School of Medicine

University of Washington School of Medicine
CVANS: Virtual Dog Laboratory

HUBIO 543
UW Restricted

CVANS MODULE

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syringelab dogsyringe

A number of generations of medical students learned practical aspects of autonomic and cardiovascular pharmacology from a traditional dog lab. In such a laboratory students were presented with an anesthetized dog. An afternoon was typically spent injecting drugs, monitoring responses and discussing the whys and hows. Many students said it was the most worthwhile afternoon of the first two years of Medical School.

Injecting drugs and monitoring BP

(click on the small images to see enlarged pictures)

Catheters

We will do a BP lab experiment in a rather traditional manner, but virtually. Following induction of surgical anesthesia (typically by IV pentobarbital) catheters are placed for IV injection of drugs on the one hand (femoral vein) and for intra-arterial monitoring of arterial blood pressure on the other hand (femoral artery).

Results from a Real Experiment

view results

In this section, recordings (courtesy of Dr. B. Raess, Indiana University, School of Medicine, Evansville, IN) made during an actual experiment are presented along with text. The electrocardiogram was used to trigger a heart rate monitor (upper tracing). The scale for heart rate is calibrated from 60 to 180 beats/min. Arterial pressure was monitored by an indwelling catheter in the femoral artery. Blood pressure is calibrated from 0 to 200 mm Hg in this and similar tracings.

The Baroreceptor Reflex

A Potent Modifier of Cardiovascular Responses to Drugs

epinephrine effects

It is important to understand that in anesthetized, but otherwise untreated animals, autonomic reflexes respond to changes in blood pressure. The reflexes can significantly alter responses to injected drugs, as will be demonstrated.

IV injection of epinephrine (EPI) elicits a prompt increase in blood pressure and pulse pressure. The increase in blood pressure is caused mainly by effects on vascular smooth muscle alpha-1 receptors. There is also a positive inotropic effect on the heart mediated by beta-1 receptors. However, the positive chronotropic effect of epinephrine on the heart was completely masked by reflex bradycardia. This is triggered by the increase in blood pressure and is mediated mainly by increased firing of the vagus (releasing acetylcholine [ACh] in supraventricular parts of the heart, including the sino-atrial [SA] node). While heart rate decreased, it also became a little irregular. This is typical when EPI is pushing the SA node 'accelerator', and the ACh from the vagus nerve is pushing the SA node 'brakes'. Also note that diastolic pressure did not increase very much in response to EPI. This is partly because of the reflex bradycardia that provides greater time for diastolic 'runoff' of blood. Also, there is an underlying vasodilation in some vascular beds mediated by beta-2 receptors. When a rapid bolus of epinephrine is injected IV, a pressor response is typical as shown here. This is different from the classical response seen upon slow IV infusion of catecholamines in humans.

Catheters

Reminder: for this injection of EPI and all similar injections, the injection of the drug from one syringe needs to be followed by a few milliliters of saline, usually from another syringe. As shown, the syringes are commonly both hooked to a '3-way' valve that allows injection of one syringe, then another. The saline injection is needed to flush the drug into the system because the tip of the IV catheter extends from the femoral vein to the inferior vena cava near the heart. Unless the catheter is properly flushed, one can end up injecting confusing mixtures of drugs 'left over' in the catheter.

The anesthetized dog weighed 22 kg. What volume of a solution containing 0.1 mg of epinephrine per ml was needed to inject a dose of 2 mcg (micrograms) per kg? You should be able to make such calculations easily. If you cannot, then you may want to check out dosage calculation.

ACh response

IV injection of ACh causes a depressor response. The response is short lived because of rapid hydrolysis of ACh by acetylcholinesterase (AChE). The depressor response is mediated mainly by activation of muscarinic receptors on endothelial cells. When activated, the muscarinic receptors augment the production of endothelial derived relaxing factor (EDRF), now known to be nitric oxide. Nitric oxide diffuses into the smooth muscle cells of the vasculature and increases the activity of guanylate cyclase. This transiently increases the production of cyclicGMP and promotes vasodilation.

Injection of ACh is also associated with an increase in heart rate. This reflex tachycardia is mediated by decreased vagal activity and increased activity of sympathetic nerves caused when blood pressure falls. Any tendency of ACh to exert a negative chronotropic effect on the SA node of the heart is thus masked by the powerful reflex connecting blood pressure and parasympathetic and sympathetic outflows.

Assuming that the solution of ACh contained 200 mcg/ml, how many ml were injected into this 22 kg dog to obtain the dose of 5 mcg/kg?

After Atropine

Atropine blocks existing vagal tone and blocks reflex bradycardia for the rest of the experiment

atropin results

There are a number of different mechanisms by which the parasympathetic arm of the baroreceptor reflex can be interrupted. You should be able to list and discuss at least four or five different mechanisms. One approach to blocking the reflex bradycardia associated with increased blood pressure is the use of atropine or other muscarinic antagonist. Injection of atropine resulted in an increase and regularization of the heart rate. The increase in heart rate demonstrates that there was substantial vagal tone to the heart in this anesthetized dog. The slight decrease in pulse pressure seen following the injection of atropine is probably related to the greater heart rate (i.e., less diastolic ventricular filling). There is little or no inhibition of ventricular muscle function by ACh. You should be able to explain why there is no increase in blood pressure following the injection of atropine.

Certain drugs, once injected, exert their effects throughout the duration of the experiment. For example, a single injection of atropine remains throughout the rest of this experiment. This is typical for antagonist drugs, in contrast to the agonist 'test drugs' (for example, EPI and ACh) which typically have short durations of action.

epinephrine after atropine

When epinephrine is injected after atropine, reflex bradycardia is missing and epinephrine exerts its expected positive chronotropic effect on the heart. The pressor response remains and is somewhat greater, particularly in the diastolic pressure (less time for diastolic 'runoff' at higher heart rates).

ACh after atropine

Injection of ACh (5 mcg/kg, same dose as previously, first arrow) after atropine results in no response. This is because muscarinic receptors are blocked by atropine. Note that there is no acceleration of heart rate, showing that all of the acceleration caused by ACh prior to atropine was dependent on reflex tachycardia. The response was elicited by the vasodilation mediated by muscarinic receptors (now blocked) on endothelial cells.

Injection of 500 mcg/kg of ACh (second arrow), a dose 100 times more than previously, causes a transient depressor response (probably very minor breakthrough of the atropine block - remember atropine & ACh is a classic example of competitive [and surmountable] pharmacological antagonism). However, the major response is an increase in both blood pressure and heart rate. This response is caused by ACh activation of nicotinic receptors on sympathetic autonomic ganglia, including the adrenal medulla. This results in activation of postganglionic sympathetic fibers innervating the vascular smooth muscle as well as fibers innervating the heart. These fibers release NE. Vascular smooth muscle constricts because the NE activates alpha receptors. Heart rate increases and heart muscle beats more forcefully because NE activates beta-1 receptors. EPI and NE released from the adrenal medulla circulate to these same receptors and also participate in the response.

You should be able to diagram the receptor locations and nerve pathways involved in this 'nicotinic response to ACh and suggest at least four different pharmacologic mechanisms that could be invoked to prevent this pressor response to ACh in the presence of atropine. The dose of ACh used to elicit this response would have been a lethal dose in the absence of atropine. Thus, atropine can be life saving in cases of excess activation of muscarinic receptors. Can you think of a practical application of this information? Also, can you suggest the relative affinities of the muscarinic and nicotinic receptors for the agonist, ACh?

Epinephrine Reversal

A Classical Observation

EPI after atropine and phenoxybenzamine

EPI is an agonist of both alpha and beta adrenergic receptors. Following administration of phenoxybenzamine (POB) (atropine already present), the alpha receptors are blocked. In such a condition the injection of EPI results in a depressor response. This is mediated by the beta-2 receptors located in some vascular beds. In the absence of POB this vasodilation is present, but the alpha receptor mediated vasoconstriction is so prominent that the beta-receptor mediated vasodilation is masked. Thus, when injected in a bolus EPI normally causes a pressor response, but in an animal whose alpha receptors are blocked EPI causes a depressor response. This change in the qualitative nature of the blood pressure response to EPI is known as 'epinephrine reversal'.

In the presence of atropine + POB, EPI also causes a prominent increase in heart rate. Some of this is due to a reflex tachycardia elicited by the depressor response of blood pressure. Some of this tachycardia is also due to EPI activation of beta-1 receptors in the SA node of the heart.

You may notice that there was a very transient increase in blood pressure when EPI was injected in the presence of atropine + POB. Do you have any idea why this occurred? Hint: think of the plumbing and the sequence of EPI access to the receptors.

One of the most spectacular successes in pharmacology has been the ability to distinguish between different kinds of closely related receptors. This tracing shows the response to EPI following atropine + POB + practolol. Practolol is a selective antagonist of beta-1 receptors. Thus, it prevents the effects of catecholamines on the heart but not on the vasculature. Injection of EPI in this condition thus results in a depressor response, but almost no tachycardia. Both the direct and reflex components of the tachycardia caused by EPI are blocked.

Can you suggest a drug that might be used to prevent the depressor response to EPI that remains in this animal?

Virtual Lab Responses

We have seen that the baroreceptor reflexes can exert significant effects on drug responses. In order to simplify things for a teaching/learning laboratory, it is common to perform bilateral vagotomy. This prevents reflex bradycardia without blocking pharmacologic receptors. In the remainder of the virtual lab you will be presented with SIMULATIONS of blood pressure responses in a 'vagotomized animal'. You will be presented with responses to 13 different test drugs or procedures.

blood pressure with epinephrine

In this virtual lab you will be presented with SIMULATIONS of BP responses to a variety of different test drugs or procedures. In this and other figures, arterial blood pressure is plotted as a function of time (sec). Thus, the figure presents a BP tracing lasting 4 min. The dark band shows the pulse pressure (systolic/diastolic) On this time scale, individual arterial pulses are not visible. The example shown is a response to IV injection of epinephrine (EPI) at 30 sec. A rapid and short acting pressor response (increased BP) is observed. It is assumed that appropriate doses of drug are given in each case. Smaller doses would give smaller responses, and bigger doses would give bigger responses; within limits.

Tracheostomy, vagotomy, etc.

Neck

A cut down in the neck is done for possible placement of an endotracheal tube. This would be used to assist breathing, if needed. In addition, carotid arteries are isolated bilaterally by BLUNT dissection (in a living animal with BP of 120/80 mm Hg pressure or thereabouts we do not want to cut even small arteries). Loose sutures are placed around both carotid arteries. Later these sutures will be used (by gently lifting) to temorarily cut off the arterial pressure to the carotid sinus baroreceptors (carotid occlusion, CO). Near the carotid arteries the vagus nerves are found and cut with a suture remaining on the distal stump. This allows one to electrically stimulate the vagus nerve going to the heart (vagal stimulation, VS).

The drugs and procedures used in the virtual lab are designed to illustrate certain fundamental aspects of the autonomic nervous and cardiovascular systems. They include drugs which are not used in humans (for example, DMPP), and they include procedures (bilateral vagotomy, for example) that could not be justified in a humans, but which help to clarify and simplify the experiment by (for example) preventing reflex bradycardia in response to drugs that exert a vasopressor effect.

In this virtual lab you have three kinds of experiments:

Control Experiment

You administer 13 standard test drugs or procedures to an dog that has no pretreatment except anesthesia and vagotomy. Learn the responses to these standard drugs and procedures. Figure out where the initial drug/receptor interaction takes place and whether the response depends on any intermediary steps, such as (for example) activation of a ganglion cell and conduction of an impulse to a nerve ending with release of neurotransmitter and interaction of that transmitter with its receptor. Make sure you understand the responses to the standard test drugs or procedures before you move onto the pretreatment experiments.

Pretreatment Experiment

You utilize a series of dogs that have had anesthesia, vagotomy and pretreatment with a drug or drugs that may modify the responses to the standard test drugs or procedures. Because you are familiar with them, the acute changes in blood pressure (increase, decrease or no change) observed in the control experiment, are summarized for you in a table. Next to these, are shown the acute changes in blood pressure caused by the same standard drugs or procedures in an animal pretreated with the drug or drugs listed at the top of the column. Each pretreatment experiment uses a different dog. Assume that the pretreatment drugs are used in doses at least adequate to elicit their most characteristic pharmacological effects. Assume also that all pretreatment drugs act throughout the experiment. The quickest way to figure this out is to notice where the response of a pretreated animal differs from the response of a control animal. Then based on the site and mechanism of the pretreatment drug(s), figure out where and how the pretreatment changes the responses. When you have this in hand, then you are ready to find out if you really understand this stuff by trying the self-evaluation experiments.

Self-evaluation Experiment

You will utilize a series of dogs that have had anesthesia, vagotomy and pretreatment with an UNKNOWN drug or drugs that may modify the responses to the standard test drugs or procedures. The acute changes in blood pressure (increase, decrease or no change) observed in the control experiment, are summarized in a table. Next to these, are shown the acute changes in blood pressure caused by the same standard drugs or procedures in an animal pretreated with the UNKNOWN drug or drugs. Each self evaluation experiment uses a different dog. Assume that the pretreatment drugs are used in doses at least adequate to elicit their most characteristic pharmacological effects. Assume also that all pretreatment drugs act throughout the experiment. As before, the quickest way to figure this out is to notice where the response of a pretreated animal differs from the response of a control animal. Then based on the pattern of blood pressure responses in the animal pretreated with the UNKNOWN drug or drugs, and from the possible choices offered, choose the minimum number of pretreatment drug(s) which would be required to account for, and still be compatible with, the observed results. If you can do this reliably, then you have a good mental image of the sites and mechanisms of action of drugs that affect the autonomic and cardiovascular systems.