Expression Of Heparanase In Mouse Oxygen-induced Retinopathy And Its Significane

ObjectiveTo investigate the expression and significance of heparanase in oxygen-induced mouse retinopathy.Methods1. To investigate the dynamic expression of heparanase in oxygen-induced mouse retinopathy.This experiment was a control experiment study. Newborn C57BL /6J mice were exposed to hyperoxia, and then returned to induce retinal neovascularization. Mice of the normal control group were kept in normal air environment all the time.1.1 Observation of the retinal neovascularizationMice were sacrificed at postnatal days 12, 13, 17,21and 30 and the retina wereprocessed for fluorescein angiography and Paraffin tissue slice with hematoxylin-eosin staining.1.2 Immunochemical stainingAt the time point of postnatal days 12,13,17,21 and 30, the expression of heparanase and perlecan was observed by immunochemical method of SABC.1.3 RT-PCRAt the same time point, the levels of heparanase, perlecan and VEGF mRNA were semi-quantified by RT -P C R.1.4 Western blotAt the same time point, the levels of heparanase protein was semi-quantified by Western blot.1. 5 AnalysisAnalysis of variance and LSD was used to compare the mRNA level of heparanase, perlecan and VEGF; analysis of variance and LSD was used to compare protein level of heparanase.Statistical differency was considered significant at a P value less than 0.05.2. To investigate the effect of HPAsiRNA on oxygen-induced mouse retinopathy40 healthy C57BL/6J newborn mice without restriction of gender were randomlydivided into HPAsiRNA treatment group(1μg /μl), control plasmid treatment group , hyperxia group and control group, Mice of the normal control group were kept in normal air environment all the time, while the animals of hyperxia group and treatment groups were put into oxygen chamber to establish retinal neovascularization model. In the HPAsiRNA treatment group (1μg/μl) and control plasmid treatment group(1μg/μl), 1μl plasmid was injected into two eye vitreous cavity at the postnatal day 12. Evaluate the retinal neovascualrization at the postnatal day 17 as follows:2.1 Evans blue angiography and histological observation and vascular endothelial cells counting2% Evens blue solution was injected into the mice's superior vena cava and the mice were killed five minutes later. The eyeballs were enucleated and fixed in 4% paraformaldehyde, then the retinas were separated and flat-mounted on the slides. Morphous of the retina vessels were observed and captured under fluorescence microscope.The t eyeballs were enucleated after the mice were killed and then fixed. After paraffin imbedding, 4μm serial slices, hematoxylin-eosin staining, select one section every 60μm (the ones that containing optic papilla were excluded) to count the endothelial cell nucleus that break through the inner limiting membrane, that was, the amount of neovascular endothelial cell, for quantitative analysis comparison.2.3 Western blotThe expression levels of heparanase protein in retina between groups were semi-quantified by Western blot.2.4 AnalysisAnalysis of variance and LSD was used to compare the protein level of heparanase of deferent groups. Statistical differency was considered significant at a P value less than 0.05.Result1. To investigate the dynamic expression of heparanase in oxygen-induced mouse retinopathy.1.1 Establishment and observation of oxygen-induced retinopathy model in miceAll the models showed retina neovascularization response. Retina flat-mounted with Evans blue angiography could clearly and completely delineate the nonperfusion region, vessel leakage, neovascularization retinopathy of the mouse model.Retinas of the normal mice observed at the postnatal day 12 to 30 had a uniform vascular distribution without obvious nonperfused area. Retinas of the oxygen- induced retinopathy models formed a large region of nonperfusion areas since the postnatal day 12, with vessels distortion and dilation, leakage, microaneurysm and new vessels generation. The response of neovascularization achieved the high point at postnatal day 17; the response lasted until postnatal day 21 and then began to retreat gradually. However, when postnatal day 30, rare responses were observed.There are little vascular endothelial cells which break through the inner limiting membrane in normal control group, but happened in all hyperxia groups. The amount of vascular endothelial cells which break through the inner limiting membrane at postnatal days17 and 21 was at most .At postnatal days 13, we discovered the inner limiting membrane becomed rough and thickening.1.2 lmmunochemical methodThe expression of HPA and perlecan presented the similar pattern by immunochemical method during the development of oxygen-induced retinopathy. They all displayed distribution at the vascular endothelial cells which break through the inner limiting membrane gan, vessel wall and ganglion cells.there is little distribution in nomal retina.1.3 RT-PCR and Western blotHPA mRNA and protein were both expressed in normal retina and In oxygen-induced retinopathy .The tendency of HPA mRNA and protein level in experiment group raised first and the decreased .and the postal day 17days was the peak time. At the postal days 13, 17and 21, mRNA level in experiment group was significant higher than control. At the postal days 13, 17, 21, and 30 proteins level in experiment group was significant higher than control .the changes of HPA correlated with the development and progression of retinal neurovascularization. Perlecan mRNA has the similar expression mode to HPA.The tendency of perlecan mRNA level in experiment group raised first and the decreased. The postal day 13 and 17days was the peak time. At the postal days 13 ,17 and 21,mRNA level in experiment group was significant higher than control.The tendency of VEGF mRNA level in experiment group raised first and the decreased. The postal day 13 and 17days was the peak time. At the postal days 13 and 17 mRNA level in experiment group was significant higher than control.2. To investigate the effect of HPAsiRNA on oxygen-induced mouse retinopathy2.1 Evans blue angiographyRetinas of the normal mice observed at the postnatal day 17 had a uniform vascular distribution without obvious nonperfused area. Retinas of the oxygen-induced retinopathy models formed a large region of nonperfusion areas at the same time, with vessels distortion and dilation, leakage, microaneurysm and new vessels generation. Retina flat-mounted with Evans blue perfusion has manifested that HPAsiRNA plasmid injected into vitreous cavity could significantly reduce retina neovascularization and vessel leakage in mice.2.2 Histological observation and vascular endothelial cells countingParaffin tissue slice with hematoxylin-eosin staining showed that, in HPAsiRNA treatment group,control plasmid treatment group ,hyperxia group, normal control group, the average counts of vascular endothelial cells which break through the inner limiting membrane were 6.88±2.32, 21.04±5.39, 32.73±6.38, 0.37±0.15. HPAsiRNA plasmid treatment group compared with control and hyperxia group, the differences were significant, P <0.01.2.3 Western blotThe HPA protein level of HPAsiRNA treatment group was lower than control plasmid treatment group and hyperxia group. The HPA protein level of the control plasmid treatment group was also lower than control plasmid treatment group and hyperxia group, P<0.01.Conclusion1. HPA and perlecan expression correlated with the development and progression of retinal neurovascularization.2. HPA may participate in the angiogenesis of oxygen induced retinopathy, and target inhibits HPA expression may became a new method to treat the proliferation retinopathy.