Authors: Shen Yumeng, Lin Zhongqiao, Liu Shiliang, Tian Kailin, Chen Yuanyuan, Shen Yin
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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 precooling 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.