Study The Effect Of HIF-1α Antisense Oligonucleotides On The Animal Model Of Retinopathy Of Prematurity

Part oneObjective To prepare an animal model of retinopathy of prematurity which can be quantization by economic and simple mean.Methods Totally 44 postnatal day 7 (7d) wistar rats were randomly assigned into 2 groups according to different oxygen inhalation modes ( n = 22). Experimental group was exposed to 75% oxygen for 5 d,then oxygen supply was suddenly stopped, followed by room air exposure for 5d. The Control group were exposed to room air in the same experimental condition. The retinal vascular development and proliferation were evaluated by stretched preparation (ADPase stained retina)and eyeball cross-section and staimed with hematoxylin and eosin(HE), the expression of vascular endothelial growth factor (VEGF) was detected by immunohisto-chemical stain.Result the central vessels became tortuous and constricted and the central avascular area increased in high-concentration oxygen environment. But Followed hypoxia environment made great vessels of retina broaden and abnormal curvature obviously , neovascularization was seen in the Experimental group . The average number of neovascular nuclei which breakthrough limiting membrane of amphiblestrodes in the Experimental group and the Control group was 33.85±0.7452;2.60±0.6806 respectively. There were significant differences between them. Stain of VEGF was weaker in the control group than the treated group.Conclusions the rat modle of retinopathy of prematurity has powerful repeatability,high constancy and can be used in quantitative study.it is a suitable mode to study the pathogenesy and therapy of ROPPart twoObjective To investigate the changes of VEGF,HIF-1αmRNA and protein levels in rat retina of normal retinal vascular development and ROP to elucidate risk factors and pathogenesis of ROP.Methods Totally 64 postnatal day 7 (7d) wistar rats were randomly assigned into 2 groups according to different oxygen inhalation modes ( n = 32). Experimental group were exposed to 75% oxygen for 5 d and stopped oxygen supply suddenly, followed by room air exposured for 5d.The Control group were exposed to room air in same experiment condition. The experimental objects were killed by cervical vertebra luxatio . eyeball cross-sectio and staimed with hematoxylin and eosin(HE), the nuclei of proliferative retinal vessels of two groups were counted. The VEGF and HIF-1αexpressions in retina were detected by immunohisto-chemical staining. The dependability between them were also analyzed.Result The average number of neovascular nuclei which breakthrough limiting membrane of amphiblestrodes in the Experimental group and the control group was 32.80±0.7678; 2.75±0.7164 respectively.There were significant differences between them.The VEGF expressions increased after hypoxia ,mainly in endochylema and nucleus of capillary endothelium of ganglionic layer of retina and inner nuclear layer.There were significant differences (P < 0. 05); there were no expressions of HIF-1αin The Control group. In Experimental group ,we can detect the expression of HIF-1αin nuclei. Linear correlation analysis of the data indicated the positive correlation was presented between the nuclei of the proliferative vessels and the VEGF levels in retina (r=0.791,p<0.01), between the nuclei of the proliferative vessels and the HIF-1αlevels in retina (r=0.951.p<0.01), between the express HIF-1αand VEGF in retina(r=0.99,p<0.01) .Conclusions Partly hypoxia in retina can increase the expression of HIF-1αand VEGF which made anormogenesis of retinal vessel, The average number of neovascular nuclei which breakthrough limiting membrane of amphiblestrodes increased obviously and the positive correlation between them . They have closely correlation with ROP.Part threeObjective Get the message how to abaissement retinal neovascularization and expressed of VEGF through injecting HIF-1αantisense oligonucleotides into rat's cavum vitreum .Methods Totally of 80 wistar rats(7d) were randomly assigned into two group , Experimental group(n=40) were exposed to 75% oxygen for 5 d,then oxygen supply was suddenly stopped,followed by room air exposure for 5d. The Control group(n=40) were exposed to room air in the same experimental condition. Both two groups were injected HIF-1αantisense oligonucleotides into rat's left cavum vitreum at 12, 14, 16d. ocul dext is own control .some of experimental groups were injected isotonic Na chloride into rat's left cavum vitreum at 12,14,16d. So we get five group examples: normalis eye(A); normalis eye+HIF-1αantisense oligonucleotides(A1); ROP eye(B); ROP eye+HIF-1αantisense oligonucleotides(B1); ROP eye+ isotonic Na chloride (B2). The retinal vascular development and proliferation of five groups were evaluated by 15 newborn rat's retina stretch preparation respectively at 12, 14, 17d who were taked from experimental group and control group(one left eye injected isotonic Na chloride at each time spot).The 20 animals which selected from experimental group and control group (each group is 10)were killed by cervical vertebra luxatio . eyeball cross-sectio and staimed with HE, Though the cross-sections,the nuclei of proliferative retinal vessels of five groups were counted. The expressions of VEGF and HIF-1αin retina were observed by immunohisto-chemical staining. The expressions of VEGF and HIF-1αin retina were detected by RT-PCR.Result Retina stretch preparation showed: The peripheral vascular pattern was clear, and a few avascular areaswere seen in group A at 12d. At 14d the avascular area more diminution. At 17d, the entire vascular pattern became completely normal; retinal vessel shape of group A1 is same as group A;In group B the central vessels became tortuous and constricted and the central avascular area increased at 12d. At 14d, neovascularization was seen, peaking at 17d; group B1 is same as group A, neovascularization decreased obviously than group B; retinal vessel shape of group B2 is same as group B. The average number of neovascular nuclei which breakthrough limiting membrane of amphiblestrodes in the groups A, A1, B, B1, B2: 2.75±0.7164;2.70±0.7327;32.80±0.7678;28.9±0.9119;32.75±0.7164 respe- ctively. There were significant differences between the groups B and the group A (P < 0.05),the same as the groups B and the group B1.There were no significant differences between the groups B and the group B2( P>0.05); the expressions of VEGF in immunohisto-chemical staining were weaker in groups A,A1 than in groups B, B2. There were significant differences ( P < 0.05); the same as the group B and the B1( P < 0.05). the expressions of VEGF mainly been seen in capillary endothelium endochylema and nucleus of ganglionic layer of retina and inner nuclear layer. There were no expression of HIF-1αin immunohisto-chemical stain in group A,A1, The expression of HIF-1αin immunohisto-chemical stain was weaker in group B1 than B,B2.There were significant differences( P < 0.05). The expressions of HIF-1αmainly been seen in capillary endothelium nucleus of ganglionic layer of retina and inner nuclear layer. The level of VEGF mRNA maintained a low level at 12,14,17d in group A,A1 . Cut down obviously in group B at 12d,and then increased at 14,17d. There were significant differences in The groupA and group B. ( P < 0. 05). The level of HIF-1αmRNA increased gradually from 12 to 17d .Conclusions Abrupt quit of oxygen supply contributes to increase expressions of HIF-1αand VEGF in rat's retina, which influence the retinal vessels development severely in neonatal rat ,and induce pathological changes of retina as ROP. We injected HIF-1αantisense oligonucleo tides into rat's left cavum vitreum can abaissement expressions of VEGF and HIF-1αin retina, thereby inhibition retinal neovascularization to cure ROP.