Objective
To determine the lipid peroxide damage in the primary cultured rabbit retinal ganglion cells induced by microwave．
Methods
Cultured rabbit retinal ganglion cells in vitro and exposed to 80 mW/cm2 of microwave for 15,30,45 min tespectively.Immediately after radiation,the morphological variation of cells was observed by optical microscope and transmission electronic microscope.Secondly,the activity of intracellular superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected.
Results
Aportion of cells congregated,with their axon disapeared after radiation.Mitochondria and endoplasmic reticulum revealed swelling under transmission electronic microscope.The content of MDA was increased obviously compared with control group while SOD decreased．The content of MDA as increased obviously compared with control group after 45 min radiation was 5.11 times,while SOD decreased.The content of MDA as in control and the ganglion cells were apparantly destroyed.
Conclusion
Microwave can induce the lipid peroxide damage in primary cultured retinal ganglion cells,and lipid peroxide effect might be one of the mechanisms of microwave retinal damage.
(Chin J Ocul Fundus Dis, 2000，16：32-34)
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 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 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 effects of form deprivation on the morphology of different types of RGC in mice.MethodsSixty B6.Cg-Tg (Thy1-YFP) HJrs/J transgenic mice were randomly assigned to form-deprived group (n=28) and control group (n=32). The right eyes of mice in the form-deprived group were covered by an occluder for 2 weeks as experimental eyes. The right eyes of mice in the control group were taken as control eyes. Before and 2 weeks after form deprivation, the refraction and ocular biometrics were measured; RGC were stained with Bra3a antibody and counted; the morphology of RGC was reconstructed with Neuroexplore software after immunohistochemical staining. The data was compared among experimental eyes, fellow eyes and control eyes by one-way analysis of variance.ResultsTwo weeks after form deprivation, the axial myopia was observed in the experimental eyes (refraction: F=15.009, P＜0.001; vitreous chamber depth: F=3.360, P=0047; ocluar axial length: F=5.011, P=0013). The number of RGC in central retina of the experimental eyes was decreased compared with the fellow eyes and the control eyes (F=4.769, P=0.035). The reconstructed RGC were classified into 4 types according to their dendritic morphology. Form deprivation affected all 4 types of RGC but in a different way. Among them, 3 types of RGC were likely contribute to form vision perception. Form deprivation increased the dendrite branches in these types of ganglion cells. However, form deprivation decreasd dendrite segment numbers in both eyes and the intersection and length insholl analyse type 4 ganglion cells which were morphologically identified as ipRGC.ConclusionForm deprivation distinguishingly affects the morphology of different types of RGC, indicating that form vision and non-form vision play different role in myopia development.
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.
ObjectiveTo observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells (RGC) after retinal ischemia-reperfusion injury (RIRI). MethodsOne hundred healthy adult male C57BL/6 mice were randomly divided into normal control group (group C), RIRI group, adeno-associated virus (AAV2)-S100A4 green fluorescent protein (GFP) intravitreal injection group (group S), RIRI+AAV2-GFP intravitreal injection group (group GIR), and RIRI+AAV2-S100A4-GFP intravitreal injection group (group SIR), with 20 mice in each group. The RIRI model was established using the high intraocular pressure anterior chamber method in the RIRI, GIR and SIR groups of mice. Eyes were enucleated 3 days after modelling by over anaesthesia. The number of retinal capillary endothelial cells and pericytes in the retinal capillaries of mice in each group was observed by retinal trypsinised sections and hematoxylin-eosin and periodic acid-Schiff staining; immunofluorescence staining was used to observe endothelial cell, pericyte coverage and RGC survival; The relative expression of Toll-like receptor 4 (TLR4), p38 MAPK and nuclear factor erythroid 2-related factor 2 (NRF2) in retinal tissues was measured by Western blot. One-way analysis of variance was used to compare data between groups. ResultsThree days after modeling, the endothelial cell to pericyte ratio in group C was compared with group S and SIR, and the difference was not statistically significant (F=106.30, P＞0.05); the SIR group was compared with group RIRI and GIR, and the difference was statistically significant (F=106.30, P＜0.000 1). Comparison of endothelial cell coverage in each group, the difference was not statistically significant (F=3.44, P＞0.05); compared with the pericyte coverage in group C, the RIRI group and the GIR group were significantly lower, and the difference was statistically significant (F=62.69, P＜0.001). Compared with the RGC survival rate in group C, it was significantly lower in RIRI and GIR groups, and the difference was statistically significant (F=171.60, P＜0.000 1); compared with RIRI and GIR groups, the RGC survival rate in SIR group was significantly higher, and the difference was statistically significant (F=171.60, P＜0.000 1). The relative expression levels of TLR4, p38 and NRF2 proteins were statistically significant among all groups (F=42.65, 20.78, 11.55; P＜0.05). ConclusionsPericytes are more sensitive to ischemia than endothelial cells after retinal RIRI in mice, and early vascular cell loss is dominated by pericytes rather than endothelial cells. The overexpression of S100A4 protein protects against loss of pericytes and RGC after RIRI by inhibiting the TLR4/p38/NRF2 signaling pathway.
Objective
To establish and evaluate a rat model of nonarteritic anterior ischemic optic neuropathy (NAION).
Methods
The rats were randomly divided into control group (n=13), sham laser group (n=11) and NAION group (n=23). The right eye was set as the experimental eye. NAION model was induced by directly illuminating the optic nerve (ON) of the right eye with 532 nm green laser, after intravenous infusion with the photosensitizing agent Rose Bengal. Sham laser treatment consisted of illuminating the ON region with 532 nm laser without Rose Bengal injection. Rats in control group underwent no intervention. The appearance of optic disc was observed with funduscope at 12 hours, 1, 3, 7, 28 days post-illumination. The histologic changes in the retina and ON of the NAION model were evaluated qualitatively with hematoxylin and eosin (HE) staining and transmission electron microscopy. The retrograde-labeled retinal ganglion cells (RGC) were counted on photographs taken from retinal flat mounts in a masked fashion.
Results
The optic disc in NAION eyes were swollen 3 days after photodynamic treatment. HE-stained longitudinal ON sections of NAION revealed vacuolar degeneration on day 3 after induction. Besides, ultrastructural study showed axonal edema and collapsed sheaths in the ischemic optic nerve at the same time point after modeling. ON edema resolved 7 days after induction. The final results revealed optic disc atrophy, extensive axonal loss, severe glial scar, and RGC death in large numbers 4 weeks after modeling. There were no aforementioned manifestations in control and sham laser group. The RGC density of the right eyes was statistically significantly lower in NAION group than that in control group and in sham laser group (t=?14.142, ?14.088; P=0.000, 0.000). The survival rate of RGC was statistically significantly lower in NAION group than in control group and in sham laser group (t=?17.048, ?16.667; P=0.000, 0.000). There was no difference of RGC density and survival rate of RGC between control and sham laser group (t=0.050, 0.348; P=0.961, 0.731).
Conclusion
A rat model of NAION was established successfully by photodynamic treatments with Rose Bengal, which induce optic nerve damage and RGC death.
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)
