Effects Of Drag-reducing Polymers On Exercise Tolerance Of Acute Ischemia Hindlimb Rats And Its Possible Mechanisms

BackgroundPeripheral arterial disease (PAD), which is caused by atherosclerotic occlusion of the arteries, is an important manifestation of systemic atherosclerosis. Clinical symptoms include numbness and tingling of limbs, intermittent claudication, rest pain, artery pulse weakening or disappearance, severe cases can cause ischaemic ulceration, infection and gangrene. Intermittent claudication, defined as pain in the muscles of the leg with ambulation, is the earliest and the most frequent presenting symptom in patients with lower extremity PAD. Although many never experience symptoms, 7～15% of people with asymptomatic PAD will develop intermittent claudication within five years. As well as, the frequency of intermittent claudication increases dramatically with advancing age, ranging from 0.6% in individuals aged 45～54 years, to 2.5% in those aged 55～64 years, to 8.8% in patients aged 65～74 years. As the disease progresses in severity patients might have pain at rest, most prominent while the legs are elevated in bed at night, and relieved by dependency. Although claudication symptoms are typically localised in the calf or the thigh, "rest pain" is characteristically in the foot. In the late stages of PAD, tissue hypoperfusion progresses to ischaemic ulceration and gangrene, and major amputation is eventually required in more than a third of these patients. And patients should be reassured accordingly only 1～3.3% of patients with intermittent claudication will require major amputation in a five year period. Mortality rates as high as 20% in several series. And patients with peripheral arterial disease, even in the absence of a history of myocardial infarction or ischemic stroke, have approximately the same relative risk of death from cardiovascular causes as do patients with a history of coronary or cerebrovascular disease. PAD, which is high cripple and mortality, has been threatening human life. Hence, explore the treatment of PAD is extremely necessary. The focus of PAD treatment is to reduce symptoms and improve quality of life, to reduce overall cardiovascular morbidity and mortality. Current treatments mainly include smoking cessation, controlling blood lipids, controlling blood pressure, controlling diabetes, antiplatelet, antithrombotic and revascularization. However the investigation for patients with claudication to relieve their exertional symptoms, improve their walking capacity, and improve their quality of life still needs further exploration.Drag reducing polymers (DRPs) are a distinct class of chemical compounds that aracterized by extraordinarily high molecular weights (greater than 106) or the ability to form molecular aggregates. The addition of very small amounts of these polymers to a fluid leads to a reduction in resistance of turbulent flow in pipes, thereby increasing flow rate at a constant pressure or by reducing driving pressure at a constant flow. This drag-reducing phenomenon was described by Toms (Toms effect) in 1948. The Toms phenomenon has been investigated and used for various industrial and engineering applications including crude oil transport through pipelines, firefighting, and reducing drag on ships and submarines. Over the years, blood-soluble drag-reducing polymers have been shown to produce positive hemodynamic effects in various acute and chronic animal models. Specifically, nanomolar concentrations of these polymers injected intravenously caused an increase in aortic and arterial blood flow and a decrease in both blood pressure and peripheral vascular resistance, with no effect on blood viscosity or blood vessel tone. DRPs were found to significantly increase collateral blood flow in rabbits and the number of functional capillaries in normal and diabetic rats; and reduce hydrodynamic resistance in both normal and adenosine dilated rat mesentery arterioles. In canine models of a subcritical stenosis in the aorta and carotid artery, injection of DRPs reduced poststenotic flow separation and flow disturbances. Increased blood flow through iliac artery stenoses and redistributed wall shear stresses in the area of aortic stenoses have also been measured in dogs. Chronic intravenous injections of DRPs diminished the development of atherosclerosis in several atherogenic animal models. And most recently it was shown that the DRPs, when used as a component of a resuscitation fluid, were able to significantly improve tissue perfusion and oxygenation and to reduce lethality in animals subjected to severe hemorrhagic shock. This study is the first to show that intravenous DRPs reduce the pressure loss between the aorta and the arteriolar compartment, resulting in a higher pre-capillary driving pressure. In summary, the present work demonstrates that blood soluble DRPs, when injected at nanomolar concentrations in animals in hemorrhagic shock, rapidly increased blood pressure andrestored microcirculatory flow, resulting in increased oxygen delivery to tissues. My research group confirmed that PEO can increase the blood flow of abdominal aorta of normal rats, and promote acute hindlimb ischemia in the recovery of skeletal muscle perfusion. This research is to explore the effect of DRPs on the exercise tolerance in acute rat hindlimb ischemia and its possible mechanism.PartⅠ Effects of Drag-reducing polymers on hemodynamics of abdominal aorta of acute ischemia hindlimb rats Objective To observe the effects of drag-reducing polymers on blood flow of abdominal aorta of acute ischemia hindlimb rats by compare with heart rate、central venous pressure and mean arterial pressure between two groups, to estimate the effects and drug safety of DRPs.Methods1. Preparation of DRPsPolyethylene oxide (PEO) is widely used in drag-reducing areas at animal experiment. In this study we tested Polyethylene oxide (PEO, with average molecular weight of 5x106Da), dissolved in saline at a concentration of 0.1% and then dialyzed against normal saline for 24 hours using a membrane with 50kDa molecular weight cutoff. After dialysis, PEO was diluted to a concentration of 50 ppm with saline and stored at-4 centigrade degress.2. Preparation for animal and hemodynamic monitoringTwelve rats were maintained in a room with controlled temperature (22～26℃, 50%humidity), and light [light and shade cycle every 12h(8:30am-8:30pm)]and were provided food and water ad libitum. All rats were accustomed to treadmill running. Exercise was performed on a rodent treadmill, beginning at 10m/min at 11% grade for 5 min/day, for 3 days, the intensity was gradually increased to 12m/min. At day5, twelve rats were anesthetized and then both two side femoral arteries were exposed、 divided and ligated. After 2 days of rest, all rats were exposed to running exercise again. At 14 days, twelve rats were anesthetized again and randomly divided into two groups. An ultrasonic flow probe was placed around the abdominal aorta (about 5mm above the common iliac artery) to monitor blood flow of abdominal aorta. Heart rate (HR), central venous pressure (CVP) and mean arterial pressure (MAP) were also monitored.3. Record of dataThe rats in experimental group (n=6) received an intravenous injection of PEO at the speed of 7mL/h for 10 minute, while in control group (n=6), normal saline was infused at the same speed and time. Record blood flow of abdominal aorta, heart rate(HR)、central venous pressure(CVP) and mean arterial pressure(MAP).ResultsCompared with control group, rats in experimental group have an significant increased of abdominal aortic flow(F=10.994, P<0.001) and peak blood flow (6.4±1.1 ml/min vs 4.9±0.3 ml/min, F=11.027, P=0.008); However, there were no signifant differences of mean arterial pressure(F=0.035, P=0.855)、central venous pressure (F=0.009, P=0.925) and heart rate(F=0.060, P=0.811) between two group.ConclusionsPEO increased the blood flow of abdominal aorta in acute rat hindlimb ischemia. However, there were no signifant differences of mean arterial pressure、central venous pressure and heart rate between two group.PartⅡ Effects of Drag-reducing polymers on exercise tolerance of acute ischemia hindlimb rats and its possible mechanismsObjectiveTo observe the effects of drag-reducing polymers by comparing with the time of exhaustive exercise, the content of blood lactic acid, serum nitric oxide level, serum superoxide dismutase activity, serum creatine kinase level, serum lactate dehydrogenase level and Lactic acid of gastrocnemius muscle between two group in an exercise tolerance of acute rat ischemia hindlimb model and meanwhile explore the possible mechanisms.Methods1. Preparation of DRPs:Polyethylene oxide (PEO) is widely used in drag-reducing areas at animal experiment. In this study we tested Polyethylene oxide (PEO, with average molecular weight of 5x106Da), dissolved in saline at a concentration of 0.1% and then dialyzed against normal saline for 24 hours using a membrane with 50kDa molecular weight cutoff. After dialysis, PEO was diluted to a concentration of 50 ppm with saline and stored at-4 centigrade degress.2. Preparation for animal and infusion channel:Eighty rats were maintained in a room with controlled temperature (22～26℃, 50% humidity), and light [light and shade cycle every 12h(8:30am～8:30pm)]and were provided food and water ad libitum. All rats were accustomed to treadmill running. Exercise was performed on a rodent treadmill, beginning at 10m/min at 11% grade for 5 min/day, for 3 days, the intensity was gradually increased to 12m/min. Day5 all rats were anesthetized and then both two side femoral arteries were exposed, ligated and divided, jugular vein of rats were separated, catheterized and fixed well, rats were washpiped daily with heparin saline. After 2 days of rest, all rats were exposed to running exercise again, beginning at 10m/min at 11% grade for 3 min/day, for 4 days, the intensity was gradually increased to 12m/min.And then rats were allowed to rest for 2 days. In day 14, rats which cannot run on treadmill or its intravenous-catheter blocked were excluded.64 rats were randomized into two groups. The rats in experimental group (n=32) received an intravenous injection of PEO at the speed of 7mL/h for 10 minute, while in control group (n=32), normal saline was infused at the same speed and time during exhaustive exercise. After rat’s energy was exhausted (Exhaustion was defined as the rats could no longer keep pace with the treadmill). Observing the time of exhaustive exercise and measuring the content of blood lactic acid、serum nitric oxide (NO) level、serum superoxide dismutase (SOD) activity、serum creatine kinase (CK) level, serum lactate dehydrogenase (LDH) level and Lactic acid (muscle LD) of gastrocnemius muscle.3. Record of dataRecord the time of exhaustive exercise and measuring the content of blood lactic acid、serum nitric oxide (NO) level、serum superoxide dismutase (SOD) activity、 serum creatine kinase (CK) level, serum lactate dehydrogenase (LDH) level and Lactic acid (muscle LD) of gastrocnemius muscle.ResultsCompared with controls, rats in experimental group had a longer time of exhaustive exercise(P<0.05), increased NO and SOD activity(P<0.05), reduced LD concentration in gastrocnemius muscle(P<0.001). However, there were no signifant differences of CK, LDH and LD concentration (P>0.05).ConclusionsPEO can prolong the time of exhaustive exercise and improve the exercise tolerance of acute ischemia hindlimb rats by increasing blood flow reserve(increased NO level), improving local tissue metabolism(reduced LD concentration in gastrocnemius muscle) as well as enhancing the antioxidant effect(improved SOD activity), which exerting an instrumental effect in anti-fatigue.