Pharma Mannual

WINTER 2013
UCSD

Phar/SOM 240/ SPPS 260

LABORATORY EXERCISE MANUAL!
SOM/Pharm 240/SPPS 260 Laboratory Protocol Winter 2012

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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.

SOM/Pharm 240/SPPS 260 Laboratory Protocol Winter
2012

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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 O
2

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 0
2
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 H
2
0.

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 pigs 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?


16
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?


17
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?


18
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

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?


19
VASOPRESSIN: Review the expected responses carefully before injecting this agent. Prior to injecting the drug

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

20
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


21

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 .


22
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


23

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 cm
5

Pulmonary VR 50 dynes-sec cm
5

Calculating Oxygen Consumption
Following formulae will permit estimation of consumption of O2 consumption in mls /
min given CO and arterial and venous O
2
content.
x 80 =
_______dynes-sec/cm
5

CO = MAP - CVP # SVR = MAP - CVP
SVR CO

x 80 =
_______dynes-sec/cm
5

CO = MPA - PCWP # SVR = MPA - PCWP
PVR CO


24

Arterial O
2
content is based on femoral arterial blood sample
Venous O
2
is based on femoral arterial sample.

[ ] [ ] 2 2
min) / ( 2
min) / (
VO AO
ml n consumptio O
L CO
!
=

Where:
[AO
2
] = arterial O
2
content in ml/L
[VO2] = venous O
2
content in ml/L

To calculate O
2
content of arterial [AO
2
] or venous [VO
2
] blood

Blood O
2
content in ml/dl =
1.39 ml/g x H
g
b g/dl x %sat.H
g
b (/100) + 0.003 ml/dl x PO
2

Data from blood gas report
H
g
b: Hemoglobin (g/dl)
%sat.H
g
b: % saturation (sO
2
) (express as decimal)
PO
2
: Oxygen saturation (mmHg)

25
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


26

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.

27

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.)

28
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

29

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

30
AUTONOMIC LABORATORY
DATA COLLECTION SHEETS

DATE: ___________

Animal Number: _________

TABLE: ___________________

Students:

________________________________________
________________________________________
________________________________________
________________________________________
________________________________________
________________________________________

Table Mentor:_________________________

31
#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:


32
#2a BARORECEPTOR REFLEX-Unloading
STEP UN-LOADING PHASE
2. TIME 0: Sodium Nitroprusside infusion..1mL/m x10m
+10 s HR BP
+20s HR BP +320s HR BP
CO CO CO CO CO CO
+180 HR BP +480 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
Run chart recorder strip starting from step just before step 2 though of recovery


33
#2a BARORECEPTOR REFLEX-Loading
STEP LOADING PHASE
2. TIME 0: Phenlyephrine infusion 1mL/m x10m
+10 s HR BP
CO CO CO CO CO CO
+180 HR BP +480 HR BP
+200 s HR BP +400 s 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

34

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?


35

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 minlonger as
required
2. Observations: HR, Rhythm, BP?


36

5. ISOPROTERENOL bolus

STE
P
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?


37
6. NOREPINEPHRINE bolus

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



38
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: ___



39
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: ___



40
9. SEROTONIN bolus

STEP SEROTONIN
1 Pre: HR:_____ BP: _____
2. TIME 0: Start Chart recorder.. SEROTONIN -bolus
3.



41
10. HISTAMINE bolus

STEP
HISTAMINE
1 Pre: HR:_____ BP: _____
2. TIME 0: Start Chart recorder.. HISTAMINE -bolus
3. Peak HR:_____ BP: _____



42

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?)

43

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: ____



44
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

45

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?


46

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