| Part 1.Neuroprotective Effect of Low-dose Chloroquine on Retinal Ganglion CellsObjective:To investigate the effects of different concentrations of chloroquine on retinal ganglion cells(RGCs)in the case of retinal damage and the protective effect of low-dose chloroquine in morphology and function of RGCs.Methods:Wild-type C57/BL6 mice(5-week-old)were randomly divided into three groups.Retinas of the first group of mice were plated and cultured in chloroquine containing 0,1,5,10,25,40,50,and 100μM,respectively.The medium was cultured for 5 days to construct a retinal explant model.After 5 days of culturing in the corresponding medium,Brn3a immunofluorescence staining of retinal explants was used to detect the survival RGCs.About the second group,a dose of 0、10、100 mg/kg of chloroquine was injected intraperitoneally to different mice for 2 days,then anterior chamber high pressure infusion(90 mmHg,60 min)was performed with normal saline to build an animal model of acute ocular hypertension.The animals were injected intraperitoneally every day until the animals were killed.The intraocular pressure was measured 7 days after the model was established,and the retinas were subjected to immunofluorescence staining of whole retinas to compare the number of remaining RGCs in mice of different experimental groups.The third group of mice were first treated with 0,10,50,100 mg/kg of chloroquine.After intraperitoneal injection of different chloroquine doses into different mice for 2 days,1μL of 5mM NMDA was injected into the vitreous cavity to establish an animal model of retinal excitotoxicity.At the histological level,immunofluorescence staining of retinas,and paraffin resection were performed.H&E staining was used to observe the damage of RGCs at 7 days after modeling.At the functional level,visual acuity response and awave,b-wave,and PhNR-wave of electroretinogram detection were used to evaluate 2(9)days and 3(10)after modeling to evaluate the function status of surviving RGCs.The density,visual acuity,and the amplitude of PhNR-wave of RGCs between groups were compared by one-way analysis of variance.Results:In the retinal explant model,compared with the control group,a low concentration of 10 μM chloroquine could double the survival of RGCs(P<0.001),and when the concentration of chloroquine reached 50 μM or more,compared with the control,the number of surviving RGCs was reduced.In the acute ocular hypertension animal model,the density of RGCs[(2129.67±91.00)mm2]after treatment with low concentration of chloroquine(10 mg/kg)was higher than that of the normal saline control(untreated)group[(1495.33±86.55)mm2],the difference was statistically significant(P<0.05).The density of RGCs decreased slightly in the high concentration chloroquine(100mg/kg)treatment group[(1233.67±33.72)mm2].In animal models of NMDA-induced excitatory injury,NMDA can significantly reduce the density of RGCs and the amplitudes of b-waves and PhNR waves of electroretinograms.After treatment with low-dose chloroquine(10 mg/kg),the density of RGCs in mouse retinas was paved.It was significantly higher than that of the control group,and the visual acuity and the amplitude of b-wave and PhNR wave were also improved,the differences were statistically significant(P<0.05).The density was lower than that of the saline control group.Conclusion:Low-dose chloroquine can reduce the death of RGCs in mouse retinal explants,NMDA model and acute ocular hypertension animal model.It can also functionally protect RGCs from NMDA induced excitotoxicity.High concentrations of chloroquine will aggravate the apoptosis of RGCs,indicating that different concentrations of chloroquine may play different roles in retina through different mechanisms.Part 2.Mechanism of Low-dose Chloroquine in Protecting Retinal Ganglion Cells from NMDA Induced ExcitotoxicityObjective: To explore the protective mechanism of low-dose chloroquine on retinal ganglion cells(RGCs)in case of NMDA-induced retinal injury.Methods: Wild-type C57 / BL6 mice(5-week-old)were randomly divided into three groups.Mice in the low-dose chloroquine group were injected intraperitoneally daily at a dose of 10mg/kg of body weight,and chloroquine solution was injected intraperitoneally every day at a dose of 100mg/kg of body weight in high-concentration chloroquine group,normal saline of equal volume was injected intraperitoneally in control group mice.After 2 days of continuous injection,intravitreal injection of 1μL of 5m M NMDA was carried out to establish an animal model of retinal excitatory injury.The mice were sacrificed 11 days after modeling,the retinas were removed,retinal RNA and protein were extracted,and QPCR and Western Blot techniques were used to evaluate the expression of inflammatory factors and Sigma1 receptor in each group of mice.Frozen sections and immunofluorescence staining were performed on retina to evaluate the glial activation reaction of retina in each group of mice.The above was used to preliminarily explore the mechanism of low-dose chloroquine protecting RGCs against excitatory toxicity of NMDA.One-way ANOVA was used for data comparison among multiple groups.Results: 11 days after NMDA modeling,the protein and m RNA levels of caspase-3 and the number of TUNEL positive cells in the retina of mice in low-dose chloroquine group were lower than those in normal saline control group,and the difference was statistically significant(P< 0.05).Low-dose chloroquine treatment reduced the m RNA levels of inflammatory factors such as IL-6,TNF-α and IL-1β compared with the control group,while the high-dose chloroquine had the opposite effect.The m RNA levels of the above inflammatory factors were higher than those of the control group,and the differences were statistically significant(P<0.05).At the same time,the positive signal,protein and m RNA level of GFAP and Iba1 positive cell number of mice retina in low-dose chloroquine group were also lower than those in other two groups.The above indexes in high-dose chloroquine group were slightly higher than those in control group.Compared with normal saline control group,the protein expression of Sigma1 receptor increased after low-dose chloroquine treatment,with statistically significant difference(P < 0.05).Conclusion: In NMDA retinal injury model,low-dose chloroquine can play a protective role on RGCs by inhibiting apoptosis of RGCs,reducing the expression of glial activation reaction and inflammatory factors after injury,increasing the expression of protection receptor σR1. |
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