Ontogenic changes of kynurenine aminotransferase I activity and its expression in the chicken retina

Kynurenine aminotransferases are key enzymes for the synthesis of kynurenic acid (KYNA), an endogenous glutamate receptor antagonist. The study described here examined ontogenic changes of kynurenine aminotransferase I (KAT I) activity and its expression in the chicken retina. KAT I activity measured on embryonic day 16 (E16) was significantly higher than at all other stages (E12, P0 and P7). Double labeling with antibodies against glutamine synthetase showed that on P7 KAT I was expressed in Müller cell endfeet and their processes in the inner retina. Since KAT I activity is high in the late embryonic stages, it is conceivable that it plays a neuromodulatory role in the retina during the late phase of embryogenesis.     

Age-dependent decrease of retinal kynurenate and kynurenine aminotransferases in DBA/2J mice, a model of ocular hypertension

The study examines age-dependent changes of kynurenic acid (KYNA) content and kynurenine aminotransferases (KAT I and KAT II) celluar expression in the retinas of DBA/2J mice. Retinas were obtained from DBA/2J mice of different ages (3, 6 and 11 months). C57BL6 mice were used as controls. As measured with HPLC, KYNA content decreased (p < 0.01) in the retinas of 6-month-old DBA/2J mice and continued to decrease (p < 0.0074) in the retinas of 11-month-old animals compared to the controls. Immunohistochemistry showed that expression of both KAT I and KAT II decreased markedly in the retinas of 11-month-old DBA/2J mice compared to controls. The impairment in KYNA biosynthesis in the retinas of DBA/2J mice may be one of the mechanisms of retinal neurodegeneration related to ocular hypertension.     

Increased activity of cyclooxygenase-2 signals early neurodegenerative events in the rat retina following transient ischemia

Following transient retinal ischemia, there is neuronal cell death, breakdown of the blood-retinal barrier, activation of microglia and infiltration by hematogenous cells. The early appearance of cyclooxygenase-2 (COX-2) following an ischemic event may be responsible for signaling some of the responses that lead to neurodegeneration. We have determined the time courses of changes in protein levels and cellular localizations of COX-2 in the rat retina after transient ischemia. In the normal rat retina, COX-2 immunoreactivity was present in neurons in the INL and ganglion cell layer (GCL). Six to 12 hr after ischemia, COX-2 immunoreactivity was upregulated/induced in horizontal cells, amacrine cells, retinal ganglion cells, displaced amacrine cells of the INL and GCL, and Müller cells. The NMDA-receptor antagonist, MK801, blocked the increased COX-2 protein level and COX-2 immunoreactivity in neurons of the INL and GCL, but did not affect the induction of COX-2 in Müller cells after ischemia. The selective COX-2 inhibitor, SC-58236, prevented apoptotic cell death and was neuroprotective against loss of retinal ganglion cells after ischemia. Following transient ischemia, the selective COX-2 inhibitor did not prevent breakdown of the blood-retinal barrier or activation of microglia. However, the selective COX-2 inhibitor reduced infiltration of hematogenous cells into the retina. These results suggest that the early, increased activity of COX-2 signals neurodegenerative events following retinal ischemia.     

Long-term glial reactivity in rat retinas ipsilateral and contralateral to experimental glaucoma

Although glaucoma is known to alter glial reactivity, the long-term effect of elevated intraocular pressure (IOP) on glial change has not been fully elucidated. This study aimed to examine how chronically elevated IOP induced by episcleral vein cauterization (EVC) in unilateral eyes affect reactivities of astrocytes and Müller cells of rats in the treated as well as contralateral eyes over time. EVC in unilateral eyes of Sprague-Dawley rats were performed to produce chronically elevated IOP. Flat mounted retina preparations were made at several points until 6 months, which were subjected to immunostaining for glial fibrillary acidic protein (GFAP). Retinal homogenates were one- or two-dimensionally electrophoresed, followed by GFAP immunoblotting. EVC significantly increased IOPs up to 27.8 from 13.1 mmHg, which gradually decreased over time. In flat mounted retinas, astrocytes lost but Müller cells gained GFAP immunoreactivity at 3 days after cauterization. The glial changes were partially reversed over time but last even after IOP normalization. In the contralateral eyes, similar glial changes gradually appeared at 1 month after EVC and thereafter. Immunoblotting demonstrated not only molecular size shifts but also alteration of isoelectric focusing of GFAP both in treated and contralateral retina as compared with age-matched control retina. EVC led to opposite reactions in astrocytes and Müller cells in terms of GFAP immunoreactivity. Late-onset glial reactivity also occurred in the contralateral retina.     

Altered expression and interaction of adherens junction proteins in the developing OLM of the Rho(-/-) mouse

Retinitis Pigmentosa (RP) represents a major cause of progressive retinal disease worldwide and comprises a heterogeneous group of inherited diseases that are characterised by primary degeneration of rod photoreceptors and secondary degeneration of cone photoreceptors in the retina. The outer limiting membrane (OLM) which allows for the interaction of photoreceptors with surrounding photoreceptors and Müller cells is compromised in many degenerative retinal diseases. Using indirect immunostaining of retinal cryosections from C-129 Wild Type (WT) and C-129 Rho(-/-) mice, we have determined levels of expression of the adherens junction associated proteins ZO-1, beta-catenin and p120-catenin at the OLM from newborn and 1, 2, 3, 4 and 5-week old animals. We have also used immunoprecipitation analysis to determine changes in the association of E-cadherin with ZO-1, beta-catenin and p120-catenin and the association of alpha-catenin with ZO-1 and beta-catenin at these time points in WT and Rho(-/-) mice. We have found that ZO-1 expression at the OLM is present in WT and Rho(-/-) mice after 2weeks, but that levels of expression at the OLM decrease after this time point in the Rho(-/-) mice. beta-catenin expression in the Rho(-/-) mice became compromised at the OLM after 3 weeks, showing a distinct change in staining pattern after 4 weeks and no staining at the OLM after 5 weeks. Moreover, we have shown that p120-catenin expression is not evident at the OLM of the Rho(-/-) mice at the 4 or 5 week time point. To complement this data, we have performed immunoprecipitation analysis on neural retinal lysates from WT and Rho(-/-) mice and herein report fluctuations in the association of E-cadherin with ZO-1, and beta-catenin, while showing that the interaction of E-cadherin with p120-catenin is not established in the retina of C-129 WT and Rho(-/-) mice until 4 weeks after birth and remains un-changed up to and including 5 weeks after birth. Meanwhile, we report that the association of alpha-catenin with ZO-1 is decreased in retinas of the Rho(-/-) from newborn animals up to and including 5 weeks after birth. We have also shown that the association of alpha-catenin and beta-catenin is not well established in WT and Rho(-/-) mice until at least 5 weeks after birth. We hypothesize that these retinal changes at the OLM may contribute significantly to the pathogenesis of retinal degenerations and may represent a unique therapeutic target for intervention in conditions involving rapid photoreceptor cell death.     

Localization of voltage-sensitive L-type calcium channels in the chicken retina

L‐type calcium channels have been associated with synaptic transmission in the retina, and are a potential site for modulation of the release of neurotransmitters. The present study documents the immunohistochemical localization of neuronal α1 subunits of L‐type calcium channels in chicken retina, using antibodies to the α1c, α1d and α1f subunits of L‐type calcium channels. The α1c‐like subunits were localized to Müller cells, with predominantly radial processes, and a prominent band of horizontal processes in the outer plexiform layer. The antibody to α1d subunits labelled most, if not all, cell bodies. The antibody to a human α1f subunit strongly labelled photoreceptor terminals. Fainter immunoreactivity was detected in the inner segments of the photoreceptors, a subset of amacrine cells, two bands of labelling in the inner plexiform layer and many ganglion cells. The differential cellular distributions of these α1‐subunits suggests subtle functional differences in their roles at different cellular locations.     

Atrial natriuretic peptide inhibits osmotical glial cell swelling in the ischemic rat retina: dependence on glutamatergic-purinergic signaling

Atrial natriuretic peptide (ANP) is a regulator of the water and electrolyte content in the brain which also mediates cell volume homeostasis. Here, we determined whether the expression of ANP in the retina of the rat undergoes changes during ischemia-reperfusion, and whether ANP affects the osmotic swelling of Müller glial cells in postischemic retinas under hypotonic conditions. Transient retinal ischemia was induced by elevation of the intraocular pressure above systolic blood pressure for 1h. At 1 and 3 days after reperfusion, there was an increased content of ANP protein in the retina, as determined by Western blotting. The increase of the retinal ANP content was markedly reduced when triamcinolone acetonide (10 mM in 2 microl vehicle) was intravitreally injected before ischemia. ANP inhibited the osmotic swelling of Müller cell somata in retinal slices. The effect of ANP was mediated by activation of NP receptors expressed by retinal neurons which evoked a release of glutamate. The stimulation of metabotropic glutamate receptors expressed by Müller cells evoked an autocrine purinergic signaling mechanism that resulted in the opening of K(+) and Cl(-) channels; the ion efflux counteracted the osmotic swelling of Müller cells. It is concluded that the expression of ANP is transiently upregulated in the postischemic retina of the rat. The increased expression of ANP may represent a part of the retinal response to transient ischemia and may inhibit cytotoxic glial cell swelling.     

NADPH diaphorase activity in the rat retina during the early stages of experimental diabetes

Background Nitric oxide (NO) plays an important physiological role in inter-cellular communication, but when produced in excess it can become toxic. Our goal was to evaluate possible involvement of NO in the development of retinopathy in diabetic rats.Methods Diabetes was induced in male albino rats by intravenous injection of streptozotocin. Some of the normal and diabetic rats were raised with added L-arginine to increase in vivo NO synthesis, or with added L-NAME to inhibit the rate of in vivo NO synthesis. NADPH diaphorase histochemistry was conducted on retinal whole mounts and transverse sections at different time intervals after induction of diabetes. The electroretinogram (ERG) was recorded to assess retinal function.Results After 6 weeks of diabetes, NADPH diaphorase amacrine cells in the diabetic retinas appeared abnormal in their morphology and the degree of staining was decreased in their processes. In contrast, NADPH diaphorase activity was augmented in Müller cells. Supplementing the rats' diet with L-arginine for 10 weeks slightly reduced NADPH diaphorase activity in amacrine cell in normal rats but had no effect on the diabetic rats. Adding L-NAME for 10 weeks did not alter NADPH diaphorase histochemistry in either normal or diabetic rats. The ERG responses were reduced by L-arginine supplementation in normal and diabetic rats, and were unaffected by adding L-NAME to the drinking water.Conclusions Our findings are consistent with the hypothesis that high glucose levels are deleterious to the rat retina and that excessive synthesis of NO may contribute to the development of diabetic retinopathy.     

Presence and distribution of l‐kynurenine aminotransferases immunoreactivity in human cataractous lenses

Purpose: To investigate the presence and distribution of l ‐kynurenine aminotransferases immunoreactivity in human and animal lenses during cataract formation. Methods: Immunohistochemistry was conducted using polyclonal antibodies against KAT I, KAT II and KAT III on sections of 26 anterior capsules from patients undergoing surgical treatment of anterior subcapsular cataract (ASC) and 22 cataractous lenses from human eyes enucleated because of choroidal malignant melanoma. Additionally, the eyes of 11‐month‐old DBA/2J mice (6 eyes) were investigated (with KAT I and II). Ten clear human lenses and four BL6 mice lenses were used as controls. Spatial immunoreactivity patterns of enzymes were compared with Periodic Acid – Schiff (PAS)‐stained sections. Results: Immunohistochemical analysis revealed presence of KAT I, KAT II and KAT III in extracellular structures of all studied types of cataract in human eyes showing specific pattern of the stain. In cortical cataract, immunoreactivity was observed on cortical lens fibres. In nuclear cataract, KAT II revealed stronger and diffused staining than KAT I. Additionally, both KAT showed more pronounced staining at the edge of small clefts. In normal human lenses, KAT I, II and III, immunoreactivity was not observed. Presence of KAT I and KAT II in the intercellular substance of DBA/2J mice cataract was observed. In BL6 mice lenses without cataract, only weak KAT I and KAT II staining was observed. Conclusions: Presence of l ‐kynurenine aminotransferases in extracellular matrix (ECM) during human cataract formation suggests that products of l ‐kynurenine pathway might be involved in mechanisms of cataractogenesis.     

Expression of glycosaminoglycans during development of the rat retina

PURPOSE: To investigate the spatiotemporal expression of glycosaminoglycans during development of the rat retina. METHODS: Hyaluronan and sulfated glycosaminoglycans, including chondroitin sulfate, heparan sulfate and keratan sulfate were detected using biotinylated hyaluronan binding protein, immunohistochemical analysis, respectively, in the rat retina at various stages of development. RESULTS: Hyaluronan was expressed in the nerve fiber layer, inner plexiform layer and outer plexiform layer during early postnatal stages (postnatal day 1-14; P1-P14) and was undetectable after P21. In contrast, hyaluronan was faintly observed in the photoreceptor layer on P7, and gradually increased up to P49. The spatiotemporal expression pattern of chondroitin sulfate was similar to that of hyaluronan. Heparan sulfate was also detected in the nerve fiber layer, inner plexiform layer and outer plexiform layer during early postnatal stages (P1-P14). In addition, heparan sulfate was expressed in the inner limiting membrane during all stages of development. Keratan sulfate was not detected in the retina at any stage of development. CONCLUSIONS: Hyaluronan, chondroitin sulfate and heparan sulfate are expressed in nerve fiber-rich layers during early postnatal stages and may regulate neurite outgrowth. In adulthood, both hyaluronan and chondroitin sulfate are expressed in the photoreceptor layer and may consist of the interphotoreceptor matrix. In addition, heparan sulfate is expressed in the inner limiting membrane throughout the various stages of development and may be associated with the structure of the inner limiting membrane.     

大麻素受体在大鼠视网膜中的表达和分布

目的 研究大麻素CB1和CB2受体在幼年、成年和老龄大鼠视网膜组织的表达和分布.方法 研究采用免疫组织化学、Western印迹法和视网膜神经节细胞的上丘逆行追踪标记技术.结果 CB1和CB2受体在成年大鼠视网膜上均有较为丰富的表达,其中CB1受体的主要表达部位是外核层（ONL）、内核层（INL）和视神经节细胞层（GCL）,以及外网状层（OPL）和内网状层（IPL）;CB2的主要表达部位是GCL、INL、ONL的细胞膜和OPL,以及Müller胶质细胞的终足和突起上.免疫荧光双标结果显示,CB1和CB2受体在几乎所有的大鼠视网膜神经节细胞均有表达.CB1和CB2受体在幼年和老龄大鼠视网膜的表达分布与成年大鼠相似.结论 大麻素CB1和CB2受体在大鼠视网膜广泛分布和表达,从幼年到老龄,其表达和分布相似,提示在出生后早期这些受体的发育已经基本完成.大麻素受体系统在视网膜信息处理的调控和视网膜神经保护中可能发挥重要作用.     

Changes in Musashi-1 subcellular localization correlate with cell cycle exit during postnatal retinal development

RNA-binding proteins, and in particular, the Musashi genes, function as essential regulators of progenitor functioning in both the developing and adult organism. In this report, we characterize the differential subcellular distribution of Musashi-1 in cells engaged in either proliferating or differentiating contexts in the developing mouse retina, and in cultured Müller glia. During retinal cell differentiation, Musashi-1 immunoreactivity shifts from exclusively cytoplasmic in retinal progenitor cells, to predominantly nuclear localization in differentiating neurons. This nuclear shift is transient, with localization in the adult retina becoming predominantly perinuclear and cytoplasmic in Müller glia and photoreceptors. A correlation between cell cycle progression and subcellular distribution of Musashi-1 is observed in passageable, adult Müller glial cells in vitro. Furthermore, treatment of Müller cultures with neuron-promoting differentiation media induces asymmetric cytoplasmic Musashi-1 immunoreactivity in dividing daughter cells. The observed shifts in subcellular Musashi-1 localization are consistent with contrasting roles for Musashi-1 during cell proliferation and differentiation. These data provide evidence that nuclear, and cytoplasmic sequestering of Musashi-1 in retinal cells is context-specific, and may contribute to downstream functioning of Musashi-1.     

Notch信号通路在视网膜细胞发育及血管生成中的作用

视网膜细胞变性和新生血管形成存在于多种常见眼病的发生和发展过程中,严重损害患者视力,甚至致盲.多项研究结果表明Noah信号通路参与调控多种器官和组织的发育及细胞分化过程,因此,有必要就Notch信号通路的组成及其在视网膜细胞发育和血管发生过程中的作用进行综述,以期为视网膜疾病的防治研究提供新的思路和作用靶点.     

Induction of nestin, Ki-67, and cyclin D1 expression in Müller cells after laser injury in adult rat retina

Purpose The purpose was to examine the expression of nestin, Ki-67, and cyclin D1 in Müller cells after laser injury in adult rat retina. Methods The right eyes of adult Brown Norway rats were treated with laser photocoagulation. The eyes were removed 3, 7, and 14 days after laser treatment. The retinas were investigated immunocytochemically by confocal microscopy. Agarose-embedded sections were immunostained with antibodies to nestin, vimentin, glial fibrillary acidic protein (GFAP), glutamate-aspartate transporter (GLAST), rhodopsin, Ki-67, and cyclin D1. Cell death was examined using terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay on agarose sections. Results Nestin expression was induced in Müller cells following laser injury. In addition, Ki-67 and cyclin D1 expression was found in the nuclei of Müller cells after the treatment. TUNEL assay demonstrated that Müller cells were not labeled; hence these cells were not apoptotic. Conclusions These results suggest that dedifferentiation and proliferation of Müller cells can be induced by laser injury in adult rat retina.     

A novel cone visual cycle in the cone-dominated retina

The visual processing of humans is primarily reliant upon the sensitivity of cone photoreceptors to light during daylight conditions. This underscores the importance of understanding how cone photoreceptors maintain the ability to detect light. The vertebrate retina consists of a combination of both rod and cone photoreceptors. Subsequent to light exposure, both rod and cone photoreceptors are dependent upon the recycling of vitamin A to regenerate photopigments, the proteins responsible for detecting light. Metabolic processing of vitamin A in support of rod photopigment renewal, the so-called "rod visual cycle", is well established. However, the metabolic processing of vitamin A in support of cone photopigment renewal remains a challenge for characterization in the recently discovered "cone visual cycle". In this review we summarize the research that has defined the rod visual cycle and our current concept of the novel cone visual cycle. Here, we highlight the research that supports the existence of a functional cone-specific visual cycle: the identification of novel enzymatic activities that contribute to retinoid recycling, the observation of vitamin A recycling in cone-dominated retinas, and the localization of some of these activities to the Müller cell. In the opinions of the authors, additional research on the possible interactions between these two visual cycles in the duplex retina is needed to understand visual detection in the human retina.     

A spatial analysis of on- and off-ganglion cells in the cat retina

Using reduced silver staining methods it was possible to stain all α-ganglion cells of the cat retina. The dendritic trees of α-cells are unistratified in either of two laminae within the inner plexiform layer. This stratification difference was shown physiologically to correspond to the on-off dichotomy. For all α-cells recorded, the dendritic field was covered by the corresponding receptive field centre. In addition the general shape of the receptive field centre corresponded to the shape of the dendritic field. The size of the dendritic tree was always smaller than the receptive field centre. The topographical distribution of on- and off-α cells could be studied. They were found to occur in about equal numbers. Both on- and off-α-cell perikarya form a regular lattice and both lattices are superimposed independently. The dendritic branches of neighbouring α-cells overlap and each retinal point is covered by the dendritic field of at least one on- and one off-α-cell.After horseradish peroxidase (HRP) injection into the lateral geniculate nucleus all β-cells were labelled. In this way it is shown that about 55% of all ganglion cells are β-cells. The mosaic of on- and off-β-cells was studied from the HRP-labelled material. It is commonly assumed that beta-cells are associated with the resolution of fine detail in the cat visual system. The mosaic of β-cells imposes some constraints and permits some predictions to be made with respect to the cat's visual discrimination.     

Regulation of brain-derived neurotrophic factor on the expression of GLAST protein and its function in retinal Müller cells of mice

Objective To study the effect of brain-derived neurotrophic factor (BDNF)on the expression of L-glutamate/L-aspartate transporter (GLAST) protein and its function in the retinal mice at 3 to 7 days postnatal were cultured by an enzymatic digestion method, and the third passage different concentrations of recombinant human BDNF (50, 75, 100, 125 and 150 ng/ml) for 24 h in group and the control group. The expression of GLAST protein was analyzed with one-way analysis of variance (ANOVA) and L-[3,4-3H]-glutamic acid uptake was analyzed with independent samples t test. Results The expression of GLAST protein in the control group was 0.151±0.025 and the expression in the BDNF group (50, 75, 100, 125 and 150 ng/ml) was 0.331±0.076, 0.413±0.110, 0.497±0.080, 0.411±0.072, and 0.319±0.084, respectively. Different concentrations of BDNF could up-regulate the expression of GLAST protein compared to the control group (F=6.793, P=0.003).The expression of GLAST protein reached a maximum when the concentration of BDNF was 100 ng/ml.L-[3,4-3H]-glutamic acid uptake for the 100 ng/ml BDNF group and control group was 81 213±significantly higher than for the control group (t=6.462, P=0.023). Conclusion BDNF can up-regulate the expression of GLAST protein and increase extracellular glutamate uptake.     

Effect of triiodothyronine on glutamine synthetase in the developing rat retina.

It has been previously demonstrated that triiodothyronine (T3) causes a precocious increase in glutamine syntehtase (GS) activity in the developing retina as measured by the glutamyltransferase (GT) reaction. To determine its distribution and the mechanism of its increased activity an immunohistochemical study was performed in retinas of 1 to 24-day-old rats given a subcutaneous injection of T3 on the day of birth. No difference was seen between T3-treated rats and controls on postnatal days 1 and 2. However, there was significantly less stain in T3-injected animals than in control animals especially in the pigment epithelium on day 5 and in Müller cells on days 8. By days 12 and 24 no difference was observed between experimental and control rats. We were thus unable to demonstrate increased synthesis of GS to correlated with its increased activity following T3 administration. On the contrary, we obtained evidence of decreased GS synthesis. It is suggested that T3 either causes both increased GT activity and decreased GS synthesis or that the T3-induced elevation of GS activity results in decreased synthesis of GS presumably through end-product inhibition.     

Glial cell-mediated spread of retinal degeneration during detachment: a hypothesis based upon studies in rabbits

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.     

The radial glia of Müller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein.

The glial response to injury was studied in the rat retina by immunofluorescence with GFA antisera. In the normal rat only the glia limitans on the inner surface of the retina was positive by immunofluorescence. Following interruption of the optic nerve or multiple penetrating wounds of the eye the radial fibres of Müller became intensely stained. It is concluded that Müller glia are similar to brain astrocytes with respect to their reaction to injury, that is they accumulate GFA protein.