The Preliminary Study On The Relation Of Rat Chronic Hypertension Retinal Injury And Hypoxia-responsive Genes Regulation

ObjectiveThe mechanism of glaucomatous retinal and optic nerve degeneration is very complicated. Someone suggest the hypothesis of hypoxia.But there is no direct evidence about retinal hypoxia.Hypoxia-inducible factor 1 (HIF-1) is an oxygen-regulated transcriptional activator that functions as a master regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1 activates the transcription of a broad variety of genes, including those encoding erythropoietin, cysteine aspartase-9, vascular endothelial growth factor, inducible nitric oxide synthase, cyclooxygenase-2 and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. So we plan to research the mRNA and protein expressions of hypoxia-inducible factor(HIF-1), inducible nitric oxide synthase (iNOS), icyclo oxygenase-2(COX-2) and cysteine aspartase-9 (Caspase-9)in nomal retina and ocular hypertention retina,investigate the relation of glaucomatous retinal degeneration and hypoxia-responsive gene regulation.Methods1. establishment of the modelan increase of IOP was induced by coagulating two episcleral veins unilaterally in rats with electric coagulator.2.hematoxylin-eosine staining 3. transmission electron microscopy.4. ERG-b wave5.Research on mRNA expression of HIF-1,iNOS,COX-2,Caspase-9 genes in nomal,chronic hypertension,using EPO retina.Use PT-PCR analysis to examine the expression of HIF-1,iNOS,COX-2, Caspase-9genes.Results were showed by average±standard, and analyze the average between several groups by SPSS 12.0 software.6.Research on protein expression of HIF-1, iNOS, COX-2, Caspase-9 genes in nomal,chronic hypertension,using EPO retina.We detected protein expression of HIF-1, iNOS, COX-2, Caspase-9 by immunochemistry and Western Blot.Results were ststistical with One Way-ANOVA and T-test by SPSS 12.0 software.Result1. coagulating two episcleral veins unilaterally in rats with electric coagulator establish the chronic hypertension model.After operation IOP elevate 140%;after 3th day IOP is 1.77 times of nomal IOP; after 7th day IOP is 2.66 times of nomal IOP; after 14th day IOP is 2.55 times of nomal IOP; after 21th day IOP is 1.99 times of nomal IOP; after 28th day IOP is 2 times of nomal IOP.Compared with control group,There was significant difference (P＜0.01). after 3th day of hypertension,ERG-b wave amplitude was lower and it was the most lower on 3th day after hypertension.2. RT-PCR, HIF-1,iNOS,COX-2,Caspase-9 genes mRNA were fanitly expressed in normal rat retina, and the expression of them increased in IOP elevation model rats with the time going on. The peak expression of HIF-1,iNOS and COX-2 were on the 7th day after IOP elevated and The peak expression of Caspase-9 was on the 7th day after IOP elevated then decreased but still maintained at a higher level. The expression among experimental groups was statisticlly different compared with the control group. The increasing trend of EPO drug treatment group weakened remarkably, The expression among EPO drug treatment groups was statisticlly different compared with the non-drug treatment group except for the 3th day group in iNOS and COX-2.3.immunohistochemistry, The expression of HIF-1,iNOS,COX-2,Caspase-9 were very faint immuno staining in nomal retina.In the glaucomatous retina Immunostaining for HIF-1, iNOS,COX-2,Caspase-9 was predominant in the inner retinal layers, mostly in the retinal ganglion cell layer and the inner nuclear. HIF-1,iNOS,Caspase-9 immunostaining was also detectable in the photoreceptor cells.In Western-Blot, the protein expression of them increased in IOP elevation model rats with the time going on. The peak expression of HIF-1,iNOS and COX-2 were on the 7th day after IOP elevated and The peak expression of Caspase-9 was on the 7th day after IOP elevated then decreased but still maintained at a higher level. The expression among experimental groups was statisticlly different compared with the control group. The increasing trend of EPO drug treatment group weakened remarkably, The expression among EPO drug treatment groups was statisticlly different compared with the non-drug treatment group except for the 3th day group in iNOS, COX-2 and Caspase-9.4. In EPO drug treatment group,the amplitude of experimental group restored to 80% of the one before experimental on the 28th day after IOP elevated, while the control group restore to 61% only.There was significant difference between the two groups(P＜0.05)Conclusion1. Because the regions of HIF-1 induction represent the areas of decreased oxygen delivery and hypoxic stress, information obtained from this study provides direct evidence that tissue hypoxia is present in the retina and optic nerve head of glaucomatous eyes2.HIF-1, iNOS,COX-2,Caspase-9 participates in signal transduction of retinal neurons during retinal injury on chronic high ocular pressure, hypoxic signaling is a likely component of the pathogenic mechanisms of glaucomatous neurodegeneration.3. Erythropoietin(EPO) protects retinal neurons from chronic high ocular pressure injury.4.as a neuroprotective agent in glaucomatous injury. Protection of EPO is in part mediated by regulating HIF-1 and its hypoxia-responsive genes.