Index
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Sr.
# |
Experiments |
Signature |
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1. |
INTRODUCTION TO
EXPERIMENTAL PHARMACOLOGY |
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2. |
STUDYING OF ANIMAL’S ETHICS AND GOOD LABORATORY
PRACTICE |
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3. |
STUDY
OF DIFFERENT LABORATORY ANIMALS AND THEIR APPLICATIONS IN EXPERIMENTAL
PHARMACOLOGY |
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4. |
STUDY
OF DIFFERENT ROUTS OF ADMINISTRATION IN ANIMALS |
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5. |
TO STUDY IN-VIVO ANTIDEPRASANT ACTIVITY ON MICE |
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6. |
TO STUDY ANTI-CONVULSANT ACTIVITY ON MICE |
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7. |
TO STUDY SEDATIVE ACTIVITY ON
MICE |
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8. |
TO STUDY ANXIOLYTIC ACTIVITY ON MICE |
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9. |
EVALUATION OF ANALGESICS BY CHEMICAL METHOD |
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10. |
MONITORING OF ADVERSE DRUG REACTION |
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11 |
STUDY OF GENERAL PRINCIPLES IN THE MANAGEMENT OF SOME COMMON POISONINGS |
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EXPERIMENT
NO. 01
INTRODUCTION
TO EXPERIMENTAL PHARMACOLOGY
Aim: To study the advantages,
disadvantages, rout of drug administration and experimental uses of different
most commonly used laboratory animals, in order to be able to understand
pharmacological research papers.
Background Information:
Pharmacology: is the branch of science
which deals with the study of drugs.
Objectives of Pharmacology:
1. To find out the therapeutic agent suitable for human
use.
2. To study the toxicity of the drugs.
3.
To study the mechanism and site of action of drugs.
Experimental Pharmacology: It is that
branch of pharmacology which deals with the effect of drugs on living system.
It can be studied under heads:
a) Preclinical Pharmacology: dealing
with effects of drugs on animals. The experiment can be carried out in whole
animal (in vivo) or in isolated organs (in vitro).
Bioassay (comprises of “bio” means living material and “assay” means
assessment at laboratory): defined as comparative assessment of relative
potency of a test compound (T) to a standard compound (S) on any living animal
or biological tissue.
b)
Clinical Pharmacology: dealing with effect of
drugs on human livings.
EXPERIMENT
NO. 02
STUDYING OF ANIMAL’S
ETHICS AND GOOD LABORATORY PRACTICE
OBJECTIVES:
At
the end of the session, the student shall be able to:
1.
Realize the importance of using animals for pre-clinical testing.
2.
Justify the need for adhering to proper standards of maintenance and care in
the use of animals for research and teaching.
3.
Understand the principles of good laboratory practice and its importance in the
conduct of experiments.
The
class is divided into two batches. One batch will be taken to the central
animal house for a visit. This will be followed by a debate on the topic
"The use of animals in research and teaching is justified", which in
turn will be followed by a role play on good laboratory practice (GLP).
Visit
to animal house – 30 minutes
Preparation
time for debate – 30 minutes
Debate
– 1 hour
Role
play 15-20 minutes
Concluding
remarks by faculty – 10-15 minutes
Batch
A will defend the use of animals in research and teaching. Batch B will speak
against the use of animals in research and teaching. Use the given printed
material for preparation in addition to your own views. Select three speakers
from each team who will speak for five minutes each. Preparation time 30
minutes. Each batch will visit the animal house and discuss the following
points at the animal house:
1.
Maintenance of stock
2.
Separate housing of species
3.
Separation of pregnant, just delivered, pups, sick animals.
4.
Maintaining room temperature
5.
Feeding practice (pellets, greens etc.,)
6.
Need to use inbred strains in experiments
7.
Disposal of dead animals
Five
or six students will perform a role-play to highlight the importance of a code
of conduct in the laboratory. The role play will focus on the rules which are
usually broken by students, for e.g. not wearing aprons, teasing animals,
lifting mice by tails and walking up and down, dropping animals, rinsing
syringes onto the floor, discarding excess drug from dropper onto the table, cooking
up data, copying from senior's record etc.
EXPERIMENT
NO. 03
STUDY
OF DIFFERENT LABORATORY ANIMALS AND THEIR APPLICATIONS IN EXPERIMENTAL
PHARMACOLOGY
Laboratory Animals: These are those
animals which can be used and maintain in the laboratory under suitable
conditions. The most common laboratory animals are; rat, mice, guinea pig,
rabbit, and hamster. Other animals used for experimental purpose are cat,
dog, monkey, pigeon etc.
Animal
selection is based on the following criteria:
1. Size: smaller animals
are preferred because they are easy to handle & less quantity of drug is
required.
2. Availability: animals which are
commonly available should be selected e.g. mice, rats, and rabbits.
3. Biological properties: animals differ in their biological response
to the studied drug.
- Guinea pig are 500 more times
sensitive to histamine than are rats & mice.
- The rat heart is known to be very resistant to
cardiac glycosides.
Characteristics and Advantages of Different
Animals:
Mouse: (Adult weight: 20-25 gm., age suitable for experiment,
2 month)
1. The smallest laboratory
animal.
2. Less drug required.
3. Easy to handle.
4. Cheap.
Most commonly used experimentally in studies related
to genetics & cancer research.
Rat: (Adult wt. 180-200 gm., age suitable for most of the
experiment 1.5 months)
1. The most commonly used
laboratory animals.
2. The most standardized of all
laboratory animals.
3. Vomiting center is absent and so drug can be
administered orally.
Used experimentally for studies on isolated tissue
preparation like uterus, stomach, heart, etc.
Guinea pig:
1. Docile animals.
2. Highly sensitive to
histamine, penicillin.
3. Required exogenous vitamin C in diet.
Preferably used in Anaphylactic and immunological
studies.
Rabbit: (Adult weight 1.5-3.0 Kg. age suitable for experiment
5-6 months)
Standard animal for pyrogen testing in parenteral
preparation.
EXPERIMENT
NO. 04
STUDY
OF DIFFERENT ROUTS OF ADMINISTRATION IN ANIMALS
In
`Practical Pharmacology`, there are seven general routes of drugs
administration.
These
will be mentioned below together with the suitable technique used in common
laboratory animals (except the inhalation and rectal routes as they are not
discussed in this manual).
A: Oral administration (Oral Gavage)
1. Rabbits:
Liquids are administered by means of catheters using a
mouth gag. Insert the mouth gag while the animal is in the sitting position, by
squeezing it, lubricate the catheter with mineral oil and then introduce it
through the hole in the mouth gag and insert it to approximately 20-25 cm. To
verify that the catheter is in the esophagus and not in the trachea, dip the
end of the catheter into a beaker containing water. Bubbling indicates wrong
position.
Solid dosage forms are administered to the animal in
sitting position by means of a plastic tube with a plunger. The tube is pressed
towards the pharynx. The plunger pushes the tablet or capsule into the
esophagus.
2. Rats, mice:
Solutions
and suspensions are administered to rats and mice by means of blunt large
needle oragastric tube (Figure 1).
Figure 1: Oral administration to rats
Disadvantages of oral administration include:
1-
The drug given orally is exposed to first pass effect.
2-
Length of GIT (late effect).
3-
Food status affected the absorption of administered drug.
4-
Acid labile drugs are not suitable to be given via this route.
B: Subcutaneous (S.C.)
The
drug is to be introduced directly underneath the skin.
1- Subcutaneous (S.C.) injection in mice and rats:
The
most usual site for injection is over the shoulders, into the loose skin over
the neck (see figure 2) but other sites with loose folds of skin can also be
used. When repeated doses of material are needed, varying the site of injection
can help reduce the likelihood of local skin reactions.
Note;
an example of a drug given subcutaneously is insulin: used for gradual release
of medication because of limited blood flow.
Figure 2: Subcutaneous
injection in mice
C: Intravenous injection
1.
Rabbits:
Shave
the hair over the ear (marginal vein), figure 3. Rub vigorously the
marginal ear vein with a piece of cotton soaked in xylol to dilate and distend
the vessels. Verify position of needle in gentle aspiration. After injection,
press cotton on to the wound by means of a clamp.
Figure 3:
Intravenous injection in Rabbits
2.
Rats and mice:
-Intravenous administration of material
can be technically difficult. The rats are to be held firmly by wrapping them
in a cloth or by placing them in a suitable animal holder (restraining device),
figure 4.
- It is common practice to stimulate
dilation of the tail veins in mice either by placing them in a warmer
environment (e.g. at 28-30 ºC) for up to 30 minutes, or by placing the tail in
warm (30-35 ºC) water for 5 to 10 seconds.
-
If the needle is not in the vein, resistance will be felt while injecting and
blanching will be observed in the surrounding connective tissue.
Figure 4: Intravenous injection in
mice and rats
D: Intraperitoneal
injection (I.P)
This is a common
method to introduce drugs into animals. The drug is injected into the
peritoneal cavity in the lower part of the abdomen, where absorption is rapid
(Figure 5).
Figure 5: Intraperitoneal injection
in mice
Advantages of this route of administration include:
1-
Large surface area for absorption.
2-
High blood perfusion.
3-
Thin layer for absorption.
E: Intramuscular Injection (I.M)
In
the rat and other small rodents, the very small muscle mass makes intramuscular
administration both technically difficult and painful for the animal because of
the distension of the muscle.
-
If intramuscular injections are necessary, they can be made into the front or
back of the thigh in all small rodents (Figure 6).
One
must always pull on back the plunger to make certain the needle is not in the
vein.
Figure 6: Intramuscular Injection in
rats
EXPERIMENT
NO. 05
TO
STUDY IN-VIVO ANTIDEPRESSANT ACTIVITY ON MICE
THEORY
What
is Depression: A serious neuronal disorder which
includes intense feeling of sadness, hopelessness, social isolation, altered
sleep patterns and suicidal thoughts is called depression.
Apparatus:
1. Forced
Swim Test (FST)
2. Tail
Suspension Test (TST)
Requirements:
1. Swiss albino mice (25-30 g weight).
2. Normal
Saline (N.S)
3. Fluoxetine
4. Video
Camera
1. Procedure
for FST
FST formerly used for evaluating antidepressant
activity, was applied. The test model consist of a
transparent glass cylinder (30 cm high × 20 cm diameter) filled with water up to 20 cm depth and maintained at 25 ± 2°C.
Mice were divided into 2 groups (each having 2 animals). Group-1 received an
i.p. dose of fluoxetine (20 mg/Kg) and Group-2 received an i.p. dose of normal
saline (10 mL/Kg). Thirty min. later to saline and fluoxetine treatment, each
mouse was allowed to swim in tank for 5 min. and duration of immobility;
swimming and climbing were recorded via digital video camera. Animal was
considered to be immobile when it remained floating over water without
struggling, making only little movements to keep its head above water. Swimming
is large horizontal movements of mice with forepaws, displacing mice body
around cylinder while climbing is vigorous vertical movements of mice with
forepaws, directed against wall of tank, leading to displacement of mice body
around cylinder. The decrease in immobility time while increase in swimming
and/or climbing behaviors show antidepressant activity.
Result.
The group-1 mice (fluoxetine), swum for
3.5 mints and were immobile for 1.5 mints as compared to group-2 (N. Saline),
swum for 2.0 mints and immobile for 3.0 mints. This proved that fluoxetine has
antidepressant activity.
2. Procedure
for TST
The Previously described tail
suspension test was followed for measuring immobility time. In this test, mice
were suspended 50 cm above floor on table’s edge by adhesive tape placed about
1cm from tip of tail. Mice were divided into 2 groups (each having 2 animals). Group-1
received an i.p. dose of fluoxetine (20 mg/Kg) and Group-2 received an i.p.
dose of normal saline (10 mL/Kg). Thirty min. later to saline and fluoxetine
treatment, each mouse was suspended and duration of immobility was recorded for
5 min. via digital video camera. Animal will be considered immobile when it
becomes motionless and hanged passively. Drug that decrease immobility time
will show antidepressant activity.
Result.
The group-1 mice (fluoxetine), were
immobile for 2.5 mints and group-2 (N. Saline), were immobile for 4.0 mints.
This proved that fluoxetine decreased immobility time of mice as compared to
normal saline group and hence showed antidepressant activity.
EXPERIMENT
NO. 06
TO
STUDY ANTI-CONVULSANT ACTIVITY ON MICE
THEORY
What
is Convulsion: A medical condition where body
muscles contract and relax rapidly and repeatedly resulting in an uncontrolled
shaking of the body is called convulsion.
Requirements:
1. Mice
(25-30 g weight)
2. Normal
Saline (N.S)
3. Diazepam
(DZP)
4. Pentylenetetrazole
(PTZ)
5. Plastic
Cage
6. Video
Camera
Procedure:
Mice
were divided into 2 groups (each having 2 animals). Group-1 was (i.p) injected
diazepam (1 mg/Kg) and Group-2 was injected (i.p) with normal saline (10
mL/Kg).
Thirty
min. later to saline and diazepam treatment, an i.p. dose of PTZ (90 mg/Kg) was
given to all animals and each animal was observed for onset time of myoclonic
jerks and tonic-clonic seizures, as well as duration of tonic-clonic seizures
for 30 min via digital video camera.
Drugs
that delayed onset of myoclonic jerks, tonic-clonic seizures and/or shortened
duration of tonic-clonic seizures are considered to exhibit anticonvulsant
effect.
The
animals were also observed for mortality (% mortality = number of mice dead
after convulsion/total number of mice used × 100).
Result.
The group-1 mice (diazepam), delayed onset
of myoclonic jerks, tonic-clonic seizures and shortened duration of
tonic-clonic seizures as compared to group-2 (N. Saline) and hence diazepam
showed anticonvulsant activity in below figure.
EXPERIMENT
NO. 07
TO
STUDY SEDATIVE ACTIVITY ON MICE
THEORY
What
is Sedation: Reduction of irritability or
agitation by administration of sedative drugs, generally to facilitate a
medical procedure or diagnostic procedure is called sedation.
Requirements:
1. Mice
(25-30 g weight)
2. Normal
Saline (N.S)
3. Diazepam
(DZP)
4. Thiopental
Sodium
Procedure:
Thiopental-induced
sleeping test was used for determining sedative activity. Mice were divided into 2 groups (each having 2 animals).
Group-1 was (i.p) injected diazepam (3 mg/Kg) and Group-2 was injected (i.p)
with normal saline (10 mL/Kg).
Thirty
min. later to saline and diazepam treatment, an i.p. dose of thiopental sodium
(50 mg/Kg) was given to all animals for induction of sleep. As thiopental
sodium was injected, mice were placed in a test arena (plastic cage) for
observing the onset and duration of sleep.
Drug
that decrease the onset of sleep while increase the duration of sleep will show
sedative activity.
Result.
The group-1 mice (diazepam), decreased the
onset of sleep while increased the duration of sleep and hence showed sedative
effect as shown in below figure.
EXPERIMENT
NO. 08
TO
STUDY ANXIOLYTIC ACTIVITY ON MICE
THEORY
What
is Anxiety: Fear of unknown having symptoms such
as uneasiness, tachycardia, sweating, trembling and palpitation is known as
anxiety.
Apparatus:
1. Elevated
Plus Maze Model (EZM)
2. Light
Dark Box Maze Model (LDB)
Requirements:
Two Swiss Albino Mice of 25-35 gm weight for each
model
Chemicals:
Diazepam (DZP), Normal Saline (N.S)
Model
No. 1
Elevated
Plus Maze Model (EPM)
1. Elevated,
plus shaped (+) apparatus with two open arms across from each other and
perpendicular to two closed arms is called EPM. Two open arms have small boundary
walls to prevent falling of mice whereas two closed arms have high wall to
enclose the arm. The entire apparatus is 50cm above the floor.
2. Two
mice will be used for EPM model
3. Mice-1
= DZM 1mg/Kg (I.P)
Mice-2
= N.S 10ml/Kg (I.P)
4. After
30 minutes of treatment, each mouse was allowed to explore EPM model for 5
minutes.
5. Following
parameters were recorded by a video camera
·
No of entries in Open
Arms
·
No of entries in Closed
Arms
·
Time spent in Open Arms
·
Time spent in Closed Arms
6. Drug
that increase No of entries and Time spent in open arms while decrease No of
entries and Time spent in closed arms will show anxiolytic activity.
Result.
The mice-1 (diazepam), increased No of
entries and Time spent in open arms while decreased No of entries and Time
spent in closed arms as compared to mice-2 (N. Saline)., hence diazepam showed
anxiolytic effect.
Model No. 02
Light Dark Box Maze Model
(LDB)
1. The
LDB apparatus has two compartments, the light compartment is 2/3 of the box and
is brightly lighted and open. The dark compartment is 1/3 of the total box and
is covered and dark. A hole of 7cm connects the two compartments.
2. Two
mice is used fo LDB model
3. Mice-1
= DZP 10mg/Kg (I.P)
Mice-2
= N.S 10ml/Kg (I.P)
4. After
30 minutes of treatment, each mouse was allowed to explore LDB for 5 minutes.
5. Following
parameters were recorded by a video camera:
·
No of entries in Light Compartment
·
No of entries in Dark
Compartment
·
Time spent in Light
Compartment
·
Time spent in Dark
Compartment
6. Drug
that increase No of entries and Time spent in Light Compartment while decrease
No of entries and Time spent in Dark Compartment will show anxiolytic activity.
Result.
The mice-1 (diazepam) increased No of
entries and Time spent in Light Compartment while decreased No of entries and
Time spent in Dark Compartment as compared to mice-2 (N. Saline), hence
diazepam showed anxiolytic effect.
EXPERIMENT
NO. 09
EVALUATION OF ANALGESICS BY CHEMICAL METHOD
OBJECTIVES:
At
the end of the practical class, the student shall be able to:
1.
Calculate and measure the exact quantity of drug to be injected to the animal.
2.
Administer the drugs by the subcutaneous and intraperitoneal
routes to mice.
3.
Identify and record chemically induced abdominal constrictions in mice.
4.
Tabulate observations and draw suitable inferences from the experiment.
Chemical Method: Acetic acid induced abdominal
constrictions
Animals: 2 Mice (if female, non-pregnant)
Apparatus:
Syringe (with 100 divisions) 26g needle
Drug
& Solutions:
Morphine sulphate 1mg/ml, acetic acid 0.6%, Saline
Procedure
Weigh 2
mice (in gm) and number them. Pre-treat one mouse with morphine
5mg/Kg sc and the other with saline (0.1ml) sc. Note time of injection.
30 minutes after injection of drug/saline, inject 0.3ml of 0.6% acetic
acid i,p. to
each mouse with 26g needle. Place the mouse on the top of a stool
for better
observation. Observe the no of
abdominal constrictions (stretching syndrome) occurring in the next 15 minutes. Record
each observation and then total. One abdominal
constriction is
taken as the complete movement
from side to side (both sides). Observe only one mouse at a
time and note the position of the tail in the mouse treated with morphine.
Number of abdominal constrictions to be noted for 15
minutes from the time of administration of acetic acid. Tabulate your observations.
Pool the data
from other groups, tabulate the data,
calculate mean and SD and use appropriate statistical tests (unpaired Student’s
t test) and
draw inferences based on the statistical
analysis.
EXPERIMENT
NO. 10
MONITORING OF ADVERSE DRUG REACTION
OBJECTIVES:
At the end of the
practical group work, the student shall be able to:
1. Define ADR
Monitoring and list the sources of ADR reports.
2. Appreciate the
importance of ADR monitoring.
3. Identify which
adverse effects should be reported and why.
4. Report an ADR
to a monitoring centre.
1. A faculty
member will give an introduction for 15 minutes on history (Thalidomide,
sulphanilamide, SMON) and current problems (how ADRs may lead to withdrawal of
drug from the market, dosage change or restricted prescription etc.,)
2. Group tasks
will be distributed. About one hour will be allocated for the group tasks. At
the end of one hour a plenary session will be held for one hour where different
groups will present their findings.
GROUP TASKS
Group No.1
1. List the
sources of ADR reports and the types of ADRs likely to be revealed from them.
2. What are the
number of patients to be exposed to a given drug to be able to detect various
ADRs with varying incidences, and various background incidences of disease.
Group No.2
1. Prepare an ADR
monitoring form and indicate the need for each component.
Group No.3 & 4
(The teacher will
give situations of a drug producing an ADR. Each group will be given 3-4
situations. Answer the following questions)
a) Will you report this ADR? If yes
why? if no why?
b) Will you decallenge? If yes why? if no why?
c) Will you rechallenge? If yes why? if no why?
Group No. 5 &
6
Fill up the ADR
monitoring forms based on the information available in the hospital record of a
patient. You are provided with (a) A patients' hospital record (given below)
(b) ADR monitoring forms from the JIPMER centre (Appendix - 2).
PATIENT'S
HOSPITAL RECORD
Name of the patient:
Shiv Kumar
Age : 30 years
Sex
: Male
Height : 6 feet, Weight :
60 Kg.
Hospital registration No: 25098
Date : 1.9.98
Diagnosis : Essential hypertension
General physical examination: Patient healthy, well oriented
in time & space, no jaundice, JVP normal
Vitals: Pulse - 70/min
Respiratory rate - 16/min
temp. afebrile
BP 150/100 mm Hg (rt. arm supine)
Systemic examination: Resp.system – normal CVS - normal
Past history _ No history of allergy No family history of diabetes or hypertension
Lab. investigation : Hb - 16 gm% TLC - 6000/cu mm Serum cholesterol- 200 mg %
Treatment : given on 1.9.98
Tab. Prazosin (minipress) 1mg
B.D.
Tab. Hydrochlorothiazide 25 mg O.D.
After one week treatment patient felt dizzy and fell
down in the bathroom and sustained superficial scalp injury. The dose of
Prazosin was reduced to 0.5 mg after checking the BP standing. (standing BP 100/70 mm Hg).
EXPERIMENT
NO. 11
STUDY OF GENERAL
PRINCIPLES IN THE MANAGEMENT OF SOME COMMON POISONINGS
OBJECTIVES:
At
the end of the session, the student will be able to:
1.
List the general supportive measures to be extended to a patient with poisoning
2.
Understand the principles of treatment of a patient with poisoning.
3.
List the steps in the management of a patient with (a) organophosphorus
poisoning (b) Illicit liquor poisoning (c) Snake-bite, (d) scorpion sting (e)
Yellow Oleander poisoning
The
most commonly encountered emergency poisonings in Pondicherry are the
following:
Plant:
Yellow Oleander Animal: Snakebite, Scorpion sting Drug:
Organophosphorous compounds, illicit liquor (methyl alcohol). An introduction
on the general supportive measures to be given to a patient with poisoning is
given by a faculty member (15 min).
Students
are divided into 5 small groups and each group is allocated a group task
(45min). A plenary is conducted after completion of group tasks. During plenary
(1-1½ hour), a small group discussion will ensue following the presentation of
each group.
GRoup tasks
(a) List the common signs and symptoms of
poisoning/bite/sting.
(b) Formulate a plan of management for
(i)
First-aid
(ii)
Definitive
treatment of a patient with the same.
(c) Briefly discuss methods to identify
poison/snake
(d) Do you need to take any precautions
during therapy?
Group
1. Yellow Oleander
Group
2. Snake bite
Group
3. Scorpion sting
Group
4. Methyl alcohol
Group
5. Organophosphorus compound
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