ObjectiveTo evaluate the protective effect of estrogen on survival of retinal ganglion cells (RGCs) after transient retinal ischemia-reperfusion (RIR) in rats.MethodsRIR was induced in 60 ovariectomized adult rats (OVX) by increasing intraocular pressure via an intracameral catheter. All of the rats were divided into two groups randomly: in experimental group, the rats underwent a subcutaneous injection with 17β-estrodiol（100 μg/kg） 2 hours before retinal ischemia; and in the control group, saline water was injected correspondingly. The number of RGCs and the thickness of the inner retinal layers were mesured by HE staining method before and 12, 24, 48, and 72 hours after reperfusion. TdT-mediated biotin-dUTP nick end labelling (TUNEL) staining technique was used to examine the apoptosis of RGCs.ResultsTwenty-four and 48 hours after reperfusion, the number of apoptotic cells in experimental group was obvious lower than that in the control group(Plt;0.05), and the number of RGCs in experimental group was higher than that in the control group(Plt;0.05).ConclusionEstrogen can protect retinal neurons from transient RIR in ovariectomized rats.(Chin J Ocul Fundus Dis, 2005,21:177-179)
Objective To observe apoptotic and proliferative characteristics of the retinal vascular end othelial cells (RVECs) of the 1～16 weeks diabetic rats and p53 and bcl-2 expressions of the rats,in order to probe the pathogenic mechanism of diabetic retinopathy(DR). Methods Models of diabetic Wistar rats were made by alloxan venous injection.The retinal blood vessels were filled by ink,the wholemounts and paraffin-embedded sections of the retinas were made,TUNEL staining and Immunohistochemical ABC staining were used,and light microscopy was taken,in succession. Results Apoptosis of the RVECs was not found.Compared with control group,the morphologic features of the RVECs and the structure of the retinal blood vessels remained unchanged.In the period from the 10th to the 16th week,the immunohistochemical stain of PCNA,BrdU,p53,and bcl-2 for RVECs revealed positive results,but there was no any sign of the RVECs stacking and proliferating or new blood vessels forming in the retinas.In control group,the reaction of immunological stain of the aforementioned parameters was negative. Conclusions No accelerated apoptosis and proliferation of the RVECs in the 1～16 week diabetic rats happen after alloxan injection.Almost all of the RVECs were stimulated to enter the cell cycle in the 10th week.Expression of p53 and bcl-2 might play an important role in stabilizing the RVECs in early stage of diabetes. (Chin J Ocul Fundus Dis, 1999, 15: 157-159)
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.
ObjectiveTo observe the inhibitory effect of endostatin (ES) on oxygen-induced retinal neovascularization.MethodsThirtyfour 7-day-old C57BL/6J mice were randomly divided into 3 groups: oxygen-exposed group (12 mice), ES group (12 mice) and the control group (8 mice). The mice in oxygen-exposed and ES group were exposed to (75±5)％ oxygen for 5 days and then back to the normal air. In ES group, 1 μg ES endostatin were injected into vitreous in one eye, while PBS was injected into the other eye as the control 12 and 36 hours after being exposed to oxygen. The mice in the control group were fed in normal circumstance. The changes of retinal neovascularization was examined by fluorescence angiography with fluorescein isothiocyanatedextran. The number of endothelial cells breaking through the internal limiting membrane (ILM) was counted and the inhibitory effects of ES on retinal neovascularization was observed.ResultsCompared with the oxygen-exposed group, the branches of retinal vessels went normal without any un-perfused area in ES group. The number of nuclei of endothelial cells breaking through ILM on each retinal crosssection decreased to (5.39±1.52), which differed much from that in the oxygen-exposed group (22.56±2.13) (plt;0.001).ConclusionES can effectively inhibit the formation of retinal neovascularization in rats and might be a new path of the treatment for proliferative retinopathy.(Chin J Ocul Fundus Dis, 2005,21:314-317)
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 observe the effects of basic fibroblast growth factor (bFGF) on the expression of heat shock protein 70 (HSP70) in ratrs retina after iscbemia/reperfusion injury.Methods The rat model of experimental retinal ischemia/reperfusion injury was made by increasing the intraocular pressure. Tweenty-four Wistar rats were divided into normal (3 rats) and operation group (21 rats) randomly. The latter group was subdivided into group 0 hour, 4, 8, 12, 24, 48 and 72 hours after reperfusion, in which the left eyes of the rats were in the ischemia/reperfusion groups and the right ones were in the treatment groups (bFGF 2 t~g intracameral injection). The expression of HSP70 was observed by strept avidin-biotin complex (SABC) immunohistochemistry. Results No HSP70 positive cells were found in normal group; a few of HSP70 positive cells were found 0 hour after reperfusion [20.8±4. 5) cells/mm2], and increased gradually until reached the peak 24 hours later [(111.2±4.4) cells/mm2] and then decreased gradually. Few HSP70 positive cells were found 72 hours after reperfusion. The amount of HSP70 positive cells increased in treatment group at all time courses, and the peak time was earlier and longer than that in ischemia group. HSP70 positive cells distributed extensively in retinal ganglion cell layer and inner nucleous layer. The difference of the amount of HSP70 positive cells between the two groups was significant (Plt;0.05) 8, 12, 24, 48 and 72 hours after reperfusion.Conclusion bFGF can enhance the expression of HSP70 in rat’s retina after retinal ischemia/reperfusion injury.(Chin J Ocul Fundus Dis，2004，20：37-39)