Objective To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism. Methods The male Wistar rats (60) were divided into control (CON) group, diabetes (DM) group and Niacin-treated (NA) group, 20 rats in each group. Rats diabetes models were induced with streptozotocin injection. Niacin (40 mg/kg·d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months. Pathological outcomes, total cholesterol (TC) and high-density lipoprotein (HDL) were evaluated at month 3. Optical microscopy was used to observe the retinal structure. The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method. The relative expressions of Claudin-5, Occludin, zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A, tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot. Results Compared to CON group, the TC content was increased and HDL content was decreased in DM group (t=4.034, 5.831; P < 0.05). Compared to DM group, the TC content was decreased and HDL content was increased in NA group (t=6.868, 3.369; P < 0.05). The retinal structure of CON group was normal. Pathological changes were found in the DM group, such as tumescent nuclei and disorganized structures. The retinal structure of NA group was similar to the control group. Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712, P < 0.05), while in NA group was decreased compared with DM group (t=16.414, P < 0.05). The mRNA expression of Occludin, Claudin-5, ZO-1 in DM group were decreased compared with CON group (t=11.422, 12.638, 12.060; P < 0.05), while in NA group were increased compared with DM group (t=5.278, 3.952, 8.030; P < 0.05). The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053, P < 0.05). The protein expression of GPR109A, IL-6, TNF-αin DM group were increased compared with CON group (t=4.915, 11.106, 6.582; P < 0.05). Compared to DM group, the protein expression of GPR109A was increased (t=5.806, P < 0.05), while the protein expression of IL-6 and TNF-α were decreased (t=10.131, 5.017; P < 0.05). Conclusion Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation.
Objective To observe the effects of high concentr at ion glucose on the calcium-activated potassium channel of rabbits′ retinal Müller cells. Methods The rabbits′retinal Müller cells were cultured in vitro under the condition of high concentration glucose, and identified by immunohistochemical staining and transmission electron microscopy. Patch-clamp technique was used to observe the changes of the calcium-activated potassium channel of retinal Müller cells caused by high concentration glucose at different time.Results High concentration glucose could inhibit the calcium-activated potassium channel of cultured retinal Müller cells in a time-dependent manner. Conclusion High concentration glucose may reduce the biological functions of Müller cells by inhibiting calcium-activated potassium channel. (Chin J Ocul Fundus Dis，2003，19：164-167)
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells. METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml. RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml. CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration （Chin J Ocul Fundus Dis,1997,13: 86-88）
Objective To explore the effects of drugs on functions of mitochondria in retinal nerve cells, and to lay a foundation of the investigation of drug protection for retinal nerve cells. Methods Cultivation of the retinal nerve cells of 8 eyes of neonatal calves was performed. The changes of fluorescent density of the mitochondria of cultured cells labeled by dye rhodamine 123 (Rh123) before and after the activation of the medicines, including ferulic acid (FA), arginine, glycine,taurine, vitamine E and brain derived neurotrophic factor( BDNF) respectively, were detected by laser-scanning confocal microscopy. Results FA with the concentration of 500 μg/ml led the diphasic variation of the fluorescent intensity of mitochondria. After scanning for 60.772 seconds when treated with FA firstly, the fluorescent intensity decreased rapidly (from 45.425±4.153 to 22.135±5.293); while after 112.774 seconds when treated secondly, the in tensity increased obviously (from 19.655±4.383 to 28.247±4.764), and after 168.773 seconds when treated thirdly the intensity still increased. After scanning for 56.457 seconds when treated with vitamin E (12.5 mg/ml), the fluorescent in tensity increased obviously (from 88.255±5.039 to 111.273±4.529), which suggested that vitamin E with the concentration of 12.5 mg/ml strengthen the fluorescent intensity. After scanning for 58.147 and 134.148 seconds when treated with BDNF(50 ng/ml) respectively, the fluorescent intensity increased obviously (from 69.115±5.038 to 77.225±5.131) which suggested that BDNF with the concent ration of 50 ng/ml led the increase of the fluorescent intensity. Glycine (2.5 mg/ml) and arginine(30 mg/ml) didn’t affect the fluorescent intensity of mitochondria, and taurine (6.25 mg/ml) caused the appreciable decrease of the fluorescent intensity . Conclusion FA, BDNF and vitamin E may promote the metabolism of retinal nerve cells via the path of mitochondria, while amino acids may adjust the activation of retinal nerve cells through other ways. (Chin J Ocul Fundus Dis，2004，20：229-232)
ObjectiveTo investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. MethodsH1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. ResultsOn the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m2. ConclusionsL-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
OBJECTIVE:To verify the safe dose of cephradine in intravitreal injection. METHODS:After injecting different doses of cephradine(100mu;g,200mu;g,250mu;g,300mu;g,400mu;g)into vitreous cavity of different group of rabbits the activities of the retinal enzymes (SDH,LDH )on different time (Id,3d, 7d ) were determined respectively, and the histological and ultrastructural changes of retinas were also observed simuhaneously. RESULTS:The activity of rellnal SDH and LDH was found to be decreased gradually with tbe icreasing of the dosage of intravitreal cephradine. The activities of SDH and LDH were found in the lowest level on tile 3rd and lsl day,but they recover to normal levels on tile 7th day after intravitreal in}eetion in 100mu;g,200mu;g groups,and still lower tban normal in the other groups. Histologically,retinal edema was found both in 100mu;g and 200mu;g groups,but degradation of retinal cells,and loss of cones and rods were round in the 250mu;g, 300mu;g and 400mu;g groups. CONCLUSION: The safe dose of intravitreal injection of cepbradlnc is 200mu;g. （Chin J Ocul Fundus Dis,1997,13:139-142 ）
Objective To investigate the protective effects of ginkgo biloba extract (EGb) 761 on retinal ganglion cells (RGC) in rats，and to establish a method to define the rat RGC using fluorogold as a fluorescence dye. Methods RGC of 12-20 day-old SpragueDawley rats were labeled by injecting fluorogold into superior colliculus. The eyeball enucleation was performed 6 days later. Retinal stretched preparation was obtained from one eye to observe the label result under fluorescence microscope, and the retina from the other eye was detached to make the cell suspension to observe the configuration of stained RGC under the contrast fluorescence microscope. The cell suspension was divided into the control group and Egb761 groups with the concentration of 0.03%,0.10%, 0.30%, 1.00%, and 3.00%. Trypan blue dye was used to evaluate cells viability and the survival rate of the large retinal ganglion cells was calculated. Results The sign of the RGC was clear after labeled by fluorogold. The characteristics of large RGC were obvious. After detachment, large RGC died quickly in the cell suspension and the fluorescence disappeared. The result of Trypan blue staining indicated that large RGC died rapidly in the cell suspension. Large RGC in EGb761 group showed significantly better survival rates than that in control group at different time sites (Plt;0.01) in a dose-dependent manner (Plt;0.01). Conclusions EGb761 has a significant protective effect on large RGC cultivated in vitro, and retrolable method to identify RGC is feasible.
Objective To investigate the effect of inducible nitric oxide synth ase (iNOS) or cyclooxygenase－2 (COX－2) on retinal neovascularization and its possible mechanism in oxygen－induced retinopathy （OIR） mouse model. Methods Retinal neovascularization was induced by oxygen with different concentration. The expression of iNOS, COX－2, matrix metalloproteinases 2 (MMP－2) and vascular end othelial growth factor (VEGF) in the retinae of experimental animals were analyzed by immunohistochemistry, realtime polymerase chain reaction and western blotting technologies. Results The inhibition of COX－2 or iNOS obviously attenuated retinal neovascularization and decreased the expression of VEGF and MMP－2. The iNOS inhibition decreased COX－2 expression, and vice versa. Conclusions COX－2 and iNOS may play a role in retinal neovascularization in OIR mouse model, which may act by regulating the expression of VEGF and MMP－2.