The Effect Of Adenosine On The Microvasculation In The Distal End Of Experimental Femoral Artery Stenosis In Rats And Its Mechanism

Objection:Establishment the experimental femoral artery stenosis model of rat.And detect the influence of the blood flow,the volume of capillary and adenosine receptor,oxide content on femoral artery distal after injected with adenosine in the tail vein.To investigate the effect and mechanism of adenosine on the distal microvessels of experimental femoral artery stenosis.It will provide a new diagnostic method for the early stenosis of the lower extremities.Methods:1.Total of thirty male Wistar rats were randomly divided into: experimental groups(femoral artery ligated)and control groups(threading without ligation under the femoral artery).And all of them were injected with adenosine in the tail vein.Another 60 rats were used for the second part of the experiment.and randomly divided into 4 groups: model saline group(A group),model adenosine group(B group),control saline group(C group),and control adenosine group(D group).In the 4 groups,And all of them were injected with physiological saline and adenosine in the tail vein after the modeling.2.The OMAG(Optical Microangiography)system was used to observe the changes of blood flow in the distal femoral artery at the 0th,4th,8th,and 12 th minutes after injection of adenosine in the model group and the control group.3.2-photon(2-photon Zeiss LSM 7MP microscope)technology was used to observe the changes of the number of microvessels distal to the femoral artery stenosis in the model group and the control group before and after 8 minutes of adenosine injection.4.The pathological staining of hematoxylin-eosin(HE)was used to observe the arrangement of skeletal muscle cells and the distribution of intermuscular blood vessels in the rat model group and the control group before and after the administration.5.Immunohistochemistry(IHC)technique was used to detect the expression of adenosine A2 a and adenosine A2 b receptor in the distal femoral artery.6.Westernblot was used to detect the protein expression of adenosine A2 a and adenosine A2 b receptors in the quadriceps muscle of each group.7.Real time fluorescent quantitative PCR was used to detect the expression level of A2 a mRNA,adenosine A2 b mRNA,endothelial nitric oxide synthase(eNOS)mRNA and inducible nitric oxide synthase(iNOS)mRNA in the quadriceps muscle of each group.8.Nitrate reductase method was used to detect the concentration of NO in the serum of inferior vena cava in the four groups.Results:1.The changes of blood flow at the distal of femoral artery stenosis in rats: Adenosine could reduce the femoral artery stenosis distal blood flow of experimental group within 8min,and the blood flow began to rise after 8 minutes.While in the control group,the distal blood flow of the femoral artery slightly increased after administration of adenosine.And the blood flow tends to be normal after 8min.2.The volume of the capillary filling of hindlimb far-end: Adenosine could reduce the volumer of microvessels distal to the femoral artery stenosis in the experimental group.While the volume of quadriceps capillaries in the control group increased after administration.3.The relative expression levels of A2 a and A2 b receptor proteins in skeletal muscle of each group: The expression of two kinds of proteins in group B was significantly lower than that in group D(P<0.05).The expression of the two kinds of proteinsin in group B also lower than that in group A(P<0.05).Compared with group C,the expression of A2 a and A2 b receptor proteins were increased in group D(P<0.05).There was no statistically significant difference between the C?D group and the group A(P>0.05).4.The relative expression levels of A2 a mRNA,A2 b mRNA,eNOS mRNA,and iNOS mRNA in skeletal muscle of each group: Compared with D group,the expression of A2 a,A2b,eNOS mRNA was significantly decreased in the groups B(P<0.05),while the expression of iNOS mRNA was significantly increased(P<0.05).Compared with group A,the results were similar in group B.Compared with group C,the expression of A2 a,A2b and eNOS mRNA was increased in group D(P<0.05),and the increase of iNOS mRN concentration was no statistically significant(P>0.05).The four genes in C and D groups were no statistically significant compared with the A group(P>0.05).5.The concentration of NO in the blood of inferior vena cava in each group: The concentration of NO in group B was slightly lower than that in group D(P<0.05);the concentration of NO in group B was significantly higher than that in group A(P<0.05).Compared with group C,the concentration of NO was significantly increased in group D(P<0.05),while there was no significant difference between group C and D compared with group A(P>0.05).Conclusion:1.Adenosine can cause blood stealing effect in the femoral artery experimental stenosis model,thereby reducing the distal blood flow and the number of microvessels in the stenosis area.But in normal femoral artery,adenosine has a certain vasodilation effect on femoral artery.2.The vasodilation effect of adenosine in normal blood vessels is activated by a large number of A2 a and A2 b receptors.However,the expression of adenosine A2 a and A2 b receptors in experimental stenotic vessels decreases significantly.3.Nitric oxide synthase participates in the process of regulating vasoconstriction or vasodilatation by adenosine.And endothelial nitric oxide synthase is mainly involved in the process of adenosine-mediated vasodilation,and inducible nitric oxide synthase is mainly involved in adenosine-mediated vascular damage.