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 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 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 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)
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.
ObjectiveTo investigate the effects of Krüppel-like factor 7 (KLF7) on the survival of retinal ganglion cells (RGCs) and electroretinogram (ERG) after retinal ischemia-reperfusion (RIR) injury in mice.MethodsA total of 126 male C57BL/6J mice were randomly divided into normal group, RIR group, normal-KLF7 group, normal-green fluorescent protein (GFP) group, RIR-KLF7 group and RIR-GFP group. At the age of 8 weeks, mice of normal-KLF7 group and RIR-KLF7 group were intravitreally injected 1ul of 1.0×1012 vg/ml adeno-associated virus overexpressing KLF7 (AAV2-KLF7-GFP). Mice of normal-GFP group and RIR-GFP group were injected adeno-associated virus of AAV2-GFP with the same titer. At the age of 11 weeks, RIR injury was induced in mice of RIR group, RIR-KLF7 group and RIR-GFP group, and intraocular pressure was measured. Retinal cryosections were used to access the efficacy of virus transfection 4 weeks after AAV2-KLF7-GFP transfer. 7 days after RIR injury, RGCs’ survival rate was observed and quantified by immunofluorescent staining. ERG was performed to observe the differences in amplitudes and incubation period of scotopic ERG a-, b-wave, oscillatory potentials (Ops), photopic negative responses (PhNR). Optomotor response was performed to observe the differences of visual acuity. Expression of KLF7 was detected by western blot 4 weeks after AAV2-KLF7-GFP transfer.ResultsCompared with normal group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR group were decreased, and the differences were statistically significant (t=12.860, 7.157, 5.735, 8.953, 4.744, 9.887; P＜0.05). With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. Compared with RIR group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR-KLF7 group were increased, and the differences were statistically significant (t=6.350, 3.253, 3.695, 5.825, 5.325, 4.591; P＜0.05). Protein level of KLF7 was up-regulated in normal-KLF7 group than those in normal group, and the difference was statistically significant (t=4.105, P＜0.01).ConclusionOverexpression of KLF7 can improve RGCs’ survival rates and preserve the electrophysiological function.
ObjectiveTo observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ, CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P＜0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.
The optic nerve belongs to the central nervous system (CNS). Because of the lack of neurotrophic factors in the microenvironment of the CNS and the presence of myelin and glial scar-related inhibitory molecules, and the inherent low renewal potentials of CNS neurons comparing to the peripheral nerve system, it is difficult to spontaneously regenerate the optic nerve after injury. Protecting damaged retinal ganglion cells (RGCs), supplementing neurotrophic factor, antagonizing axon regeneration inhibitory factor, and regulating the inherent regeneration potential of RGCs can effectively promote the regeneration and repair of optic nerve. Basic research has made important progress, including the restoration of visual function, but there are still a lot of unsolved problems in clinical translation of these achievements, so far there is no ideal method of treatment of optic nerve injury. Therefore, it is rather urgent to strengthen the cooperation between basic and clinical research, to promote the transformation of basic research to the clinical applications as soon as possible, which will change the unsatisfactory clinical application status.
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.