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.
Objective To observe the neuroprotective effect of alpha;-lipoic acid (ALA) on cultured retinal ganglion cells (RGC-5) under elevated pressure in vitro. Methods Cultured RGC-5cells were divided randomly into 4 groups, including normal control group (group A), negative control group (group B), elevated pressure group (group C) and elevated pressure + ALA group (group D). The cells of group A and C were not intervened with ALA. The cells of group B were treated with 200 mu;mol/L ALA. The cells of group D were treated with different concentrations of ALA (50, 100, 200 mu;mol/L) for one hour. Then cells of group C and D were exerted to 50 mm Hg (1 mm Hg=0.133 kPa) for 24 hours, while the cells of group A and B were exerted to normal pressures for 24 hours. The cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay and apoptosis was evaluated using 4prime;,6-diamidino-2-phenylindole (DAPI) staining. Expression of MnSOD was determined by realtime polymerase chain reaction (RT-PCR) and Western blot, respectively. Results The cell viability of group B was (65.6plusmn;3.4)%, which lower than that in group D of three concentrations of ALA［(75.1plusmn;3.3)%, (81.8plusmn;2.9)%,(87.9plusmn;3.1)%］, the differences were significantly (t=5.108, 10.007, 12.513;P＜0.05). DAPI staining revealed that characteristic apoptotic changes, such as chromatin condensation,convoluted nuclei with cavitations, fragmentation of the nucleus, and apoptotic bodies appeared in RGC-5cells after 24 ours pressure. There was almost no evidence of apoptosis in group D. RT-PCR and Western blot analysis revealed that the expression of MnSOD mRNA and protein were weakly expressed in group C compared with control A (t=22.045，26.979;P＜0.01). Compared group C with group D, the level of MnSOD mRNA and protein in group D increased significantly (t=9.171, 12.267, 23.567, 7.723, 12.009, 28.198;P＜0.05).In addition, the presence of ALA was found to inhibit hydrostatic pressure induced damage of RGC-5cells in a dose-dependent manner (F=134.273,194.597;P＜0.01). Conclusion ALA can effectively improve the expression of MnSOD in RGC-5cells under the condition of elevated pressure, enhance the ability of RGC-5cells against oxidative damage.
Objective To observe the protective effect of ultrasound microbubble contrast agentmediated transfection of brain-derived neurotrophic factor(BDNF) into the retina and visual cortex on retinal ganglion cells (RGC) after optic nerve injury. Methods A total of 88 male Sprague-Dawley (SD) rats were randomly divided into normal group (group A, eight rats), sham operation group (group B, 16 rats), control group (group C, 16 rats), eyes transfection group (group D, 16 rats), brain transfection group (group E, 16 rats), combined transfection group (group F, 16 rats). The optic nerve crush injury was induced, and then the groups B to F were divided into one-week and two-week after optic nerve injury subgroup with eight rats each, respectively. The rats in group B and C underwent intravitreal and visual cortex injection with phosphate buffered solution respectively. The rats in group D and E underwent intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids respectively. The rats in group F underwent both intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids at the same time. The ultrasound exposure was performed on the rats in group D to F after injection with the mixture solution of microbubbles and BDNF plasmids. One and two weeks after optic nerve injury, RGC were retrogradely labeled with Fluorogold; active caspase-3 protein was observed by immunohistochemistry and the N95 amplitude was detected by pattern electroretinogram (PERG). Results Golden fluorescence can be observed exactly in labeled RGC in all groups，the difference of the number of RGC between the six groups and ten subgroups were significant（F=256.30，65.18；P＜0.01）. Active caspase-3 in ganglion cell layer was detected in group C to F, but not in group A and B. The difference of the N95 amplitude between the six groups and ten subgroups were significant（F=121.56，82.38；P＜0.01）.Conclusion Ultrasound microbubble contrast agent-mediated BDNF transfection to the rat retina and visual cortex can inhibit the RGC apoptosis after optic nerve injury and protect the visual function.
Objective To investigate the protective effect of blocking the signal path of p38 mitogen activated protein kinase on blood retinal barrier (BRB) and retinal ganglion cells (RGC) in early diabetic rats.Methods A total of 60 Wistar rats were divided into the control and diabetes group, with 30 rats in each group. Diabetes was induced in rats in diabetes group by peritoneal injection of streptozotocin (STZ);the plasma glucose level of ＞16.7 mmol/L indicated that the diabetes model was set up successfully.The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution. IgG leakage method was used to measure the damage of BRB function and vascular leakage. The expression and localization of caspase-3 and vascular endothelial growth factor (VEGF) in retina of diabetic rats were examined by immunohistochemistry analyses.Two weeks after the establishment of the diabtes model, the rats in diabtes group underwent intravitreal injection with SB203580, a p38 inhibitor;six weeks after the injection, the expression of caspase-3 and VEGF was detected, and the number of apoptosis RGC was counted via immunofluorescence technique.Results In the contral group, IgG staining located in the blood vessels with little leakage; while the IgG leakage was much more obvious in the diabetes group eight weeks after the establishment of the model. Six weeks after intravitreal injection with SB203580, the leakage decreased in diabtes rats. The results of semiquantitative analysis and fluorescence immunohistochemistry showed that compared with the results in diabetes rats 8 weeks after intravitreal injection (2.9 times much more than that in the control group), the fluorescence expression of VEGF decreased in diabetes rats six weeks after intravitreal injection (1.8 times much more than that in the control group).The apoptisis RGC number in rats 6 weeks after intravitreal injection of SB203580 was much less than that in rats without intravitreal injection (t＝5.731, Plt;0.01). Conclusions SB203580 can alleviate the disruption of BRB and apoptosis of RGC in early diabetes rats, which suggests that p38 MAPK pathways appear to be directly involved in the pathogenesis of early diabetic retinopathy.
Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 SpragueDawley（SD）rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi.Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope.Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat mounts. The density of microglial cells which phagocytosed DiI particles in the RGC layer significantly increased in the 3month diabetes group（P＜0.01）. The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group（P＜0.05）. However there were more microglial cells in the RGC layer in the 3- month diabetes group than the 1-month diabetes group（P＜0.0001）. Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats.
Objective To investigate the protective effects of estrogen on rabbit retinal damages induced by chronic ocular hypertension.Methods A total of 18 white New Zealand female rabbits were randomly divided into ovariectomized (OV) group and sham OV (SOV) group. Bilateral ovaries were remove in OV group while only the adipose tissue around ovarian were remove in SOV group. Chronic ocular hypertension was induced by anterior chamber injection of carbomer. Retinal cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL), the expression of bcl-2, bax were detected by immunohistochemistry. The images were captured under microscope and analyzed with computer-image-analysis system. Results Four, six and eight weeks after ocular hypertension modeling, the OV retinas have less retinal ganglion cells, thinner optic nerve fiber layer and inner nuclear layer and more TUNEL positive cells (t=3.285，4.012，3.624；P＜0.01). The OV retinas also have less bcl-2 expression (t=4.256，3.867，3.459；P＜0.01), more bax positive cells (t=3.211，3.625，3.253；P＜0.01). Bcl-2 expression was negatively correlated with TUNEL positive cells indicating bcl-2 can inhibit apoptosis. Bax expression was positively correlated with TUNEL positive cells indicating bax induce apoptosis.ConclusionEstrogen has a neuroprotection role to rabbit retina under chronic ocular hypertension by reducing 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.
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 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 construct expression plasmid of the fusion protein of brainderived neurotrophic factor (BDNF)green fluorescent protein (GFP), and observe its characteristics.Methods BDNF cDNA segment was inserted into plasmid pcDNA3.1/ NT-GFP-TOPO and in the same reading frame with GFP. After verified by sequencing, the BDNFGFP plasmid was transferred into cultured Schwann cells by electroporation. And the expression of BDNFGFP fusion protein was observed by immunohistochemistry and Western blotting. The neuralprotective function of the fusion protein was evaluated by transferring the plasmid into adult rat retinas with transected optic nerve.Results The sequence of BDNFGFP plasmid was verified correctly by autosequencing. The results of Western blotting showed that the BDNF-GFP fusion protein expressed a brand with the relative molecular mass of 41times;103. Seven days after the optic nerve was transected, the number of survival retinal ganglion cells (RGC) in BDNF-GFP group and GFP group was （1201plusmn;286） and（482plusmn;151）cells/mm2, respectively; and the survival rate was （51.39plusmn;12.24）% and （20.62plusmn;6.46）% , respectively. Twentyeight days after the optic nerve was transected, the number of survival RGC in the two groups was （715plusmn;71） and （112plusmn;24）cells/mm2, respectively; the survival rate was（30.59plusmn;3.04）% and (4.79plusmn;1.03）% respectively. The differences of the survival rate of RGC between the two groups were significant (t=3.144，11.378；Plt;0.01).Conclusion BDNF-GFP fusion plasmid can express a fusion protein which emit green fluorescence and has the biological activity of BDNF.