Objective To study the effects of several neurotrophic factors and growth factors on the survival of human retinal ganglion cells(RGC)in vitro. Methods RGC were isolated from donor eyes and cultured.RGC in cell culture were identified by morphologic criteria and immunocytochemical staining.Various neurotrophic factors and growth factors were added individually to the cultures.Numbers of RGC in wells in which these agents had been added were compared with those from control wells(cultures without supplements). Results No or very few RGC were present in cell cultures containing medium without supplements or those supplemented with neurotrophin-3(NT-3),nerve growth factor (NGF),epidermal growth factor(EGF)amd plateletderived growth factor(PDGF).Numbers of RGC(per 10 fields)in cell cultures containing brain derived neurotrophic factor(BDNF),ciliary neurotrophic factor(CNTF),neurotrophin-4/5(NT-4/5)and basic fibroblast growth factor(bFGF)wer 4.08,1.23,2.63 and 2.65,respectively,significantly more than found in the control cultures. Conclusions BDNF,NT-4/5,bFGF,CNTF improve survival of human RGC in vitro,while NGF,NT-3,EGF and PDGF do not. (Chin J Ocul Fundus Dis, 1999, 15: 149-152)
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)
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
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 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)
Primary or secondary death of retinal ganglion cells (RGC) is a common outcome in various optic neuropathies, often resulting in severe visual damage. The inherent characteristics of RGC include the continuous upregulation of intracellular growth-inhibitory transcription factors and the downregulation of growth-inducing transcription factors during cell differentiation. Additionally, the external inhibitory microenvironment following RGC damage, including oxidative stress, chronic inflammation, lack of neurotrophic factors, high expression of myelin proteins, and the formation of glial scars, all restrict axonal regeneration. Both intrinsic and extrinsic factors lead to the death of damaged RGC and hinder axonal regeneration. Various neuroprotective agents and methods attempt to promote neuroprotection and axonal regeneration from both intrinsic and extrinsic aspects, and well knowledge of these neuroprotective strategies is of significant importance for promoting the neuroprotective experimental research and facilitating its translation into clinical practice.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
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.
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.