than centrally as is the case in other vertebrates. Here we describe the topographical spread of cell differentiation for ganglion cells, double cones and rods in the zebrafish retina between 36 and 72 hours postfertilization (hpf), by using immunohistochemical markers in retinal wholemounts. Staining for all three cell types commenced within the ventral retina on the nasal side of the optic nerve and choroid fissure, at 38 hpf for ganglion cells and 50 hpf for double cones and rods. Within 3 to 4 hours, the staining of ganglion cells and double cones spread in a continuous wave-like fashion into the nasal region of the ventral retina. After this time, the staining patterns for ganglion cells and double cones progressed dorsally into the central and temporal retina. Finally, stained somata of ganglion cells were observed within the temporal-ventral region by approximately 48 hpf, more than 8 hours later than the first ganglion cells within the nasal retina. The topographical spread of double cone staining was slightly less orderly. After staining had extended into the nasal retina between 50 and 54 hpf, a small group of stained double cones often appeared at the temporal edge of the choroid fissure by 56 hpf, simultaneously with initial staining observed dorsal and temporal to the optic nerve. The topographical spread of rod staining in the ventral retina was more symmetrical. After rod staining appeared near the nasal edge of the choroid fissure at 50 hpf, rods accumulated within a localized patch nasal to the fissure. Approximately 5 hours after initial rod staining, scattered rod staining appeared on the temporal side of the choroid fissure (approximately 55-57 hpf). Rods increased rapidly within the ventral retina, and a dense symmetrical patch extended out from the choroid fissure into the nasal and temporal regions of the ventral retina by 70 hpf. A scattered pattern of rod staining also occurred within the dorsal retina at this time.

Animals↗