The Action Of TSP1in Mouse Ischemic Reperfusion Model And The Meaning In Diabetic Retinopathy

Diabetic has been a monster for lots of working age people.Especially for the rapid lifepace and the fast food, higher calories food structure. And as a result, in China, diabeticretinopathy has been the4thblind disease nowadays.The most possible reason for theblindness is the angiogenesis of DR. The typical clinical sign is the aneurysm,angiogenesisand the vitrous hemarrage,the proliferative membrane and the retinal detachment. These arethe late stage and final results of DR and hard to deal with. So to cure the patient in theearlier stage will be more important. Before these ultimate injuries, the apoptosis of retinalendothelial cells and ganglion cells,the increase of inflammatory genes may be the mainmechanism of DR. So lots of Diabetic Retinopathy research center just focus on theinflammation and apoptosis mechanism through animal DR model. However, as we knowthere are lots of method of making the DR model, the common problem is time costing.Especially the STZ injection,which is the most reliable one so far, but need6months or so.Therefore, we want to find a quick method to replace DR model in some extend. So wechoose IR.The IR model means ischemic reperfusion, in detail is after a period of ischemia,getting rid of the reason and the blood will go back and perfuse the tissue again. For therewill be double injury in this duration. One is the ischemia, the other is the reperfusion. Forischemia will cause abnormal metabolic trash including the free radicals, for lack of oxygenand blood. Furthermore, reperfusion can bring lots of oxygen which will react with the freeradical, and generate lots of superoxygen. The result will be the inflammation and apoptosis,and lots of cells dysfunction and lost. So there will be lots of similarities between DR and IR.We think IR model will be a valuable model for the research of DR. We are going tocompare the pathologic injury between IR and DR, then we can track the mechanism of DRin IR model. In this procedure, We found that TSP1, known as an innate angiogenesisinhibitor during some eye diseases, which increase in the early stage of IR injury. So thehypothesis is TSP1must have some important function in IR model, even in DR model. Sowe decide to use the TSP1KO mice to make the IR model to see what will happen withoutTSP1. To our excitement, TSP1can induce the apoptosis and inflammation, deteriorate thesuperoxygen damage, increase the attachment of leucocytes, break down the blood retinalbarrier. And we hope TSP1will do something in DR model. 1,We made the Diabetic model successfully by injecting the STZ. And we did someexperiments about inflammation, the apoptosis of retinal endothelial cells and ganglion cellsand the attachment ability of leucocytes, the BRB function, etc.We found the dose of STZ will be a most reasonable one for the DR model. Also wefound the damage will be worse according the time duration of higher glucose. We foundthe TSP1will increase after the2months STZ injection model. So the hypothesis is TSP1have some reaction during the DR injury. SO we want to use IR model to mimic the damageof DR.2,We made the ischemic reperfusion model successfully by increasing the intraocularpressure. Also we compare the pathogenic damage of IR with DR and found they are alikeon inflammation, apoptosis, leucocytes attachment, BRB dysfunction, superoxygen damage.Also TSP1can promote the procedure of inducing apoptosis, inflammation, oxygen stress.We use a pressure of90mmHgby increasing the height of liquid bottle and the durationis90minutes.In this condition,the IR model will be stable and modest damage which issimilar with the DR. And we found the inflammatory gene increased, the same with theapoptosis. So we got the conclusion the major mechanism during IR is inflammation andapoptosis.In the TSP1KO group,the degeneration of ganglion cells and endothelial cells decreased.It is because TSP1can promote the apoptosis in IR.To testify the ability of inducing apoptosis of TSP1in IR, we design to mimic the IRmodel in vivo by putting the cells in the circumstance first absence of oxygen then thenormal oxygen.3,We culture the HREC, by doing so, we can testify the action of TSP1to endothelialcells.First we put HREC into the1%O2chamber, after sometime, then transfer HREC to thenormal oxygen chamber. We found the cell concentration is1×105,4hours hypooxygen thennormoxygen will be the best choose. TSP1will increase2folds and the result of cell deathdetection increased3folds. Which means TSP1will increase during this procedure and hasintimate relation with cell death.Next we use RNAi to knock down TSP1. We found furthermore, TSP1KO will protectthe cell by decreasing30%of cell death. To observe the effect of TSP1with higher glucose, we stimulated HREC with differentconcentration of glucose. Also we use the RNAi to find TSP1still have protective action ofHREC in higher glucose.The conclusion is we set up a ideal model of DR and IR, and we found lots ofsimilarity between them in the pathogenic injury. For IR is a much quicker model comparedwith DR. So it will help a lot if we replace DR with IR in exploring the mechanism andtherapy of DR. Another important thing is TSP1can induce apoptosis, inflammation,deteriorate the oxygen damage, break down BRB. We also utilize HREC IR model to makesure the action of TSP1. Thus, we think TSP1will be a novel direction in both IR and DR.