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Background Studies show that retinal neurodegeneration may precede retinal microvascular changes in diabetes mellitus.The apoptosis of retinal ganglion cells (RGCs) is an early finding in retinal neurodegeneration.Toll-like receptor 4 (TLR4) is proved to be up-regulated in diabetic rats retina.However,the impact of TLR4 on RGCs damage in retinal neurodegeneration is poorly understood.Objective The aim of this study was to investigate the expressing change of TLR4 induced by high glucose in RGCs in order to offer a basis for the prevention diabetic retinal neurodegeneration and the study on targeting drugs.Methods RGCs were isolated and purified from the retinas of SPF SD rats aged postnatal 1-3 days by using papain digestion method and then were identified by immunofluorescence technology to detect the expression of Brn3a,a specific marker of RGCs.The cells were divided into normal control group and 10,20,30 mmol/L glucose groups.The expressions of TLR4 mRNA and protein in the ceils were detected by real-time fluorescence quantitative PCR and Western blot analysis in 24 and 48 hours after addtion of glucose.All procedures performed in studies were in accordance with the Association for National Institutes of Health (NIH) Statement for the Care and Use of Laboratory Animals recommendations.The protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University.Every effort was made to minimize animal discomfort and stress.Results The normal cells grew well with the shape of near roundness after inoculaton.The cells were gradually enlarged and clustered with obvious axons and dendrites 24 hours after purifying.Brn3a showed the positive expression in cultured cells.At 24 hours and 48 hours after glucose culture,the cell structures were gradually invisible in most cells.The expressions of TLR4 mRNA in the cells were 0.945 ±0.237,1.180±0.193 and 0.827±0.213 at 24 hours and 1.509±0.422,2.433±0.617 and 1.435±0.410 at 48 hours after culture in the 10,20 and 30 mmol/L glucose groups,respectively,which were significantly higher than 0.600±0.099 and 0.724±0.302 in the normal control group (all at P＜0.01).The expressions of TLR4 protein in the cells were 0.442±0.147,0.626±0.128 and 0.330±0.153 at 24 hours and 0.464±0.121,0.930±0.441 and 0.394±0.158 at 48 hours after culture in the 10,20 and 30 mmol/L glucose groups,respectively,which were significantly higher than 0.090±0.050 and 0.094±0.070 in the normal control group (all at P＜0.01).Conclusions A large number of RGCs die in a high-glucose environment in vitro,meanwhile,the expression of TLR4 up-regulates in the cells,indicating that TLR4 maybe participate in the damage of RGCs induced by high glucose.
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Background The establishment of chronic ocular hypertension is a basis for the research of glaucoma.Previous laser photocoagulation method to establish ocular hypertension model showed obvious fluctuation of intraocular pressure (IOP) and complications and need repeatedly photocoagulation.Improvement of modeling method is of important significance for glaucoma.Objective This study was to establish chronic ocular hypertension rat models by transgoniscope laser photocoagulation to trabecular meshwork and to compare this method with previous translimbal laser photocoagulation.Methods Thirty-six 8 to 12-week-old clean grade Fischer344 rats were collected and divided into normal control group,translimbal laser photocoagulation group and transgoniscope laser photocoagulation group,12 rats for each group.Five hundred and thirty-two nm YAG laser was used to photocoagulate trabecular meshwork translimbally in the right eyes of rats in the translimbal laser photocoagulation group,with the laser power 440-500 mW and spots 40-60,and the photocoagulation was perfored transgoniscopely in the right eyes of rats in the transgoniscope laser photocoagulation group,with the laser power 800-850 mW and spots 100-120.IOP was measured by using Tonolab tonometer in all the rats after modeling.The rats were sacrificed 3 weeks after modeling and retinas were isolated,the Tuj-1 positive retinal ganglion cells (RGCs) were counted by immunofluorescence technology.The use and care of the animals followed the Statement of ARVO.Results The successful rate of establishement of models was 75％ in the translimbal laser photocoagulation group and 100％ in the transgoniscope laser photocoagulation group.The mean IOP was (11.0±1.3),(23.4±12.6) and (25.3± 4.9) mmHg,and the peak IOP was (12.3 ± 1.0),(50.5 ± 7.3) and (44.3 ± 12.3) mmHg in the normal control group,transgoniscope laser photocoagulation group and translimbal laser photocoagulation group,respectively,with significant differences among the groups (F=25.496,80.762,both at P＜0.001),and the mean IOP was significantly higher in the transgoniscope laser photocoagulation group and translimbal laser photocoagulation group than that in the normal control group (all at P＜0.001),and no significant differences in the mean and peak IOP between transgoniscope laser photocoagulation group and translimbal laser photocoagulation group (P=1.000,P=0.195).The numbers of Tuj-1 positive RGCs in the retinas were (2 048.2± 148.5),(645.2 ± 177.1) and (1 223.7 ± 148.6)/mm2 in the normal control group,transgoniscope laser photocoagulation group and translimbal laser photocoagulation group,showing a significant difference among the groups (F=98.767,P＜0.001).The number of Tuj-1 positive RGCs was considerably reduced in the transgoniscope laser photocoagulation group and translimbal laser photocoagulation group compared with the normal control group and the number of Tuj-1 postive RGCs was low in the translimbal laser photocoagulation group compared with the transgoniscope laser photocoagulation group (all at P＜0.001).Conclusions Transgoniscope laser photocoagulation targeting trabecular meshwork can induce chronic ocular hypertension and RGCs losing.However,its pattern is different from translimbal laser photocoagulation.Transgoniscope laser photocoagulation has a higher successful rate of chronic ocular hypertention than that of translimbal laser photocoagulation.
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Background Optic nerve crush (ONC) model is an available tool in the basic research on the mechanism and treatment of optic nerve injury.The opening optic nerves sheath crushing and via bulbar conjunctiva lateral canthus optic nerves crushing are frequently used ONC modeling methods.However,the comparison between these models is not elaborated.Objective This study was to compare the outcomes between opening sheath ONC model and via bulbar conjunctiva lateral canthus ONC model.Methods Twenty-four male SD rats were randomized into four groups.ONC models were established via superorbital rim opening sheath to crushing optic nerve for 20 seconds in the opening sheath ONC group,or via bulbar conjunctiva lateral canthus cutting to crush optic nerve for different time in the via bulbar conjunctiva ONC 20-second,40-second and 60-second groups,respectively.All models were monocular created in the rats,and the fellow eyes served as controls.In 14 days after modeling,flash visual evoked potential (F-VEP) were recorded,optic nerve and retinal sections were prepared in the rats.The histopathology of the samples was examined by hematoxylin and eosin staining.The expression of Brn-3α in the retinal ganglion cells (RGCs) was detected by immunofluorescence technique and the number of Brn-3α+ RGCs was counted.The modeling procedure and outcomes were compared between the two approaches.Results The latencies of P1 waves were significantly extended in the opening sheath ONC group,via bulbar conjunctiva ONC 20-second,40-second and 60-second groups in comparison with the corresponding control eyes (t =-11.64,-8.04,-6.50,-10.84,all at P＜0.01).The P1 latencies were longer in the opening sheath ONC group than those in the via bulbar conjunctiva ONC 20-second,40-second and 60-second groups (P =0.01,0.02,0.05),but no significant differences were found in the amplitudes of P1 waves between model eyes and corresponding control eyes (all at P＞0.05).The Brn-3α+ RGCs numbers were evidently decreased in the model eyes in comparison with the fellow control eyes.The Brn-3 α + RGCs numbers were (13.60 ± 2.14),(18.74 ± 3.61),(15.84 ± 2.31) and (14.58 ± 3.23)/field in the opening sheath ONC group,via bulbar conjunctiva ONC 20-second,40-second and 60-second groups,which reduced to 47.49％,67.70％,56.69％ and 50.17％ of the fellow eyes,respectively.No significant differences were seen in the Brn-3α+ RGC numbers between the opening sheath ONC group and via bulbar conjunctiva ONC 40-second or 60-second groups (both at P＞0.05).The disorder of glial cell arrangement,vacuolization of the cell matrix and infiltration of inflammatory cells were displayed in various model groups,with the prominent findings in the opening sheath ONC group.Conclusions Compared with the via bulbar conjunctiva ONC models,the morphological and functional damage of optic nerve is more obvious,and the survival rate of RGCs is lower in the opening sheath ONC models.
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Retinal ganglion cells (RGCs) apoptosis is the ultimate pathway of many disorders involved in the retina and optic nerve,and contributes to irreversible visual loss.RGCs are vulnerable to various stimulates including ocular trauma,intraocular hypertension,endoophthalmitis and neurotoxin,and eventually undergo apoptosis.Those external stimulates can activate multiple intracellular signal pathways,and regulate apoptosis associated genes.This paper reviewed the recent findings regarding external stimulates,intracellular signaling pathways and gene regulation involved in the pathological retinal ganglion cells death,in order to provide potential targets for the development of new specific therapies for disorders associate with RGCs degeneration.
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Objective To observe the protective effect on retinal ganglion cells (RGC) and the safety of intravitreal injected acteoside in rats.Methods A total of 50 male Sprague Dawley rats with the weight of 190-210 g were used in this study.Fifteen rats were used for safety experiment of intravitreal injection of acteoside.The rats were divided into group A,B,C,control group and blank group,three rats in each group.The rats in group A,B and C were received intravitreal injection of 5 μl acteoside at the concentration of 1,2,and 5 mg/ml,respectively.Phosphate buffer solution (PBS) was injected in rats of control group.No treatment was performed for blank group.The retinal structure was examined by hematoxylin-eosin (HE) staining of retinal frozen sections at one,two and three weeks after injection.The retinal ultrastructure was examined by ultrathin section under transmission electron microscope at one and three weeks after injection.Others 35 rats were used for experiment of protective effect of acteoside on RGC.The rats were divided into operation group A and B (n=8),sham operation group C and D (n=8),and blank group (n=3).The optic nerve of rats in operation group was clamped for 10 seconds after optic nerve exposure,while the optic nerve of rats in sham operation group was exposed only.The rats in operation group A and B were received intravitreal injection with 5 μl acteoside (1 mg/ml) and 5 μl PBS respectively.The rats in sham operation group C and D were received intravitreal injection with 1 μl acteoside (1 mg/ml) and 1 μl PBS respectively.No treatment was performed for blank group.The retinal structure was examined by HE staining of retinal frozen sections at one,two and four weeks after injection.Immunohistochemistry was used to measure the expression of growth associated protein 43 (GAP-43).RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Software of SPSS 13.0 was used for the data statistical analysis in this study.Results In the safety experiment of intravitreal injected acteoside,there was no abnormity of cornea,anterior chamber,lens,vitreous cavity and retina after injection.At one,two and three weeks after injection,the retina structure was normal without significant apoptosis,necrosis and inflammatory cell infiltration.The ganglion cell layer showed slightly edema; there was no obvious change of retinal ultrastructure after injection of acteoside with 5 mg/ml and 2 mg/ml,but slight change with the format of 1 mg/ml.Transmission electron microscopy showed that intravitreal injection of 5μl acteoside at the concentration of 2 or 5 mg/ml can induce significant changes of micro-structures of retina,while injections at 1mg/ml can only induce minor changes.In the experiment of protective effect of acteoside on RGC,light microscope revealed that the cell showed typical changes of apoptosis in operation group,but not in sham operation group and blank group.At the first and second week after injection,compared with the sham operation group and blank group,the RGC number was decreased in operation group.The difference of RGC numbers between operation group A and B was statistically different (F=26.206,P＜0.05).The RGC numbers in operation group continues to decrease at the fourth week after injection,there was obvious difference compared with the first and second week after injection (F=17.364,P＜0.05),but there was no difference of RGC numbers among sham operation intra-group and between sham operation group and blank group at all the time points.Immunohistochemistry showed that at the first week after injection,the integrated absorbance (IA) value in operation group was higher than that in other groups (F=33.466,P＜0.05) ; there was no difference of IA value between operation group A and B.At the second week after injection,IA value in operation group A had slightly declined,but higher than that in operation group B (F=14.391,P＜0.05).At the fourth week after injection,IA value in operation group A declined further,but also higher than that in other groups (F=4.178,P＜0.05).TUNEL showed that on the first week after injection,RGC apoptosis rate in operation group was increased than that in other groups (F=15.365,P＜0.05).At the second week after injection,RGC apoptosis rate in operation group was decreased,and it in operation group A was lower than that in operation group B (F=15.365,P＜0.05).At the fourth week after injection,RGC apoptosis rate in operation group was decreased obviously,there was no difference compared with other groups (F =2.057,P ＞ 0.05).There was no difference of RGC apoptosis rate between sham operation group and blank group at all the time points.Conclusion Intravitreal injection of 5 μl acteoside (1mg/ml) is safe for rat retina,and can up-regulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats.
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OBJETIVO: Avaliar a espessura da camada de fibras nervosas da retina em olhos amblíopes e comparar com olhos normais e certificar se há correlação com a redução da acuidade visual. Além disso, este estudo se propõe avaliar a eficácia e eficiência em uma série de casos do protótipo de um equipamento nacional de magnificação para leitura. MÉTODOS: Participaram deste estudo 30 pacientes na faixa etária entre 9 e 80 anos (17 do sexo masculino). Foi desenvolvido um aparelho portátil, patenteado pela Unifesp (PI#020050145260), com um sistema de captura de imagens acoplado a um monitor de 5,6 polegadas proporcionando um aumento de 15 x. Foram analisadas a eficácia da acuidade visual e a eficiência de leitura após a utilização do protótipo proposto. RESULTADOS: Seis pacientes (20 por cento) apresentaram AV 8M, 12 pacientes (40 por cento) apresentaram AV 6M, 7 pacientes (23,3 por cento) apresentaram 5 M, 5 pacientes (16,7 por cento) apresentaram 4M. A média de acuidade visual antes da utilização do SLP medida pela tabela LHNV-1 logMAR foi de 5,75M e após a utilização 100 por cento dos pacientes atingiram a eficácia de AV J1. CONCLUSÃO: O protótipo do SLP mostrou-se um recurso alternativo no processo de inclusão social das pessoas com baixa visão com diferentes níveis de resíduo visual. Também pode proporcionar incentivo psicológico, permitir conforto, mobilidade e independência àqueles que necessitam de uma leitura mais prolongada e maior distância de trabalho.
OBJECTIVE: To compare the thickness of the retinal nerve fiber layer (RNFL)and the macular thickness of the amblyopic eye with those of the non-amblyopic eye in patients with unilateral amblyopia using optical coherence tomography (OCT). METHODS: OCT was performed for13 patients with unilateral amblyopia who had no neurologic disease. Nine male andfour female patients, whose ages ranged from 23 to 63 years, were enrolled in the study. The RNFL thickness average analysis program was used to evaluate mean superior, inferior, temporal, and nasal thickness. The data for all clock quadrants (12 values averaged) were identified as the overall RNFL. The retinal thickness analysis program was used to evaluate macular scans. Data were compared using the Man n-Whitney U test. The mean age ( standard deviation) was 35,43years. RESULTS: There were 13 eyes with amblyopia; this group had visual acuity 0,1 logMAR or better in the best eye. OCT parameters including the RNFL thickness in all quadrants, overall RNFL thickness and macular thickness showed no significant differences between the two groups (p >0,5). CONCLUSION: Assessment of RNFL thickness and macular thickness with OCT revealed no difference between the two eyes of patients with unilateral amblyopia.