Experimental Pharmacology

1. Effect of drug’s on frog’s heart perfusion
Object: To study the effect of various drugs on isolated perfused frog’s heart
Apparatus: Kymograph, frog board, dissection set, perfusion apparatus
(Mariotte bottle, rubber tubing and a venous cannula), iron stand, clamps,
universal lever, thread, pins, tuberculin syringe, pithing needle, frog’s ringer
solution.
Experimental Animal: Frog
Drugs to be studied:
Drug Concentration Dose
Adrenaline 1:10,000 0.1-2 ml
Acetyl Choline 1:10,000 0.1-2 ml
Calcium Chloride 1:100 0.1-2 ml
Potassium Chloride 1:100 0.1-2 ml
Atropine 1:5000 0.1-2 ml
Experimental SetUp:
 A medium sized frog is stunned, pithed and clamped on frog’s board on its
back.
 Abdominal and thoracic walls are cut by a midline incision. Bones of the
pectoral girdle are cut open and the heart is exposed without injuring it.
 Thoracic cavity is widened by stretching all the four limbs on the sides.
 The heart is now gently freed from the pericardium and a few drops offrog’s
Ringer solution are poured over it.
 One branch of the truncus arteriosus (5) is tied firmly with the help of a thread
while the other branch is cut open for the perfusion fluid to come out.

2.  Heart is then lifted up to visualize sinus venosus. Once it is identified a ligature
is passed beneath sinus venosus and a venous cannula is inserted into it. It is
kept in position by tying a ligature around it.
 The venous cannula is connected to the perfusion bottle containing frog’s ringer
solution with rubber tube.
 The circulation of fluid of frog’s Ringer solution inside the heart is from sinus
venosus to right auricle ventricle arch of aorta out of the cut end of the
aorta (First of all the fluid goes from the reservoir to the venous cannula and
then sinus venosus).
 The rate of perfusion is kept 30-40 drops/min. The perfusion pressure is
adjusted by altering the height of the reservoir above the level of the heart.
 Now a pin hook is passed through the apex of the ventricle and the pin is
attached to universal lever to write on smoked drum.
Parameters to be studied:
1) Heart Rate: This is counted by counting the number of times lever
comes down per min.
2) Rhythm of heart: Note whether regular or irregular.
3) Force of contraction:It is calculated by observing the height of the
tracing (amplitude). Down going lever indicates systole. Up going lever
indicates diastole.

3. 4) Tone : It is the state of partial contraction of muscle at rest. This is
denoted by baseline of curves. A rise in baseline indicates increase in
tone.
Recording:
 First the drum is started at the minimum speed.
 A small tracing is recorded. Drugs to be studied are injected in a dose of
0.1 ml with the help of tuberculin syringe in the rubber tube near to the
venous cannula and the effect is recorded till the heart becomes normal
again.
 In this way, effect of Adrenaline, Acetyl Choline, Calcium Chloride &
Potassium Chloride and atropine is recorded.
 Keep at least 5 minutes gap between administrations of each dose of the
drug. If the effect is not significant then, the dose may be increased.
 After recording effect of all drugs, effect of Acetyl Choline and
Potassium Chloride are recorded on atropinized heart.
Precautions:
 Syringe should be washed before injecting each drug.
 Drugs should be injected near to the cannula.
 Over perfusion must be avoided.
 Control tracing must be recorded before and after effect of each drug is
recorded.
 Heart must be kept wet by continuously pouring Ringer solution
 No air bubbles should be present in the tube.
 Heart should not be injured.
ObservationTable:
Drugs Effects on Inference
Heart Rate Force of
Contraction
Rhythm
1) Adrenaline Regular It is a sympathomimetic drug. It acts via β1
and β2 receptors to stimulate the heart.

4. 2) ACh
Heart may stop in
diastole (at higher
doses)
It is a parasympathomimetic drug. It acts by
muscarinic (M2) receptors and causes
cardiac depression.
3) Calcium
Chloride
(1:100)
No effect Systolic
force of
contraction
The heart may
stop in systole.
It is a directly acting cardiac stimulant. On
the dose and concentration it causes calcium
rigor and systolic arrest.
4) Potassium
Chloride
(1:100)
Heart may stop in
diastole if dose is
It is a directly acting cardiac depressant. On
the dose and concentration, diastolic arrest
may occur.
It decreases the resting membrane potential,
fibres become inexcitable.
5) Atropine May No change Regular It is an antimuscarinic drug. Acts
by blocking M2 receptors on the heart.
6) Atropine +
ACh
No effect No effect Regular Atropine bocks the action of Acetyl Choline
7) Atropine +
Potassium
Chloride
Heart may stop in
diastole
Effect of Potassium Chloride is not blocked
by Atropine as it is a directly acting cardiac
depressant.