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 investigate the effect of inducible nitric oxide synth ase (iNOS) or cyclooxygenase－2 (COX－2) on retinal neovascularization and its possible mechanism in oxygen－induced retinopathy （OIR） mouse model. Methods Retinal neovascularization was induced by oxygen with different concentration. The expression of iNOS, COX－2, matrix metalloproteinases 2 (MMP－2) and vascular end othelial growth factor (VEGF) in the retinae of experimental animals were analyzed by immunohistochemistry, realtime polymerase chain reaction and western blotting technologies. Results The inhibition of COX－2 or iNOS obviously attenuated retinal neovascularization and decreased the expression of VEGF and MMP－2. The iNOS inhibition decreased COX－2 expression, and vice versa. Conclusions COX－2 and iNOS may play a role in retinal neovascularization in OIR mouse model, which may act by regulating the expression of VEGF and MMP－2.
Objective To investigate the effects of intravitreal injection of matrix metalloproteinase-3 (MMP-3) on the vitreoretinal adhesion and the vitreous gelatin. Methods Twenty-four pigmented rabbits were randomly divided into 3 experimental groups（group A, B, and C）and one control group with 6 rabbits (12 eyes) in each. Different concentrations of 0.1 ml MMP-3 (5,10, 20 ng in group A, B, and C, respectively) and equivalent dose of balanced salt solution were intravitreally injected to the rabbits, respectively. Clinical examinations (such as gross observation, slit-lamp biomicroscopy, indirect fundus ophthalmoscopy ), electroretinography (ERG) and fundus fluorecein angiography (FFA) were taken before and after injection. Results One week after injection, posterior vitreous detachment (PVD) and focal vitreous liquefaction were recognized clinically for the first time in 1 eye in group B. By the end of this study, clinically detected PVD developed in 1 eye in group A, 3 eyes in group B, but the synchisis developed slowly, and no liquefaction or PVD occurred in control group. As for the histological examination, partial PVD was observed in 1 eye in group A and 3 eyes in group B 60 minutes after injection. All of the eyes in group A and B showed partial PVD 1 week after injection, and the area of PVD enlarged in contrast with before. Complete PVD were recognized in 1 eye in group A and 3 eyes in group B 15 weeks after injection, and the cleavage was narrow and limited. In group C, inflammatory cell infiltration in the inner layer of retina, destruction of retinal structure, and fluorescein leakage at late phase was found in the early period after injection. Conclusions MMP-3 is effective in disrupting the adhesion of the posterior hyaloid to the inner limiting membrane leading to PVD, and helpful to some extent in producing vitreous liquefaction. The dose of 10 ng MMP-3 is safe and effective for the rabbits eyes, which may be used as an promising assistant of vitreous surgery. (Chin J Ocul Fundus Dis，2004，20：67-132)
Objective To investigate the expression of nuclear factor (NF)-κB and intercellular adhesion molecule (ICAM)-1 in rat′s retina injured by ischemia-reperfusion, and the effect of pyrrolidine dithiocarbamate (PDTC) on the expression of NF-κB and ICAM-1. Method The model of retinal ischemia-reperfusion was set up in 60 SD rats, which were divided into two groups with 30 rats in each: ischemia-reperfusion group and ischemia-reperfussion with injection of PDTC group. The left cephalic artery of each rat was ligated, and the right side was the control. Every group was subdivided into group 1 hour, 6, 12, 24, 48, and 72 hours after ischemia-reperfusion injury, and with 5 rats in each group. mRNA of NF-κB and ICAM-1 mRNA was measured by in situ hybridization (ISH) method in rat′s retina. Every rat underwent electroretinography (ERG) at the corresponding time before executed by neck breaking. Results In ischemia-reperfusion group, expression of NF-κB and ICAM-1 was detected at the 6th hour after ischemia-reperfusion, reached the highest level at the 24th hour, and weakened gradually later. In ischemia-reperfusion with injection of PDTC group, expression of NF-κB and ICAM-1 was detected at the 12th hour after ischemia-reperfusion, and reached the highest level at the 24th hour but lower than that in ischemia-reperfusion group. No expression of NF-κB and ICAM-1 was found in the control group. The relative recovery rate of ERG a and b wave amplitude in ischemia-reperfusion groups was lower than that in ischemia-reperfusion with injection of PDTC group at every stage(P＜0.01 ). The lowest relative recovery rate of ERG a and b wave amplitude in different stages in both of the 2 groups was at the 24th hour(P＜0.01). Conclusions NF-κB and ICAM-1 may play an important role in retinal ischemia-reperfusion injury, as the inhibitor of NF-κB, PDTC may relieve the retinal ischemia-reperfusion injury. (Chin J Ocul Fundus Dis，2004，20：175-178)
Objective To explore the effect of ischemia-reperfusion injury on the retinal functions of rats. Methods Seventy Wistar rats were selected, 20 of which were selected randomly and divided into two groups (control group and single-irrigated group). The rats were anesthetized and their anterior chambers of the right eyes were cannulated with a 7-gauge needle connected to a reservoir containing ringers balanced salt solution, which was maintained at the same level o f the eye for 1 hour. After that, ERG was recorded in both eyes of all rats. All the left rats were divided randomly into 10 groups and they were treated as the single-irrigated group. Retinal ischemia was induced by raising the reservoir to a height of 150 mm Hg. One hour later except the single ischemia group, all o f t he groups resumed perfusion after 3,6,12,and 24 hours and 3,5,7,14,and 21 days s eparately. ERG was recorded in both eyes of all rats.Results There was no difference in the results of ERG between left and right eyes in either the control group or the single-irrigated group. All the waves of ERG vanished in the single-ischemia group after 1 hour. In the ischemia-reperfusion groups, the waves of ERG partly recovered and the amplitude reduced persistently and progressively.Conclusion Ischemia-reperfusion injury may affect the function of the retina persistently and progressively. (Chin J Ocul Fundus Dis，2003，19：201-268)
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 establish rabbit models of mixture-infectious endophthalmitis induced by exogenous Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Methods A total of 84 eyes of 42 New Zealand white albino rabbits were randomly divided into 4 groups. There were 21 eyes in each group. Rabbit eyes in group 1, 2, 3 and 4 received an intravitreal injection of 0.1 ml of mix bacterium (2times;104 CFU/ ml, including 103 S. aureus and 103 E. coli), S. aureus (104 CFU/ ml), E. coli (104 CFU/ml), and sterilized saline respectively. The eyes were examined by slit-lamp microscopy, ophthalmoscopy, A/B scan, electroretinography (ERG) and bacterial culture of vitreous humors at the timepoints of 6, 12, 24, 48 and 72 hours, and 4, 7, 10, 14 days after intravitreal injection. All eyeballs were then enucleated for histopathological examination. Results Various degrees of inflammatory reactions were presented in the 3 experimental groups after the injection, and the development trend of the disease was nearly the same. In group 1 active intraocular inflammation like anterior chamber exudates, started at 12 hours after injection (which was early than that in group 2 and 3), aggravated between 48 and 72 hours, alleviated slowly from 4 to 7 days, and was obviously better after 10 to 14 days while the corneal neovascularization and vitreous gray opacity begun to form. The bacterial culture was positive in group 1 (100％, 6 hours to 14 days after injection), group 2 (100%, 6 hours to 3 days after injection) and group 3 (100% from 6 hours to 7 days, and 67.67% at 14 days after injection). It was negative for group 2 (7 to 14 days after injection) and group 4 (6 hours to 14 days after injection). The amplitude of ERG b wave dissapeard in group 1 to 3, and decreased less than 30% in group 4 from the 48th hour after injection. Histopathological examination revealed that all intraocular structures infiltrated with inflammatory cells. Conclusion Complicated endophthalmitis rabbit models can be successfully established by intravitreal injection with S. aureus and E. coli.
Objective To investigate the expression and significance of inducible co-stimulator (ICOS) in experimental autoimmune uveoretinitis (EAU). Methods EAU was induced in 24 Lewis rats (immune group) by immunization with retinal S-antigen (50 mu;g) and complete Freundprime;s adjuvant, and another 4 rats were in the control group. Anterior segment of the ratsprime; eyes were observed by split microscope every day. Immunohistochemical staining was performed using polyclonal antibodies to ICOS on the sections of the spleen which were obtained from the rats in immune group at the 7th, 12th, 15th and 21st days after immunisation respectively. Western blotting was performed to investigate the dynamic expression of ICOS protein in the spleen. The same procedures were made at the corresponding time points in the rats in control group. Results A few ICOS positive cells were observed in the normal spleen. The number of ICOS positive cells in immune group increased obviously at the 7th and 12th days after immunization, reached the peak at the 15th day, and decreased at the 21st day which was still higher than that in the control group. The result of Western blotting showed that the dynamic changes of ICOS protein was identical with the changes of positive-cell number detected by immunohistochemistry. Conclusions The enhanced expression of ICOS happens before EAU occurs, which increases when the inflammation occurs and deteriorates, and decreases at the alleviative stage of EAU. It suggests that ICOS participates in the formation, development and disappearance of EAU and plays an important role in the incidence of EAU. (Chin J Ocul Fundus Dis, 2005,21:114-117)
ObjectiveTo investigate the effect of nerve growth factor (NGF) on recuperate of optic nerve after contusion by clamping in adult rabbits. MethodsSixteen adult rabbits were randomly divided into NGF and the control group with 8 rabbits in each group. After the optic nerve of the right eyes was clamped，tissue engineering nerve containing 0.06 ml NGF（concentration: 5×10-4 g/L, NGF group） and 0.06 ml of PBS (control group) was immediately transplanted into the injured eyes respectively, and 0.02 ml NGF（concentration: 5×10-4 g/L, NGF group）and 0.02 ml of PBS (control group) were injected into the vitreous of right eyes respectively. Flash visual evoked potential (FVEP) test was performed on the eyes 1 day, 2 weeks and 8 weeks after the injury. The number of retinal ganglion cells (RGCs) and changes of optic nerves were observed by light microscopy and electron microscopy at the 8th week after contusion,and a computer-image-analysis system was used to count the optic nerve axons.ResultsThe ratio of amplitude of FVEP of the injured and healthy eyes was 0.765±0.150 in NGF group and 0.494±0.108 in the control at the 2th week after injury with a significant difference between the two groups (Plt;0.05); and was 0.581±0.138 and 0.409±0.119 respectively at the 8th week after contusion with statistical difference between the two groups (Plt;0.05). The results of light microscopy and electron microscopy showed that degeneration of RGCs and optic nerves in the NGF group was lighter than that in the control group 8 weeks after injury, while the amount of optic nerve axons was (10 955±608.7) axons/ mm2 in the NGF group and (7 898±608.8) axons/mm2 in the control with statistical difference between the two groups (Plt;0.05). ConclusionNGF may redound to the survival of RGCs and regeneration of the axons in some degree, which can promote the recuperation of optic nerve and visual function. (Chin J Ocul Fundus Dis, 2005,21:253-257)
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.