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.
ABSTRACT
Background The conventional drugs for preventing and treating graft rejection have the risks of inducing adverse responses.Researches showed that resolvinE1 (RvE1) can regulate Th1 cell-mediated immunoreaction.However,whether RvE1 has an inhibit effect on high-risk corneal graft is unclear now.Objective This study was to investigate the effects of RvE1 on immune rejection in high-risk corneal grafting mouse models.Methods SPF BALB/c mice were used as recipients,C57BL/6 mice were as donors.Ninety BALB/c mice were divided into corneal allograft group,corneal allograft+RvE1 group and corneal autograft group according to random number table.High-risk corneal graft models were established by corneal suturing for 14 days and followed by penetrating keratoplasty in recipients.Allograft keratoplasty was performed on the right eyes in the mice of corneal allograft group and corneal allograft+RvE1 group,and self-corneal graft rotated 180°was transplanted on the right eyes in the mice of autograft group.Normal saline solution of 10 μl was subconjunctivally injected after surgery once per day for 7 days in the corneal allograft group and corneal autograft group,and 10 μl RyE1 (1 μg) was used in the same way in the corneal allograft+RvE1 group.The recipient eyes were examined for potential rejection signals with slit lamp microscope and calculated the mean survival time and rejection index (RI).The histopathology was examined 21 days after modeling by hemotoxylin and eosin staining.The expressions of CD4 and interferon-γ (IFN-γ)in the corneas were detected by immunohistochemistry.Th1 cell (CD3+CD8a-IFN-γ+) percentage in draining lymph nodes were measured by flow cytometry.The mRNA expression levels of interleukin-2 (IL-2),tumor mecrosis factor-α (TNF-α),IFN-γ and T-bet were detected by real-time fluorescence quantitative PCR.Results The mean survival time of grafts was (28.5± 1.7) days in the corneal allograft group,and that in the corneal allograft+RvE1 group was (14.0±1.6) days,showing a significant difference between them (t =4.14,P＜0.001),while the survival rate was 100％ at 50 days after modeling in the corneal autograft group.Corneal edema and inflammatory cell infiltration were slight in the corneal allograft+RvE1 group and corneal autograft group compared with corneal allograft group.CD4 was positively expressed in corneal tissue,and IFN-γ was expressed in corneal epithelium.The CD4+ and IFN-γ+ cell number was decreased in the corneal allograft+RvE1 group and corneal autograft group compared with corneal allograft group under the fluorescence microscope.The percentages of Th1 cells in lymph cells of corneal allograft +RvE1 group and corneal autograft group were (1.07 ±0.25) ％,(0.85 ±0.12) ％,respectively,which were significnatly lower than (1.56±0.20) ％ in the corneal allograft group (both at P＜0.05).The expressions of IL-2,TNF-α,IFN-γ and T-bet mRNA in the corneal tissue in the corneal allograft group were higher than those in the corneal allograft+RvE1 group and corneal autograft group (all at P＜0.05).Conclusions RyE1 inhibits graft rejection in high-risk allograft mouse models probably by down-regulating the Th1 cell percentage in lymph cells and the expression of inflammationrelated cytokines in corneal grafts.
ABSTRACT
Background Retinal neovascularization is associated with various disorders.Studying the pathogenesis of retinal neovascularization is of important significance.Oxygen-induced retinopathy(OIR) mouse model is a common animal model for the study of retinal neovascular diseases.However, conventional modeling methods usually cause high animal mortality and low rate of success.Objective This study aimed to establish a modified method of mouse OIR model.Methods Eighty 1-week-old SPF C57BL/6J mice were randomly divided into normal control group and OIR group with 40 mice for each.The newborn mice of the normal control group were kept in a normal air environment with their breast-feeding mothers, but the mice of postnatal 2 days (P2) in the OIR group were raised with two litters per cage until P7.The P7 mice exposed to oxygen tank containing 80％ oxygen together with one or another mother mouse alternately daily for 5 days and then returned to the normal air environment.The success rate of modeling,mortality rate of maternal mice and survival rate of immature mice were evaluated.The mixed solution of fluorescein isothiocyanate (FITC) and PBS with 4％ paraformaldehyde was infused into the hearts of P12, P14,P17 and P21 mice and the eyeballs were obtained after the mice were sacrificed for histopathological examination of retinas and preparation of retinal flatmounts.The number of vascular endothelial cells extending inner limiting membrane was counted and the distribution of retinal vessels was evaluated.The use and care of the animals complied with the Statement of ARVO.Results The survival rate of the neonatal mice was 100％ both in the normal control group and the OIR group,and the survival rate of maternal mice was 85.7％ in the OIR group.Retinal new vessels were found in the mice of the OIR group,with the success rate of modeling 100％.The retinal vessels distributed from optical disc toward periphery in P14 mice in the normal group.However,in the OIR group,non-perfusion area at the posterior pole was seen in P12 mice,new blood vessels at the periphery were found in P14 mice, neovascularization at the junction area between vascular area and non-perfusion area as well as leakages were exhibited in P17 mice,and less non-perfusion area and new vessels were seen in P21 mice.Retinal inner limiting membrane was smooth in the mice of the normal group, and the vascular endothelial cell nucleus extending inner limiting membrane were seen in P12 mice and peaked in P17 mice.The vascular endothelial cell nucleus were (11.44±2.01), (31.24±1.50) and (9.23-±1.12)/slide in P14, P17 and P21 mice in the OIR group,which were significantly more than (0.27±0.14) , (0.30±0.11) and (0.32±0.16)/slide in P14, P17 and P21 mice in the normal group (t=47.90,61.30,40.70,all at P＜0.05).Conclusions The method of OIR modeling is modified by alternating maternal mice,exposing to 80％ oxygen-nitrogen mixture gas and cohabitating immature mice.Modified modeling method is simple with the low death rate of maternal mice and stable OIR phenotype.
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Objective To observe the protective effect of N-acetyl-cysteine (NAC) on the injury of irradiation-in-duced bone marrow mononuclear cells (BMMNCs), and explore the possible mechanism. Methods There were 3 groups in the study:control group, irradiation group (doses of irradiation were 1 Gy and 4 Gy) and irradiation with NAC group (NAC was cocultured with BMMNCs half hour before irradiation). The 2×106/mL BMMNCs and the RPMI-1640 medium or 2×10-5 mol/L NAC were added into the 2 mL EP tubes according to the different requirement of groups. The tubes were then cul-tured in the 37℃CO2 incubator for 30 min and irradiated with 1 Gy and 4 Gy. The viability of BMMNCs was measured by bioluminescence. The level of reactive oxygen species (ROS) was measured by DCFH-DA, and the ability of colony-forming units was detected by CFU-GM. Results After 4 Gy irradiation exposure, the cell viability of BMMNCs was significantly lower in irradiation group (284 296.7±16 541.2) than that of control group (848 586.7±61 404.4). After 1 Gy irradiation expo-sure, the level of ROS was higher in irradiation group (6 750.0±103.5) than that of control group (5 710.7±56.2). The number of colony-forming units per 105 cells after irradiation exposure was (626.7±51.3), which was significantly lower than that of control group (986.7±100.7). Compared to irradiation group, the cell viability of BMMNCs increased to (352 770.0±23 466.1) in irradiation with NAC group. The level of ROS decreased to (5 430.0±61.0), and the number of colony-forming units per 105 cells increased to (773.3 ± 49.3). Conclusion NAC has protective effect on irradiation-induced injury in BMMNCs, which may be related with the decreased level of ROS. NAC can play the role of positive control for the following work.