| BackgroundPeripheral vascular disease is a common disease, the course of which includes atherosclerosis, thrombosis, inflammation which result in arterial stenosis and occlusion, the pathogenesis of which includes blood microcirculation, rheology, platelet function, coagulation mechanisms, immune and metabolism, etc.. Large vessel stenosis or occlusion causes ischemic injury in skeletal muscle in its supply area, leading to rest pain, intermittent claudication, limb gangrene and a series of clinical symptoms.Thrombolysis, surgery, interventional thrombectomy and other methods play a role in opening the great vessels. However,there is a lack of good treatment, for major vascular injury in the microcirculation. Therapeutic angiogenesis by human vascular delivery to ischemic tissue growth factor, gene or recombinant protein, to promote the angiogenesis in ischemic tissue, thereby improving ischemic tissue ischemia and hypoxia. In recent years the study has achieved some results, but these growth factors in the induction of angiogenesis in the process, it does not control angiogenesis-promoting effect leading to many complications. So current clinical application is still limited. The study for other treatments options has become important.Neovascularization includes angiogenesis and arteriogenesis.The driving force of angiogenesis is hypoxia, while that of arteriogenesis is the increase of blood flow shear stress. In the model of acute femoral artery occlusion in pig, at the distal of occlusion made an action-venous fistula by human, that can induce more collateral circulation of ischemic tissue, and the situation of blood perfusion was much better than that of vascular growth factor. It showed that increasing the blood flow in ischemic tissue shear stress can significantly promote the formation of small arteries. So, can promoting the formation of small arteries by increasing blood flow shear stress to the treatment of ischemic disease?In 1948, British scientists B.A Toms reported the Toms phenomenon that a special class of soluble polymers can reduce resistance to turbulent flow without influence on the fluid viscosity. These polymers so be called Drag-reducing polymers (DRPs).Since discovery of the Toms phenomenon, DRPs has been used in mediacal research, petroleum, fire fighting, irrigation, navigation and other areas widely.In the medical field, it has been already proved that DRPs can improve the prognosis of acute myocardial ischemia in animals, improve the microcirculation in hemorrhagic shock and diabetic animals, such as the role of the microcirculation, DRPs increases blood flow through to reduce atherosclerotic plaque formation. In the area of ischemic cardiovascular disease, atherosclerosis, hemorrhagic shock and other circulatory system diseases, DRPs shows a great prospect of development. My research group confirmed that PEO can promote acute hindlimb ischemia in the recovery of skeletal muscle perfusion, and directly observed that PEO increased 30% of shear stress of arterial wall. But with the exception of DRPs intervention atherosclerotic plaque studies, all studies are limited to acute vascular occlusion, stenosis, hemorrhagic shock and hemodynamic changes, the research about chronic ischemic disease has not been reported. How can we use PEO to increase shear stress to increase blood flow in order to treat the ischemic disease?The study was to quantitative blood flow of microcirculation with ultrasound imaging,we observed the effect of PEO on blood perfusion of chronic ischemia skeletal muscle by totally occluded peripheral arteries and hypothesised that DRPs can improve blood flow of ischemia tissue and promote arteriogenesis by increasing blood shear stress. PartⅠ:Quantitation of the absolute blood flow of hind limb skeletal muscle in ratsObjectiveEstablish a method of "quantitative absolute blood flow of skeletal muscle in rat with contrast-enhanced ultrasonography,"and evaluate the stability and repeatability of the method.Methods1.Animal preparation12 SPF male Wistar rats were anesthetized with 3% pentobarbital intra-peritoneally, and fixed on the rat board after skin of precordial and left hind limb was prepared.2.Ultrasound contrast agent"Quanfuxian"(Nanfang Hospital, Southern Medical University), a suspension of perflutren-filled albumin microbubbles with a mean diameter of 2.0 to4.0μm at a concentration of 1.0 to 2.0×109 microspheres/ml, was diluted with 3ml 0.9% sodium chloride solution and shake stand before experiments.3. Method of imagingSequoia512 ultrasonic diagnostic apparatus (17L5 probe) was used. The probe positioned in the left ventricular short axis view, contrast agent infusion of "Quanfuxian" through an infusion pump at a steady rate of 20μl/min In contrast pulse sequence mode (Coherent Pulse Sequence, CPS), R wave triggered intermittent imaging; then the ultrasound probe was positioned in the middle of rat hind limb (the midpoint of the patella and ankle), the parameters remain the same, to time-triggered (the interval was 500ms) imaging, When the intravenous contrast agent (200μl/min) to the filling of steady state is stable, with high mechanical index contrast agent microbubble destruction to access skeletal muscle after contrast agent within the process of re-filling all the images.7 days after the experimental procedures, repeat angiography was undertaken in accordance with the above mentioned steps.4. Calculation of skeletal muscle blood flow All the contrast perfusion images were analyzed offline with the software MCE (OHSU, USA). ALV was stand for the average intensity of left ventricular cavity in steady state, Am was stand for platform intensity of skeletal muscle,βm was stand for refill rate of contrast agent.When left ventricular angiography and skeletal muscle with the same dose of contrast agent, local tissue blood flow wasβm*Am/ALV/ρm, Where Am/ALV as the standard platform strength of the acoustic was standardized blood vessels of rat hind limb skeletal muscle.In this experiment, the concentration of contrast agent in the contrast ehanced ultrasound of skeletal muscle is 10 times of that of heart chamber, whileρm was stand for the rat muscle density (ρm=1.05g/ml), namely, the absolute skeletal muscle blood flow was 60*βm*Am/(ALV* 10*ρm* 1.05) (unit:ml·g-1·min-1).ResultsThe imagings of hind limb skeletal muscle and cardiac chambers of rats were good. Comparison of two measurements of different time, volume of skeletal muscle capillaries, blood flow velocity and standardized calculation of skeletal muscle blood flow has no statistically significant differences (P> 0.05). All the goodness of fit curve were good coefficient and the determination was 0.944±0.046 (0.789-0.985), P<0.01.ConclusionsThe methods of quantification of absolute blood flow in skeletal muscle has a good repeatability and can be regarded as a new method to quantify absolute blood flow in vivo.PartⅡ:The effect of DRPs on chronic ischemic hind limb perfusion of skeletal muscle in ratsObjectiveEstablished chronic ischemia model of hind limb in rat and treatsd by intravenous drug, measured blood flow with contrast enhanced ultrasound and studied vascular changes in immunohistochemical methods, evaluated the effect of DRPs on blood flow perfusion of chronic ischemic hindlimb skeletal muscle, and explored the mechanism of increasing blood flow initially,in order to build a foundation of new treatment for ischemic diseases.Methods1. Preparation of DRP:PEO dissolved in saline at a concentration of 0.1%(10ppm) and then dialyzed against saline for 24 hours using a membrance with 50KDa molecular weight cutoff.The solution of PEO was sterilized using very slow filtration through 0.2μm filter. The molecular weight of PEO in solution was 4.99×106 Da using gel permeation chromatography.The PEO solution of PEO was diluted into 50ppm with saline prior to use.2. Preparation of infusion channel:Jugular vein of rats were separated under anesthesia and inserted catheter that the catheter was fixed with sutures in multiple subcutaneous and fixed the boot to the back incision. Caged rats were divided and washpiped daily with 0.1ml of heparin saline (10iu/ml).3. Model of chronic ischemia:3 days after the infusion channel establishment, the rats were anesthesiaed intraperitoneally with 3% sodium pentobarbital.1 cm long incision of the right hind limb of rats were made in sterile conditions along the lower edge of the inguinal ligament, separation of the right femoral artery and its branches were ligated with 5-0 fine silk thread and cut off, to avoid harming vein and nerve, the left hind were not deal with.4. Method of delivery:The rats were randomly divided into two groups.1,3,5,7,9,11,13 days after operation,the rats were administrated in conscious unrestrained state through the infusion channel, the treatment group of DRP (30min,1.4ml/h, blood volume to body weight, the blood concentration of PEO 2μg/ml), to be the same speed as the control group received saline at the same time,at the end of treatment washpiped with 0.1ml heparin saline (10iu/ml), infusion catheter was closured with a small steel wire. 5. Measurement of blood flow of skeletal muscle with CEU:On the day of pre-operation and the 1st day,3rd day,5th day,7th day,14th day,21th day and 28th day after operation, blood flow of skeletal muscle was measured with CEU,the method was as same as the first part.6. Measurement of blood flow reserve:On the 28th day, after the contrast echocardiography test of rest state,continued infusion of perfluoropropane (200ul/min), kept the same imaging pattern, stimulated hindlimb skeletal muscle of rats with temporary pacemaker in frequency of 120 beats /min.2 minutes later, began to take figure. Analyze images offline with MCE software, recorded the parameter A,βand A×β.Flow reserve is the pacing of the A xβvalue of pacing in front of A×βvalue ratio.7. Pathology:After ultrasound imaging,the experimental rats were sacrificed for HE staining andα-actin antibody staining.ResultsAfter 4 weeks, blood flow of ischemic hind limb of DRPs group was significantly higher than that of saline control group (0.319±0.044 vs 0.211±0.040 ml/g/min, P= 0.001). Blood flow reserve of ischemic hind limb of DRPs group was also higher than that of saline control group (3.227±0.941 vs 1.867±0.551, P= 0.012).Blood volume of ischemia hindlimb skeletal muscle of DRPs group was significantly increased compared with the saline group (0.446±0.111 vs 0.310±0.059, P= 0.024).In HE staining, non-ischemic hindlimb skeletal muscle cells in rats was in normal morphology, not congestive and hydropic, while ischemic hindlimb skeletal muscle cells, both in saline group and DRPs groups, could be found inflammatory cells. In saline group, skeletal muscle cells were hydropic, while the cells of DRPs group was no significantly hydropic.Immunohistochemistry showed that the number of arterioles per high power microscope in DRPs group was more than that of the saline group (9.25±1.72 vs 6.23±0.86, P= 0.026). ConclusionsDRPs can increase blood flow and blood flow reserve of chronic ischemic hind limb, the immunohistochemistry suggested the number of DRPs groups of small arteries was more than the saline group, indicating that DRPs may promote arteriogenesis. |
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