ABSTRACT
Objective To explore whether brimonidine has a protective effect on retinal ganglion cells (RGCs) through improving mitochondrial function under the oxidative stress.Methods Mouse RGC-5 cells were cultured in DMEM medium containing low concentration of glucose (1 g/L),10％ fetal bovine serum and 100 U/ml penicillin-streptomycin solution.The cells were divided into normal control group,H2O2-treated group and brimonidine+ H2O2 group.H2O2 at the concentration of 800 μmol/L was added into the medium in the H2O2-treated group,and 1 μmol/L brimonidine was added into the medium for 2 hours prior to the addition of H2O2 in the brimonidine+H2O2 group.The cells were sequently cultured for 24 hours.The morphology of the cell nucleus was examined by Hoechst fluorscence staining.The expressions of apoptosis-related protein in the cells were detected by Western blot assay.Mitochondrial membrane potential was assessed by JC-1 staining.Results The cell nuclei showed round or oval in shape with consistent size in the normal control group.The pycnosis and karyorrhexis of the cell nuclei were seen in the H2O2-treated group,and less abnormal nuclei were found in the brimonidine+H2O2 group.The relative expression level of bcl-2 protein in the cells was 0.76±0.15,0.50±0.13 and 0.75±0.17 in the normal control group,H2O2-treated group and brimonidine + H2O2 group,respectively,and the expression of bcl-2 protein in the H2O2-treated group was significantly lower than that in the normal control group and brimonidine+H2O2 group (both at P＜0.05).The relative expression level of bax protein in the cells was 0.65±0.13,0.83±0.07 and 0.70±0.10 in the normal control group,H2O2-treated group and brimonidine+H2O2 group,respectively,and the expression of bax protein in the H2O2-treated group was significantly higher than that in the normal control group and brimonidine+H2O2 group (both at P＜0.05).A strong orange fluorescence was seen in the mitochondrial membrane of RGC-5 in the normal control group with a coexpression with the green fluorescence of cell membrane.In the H2O2-treated group,the orange fluorescence intensity in the cells was evidently weakened,and the number of JC-1 responsed cells was considerably increased and the orange fluorescence intensity was enhanced in the brimonidine + H2O2 group.Conclusions Brimonidine can prevent RGCs from oxidative-stress damage by improving the mitochondrial function and therefore play a potential neuroprotective effect on optic nerve.
ABSTRACT
Background Acute retinal ischemia anoxic injury is common in eye disorders,such as acute glaucoma,central retinal artery occlusion and ischemic optic neuropathy,etc.This will cause retinal ischemia anoxic injury and induce retinal ganglion cells (RGCs) death in addition.Endogenous cannabinoid (CB) and its receptors are involved in the central nervous system injury,ischemia,inflammation,and poisoning and other physiological and pathological process.Objective This study was to investigate the effect of CB on RGCs damage induced by oxygen-glucose deprivation (OGD).Methods The eyeballs were obtained from 6-week-old normal C57BL/6J mice to prepare retinal frozen sectionsand the expression and distribution of cannabinoid receptors (CB1R and CB2R) in RGCs was detected by immunofluorescence staining.The eyeballs of ten newborn C57BL/6J mice (postnatal 0-3 days) were obtained after immersed by 75％ alcohol and the retinas were isolated in preeooling DMEM for the primary culture of RGCs.The cells were identified by detecting the expression of Brn3a,a marker of RGCs,with immunofluorescence staining.Then the cells cultured for 14 days were divided into normal control group (in complete culture medium+95％ air+5％ CO2) and OGD group (in glucose-free medium+95％ N2 +4％ CO2 + 1％ O2) for 20 hours.The mitochondrial damage and RGCs morphology changed were evaluated by JC-1 staining to observe the mitochondrial membrane potential change.SR141716A (CB1R antagonist,1 μmol/L),SR144528 (CB2R antagonist,1 μmol/L) and 5 or 10 μmol/L WIN 55212-2 (CB1R and CB2R agonist) were added,and the survival rate of RGCs was assayed MTT.Results CBR was positively expressed in various layers of normal mouse retinas.The cells in the normal control group showed uniform size and polygon in shape with the long and thin axons,and the expression of Brn-3a was seen in the cells.However,in the OGD group,cell shrinkage and fragments were found and most of the axons disappeared.The expression of Brn-3a was evidently weakened.The fluorescence intensity of JC-1 was evidently weakened in the OGD group compared with the normal control group,showing the reduce of mitochondrial membrane potential.MTT assay showed that the survival rate of RGCs was (100.00± 13.87)％,which was significantly higher than (89.52-± 18.16)％ in the normal control group (q =8.065,P =0.008).The mean survival rates of RGCs were (116.63±22.21)％ and (112.61 ±19.02)％ in the cells treated by SR141716A and SR144528,and that in the normal cells was (89.52 ± 18.16)％ in the OGD group,with significant differences between SR141716A-or SR144528-treated cells and normal cells (q =29.780,17.391;both at P＜ 0.01).Conclusions Hypoxia and glucose-free up-regulate the expression of CB and activate CB pathway.Inhibition of activation CBR process has a neuroprotection effect under the Hypoxia and glucose-free condition.