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 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 protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro. MethodsNew Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method. ResultsThe survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11± 0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant (P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.001); the difference between ET medium dose group and ET high dose group was statistically significant (P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant (P＜0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group (P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant (P=0.007). ConclusionET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.
ObjectiveTo investigate the effect of erigeron breviscapus (EBHM) on ocular hypertension and the protective effect of retinal ganglion cells (RGCs) in rats by regulating mitogen activated protein kinase (MAPK) signaling pathway.MethodsSixty male Sprague-Dawley rats were divided into control group, model group, low-dose EBHM group (group A), medium-dose EBHM group (group B), and high-dose EBHM group (group C) by random number table method. There were 12 rats in the group, the left eye was used as the experimental eye. The rats of model group, group A, group B, and group C were infused with normal saline through the anterior chamber to construct an acute ocular hypertension model; the control group was given general anesthesia only. Then, 2-30 days after modeling, rats in the control group and model group were given 3 ml of normal saline once a day; rats in group A, group B, and group C were given 0.30, 0.45, and 0.60 g/100 g EBHM by intragastric administration, respectively, 1 time/d. The rat intraocular pressure was measured before modeling and 1, 14, and 30 days after modeling, and the proportion of high intraocular pressure model was measured. Thirty days after modeling, hematoxylin-eosin (HE) staining was used to observe the pathological changes of retinal tissue; immunofluorescence staining was used to detect the changes in the number of RGCs; real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect p38 in the retinas of rats in each group. The relative expression of MAPK and Caspase-3 mRNA; western blot was used to detect p38MAPK and phosphorylation in the retina of rats in each group relative expression of phosphorylate-p38MAPK (p-p38MAPK) and Caspase-3 protein. One-way analysis of variance was used for multi-sample comparison, and SNK-q test was used for comparison between two samples.ResultsOne day after modeling, none of the rats in the control group developed acute ocular hypertension, and the other groups were successfully modeled. Compared with the model group, the rates of acute ocular hypertension at 14 days after modeling in groups B and C were lower (χ2=98.701, P＜0.05), and the rates of acute ocular hypertension at 30 days after modeling in groups A, B, and C were 0. There was no statistically significant difference in the rates of acute ocular hypertension between 14 and 30 days after modeling in the A, B, and C groups (P＞0.05). The results of HE staining showed that the structure of the retina in the control group was complete, and the layers were clearly visible; the RGCs count was not abnormal, and the morphology was plump and round. The retina of rats in the model group became thinner; the number of RGCs was greatly reduced, the morphology was vacuolated, and the arrangement was sparse. The retina of rats in groups A, B, and C became thicker, and the number of RGCs increased, and the retina structure in group C was better restored. The results of immunofluorescence staining showed that the RGCs counts of rats in groups A, B, and C were higher than those in the model group, and the difference was statistically significant (F=297.514, P＜0.05); pairwise comparison between groups, group A was lower than that of group B and C Group (q=2.842, 5.263), group B was lower than group C (q=2.457), the difference was statistically significant (P＜0.05). The results of RT-qPCR and Western blot showed that compared with the model group, the relative expression of Caspase-3 mRNA (F=267.912) and protein (F=692.279) and the relative expression of p-p38MAPK protein in the retina of rats in groups A, B and C. The expression level (F=150.061) all decreased, and the difference was statistically significant (P＜0.05); pairwise comparisons between groups showed that Caspase-3 mRNA (q=6.977, 15.642) and protein (q=6.997, 15.642) relative expression levels and p-p38MAPK protein (q=12.443, 24.358) relative expression levels are lower than groups A and B, group B was lower than group A (q=11.678, 12.471, 10.204), the difference was statistical academic significance (P＜0.05).ConclusionsEBHM can significantly reduce intraocular pressure in rats with acute ocular hypertension, increase RGCs counts, and reduce retinal damage. Its regulatory mechanism may be related to the MAPK pathway.
ObjectiveTo investigate the effect of DJ-1 encoded by Park7 gene on retinal ganglion cells (RGC) and visual function after optic nerve crush injury (ONC) in mice.MethodsThirty-seven and 116 healthy male C57BL/6J mice were randomly divided into group normal, group ONC 2d, group ONC 5d, group ONC 7d and group control, group Park7, group Park7-ONC, group ONC and group green fluorescent protein (GFP)-ONC. Group ONC 2d, group ONC 5d and group ONC 7d were sacrificed on the 2nd, 5th and 7th day after the establishment of ONC model, and the follow-up experiments were carried out. The mice in group Park7 and group Park7-ONC were injected 1 μ recombinant adeno-associated virus (rAAV) with knocking down Park7 gene into vitreous cavity, and 1 μ l rAAV with only GFP was injected into vitreous cavity of mice in group GFP- ONC, and virus transfection was observed 4 weeks after injection. The injury of ONC was perfomed at 23 days after vitreous injection in group ONC, group Park7-ONC and group GFP-ONC, and the samples were taken for follow-up experiment 5 days after modeling. The average density of RGC was observed by immunofluorescence staining, the latencies and amplitudes of a-wave, b-wave and photopic negative response (phNR) and the amplitude of oscillatory potential (OPs)were detected by full-field flash electroretinogram,and the visual acuity of mice was measured by optomotor response (OMR). The relative expression levels of DJ-1, Bax and B lymphoblastoma / leukemia-2 (Bcl-2) protein in the retina of mice in each group were detected by Western blot. One-way ANOVA was used to compare the data between groups, and t-test was used for pairwise comparison between groups.ResultsCompared with the normal group, the relative expression of DJ-1 protein in the retina of the ONC 2 d group and ONC 5 d group increased significantly, and the difference was statistically significant (t=16.610, 5.628, P＜0.01,＜0.05). Four weeks after virus transfection, strong GFP expression was seen in the RGC layer and inner plexiform layer of the retina of mice in the Park7 group. Compared with the control group, the RGC density of the retina in the ONC group decreased significantly, and the difference was statistically significant (t=16.520, P＜0.000); compared with the ONC group, the RGC density of the retina in the Park7-ONC group decreased significantly, and the difference was statistically significant (t=6.074, P＜0.01). With the increase of stimulus light intensity, the dark adaptation a wave and b wave latency of the mice in the control group gradually shortened, and the amplitude gradually increased. The stimulus light intensity was 3 cd·s/m2. There was no statistically significant difference in the dark adaptation a wave and b wave latency and amplitude of the control group, Park7 group, Park7-ONC group, ONC group, and GFP-ONC group (Incubation period: F=0.503, 2.592; P=0.734, 0.068. Amplitude: F=0.439, 1.451; P=0.779, 0.247). Compared with the control group, the Ops and PhNR amplitudes of the ONC group mice were significantly decreased (t=15.07, 12.80; P＜0.000,＜0.001). Compared with the ONC group, the Ops and PhNR amplitudes of the mice in the Park7-ONC group were significantly decreased (t=4.042, 5.062; P＜0.05,＜0.01); there was no statistically significant difference in the PhNR latency of the mice in each group (F=1.327, P=0.287). Compared with the control group, the visual acuity of the mice in the ONC group was significantly decreased, and the difference was statistically significant (t=23.020, P＜0.000); compared with the ONC group, the visual acuity of the mice in the Park7-ONC group was significantly decreased, and the difference was statistically significant (t=3.669, P＜0.05). Compared with the control group, Park7-ONC group and ONC group, the relative expression of DJ-1 protein in the mouse retina was significantly down-regulated, and the difference was statistically significant (t=47.140, 26.920; P＜0.000,＜0.000). There was no significant difference between ONC group and GFP-ONC group (t=0.739, P=0.983). Compared with the ONC group, the relative expression of Bax protein in the mouse retina of the Park7-ONC group was significantly increased, and the relative expression of Bcl-2 protein was significantly reduced. The differences were statistically significant (t=5.960, 9.710; P＜0.05,＜0.05); the relative expression ratio of Bcl-2/Bax in the Park7-ONC group was significantly lower than that in the ONC group, and the difference was statistically significant (t=13.620, P＜0.01).ConclusionThe expression of DJ-1 encoded by Park7 gene is down-regulated after Park7 gene was knocked down, which aggravates the RGC damage and the decrease of retinal electrophysiological response and visual function in ONC injury mice.
Transsynaptic retrograde degeneration of optic neuropathy (TRDON) refers to the degeneration and/or apoptosis of presynaptic neurons (retinal ganglion cells) caused by damage to the lateral geniculate body and post-geniculate visual pathway. At present, the pathogenesis of TRDON is secondary apoptosis of Pβ-type retinal ganglion cells, resulting in the atrophy of optic tract, thinning of the retinal nerve fiber layer and retinal ganglion cell layer thickness and declining of retinal microvascular density, which are consistent with the visual field defect attributed to the primary disease. Of which, the thinning of the retinal ganglion cell layer thickness is considered as the characteristic of TRDON. Now, there is little understanding and related research on TRDON in China. Clinicians should pay attention to the characteristics and severity, occurrence time and location of the above structural changes in these patients through optical coherence tomography, and monitor the activity and progress of the lesions, so as to determine the cut-off point for drug intervention and the drug targets for developing new treatment methods, and bring benefits for patients in partial visual function recovery and disability reduction.
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 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 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 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)