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 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.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.