Differential cell death and Bcl-2 expression in the mouse retina after glutathione decrease by systemic D,L-buthionine sulphoximine administration

Glutathione (GSH) plays a critical role in cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH decrease using [D, L]-buthionine sulphoximine (BSO) induces retinal cell death, but the underlying mechanisms of this are still unclear. Here, we demonstrated that retinal GSH level is closely related to retinal cell death as well as expression of an anti-apoptotic molecule, Bcl-2, in the retina. We induced differential expression of retinal GSH by single and multiple administrations of BSO, and examined retinal GSH levels and retinal cell death in vivo. Single BSO administration showed a transient decrease in the retinal GSH level, whereas multiple BSO administration showed a persistent decrease in the retinal GSH level. Retinal cell death also showed similar patterns: transient increases of retinal cell death were observed after single BSO administration, whereas persistent increases of retinal cell death were observed after multiple BSO administration. Changes in the retinal GSH level affected Bcl-2 expression in the retina. Immunoblot and immunohistochemical analyses showed that single and multiple administration of BSO induced differential expressions of Bcl-2 in the retina. Taken together, the results of our study suggest that the retinal GSH is important for the survival of retinal cells, and retinal GSH appears to be deeply related to Bcl-2 expression in the retina. Thus, alteration of Bcl-2 expression may provide a therapeutic tool for retinal degenerative diseases caused by retinal oxidative stress such as glaucoma or retinopathy.     

Plasma cortisol in men--relationship with atherosclerosis of retinal arteries

The production of cortisol increases in acute stress but the effects of chronic stress on plasma cortisol are still controversial. Stress on the other hand plays a role in coronary artery disease (CAD) and carotid atherosclerosis. Since there is no data about plasma cortisol and atherosclerosis of the retinal arteries, the purpose of this study was to explore the relationship between plasma cortisol in 101 adult males with the degree of their retinal vessels atherosclerosis. The results were compared with those in 47 matched apparently healthy men with no retinal vessels changes. The atherosclerotic changes of retinal vessels were determined by direct ophthalmoscopy and graded (1-4) according to Scheie. Morning plasma cortisol levels were determined by radioimmunoassay using commercial kits. The results were compared by using chi-square test. No association between morning plasma cortisol concentrations and retinal vessels atherosclerosis could be found. The results of this study do not support a role for physiological levels of plasma cortisol in the development of atherosclerosis, at least of the retinal arteries, in men.     

PKC/MAPK signaling suppression by retinal pericyte conditioned medium prevents retinal endothelial cell proliferation

Little is known about the regulation mechanism of endothelial cell proliferation by retinal pericytes. The purpose of this study was to elucidate the suppression mechanism of retinal capillary endothelial cell growth by soluble factors derived from retinal pericytes. Conditioned medium of retinal pericytes (rPCT1-CM) suppressed ischemia-induced retinal neovascularization. The growth and DNA synthesis of TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line, were suppressed in a concentration-dependent manner by concentrated rPCT1-CM. The number of human cultured endothelial cells was also reduced by rPCT1-CM. These results provide the first evidence that CM from the cultivation of pericytes alone can inhibit retinal neovascularization in vivo and in vitro. Although the growth reduction of TR-iBRB2 cells was only partly reversed by treatment of rPCT1-CM with antibodies to transforming growth factor-beta1, it was completely lost by heat-treatment of rPCT1-CM, suggesting that anti-angiogenic factors are soluble proteins. The levels of expression of G1/S-phase-related proteins, such as cyclin D1, cyclin-dependent kinase (cdk)4, cdk6, and proliferating cell nuclear antigen, were reduced and a cdk inhibitor, p21(Cip1), was induced in rPCT1-CM-treated TR-iBRB2 cells. Moreover, phosphorylated p44/42 mitogen-activated protein kinase (p44/42 MAPK) in TR-iBRB2 cells was reduced by rPCT1-CM treatment and phosphorylated protein kinase C (PKC)alpha/betaII, which is upstream of p44/42 MAPK, was also suppressed. In conclusion, CM from retinal pericytes suppresses PKC-p44/42 MAPK signaling, inhibits endothelial cell growth, and prevents retinal neovascularization. Anti-angiogenic factors derived from retinal pericytes are likely to play a critical role in the regulation of retinal endothelial cell growth.     

The influence of retinal on complement-dependent immune damage to liposomes

Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.     

Is lipoprotein(A) a risk factor for atherosclerosis of the retinal arteries?

Elevated plasma Lp(a) has been linked to development of coronary artery disease (CAD). There is no data about plasma Lp(a) and atherosclerosis of the retinal arteries. Therefore the purpose of this study was to assess the risk of retinal vessels atherosclerosis conferred by elevated plasma Lp(a) levels in 73 adult males. The results were compared with those in 45 matched apparently healthy males with no retinal vessel changes. The atherosclerotic changes of the retinal vessels were determined by direct ophthalmoscopy and graded (1-4) according to Scheie. Plasma levels of Lp(a) were measured by radial immunodiffusion. The results were compared using chi-square test. Although a very weak correlation between plasma Lp(a) levels and the incidence of retinal atherosclerosis was found, no significant association between the degree of atherosclerotic changes and plasma Lp(a) levels could be proven. Thus it could be concluded that plasma Lp(a) level is not a significant risk factor for atherosclerosis of the retinal arteries.     

Global and Ocular Hypothermic Preconditioning Protect the Rat Retina from Ischemic Damage

Retinal ischemia could provoke blindness. At present, there is no effective treatment against retinal ischemic damage. Strong evidence supports that glutamate is implicated in retinal ischemic damage. We investigated whether a brief period of global or ocular hypothermia applied 24 h before ischemia (i.e. hypothermic preconditioning, HPC) protects the retina from ischemia/reperfusion damage, and the involvement of glutamate in the retinal protection induced by HPC. For this purpose, ischemia was induced by increasing intraocular pressure to 120 mm Hg for 40 min. One day before ischemia, animals were submitted to global or ocular hypothermia (33°C and 32°C for 20 min, respectively) and fourteen days after ischemia, animals were subjected to electroretinography and histological analysis. Global or ocular HPC afforded significant functional (electroretinographic) protection in eyes exposed to ischemia/reperfusion injury. A marked alteration of the retinal structure and a decrease in retinal ganglion cell number were observed in ischemic retinas, whereas global or ocular HPC significantly preserved retinal structure and ganglion cell count. Three days after ischemia, a significant decrease in retinal glutamate uptake and glutamine synthetase activity was observed, whereas ocular HPC prevented the effect of ischemia on these parameters. The intravitreal injection of supraphysiological levels of glutamate induced alterations in retinal function and histology which were significantly prevented by ocular HPC. These results support that global or ocular HPC significantly protected retinal function and histology from ischemia/reperfusion injury, probably through a glutamate-dependent mechanism.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.     

视网膜神经节细胞的纯化和体外存活

We had used a specific anti-Thy 1.1 antibody binding method and a nylonmembrane sieve method to isolate and purify retinal ganglion cells from neonatal rats in order to compare the effect of tectal extract on these purified cells retinal ganglion cells. Isolated retinal cell suspension with retinal ganglion cells retrograde-prelabelled with Fast Blue were seeded on culture dishes coated with the specific anti-Thy 1.1 antibody for 30 minutes before nonadherent cells were removed. The percentage purity of the adherent retinal ganglion cells determined microscopically to be 95%. However, the percentage purity of the Fast Blue-labelled retinal ganglion cells recovered using the nylon membrane of pore size 15 microns was only 60 +/- 5%. Retinal ganglion cells purified by both methods could survive and grow into large, active neurons with neurite outgrowths in the presence of tectal extract. A MTT colorimetric microassay was used to quantify the survival growth activity of these purified retinal ganglion cells after culture for 24 hours. The result showed that the optical density ratio (+Te/-Te) of the retinal ganglion cells purified by anti-Thy 1.1 antibody binding method was 12.3 (0.111/0.009) and by the nylon membrane method was 6.4 (0.102/0.016), and the optical density ratio of the non-purified retinal cells was 3.8 (0.095/0.025), p less than 0.01 for all 3 sets of results. It was concluded that in the absence of other cells, the purified retinal ganglion cells responded specifically to the trophic activity in tectal extract, the purer the retinal ganglion cells and the clearer the effect.     

Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a co-culture system using retinal capillary endothelial cell line (TR-iBRB2)

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.     

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.     

Inhibitory role of miR-203 in the angiogenesis of mice with pathological retinal neovascularization disease through downregulation of SNAI2

BackgroundPathological retinal neovascularization is a disease characterized by abnormal angiogenesis in retina that is a major cause of blindness in humans. Previous reports have highlighted the involvement of microRNAs (miRNAs) in retinal angiogenesis. Therefore, we aimed at exploring the mechanism underlying miR-203 regulating the progression of pathological retinal neovascularization.MethodsInitially, the mouse model of pathological retinal neovascularization disease was established and the hypoxia-induced human retinal microvascular endothelial cells (HRMECs) were generated. Then, miR-203 and SNAI2 expression in HRMECs and retinal tissues was examined. Subsequently, the effects of miR-203 and SNAI2 on viability, migration, apoptosis and angiogenesis of HRMECs were investigated, with the expression of Bax, Ki-67, MMP-2, MMP-9, VEGF and CD34 measured. Finally, the regulation of miR-203 or SNAI2 on GSK-3β/β-catenin pathway was determined through examining the levels of phosphorylated p-GSK-3β and β-catenin.ResultsPoorly expressed miR-203 and highly expressed SNAI2 were found in HRMECs and retinal tissues of pathological retinal neovascularization. Importantly, overexpressed miR-203 or silencing SNAI2 inhibited viability, migration and angiogenesis but promoted apoptosis of HRMECs, evidenced by elevated Bax expression but reduced expression of Ki-67, MMP-2, MMP-9, VEGF and CD34. Moreover, overexpression of miR-203 was found to repress the GSK-3β/β-catenin pathway by downregulating SNAI2.ConclusionCollectively, this study demonstrated that overexpression of miR-203 suppressed the angiogenesis in mice with pathological retinal neovascularization disease via the inactivation of GSK-3β/β-catenin pathway by inhibiting SNAI2, which provided a novel therapeutic insight for pathological retinal neovascularization disease.     

Altered retinal metabolism in diabetes. I. Microanalysis of lipid, glucose, sorbitol, and myo-inositol in the choroid and in the individual layers of the rabbit retina

Total lipid, sucrose, glucose, sorbitol and myo-inositol contents in individual layers from normal and alloxan-diabetic rabbit retinas were measured using gravimetric and enzymatic microtechniques. Pure samples of nine retinal layers were microdissected from freeze-dried retinal cryosections. The lipid content was measured by loss of weight after ethanol and hexane extraction. Retinal lipid varied 3-fold across the retina and was not influenced by diabetes. Sucrose, glucose, sorbitol, and myo-inositol were measured with fluorimetric microassays. Sucrose infused intravenously prior to sampling the tissue did not traverse the outer blood retinal barrier of the normal or the diabetic retina. In both normals and diabetics, glucose followed a diffusional curve, with outer and inner retinal concentrations, respectively, equal to choroidal blood and vitreal glucose concentrations. Sorbitol was elevated in all retinal layers of diabetic animals. The peak sorbitol concentrations, of 2 mmol/kg defatted dry weight in diabetics, were not high enough to be osmotically significant. Retinal myo-inositol, of the order found in brain tissue (10-30 mmol/kg defatted dry tissue), was decreased by 22-40% in all retinal layers of the diabetics. The results indicate that diabetes affects the metabolism of retinal structures independently of the small blood vessel disease that is the hallmark of diabetes of long duration. It is conceivable that primary intraretinal metabolic alterations cause, aggravate, or perpetuate the well known degenerative processes that occur in retinal blood vessels in diabetes.     

Comparative analysis of the uptake and expression of plasmid vectors in human ciliary and retinal pigment epithelial cells in vitro.

The retinal pigment epithelium is uniquely suited to gene therapy that uses lipid-mediated DNA transfer due to its high phagocytic activity in situ. We compared the relative efficacy of phagocytosis on the uptake of labeled plasmid vectors by retinal pigment epithelial and ciliary epithelial cells in vitro. Relative levels of endocytosis were then compared with the efficiency of marker transgene expression in these cells. Human retinal pigment epithelial and ciliary epithelial cells from a single donor were isolated and expanded in vitro. Polyplex-mediated transfections were performed using a rhodamine-labeled expression vector for green fluorescent protein. Rhodamine-labeled endosomes were examined by fluorescence microscopy at different time points. Rhodamine labeling and green fluorescent protein expression were analyzed by flow cytometry 48 h after transfection. These gene transfer studies showed that expression of transgenes does occur in both human retinal pigment epithelial and ciliary epithelial cells in vitro. Endocytosis of labeled plasmid vectors occurs at a significantly higher number and density in retinal pigment epithelial cells than in ciliary epithelial cells (P < 0.04). However, the efficiency of marker transgene expression is similar in the two cell types. These studies demonstrate that the higher intrinsic phagocytic activity does not enhance the efficacy of transgene expression in retinal pigment epithelial cells in vitro. Both human retinal pigment epithelial and ciliary epithelial cells are competent recipients for lipid-mediated gene transfer, and transgene expression occurs at similar levels in both cell types.     

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.     

Basic Fibroblast Growth Factor Expression is Implicated in Mesenchymal Stem Cells Response to Light-Induced Retinal Injury

Neurotrophic factors are involved in neuroprotection and its expression in mesenchymal stem cells (MSCs) may change during light-induced retinal injury. In this study, neurotrophic factor expression in MSCs was investigated after stimulation by supernatants of homogenized retina (SHR) from normal and light-injured rats. Conditioned media from control MSCs (CM-MSCs), MSCs stimulated by normal SHR (CM-NSHR), and MSCs stimulated by light-injured SHR (CM-ISHR) were examined regarding their ability to prevent degeneration of retinal explants. Basic fibroblast growth factor (bFGF) in MSCs was knockdown by lentivirus-mediated mRNA interference. Transfected MSCs were stimulated by SHR, and retinal preservation was reevaluated in the resultant conditioned media. We detected significant up-regulation of bFGF in CM-ISHR, accompanied by superior retinal neurotrophic effects in CM-ISHR over CM-NSHR and CM-MSCs. Down-regulation of bFGF in MSCs effectively inhibited this protective effect. Adding neutralizing antibody against bFGF to CM-ISHR also induced a similar effect. It is thus concluded that retinal injury may enhance neurotrophic factor expression in MSCs and promote the repair process. bFGF may play a critical role in MSCs’ response to retinal injury.     

兔眼孔源性视网膜脱离手术复位后视功能以及超微结构的变化

目的对比研究兔眼视网膜脱离后选择不同时期手术复位视功能的变化情况,为临床手术时机的选择及预测术后视功能的恢复情况提供理论与实验依据。方法利用家兔制备孔源性视网膜脱离模型,成模后1 d、7 d1、4 d时经手术达解剖复位,采用多焦视网膜电流图检测复位后视网膜的功能,数据处理应用SPSS软件。制备组织病理学切片。结果多焦视网膜电流图显示1 d、7 d、14 d的RRD手术复位后P1波平均象限反应密度(QAP1,nV/deg2),P1波幅值(AP1,μV),N1波幅值(AN1,μV),P1波潜伏期(TP1,ms),N1波潜伏期(TN1,ms)各项数值差异有显著性(P<0.05)。光镜电镜显示视网膜脱离复位后组织病理学改变。结论视网膜脱离手术复位后视网膜细胞功能的恢复与脱离时间有明显的负相关性;多焦视网膜电流图对于局部视网膜功能的评价有重要意义;组织病理学研究提供了解释视功能变化的重要依据。     

Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-d-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p–c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.     

Chitosan Oligosaccharides Attenuates Oxidative-Stress Related Retinal Degeneration in Rats

This study investigated the therapeutic potential and mechanisms of chitosan oligosaccharides (COS) for oxidative stress-induced retinal diseases. Retinal oxidative damage was induced in Sprague-Dawley rats by intravitreal injection of paraquat (PQ). Low-dose (5 mg/kg) or high-dose (10 mg/kg) COS or PBS was intragastrically given for 14 days after PQ injection. Electroretinograms were performed to determine the functionality of the retinas. The surviving neurons in the retinal ganglion cell layer and retinal apoptosis were determined by counting Neu N-positive cells in whole-mounted retinas and TUNEL staining, respectively. The generation of reactive oxygen species (ROS) was determined by lucigenin- and luminol-enhanced chemiluminescence. Retinal oxidative damages were assessed by staining with nitrotyrosine, acrolein, and 8-hydroxy-2''-deoxyguanosine (8-OHdG). Immunohistochemical studies were used to demonstrate the expression of nuclear factor-kappa B (NF-κB) p65 in retinas. An in vitro study using RGC-5 cells was performed to verify the results. We demonstrated COS significantly enhanced the recovery of retinal function, preserved inner retinal thickness, and decreased retinal neurons loss in a dose-dependent manner. COS administration demonstrated anti-oxidative effects by reducing luminol- and lucigenin-dependent chemiluminenscense levels and activating superoxide dismutase and catalase, leading to decreased retinal apoptosis. COS markedly reduced retinal NF-κB p65. An in vitro study demonstrated COS increased IκB expression, attenuated the increase of p65 and thus decreased NF-κB/DNA binding activity in PQ-stimulated RGC-5 cells. In conclusion, COS attenuates oxidative stress-induced retinal damages, probably by decreasing free radicals, maintaining the activities of anti-oxidative enzymes, and inhibiting the activation of NF-κB.     

A COMPLETE RECONSTRUCTION OF THE NEURAL RETINA OF CHICK EMBRYO GRAFTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

When dissociated neural retinal cells of 6-to 10-day-old chick embryos were grafted as a pellet onto the chorio-allantoic membrane and allowed to develop, complete retinal structures were reconstructed. Especially when the retinal cells of 6-day-old embryos were used, well orientated retinal structures, which possesed three nuclear layers and two plexiform layers, were formed. The fundamental steps in this complete reconstruction were as follows; rosette formation, formation of a fibrillar lumen, differentiation of receptor and ganglion cells, fusion of the fibrillar lumen, fusion of the receptor lumen and finally the formation of a three-layered neural retina. Reconstruction by the retinal cells of older embryos was less complete. This stagedependent difference in the capacity for reconstruction was due to a difference in the ability to form well developed rosettes at an early phase of the process of reconstruction.