Protein Kinase B (Akt) Promotes Pathological Angiogenesis In Murine Model Of Oxygen-induced Retinopathy

Chapter one:Expression of protein kinase B (Akt) in mice with oxygen-induced retinopathy.Objective:To explore the changes and significance of expression of protein kinase B(Akt) mRNA and Akt(or pAkt) protein in mice with oxygen-induced retinopathy.Methods:Fifty four 7-day-old C56BL/6 mice were randomly divided into model group(27 mice) and normal group(27 mice). On the 7th day after the birth, the mice in model group were raised in the box full of (75%±2%) and then under the normal atmosphere condition on the 12th postnatal day. At the 17th day,the model and normal groups mice were angiographied with Fluorescein-Dextran respectively.The expression of Akt in model group were detected by reverse transcription-polymerase chain reaction and western blot on the 7th,9th and 11th postnatal day,when under hyperxic condition, and on the 12th, 14th,16th,17th and 23th, when under relative hypoxic condition.Results:Normal groups mice with normal retinal vessel structure,the model groups mice with neovascular tufts appear as hyperfluorescence at the junction between the perfused and non-perfused area. The expression of Akt mRNA and Akt/p-Akt protein decreased after hyperxia, and increased after relative hypoxia, reached the peak at the P17, and then decreased.Conclusions:The expressions of Akt mRNA and protein in mice with oxygen-induced retinopathy are related to retinal vessel loss and angiogenesis. Chapter two:Use of active recombinant Akt in the hyperoxic condition can prevents retinal vessel lossObjective:To examine whether active recombinant Akt treatment during the hyperoxic condition of oxygen-induced retinopathy (when retinal Akt levels are suppressed) helps to prevent retinal vessel loss.Methods:At P6 and P7, mice were anesthetized with intraperitoneal injection of pentobarbital sodium, A 32-gauge Hamilton needle and syringe were used to deliver 1?l\recombinant Akt into the vitreous cavity. Control PBS was injected into littermates.Mice were raised in the box full of (75%±2%)oxygen. After oxygen exposure, eyes from P8 mice were perfused with FITC-dextran in order to observe the changes of retinal non-perfusion areas.Results:At P8, mice treated with recombinant Akt had a decresed capillary-free compared with PBS-injected littermate controls. Differences are statistically significantConclusions:Correcting retinal Akt deficiency during the first phase of retinopathy with exogenous Akt protects the postnatal mouse retina from hyperoxia-induced vessel loss. Chapter three:Use of Akt inhibitor in the hypoxic condition can prevent retinal neovascularization, but can not prevents retinal vessel loss.Objective:To examine whether Akt inhibitor treatment during the second phase of oxygen-induced retinopathy (when retinal Akt levels are increased) helps to prevent retinal neovascularization.Methods:7-day-old C56BL/6 mice were exposed to75%±2% oxygen for 5days, then returned to the room air to induce rentinal neovascularization. At P12,1.5?l Akt inhibitor were injected into vitreous cavity. Control PBS was injected into littermates. Retinal neovascularization was analyzed with FITC-dextran and histological analysis at P17.Results:Mice treated with Akt inhibitor during the second phase had a reduced pathologic neovascular tufts and fluorescence leakage compared with PBS control. The histological analysis shows that neovascular nuclei protruding into viteous cavity were decreased compared to the control eyes.For the area of capillary-free, no differences were seen in capillary-free area between Akt inhibitor and PBS treatmentsConclusions:Akt inhibitor treatment during the second phase of retinopathy protects the retina from vessel pathological proliferation, but cannot protects the retina from vessel loss.