实验课题的开展与质量控制.ppt

weil institute of critical care medicine, rancho mirage, ca the keck school of medicine of the university of southern california,实验课题的开展与质量控制,june 2009, china,shijie sun, md, fccm, faha,选题 目标与假设-hypothesis no hypothesis no protocol 实验方案-protocol no protocol no experiment 实验-experiment no experiment no data 实验数据和结论-data and conclusion no data no conclusion,实验课题的建立,阅读,学术会议,交流,探讨,alone we are smart, together we are brilliant.,目的与假设-hypothesis no hypothesis no protocol,实验方案-protocol no protocol no experiment,protocol,the effect of epinephrine during hypothermic cardiopulmonary resuscitation shijie sun, md; giuseppe ristagno, md; fengqing song, md, phd; cristina santonocito, md; yi shan, md; tao yu, md; yongqin li, phd; max h. weil, md, phd; wanchun tang, md weil institute of critical care medicine rancho mirage, ca 92270,example,标题,example,1. specific aims and hypothesis 1.1 specific aims to test the effects of epinephrine, administered during cpr and concurrent therapeutic hypothermia on initial success of resuscitation, post-resuscitation myocardial and neurological function, and duration of survival in a rat model of cardiac arrest. 1.2 hypothesis therapeutic hypothermia decreases systemic oxygen consumption and metabolic rate. this study investigates the hypothesis that hypothermic cpr will minimize the detrimental effects of epinephrine. therefore, when epinephrine is administered during therapeutic hypothermia cpr, it will not only improve immediate resuscitation, but minimize the severity of myocardial ischemic injury and therefore the severity of post resuscitation myocardial dysfunction, reduce post resuscitation cerebral ischemic injury, and improve post resuscitation 72 h survival and in contrast to epinephrine administered during cpr without hypothermia.,目标和假设,目标,假设,2. background and significance 2.1 post resuscitation myocardial and neurological dysfunction patients who are successfully resuscitated following cardiac arrest often present with what is now termed “post resuscitation disease” (1). most prominent are post resuscitation myocardial failure and ischemic brain damage. although post-resuscitation myocardial dysfunction has been implicated as an important mechanism accounting for fatal outcomes after cardiac resuscitation (2-4), morbidity and mortality after successful cardiopulmonary resuscitation largely also depends on recovery of neurologic function. 30% of survivors of cardiac arrest in fact manifest permanent brain damage (5-7). the greatest post resuscitation emphasis has been on long-term neurologically intact survival (8). 2.2 epinephrine epinephrine has been the preferred adrenergic agent for the treatment of human cardiac arrest for more than a century (9, 10). it is a powerful agonist of 1 and 2, 1 and 2adrenergic receptors. epinephrine can increase coronary and cerebral perfusion pressure during cpr, therefore improve initial rosc and early survival rates. however, many study demonstrated that the major adverse effects of epinephrine due to its 1 and adrenergic actions, include ventricular dysrhythmias and disproportionate increases in myocardial oxygen consumption and metabolic rate, thereby increase the severity of myocardial ischemic injury and the severity of post resuscitation myocardial dysfunction (11-15). in animal studies, this effect also results in increased severity of ischemic injury during low flow states of circulatory shock and cardiac resuscitation (12, 14, 15). based on both beneficial and toxic physiologic effects of epinephrine administration during cpr, in 2005 american heart association guideline for cardiopulmonary resuscitation and emergency cardiovascular care, epinephrine had been changed from the drug of first choice to class iib in the management of cardiac arrest (16).,example,背景和意义,2.3 therapeutic hypothermia therapeutic hypothermia following return of spontaneous circulation (rosc) has been advocated for decades prior to its clinical acceptance (17). therapeutic hypothermia has been in fact used routinely in the operating room since the early 1950s for patients undergoing cardiac surgery (18) and more recently for neurologic surgery (19). more than a decade ago it was reported that young and healthy people who underwent accidental deep hypothermia with cardiac arrest were able to survive with no or minimal cerebral impairment even after prolonged cardiac arrest (20). in 1996, safar induced hypothermia by instilling ringers solution maintained at a temperature of 4c for 12 hours into the abdominal cavity of dogs after resuscitation from cardiac arrest. functional recovery was associated with minimal histological brain damage (17). two of the largest randomized clinical trials on systemic hypothermia published in 2002 (21, 22) have objectively demonstrated improvements in neurological outcomes and within a short 5 years, this therapeutic intervention has finally proven to be neuroprotective (8, 21, 22). on the basis of these studies, current guidelines of the international liaison committee on resuscitation (ilcor) are recommending the use of therapeutic mild hypothermia for all unconscious patients after cardiac arrest, specifically ventricular fibrillation victims (8, 23). therapeutic hypothermia reduces metabolic rate and therefore oxygen consumption (24, 25). the physiologic effect of therapeutic hypothermia on the injured brain is that for each 1 c decrease in temperature, the cerebral metabolic rate decreases by 6-7% (26). for the cardiovascular system, therapeutic mild hypothermia on the unconscious patients decreases heart rate and increases systemic vascular resistance, whereas stroke volume and mean arterial blood pressure were maintained (22). also contract with the risk of vf in accidental hypothermia patients, with therapeutic mild hypothermia cardiac arrhythmias are rarely seen, even in patients with myocardial ischemia (21, 22). there are more recent observations of increased myocardial contractility following cooling treatment after cardiac arrest in animal experiment studies. in isolated hearts perfused and exposed to mild or moderate hypothermia during 120 minutes of ischemia, moderate hypothermia suppressed anaerobic metabolism during ischemia and significantly diminished left ventricular end-diastolic pressure at the end of ischemia. therefore, hypothermia contributed to the preservation of myocardial function, coronary flow, and oxygen consumption compared with normal control hearts and myocardial integrity (27). interesting data, highlighting improvements of myocardial contractility in animals that received hypothermia treatment following cardiac arrest, have been recently introduced by zhao et al (28). in those experiments, the hypothermic cardiovascular reperfusion resulted in considerably greater cardiac output with concomitant lesser systolic and diastolic myocardial dysfunction during the post-arrest period.,example,example,3. study design and methods 3.1 study design our established rodent model of cardiac arrest and resuscitation is utilized. sprague-dawley rats weighing between 450-550 g are the experimental subjects. the relatively large size of these breeder rats allows for ease of endotracheal intubation and vascular catheterization. this rodent model of cpr provides us with a special asset because it has proven both efficient and cost redeeming over more than 20 years of research in our laboratory. we propose to investigate the effects of epinephrine in association or without therapeutic hypothermia on the rate of success of resuscitation, post resuscitation myocardial and cerebral functions, and on survival when these agents are administered during cpr. the independent variables are the adrenergic agents and the temperatures (37c0.2 vs 32c0.2). epinephrine (20 g/kg) is compared with saline placebo. the doses selected are based on earlier studies in our laboratories. the project therefore involves a 2 x 2 design with equal number of animals assigned at random to each of 6 groups, for a total of 30 animals as shown on table. table: study design number of animals setting of temperature (c) epinephrine saline placebo 370.2 6 6 320.2 6 6 8 minutes untreated vf and 8 minutes of cpr prior to defibrillation attempts.,“the independent variables are the adrenergic agent and the temperatures (37c0.2 vs 32c0.2)”,设计和方法,设计,the primary dependent variables include restoration of spontaneous circulation (rosc), post resuscitation myocardial function, 24, 48, and 72 h neurological deficits by the method of hendrickx hl et al (29), and survival. we have demonstrated that the energy of electrical defibrillation effects post resuscitation myocardial function (30). accordingly, the total defibrillation energy required for restoration of spontaneous circulation after the pharmacologically intervention, is included as an additional dependent variable. measurements of time coincident diastolic aortic and right atrial pressure allow for estimation of cpp. coronary perfusion pressure generated by precordial compression with a miniaturized pneumatic chest compressor of our design has been refined by us such that it may be adjusted to maintain a pressure of 24 2 mm hg prior to administration of the adrenergic agents. this target of cpp of 24 mmhg was selected on the basis of our earlier and large experience. after precordial compression is adjusted to achieve these minimized cpp, no further adjustments are made following the administration of the adrenergic drugs. myocardial systolic and diastolic functions are measured or confirmed by echo-doppler technique. cardiac output is calculated as the product of trans-aortic flow time velocity integral, aortic valve diameter and heart rate. left ventricular systolic function is estimated by measurements of left ventricular end systolic and end diastolic volume (lvesv /lvedv), based on fractional area change (fac) and resulting stroke volume (sv) and ejection fraction (ef). left ventricular diastolic function is determined by measurements of lvedv and wall thickness (lvpsw /lvpdw) (31, 32). end-tidal co2 is measured continuously such as to confirm appropriate ventilator settings prior to and following reversal of cardiac arrest. during the low flow state of cardiac arrest (with gasping) and under conditions of precordial compression, etco2 has been shown by us to serve as a quantitative indicator of relative pulmonary blood flow, i.e. cardiac output on this model (33). we recognize an additional incentive for measurement of etco2. as cited above, b-adrenergic effects of epinephrine produce v/q abnormalities that are reflected in the etco2. the etco2 measurement itself, like cpp, is of itself predictive of the success of cardiac resuscitation and has the added advantages that it is non-invasive and is achieved with ease. it was originally documented by us on this model and later confirmed by us on pigs and subsequently on patients (34, 35). as such it has come into current routine clinical practice in settings of cardiac arrest and circulatory shock. arterial and mixed venous blood gases are measured prior to, during, and after resuscitation from cardiac arrest, together with arterial blood lactic acid. these quantitate, in part, pulmonary gas exchange and oxygen delivery, and veno-arterial ph and pco2 gradients, with special focus on the effects of the adrenergic drugs. we routinely measure lactate on 30 l arterial blood to assess the metabolic effects of systemic hypoperfusion. all of these measurements are performed on a total of 300 l of blood. blood is quantitatively replaced by transfusion from a donor rat of the same colony and such has had no adverse effects. from arterial and venous blood gases o2 consumption is calculated based on the following the fick equation: vo2 = co * (cao2 - cvo2),example,the primary dependent variables include restoration of spontaneous circulation (rosc), post resuscitation myocardial function, 24, 48, and 72 h neurological deficits by the method of hendrickx hl et al (29), and survival.,example,3.2 methods animal preparation male sprague-dawley retired breeder rats weighing 450-550 g are fasted overnight except for free access to water. the animals are anesthetized by intraperitoneal injection of pentobarbital (45mg/kg) and additional doses (10 mg/kg) are administrated at intervals of approximately one hour or when required to maintain anesthesia, except that no anesthetic agents are administered for 30 min before induction of cardiac arrest. procedures for vascular catheterization, hemodynamic measurements, blood sampling, monitoring of etco2, induction of vf and precordial compression are as previously described (33). the trachea is orally intubated with a 14 g cannula mounted on a blunt needle with a 145 angled tip. end-tidal co2 is continuously monitored with a side-stream infrared co2 analyzer (end-til il 200; instrument laboratory, lexington, ma). a conventional lead ii ekg is continuously monitored. animals are breathing room air spontaneously. a polyethylene catheter (pe-50; becton-dickinson, franklin lakes, nj) is advanced into the descending aorta from the surgically exposed left femoral artery for measurement of arterial pressure and baseline blood gas. through the left external jugular vein, another polyethylene catheter (pe-50; becton-dickinson, franklin lakes, nj) is advanced into the right atrium for measurement of right atrial pressures. aortic and right atrial pressures are measured with reference to the midchest with high-sensitivity transducers (model 42584-01, abbott critical care systems, north chicago, il). a polyethylene catheter (pe-50; becton-dickinson, franklin lakes, nj) is advanced from the left femoral vein into the inferior cava vein for the treatment infusion. a thermocouple microprobe, 10 cm in length and 0.5 mm in diameter (9030-12-d-34, columbus instruments, columbus, oh), is inserted into the right femoral artery, advanced to the aortic valve and then withdrawn to the more distal ascending aorta. blood temperature is measured with this sensor. a 3-f pe catheter (model c-pms-301j, cook critical care; bloomington, in) is advanced through the right external jugular vein into the right atrium. a pre-curved guide wire supplied with the catheter is then advanced through the catheter into the right ventricle confirmed by endocardial electrocardiogram. all the catheters are flushed intermittently with saline containing 2.5 iu/ml of crystalline bovine heparin.,方法,动物的准备,experimental procedures ventricular fibrillation is induced and trough the guidewire into the right ventricle. a progressive increase in 60 hz current to a maximum of 3 ma is delivered to the right ventricular endocardium, and current flow is continued for 3 min such as to prevent spontaneous defibrillation. mechanical ventilation is stopped after onset of vf. precordial compression is begun 8 min after onset of vf with a pneumatically driven mechanical chest compressor as previously described (33). coincident with start of precordial compression, the animal is mechanically ventilated. tidal volume is established at 0.65 ml/100 g animal weight, at a frequency of 100/min, and with a fio2 of 1.0. precordial compression is maintained at a rate of 200 min-1 and synchronized to provide a compression/ventilation ratio of 2:1 with equal compression-relaxation duration (i.e. 50% duty cycle). depth of compression is initially adjusted such as to secure a cpp of 22 2 mm hg. animals are randomized to receive right atrial injection of either epinephrine (20 g/kg), or saline placebo over a 30 sec interval beginning 5 min after start of precordial compression. resuscitation is attempted with up to 3, two (2) joule countershocks, after 8 minutes of cpr. restoration of spontaneous circulation is defined as the return of supraventricular rhythm with a mean aortic pressure of 50 mm hg for a minimum of 5 min. in animals randomized to therapeutic hypothermia, body cooling is initiated after the 8 minute of cardiac arrest such to achieve a blood temperature of 34c0.2 before administration of treatment and further reduce to 32 c0.2. rapid cooling is perfomed externally with the aid of ice packs and an electrical fan (36). for those animals not subjected to cooling, a blood temperature is maintained at 37c0.2. once reached the target temperature, it is maintained with the aid of blanketrol ii (csz, cincinnati, usa) until 4h after rosc. following rosc, mechanical ventilation is continued with 100% inspired oxygen for 1 h, and then maintained with 21% inspired oxygen the following 3 hrs at which time the animals have uniformly recovered from anesthesia. all catheters including the endotracheal tube are then removed. the animal is continuously observed by the investigators for an additional 2 h. butorphanol (0.4 mg/kg) is injected intramusculary if discomfort is identified. the status of the animal is then evaluated at 4 h intervals by a participating investigator for a total of 72 h. levels of consciousness, brain stem function and overall performance are examined and scored according to the method of hendrickx hl et al (29) (neurological deficit score when normal = 0 and coma = 500). the animal is euthanized by intraperitoneal injection of pentobarbital (150 mg/kg) after 72 h. at autopsy, organs are inspected for gross abnormalities, including evidence of traumatic injuries consequent to cannulation or precordial compression.,实验程序,measurements aortic, and right-atrial pressures, electrocardiographic tracings, and etco2 are continuously recorded on a pc-based data-acquisition system supported by codas software (dataq, akron, oh). cpp is calculated as the difference between decompression diastolic aortic and time-coincident right-atrial pressure measured at the end of each minute of precordial compression. b