Objective To observe the effect of persistent flickering stimulus on the structure and function of retina in guinea pigs during a developmentally sensitive period.Methods Twenty-four two- week-old guinea pigs were randomly divided into flicker light (FL) group and control group, with 12 guinea pigs in each group. Animals in FL group were raised under 500 Lux illumination with a duty diurnal cycle of 50% at a flash rate of 0.5 Hz. Animals in control group were reared under steady 500 Lux illumination. Light emitting diode (LED) lamps were used for lighting under a 12-hour light/12-hour dark cycle. After the collection of fundus photographs and electroretinograms recorded at week 12, eyeballs were taken out, three dimensions were measured, and histopathological changes were examined.Results Compared to control group, tessellated fundus in FL group appeared more prevalent; implicit time of ldquo;ardquo; waves were prolonged in electroretinogram; the eyeballs were increased in horizontal, vertical, axial dimensions by (0.89plusmn;0.30), (0.69plusmn;0.20) and (0.96plusmn;0.30) mm respectively, the differences between two groups were statistically significant (t=12.7,11.9,15.8;P＜0.05). The gap of sclera collagen fiber was slightly widened.The photoreceptor layer was more likely to develop a disordered outer segment, which contained deciduous disc membranes.Conclusion Persistent flickering stimulus is attended by development of excessive ocular enlargement,which could affect the retinal structure and function of photoreceptors.
Objective To investigate the protective effect and mechanism of intravitreal injection with cyclosporin-A(CsA) on blood-retinal barrier (BRB) in diabetic rats. Methods A total of 48 Sprague-Dawley male mice at the age of 8-10 weeks were divided into normal group, diabetes mellitus (DM) group, CsA group and DMSO group, with 12 rats in each group. The rats in DM, CsA group and DMSO group were induced with streptozotocin (STZ) injection creating a diabetic retinopathy model. The same volume of citric sodium citrate buffer was injected into the rats in the normal group. Immunohistochemical staining was used to detect the BRB permeability, Western blot was used to measure the protein expression of intercellular adhesion molecule (ICAM-1). Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of vascular endothelial growth factor 72 hours after injection. Results Compared with the normal group, the BRB permeability, ICAM-1 and VEGF expression were significantly increased in DM group (F=29.350, 29.240, 9.658; P＜0.01). Compared with the DM group, the BRB permeability, ICAM-1 and VEGF expression were significantly decreased in CsA group (t=3.174, 5.000, 3.352; P＜0.05); but there was no obvious change of above indexes in DMSO group (t=0.420, 0.561, 0.312; P＞0.05). Conclusion Intravitreal injection of CsA has protective effects on BRB in diabetic rats. Down-regulated expression of ICAM-1 and VEGF may be the mechanism.
Objective To observe the effects of CXCR4 inhibitor (AMD3100) combined with anti-vascular endothelial growth factor (VEGF) antibody on experimental choroidal neovascularization. Methods Choroidal neovascularization (CNV) was induced in 48 BrownNorway (BN) rats by Krypton red laser photocoagulation, and those rats were randomly divided into AF564 group (group A), AMD3100 group (group B), combined treatment group (group C) and PBS group (group D), 12 rats in each group. Left eyes were the experimental eyes. The rats of group A-D received intravitreal injection of 5mu;l of AF564, AMD3100, AF564/AMD3100 and PBS after laser photocoagulation respectively. Fourteen days after photocoagulation, fundus fluorescein angiography (FFA), pathological section analysis and choroidal vascular wholemount were used to observe the degree of fluorescein leakage, the relative thickness and areas of CNV. Results Fourteen days after photocoagulation, the scores of fluorescein leakage in group A - D were 2.16plusmn;0.91, 2.16plusmn;0.91, 1.92plusmn;1.03, 1.39plusmn;0.93 respectively. Fluorescein leakage in group A - C was obviously reduced compared to group D (F=12.91,P＜0.001), while fluorescein leakage in group C was reduced compared to group A and B (F=9.21,P＜0.05). The CNV relative thicknesses in group A-D were 1.82plusmn;0.11, 1.90plusmn;0.22, 1.12plusmn;0.12, 2.82plusmn;0.29 respectively. Group A -C had thinner CNV compared to group D (F=5.92，P＜0.001), while group C had thinner CNV compared to group A and B (F=5.16, P＜0.05). The CNV areas in group A -D were (8204plusmn;122), (9332plusmn;211), (6533plusmn;101), and (13644plusmn;255) mu;m2 respectively. Group A -C had smaller CNV area compared to group D (F=147.50，P＜0.001), while group C had smaller CNV area compared to group A and B (F=112.60, P＜0.05). Conclusion Combined treatment with CXCR4 inhibitor and anti-VEGF antibody can inhibit laser-induced CNV significantly.
Objective To observe the concentration of vancomycin and the changes of pharmacokinetic parameters in rabbit vitreous with endophthalmitis in different pathological conditions.Methods Eighty-one adult healthy rabbits were randomly divided into endophthalmitis with lens group (group A), aphakic endophthalmitis group (group B), aphakic endophthalmitis and vitrectomy group (group C), 27 rabbits in each group. The right eyes of all rabbits received intravitreal injection of 1 ml (10 mg/ml) vancomycin. Three rabbits from each group were sacrificed at 0.5, 2.0, 4.0, 6.0, 12.0, 24.0, 48.0, 72.0, 84.0 hours after the injection. The eyes were harvested to collect the vitreous. The vitreous concentrations of vancomycin in all the groups were detected by high performance liquid chromatography (HPLC-UV). The pharmacokinetic parameters including the area under the curve (AUC) of the concentration-time graph, clearance rate (CL), half-life period (t1/2) and peak concentration (Cmax) were calculated by 3p 97 pharmacokinetic software. Results The concentrations of vancomycin in the group A were always higher than the therapeutic drug levels after injection. In the group B and C, the concentrations of vancomycin remained significantly high at 0.5, 2.0, 4.0, 6.0 and 12.0 hours after injection, decreased quickly at 24 and 48 hours after injection, below the minimal inhibitory concentrations at 72 hours after injection. The differences were statistically significant among group A, B and C (t=4.968, 5.232;P＜0.05), but not statistically significant between group B and C (t=1.279, P＞0.05). The AUC were 15 790.61,7643.94, 7443.44 mu;g/(ml?h), CL were 0.063, 0.131, 0.134 ml/h, t1/2 were 13.49, 7.15, 6.93 hours and Cmax were 711.56, 648.45, 667.74 mu;g/ml in the group A, B and C, respectively. In the group A, the CL was lower (t=2.963, 3.097; P＜0.05) and t 1/2 was longer (t=3.315, 3.481; P＜0.01) than those in the group B and C, but there was no significant difference on Cmax (t=1.687,1.214;P＞0.05). Conclusion The pharmacokinetic parameters of vancomycin in rabbit vitreous with endophthalmitis varied between different pathological conditions
Objective To observe the expression of C9 in laser-induced choroidal neovascularization (CNV) and the inhibitory effect of cobra venom factor (CVF) on CNV. Methods Thirty-six C57BL/6J mice were randomly divided into control group (n=12), laser photocoagulation group (n=12) and CVF group(n=12). The mice in control group had no interference treatment. CNV of the latter 2 groups were induced by photocoagulation. The mice of CVF group received intraperitoneal injection of CVF (0.1 mu;g/g) 2 days before and once a day after laser treatment. Six days after photocoagulation, the examination of fundus fluorescein angiography was used to confirm that CNV existed in the mice of laser the photocoagulation group. Then at 1, 3, 5, 7 days, the C9 expression was observed using SABCFITC histochemical stain and HE stain. The absorbance was measured by Image-Pro Plus 6.0 image software. Results Only in the laser group had formed CNV. At different time points, C9 expression was observed in the laser photocoagulation group but not in the CVF group. The A value showed a significant difference among 3 groups at different time points (F=146.045, P=0.000). The significant difference of A value exited in each group at different time points (F=9725, P=0.000). Conclusions There is C9 expression in laser-induced choroidal neovascularization. CVF can inhibit CNV formation by inducing complement consumption.
Objective To observe the inhibitory effect of intravitreal injection of triamcinolone acetonide (TA) on oxygen-induced retinal neovascularization, and to investigate its mechanism. Methods A total of 48 C57BL/6 mice at the age of 7 days were divided into normal group (groupA,n=6), highoxygen group (group B, n=6), TA control group (group C,n=18) and TA highoxygen group (group D,n=18). The retinal neovascularization of group B and D were induced by oxygen. One eye of each mouse of group C and D received an intravitreal injection 2 mu;l (20 mu;g /mu;l) of TA, and the same volume of BSS was injected into the other eye of the mice as BSS control group (group E) and BSS highoxygen group (group F). At postnatal day 17, the retinas were collected and the number of the endothelium cell nuclei of new vessels beyond the inner limiting membrane (ILM) was counted on HE-stained paraffin retina sections. The expression level of vascular endothelial growth factor (VEGF), stromal cellderived factor 1 (SDF-1) and CD14 were measured by immunohistochemical staining. The mRNA expression of VEGF and SDF-1 were detected by real-time RT-PCR. Results The numbers of the endothelium cell nuclei of new vessels beyond the ILM in group A - F were 0, 675, 0, 0, 110 and 688 respectively. In group A and D, it decreased than that in group B and F respectively (t=30.62, 19.532; P＜0.05). There was no difference of VEGF, SDF-1 and CD14 expression between group C and E (t=0.161, 0.284, 0.223; P＞0.05), but the differences were statistically significant between group D and F(t=-2.264, -2.358, -4.897;P＜0.05). There was no difference on mRNA level of VEGF and SDF-1 between group C and E(t=-0.497,-0.709;P＜0.05), but the differences were statistically significant between group D and F(z=-5.137,-4.411;P＜0.05). Conclusion Intravitreal injection with TA can inhibit oxygen-induced retinal neovascularization, down-regulated expression of VEGF and SDF-1 may be the mechanism.
Objective To investigate the possible effects of phosphorylated signal transduction and activator of transcription3 (STAT3) in the formation of choroidal neovascuarization (CNV) induced by photocoagulation in rats. Methods The CNV model in rats induced by photocoagulation was established, and the expression of phosphorylated STAT3 at the early stage in CNV were observed by immunofluorescence. To set up the hypoxia model, the specific inhibitor of Janus kinase 2 (JAK2), AG490 was mixed into cell culture fluid and then cultured for 0,1 hour,3,6,12,and 24 hours.Retinal pigment epithelial (RPE) cells proliferation activity were detected by flow cytometry (FCM).the expression of hypoxiainducible factor (HIF)1α and vascular endothelial grow factor (VEGF) mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR); the expression of HIF1α protein was detected by Western blot; the content of VEGF in the supernatant of cell culture fluid was measured by enzyme linked immunosorbent assay (ELISA). Results Phosphorylated STAT3 highly expressed in CNV areas in rats 3 days after the photocoagulation. The proliferation activity of human RPE cells under hypoxia condition significantly decreased after inhibition of JAK2/STAT3 signal transduction pathway (t=1.472, 3.566,2.391,6.420; P=0.054,0.038,0.042,0.016). The expression of HIF-1α and VEGF mRNA increased gradually with increasing time of hypoxia;while the expression of HIF1α and VEGF mRNA and the activation of HIF1α protein in cultured human RPE cells with the JAK/STAT3 signal transduction pathway blocked by AG490 were suppressed obviously under hypoxia condition (t=0.07,0.02,0.01, P<0.05); the content of VEGF in RPE cells supernatant decreased significantly (t=1.330,1.106,2.828,7.742,5.610,6.894; P=0.082,0.063,0.014,0.002,0.016,0.011). Conclusion STAT3 may be involved in CNV formation, which may partly dependent on JAK2/STAT3 signal transduction pathway regulating the expression of HIF-1α and VEGF in RPE cells.
Objective To observe the inhibitory effects of construction of complement factor (CFB)small interference RNA (siRNA) on choroidal neovascularization（CNV）induced by photocoagulation in rats.Methods We constructed an expression vector of CFBsiRNA by cutting CFBsiRNA and plasmid pRNATU61/Neo. Experimental CNV was induced by photocoagulation in 42 Brown Norway rats. After the model was set up, the rats were randomly divided into tail intravenous injection, vitreous injection, subretinal injection, and control group; each group except the control one had a corresponding blank plasmid control group. CFBsiRNA was injected 1, 3, and 5 days respectively after photocoagulation in the injection groups; the dosage was 50, 20, and 10 mu;g in tail intravenous injection, vitreous injection, and subretinal injection group respectively, while no injection was give to the control group after photocoagulation. Before and 14 days after photocoagulation, fundus fluorescein angiograoph (FFA) was performed and CNV development was judged by the leakage; the expression of vascular endothelial growth factor (VEGF) and factor Ⅷ were detected by immunohistochemistry. Results The leakage of fluorescein was obvious lower in tail intravenous injection group than that in the control group (chi;2=15.1620,Plt;0.05). The expression of VEGF and factor VIII in tail intravenous injection group at different time points after photocoagulation didnprime;tdiffer much (F=20.35,18.33; Pgt;0.05); while was apparently lower than that in the other groups at different time points (F=77.96,55.68; Plt;0.05).All of the groups, except tail intravenous injection group, had higher expression of VEGF and factor VIII 14 days after photocoagulation compared with that 7 days after photocoagulation (F=60.89, 61.12; Plt;0.05).Conclusions Constructed CFBsiRNA can inhibite CNV by downregulating the expression of VEGF and factor Ⅷ.
Objective To observe the inhibition effect of selective cyclooxygenase2 inhibitor（celecoxib）on the experimental choroidal neovascularization（CNV）. Methods Thirty 8-10 weeks old healthy male Brown-Norway（BN）rats were randomly divided into the control, laser and celecoxib group,with 10 rats in each group. At the dosage of 50 mg/kg, celecoxib was gavaged twice per day. After 7 days, experimental CNV was induced by Krypon laser on laser group and celecoxib group. Fundus fluorescein angiography (FFA) was performed on days 3, 7,14,21,30 after laser photocoagulation.On days 21 after photocoagulation, 5 rats in each group were sacrificed and the relative thickness of CNV membranes, the expression of COX-2, vascular endothelial growth factor(VEGF) and matrix metalloproteinase-2(MMP-2) were studied by histopathologic or immunohistochemistry examination.Results On days 21 after photocoagulation, the incidence of CNV in the celecoxib group is significantly lower than that in the laser group (chi;2=7.1068,P=0.0077); the relative thickness of the CNV membranes in the celecoxib group is reduced 41.38% compared to the laser group, the difference is statistically significant (t=16.760 0，P=0.0000).COX-2,VEGF and MMP-2 expression in the CNV membrane of celecoxib group were significantly lower than in control group (t=5.710 0,5.840 0, 8.020 0; P=0.000 0); the COX-2, VEGF and MMP-2 expressions in choroid and retina of control group were weak. Conclusion Prophylactic celecoxib can reduce the expression of VEGF and MMP-2 by inhibiting COX-2, and prevent the CNV induced by laser photocoagulation.
Objective To investigate if insulin can affect the expression of vascular endothelial growth factor (VEGF) in the retina of streptozotocin-induced diabetic rats. Methods A total of 60 male SpragueDawley rats were randomly divided into sodium citrate buffer control group (CIT-CON, n=30) and STZ-induced diabetic group (STZ-DM, n=30). At the 16th week, 24 rats from CIT-CON group at random were randomly divided to group A (sodium citrate buffer control group, n=12) and group B (sodium citrate buffer plus insulin group, n=12). The remaining 6 rats from as CIT-CON group served as negative control. At the same time, 24 rats from STZDM group at random were randomly divided to group C (STZinduced diabetic group, n=12) and group D (STZ-induced diabetic plus insulin group, n=12). The remaining 6 rats from STZ-DM group also served as negative control. 4 IU of insulin was injected subcutaneously to rats of group B and D. Immunohistochemistry, Western blot and Real-time polymerase chain reaction (RT-PCR) were used to measure the expression level of VEGF protein and mRNA respectively. RESULTS Insulin significantly increased the VEGF mRNA (7.71plusmn;0.25 vs 5.36plusmn;0.37, t test Plt;0.05) and protein expression (0.4925plusmn;0.0122 vs 0.4272plusmn;0.0110, t test Plt;0.05) in the retina of CITCON rats. However, in retina of STZDM rats, insulin had no effect on VEGF mRNA (8.92plusmn;0.27 vs 9.05plusmn;0.28, t test, Pgt;0.05) and protein expression (0.5152plusmn;0.0109 vs 0.5099plusmn;0.0100, t test Pgt;0.05). Conclusions Insulin had no effect on VEGF expression in the retina of STZ-DM rats.