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WINTER 2013 UCSD Phar/SOM 240/ SPPS 260 LABORATORY EXERCISE MANUAL©

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Page 1: Pharma mannual

WINTER 2013

UCSD

Phar/SOM 240/ SPPS 260

LABORATORY EXERCISE MANUAL©

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INTRODUCTION The teaching of Pharmacology at most American and European medical schools has long utilized a live preparation in order to facilitate an understanding of the clinical relevancy of autonomic and autocoid agents and pharmaceuticals designed to interfere with or mimic these agents. The Pharmacology Canine laboratory has been an integral part of the teaching of Pharmacology (and Physiology) since the inception of the medical school in 1968, as has the Physiology Cardiology laboratory. These two laboratories have, since 1968, been designed to be complementary to each other in the learning process, and while some procedures may appear similar, in fact the teaching aspects of the laboratories are quite different. The Pharmacology faculty, both PhDs and MDs, believe that no textbook or computer program can, at this time, substitute for the experiences gained from the laboratory. The Pharmacology laboratory is not designed merely to illustrate drug responses (although visualization and direct experience is still the primary method by which physicians learn the "art" of medicine and particularly of diagnosis). Rather, this laboratory has, from its inception, been designed as a clinical problem solving exercise. Almost all of the agents used in the laboratory underlie the symptomatology associated with most diseases that afflict mankind. Rather than presenting you with a diseased preparation, such as would be encountered in the clinical situation, we utilize as unknown, agents that cause the symptoms of disorders. In some cases the unknown agent is an antagonist of an endogenous autonomic or autocoid system, in other cases the unknown is an agonist. Nevertheless, for each agent the symptoms of various disorders can be linked to excessive or reduced action of one or more of the agents used in this laboratory. In 1997 the Department of Anesthesiology joined with the Department of Pharmacology in the teaching and organization of this laboratory. Almost all of the pharmacological systems which are used in the pharmacology laboratory are encountered on a daily basis by practicing anesthesiologists. In addition, and building on the cardiovascular physiology you have learned in the cardiology canine laboratory, clinical anesthesiologists have brought to this laboratory direct, hands-on clinical experiences associated with the acute care setting, specifically the application of PEEP (positive end expiratory pressure) and exposure to both its clinical advantages and potential disadvantages. Understanding when to apply PEEP and its effects are important lessons that will be immediately evident when you reach the wards. The clinical anesthesiology faculty bring to your experiences the opportunity to appreciate how endogenous agents affect both pulmonary perfusion (and thereby ventilation) and systemic hemodynamics, and thereby how symptoms of some disorders express themselves in ventilatory and hemodynamic observables. The practice of Anesthesiology, as a clinical specialty in the acute setting, utilizes many of the basic and clinical principles of pharmacology, especially autonomic, cardiovascular and pulmonary pharmacology, in addition to the management of surgical pain. While the initial participants in the course were primarily medical students, in 2005-2006, there was an increased participation by graduate students and by pharmacy students.

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The experiences gained in these laboratories are, we believe, are an important opportunity in education. Many of our alumni have returned to tell us that these first year laboratories provided some of the most important clinically relevant learning experiences in the first two years of medical school. For the pharmacy students and graduate students, the course is significant as it emphasizes the role of large organ system models in research and drug development and approval. In 2012, with the capabilities provided by the new Telemedicine facilities, the move to the porcine model, initiated several years earlier was completed. General comments: Since this laboratory is a clinical experience, we require that you treat the preparation as if you would treat a human patient. Have respect for the animal and conduct the monitoring of the preparation throughout the laboratory as we have instructed you to do. This will require one member of each team to always be monitoring the preparation, e.g., measure and record body temperature, anesthetic administered, depth of anesthesia, degree of blood oxygenation, etc. Misadventures can happen, as they do in the clinical setting with patients, and since these laboratories are, for most of you, your first real experience in a surgical setting where acute pharmacology is practiced, and decisions must be made in "real time", observe and be involved in all aspects of the laboratory so that you will minimize any mistakes later. This laboratory is also of considerable value in observing biological variation since animal preparations, like patients, do not show identity of responses. Compare the responses of your preparation with those of colleagues from other tables. Every student should participate in every part of the laboratory: insertion and placement of the Swan-Ganz catheter for wedge pressure, measurement of cardiac output, determination of cardiovascular observables - pressures and heart rate, monitoring of the status of the preparation, agent administration, recording actions of the team, determining baroreflex function, etc. The faculty, clinical and preclinical, laboratory staff and veterinarians are all present to help make this a true learning experience. The laboratory may not be the most comfortable experience you have, but what you learn during this laboratory will -- we are quite certain – provide the student with insights and experiences that will stand them importantly in their future career in research and medical practice. Take full advantage of the opportunity and feel free to ask questions of the faculty and staff present.

OBJECTIVES OF THE LABORATORY The primary objectives of the Pharmacology & Anesthesiology Laboratory are the following: 1. To emphasize and discuss principles of autonomic and autocoid pharmacology with an

emphasis on the cardiovascular, pulmonary and gastrointestinal systems and the receptors mediating observed clinical responses, in a setting analogous to that found in an acute care environment.

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2. To implement and observe the effects of positive end expiratory pressure on cardiac output and

venous P02. 3. To evaluate and differentiate the effects of known endogenous agents on cardiovascular

hemodynamics, cardiac output, heart rate, and high-pressure baroreceptor activation or inhibition.

4. To use your powers of observation and deduction in a clinical problem solving exercise

to identify two "unknown" agents and to understand why and how they yield the physiological effects observed. 5. It is essential that you read and prepare for this exer e. Many procedures and observations will be undertaken and lack of prior understanding will mean that you will diminish the impact of the experience for your self and for your fellow students. Moreover, failure to prepare is inexcusable as it represents a lack of respect for the animal and for the effort put forth to provide you with this experience. Read through the 5 components of this laboratory protocol and consider any questions posed in the syllabus before you get to the laboratory. If you have questions, write them in your notebook for discussion during or following the laboratory. A review meeting will be held at the class meeting on the following meeting of the class session. Here each group will present the answers or display the graphs based on the data that they have obtained. Be prepared to present at this time.

The five parts are as follows: a. Preparation and clinical maintenance and oversight of the subject. b. Examination of the clinical effects of PEEP especially effects on cardiac output.

c. Assessment of baroreflex status prior to pharmacological blockade. d. Evaluation and assessment of autonomic agents in the acute care setting. e. Test of identification of unknown autonomic or autocoid.

6. Complete in advance the "Observation Form - Expected Responses "page (p21) indicating "predicted" direction of change and magnitude (we recommend your using 1, 2 or 3 arrows for magnitude of response and direction) -- see back of protocol. There is a second copy for use during the laboratory.

7 . Turn in 1 report per group (table) of your findings from the two unknowns assigned your

group with an explanation of your conclusions. All students must sign off on the conclusions. Prepare to discuss your results in the class discussion at the end of the laboratory.

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GENERAL INFORMATION 1. Calibration of the pressure gauges Two pressure gauges are used during this laboratory - for systemic femoral artery pressure, and pulmonary artery wedge pressures. These systems are similar to those used in most intensive care units. The transducer is essentially a very thin metal diaphragm that is deformed by pressure exerted on the diaphragm through a fluid column. Thus, one side of the diaphragm is "hydraulically coupled" by the catheter to the fluid column (blood stream or water). On the other side of the

diaphragm are "bonded piezoelectric devices" that change their electrical resistance when deformed. The electric signals from these devices are amplified and put through a "bridge balancing circuit" which results in electrical signals that displace the pen proportionately on the strip chart recorder. The sensitivity and range can be adjusted to maximize the signal and pen displacement. In general calibration will have been accomplished prior to the laboratory. .

• Flush all air from the transducer housings. Air bubbles in the fluid column act as absorbers of the pressure waves and dampen out the effect being exerted on the diaphragm.

• Fill the catheters/stopcocks with heparinized saline. Make certain the stopcock is closed to

the catheter. • Make sure you know how to read the recorder, the scale on the chart

2. Data monitor Different recording systems may be employed. However, the monitors provide a continuous ongoing read of the BP, HR as well as endtidal gases. Generally the data are recorded when the machine is activated to pvodie astrip of hear ate or BP with the other vital signs being printed on the edge of the strip. The strip is typically run briefly (5 -10 sec prior to a treatment and allowed to run continuously until the effect has resided. Always get a control pre-injection tracing. Record pre-injection values and maximum (or minimum) changes at peak response. 3. Drug Delivery You will administer pharmacological agents in three ways during this laboratory: 1. Bolus injection: All drugs are diluted so that the animal's weight (kg) x 0.1 = dose (in ml). The bolus is injected into the venous catheter, followed immediately by a flush with 5 ml of

saline (not heparinized saline) through the other port on the venous

catheter's stopcock. This insures total and rapid delivery of the drug into the systemic venous return to the heart. 2. Staggered partial injection: This method approximates a slow infusion of the drug and is used for agents that have "green labels". The calculated dose is divided into 4-5 doses with each individual dose administered 1/minute each followed by a 5-6 ml saline flush. It is essential that each 1/4 - 1/5 of the dose is followed by a flush of saline, otherwise the drug accumulates in the catheter and is administered as a bolus with the final fraction of dose. 3. Subcutaneous injection: On a ventral surface free of hair (or shaved such as the abdomen or femoral triangle) a small bolus of the drug is administered immediately below the skin. The volume of drug administered in this manner is usually less than 1 ml. 4. Infusion: In this case, the drug is diluted into a saline bag and administered via a drip into the venous or pulmonary artery.

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PRESTUDY - PREPARATION Staff surgical preparation Prior to your arrival in the laboratory, the clinical staff will have anesthetized the pig with propofol delivered though an ear vein (10 mg/kg). The pig will be intubated and then placed on a closed circuit pressure regulated ventilator with a vaporizer delivering isoflurane at approximately1-1.2%. Your initial clinical assessment of the preparation: Each table team of students will meet with the instructors assigned to their table and review the status of the preparation. • Check on the state of arousal as instructed during the initial briefing and check on the eye's blink reflex.

Be alert for signs of the pig shivering or any spontaneous movement or resisting the respirator (e.g. active diaphragmatic movements).

• Compress the ankle between the digits of the paw briskly between thumb and forefinger and look for evidence of limb retraction.

• Blink reflex: Touch the lid or cornea lightly with a cotton wisp or stiff suture to evoke lid movement. See Appendix 4

If you feel the animal needs more anesthetic any time during this session, contact one of the staff or the Veterinarian.

• Check that the preparation is adequately ventilated based on color of the oral mucosa. • Feel for the femoral pulse and determine and record baseline heart rate from the pulse. • Review the recorder setup, and determine and record either from the EKG profiles or pulsatile pressure

waveforms baseline heart rate and systolic and diastolic pressures. • The animals will have a pulse oximeter on the ear. recording O2 saturation (and heart rate). • Record temperature • Place hand between animal and heating pad to make certain the pad is not overly hot to the touch. • At this time, one member of each group is designated to record blood pressure and body temperature at 15

in intervals. This responsibility should rotate every hour or so. This information is charted on a form provided to each table. The time and dose of supplemental anesthetic must be recorded on this sheet.

• Keep the animal covered as much as possible with surgical drapes to conserve body heat. An underbody heating pad is typically turned on all of the time. Care should be taken to insure that the animal does not suffer burns from the heating pad. This likelihood can occur when the mats between the animal and the heating pad become damp. The observer can assess this risk by simply placing their hand between the animal and the pad.

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ANESTHESIA The pigs will be induced by the staff with propofol. This initial induction permits the animals to be stable for a period of up to 10-20 min. and permit time for the initial intubation and equilibration with the gaseous anesthetic.. During this initial phase the animal will be monitored regularly (every 10 min) for depth of anesthesia by examining pupil size, blood pressure and respiratory effort. Under adequate anesthesia, the pupils will be constricted, the pulse and BP stable. The animal is being positively ventilated, but efforts to breath spontaneously (e.g “bucking” the respirator) is a sign of anesthetic lightening. SURGICAL PROCEDURES Overview: The preparation is a closed chest, anesthetized pig with the right external jugular vein and femoral vessels cannulated. You will be responsible for placing arterial and venous catheters in the femoral vessels and placing a Swan-Ganz thermodilution catheter -- via the right external jugular vein -- advancing through the right atrium and ventricle and into the pulmonary artery to obtain both cardiac output and wedge pressures during the designated procedures. Femoral vein cannulation Purpose: An intravenous route such as this must be established to provide easy and rapid access for further administration of anesthetic, fluids or drugs as required. • Inspect and prepare the femoral vein catheter with a

stopcock. Make sure the catheter is filled with heparinized saline and that the stopcock is closed off towards the catheter end.

• Next, locate the femoral triangle (recall the procedure used in the cardiology laboratory for locating the incision site).

• Make an approximate 3-inch incision over the artery extending from the inguinal ligament along the ventral surface of the leg.

• Use "blunt dissection" with blunt forceps and Mayo scissors and carefully dissect down to the muscle and aponeurosis layer. Incise the aponeurosis at the muscular junction and below should lie the femoral vein, artery and nerve. Work carefully to avoid damage to the nerve. Incise the sheath by blunt dissection spreading parallel to the vessels.

• Clean at least a 2 cm segment of both artery and vein -

note that there may be branches of both vessels, especially in female preparations. Take care not to tear these branches.

• Place loose sutures both proximally (towards the heart) and distally (away from the heart) on both artery and vein. Place the sutures for the artery lying to one side away from the field of work and turn first to the vein. It is normally best to insert the vein catheter first to insure you have a port for fluid administration should there be bleeding; however, a port already exists through the jugular vein.)

• Turning your attention to the vein, ligate the vein distally (making sure the ends of the suture are sufficiently long to tie down the catheter).

• While maintaining tension on the proximal tie to minimize back-bleeding, make a small cut with the Mayo scissors. (The incision should not be too close to the proximal tie since you want to have some flexibility in surgical approach should

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another incision be necessary.) • Carefully advance the femoral catheter past the

"tensioned" ligature (tension needs to be released as you advance past the ligature) into the vein through the incision. (Before inserting the catheter you should make sure that the incision is into the lumen - by inspecting for the glistening white intimal surface.)

• Advance the catheter about 6 cm into the vessel

towards the heart and tie the proximal ligature securely over the vein and cannula.

• Secure the cannula with a second tie using the ends of the suture on the distal end of the vessel.

• Withdraw some blood to insure patency and then flush with heparinzed saline.

Femoral artery cannulation • Cannulate the femoral artery in a similar manner. • It is essential that the femoral artery be well exposed

so that the incision into the artery can be made a good distance 2-3 cm) from the proximal ligature.

• First, tie down the distal ligature leaving sufficient suture to anchor the distal end of the catheter.

• Place tension on the proximal ligature to control the 150 mm of Hg pressure head. (Too much tension can tear the artery after it is cut. The artery is strong but not infinitely so.

• Make a small cut with the Mayo scissors near the distal ligature. (Again, check that the incision is into the lumen)

• While holding "strong" tension on the proximal ligature, advance the catheter up to the "tensioned" ligature. Using a smooth motion, release some tension on the proximal catheter and advance the catheter past the ligature and up at least 10 cm into the artery. In this manner, the catheter tip can be advanced well into the femoral artery prior to loosening the proximal ligature leading to minimal blood loss.

• Now, another student should move in and hold the catheter to prevent the pressure from "blowing it back out", while the first student ties down the proximal ligature around the catheter.

• (The artery catheter must be tied tightly - double-knotted using a reverse knot placement - to insure it does not slip back out) Immediately tie the distal ligature to anchor the arterial catheter. You should be able to see the pulse waves in the heparinized saline.

• Now, inspect the pressure transducer. The arterial pressure gauge is calibrated for 0-200 mmHg (5

mmHg/division). It should be filled with fluid and, most importantly, there should be no air bubbles that would dampen out the pulsatile pressure waveforms.

• Connect the transducer. Attach a syringe filled with heparinized saline onto the stopcock proximal to the transducer (assuming there are now 2 stopcocks in series - one originally on the catheter and the second originally on the transducer).

• With access to the transducer shut off (the syringe is "pathed" to the catheter stopcock) open the path from the artery to the syringe. You can now check that you have arterial pressure by pulling back slowly on the syringe permitting blood to enter the catheter.

• Then, clear the blood from the catheter with 5 - 7 ml of heparinized saline. Open the "path" from the artery to the pressure transducer by switching the syringe stopcock. Immediately inspect for leaks (blood flowing back into the catheter, leaky connection, faulty pressure transducer, faulty stopcock, etc...problems happen!). Check the tracing on the recorder. There should be a strong pulse pressure.

• Now, if everything is okay - wet some gauze with saline, pack gently the femoral triangle area, leaving access to the catheters, and drape the preparation.

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YOU WILL BE MONITORING FEMORAL ARTERY PRESSURE

Connection of ECG leads The OLR staff will have connected four ECG leads to each of the animal's upper and lower extremities. Check and monitor Lead II, III or AVF for the duration of the laboratory. Identify P-wave, QRS complex and T-wave in the lead selected. Insure that the leads do not get disconnected.

YOU WILL BE MONITORING HEART RATE AND CARDIAC RHYTHM Insertion of the Swan-Ganz catheter: You may have seen the placement of Swan-Ganz catheter previously. Here, every student should take the opportunity to float the catheter. This is a procedure that is frequently performed on humans.

• First, inspect and review the multi-lumen catheter. • Check that you understand what are all the ports and especially which lumens and ports will

be used during today's laboratory. Insertion of the Swan-Ganz catheter:

• Place stop cocks on all lumens and fill all ports with heparinized saline. • Test the Swan-Ganz balloon for leaks (inflate balloon with no more than 1.5-ml air using a special syringe. before inserting the catheter. • Connect the disal lumen catheter to the low pressure transducer. The Swan-Ganz gauge is calibrated for 0-40 mmHg for 40 divisions (1 mm Hg/division). The transucer should be approximately at the level of the heart. • Check the zero calibration on the pressure transducer by opening it up to atmospheric pressure. This is zero pressure. • Identify the right jugular. Perform a cut down in the neck and dissect a 2cm length of jugular. Pace ligatures proximal and distal. Retract the proximal ligature and then tie off the jugular dista l(from the heart).

• A small incision is made though which the catheter is passed.

• Insert the heparin-saline primed Swan-Ganz catheter into the jugular vein with the balloon collapsed in the fashion described above for the femoral vein Once

inserted tie the proximal ligature loosely so that the catheter is able to moved in and out with out leakage.

• When in the superior vena cava, inflate the balloon with 1.5 cc of air and continue to advance it until it lies in the right ventricle. NEVER INFLATE THE BALLOON WITH MORE THAN 1.5 CC OF AIR. A special syringe is available that precluded over filling.

• Advance the catheter through the right atrium and into the right ventricle by monitoring pressure waveforms though the distal port. How do you know when you have entered the right ventricle

• Continue on and float (advance) the catheter into the pulmonary artery. How do you know when you have entered the pulmonary artery?

• With the balloon inflated, entry into the pulmonary artery is evidenced by a marked

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change in wave form, The average pressure drops indicating that the catheter is "wedged" and that a pulmonary capillary wedge (PCW) pressure is being recorded.

• Once wedge pressures have been examined, deflate the

balloon, back off the catheter slightly so it rests within the pulmonary artery and flush the catheter periodically with heparinized saline to maintain patency.

YOU WILL MEASURE RIGHT ATRIAL (RA) PRESSURE (The pressure you see just before you enter the right ventricle). RIGHT VENTRICLULAR PRESSURE (RV), PULMONARY ARTERY (PA) AND PULMONARY CAPILLARY WEDGE(PCW) PRESSURE.

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EXPERIMENTAL PROTOCOLS: There are 5 components to this laboratory: 1. Preparation of Pig 2. PEEP 3. Baroreceptor reflex assessment 4. Autonomic agents 5. Unknown agents. All tables will do all parts. Some tables will do PEEP first and Baroreceptor function second, and the other tables will do the two in reverse order. I. MEASUREMENT OF CARDIAC OUTPUT AND HEMODYNAMIC EFFECTS OF PEEP Measurement of cardiac output by thermal dilution: You will estimate cardiac output (CO) by following the time course of temperature drop of the blood (measured by the terminal thermistor in the catheter) when 5 ml of ice cold (4 degrees C) saline is rapidly injected into the chamber upstream of the thermistor. Procedure for cardiac output measurement An ice bucket has been placed at each table with several 5-ml syringes and a beaker of ice cold saline. The syringes are filled and kept in ice to keep them cold. First, review with your instructor which port you will inject the saline into and where the thermal sensor (thermistor) is located. You should have found this out in the earlier review of the multi-lumen Swan-Ganz catheter. The measurement requires coordination among up to 3 students - one injecting,

one providing cold syringes and activating the monitoring device, one recording data on the chart recorder. • When the syringe is in place on the Prox. Injectate port, , the "activator" (with their finger on the machine start button) quickly counts down from 3, pushes the start button and the injector administers (with a strong push injection) the 5-ml ice-cold saline. The machine then goes into a sequence of operations that lead to a display CO - This procedure is immediately repeated up to two more times to get a stable and reproducible CO.

Note : Concept behind CO measurement: Since heat loss of the blood must equal heat gain of the cold saline and if we assume that passage from one cardiac chamber to the next assures mixing, then, the product of the mean temperature drop and the time over which the temperature drops and return to 37 degrees occurs, represents the area under a temperature vs. time curve. This area can yield an average value (temp drop) over a known time, and can be used to calculate how much the original saline must have been diluted in that time. Correcting to 1 minute gives CO (volume/min). You will employ an electrical device that will integrate the area under this curve automatically and calculate how much blood must have been cooled by the 5 ml of iced saline giving you directly cardiac output. Who were Swan and Ganz?

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Effects of peep on cardiac output and venous O2 Control values and Control blood gas samples • Obtain control values of systolic, diastolic and mean arterial pressure, heart rate, pulmonary artery

pressure and cardiac output. Obtain three values for cardiac output and average them. The three values should be within a total range of about 40%. Calculate pulmonary and systemic vascular resistance (PVR, SVR).

• Take both an "arterial" and a "mixed venous" blood samples from the femoral artery and Swan-Ganz (balloon deflated) catheters, respectively, at the same time. Remove any bubbles, cap and label the syringes (A1 and V1 and add your table #), and store them on ice until they can be taken for 02 analysis.

Note: Blood is withdrawn from the catheters until a 'fresh" sample is at the tip of the catheter. The desired blood samples are obtained in the specific syringes, immediately capped and placed on ice, and the withdrawn blood re injected and catheters flushed with heparinized saline. • NEXT, increase the PEEP on the expiratory port of the ventilator from the control level to

15-cm H20. Note: End expiratory pressure is increased by PLACING A CALIBRATED BALL VALVE INTO THE VENTILATER OUT LET. • Mark on the chart recorder when PEEP was commenced. Monitor the recorder and observe changes in

arterial and right heart pressures. • After a new steady state (ca. 5-min) is achieved, take a second mixed venous blood sample for 02 analysis

(label it V2, table #). • Immediately, repeat triplicate measurements of cardiac output with the thermal dilution apparatus. • Return PEEP to the control levels. Do not maintain PEEP for prolonged periods of time. Additionally,

monitor the oxygenation of your preparation through oral mucosa color. Calculate : 6. Systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). (See Appendix 1 at

end of handout for worksheet.) 7. Oxygen consumption. (See Appendix 2 at end of handout for worksheet.)

QUESTIONS TO BE ANSWERED & DISCUSSED IN THIS PHASE OF THE LABORATORY: 1. Using the Fick equation, calculate your pig’s oxygen consumption. 2. What happened to blood pressures and cardiac output when PEEP was increased and why?

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Questions continued: 3. What was the effect of PEEP on mixed-venous 02 content? Why? 4. What were the pulmonary vascular resistance and systemic vascular resistance? How does it compare with the standard values for this preparation? What factors might contribute to differences? II. MEASUREMENT OF BARORECEPTOR REFLEX In this protocol, you will determine the gain of the high pressure baroreceptor reflex

• You will be injecting two agents that both

raise and lower arterial pressure. For transient acting agents, and in the absence of specific blockers, the baroreceptor response will adjust heart rate accordingly. You will develop a baroreflex curve by plotting Heart Rate (BPM) versus MAP. It is important that one uses "pure" vasodilators or vasoconstrictors rather than agents that can have a direct effect on heart rate.

• The "unloading" limb of the baroreflex curve is obtained injecting the 10 mL syringe by hand at the rate of approximately 1 mL / 10 sec of nitroprusside. Continue the injection until

the mean arterial blood pressure falls by about 30-50 mm under baseline (or approximately 60 mm MAP) which ever happens first. Then terminate injection. This will provide the "unloading" limb.

• Measure cardiac output at this time. • Allow the blood pressure to return to baseline. The

"loading" limb of the baroreflex curve is obtained injecting the 10 mL syringe by hand at the rate of approximately 1 mL / 10 sec of phenylephrine. Continue the injection until the mean arterial blood pressure rises by about 50 mm over baseline (or 200 mm) which ever happens first. Then terminate injection.

• Measure cardiac output at this time.

Calculate: • Using Work Sheet in Appendix 3, plot heart rate against the mean arterial pressure

(MAP). We can thus estimate the "gain" of the baroreflex.

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QUESTIONS TO BE ANSWERED & DISCUSSED IN THIS PHASE OF THE LABORATORY: Q: Does the "return" phase of the MAP - HR relationship track the initial loading phase? Q: Which receptors are being activated by phenylephrine and which signaling pathway leads to the observed direct response and the indirect response? What about sodium nitroprusside? Q: Discuss effect of lowering and raising peripheral vascular resistance on cardiac out put. Q: What is a normal baroreceptor reflex and what is a "blunted" baroreceptor reflex? What would be the effect of a blunted baroreflex on the response to (a) a sympathomimetic drug and (b) a vasodilator?

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III. PHARMACOLOGIC EFFECTS OF AUTONOMIC & AUTOCOIDS You will want to observe all the organism responses to each agent. Monitor respiration, cardiovascular hemodynamics (BP & HR), signs of skeletal muscle activity, gastrointestinal activity, cardiac activity (EKG) and any other parameters available to you. Inject each [dose (in ml) = wgt (in kg) x 0.1] rapidly and flush in with 5 m l of saline. Observe the full time course of the effect at a moderate paper speed, collecting a few seconds of high-speed recording at the peak response. Allow for full recovery between injections (about 4 min). ACETYLCHOLINE: Inject a low dose of acetylcholine as a bolus. At the beginning of the response run the recorder at 10 mm/sec and at the time of maximal response, run the recorder at 25 mm/sec but only for 2 - 4 seconds. Measure the changes in RR and PR interval occurring early and late during the response. Q: What receptors at what locus have been occupied to produce the change in blood pressure? Q: What is/are the origin(s) of any changes in heart rate? REPEAT FOR HIGH DOSE ACETYLCHOLINE. At the beginning of the response and at the time of maximal response, run the recorder at 25 mm/sec for a few seconds. Measure the changes in RR and PR interval occurring early and late during the response. Q: What are two ionic mechanisms of action of acetylcholine on AV nodal tissue? How do they explain conduction slowing and block? Q: How would acetylcholine influence automaticity as well as conduction? Q: What factors influence the duration of the effects of ACh?

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ISOPROTERENOL: With the recorder running at 10 mm/sec, administer isoproterenol intravenously. When you have achieved a peak response, get 2 - 4 sec at 25 mm/sec. At peak response, measure cardiac output by thermodilution. Q: What receptors are being activated in the heart and vasculature? Q: Which responses are direct and which are indirect? What about reflexes? Q: What are the ionic mechanisms of action of isoproterenol on AV nodal tissue? On the SA node? and on the RR and PR intervals? Q: How would isoproterenol influence automaticity as well as conduction? Note the duration of the response. Q: Would you expect pulmonary airway effects and did you observe any? NOREPINEPHRINE: With the recorder running at 10 mm/sec, administer the agent intravenously. At peak response measure cardiac output by thermodilution. Q: Which receptors are being activated in the heart and vasculature? Q: Which responses are direct and which are indirect? Q: Can you detect a cardiac output contribution to the systemic arterial pressure? Q. What are the ionic mechanisms of action of norepinephrine on AV nodal tissue, on the SA node and the RR and PR intervals?

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EPINEPHRINE: With the recorder running at 10 mm/sec, administer the agent intravenously. At peak response measure cardiac output by thermodilution. Q: What receptors are activated to explain the responses observed? Compare with responses to isoproterenol and norepinephrine. Q: Which portions of the responses are direct? Which are indirect (reflex) responses? Q: What accounts for the different durations of action of these catecholamines? ANGIOTENSIN II: With the recorder running at 10 mm/sec, administer the agent intravenously. At peak response measure cardiac output by thermodilution. You may observe more than one phase of peak response, which will be noted most readily if you do not run the paper too fast. Q: At what receptor(s) does Ang II act? Which receptors are you likely observing responses for? Q: Why are its responses so prolonged? Q: If there is more than one phase to the pressor response, can you explain the origin of the second phase? How might you test your hypothesis in the laboratory today? Q: How is the action of angiotensin II terminated? Q: Vasoconstrictor agents can release NO, did you observe any being released? What might you predict would be observable - if you could observe it?

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SEROTONIN: Review the expected responses carefully before injecting this agent. Prior to injecting the drug obtain a baseline cardiac output. With the recorder running at 10 mm/sec, administer the agent intravenously. Q: Explain the observed blood pressure and heart rate responses? What is this reflex? Q: Was there an observable effect on gastrointestinal motility? If so, how do you explain it? Q: Was there any evident effects on superficial (skin) blood flow? HISTAMINE: Review the expected responses carefully before injecting this agent. Prior to injecting the drug obtain a baseline cardiac output. With the recorder running at 10 mm/sec, administer the agent intravenously. Q: Explain the observed blood pressure and heart rate responses? What receptor subtypes are involved? Q: Was there an observable effect on gastrointestinal motility? Q: What are the physiologic sites of histamine synthesis, storage and release? Q: What prominent and physiologically important effects of histamine are we not assessing? TRIAD SUBCUTANEOUS INJECTIONS: Get bradykinin, serotonin and histamine and inject subcutaneously 0.2 ml of each in the same area of the abdomen - at least 2 inches apart. Q: Compare the initial, mid and terminal phases of the observed responses. What do you expect to see and how do you explain the similarities or differences among the three agents?

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VASOPRESSIN: Review the expected responses carefully before injecting this agent. Prior to injecting the drug obtain a baseline cardiac output. With the recorder running at 10 mm/sec, administer the agent intravenously. Q: Explain the observed blood pressure and heart rate responses? What is this reflex? Q: There has been an increasing emphasis upon the use of Vasopressin in cardiac resusitation. How do its actions differ from epinephrine. Are these differences favorable as compared to those produced by epinephrine. IV. IDENTIFICATION OF UNKNOWN AUTONOMIC & AUTACOID AGENTS Using your knowledge of autonomic pharmacology and physiology, autacoid pharmacology and physiology and your capacity to inject known agonists and antagonists, you must now identify two unknown agents. The first unknown will be an agonist, the second is an antagonist. Drugs in this section will be injected via the femoral venous cannula. REMEMBER: DO ANTAGONIST LAST!!!!! DO NOT BOLUS ADMINISTER ANY AGENT WITH A GREEN LABEL!!!! EXPERIMENTAL PROTOCOL: 1. Inject the first unknown agent, an agonist, as a bolus. Observe the CV and GI responses carefully. Can you make any educated guesses about the class or identity of the unknown? 2. Using your knowledge of cardiovascular pharmacology and available agonists and antagonists (see list below) identify the first unknown. You should avoid using a specific antagonist to test your hypothesis until you have injected your second unknown, which is an antagonist. You will be expected to achieve identification by mimicry, by pharmacologic blockade, if possible, and by comparison with and elimination of agents with similar actions. You must present the evidence to support your interpretation of the unknown agent. Think of the responses you are observing as symptoms and try to deduce which of the agents below would, in pathological excess, cause these symptoms. 3. Repeat for your second unknown, an antagonist, but inject the antagonist slowly (in divided doses over several minutes). The antagonists are not metabolized as rapidly as the physiologic agonists and their congeners; these antagonists have half-lives of hours rather than minutes. The unknowns will be chosen from the following agents, all of which are available to you as knowns (at the

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center table): AGONISTS epinephrine norepinephrine isoproterenol phenylephrine acetylcholine methacholine histamine nicotine angiotensin II bradykinin serotonin methoxamine ANTAGONISTS phentolamine atropine hexamethonium propranolol cocaine neostigmine diphenhydramine metoprolol succinylcholine

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FORM FOR PREDICTED RESPONSES Agent Action Systolic Diastolic Mean dP/dt GI Secretory Skeletal Press Press Art Press motility activity muscle acetylcholine methacholine . nicotine atropine neostigmine Sucynlcholine norepinephrine epinephrine isoproterenol . histamine serotonin phenylephrine . cocaine bradykinin angiotensin II . phentolamine propranolol diphenhydramine adenosine nitroprusside Vasopressin .

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CANINE LABORATORY DATE: GROUP: ____________ ___ FORM FOR RECORDING OBSERVED UNKNOWN RESPONSES

Agent Action Systolic Pressure

Diastolic Pressure

MeanArt press

dP/dt GI Motility

Secretory activity

Skeletal muscle

Unknown #1

Unknown #2

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SVR / PVR Following formulae will, with data available from the arterial line, the pulmonary artery catheter and Swan-Ganz, allow calculation of resistance of the peripheral (systemic) vascular bed (SVR) and pulmonary vascular bed (PVR).

ABBREVIATION / SOURCE OF MEASUREMENTS

CO = cardiac output: Swan-Ganz-thermal dilution PCWP = pulmonary capillary wedge pressure: Swan Ganz

CVP = central venous pressure: Swan Ganz tip prior to RV insertion. MAP = mean arterial pressure: Arterial line MPA = pulmonary artery pressure: Distal lumen of Swan-unwedged. (Note: Mean pressures ≈ diastolic pressure + 1/3 pulse pressure) Normal PIG values: Systemic VR 1500 dynes-sec cm5 Pulmonary VR 50 dynes-sec cm5

Calculating Oxygen Consumption Following formulae will permit estimation of consumption of O2 consumption in mls / min given CO and arterial and venous O2 content.

x 80 = _______dynes-sec/cm5

CO = MAP - CVP → SVR = MAP - CVP SVR CO

x 80 = _______dynes-sec/cm5

CO = MPA - PCWP → SVR = MPA - PCWP PVR CO

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Arterial O2 content is based on femoral arterial blood sample Venous O2 is based on femoral arterial sample.

[ ] [ ]22

min)/(2min)/(VOAOmlnconsumptioOLCO

!=

Where: [AO2] = arterial O2 content in ml/L [VO2] = venous O2 content in ml/L

To calculate O2 content of arterial [AO2] or venous [VO2] blood

Blood O2 content in ml/dl = 1.39 ml/g x Hgb g/dl x %sat.Hgb (/100) + 0.003 ml/dl x PO2 Data from blood gas report Hgb: Hemoglobin (g/dl) %sat.Hgb: % saturation (sO2) (express as decimal) PO2 : Oxygen saturation (mmHg)

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Appendix 3: Baroreceptor loading and unloading 1. Take polygraph trace during nitroprusside and then phenylephrine infusion and mark a line though the BP trace and the EKG trace at intervals corresponding to approximately 10 mmHg changes in Mean BP. Run it fast enough to see hear beats to permit beat to beat rate calculation. Note a simpler method is to take the reading off of the pulse oximeter. Set recorder to indicate “mean” (or “AVG”) to give the mean e.g. [2/3(Sys-Dia) + Dia]. The adjacent schematic shows a cartoon for the nitroprusside and phenylephrine parts of the study. 2. Count number of heartbeats in 10 sec interval around vertical line and calculate beats /min. Fill in table below. 3. Plot HR vs BP on the following graph. Slope of line indicates gain of baroreceptor reflex. Note that you will have two tracings, the one comprised of data generated during the infusion of either agent and the second composed of data derived from the recovery after each injection. Data Analysis Table Nitroprusside (Unloading baroreceptor)

Pre | Nitroprusside Infusion → | Recovery → BP HR

Phenylephrine (Loading baroreceptor)

Pre | Phenylephrine Infusion → | Recovery → BP HR

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Appendix 4: COMMENTS ON ANESTHETIC DEPTH. Surgical interventions, such as those employed in the present series of studies are under taken in the "anesthetized" animal. The anesthetic state is not absolute but is a graded condition. Different anesthetics show different profiles of effect. In the present work, we are using sodium pentobarbital, a barbiturate with a relatively long half-life (approximately 1.5 hrs). This allows the titration of a relatively stable depth of effect with single (repeated) bolus injections. It is of importance to note that the anesthetic will wane during the course of a period of 4-6 hrs and there will be the need to supplement as signs of lightening of the anesthetic state are observed. While individual differences in the sensitivity of the animal or human may exist with respect to the specific dose, increasing blood levels will be associated with the disappearance of specific components of the functionality of the organism.

• Normal alerting response to the environment, normal organized motor function. • In-coordinated motor response to a strong stimulus. • Loss of motor tone / Loss of consciousness. • Loss of response to ongoing non noxious stimulation • Loss of the blink reflex (evoked by lightly touching the eye lid or the cornea ) • Loss of response to a surgical stimulus stimuli applied to the skin or viscera • Loss of spontaneous respiratory movements. • Iso-electric EEGs.

When the depth of anesthesia is appropriate, the organism (pig or human) will show no organized response to even an intense surgical stimulus, but retain the predominant components of autonomic function (e.g. baroreceptor reflexes). The present studies are carried out in just such a state. As outlined above, as the anesthetic is cleared , the animal shows a progressive reappearance of these signs. Thus, periodic monitoring of the animal by assessing the presence of the blink reflex, the presence of increased muscle tone in response to the pinching of the toe, are important indicators of the depth of the anesthetic state of the animal. If these reappear, the animal is still in an adequately anesthetized state, e.g. it is still unconscious, but it serves as a sign signaling the need for additional anesthetic in the next 10-20 min. it should be stressed that as long as these signs are followed and their absence verified, it can be safely assured in humans and animals that there is an adequate anesthetic state.

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GROUP SUMMARY REPORT: Group Number: ________________ Laboratory Date: ________________ Report Date: ______________ Date report received in Pharmacology Department Office: ______________ Name of Clinical Teaching Fellow assisting: ________________________________ Name of Pre-Clinical Teaching Fellow assisting: ______________________________ NAMES OF GROUP MEMBERS SIGNATURES UNKNOWN #1 CODE NUMBER: ________________

YOUR IDENTIFICATION: ________________

UNKNOWN #2 CODE NUMBER: ________________

YOUR IDENTIFICATION: ________________

Briefly and systematically discuss the sequence of steps leading to the identification of Unknowns #1 and #2. List the elements of the "Proof of Principle" used to conclusively establish the identity. If your conclusion was not correct, discuss briefly what contributed to the mis-identification. (Use the other side or another page as needed.)

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Facilitator Evaluation Pharmacology laboratory

Please fill out one for each facilitator at your table (normally 2)

Facilitator Name: (Anesthesiologist)__________________________Table #_____ Facilitator Name: (Pharmacologist) __________ _________ _Table #__ . Knowledge of material: NONE LOW MEDIUM HIGH Ability to communicate material: NONE LOW MEDIUM HIGH Level of interaction with student: NONE LOW MEDIUM HIGH Enthusiasm: NONE LOW MEDIUM HIGH Overall effectiveness: MARGINAL MODERATE EXCEPTIONAL SUPERIOR COMMENTS?

Please circle appropriate ranking for facilitator activity

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Facilitator Evaluation. Pharmacology laboratory:

Please fill out one for each facilitator at your table (normally 2) Facilitator Name: (Anesthesiologist)__________________________Table #_____ Facilitator Name: (Pharmacologist) __________ _________ _Table #__ . Knowledge of material: NONE LOW MEDIUM HIGH Ability to communicate material: NONE LOW MEDIUM HIGH Level of interaction with student: NONE LOW MEDIUM HIGH Enthusiasm: NONE LOW MEDIUM HIGH Overall effectiveness: MARGINAL MODERATE EXCEPTIONAL SUPERIOR COMMENTS?

Please circle appropriate ranking for facilitator activity

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AUTONOMIC LABORATORY DATA COLLECTION SHEETS DATE: ___________ Animal Number: _________ TABLE: ___________________ Students: ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Table Mentor:_________________________

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#1 Data Collection sheet: PEEP STEP 1 Pre: CO: CO CO HR: BP: PA Press_____ PAWpress_____ 2. Pre: Draw Blood Arterial (A1) /mixed venous

(V1) 3. Time: Insert PEEP VALVE 4 +2 m CO: CO CO HR: BP: 5. +5 m CO: CO CO HR: BP: 6. +5 m Draw Blood Arterial (A2)/mixed venous (V2) 7. Remove peep valve 8. +10 m CO: CO CO HR: BP: NOTE. Run chart recorder strip starting from just before step 3 to step 7 Immediately upon collection of the sample A1/V1 and later the A2/V2 sample, take to technician for blood gas measurement. COMMENTS/Observations:

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#2a BARORECEPTOR REFLEX-Unloading STEP UN-LOADING PHASE 1 Pre: CO: CO CO HR: BP: 2. TIME 0: Sodium Nitroprusside infusion..1mL/m x10m +10 s HR BP +20s HR BP +320s HR BP +30s HR BP +330s HR BP +40s HR BP +340s HR BP +50s HR BP +350s HR BP +60s HR BP +360s HR BP +70s HR BP +370s HR BP +80s HR BP +380s HR BP +90s HR BP +390s HR BP +100s HR BP +400s HR BP CO CO CO CO CO CO +110s HR BP +410s HR BP +120s HR BP +420s HR BP +130s HR BP +430s HR BP +140s HR BP +440s HR BP +150s HR BP +450s HR BP +160s HR BP +460s HR BP +170s HR BP +470s HR BP +180 HR BP +480 HR BP +190s HR BP +490s HR BP +200s HR BP +400s HR BP +210s HR BP +510s HR BP +220s HR BP +520s HR BP +230s HR BP +530s HR BP +240 HR BP +540 HR BP +250 HR BP +550 HR BP +260 HR BP +560 HR BP +270 HR BP +570 HR BP +280 HR BP +580 HR BP +290 HR BP +590 HR BP 300 HR BP +600 HR BP 310 HR BP +610 HR BP Run chart recorder strip starting from step just before step 2 though of recovery

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#2a BARORECEPTOR REFLEX-Loading

STEP LOADING PHASE 1 Pre: CO: CO CO HR: BP: 2. TIME 0: Phenlyephrine infusion 1mL/m x10m +10 s HR BP +20s HR BP +320s HR BP +30s HR BP +330s HR BP +40s HR BP +340s HR BP +50s HR BP +350s HR BP +60s HR BP +360s HR BP +70s HR BP +370s HR BP +80s HR BP +380s HR BP +90s HR BP +390s HR BP +100s HR BP +400s HR BP CO CO CO CO CO CO +110s HR BP +410s HR BP +120s HR BP +420s HR BP +130s HR BP +430s HR BP +140s HR BP +440s HR BP +150s HR BP +450s HR BP +160s HR BP +460s HR BP +170s HR BP +470s HR BP +180 HR BP +480 HR BP +190s HR BP +490s HR BP +200 s HR BP +400 s HR BP +210s HR BP +510s HR BP +220s HR BP +520s HR BP +230s HR BP +530s HR BP +240 HR BP +540 HR BP +250 HR BP +550 HR BP +260 HR BP +560 HR BP +270 HR BP +570 HR BP +280 HR BP +580 HR BP +290 HR BP +590 HR BP 300 HR BP +600 HR BP 310 HR BP +610 HR BP

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3. ACETYLCHOLINE: Low dose of acetylcholine as a bolus. STEP 1 Pre: Start Chart.. obtain 10-15 sec baseline

Inject drug … run chart for about 1-2 min. 2. Observations: HR, Rhythm, BP?

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4. ACETYLCHOLINE: H dose of acetylcholine as a bolus. STEP 1 Pre: Start Chart.. obtain 10-15 sec baseline

Inject drug … run chart for about 1-2 min…longer as required

2. Observations: HR, Rhythm, BP?

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5. ISOPROTERENOL – bolus STEP

Isoproterenol

1 Pre: CO:_____ CO____ CO_____ HR:__ BP:___ 2. TIME 0: Start Chart recorder……..Isoproternol-bolus 3. Peak CO:_____ CO__ CO_____ HR:___ BP: ___

4. Observations: HR, Rhythm, BP, CO?

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6. NOREPINEPHRINE – bolus STEP

Norepinephrine

1 Pre: CO:___ CO___ CO____ HR:__ BP: ___ 2. TIME 0: Start Chart recorder…..Norepineprine -bolus 3. Peak CO:___ CO___ CO__ HR:____ BP: ___

4. Observations: HR, Rhythm, BP, CO?

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7. EPINEPHRINE – bolus STEP Epinephrine 1 Pre: CO____ CO___ CO____ HR___ BP: ___ 2. TIME 0: Start Chart recorder…..Norepineprine -bolus 3. Peak CO:____ CO___ CO____ HR:____ BP: ___ 4. Observations: HR, Rhythm, BP, CO?

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8. ANGIOTENSIN II – bolus STEP ANGIOTENSIN II 1 Pre: CO____ CO___ CO___ HR:___ BP: ___ 2. TIME 0: Start Chart recorder….. ANGIOTENSIN II -bolus 3. Peak CO:_____ CO____ CO____ HR:___ BP: ___ 4. Observations: HR, Rhythm, BP, CO?

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9. SEROTONIN – bolus STEP SEROTONIN 1 Pre: HR:_____ BP: _____ 2. TIME 0: Start Chart recorder….. SEROTONIN -bolus 3. 4. Observations: HR, Rhythm, BP, CO?

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10. HISTAMINE – bolus STEP HISTAMINE 1 Pre: HR:_____ BP: _____ 2. TIME 0: Start Chart recorder….. HISTAMINE -bolus 3. Peak HR:_____ BP: _____ 4. Observations: HR, Rhythm, BP, CO?

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11. TRIAD SUBCUTANEOUS INJECTIONS:

Get bradykinin, serotonin and histamine and inject subcutaneously 0.2 ml

of each in the same area of the abdomen - at least 2 inches apart.

COMMENTS

(Wheal/ flare?)

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12. VASOPRESSIN – bolus STEP Norepinephrine 1 Pre: CO:___ CO____ CO____ HR:___ BP: ____ 2. TIME 0: Start Chart recorder…..VASOPRESSIN -bolus 3. Peak CO:___ CO____ CO____ HR:___ BP: ____

4. Observations: HR, Rhythm, BP, CO?

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13. UNKNOWN AGENT 1 STEP UNKNOWN 1 Pre: CO:____ CO____ CO____ HR:___ BP: ___ 2. TIME 0: Start Chart recorder….. UNKNOWN -bolus 3 Peak CO:____ CO___ CO____ HR:___ BP: ____ Observations: HR, Rhythm, BP, CO?

14. UNKNOWN AGENT 2

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STEP UNKNOWN 1 Pre: CO_____ CO___ CO____ HR:___ BP: ___ 2. TIME 0: Start Chart recorder….. UNKNOWN –bolus 3 Peak CO:_____ CO____ CO___ HR:___ BP: ___ Observations: HR, Rhythm, BP, CO?

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