Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage. Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted. Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05). Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
Objective To observe the degradation regulation of ubiquitinproteasome inhibitor nuclear factor kappa;B（NF-kappa;B）and its inhibitory signal protein Ikappa;B kinase in earlier period diabetic retinopathy（DR）,and the effects on retinal ganglion cells (RGC) apoptosis.Methods Forty healthy adult Wistar rats were randomly divided into control (group A）,DR（group B）,DR+lowconcentration MG132 treated （group C）and DR+high concentration MG132 treated（group D）groups,10 rats in each group.After 6 and 8 weeks,the results of body masses and fasting blood glucose (FBG) were detected,the expression of NF-kappa;B and Ikappa;B were observed by immunohistochemistry respectively.RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Results The expression of NF-kappa;B was upregulated in group B compared with group A,its expression decreased in group D compared with group B; but the expression of Ikappa;B was contrary to NF-kappa;B; RGC apoptosis was followed a similar pattern with the expression of NF-kappa;B; the differences among them were statistically significant (P＜0.01).Compared the expression of NF-kappa;B，Ikappa;B and RGC apoptosis in group C and D, there were no statistically significant differences(P＞0.05).Conclusion Ubiquitin-proteasome inhibitor MG132 can block the activation of NF-kappa;B,inhibit ubiquitination of Ikappa;B degradation and RGC apoptosis.
Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adenoassociated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo.Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A,B,C,D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 mu;l phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 mu;l recombinant adenoassociated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5 mu;l rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects.Results Green fluorescence can be observed exactly in labeled RGC in B，C，and D groups. The AOD and transfection rate in group D was （95.02plusmn;7.25）% and（20.10plusmn;0.74）% , respectively; which were higher than those in group B and C (F=25.970，25.799；P＜0.01). The difference of the number of RGC among the four groups was not significant(F=0.877,P＞0.05). Conclusion Under the condition of low frequency and with certain energy, ultrasoundmediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.
ObjectiveTo investigate the effect of intravitreal injection of neural stem cells (NSC) derived from human umbilical cord mesenchymal stem cells (hUCMSC) on the expression of brain-derived neurotrophic factor (BDNF) and the number of retinal ganglion cells (RGC). MethodsFifty-two adult male Sprague-Dawley rats were randomly divided into normal group (group A) and diabetes mellitus group which received intraperitoneal injection of streptozocin to make diabetic rat models. One month after the diabetic rat models were confirmed successfully, diabetic rats were randomly divided into diabetic group (group B), hUCMSC group (group C) and hUCMSC-induced NSC group (group D). And thirteen diabetic rats were included in each group. Immuno-cytochemistry was applied to observe BDNF and thymosin-1(Thy-1) staining in the retina. Then mean integrated absorbance of the staining region on the retina slices were analyzed by Image-Pro Plus 6.0. The number of Thy-1 labeled RGC was record. ResultsBDNF and Thy-1 were positive on the retina slices from group A. The staining intensity from group B became weak and the expression of BDNF and Thy-1 gradually decrease with time (P < 0.05), and those from group C and group D were positively (P < 0.05), especially in group D (P < 0.05). The BDNF expression and Thy-1 labeled RGC were the same between group B and C (P > 0.05) at 2 weeks after injection, but were significant different for other time points (P < 0.05).Significant positive correlation between the expression of BDNF and the number of RGC were found by the Pearson correlation analysis (r=0.964, P < 0.05). ConclusionIntravitreal injection of hUCMSC-derived NSC to diabetic rat may protect the retina by promoting the expression of BDNF and increasing the number of RGC.
Objective To establish a purified model of rat retinal ganglion cells （RGCs） cultured by serum-free medium，and provide a good cell model to investigate the damage of RGCs in glaucoma，retinal ischemia，and degenerative retinopathy. Methods Two monoclonal antibodies，anti-rat SIRP（OX-41）against rat macrophage and antibody against rat Thy-1（OX-7），were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley（SD）rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor（CNTF） meeting the neuronal cellrsquo;s special requirements.Photomicrographs illustration，immunfluorescence staining of Thy-1，calcein-acetoxymethyl ester（calcein-AM）fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells，91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day，many cells had uniform configuration，large body，and long protuberances. At the 14th day，over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining，which stained the living cells only，showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size，good configuration，and high purity，which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents. (Chin J Ocul Fundus Dis, 2006, 22: 200-203)
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 mu;l 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 mu;l acteoside (1 mg/ml) and 5 mu;l PBS respectively. The rats in sham operation group C and D were received intravitreal injection with 1 mu;l acteoside (1 mg/ml) and 1 mu;l 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 dUTPbiotin nickend 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 5 mu;l 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 intragroup 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 mu;l acteoside (1 mg/ml) is safe for rat retina, and can upregulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats.
ObjectiveTo observe the correlation between the thickness of foveal ganglion cell-inner plexiform layer (GCIPL) and visual field mean defect before and after gamma knife treatment in patients of sellar region tumors with optic chiasmal compression. MethodsThis was a prospective case series. 72 eyes of 37 consecutive patients suffering from optic chiasmal compression of sellar region tumors treated with gamma knife were enrolled in the study. According to the change of visual field before and after gamma knife treatment, the patients were divided into three groups. There were 13 eyes of 7 patients in group 1 with no vision defect pre-and post-treated, 34 eyes of 17 patients in group 2 with improvement of visual field defect after treatment, 25 eyes of 13 patients in groups 3 with no improvement or reorganization of visual field defect after treatment. Overall average thickness of GCIPL, and of the superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal retina were measured with the Cirrus high-definition spectral domain optical coherence tomography, and mean deviation (MD) with the Humphrey field analyzer before and 6 months after treatment. There was no significant difference in MD values between group 2 and 3 pre-treated (t=1.471, P=0.084). There was significant difference between all the groups in total average value of GCIPL thickness and the 6 quadrant GCIPL thickness values pre-treated (P < 0.05). Logistic regression model was applied to analysis of the correlation between GCIPL thickness and the improvement of visual field after treatment. ResultsThe MD values of the group 1, 2 and 3 were (-2.96 ±0.75), (-10.24 ±1.31), (-20.2 ±5.88) dB at 6 months after treatment. There was significant difference between group 2 and 3 of MD value after treatment (t=6.974, P=0.000). In group 1, there was no significant difference in mean GCIPL thickness and the 6 quadrant GCIPL thickness values between pre-and post-treated (t=0.882, P=0.395).The mean thickness of GCIPL, superonasal and inferonasal GCIPL was increased than pre-treated in group 2, and the difference was statistically significant (t=2.438, 4.630, 4.457; P=0.035, 0.001, 0.001). The mean thickness of GCIPL, superonasal and inferonasal GCIPL was decreased than pre-treated in group 3, and the difference was statistically significant (t=-2.387, -4.603, -4.975; P=0.041, 0.002, 0.001).Logistic regression analysis showed that the greater of the value of average GCIPL thickness of patients with visual field defect pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between the value of superonasal or inferonasal GCIPL and the improvement of the visual field post-treated (OR=5.374, 4.693; P=0.000, 0.000). There was no significant correlation between the value of superotemporal or upper or lower or inferotemporal GCIPL and the improvement of the visual field post-treated (OR=1.058, 1.101, 1.074, 1.056; P=0.183, 0.080, 0.162, 0.186). ConclusionsIn patients with optic chiasmal compression of sellar region tumor, the greater of the average GCIPL thickness pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between superonasal or inferonasal value of the GCIPL thickness and the improvement of visual field defect post-treated.
ObjectiveTo investigate the protective effects of different concentrations of chloroquine on RGC in n-methyl-d-aspartate (NMDA) injured mice and its possible mechanisms.MethodsFifty-four healthy male C57/BL6 mice were randomly divided into three groups, 18 in each group. The mice in low-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 10 mg/kg daily. Mice in high-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 100 mg/kg, and the mice in control group were intraperitoneally injected with the same volume of PBS. NMDA intravitreal injection was performed 2 days after intraperitoneal injection, 5 nmoles NMDA was injected into the left eye, and the same volume of PBS was injected into the right eye as a control. The RGC staining of retinal plaques were performed 7 days after NMDA injection, and the number of alive RGC was calculated. The visual acuity and electroretinogram were used to evaluate the electrophysiological functions of RGC at 9 and 10 days after modeling. Real-time quantitative PCR and retinal frozen sections and glial fibrillary acidic protein (GFAP) immunofluorescence staining were performed 11 days after NMDA injection to evaluate the glial activation of the retina. The density, visual acuity, and the amplitude of PhNR-wave of RGC between groups were compared by one-way analysis of variance.ResultsAt 7 days after NMDA injection, the density of RGC in retinal patch of low-dose chloroquine group was significantly higher than that of intraperitoneal injection of PBS control group (F=54.41, P＜0.01). The density of RGC in retinal patch of high-dose chloroquine group was lower than that of control group (F=1.18, P＞0.05). The visual acuity was higher than control group, and the difference was statistically significant (F=9.10, P＜0.05). The amplitude of PhNR-wave was significantly higher in low-dose chloroquine group than that of the control group (F=17.60, P＜0.01). The mRNA level of inflammatory factor and GFAP positive signal was also significantly lower than that of the control group (F=23.66, P＜0.05). The amplitude of PhNR-wave, the expression of GFAP (F=110.20, P＜0.01) and the mRNA level of inflammatory factors (F=167.60, 17.78; P＜0.01) in the high-dose chloroquine group were higher than the other two groups, and the differences were statistically significant.ConclusionsIn NMDA injury retinal model, low-dose chloroquine significantly increased the survival and physiological function of RGC, and the mechanism may be related to the inhibition of glial activation and inflammatory response. High-dose of chloroquine would aggravate the apoptosis of RGC.
Objective To evaluate the inhibiting effect of adenosine on rat retinal ganglion cells (RGC) death induced by P2X7 and N-methyl-D-aspartate (NMDA) receptor. Methods (1) Long-Evan neonatal rats were back labeled with aminostilbamidine to identify RGC. The viability of RGC affected by P2X7 excitomotor BzATP (50 mu;mol/L), glutamate receptor excitomotor NMDA (100 mu;mol/L) and adenosine (300 mu;mol/L) was detected. (2) RGC from the retinae of unlabeled neonatal rats were cultured in vitro. After labeled with Fura-2 methyl acetate, an intracellular calcium indicator, the effect of BzATP, NMDA and adenosine on intracellular Ca2+ level was detected byCa2+ imaging system. Results Both BzATP (50 mu;mol/L) and NMDA(100 mu;mol/L) could kill about 30% of the RGC. Cell death was prevented by adenosine (300 mu;mol/L) with the cell viability increased from (68.9plusmn;2.3)% and (69.9plusmn;3.2)% to (91.2plusmn;3.5)% (P＜0.001) and (102.1plusmn;3.9)% (P＜0.001), respectively. BzATP (50 mu;mol/L) led to a large, sustained increase of intracellular Ca2+ concentration to (1183plusmn;109) nmol/L. After the adenosine intervened, Ca2+ concentration increased slightly to (314plusmn;64) nmol/L (P＜0.001). Conclusion Adenosine may prevent RGC death and increase of intracellular Ca2+ concentration from P2X7and NMDA receptor stimulation. (Chin J Ocul Fundus Dis, 2007, 23: 133-136)
Objective To observe the protective effects of Na2SeO3 on the damage of retinal neuron induced by microwave. Methods Cultured fluids of retinal neuron were divided into 4 groups,including 1 group of control, according to the concentration of Na2SeO3 in cultured fluid and then exposed to 30 mW/cm2 microwave for 1 hour.The targets of lipid peroxidation and the concentration of selenium in cells were measured.Apoptosis detection was taken by TUNEL detection kit. Results The activity of SOD and GSH-Px rised,meanwhile the content of MDA and the amount of apoptosis cells decreased in 1times;107 mol/L group compared with the group without Na2SeO3.The other groups was superior in antioxdant capacity to 1times;107 mol/L group. Conclusion Na2SeO3 might be possessed of the effect of protecting the damage of retinal neuron induced by microwave. (Chin J Ocul Fundus Dis,2000,16:97-99)