ANNAMALAI UNIVERSITY

INSTITUTE OF PHARMACEUTICAL TECHNOLOGY

PHARMACOLOGY - PRACTICAL

GENERAL INSTRUCTIONS TO THE STUDENTS

1. Pharmacological laboratory experiments provide opportunity to observe what students studied

in texts. To make the best of the experiment student must read the theory and technique of the
experiment well before entering the laboratory hall.

2. The students should submit the record notebooks as soon as they enter the class. The record
work of the previous experiment will be valued by the teacher and the record will be returned to
the students at the end of the class. The cumulative marks go into the sessional marks for the
practical.

3. Students must maintain separate observation note book.

4. Students must come with a clean apron in every practical class.

5. Students are responsible for work missed, so they should arrange with their instructor to make
it up as soon as possible.

6. In case students are in batch, they should do their full share. The division of labour naturally
does not excuse any member of the batch from the necessity of being acquainted with the general
purpose, progress and the results of the experiments.

7. The students should leave the laboratory only after switching off the instruments and
computers.
Expt. No :1
COMMON LABORATORY ANIMALS USED IN THE EXPERIMENTAL
PHARMACOLOGY

HANDLING OF LABORATORY ANIMALS

The animals must be handled with utmost care so that it does not suffer any pain and due
regard is paid towards the health and well being of the animals. Even when they are killed at the
end of experiment, it should be done by a humane method. That is called euthanasia which
means painless killing.

FROG
Frog belongs to the class amphibian. The isolated preparation of frog need not be
maintained at 37° C and the experiments are performed at room temperature. In frog, adrenaline
is the neurotransmitter for sympathetic nervous system. Frogs are commonly used in the study of
the actions of drugs on the central nervous system, on the heart, on the neuromuscular junction as
well as to determine the retinal toxicity of the drug.

RATS (Rattus norvegicus)

Albino rats are one of the commonest laboratory animals because of its small size and
greater sensitivity to most drugs. Rats don't vomit because they lack vomiting centre. They don't
have gall bladder.
Rat as an experimental animal:
 As they can be properly trained for various type of work performances including the
development of the conditioned reflexes they are mainly used for the testing of the
psychopharmacological agents.
 These are mainly used for the study of the drugs on the blood pressure both in acute as
well as chronic experiments.
 The rats are mainly used by the scientists like shay et.al for the study of gastric secretion
by the pyloric ligation method as the results obtained were reproducible
 The rats are mainly used for the study of analgesics by radiant heat method application on
the tail and they are also used for toxicity studies of both acute and chronic type.
  The rats are suitable for the testing of drugs for teratogenicity and carcinogenicity.
Tissues of rat used: Various isolated tissues such as uterus, ileum, stomach, and colon are
employed for the study of various drug actions.

GUINEA PIG (Cavia porcellus)

They are highly sensitive to histamine.
Guinea pig as an experimental animal: Guinea pig" is often used as a metaphor for a subject of
scientific experimentation. In the past they had been used to isolate different bacterial strains, but
in modern labs they have been replaced by mice and rats, which reproduce more quickly.
 It is mainly used for the evaluation of the bronchodilator drugs against experimentally
induced asthma (histamine or acetylcholine aerosols).
 It is also used in the field of immunology particularly in the area of delayed
hypersensitivity by using various antigens such as egg albumin, horse serum etc:
 They are employed widely in the studies of the local anaesthetics as well as the bioassay
of digitalis.
 They are suitable for hearing experiments as they have sensitive cochlea.
 They are suitable for experiments on oxygen consumption.
 It resembles man in that it also needs an exogenous source of vitamin C and so it is useful
in the study of its metabolism.
 Being a suitable host for the Mycobacterium it is also suitable for tuberculosis studies.
Various isolated tissues of Guinea pig that are useful are:
Ileum, tracheal chain, vas deferens etc: the terminal ileum is most sensitive for the preliminary
screening of the spasmodic and antispasmodic compounds and suitable for the detection and
assay of histamine and related compounds.

MOUSE (Mus musculus)

Albino mice are the smallest laboratory animals, which can be breed uniformly. They are
cheap and easy to handle.
Mice as an experimental animal :
 Mice are widely useful in acute toxicity studies.
 They are also used in the assay of insulin and analgesics and also for the general
 screening of the chemotherapeutic agents specially bred mice are mainly useful in the
study of problems in genetics and cancer
 They are most frequently used for testing of drugs due to teratogenicity
 The Nude mice which lack thymus gland is mainly useful in the study of the tissue
immunity and transplantation research.
Tissues of mice used:
The vas deferens and the ileum are the only tissues used in mice for the experiments because it is
small and delicate.

RABBITS (Oryctolagus cuniculus)

Rabbits are docile animals. In rabbit the gene for atropinesterase is linked with the colour
of the fur. The occurance of atropinesterase is linked with the colour of the fur. Atropinesterase
grately shortance the pharmacological actions of atropine.
Rabbit as an experimental animal:
 Rabbits are mainly used for the pyrogen testing in intravenous fluids.
 The agents which affect the capillary permeability are mainly studied by injecting the
substances intracutaneously and followed by intravenous injection of the dyes like Evans
blue.
 Insulin, and other antidiabetic drugs, curare and sex hormones are tested in the rabbit.
 As its skin is relatively sensitive to irritants it can be used for testing topical agents.
 It is used in serological work and for screening of the embryo toxic agents.
 It is mainly used for the research on reproduction as ovulation is nonspontaneous and its
semen is also collected easily.
 The convenience of injecting into and withdrawing of blood from the ear marginal vein is
helpful in bioavailability studies
Tissues of rabbit used are:
Isolated Heart, Jejunum, and Ileum are some of the preparations routinely used for the
testing of the drugs.

CAT
Cats are common among the carnivores. The cat has a highly developed nictitating
membrane, which is contracted by stimulation of cervical sympathetic trunk and also by number
of drugs like adrenaline, histamine etc. Contraction of nictitating membrane are recorded for the
investigation of ganglionic blocking agents. In cat morphine produces excitation of C.N.S.
Cat as an experimental animal:
 Cats are employed in acute experiments for the study of drugs affecting the blood
pressure.
 Both anaesthetized preparations and spinal preparations are used, the latter being used
particularly for the assay of catecholamines.
 Contractions of the nictitating membrane are recorded for the investigation of ganglionic
blocking action of the drugs.
 Cats are essential in the study of the nerve centers in the brain.
 Because they can produce the methaemoglobinemia, cats have been found to be most
suitable for the toxicity of compounds like acetanilide.
 Cats are often the model of choice for neurological research, as well as studies on
hearing, balance, movement and motor neuron research related to spinal cord injury. Due
to anatomical similarities in brain structure they have been used for mapping studies.
 They can be also used as models for the viral disease syndromes.

DOG (Canin familiaris)

Dogs are useful among large laboratory animals, because they can be tamed trained
without much difficulty.
Uses:
 It is used for the study of the hypoglycemic drugs as it develops spontaneous diabetis
because the pancreas in it is diffuse.
 Study of Antihypertensives
 Anaesthetised dogs are routinely used in the study of drugs on blood pressure.
 Study of cholinergic and anticholinergic drugs
 Neuromuscular blocking drugs.
 Ganglion blocking agents
 Toxicity studies
Tissues used in experiments.
Chronically prepared gastric fistula and pouches by earlier operations are also employed for the
study of gastric secretion in the dog.

MONKEY
Monkeys and apes belong to the primates, the highest order of mammals, which includes
man. Both structurally and functionally monkeys and apes closely resemble man. Primates are
used in the field of virology, parasitology, immunology, nutrition, reproduction etc.
Expt. No :2
STUDY OF BASIC EQUIPMENT USED IN EXPERIMENTAL PHARMACOLOGY

ELECTRIC RECORDING DRUM

It consists of an electrically driven gear box with a vertical spindle carrying a cylinder of
15cm*15cm. This can be locked up in any position by a small lever. The gear box has a clutch
and control which determines the final speed of the spindle. The choice of drum speed is
specified in terms of cylinder revolutions per minute or mm/sec. The smoke glazed paper is
attached on the surface of the cylinder act as a writing surface.
The vertical spindle carries two horizontal contact arms. When the drum rotates these
arms briefly close a contact marker mounted on the base. This finds extremely useful in frog
sciatic nerve- muscle preparation. When the contact block is connected to the stimulator, it can
initiate the stimulus at particular position of the cylinder.

STUDENT ORGAN BATH

This apparatus is designed by a Russian scientist Rudolph magnum. It is made up of
Perspex glass. It contains a cylindrical inner bath. The inner bath is always filled with
physiological salt solution where an isolated organ such as frog rectus muscle or intestine is
suspended. The outer bath is filled with water and the temperature is maintained at 37 C with the
help of the heater and thermostat control. For all mammalian preparations it is necessary to
maintain the temperature at 37 C since the tissue will work only at that temperature.
One end of the tissue is tied to the tip of the tissue holder and the other end is connected
to the free arms of the simple lever. Atmospheric air is pumped through the opening of the tissue
holder. By the help of an aerator, the air is supplied to inner bath for two purposes namely
1. To supply oxygen to the tissues.
2. To stir the drug solution in inner bath.

WRITING LEVERS
These are made up of straw like aluminium, stainless steel or light balsam of wood. They
are light but rigid. The purpose is to record the movement of tissue and to magnify the response.
The magnification of lever is equal to the distance between the writing point and the fulcrum
divided by the distance between the point of attachment to the tissue and fulcrum
Before attaching any tissue to a lever it should be balanced by attaching sufficient weight
at the end of the shorter arm.
SIMPLE LEVER
It consists of a light aluminium or stainless steel with a light celluloid writing
point (stylus). It writes tangentially on drum and leads to curve trace. This is mostly used to
record isotonic contraction of smooth muscle or skeletal muscle like rectus abdominus. It writes
tangentially on the drum and leads to a curve trace.
FRONTAL WRITING LEVER
It is so designed that the actual shortening of the muscle is modified in linear
proportion on the drum. A long frontal - wringing point made of aluminium is attached to the end
of the strip by means of hinge. The lever writes frontally and thus gives a straight line rather than
curve. It is used in smooth muscle contraction experiments.
GIMBAL LEVER
With this lever the pressure of the stylus is effected by the force of gravity. The
friction between the lever and the paper is kept constant as the writing lever always has a
tendency to fall towards the writing surface like simple lever it also gives curvilinear trace useful
for recording contractions given by rectus muscle.
STERLING 'S HEART LEVER
It consists of a frame carrying a light lever arm with holes and notches supported
by a fine adjustable hook. Because of the spring action it is used where the response is very
rapid. Example: Contraction of heart muscle.

CANNULA
VENOUS CANNULA
Specially made in various sizes for vein into which it can be used.
ARTERIAL CANNULA( Francois-Frank cannula) It is used for recording arterial blood
pressure. It consists of bulb and side tube too. Side tube is useful to remove blood if clotted or to
remove air bubble.
SYME 'S CANNULA
It is used in isolated frog heart experiment.
 TRACHEAL CANNULA
It is made of metal or glass. It is in 'Y' , 'T' , or 'Z' in shape. It is used for artificial
respiration purpose by inserting the one arm into trachea of an animal and the other end
connected to a respiratory pump. It is also used to record the respiratory volume.

MAREY 'S TAMBOUR

An aluminium piece rests on a diaphragm which is tied to a small hallow steel cup. On
this piece, writing lever just slides. The entry of air into the cup displaces the aluminium piece
and writing lever. This is used for recording respiratory and spleenic volume changes. The
improved model is Brodie's tambour.

ONCOMETER
The spleenic oncometer is a Perspex chamber which can house spleen and its blood
vessels. The contraction elicited by it is transmitted to a piston or a tambour through a
polyethylene tube by means of an air displacement technique.

MERCURY MANOMETER
It is used for record mean arterial blood pressure of dog or cat. It consists of 'U' shaped
glass tube of 320 mm long and 5 cm wide. The two vertical limbs are half filled with mercury.
Since the mercury is displaced equally upon one limb and down in the other. The displacement
value recorded must be multiplied by 2. To prevent this calculation a mm scale with doubled
value is fitted with manometer. So that, there can be read directly in mm of mercury. The one
limb of the 'U' tube carries steel wire with stylus and other limb of the column has the side tube
which is connected to arterial cannula. The upper end of which is connected reservoir containing
anticoagulant fluid. At the junction of the side tube a three way stop cock connects or
disconnects the arterial cannula and the manometer from reservoir bottle

CONDON'S RAT B.P. MANOMETER

It is used for recording B.P. for rats and other small animals. It consist of 280mm*2.5mm
glass tube connected to a piece of polyethylene tubing to a mercury reserve of 2cm diameter for
a large change of mercury level in glass tube only a very small change will occur in reservoir.
The scale is calibrated to take the small change into account and into accurate pressure reading
between 0 to 250 mm of mercury may be obtained. This manometer is twice as sensible as
normal 'U' shaped manometer. A length of polyethylene tubing and a fine glass cannula connects
the free arm of reservoir.

CLAMPS
The clamps are used to hold the apparatus. The types of clamps are
1) Universal clamp, 2) Open sided X block, 3) Close sided X block
4) Thermometer clamp

MUSCLE GRIP
It is used to hold the muscle.

STANDARD ELECTRO STIMULATOR

This is used to give stimuli to a muscle or a nerve, we can select the pulse width. The
width between 2 pulses measured in millisecond. Frequency is measured as pulse per second or
hertz. The strength of the current measured in volts. The stimulator can be used to produce a
single shock or pulses of shocks.

ELECTRODES
The electrodes used are 1) Simple electrode 2) Unipolar electrode 3) Bipolar electrode
4) Phrenic Nerve electrode.

A.C. TIME CLOCK

This is a interval timer which gives signal at a desired time 5,10,20,30 or 60 sec. It is
used in conjugation with magnetic time marker.

ANALGESIOMERTER
It is used in screening of drugs for analgesic activity. It has a stainless steel plate heated
with a help of heater. The temperature can be adjusted by thermostat control.
Expt. No :3
PHYSIOLOGICAL SALT SOLUTIONS USED IN EXPERIMENTAL PHARMACOLOGY

Physiological salt solution contains various ions similar to the amounts found in blood
plasma and extra cellular fluids. Physiological salt solution maintains the functions of different
tissues.
Sodium ions
It maintains the isotonicity of the fluid. In the absence of sodium ions the cell is incapable
of conducting action potentials. Sodium ion affect the contraction of muscle indirectly by
influencing the up taking of calcium.
Potassium ions
It is also necessary to produce the action potentials. The lack of potassium ions increases
the uptake of calcium. Excess of potassium has depolarising action.
Calcium ions
It plays a role in maintaining the excitable property of the membrane. Deficiency in
calcium causes repetitive action potentials and gradually the membrane become inexcitable. The
excess calcium ions facilitate coupling and contractility.
Magnesium ions
Magnesium relaxes the tissue when the tone of contractility raised or stimulated by the
drugs. calcium ions and magnesium ions are antagonistic to each other.
Glucose
It serves as a source of energy for the isolated tissues.
Chloride ions
It plays a part in maintaining osmotic pressure.
Bicarbonate and phosphate ions
These are the buffering ions which serve to buffer the solution.
Aeration of the solution:
Isolated tissue needs oxygen to maintain the function. Aeration supply oxygen.
Composition of physiological salt solutions g/l
Salts Krebs Krebs - Locke Tyrode Ringer Clerk frog
Hensetal Ringer
NaCl 5.5 6.87 9 8 9 6.5
KCl 0.35 0.4 0.42 0.2 0.42 0.14
MgCl2 - - - 0.1 - -
MgSO4 0.11 0.14 - - 1 -
CaCl2 0.28 0.24 0.24 0.24 0.24 0.12
NaHCO3 2.1 2.1 - 1 - -
NaH2PO4 - 0.4 - 0.05 - 0.05
K2H2PO4 - - - - - 0.05
Glucose - 2 1 1 1 2

Expt.No: 4
ANAESTHESIA AND EUTHANASIA

The scientists should ensure that the procedures, which are considered painful, are
conducted under appropriate anesthesia as recommended for each species of animals.

It must also be ensured that the anesthesia is given for the full duration of experiment and at
no stage the animal is conscious to perceive pain during the experiment. If at any stage during
the experiment the investigator feels that he has to abandon the experiment
or he has inflicted irreparable injury, the animal should be sacrificed. Neuromuscular
blocking agents must not be used without adequate general anaesthesia.

In the event of a decision to sacrifice an animal on termination of an experiment an approved

method of euthanasia should be adopted and the investigator must ensure that the animal is
clinically dead before it is sent for disposal. The data about large animals, which have been
euthanised, should be maintained.
Anaesthesia

Sedatives, analgesics and anaesthetics should be used to control pain or distress under
experiment. Anaesthetic agents generally affect cardiovascular, respiratory and
thermoregulatory mechanism in addition to central nervous system.

Before using actual anaesthetics the animals is prepared for anaesthesia by over night fasting
and using pre-anaesthetics, which block parasympathetic stimulation of cardio- pulmonary
system and reduce salivary secretion. Atropine is most commonly used anti-cholinergic agent.
Local or general anaesthesia may be used, depending on the type of surgical procedure.

Local anaesthetics are used to block the nerve supply to a limited area and are used only for
minor and rapid procedures. This should be carried' out under expert supervision for regional
infiltration of surgical site, nerve blocks and for epidural and spinal anaesthesia.

A number of general anaesthetic agents are used in the form of inhalants. General
anaesthetics are also used in the form of intravenous or intra-muscular injections such as
barbiturates. Species characteristics and variation must be kept in mind while using an
anaesthetic. Side-effects such as excessive salivation, convulsions, excitement and
disorientation should be suitably prevented and controlled. The animal should remain under
veterinary care till it completely recovers from anaesthesia and postoperative stress.

Euthanasia

Euthanasia is resorted to events where an animal is required to be sacrificed on

termination of an experiment or otherwise for ethical reasons. The procedure should be
carried out quickly and painlessly in an atmosphere free from fear or anxiety. For
accepting an euthanasia method as humane it should have an initial depressive action on the
central nervous system for immediate insensitivity to pain. The choices of a method will
depend on the nature of study, the species of animal to be killed (Annexure- 6). The method
 should in all cases meet the following requirements:

a. Death, without causing anxiety, pain or distress with. minimum time lag
phase. . . .
b. Minimum physiological and psychological disturbances.
c. Compatibility with the purpose of study and minimum emotional effect on the
operator.
d. Location should be separate from animal rooms and free from
environmental contaminants.

Tranquilizers have to be administered to larger species such as monkeys, dogs and cats before
an euthanasia procedure.

HUMANE WAYS OF KILLING THE ANIMALS: -

(a) Physical methods
(b) Chemical methods

(a) PHYSICAL METHODS:-

The common method is, breaking the spinal card in cervical region or damaging the
brain itself. This is done in small animals with relatively thin skull. Mice and guinea pigs may be
quickly and painlessly killed by bringing the head suddenly against a hard object such as edge of
sink. But the method required experiences. Breaking the cervical cord can kill birds. The legs are
held in the left hand and the head in the right hand. Then the neck is quickly extended and bent
back with a sharp jerk.

(b) CHEMICAL METHODS:-

(i) Volatile agents: An overdose of some volatile agents such as ether, chloroform, nitrous
oxide etc is commonly employed. Ether alone is too irritant and exciting for large
animals. Chloroform is suitable for all the animals except dog.
(ii) Non-volatile agents: 20% of 5ml/kg of saturated magnesium sulphate by intravenous
route is used.
DISPOSAL OF ANIMALS: -
 The best way of disposing the animal is to burn them in the incinerator. But before this,
make sure that the animal is dead in order to ensure against the possibility of accidentally
burning a live animal. Animal should not be kept in the incinerator unless one of the following
conditions is applied:
(1) The body is cold, still and rigor mortis has set in
(2) The animal has been decapitated
(3) Complete necropsy has been performed
After confirming that the animal is dead, the animal has to be buried deep in the soil with salt.

Expt. No:5
DEMONSTRATE THE EFFECT OF VARIOUS DRUGS ON RABBIT EYE
AIM
The aim of the experiment is to demonstrate the effect of various drugs on rabbit eye.
ANIMALS USED: RABBITS
DRUGS USED:
1. Physostigmine – 5 microgram/ml
2. Atropine – 10 microgram/ml
3. Adrenaline – 1 microgram/ml
4. Ephedrine – 5 microgram/ml
5. Lignocaine – 10 microgram/ml
6. Normal saline (0.9% NaCl)
PRINCIPLE
The parameters like corneal reflex, light reflex and pupillary diameter are studied before
and after adding drug .
Corneal reflex:
It is studied by touching the cornea with a cotton probe. The rabbit immediately closes the eye
lid. Loss of corneal reflex show local sensation is lost.
Light reflex:
It is studied by directing the light rays into the rabbit eye. The pupillary diameter is reduced. The
loss of light reflex denote the loss of parasympathetic control on iris.
Pupillary diameter:
The diameter of the pupillary size is measured by scale. Miotics are drugs which decrease
pupillary diameter. Mydriatics are drugs which increase pupillary diameter
Intra ocular Tension: It is measured by Tonometer. It is the pressure in the aqueous humor. It is
increased in eye disorder Glaucoma. It causes irreversible loss of vision.

PRINCIPLE:
1. Physostigmine decrease the metabolism of Ach by inhibiting the enzyme acetylcholine
esterase. It is miotic drug. It decrease the pupillary diameter by contracting the circular
muscle fibers of the iris muscle. It decrease the IOT. It facilitate the drainage of aqueous
humor. Thus it is used in the treatment of glaucoma.
2. Atropine is anticholinergic drug. It competitively block the muscarnic receptors. It relax
the circular muscle fibers of the iris and cause mydriasis. It abolish the light reflex. It
relax the ciliary muscle and produce cycloplegia(paralysis of accomadation)
3. Adrenaline is mydriatic drug. It contract the radial muscle fibers of the iris. It moderately
decrease the IOT.
4. Ephedrine produces mydriasis like adrenaline.
5. Lignocaine abolish the corneal reflex. It produces loss of sensation. It is an local
anaesthetic drug.

PROCEDURE
Keep the rabbits in a uniformly and moderately illuminated room. The parameters like
corneal reflex, light reflex, pupillary diameter and IOT are cheched on both eyes of the rabbits.
The right eye of the rabbits are kept as control. The drug solutions are instilled in the conjuctival
sac of the left eye by formed gently pulling the lower eye lid .
After five minutes later the corneal reflex, light reflex and pupillary diameter are checked
on both eyes and tabulated.

INFERENCE
Local anaesthetic drugs produces loss of corneal reflex.
Miotics like cholinergic agents decrease the pupillary diameter and decrease the IOT.
Myriatics like anticholinergic agents increces pupillary diameter and abolishes the light reflex
whereas the sympathomimetic agents do not affect the light reflex.

Study of various drugs in rabbit eye

 Miosis and mydriasis
Autonomic innervations of eye

S.N Drugs Before adding drug After adding drug infer

o Right eye Left eye Right eye Left eye ence
cr lr p io cr lr p io cr lr p io cr lr p io
d t d t d t d t
1 Physostigm
ine
2 Atropine
3 Adrenaline
4 Ephedrine
5 Lignocaine
EXPT. NO. 6
STUDY OF EFFECT OF DRUGS ON CILIARY MOVEMENT OF FROG'S
OESOPHAGUS
AIM:
Demonstrate the action of drugs on ciliary movement of frog oesophagus.
Animals: Frog
Drugs and solutions:
Acetylcholine 100µg/ml
Physostigmine 100µg/ml
Atropine 1µg/ml
Frog Ringer
Apparatus: A pair of scissors, forceps, poppy seeds, cotton, droppers, frog board, stop-watch
PRINCIPLE:

The frog buccal cavity and oesophagus is lined with ciliated epithelial cells. Acetyl choline is
released as local hormone and produces ciliary movement by acting through muscarnic
receptors. Cholinergic drug increase the ciliary movement and anticholinergic drugs decrease the
movement.

PROCEDURE

Pith a frog. Slit open the oesophagus from the buccal cavity to the stomach. Wipe the blood
gently using a cotton swab dipped in Frog Ringer solution, proceeding from cephalic to
caudal end. Moisten the surface with Ringer solution. Place two pins at a distance of 2-3 cm.
Place one seed on the groove near the pin at cephalic end. Start the stopwatch and observe the
time taken for the seed to reach the pin at the caudal end. Take 2 such readings and calculate
the average. Repeat the experiment using acetylcholine, physostigmine and atropine. Take
control readings with Frog Ringer between the drugs. Tabulate the results

Result:
 Time taken to travel
S.no drug Inference
1 2 3 mean
1 Saline
2 Acetyl choline
3 Physostigmine
4 Atropine

Ciliary movement of frog’s oesophagus

Expt. No: 7
EFFECT OF CARDIAC STIMULANTS ON ISOLATED FROG'S HEART

AIM: Record the contractions of isolated frog's heart on a kymograph and demonstrate the effect
of cardiac stimulant drugs on it and show the site of action.
Animal: Frog
Drugs and solutions:
1. Adrenaline HCl 10 g/ml
2. Noradrenaline 10 g/ml
3. Isoprenaline 10 g/ml
4. Calcium chloride 10 mg/ml
5. Propranolol HCl 1 mg/ml
6. Frog Ringer

Apparatus: Kymograph, smoked drum, Starling's heart lever, syme’s cannula, , syringes,
needles.
Preparation:
A large sized Frog is pithed . The heart is exposed by dissecting the thoracic cage on the venral
side. Pericardium is removed carefully without injuring the heart. The postcavel vein is identified
by lifting the heart and putting it on the other side. A wet thread is passed below the post cavel
vein and a slit is made on the vein. The syme’s cannula is inserted into the slit and tied firmly.
The entire heart is dissected out from the animal body. The side tube of the venosus. Insert a
curved needle in the apex of the heart and attach it to the transducer / Starling's heart lever.
Record the contractions.
PROCEDURE
1. Note the normal heart rate, by counting each upstroke (systole) and downstroke (diastole)
of the moving drum together as one beat for 1 minute, force of contraction (by measuring the
amplitude or height of the contraction from the baseline with a scale).
2. Inject 0.2 ml of drugs 1-4 in succession (cardiac stimulants) in the tube through which the
heart is being perfused and record the responses. A control reading (without addition of any
drug) should be taken before and after each drug response. The next drug response should be
recorded only after the heart rate has returned to the approximate original value. In case the heart
stops because of systolic or diastolic arrest produced by a cardiac depressant the drum should be
stopped and re-started only when the heart is contracting. In case adequate response is not
observed use a higher dose.

3. Inject 0.2ml of propranolol (depressant) and note its response. inject adrenaline (same dose as
injected previously) and note whether its effect is adequately blocked. In case sufficient
blockade is not obtained repeat the procedure with 0.4ml propranolol.
4. Inject calcium chloride immediately after adrenaline effect has been blocked, and note
whether its effect has been blocked or not.
5. Tabulate your observations.
OBSERVATION
s.no drug Heart rate Force of contraction inference
1 Basal
2 Adrenaline
3 Calcium chloride
4 Propronolol
5 Propronolol + adrenaline
6 Propronolol + CaCl2

RESULT:

Expt. No:8
EFFECT OF CARDIAC DEPRESSANTS ON ISOLATED FROG'S HEART
AIM: Record the contractions of isolated frog's heart on a kymograph and demonstrate the effect
of cardiac depressant drugs on it and show the site of action.
Animal: Frog
Drugs and solutions:

1. Acetyl choline 10 g/ml

2. Potassium chloride 10 mg/ml
 3. Atropine sulphate 100 g/ml
4. Frog Ringer
Apparatus: Kymograph, smoked drum, Starling's heart lever, syme’s cannula, , syringes,
needles.
Preparation:
A large sized Frog is pithed . The heart is exposed by dissecting the thoracic cage on the venral
side. Pericardium is removed carefully without injuring the heart. The postcavel vein is identified
by lifting the heart and putting it on the other side. A wet thread is passed below the post cavel
vein and a slit is made on the vein. The syme’s cannula is inserted into the slit and tied firmly.
The entire heart is dissected out from the animal body. The side tube of the venosus. Insert a
curved needle in the apex of the heart and attach it to the transducer / Starling's heart lever.
Record the contractions.
PROCEDURE
1. Note the normal heart rate, by counting each upstroke (systole) and downstroke (diastole)
of the moving drum together as one beat for 1 minute, force of contraction (by measuring the
amplitude or height of the contraction from the baseline with a scale).
2. Inject 0.2 ml of drugs 1&2 in succession (cardiac depressants) i.e. acetylcholine and
potassium chloride after taking control readings in between drug responses. Note also the condi-
tion of the heart during diastole arrest.
3. Inject 0.2 ml of atropine and note its response. Normally no response is seen because it is an
in vitro preparation and more over atropine has no intrinsic activity of its own. Inject
acetylcholine (same dose as given earlier) and note whether effect is completely blocked. In case
sufficient blockade is not obtained, repeat the same procedure with 0.4ml of atropine.
4. Finally inject potassium chloride after the effect of acetylcholine has been blocked by
atropine and note whether the effect is blocked. There should be no blockade of KCl effect.
5. Tabulate your observations.

OBSERVATION
s.no drug Heart rate Force of contraction inference
1 Basal
2 Acetyl choline
3 Potassium chloride
4 Atropine
5 Atropine + ACh
6 Atropine + KCl

RESULT:

Expt. No. 9
DEMONSTRATE THE EFFECT OF DRUGS ON SPONTANEOUS MOTOR ACTIVITY
BY USING ACTOPHOTOMETER.
AIM
To study the effect of drugs on spontaneous motor activity and to evaluate the nature of
drugs on central nervous system.
MATERIALS USED
Animal:Rats
Drug solution: Amphetamine 1mg/ml, Phenobarbitone 1mg/ml.
Actophotometer.
PROCEDURE
Rat weighing approximately about 150 gms is placed inside the Actophotometer for a
period of 5 mins duration. The reading in the counter is noted as initial reading. The drug
phenobarbitone solution at the dose of 30 mg/kg is injected by intra peritonial route of
administration. After30 mins the animal is placed inside the actophotometer for the another 5
minutes duration. The reading is noted as final reading. The % of CNS depressant activity is
calculated.

Rat weighing approximately about 150 gms is placed inside the Actophotometer for a period of 5
mins duration. The reading in the counter is noted as initial reading. The drug amphetamine
solution at the dose of 30 mg/kg is injected by intra peritonial route of administration. After 30
mins the animal is placed inside the actophotometer for the another 5 minutes duration. The
reading is noted as final reading. The % of CNS stimulant effect is calculated.
REPORT
The spontaneous activity is decreased with phenobarbitone . Thus the drug is CNS
depressant. The percent decrease in CNS activity is

The spontaneous activity is increased with amphetamine . Thus the drug is CNS stimulant. The
percent increase in CNS activity is

OBSERVATION
S.No. DRUG TREATMENT Activity Inference
1 Control
2 Amphetamine
3 Phenobarbitone
 Effect of drugs on Rat’s isolated phrenic nerve diaphragm preparation.

AIM: To study the effects of drugs on the twitches of the Striated muscle of the Diaphragm in
response to electrical stimulation of the phrenic nerve.
REQUIREMENTS: Electrodes, Pipette, Kymograph, Organ bath, Carbogen.
PHYSIOLOGICAL SALT SOLUTION: Kreb’s Henseleit solution.
DRUGS: Tubocurarine, suxamethonium and neostigmin
PRINCIPLE: The Rat phrenic nerve diaphragm preparation is mainly useful for the screening
of skeletal muscle relaxants. The phrenic nerve extends from upper cervical segments of the
spinal cord and their axons extend all the way to the diaphragm. Rhythmic phrenic nerve activity
from the central nervous system is a source of diaphragm electrical activity. The phrenic nerve
supply is the only nerve supplying to the diaphragm. Both the sides receive a nerve running from
the cervical vertebrae to the side of the subclavian artery on both the sides of the body.

Drugs like curare which acts at neuromuscular junction and block nicotinic receptors thereby
reducing the effect of phrenic nerve stimulation. Anti-cholinesterase agents potentiates the
effects of acetylcholine. Effect of curare is reversible and doses can be applied every 10min and
the standards and unknown solutions compared accurately with one another. This preparation
can therefore be used for the Bioassay of curare. This can also be used to study the effects of
drugs on acetylcholine release(aminoglycoside antibiotics and botulinum toxin).

Suxamethonium produces depolarizing type of neuromuscular blockade. It causes persistant

activation of excitatory neuromuscular acetylcholine nicotinic receptors. It cause transient
twitching of muscle and then paralysis. It is not reversed by anticholinesterases.

Tetrodotoxin is a sodium channel blocker. It decreases the conduction of nerve as well as muscle
stimulation and not reversed by neostigmin.

PROCEDURE: Rat is killed by blow on the head. Throat is cut and is left to bleed as much as
possible. The fur and the skin are removed from the middle of the chest, and the left phrenic
nerve is dissected and removed by inserting blunt scissors to the chest wall and the ribs are cut
through alongside the base of the sternum and the thorax is examined for the chest to be clear on
the inside. The phrenic nerve is attached to the membrane. This must be dislodged by raising the
wall or by gently prodding with the blunt forceps. The ribs are cut through round towards the
animal’s flank. The upper part of the thorax removed completely and the phrenic nerve can be
seen clearly which runs from the diaphragm right up to the thymus gland.
An incision is made in the abdominal wall just below the diaphragm and two cuts are made and
this produces a fan shaped segment of the muscle and which has a junction of the phrenic nerve
about 2 to 4 mm from the apex and this segment is lifted gently and no attempt should be made
to dissect the nerve completely clean from the membrane attached to it.
This nerve is cut just below the thymus and the preparation is transferred to a flat dish containing
Kreb’s solution.
A thread attached securely to a tendonous part which forms the apex. A second thread of
preferably with a different color is attached to the cut end of the nerve with a knot and the
membrane which attaches to the muscle should be cut off, so the nerve is free from the
membrane. The preparation should be then hanging securely partly submerged while the thread
attached to the nerve and then the preparation is lowered fully and the nerve is drawn gently into
the electrode and care must be taken. The nerve should not be subjected to any tension and the
nerve is aerated with 95% of oxygen and 5% of CO2 (carbogen) and the experiment is carried out
at 370C in a 30-40 mm diameter bath and the volume of fluid in bath is to be around 40 ml. the
volume of the fluid is maintained at the same volume ensuring the nerve to be moist and the
effect of various drugs like neuromuscular blockers and the contraction of the muscle is recorded
with a light spring loaded lever with a sideway writing point and the nerve is usually stimulated
at the rate of 12 shocks/min and the bigger stimulation can be obtained by increasing the duration
of the stimulus and it is necessary to arrange the time cycle for administration of the drug.

NOTE: The effect observed on this preparation is neuromuscular block and it is necessary to
arrange the time cycle so that the drug has plenty of time in which to act; it is likewise necessary
to allow plenty of time for recovery. If the drug is allowed 3 min to produce its effect, it will
probably not be possible to add a dose more than once in 10min so, after recording the normal
contraction for 1min and the action of drug for 3 min, it is necessary to allow 6 min time for
recovery after the drug has been washed out. If the drug is allowed longer to act (5 min or even 8
min), smaller doses of drug may be used and the time necessary for recovery is not unduly
longer.
The above discussion would give a time cycle as follows:
0 min-start kymograph
1 min-add drug
6 min-stop kymograph and wash preparation
8 min-wash the preparation
10 min- start kymograph
But sometimes it may be necessary to allow longer free recovery if the contraction have not
returned to their original height or size.
OBSERVATION
S.No drugs Muscle tone Inference
1 Normal
2 d-Tubocurarine
3 suxamethonium
4 tetrodotoxin
5 Neostigmin
6 Neostigmin + d- Tubocurarine
7 Neostigmin + suxamethonium
8 Neostigmin + tetrodotoxin
RESULT:

EXPT NO:
EFFECT OF ADRENERGIC DRUGS ON MEAN BLOOD PRESSURE, HEART RATE
AND RESPIRATIOY RATE OF DOG AND DALES VASO MOTOR REVERSAL
AIM: To demonstrate the effect of adrenergic agonist and antagonist on mean blood pressure,
heart rate and respiratioy rate of dog and to demonstrate the Dale’s vaso motor reversal.
EXPERIMENTAL SET UP:
A dog is weighed and anaesthetized using intravenous chloralose (100 mg/kg of body weight). It
is fixed on a dog table in supine position. Right femoral vein is cannulated with a catheter to
inject drugs. Its neck is dissected to expose carotid arteries and vagus nerves. Left common
carotid is inserted with an arterial cannula connected to a mercury manometer or to a pressure
transducer, which is connected to a polyrite/physiograph. A chart recorder or a kymograph is set
up to receive signals from the polyrite/physiograph to produce graphical representation of
changes in blood pressure. The left side vagus is cut into central and peripheral ends for applying
electrical stimulation later in the experiment. The right common carotid is identified and exposed
for later use.
Drugs are injected (one by one) into the femoral vein and BP is recorded on the chart.
The heart rate (beats/min) is counted and noted on the chart. Drug administration is also marked.
Principle:
Nature Drug name Remarks
(Dose in µg/kg)
for BP exp in
dogs
Agonist Epinephrine: Also known as adrenaline. It stimulates the alpha and beta
Dose : 2 adrenergic receptors. Conventional doses will increase the BP
Range : 1 - 3 followed by a short fall before reaching the basal level
(biphasic response due to alpha and beta receptor responses).
To see the beta action alone, use a low dose such as 0.1 µg per
kg of body weight. The heart rate decreases due to vagal
reflex.
Agonist Norepinephrine Also known as noradrenaline. It stimulates mainly the alpha
Dose : 3 and beta1 receptors. The heart rate is generally reduced due to
Range : 2 - 5 vagal reflex in response to increased BP. Hence if the dog is
pretreated with a muscarinic blocker (atropine),
norepinephrine will show an increase in the heart rate.
Agonist Isoprenaline Also known as isoproterenaol. It is a potent, non-selective beta
Dose : 3 adrenergic stimulant. It increases the systolic BP (sometimes it
Range : 2 - 5 remains unchanged) but decreases the diastolic BP. Because
the decrease is more pronounced than the increase, the mean
arterial pressure typically falls.
Agonist Ephedrine It acts on both alpha and beta receptors and in addition
Dose : 100 enhances the release of norepinephrine from sympathetic
Range : 100 - 200 neurons. It increases the BP and heart rate.
Antagonist Phentolamine This drug, an alpha blocker, reduces BP and also affects the
Dose : 1000 alpha components of other drugs.
Antagonist Propranolol It is a beta blocker which reduces BP and heart rate. It affects
Dose : 1000 the beta components of other drugs.
 BIOASSAY OF HISTAMINE USING GUINEA PIG
ILEUM
AIM:
To determine the concentration of the given unknown sample of histamine by four point
bioassay using guinea pig ileum.
REQUIREMENTS:
 Animal – guinea pig [400g body weight]
 Drug - histamine stock solution [1mg/ml.]
 Sample solution.
  Tyrode solution.
 Student’s organ bath.
DISCUSSION:
Histamine is an autocoid having proformed physiological effect in the body. Tissues rich in
histamine are skin, gastric and intestinal mucosa, lungs, liver and placenta. Besides the triple
response caused by it, histamine has spasmogenic response on intestinal smooth muscle by
acting on H1 receptor it causes the contraction of intestinal smooth muscle. Guinea pig ileum is
highly sensitive to histamine and thus is very commonly used for the assay of histamine.
Overnight fasted animals are used to get better response of drugs on intestinal smooth muscle.
Histamine acts on histaminergic receptors [H1.H2, H3] which are Gprotein coupled
receptors. H1 receptors acts through phospholipase: IP3-DAG pathway while H2 receptors acts
through adenylyl cylase: c AMP pathway. H3 receptors has been described as presynaptic
receptor present in histamine containing nerve terminals which regulates the release and
synthesis of histamine through an auto inhibitory feed back mechanism by decreasing calcium
influx to the cell.
PROCEDURE:
ISOLATION OF THE TISSUE:
a. Guinea pig was fasted over night and sacrificed by head blow and carotid
bleeding.
b. The abdomen was cut open to trace the ileocecal junction.
c. The terminal ileum was cut after discarding 10 cm of the tissue nearest to the
ileocecal junction [because of the presence of excitatory alpha adrenoceptors near
the junction] and immediately placed in watch glass containing tyrode solution.
d. The mesentry was trimmed and the contents of the ileum were cleaned by pushing
the tyrode solution into the lumen of the ileum.
e. A piece of ileum of about 2-3 cm long was taken and thread was tied to top and
bottom ends without closing the lumen.
f. The tissue was mounted in organ bath containing tyrode solution maintained at
32-35 degree centigrade and bubbled with oxygen or air.
g. The tension of 500mg was applied and the tissue was allowed to equilibrate for 30
mins before the drugs were added.
 h. The recording of the drug response was started after the stabilization of the tissue.
The response was taken in a cyclic way. One cycle consists of 30 seconds of
recording in the absence of drug and 30 seconds of response in the presence of
drug. After each cycle, the tissue was washed 2 to 3 times. After obtaining the
ceiling response the tissue was again stabilized for half an hour. Then the drug
response for the sample of unknown concentration was taken.
i. The two responses falling in straight line portion of the DRC was taken giving
response 1:2.
1. That is, S1 & S2, T1&T2.
j. The response was taken in randomized manner to avoid the sequential error.

S1 S2 T1 T2
S2 T1 T2 S1
T1 T2 S2 S1
T2 S1 S2 T1

CALCULATION:
1) The response which is high is measured for each dose and an average response is taken
for each dose.
2) The potency of the drug is calculated by following formula.
= X1 / Y1 antilog [T2-S2 / T2 -T1 + T1-S1 / S2-S1 x log X2 / X1]
Where, X1 = lowest dose of the standard
Y1= lowest dose of the test.
X2 = highest dose of the standard.
T2 = the response produced by the highest test dose.
T1 = the response produced by the lowest test dose.
S1 = the response produced by the lowest standard dose.
S2 = the response produced by the highest standard dose.
After knowing potency of the unknown drug the concentration is determined by the following
formula.
Concentration = std. concentration x potency of unknown drug x dilution factor.
REPORT:
The concentration of the given unknown sample of histamine was found to be
………………