RESUMO
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.
RESUMO
Background Researches showed that platelet-derived growth factor (PDGF) modulate the expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase (MMP/TIMP) in cells,but the association of expression of MMP/TIMP in retinal pigment epithelial (RPE) cells and the dose and active time of PDGF is unclear.Objective This study was to observe the effects of PDGF on the expressions of MMP-2,MMP-9 and TIMP-1 in cultured RPE cells in vitro.Methods RPE cell line,ARPE-19,was calculated in vitro,and the cells were divided into 5 groups when they reached 70％-80％ confluence.Different concentrations (0,0.1,1,10,50 mg/L) of PDGF was added into the medium respectively for 36 hours,and the expressing levels of mRNA and protein of MMP-2,MMP-9 and TIMP-1 were detected by reverse transcription PCR (RT-PCR) and Western blot assay.In addition,RPE cells in PDGF group were treated with 10 mg/L PDGF for 24,36,48 hours respectively to detect the expressions of mRNA and protein of MMP-2,MMP-9 and TIMP-1 in the cells and to compare with the control group without PDGF.Results PDGF stimulated proliferation of RPE cells in a dose-and time-dependent manner.As the increase of the PDGF concentrations,the expression values of MMP-2 mRNA and MMP-9 mRNA in RPE cells were gradually elevated,with a statistically significant difference among various groups (MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =8.465,P=0.003),and the expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the 1,10,50 mg/L PDGF groups compared with 0 mg/L PDGF normal control group (all at P＜0.05).Also,the expression values of MMP-2 and MMP-9 proteins in RPE cells were gradually elevated with the increase of PDGF concentrations,showing statistically differences among the groups (MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000).Compared with the 0 mg/L PDGF group,MMP-2 and MMP-9 expression levels in the 1,10,50 mg/L PDGF groups were significantly up-regulated (all at P＜ 0.05).However,the expression levels of TIMP-1 mRNA and protein group in the cells were not significantly different among various groups (mRNA:F =0.143,P =0.962 ; protein:F =1.955,P =0.178).The expression levels of M MP-2 mRNA,M MP-9 mRNA in the cells were increased in the PDGF group compared with the control group at different time points (MMP-2 mRNA:Ftime =83.250,P=0.002 ; MMP-9 mRNA:Ftime =6.785,P =0.019).Also,the expression values of MMP-2 and MMP-9proteins in RPE cells were increased in the PDGF group compared with the control group at different time points (MMP-2:Ftime =1 l.185,P =0.041 ; MMP-9:Ftime =968.413,P =0.000).The expression levels of MMP-2 and MMP-9 mRNAs and proteins were significant between the two groups at different time points (all at Pgroup =0.000;all at Ptime＜0.05).While the expression changes of TIMP-1 werc not significant between the two groups and among various time points (all at P＞0.05) Conclusions PDGF up-regulates MMP-2 and MMP-9 expressions in RPE cells in a dose-and time-dependent manner.But,PDGF dose not alter the expression of TIMP-1.These results indicate that PDGF disrupt the balance of MMP/TIMP,which may damage the extracellular matrix and therefore facilitate the migration of RPE cells in the pathogenesis of proliferative vitreoretinopathy.
RESUMO
Objective To explore the image characteristics and clinical application of the optical coherence tomography (OCT) in patients with epiretinal membranes of the macular(ERMM). Methods 38 patients, who were diagnosed as or suspected as ERMM, were examined with OCT before and after operation from November, 2002 to October, 2003 in our hospital. Results Macular epiretinal membranes were visible on OCT as high reflective tissues, which were thin or thick, and contiguous to or anterior to the retinal surface. In most fovea, the depth decreased and the thickness increased. ERMM disappeared after operation. Conclusion OCT can display the macular epiretinal membrane and the pathological changes of macular tissues before and after operation. OCT can provide accurate information on the clinical diagnosis and operative efficacy of ERMM.