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PubMed1997

Detection of vascular endothelial growth factor (VEGF) protein in vascular and non-vascular cells of the normal and oxygen-injured rat retina.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell cytokine that can be up-regulated by hypoxia. There is evidence that VEGF is a significant mediator in retinal neovascular diseases and other disorders in which hypoxia is believed to influence the pathogenesis. Here we demonstrate the spatial relationships among areas of retinal non-perfusion, VEGF protein and vascular endothelial cells throughout the retina, and relate these results to cellular distribution of VEGF in cross section. Newborn albino rats were oxygen-injured by cycles of alternating 50% and 10% oxygen for 14 days and then placed in room air. On days 16, 21 and 26, oxygen-injured and control (raised in room air) rats were sacrificed, enucleated and retinas were dissected and fixed for whole mount immunostaining for VEGF or embedding in glycol methacrylate for VEGF immunohistochemistry. Intact eyes taken on days 16 and 20 were processed similarly. Vascular endothelial cells were demonstrated by staining whole-mounted retinas for adenosine diphosphatase (ADPase) activity. Preretinal neovascular growths (i.e., abnormal vessels extending from the retina into the vitreous) were VEGF-positive. There was also a pan-retinal distribution of non-endothelial cells that were VEGF-positive in both room air and oxygen-injured rats, with stronger immunostaining in day 16 oxygen-injured retinas. In cross-section, VEGF staining was confirmed in preretinal growths, normal retinal vessels, cells in the inner nuclear layer (primarily Müller cells) and ganglion cells. Retinas which had been incubated with nonimmune IgG or absorbed anti-VEGF antibody showed little or no staining. In conclusion, we have identified cells of the inner retina which express VEGF. The production of VEGF by these cells--in particular, Müller cells--may promote preretinal neovascularization in oxygen-injured eyes. We have found, moreover, that the combination of immunohistochemistry and ADPase staining of whole mount preparations is a unique and powerful tool for evaluating relationships between presumed areas of retinal ischemia, VEGF (and other cytokines) and retinal blood vessels, within an entire retina. This approach can be used to study any proliferative retinal disorders in which VEGF is a potential component of the pathogenesis.

Animals↗
PubMed1995

Morphological changes of the sensory retina following the occlusion of ciliary arteries in rabbits.

The whole retina, except for the medullary fiber zone in a rabbit eye, is supplied by choroidal circulation. Therefore, the histopathological changes of the sensory retina due to choroidal circulatory disturbance in rabbits may be comparable to that of the human sensory retina in the case of ophthalmic artery occlusion. This study was carried out to evaluate the histopathological changes of the ischemic retina secondary to the occlusion of choroidal circulation. The experimental occlusion of all posterior ciliary arteries and anterior ciliary arteries in the horizontal rectus muscle of rabbit eyes was performed and the subsequent histopathological changes of the sensory retina were observed by transmission electron microscopy. The morphological changes of the sensory retina following the occlusion of the ciliary arterial system are as follows: severe loss of the inner and outer segments of the photoreceptor, mild to moderate degeneration of the ganglion cells, and excellent preservation of the Müller's cell fibers and the extension of the cytoplasmic villous processes to the cytoplasmic vacuolar spaces of other degenerated cells. These findings indicate that the Müller's fibers in the ischemic condition of retina might contribute to the formation of gliosis or scarring of a damaged retina.

Animals↗
PubMed1999

Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina.

PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.

Animals↗
PubMed1999

Release of endogenous ascorbic acid preserves extracellular dopamine in the mammalian retina.

PURPOSE: To investigate whether the inhibitory effect of nitric oxide (NO) on dopamine release from the retina is due to chemical oxidation of dopamine in the extracellular medium rather than to an inhibitory effect on dopamine release from retinal neurons. METHODS: Dopamine was incubated in Krebs bicarbonate medium and its rate of chemical degradation measured by high-performance liquid chromatography (HPLC). The effects of NO donors and antioxidants on dopamine were assessed by comparing dopamine degradation in the presence and absence of drug. The effects of NO donors on the K-evoked release of [3H]dopamine were measured from isolated superfused rabbit retinas. The release of ascorbic acid from the isolated rat retina and from an eyecup preparation in anesthetized rabbits was measured by HPLC. RESULTS: After 10 minutes' incubation in Krebs bicarbonate medium, the dopamine concentration decreased by 20%. This decline increased to 80% in the presence of S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP). The increased rate of dopamine degradation was abolished if retina was incubated in the medium and then removed before the incubation of dopamine. The protective effect of preincubation with tissue was lost in the presence of ascorbate oxidase suggesting the release of ascorbic acid. HPLC analysis confirmed a substantial release of ascorbic acid from both rabbit and rat retinas. The K-evoked release of [3H]dopamine from the rabbit retina was inhibited by SNP. CONCLUSIONS: NO can rapidly, oxidize dopamine in physiological medium, but in the presence of retina, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent this chemical reaction. Thus, the inhibitory action of NO on dopamine release results from an action on retinal neurons. Ascorbate release in the retina may have an important physiological role in prolonging the life of dopamine, which often has to diffuse long distances from axons in the inner plexiform layer to receptors in other retinal layers.

Animals↗
PubMed2000

Expression patterns of connexin genes in mouse retina.

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase-polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43-positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild-type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin-deficient mice to cells in different regions of the retina.

Animals↗
PubMed2001

Distribution of OX2 antigen and OX2 receptor within retina.

PURPOSE: OX2 is a member of the immunoglobulin superfamily expressed on a broad range of tissues including neurons of the central and peripheral nervous systems, thymocytes, and endothelium. The recently identified OX2 receptor (OX2R) is restricted to the surfaces of myeloid lineage cells, including microglia. Functional data have implicated the OX2-OX2R interaction as a myeloid downregulatory signal. The purpose of this study was to determine the distribution and extent of expression of OX2 and its receptor within the retina, a tissue developed to restrain immune-mediated inflammatory damage. METHODS: OX2 and OX2R monoclonal antibodies (mAbs) were used to determine OX2 and OX2R protein expression, respectively, by flow cytometry of isolated myeloid-derived cells from normal and inflamed rat retina and by immunohistochemistry of serial sections of rat retina. For comparison, distribution of OX2 was documented using species-specific monoclonal antibodies in mouse and human retina. No OX2R mAbs are available for mouse or human detection. RESULTS: OX2 was expressed on retinal vascular endothelium and glial fibrillary acidic protein (GFAP)-negative neurons in retina and optic nerve and on a subpopulation of CD45(+) perivascular and juxtavascular cells. Within normal retina, OX2R was not detected on myeloid-derived cells. During experimental autoimmune uveoretinitis (EAU), expression of both OX2 and OX2R was noted on infiltrating leukocytes. CONCLUSIONS: Taking these results of the distribution of OX2 in normal and OX2R in inflamed retina with other functional data of OX2-OX2R interaction, it is suggested that the OX2-OX2R interaction has the potential to contribute to a novel pathway that suppresses and limits immunologic inflammatory damage within the retina.

Animals↗
PubMed2001

Expression of Drosophila omb-related T-box genes in the developing human and mouse neural retina.

PURPOSE: To examine the role of Drosophila optomotor blind (omb)-related T-box genes in development of human and mouse retina. METHODS: Mouse Tbx2, Tbx3, and Tbx5 and human TBX2 cDNAs were isolated from retinal cDNA libraries by hybridization to the Drosophila omb gene. Gene expression patterns in developing retina were analyzed by in situ hybridization. RESULTS: TBX2/Tbx2, TBX3/Tbx3, and TBX5/Tbx5 were expressed asymmetrically across the embryonic neural retina with highest levels of mRNA within dorsal and peripheral retina. The dorsoventral gradient of TBX2 expression disappeared before the ganglion cell layer (GCL) formed. Its expression then became restricted to the inner neuroblastic retina and later to the GCL and inner nuclear layer (INL). The dorsal expression domains of TBX5/Tbx5 and TBX3/Tbx3 were maintained during formation of the GCL. As the retina matured, TBX3/Tbx3 expression was restricted to the INL, and TBX5/Tbx5 was expressed within the GCL. CONCLUSIONS: The expression pattern of TBX2, TBX3, and TBX5 within the developing retina supports the idea that the encoded transcription factors play a role in providing positional information important for topographic mapping and in differentiation of distinct cell types across the laminar axis of the retina.

Animals↗
PubMed2002

Accumulation of topically applied porcine insulin in the retina and optic nerve in normal and diabetic rats.

PURPOSE: To explore the pharmacokinetics of topical insulin administration in relation to retinal and optic nerve retention. METHODS: Insulin eye drops (approximately 15 microL: 0.75% porcine insulin + 0.5% permeation enhancer) were applied to the eyes of normal and diabetic rats. The rats were killed at various intervals up to 16 hours, and the retinas and optic nerves from both eyes were analyzed for the presence of insulin in an ELISA. The extent to which systemically absorbed insulin accounted for the findings of insulin in the retina was explored by examining the effects of intravenously injected insulin on retinal insulin levels and by examining the effects of eye drop administration in decapitated rats. RESULTS: Insulin levels rose significantly and peaked in the retina of normal rats 20 minutes after eye drop application (0.7 pg/microg; P < 0.00001). Levels in diabetic retinas peaked at 60 minutes (0.66 pg/microg; P < 0.004) and remained elevated for a longer period than in normal rats. The contralateral retina showed delayed accumulation of lesser amounts of insulin in both normal and diabetic rats. Significant elevations also occurred in the optic nerves in normal and diabetic rats, with concentrations reaching 13 pg/microg in normal rats at 20 minutes and 26 pg/microg in diabetic rats at 5 hours. Topical insulin application resulted in a decrease in serum glucose concomitant with an increase in serum porcine insulin. It did not appear, however, that the systemic absorption of insulin contributed to the accumulation of insulin in the ipsilateral retinas, for two reasons: The intravenous injection of a high concentration of insulin did not appreciably influence retinal insulin levels, and the application of insulin eye drops to decapitated rats still resulted in the accumulation of insulin in the retina. CONCLUSIONS: These results led to the conclusion that topically applied insulin accumulates in the retina and optic nerve in normal and diabetic rats, with levels remaining elevated longer in diabetic animals. It did not appear that systemically absorbed insulin, resulting from ocular drainage, contributed to this effect.

Absorption↗
PubMed2002

[Morphological changes in the parr masou salmon Oncorhynchus masou retina in experimental change of geomagnetic field].

Topography of photoreceptor cells in young salmon Oncorhynchus masou retina, their properties and morphology of cellular organelles in external and internal segments of photoceptors have been first described. Morphological changes of retina cells were analysed in day and night time, and also in the experiment for indemnification of geomagnetic field (GMF) in the body of the aquarium. A comparison of retina structure in fishes of night and day time controls with that in experimental fishes has shown that the external cone segments in the latter occupy, in relation to the external limiting membrane, an intermediate position, characteristic of retinae exposed to twilight lighting. It is supposed that GMF indemnification was equivalent to weak light pulse, which, however, could considerably change melatonin production by retina photoreceptor cells. Thus, at experimental indemnification of GMF, retina sensitive cells demonstrate typical retinomotor response. Some ultrastructural changes in retina cells were also detected, in particular, size changes in ribbon synapses in rod and cone terminations. In addition, nematosomes appeared in the internal nuclear layer, and in the spinules, i.e. digitiform invaginations of terminal dentrites of horizontal cells into cone nervous terminations, the quantity of an electron dense material was noticeably magnified in comparison with a night control. The noted changes testify, in our opinion, to essential modifications in metabolic processes of retina photoreceptors under effect of GMF variations, in particular, to changes in retinal melatonin synthesis.

Animals↗
PubMed2003

Increased expression of ceruloplasmin in the retina following photic injury.

PURPOSE: Oxidative stress plays a role in the photic injury model of retinal degeneration and in age-related macular degeneration. Our preliminary microarray analysis of retinal gene expression upon photic injury suggested increased expression of ceruloplasmin, a ferroxidase that could reduce retinal oxidative stress. Patients with acerul oplasminemia have retinal degeneration, indicating that ceruloplasmin is necessary for maintenance of retinal health. The purpose of this study was to determine whether retinal ceruloplasmin is upregulated following photo-oxidation, to localize ceruloplasmin protein, and to determine which ceruloplasmin isoform is present in the retina. METHODS: Balb/c mice were exposed to bright white light for seven hours. TUNEL labeling was used to detect photoreceptor apoptosis. At several intervals after the light injury, retinal ceruloplasmin was studied by quantitative PCR, immunohistochemistry, and western analysis. Expression of the secreted and expression of the membrane-anchored glycosyl phosphatidyl inositol (GPI) linked forms of ceruloplasmin were assesed in rat retina using primers specific for each form. Vitreous ceruloplasmin was detected by immunohistochemistry in Balb/c mouse eyes and by western analysis of aspirated vitreous from post-mortem human eyes. RESULTS: Retinal ceruloplasmin mRNA was upregulated eight-fold following photic injury. Ceruloplasmin protein was detected throughout normal retinas by immunohistochemistry, with a specific increase in Muller cell labeling following photic injury. Western analysis confirmed an increase in ceruloplasmin protein following photic injury and revealed eight-fold more ceruloplasmin protein in normal retina than in brain. The mRNAs for both the secreted and GPI linked forms of ceruloplasmin were detected by RT-PCR in the retina. Ceruloplasmin protein was detected by western analysis of normal human vitreous and was increased in mouse vitreous following photic injury. CONCLUSIONS: Ceruloplasmin, a retinal ferroxidase, is upregulated at the mRNA and protein levels upon light damage. The increased protein is primarily in Muller cells. Ceruloplasmin is considerably more abundant in retina than in brain. The retina expresses both the GPI-linked and secreted forms of ceruloplasmin, and since vitreous ceruloplasmin increases following photic injury, some of the retinal ceruloplasmin may be secreted into the vitreous. Ceruloplasmin may protect the retina from oxidative stress by decreasing the amount of ferrous iron available to produce reactive oxygen species.

Aged↗
PubMed2003

Differential distribution of vesicle associated membrane protein isoforms in the mouse retina.

PURPOSE: Many proteins associated with synaptic vesicle exocytosis are differentially distributed among synapses in the retina and elsewhere in the central nervous system. The synapse-specific distribution of these proteins and their isoforms is thought to contribute to synapse-specific functional differences. Vesicle-associated membrane protein (VAMP, also known as synaptobrevin) is an integral synaptic vesicle membrane protein that is part of the fusion core complex needed for docking and fusing of synaptic vesicles at the synaptic active zone. Two VAMP isoforms have been identified that are considered to be synaptic, VAMP-1 and VAMP-2, however their distributions among the various synapses in the mammalian retina have not been characterized. METHODS: Single- and double-labeling immunocytochemistry was used to investigate the distribution of the synaptic VAMP isoforms, VAMP-1 and VAMP-2, in the mouse retina. RESULTS: VAMP-2 was the predominant isoform in both synaptic layers. Double-labeling studies using conventional and ribbon-synapse-specific markers showed that VAMP-2 was broadly distributed among conventional and ribbon synapses. In contrast, the distribution of VAMP-1 was very limited. In the outer retina, only weak labeling was present in photoreceptor terminals. In the inner retina, labeling for VAMP-1 was found in the dendrites, cell bodies, and axons of some ganglion cells, as demonstrated by double labeling with the ganglion cell markers, microtubule-associated protein-1 and Brn-3a. VAMP-1 labeling did not colocalize with amacrine or bipolar cell markers, nor did it colocalize with other pre-synaptic markers, suggesting that VAMP-1 is not associated directly with neurotransmitter release in the inner retina. Labeling for VAMP-1 identified a set of large ganglion cells that ramified in the mid-IPL (inner plexiform layer), suggesting that they may show ON-OFF responses. Some of these cells had cell bodies displaced to the inner nuclear layer. The dendrites of the large VAMP-1-immunoreactive ganglion cells did not co-stratify with the cholinergic plexuses of the starburst amacrine cells (labeled for choline acetyltransferase) and therefore are unlikely to show directional selectivity. However, these cells are likely to receive input from bipolar cells and a population of putative glutamatergic amacrine cells. CONCLUSIONS: VAMP-1 and VAMP-2 are differentially distributed among the synapses of the mouse retina. VAMP-2 is the predominant isoform and is widely expressed at ribbon and conventional synapses in both plexiform layers. VAMP-1 expression in the mouse retina is much more limited and is not restricted to presynaptic terminals. In the OPL, VAMP-1 is co-expressed with VAMP-2 presynaptically in photoreceptor terminals. However, VAMP-1 expression in the IPL is associated with ganglion cells and does not appear to be localized to presynaptic terminals. VAMP-1 is a specific marker for a set of large ganglion cells and displaced ganglion cells that ramify in the mid-IPL and are likely to have ON-OFF physiology.

Animals↗
PubMed2003

[Relationship between vitrectomy and the morphology and function of the retina].

Pathological processes in the vitreous will be reflected in the morphology and function of the retina, and these processes can originate from sources outside the vitreous. The purpose of vitreous surgery is to remove the qualitatively and/or morphologically diseased vitreous. Successful vitrectomy will be manifested by an improvement in the structure and/or function of the retina. We have evaluated the morphology of the vitreoretinal interface, and the function of the retina before and after vitreous surgery. Plasmin-assisted vitrectomy was used in some cases to remove the diseased vitreous more efficiently and less invasively. The effect of this procedure was assessed by examining the morphology and function of the retina. First, the relationship between the qualitative and structural abnormality of the vitreous in macular diseases was studied. In aphakic/pseudophakic eyes with cystoid macular edema, there was a depression of retinal function over the entire retina which may have been caused by chemical mediators released into the vitreous. These mediators may have been produced by inflammation in the anterior segment of the eye. In eyes with an idiopathic macular hole, optical coherence tomographic (OCT) images suggested that the progression of the macular hole might depend on a balance between foveal adhesion and the posterior vitreous. Second, the efficacy, surgical damage, and limitations of vitreous surgery were investigated. The recovery of macular function was assessed by focal macular electroretinograms (FMERGs) after vitrectomy for epiretinal membrane, choroidal neovascularization, and diabetic macular edema. The concurrent examination by optical coherence tomography (OCT) suggested that a decrease in retinal thickness contributed to the functional recovery. Macular functional recovery was delayed and limited after macular translocation, diabetic macular edema, and internal limiting membrane peeling. Third, we studied the effect of plasmin-assisted vitrectomy on the retina. The plasmin was used to remove the vitreous more completely and less invasively. In rabbits, ERG, OCT, and histological examinations demonstrated that the use of commercially-available plasmin at a concentration used on human patients resulted in temporary adverse effects on the retina. For human patients, we purified the plasmin from the patients' serum at the Nagoya University Hospital and the activity was about the same as in previous reports. The purified plasmin was approved by our hospital's institutional review board, and written informed consent was obtained from each patient. Patients with macular edema, idiopathic macular hole, and epiretinal membrane without posterior detachment underwent plasmin-assisted vitrectomy and were evaluated morphologically and electrophysiologically. The efficacy of the plasmin in separating the vitreo-retinal interface was demonstrated by an occasional spontaneous posterior vitreous detachment with or without core vitrectomy, and the presence of less vitreous cortex attached to the internal limiting membrane that was removed during vitrectomy. This was the first histological demonstration of the effectiveness of plasmin in the living eye. Full-field ERGs before and after surgery demonstrated clear evidence that no alteration of retinal functional had occurred, although we did detect a possible osmotic effect by an increase in OCT-determined retinal thickness by the high-molecular weight autologous plasmin. In future studies, the proper concentration and reaction time for each condition of the vitreous should be determined. In conclusion, the pathophysiology of the vitreous should reflect the retinal function. The recovery of the structure of the retina is important for the recovery of retinal function. This should always be the primary goal of surgeons who perform quality surgery.

Animals↗
PubMed2005

[Erythropoietin administration protects retina against light-induced retinal degeneration].

OBJECTIVE: To study whether recombinant human erythropoietin can pass through mice blood-retina barrier and the protective role in light-induced damage in retina. METHODS: After the injection of rHEPO, the content of rHEPO in 24 BALB/c mice retina was examined by enzyme linked immunosorbent assay (ELISA). 24 BALB/c mice were used to establish a light-induced damaged model, the difference of retina in rHEPO group and control group was compared using light microscope and TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: The amount of retinal rHEPO in four deferent time points was (0.68 +/- 0.24) mU, (1.87 +/- 0.37) mU, (0.96 +/- 0.24) mU, (0.47 +/- 0.13) mU in 100 microg retinal total protein respectively by ELISA assay, there were statistical significances among groups. The density of rHEPO in the retina reached its peak at 4th hour after injection. Histology analysis: rHEPO group, at the 12th hour after light exposure the inner segment became condensed and disorganized. At the 36th hour the retina disorganized and vesiculated were seen in outer segments. At the 72nd hour the inner and outer segments were damaged more seriously and the outer nuclear layer became thinner and denser. On the 7th day, the retinal outer nuclear layer became thinner and condenses. rHEPO group showed a minimal damage in every time points but outer nuclear layer disorganized and vesiculated in inner and outer segments. No obvious changes in retinal thickness. The apoptotic cells were detected by TUNEL. At the 12th hour after light exposure, there were the apoptotic cells in outer nuclear layer near outer plexiform layer. At 36th hour the numbers of apoptotic cells were increased, however at the 72nd it was decreased obviously, only a few scattering apoptotic cells were revealed in the outer nuclear layer. Numbers of apoptotic cells between the rHEPO group and control group in outer nuclear layer were statistical significance (P < 0.01). CONCLUSIONS: rHEPO can pass through the mice blood-retina barrier and rHEPO has neuroprotective effect on mice retina. rHEPO may be used to treat degenerative retinal diseases.

Animals↗
PubMed2006

Expression and sequences of genes encoding glutamate receptors and transporters in primate retina determined using 3'-end amplification polymerase chain reaction.

PURPOSE: Our long-term goal is to compare how expression of glutamate receptor and non-vesicular transporter subunits differs between single neurons in the primate retina. Here we set out to ascertain general expression in the retina of Macaca fascicularis using a robust technique suitable for both levels of analysis. We constructed full-complement cDNAs from whole retina RNA using a protocol optimized for detection of even low-abundance transcripts and for transcripts of various lengths. We probed these libraries for expression of genes encoding the AMPA- (GluR1-4) and kainate-sensitive (GluR5-7, KA1-2) ionotropic glutamate receptors, the metabotropic glutamate receptors (mGluR1-8), and five non-vesicular glutamate transporters (EAAT1-5) in the macaque retina and brain and determined large portions of coding sequences for each. We also asked whether each gene can be detected in cDNA generated from a limited amount of RNA extracted from an aldehyde-fixed retinal slice, a technique useful for probing gene expression in tissue used for histological studies. METHODS: We constructed full-length cDNA from RNA harvested from the macaque retina using a modified version of the 3'-end amplification (TPEA) technique of Dixon et al. With this technique, the 3' region is amplified arbitrarily using multiple primers to produce amplified cDNA containing as diverse and complete sample of genes as possible. We probed the cDNA for expression of glutamate receptors and non-vesicular transporters using gene-specific RT-PCR and assembled sequences from the reaction products using a series of overlapping primer pairs. We also used TPEA to compare expression in a small amount of RNA extracted from a fixed retinal slice. RESULTS: Macaque retinal cDNA created using TPEA contains a high abundance of transcripts of various lengths. Gene-specific PCR using primers designed against human sequences indicates expression for all GluR, mGluR, and non-vesicular transporter subunits for which we probed. Expression patterns were similar between two different macaque retinas, but different in brain. Several differences also exist between macaque and human brain. For some subunits, this is the first demonstration of expression in the macaque retina. The expression pattern obtained probing cDNA libraries generated from fixed tissue RNA with a different primer set was similar to that for fresh tissue. We sequenced between 1865 (mGluR8a) and 3697 (mGluR1) total nucleotides for each macaque gene and obtained complete coding sequences for GluR1-7, mGluR3 and mGluR4. A comparison with the corresponding human sequences reveal that the coding region of macaque GluR6 demonstrates the highest homology with only 26 nucleotide substitutions (99% homology), while mGluR4 demonstrates the lowest with 68 substitutions (97.5%). CONCLUSIONS: Neural tissue cDNA created using TPEA contains diverse transcripts of varying size, abundance, and homology. We established using TPEA the expression of all nine AMPA- and kainate-sensitive GluRs, all eight mGluRs and all five non-vesicular glutamate transporters and found that expression differs between macaque retina, macaque brain and human brain. The nucleotide sequences for the coding regions differed moderately between the human and macaque genes. We also found a similar expression pattern in a smaller amount of RNA extracted from a fixed retinal slice. Thus, this technique could be useful for comparing gene expression in cells extracted from fixed tissue pre-labeled using specific markers.

3' Flanking Region↗
PubMed2006

Characterization of gene expression profiles of normal canine retina and brain using a retinal cDNA microarray.

PURPOSE: Construction of a canine retinal custom cDNA microarray for comprehensive retinal gene expression profiling and application for the identification of genes that are preferentially expressed in the retina and brain lobes using a brain pool reference tissue. METHODS: A cDNA microarray was constructed utilizing clones obtained from a normalized canine retinal expressed sequence tag library. Gene expression profiles were analyzed for normal retina, as well as the cortex of the frontal, occipital, and temporal brain regions. Each sample was studied against a reference sample of pooled brain RNA. Data from a quantified scanned image were normalized using the loess subgrid procedure. Retina-enriched genes were identified using the Significance Analysis of Microarrays (SAM) algorithm, and confirmed by northern blot analyses for selected genes. Differences between biological samples were displayed using principal component analysis (PCA). RESULTS: Expression profiles for each tissue set were analyzed against the common reference of pooled brain. Changes in expression between the sample and the reference were higher in the retina (27.9%) than the individual brain tissues (2-6.6%). Furthermore, all individual retinal samples were clearly separated from any of the hybridizations using brain tissue in the PCA. The accuracy of observed changes in expression has been confirmed by northern blot analysis using five randomly chosen genes that represented a wide range of different expression levels between retina and brain. CONCLUSIONS: We have established an accurate and robust microarray system suitable for the investigation of expression patterns in the retina and brain. Characterization of the gene expression profiles in normal retina will facilitate the understanding of the processes that underline differences between normal and diseased retinas.

Animals↗
PubMed1990

Distribution of photoreceptor subtypes in the retina of diurnal and nocturnal primates.

We have used antibodies specific to either the red/green-or blue-sensitive cones in order to compare their ratio and distributions to that of the rods in the retinae of 3 primate species that differ in their capacity for color vision. We have found that the monoclonal antibody CSA-1 (Johnson and Hageman, 1988) and the polyclonal antibody 4942A, specific to the red- and green-cone opsin (Lerea et al., 1989), applied to retinal whole-mounts labeled approximately 90% of all cones in the diurnal Old-World rhesus monkey (Macaca mulatta) and all of the cones in the nocturnal New-World owl monkey (Aotus trivirgatus) and nocturnal prosimian bushbaby (Galago garnetti). The polyclonal antibody 108B, specific to the blue-cone opsin (Lerea et al., 1989), labeled about 10% of the cones across the entire surface of the rhesus monkey retina, but failed to label any cones in the retina of the 2 nocturnal species. Only the retina of the rhesus monkey possessed an all-cone foveola in which the density of cone inner segments was 17-fold greater than that in the fovea of the owl monkey or bushbaby retina. Surprisingly, the density of cones per unit area outside of the fovea was comparable in all 3 species. Rod density in the dorsal retina was elevated in all animals examined, but was 2-3 times greater in the nocturnal species than in the rhesus monkey retina. Application of the photoreceptor-class-specific antibodies may provide further insights into the evolution and development of wavelength sensitivity in the retina, as well as enhance our understanding of normal and abnormal color vision in humans.

Animals↗
PubMed1987

Histone proteins in fetal and adult human retinas.

The DNA-binding histone proteins from the human retina are described for the first time. Retinas were obtained from male and female donors ranging in age from 11 to 72 years old. Retinas from two human fetuses at approximately four months of gestation were also examined for their histone content. Histones extracted from purified nuclei were separated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Eight histone fractions were identified in all retinal samples, and were quantified by scanning transmittance densitometry. These fractions included three subfractions of linker histones (H1a, H1b, H1(0)), four nucleosomal core histones (H2A, H2B, H3, H4), and the modified core histone, A24 (uH2A), comprised of H2A covalently bound to the non-histone protein, ubiquitin. When fetal and adult retinal histones were compared, the relative amounts of the linker histone subfractions proved to be different. In the adult retinas, H1a diminished in amount whereas H1b and H1(0) were increased. No alterations were detected in the core histones. The developmental changes in linker histones may be related to modifications of chromatin compaction which accompany cell differentiation. Species differences in the pattern of linker histones were detected when the nuclear proteins of human and mouse retinas were compared. Non-histone nuclear proteins are more abundant in the adult human retina than in the mouse retina or the fetal human retina.

Adult↗
PubMed1986

Melatonin-binding in the frog retina: autoradiographic and biochemical analysis.

Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.

Animals↗