Objective To observe whether theograde axial flow of retinal ganglion cells (RGC) in diabetic rats at the early stage was damaged. Methods Diabetic model was induced by streptozotocin in 6 adult male Sprague-Dawley (SD)rats. Fluorogold (FG) was injected to the superior colliculi 4 weeks later.Streched preparation of retina was made 12 and 72 hours after the injection, and was stained after photographed by fluorescent microscope. The proportion of RGC with different sizes labeled by FG was calculated. Other 6 normal adult male SD rats were in the control group. Results Twelve hours after injection with FG, there was no difference of the total number of RGC in experimental and control group, but the ratio of small RGC was lower in experimental group than that in the control group; 72 hours after injection with FG, The number of RGC, especially the small RGC, decreased obviously in experimental group compared with the control group. Conclusion The speed of the retrograde axial flow of RGC in diabetic rats at the early stage is affected, and the small RGC are damageable. (Chin J Ocul Fundus Dis, 2006, 22: 4-6)
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 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 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 observe the effects of testosterone on optic nerve an d retinal ganglion cells (RGC) in experimental autoimmune encephalomyelitis (EAE ). Methods:Fourty one female Wistar rats were randomly divide d into 3 groups: the normal group (10 rats), the untreated control group (15 rats) and the testos terone group (16 rats). The rats in the first two groups were fed with 1% ethano l every day, and the rats in the testosterone group were fed with methyltestoste rone (0.25 mg/kg) every day. On the 20th day, EAE model was induced in the untre ated control group and the testosterone group by injecting guinea pig spinal cor d homogenate in complete Freund's adjuvant and bordetella pertussis vaccine. RGC were labeled with flurogold (FG) by injecting it in superior colliculus and lat eral geniculate body 7 days before establishing EAE model. All rats were fed wit h drugs continuously, and after 1430 days, rats in normal group and rats in un t reated control and testosterone groups who had symptoms within 48～72 hours were observed by light microscopy and flash visual evoked potential (FVEP) to detect the functional and morphological changes of optic nerve. The number of RGC was counted by fluorescence microscopy，and apoptosis of RGC was observed by termina l deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling (TUN E L) Results:EAE rats presented weakness or paralysis of tail a nd hind limbs 10 days after establishing EAE model. Compared with the rats in the untreated contr ol group, the rats in the testosterone group had longer disease delitescence and lower clinical score (P=0.042). Extensive demyelination of optic nerves wi th the circuitous configuration was found in the untreated control group; while mild demyelination of optic nerves with regular figure was found in the testosterone group. In the testosterone group, the latency of N1、P and N2 wave was shorter w hile the amplitude ofN1-P and P-N2was higher than that in the untreated cont rol group (Plt;0.05). The number of RGC was (2284plusmn;132), (934plusmn;78, and (1725 plusmn;95)cells/mm2 in the normal, untreated control and testosterone groups, respectively; w hich was higher in testosterone group than that in untreated control group (P=0.028). The number of TUNEL positive cells was (4.02plusmn;0.16), (24.44plusmn;2.22), and (9.84plusmn;2.36) cells per high power field (times;400) in the 3 grou ps, respectively; wh ich was less in testosterone group than that in untreated control group (P＝0.025). Conclusions:Testosterone may reduce the incidence and clinical score of EAE, inhibit the apoptosis of RGC, alleviate the demyelinatio n of optic nerves, and improved the conduction function of optic nerves.
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 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.