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Background Retinal neovascular diseases affect visual function.Although many drugs have been used to manage the visual diseases,their effectiveness is less than satisfactory.Studies showed that ursolic acid has multiple biological effects including anti-vascularization.However,the effect of ursolic acid on retinal neovascular diseases is unclear now.Objective This study was to observe the inhibitory effect of ursolic acid on the high oxygen-induced mouse retinal neovascularization after intravitreal injection.Methods Sixty clean 7-day-old C57BL/6J mice were divided into the blank control group,PBS control group,positive control group (triamcinolone) and low,moderate and high dose (1.5,3.0 and 6.0 μg) ursolic acid groups randomly.The blank control group mice were raised in normal environment,and the mice from other groups were fed in the environment with O2 concentration at (75±2)% for 5 days together with the maternal mice.The mice then were back to the normal air environment to induce retinal neovascularization.Then,the drugs were intravitreally immediately injected in the mice of the different groups.The mice were sacrificed at the 17-day old for the preparation of retinal sections.Retinal new blood vessel was examined by haematoxylin and eosin stain under the light microscope,and the number of vascular endothelial cell nucleus breaking the inner limiting membrane was counted.The gene expressions of vascular endothelial growth factor (VEGF),cyclooxygenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2) in the mouse retinas were quantitatively assayed using reverse transcription PCR.Results The number of endothelial nuclei newly-generated vessel breaking internal limiting membrane in the mice of PBS control group was (18.65±3.24)/field,which was more than (0.78±0.11)/field of the blank control group obviously (t =2.24,P<0.05).The number of endothelial nuclei newly-generated breaking internal limiting membrane in the moderate-or high-dose ursolic acid group was less than that of moderate group obviously,it was statistically significant(P<0.05).The number of vascular endothelial cell nuclei breaking internal limiting membrane in high high-dose group was (13.32 ± 1.87)/field and (8.93 ± 1.09) /field,showing significant decreases in comparison with the PBS control group and low-dose ursolic acid group (18.65±3.24)/field (15.44±2.02)/field (all at P<0.05).However,no significant difference were seen in the number of new vascular endothelial cell nucleus between the high-dose ursolic acid group and the positive control group(9.14±1.13)/field (t=1.17,P>0.05).The relative expressions of COX-2 mRNA,VEGF mRNA and MMP-2 mRNA in the mouse retinas were higher in the PBS control group than those in the blank control group (t =13.45,12.49,14.32,all at P<0.05),and those in the moderate-dose or high-dose ursolic acid group were lowed in comparison with the PBS control group and the low-dose ursolic acid group (all at P<0.05),but there were no significant differences between the high-dose ursolic acid group and the positive control group (all at P>0.05).Conclusions Ursolic acid can suppress retinal neovascularization by down-regulating the expressions of VEGF,COX-2 and MMP-2 in oxygen-induced retinopathy of mouse in dose-dependent manner.
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Background Retinopathy of prematurity (ROP) causes blindness due to retinal vasculopathy caused by abnormal oxygen dynamics.Studies have clarified the pivotal role of vascular endothelial growth factor (VEGF) in the development of ROP,while the studies on the role of VEGF receptor (VEGFR) in ROP are fewer.Objective This study was to evaluate the relationship between the expressions of VEGFR-1 and VEGFR-2 in retina and post-birth time in the mice with oxygen-induced retinopathy (OIR).Methods Sixty 7-day-old (P7) SPF C57BL/6J mice together with lactating female mice were fed in the environment with (75±2)% oxygen concentration for 5 days and then returned to normal air to establish OIR models,and other 60 matched mice were kept in normal air environment as the control group.At P8,P11,P12,P13,P14,and P17,both eyes of each mouse were enucleated to prepare the retinal sections.Retinal blood vessels were examined by hematoxylin and eosin stain under the light microscope.Real-time fluorescence quantitative PCR and immunohistochemistry were used to detect the expressions of VEGFR1 mRNA and VEGFR2 mRNA and their proteins in mouse retinas,respectively.The use and care of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Many vascular endothelial cell nucleus broke through the internal limiting membrane were seen with disorganized cell proliferation under the internal limiting membrane and retinal neovascularization bud in the P13,P14 and P18 mice in the OIR group,but no similar manifestation was found in the mice of the normal control group.The relative expression values of VEGFR-1 mRNA in retinas were significantly higher only in P12,P13,P14 and P18 mice in the OIR group than those in the normal control group (P=0.046,0.000,0.000,0.042),but the expression values of VEGFR-2 mRNA were all increased in the retinas of P8,P11,P12,P13,P14 and P18 mice in the OIR group,showing significant differences in comparison with the normal control group (all at P=0.000).No considerable difference was found in the expression level of VEGFR-1 protein in P8,P11,P12,P13,P14 and P18 mice between the two groups (M=50.00,36.00,41.00,31.00,28.00,36.00,all at P> 0.05).The expression levels of VEGFR-2 protein in retinas of P8 and P11 were close between the two groups (all at P>0.05).Gradually attenuated expressions were seen in VEGFR-2 protein in P12,P13 mice of the normal control group,however,the expressions were enhanced in the OIR group,with significant differences between the two groups (all at P<0.01).Enhanced expressions of VEGFR-2 protein were found in P14 mice in both groups,but stronger expressions were in the OIR group (P<0.01).The positive response of the expression of VEGFR-2 protein was weaker in P18 mice in the normal control group but peaked in the OIR group,showing significant difference between them (M=20.11,P<0.01).Conclusions VEGFR participates in the neovascularization of OIR mouse,and the effect of VEGFR-2 is stronger than that of VEGFR-1 in the development of OIR neovascularization.
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Background Researches indicated that etiology and epidemiology of pertussis toxin (PTX)dependent experimental autoimmune uveoretinitis(EAU)model are very different with human uveoretinitis owing to the influence of PTX on immune.Our previous study has established lipopolysaccharide (LPS),an endotoxin,which instesad of PTX,mediated EAU model.However,the exact roles of LPS and PTX in EAU still remained unclear.Objective This study was to investigate the roles of LPS and PTX in EAU model.Methods Twenty SPF C57BL/6(H-2b) mice were assigned to 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group using random number table method.The mice were immunized with interphotoreceptor retinoid-binding protein 1-20(IRBP 1-20) emulsified in complete Freund adjuvant (CFA),and concurrently with or on day 7 postimmunization,LPS or PTX was injected in the footpad or intraperitoneally respectively.Delayed-type hypersensitivity (DTH) of the mice was evaluated by measuring the ear thickness 48 hours after IRBP was injected into the ear pinna,and lymphocyte proliferation was assessed by tritiated thymidine uptake.Retinal histopathological examination was performed and scored based on criteria of Caspi.The use and care of experimental animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Serious infiltration of inflammatory cells,disorder of entire retinal structure and retinal folds were seen in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group on 21 days after injection of PTX or 14 days after injection of LPS,and severe vitritis and a few granuloma-like lesions were found in the 0 d-PTX-EAU group.However,only mild vasodilatation or less retinal folds were found in the 7 d-PTX-EAU group and 0 d-LPS-EAU group.The pathological scores in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group were higher than those of the 7 d-PTX-EAU group and 0 d-LPS-EAU group (all at P < 0.05).The ear thickness was (62.600 ± 3.362) μm,(60.000±2.345) μm,(30.400± 1.817) μm and (32.800 ± 1.643) μm in the 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group,showing a significantly difference among the 4 groups (Fgroup =259.751,P=0.000),and the ear thicknesses of 0 d-PTX-EAU group and 7 d-PTX-EAU group were significantly higher than those of the 0 d-LPS-EAU group and 7 d-LPS-EAU group (all at P<0.05).The lymphocyte proliferation was strongly enhanced in PTX-EAU groups,and the radiation count per minute (cpm) was (16 150.000±799.218)/min and (16 120.000±729.383)/min in the 0 d-PTX EAU group and 7 d-PTX EAU group,and (8 348.000±258.979)/min and (8 540.000±81.548)/min in the 0 d-LPS EAU group and 7 d-LPS EAU group respectively,with a significant difference among the PTX-EAU groups and LPS-EAU groups (Fgroup =316.978,P=0.000).Conclusions LPS and PTX play different roles during the EAU formation.LPS may be involved in the breakdown of blood-retina barriers (BRB).
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Background Spectral-domain optical coherence tomography (SD-OCT) can quantitatively analyze some ocular parameters in vivo.Although human ocular parameters have been obtained by SD-OCT,few studies were performed in animal experiment.Objective This study was to investigate the anterior and posterior segment parameters of C57BL/6 mice and pigmented rabbits using SD-OCT in vivo.Methods Some anterior and posterior segment ocular parameters,including the central corneal thickness (CCT),anterior chamber depth (ACD),white-to-white (WTW),optic nerve head (ONH) depth/width and retinal thickness,were measured in 8 eyes of 4 health SPF C57BL/6 mice and 12 eyes of 6 health SPF pigmented rabbits using SD-OCT.Results For C57BL/6 mice,Cornea,iris,lens in pupil area were clearly exhibited by SD-OCT.Mean CCT,ACD and WTW were (96±9)μm,(460±8) μm and (2.86 ± 0.41) mm pre-mydriasis,respectively,the corresponding values of post-mydriasis were (96±8) μm,(356±20)μm and (2.87±0.62)mm.There were no statistical differences of CCT and WTW between pre-and post-mydriasis (t =0.478,P =0.647 ; t =-0.737,P =0.485).ACD of post-mydriasis was significantly shallower than that of baseline (t =-13.022,P<0.001).For the pigmented rabbits,the thickness of corneal thinnest point,retinal thickness,ONH depth and width were (370 ± 10) μm,(175 ± 4) μm,(1.35 ± 0.51) mm and (4.52±0.82) mm,respectively.Conclusions As a non-contact and non-invasive technology,SD-OCT can provide not only high resolution cross-sectional ocular images,but also high precise quantitative parameters for both C57BL/6 mouse and pigmented rabbit in vivo.
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Background Diabetic retinopathy (DR) is a common ocular complication of diabetes,and its pathogenesis is associated with a variety of factors.c-Jun N terminal kinase (JNK),one of the genes involving in apoptosis,plays an important role in the pathology of diabetes,and relative research is catching increasing interests in recent years.Objective This study was to quantify the expression of JNK3 in retinas of DR murine.Methods Forty-eight SPF male C57BL/6 mice were randomly divided into the diabetes group and the normal control group.Diabetic mouse models were establishend by intraperitoneal injection of 1% streptozocin (STZ) dissolved by sodium citrate buffer,and equvilant volume of sodium citrate buffer was used in the same way in the mice of the control mice.The left eyeballs were obtained 2,4,8 weeks after modeling and the retinas were collected.Real-time quantitaive PCR was perfored to detect the expression of JNK3 mRNA in retinas.The use and care of the experimental mice complied with the Administration of Experimental Animals in Kunming Medical College.Results Blood glucose levels were significantly higher in 2,4,8 weeks after modeling in the diabetic group compared with the normal control group (t=-5.675,-5.498,-5.347,all at P<0.01).The relative expression levels of JNK3 mRNA (A value) in the retinas were significantly different between the groups at various time points (Fgroup =102.345,P<0.05 ; Ftime =131.679,P< 0.05).The relative expression levels of JNK3 mRNA in the retinas were 3.21 ±0.14 and 5.43 ±O.37 in 4 and 8 weeks after modeling in the diabetic group,which were significantly elevated in comparison with the normal control group (2.54±0.42 versus 2.26±0.67) (t =4.073,23.399,both at P<0.05).Compared with the second week and fourth week,the relative expression levels of JNK3 mRNA in the retinas in the eighth week were significantly raised in the diabetic group (t =10.756,16.857,both at P < 0.05).Conclusions JNK3 expression in the retina upregulates in diabtic mice in a time-dependent manner.JNK3 is paopably involved in the pathogenesis and development of DR.