Laboratory Manual

Pharmacology I&II
(PHL 313 & 322)

Dr/ Mohamed N Ansari

Dr/ Majid A Ganaie

Department of Pharmacology
College of Pharmacy
Salman bin Abdulaziz University

1435-1436 / 2014 – 2015

Lab No. TABLE OF CONTENTS PAGE

Pharmacology I

Laboratory Orientation 2-5

1 Study of Different Laboratory Animals and Their Application 6-7

2 Study of Different Stages of Anesthesia 8-9

3 Study of Some Basic Instruments Used for Isolated Tissue 10-11

Experiments

4 Routes of Drug Administration to Laboratory Animals 12

5 To Evaluate the Analgesic Potency of Drug by Formalin Test 13-15

6 To Evaluate the Analgesic Potency of Drug by Tail Flick Method 16-17

7 To Evaluate the Analgesic Potency of Drug by Hot Plate Method 18-19

8 Screening of Different Diuretics Using Laboratory Animals 20-21

9 Screening of Anti-inflammatory Drug Using Hind Paw Method 22-23

Pharmacology II 24

1
 LABORATORY ORIENTATION

Introduction:

The laboratory portion of this course is designed to study the different laboratory
animals, their applications and screening of different pharmacological agents
thoroughly than it is presented in lecture. Core learning will come from practical and
tissue studies. This method of ‘hands on’ learning should also enhance and strengthen
the knowledge you gain in lectures.

At times you will be working individually, in pairs or in groups of three or four. Each
lab period is loosely structured to begin with a short introduction to the exercise that
highlights the activities of the day, what materials are available for use and any changes
in procedures. After that you will work independently to learn the material.

There is never enough time in lab to go over each and every item that you are assigned.
The lab is a designated a time when you have access to materials that you will not have
available during home study time. Some of the information assigned in lab you can
learn at home, particularly animals, instruments, method, procedure, application,
mechanism of action of drugs etc.

General Lab Rules:

1. Read the lab exercise before you come to lab. There is not time to review every aspect
of each exercise and still give you time to work on your own. I will assume that you
know what the exercise covers in general and I will only review changes or specific
materials that you will use.

2. Before each lab, use the terminology list to mark the items in your manual’s text and
illustrations that you are responsible for learning.

2. Read and memorize the laboratory safety rules of the lab below. The preservatives are
irritants and some of you may be allergic to them. Gloves must be used during
dissections and will be provided. Your dissecting tools will be provided for you as well.

2
 Pharmacology Laboratory: Safety, Procedures, Emergencies

These are minimum safety requirements. Instructors may institute additional policies at
their discretion. 1. No open food or drink is permitted at any time, whether a lab is in
progress or not. No eating, drinking, candy, cough drops, chewing gum or tobacco is
permitted. All beverage and food containers must be put away in a
backpack/bag/purse or left outside of the lab. There are shelves outside in the hallway
to store food and beverages during lab. Never taste anything at all while in the lab
rooms, unless it is a part of the lab activity (such as PTC paper). Also, do not apply
cosmetics in lab (this includes lip balm).

2. Visitors under 16 years of age and children are not allowed in the lab rooms at any
time. Visitors over 16 may be allowed at the instructor’s discretion, as long as the lab
activity does not involve hazardous materials.

3. Know the locations of the eye wash and shower stations, fire alarm, fire extinguisher,
first aid kit, and emergency exits. Do not block access to these with trash cans, recycle
bins, etc.

4. Safety instructions are given at the beginning of each lab period. Always arrive on
time so that you know what you are supposed to do and are informed of any specific
safety concerns or safety equipment associated with the day’s lab activity.

5. Wear any required personal protective equipment (lab coat, apron, goggles, etc).

6. Stash book bags safely so that they won’t trip people.

7. Report all illnesses, injuries, breakages, or spills to your laboratory instructor

immediately.

8. Clean broken glass (glass that is not contaminated with any chemical reagents, blood,
or bacteria) can be swept up using the dust pan and placed in the broken glass
container. If the glass is contaminated in any way, keep the area clear to prevent
tripping or laceration hazards, and consult your instructor for proper disposal
guidance. A broken glass flow-chart is available in the lab to help you decide what to
do.

9. Notify your instructor if any of the equipment is faulty.

10. Clean up your entire work area before leaving. Put away all equipment and supplies
in their original places and dispose of reagents and infectious materials in the
designated receptacles. Disinfect your work surface if the lab activity involved any

3
infectious materials. Otherwise, wipe the entire work surface down with a clean, wet
paper towel (no soap).

11. Use the appropriate waste containers provided for any infectious or hazardous
materials used in lab.

12. Safety information reagents used in the lab activities can be found in the Material
Safety Data Sheets (MSDS), which are available in a binder in the lab. Know the location
of the MSDS binder. We (faculty and students) should be fully aware of the properties
of the reagents we are using. Please use the MSDSs. If you cannot find the MSDS for the
reagent you are using in lab, inform your instructor. They are also relatively easy to find
online. A Google keyword example is “Sodium Chloride MSDS.”

13. Use caution with the lab chairs. Because they are on casters, they can roll away when
you are standing at your workstation. Make sure your chair is where you expect it to be
before sitting down. Do not use your chair as a means of moving from one part of the
lab to the other.

14. Wash your hands before leaving the lab room.

15. If class is held at an alternate location (e.g. a field trip), you will be expected to
conduct yourself appropriately and follow lab safety rules where applicable.

GENERAL OBJECTIVES OF THE COURSE

At the end of the practical training in general, and experimental pharmacology the
learner shall be able to:

1. List the various dosage forms and enumerate their advantages and disadvantages.
2. Advise patients about the proper use of medication devices, storage of medicines
etc.
3. Retrieve drug information from appropriate sources.
4. Appreciate the role of good laboratory practice in promotion of rational diagnostics,
therapy, and experimentation.
5. Realize the cardinal role of ethics in experimentation.
6. Order monitoring of drug levels where indicated and take appropriate remedial
measures.
4
7. Prescribe rationally and in an individualized pattern.
8. Plan and carry out experiments to demonstrate the effect of drugs in experimental
animals and isolated tissues.
9. Critically appraise drug advertisements.
10. Apply fundamental statistical tests to experimental data and interpret results.

5
 LAB 1: STUDY OF DIFFERENT LABORATORY ANIMALS AND THEIR
APPLICATION

AIM: To study the advantages, disadvantages and experimental uses of different most
commonly used laboratory animals.

BACKGROUND INFORMATION:
PHARMACOLOGY is the branch of science which deals with study of drugs on living
systems.
EXPERIMENTAL PHARMACOLOGY: deals with study of effect of various
Pharmacological agents on different animal species.
OBJECTIVES OF PHARMACOLOGY:
▫ To find out the therapeutic agent suitable for human use
▫ To study the toxicity of the drugs
▫ To study the mechanism and site of action of drugs

LABORATORY ANIMALS: Animals those can be breaded and handled in laboratory.

Examples: Rat, Mice, Guinea pig, Rabbits, Frogs, Cat, Dog, Monkey, Pigeon etc.

1. RATS (Rattus norvegicus )

• Albino rats of Wistar strain are commonly used
• Other strains –
▫ Wistar kyoto rat
▫ Sprague Dawley rat
▫ Biobreeding (BBDP) rat
▫ Long-Evans rat
▫ Zucker rat
▫ Hairless rats (Rowett nude, Fuzzy, Shorn)
▫ RCS rats

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2. MICE( Mus musculus)
• Swiss albino mice are commonly used species
• Other strains are – Balb/C and C-57
ADVANTAGES AND CHARACTERISTICS:
▫ Smallest
▫ Less drug required
▫ Easy to handle
▫ Cheap

3. GUINEA PIGS ( Cavia porcellus)

ADVANTAGES AND CHARACTERISTICS:
▫ Docile animals
▫ Highly susceptible to TB and anaphylaxis
▫ Highly sensitive to histamine, penicillin
▫ Required exogenous vitamin C in diet

4. RABBITS (Lupas cuniculus)

ADVANTAGES AND CHARACTERISTICS:
▫ Docile animal with large ears
▫ New Zealand white strains are widely used
▫ It has huge caceum and long appendix
▫ Enzyme atropine esterase is present in rabbit liver and plasma so it can
tolerate large doses of belladona (atropine)

5. FROGS (Rana tigrina)

ADVANTAGES AND CHARACTERISTICS:
▫ Used before 200 years
▫ Easily available during rainy season
▫ Amphibian animal and safe to handle
▫ Cannot breed in laboratory

7
 LAB 2: STUDY OF DIFFERENT STAGES OF ANESTHESIA

AIM: To study the different stages of anesthesia and calculate onset and duration of
action of different anesthetic agent.
ANIMALS: Wistar albino rats
APPARATUS: Desiccator, Syringes, needle
DRUGS AND SOLUTIONS: Diethyl ether, chloral hydrate
BACKGROUND INFORMATION:
General anesthesia was absent until the mid-1800’s. William Morton administered ether
to a patient having a neck tumor, removed at the Massachusetts General Hospital,
Boston, in October 1846. The discovery of the diethyl ether as general anesthesia was
the result of a search for means of eliminating a patient’s pain perception and responses
to painful stimuli.
Anesthesia is defined as partial or complete loss of sensation with or without loss
of consciousness as a result of disease, injury, or administration of an anesthetic agent,
usually by injection or inhalation.
STAGES OF ANESTHESIA:
 STAGE 1 (ANALGESIA/ONSET/INDUCTION):
 STAGE 2 (EXCITEMENT/DELIRIUM):
 STAGE 3 (Surgical Anesthesia):
 STAGE 4 (Impending Death/ Stage of Danger):

PROCEDURE:
Two Wistar albino rats will be weigh and keep in different cages. One rat will be
injected by intraperitoneal route with chloral hydrate in a dose of 400 mg/kg and
another rat will be inhaled by diethyl ether. Onset and duration of action of both
anesthetic agents will be recorded. Above mentioned different stages of anesthesia will
be observed.

8
RESULTS:
S. No. Name of Anesthetic Onset of Duration of
agent action Action

CONCLUSION:

9
LAB 3: STUDY OF SOME BASIC INSTRUMENTS USED FOR ISOLATED TISSUE
EXPERIMENTS

AIM: To study the basic instruments used for in-vitro experiments.

BACKGROUND INFORMATION:

 Isolated tissue preparations are commonly used to study the effects of drugs on
specific type of receptors. These preparations are also used:
 For bioassay of drugs,
 Characterization of specific receptor or its subtypes,
 To determine concentration response curve of an agonist,
 To study antagonism of drug and in new drug discovery.
 The in vitro isolated preparation represents an isolated organ or a piece of living
tissue from a freshly killed animal.
 Optimum ionic environment
 Adequate supply of nutrition and oxygen
 And a stable temperature
 These basic requirements should be provided if one has to maintain the
isolated tissue in living state.

BASIC REQUIREMENTS:
 Physiological Salt Solutions
 Instrumentation
 Procedures and drugs – to render the animal unconscious
 Tissue: isolated or whole

PHYSIOLOGICAL SALT SOLUTIONS:

 Optimal pH 7.0 – 7.2
 Commonly used PSS are:
 Frog Ringer (for heart, rectus abdominis and other preparations of frog)
 Tyrode (for guinea pig ileum, rat ileum, rabbit ileum etc.)
 De Jaon(for rat uterus)
 Kreb’s solution (for rat fundus strip, tracheal preparations, vas deferens)
etc.

ISOLATED ORGAN BATH:

 The apparatus providing the basic requirements of life to the tissue and
facilitating the record of response.

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  Outer water bath of perspex or glass
 Inner organ bath (single or multiple)-glass (15-100 ml): Organ tube
 Tissue holder cum Aeration (oxygen) tube
 Glass coil connected to organ bath, Marriott's bottle:
Reservoir for PSS
 Electric heater
 Preheating coil
Sherrington Recording Drum and Drum Cylinder:
 It is an instrument on which physiological responses are
recorded.
 Heavy base
 Base hoofs (legs) with adjustable leveling screws
 Gear rod
 Vertical shaft
 Drum Cylinder
 Brass or iron
It has 8 variable speeds. The speed is measured in mm per second. Eight variable
speeds are 0. 12, 0.25, 1.25, 2.5, 12.5, 25, 320 and 640 mm per second.

Levers:
 Isometric contractions: change in tension (force). E.g.
spring lever
 Isotonic contractions: change in length at uniform
tension. E.g. isotonic frontal writing lever (light
aluminum or stainless steel rod)

Useful Hints for Students:

 Step by step
 Setup the assembly first
 Assembly should be on right hand side and drum should be on left hand side
 The drum should move from right to left or away from the writing point
 The speed of drum should be minimum
 The writing lever should be horizontal and the writing point should touch the
drum.
 All tracing should begin after the overlap of the paper.
 Always start with the minimum concentration of drug.
 Write the name & amount of drug. Name of experiment and student with
student ID and signature of teacher before varnishing.

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LAB 4: ROUTES OF DRUG ADMINISTRATION TO LABORATORY ANIMALS

AIM: To demonstrate different routes of drug administration using Wistar albino rats.

ANIMALS: Wistar albino rats

APPARATUS: Disposable needle and syringes

DRUGS AND SOLUTIONS: Normal saline

BACKGROUND INFORMATION:

The possible routes of drug entry into the body may be divided into following classes:
I. ENTERAL ROUTE
1. Sublingual: under the tongue
2. Oral administration (P.O.)
3. Rectal or vaginal
II. PARENTERAL ROUTE: administration of medications by needle
1. Intravenous (I.V.): into vein Fastest
2. Subcutaneous (S.C.): in the subcutaneous tissue Slowest
3. Intramuscular (I.M.) Medium
4. Intraperitoneal (I.P.) : into the peritoneum (body cavity)
5. Intraarterial: direct inject into artery
6. Intradermal: under the epidermis or into dermis
7. Intraosseous: into the bone
III. PULMONARY ROUTES: Inhalation into lungs
IV. TOPICAL
I. Nasal
II. Skin
III. Eye

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 LAB 5: STUDY OF ANALGESICS BY CHEMICAL METHOD

AIM: To evaluate the analgesic activity of drugs by using formalin test.

Chemical Method: Formalin induced writhing test

Animals: Wistar albino Rats
Apparatus: Syringe (with 100 divisions) 26g needle, stop watch
Drug & Solutions:
Aspirin 100 mg/kg, p.o.
Formalin 10 %, 0.1 ml
Normal Saline 0.9%
BACKGROUND INFORMATION:
 Analgesics: painkillers, a drug that selectively relive pain by acting in the CNS
and peripheral pain mechanism without significantly altering consciousness.
 Types of analgesics
1. Opioid or narcotic analgesics: relive visceral pain.
Uses: deep pain e.g. Cancer, myocardial infarction, anginal pain
Examples: 1- natural (as morphine, codeine) 2- semi synthetic (heroin) 3-
synthetic (pethidine) 4- endogenous opiates (endorphins & encephalin)
2. Non opioid or non-narcotic analgesics: relieve somatic pain.
Uses: dull pain e.g. Headache, toothache, backache
Examples: aspirin, paracetamol, diclofenac, piroxicam

PROCEDURE:
Rats weighing 180-300 gm will be administer with 0.1 ml of 10%
Formalin into the dorsal portion of the front paw. The test drug is
administered simultaneously either s.c. or orally. Each individual
Rat is placed into a clear plastic cage for observation. Reading is
taken for a period of 60 minutes. Pain response is indicated by
elevation or favoring of paw. Analgesic response is indicated if
both paws are resting on the floor with no obvious favoring of
injected paw.

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PAIN RATING SCALE:

0 1 2 3

Both forepaws are The injected paw is The injected paw is The injected paw is
placed on the floor rested lightly on elevated and not in licked, bitten or
and weight is the floor or on contact with any shaken while the
evenly distributed. another part of the surface. The uninjected paw is
animal body and uninjected paw is not.
little or no weight placed firmly on
is placed on it. floor.
Attempt to sleep Attempts to sleep
by curling up with off by curling up
both paws off the with only the
floor is also given injected paw off
this rating. the floor, even
when it is tucked
under the body is
also given this
rating.
Normal grooming This behavior is
(during which both quite distinct from
paws are elevated), normal grooming
licking and rearing although transition
(Forepaws are not between the two is
in contact with common.
floor, although
forepaws may be
in contact with the
wall).
During locomotion During locomotion
there is no there is not
discernible obvious limp.
favoring of the
injected paw

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EVALUATION:
 Shows the prolongation of latency time by comparing value before and after
administration of test compound.
 Comparison done by statistical analysis using t-test.

RESULTS:

RESOURCES AND HINTS FOR TEACHERS:

1. Check work books after students calculate the volume of drug to be injected. Check
volumes in syringes prior to injection. Restrain the animal for the student to inject.
Timings of injections should be noted. Only one animal to be observed at a time.
2. Formalin should be freshly prepared for each class.
3. The experiment can be completed in 90 minutes. This can be followed by a small
group discussion on evaluation of analgesics in humans.

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 LAB 6: TO EVALUATE THE ANALGESIC POTENCY OF DRUG BY TAIL FLICK
METHOD

AIM: To evaluate the analgesic activity of drugs by using Tail flick analgesiometer.

Method: Thermal
Animals: Wistar albino Rats
Apparatus: Oral feeding needle, Tail flick analgesiometer, Mouse restraint
Drug & Solutions:
Aspirin 100 mg/kg, p.o.
Normal Saline 0.9%
BACKGROUND INFORMATION: The tail flick assay
is a pain receptive assay in which a mouse is placed
within a restraining tube with its tail protruding.
PRINCIPLE: Record the response of an animal to a painful stimulus before and after
administration of analgesics.
PURPOSE: The method is based on the reaction of the rat to heat stimulus applied to a
small area of the tail. The time until this response occurs is prolonged after
administration of analgesics.

PROCEDURE:
Albino rats of either sex weighing between 100-150 g are used.
The rat is held and its tail is placed on a level surface, a radiant
heat is applied to the tail at a point not more than 3 cm from its
tip. After an interval the animal withdraws its tail with a
sudden and characteristic flick. Time of flicking is recorded,
before drug administration. The reaction time are thereafter
recorded after 15, 30, 45 and 60 minutes after administration of
the test drug

16
OBSERVATIONS TABLE:
Reaction time in second

Rat No. Control Minutes after drug administration

15 30 45 60

1.

2.

3.

4.

5.

6.

EVALUATION:

 Shows the prolongation of latency time by comparing value before and after
administration of test compound.

 Comparison done by statistical analysis using t-test.

RESULTS:

RESOURCES AND HINTS FOR TEACHERS:

1. Check work books after students calculate the volume of drug to be injected. Check
volumes in syringes prior to injection. Restrain the animal for the student to inject.
Timings of injections should be noted. Only one animal to be observed at a time.
2. The experiment can be completed in 60 minutes. This can be followed by a small
group discussion on evaluation of analgesics in humans.

17
 LAB 7: TO EVALUATE THE ANALGESIC POTENCY OF DRUG BY HOT PLATE
METHOD

AIM: To evaluate the analgesic activity of drugs by using Hot Plate analgesiometer.

METHOD: Thermal
ANIMALS: Wistar albino Rats
APPARATUS: Oral feeding needle, thermostatically controlled Hot Plate
DRUG & SOLUTIONS:
Aspirin 100 mg/kg, orally
Normal Saline 2 ml/kg, orally
BACKGROUND INFORMATION: Eddy’s Hot Plate: Animal is placed on hot plate
and time for jumping from plate is noted before and
after administration of drug.

Hind paw licking (4-6 sec) or jump response (2-3 sec)

PRINCIPLE: Record the response of an animal to a

painful stimulus before and after administration of
analgesics.

PURPOSE: The paws of rats are very sensitive to heat at temperature. So the responses
are jumping, withdrawal of paws and licking of the paws. The time until this response
occurs is prolonged after administration of centrally acting analgesics.

PROCEDURE: Wistar albino rats of either sex with weight of about 200-250 g. The hot
plate consists of electrically heated surface with controlled temperature 55±1°C.
Animals are place on hot plate and time of flicking or jumping is recorded by stop
watch, before drug administration. The latency time is recorded after 15,30,45,60
minutes following oral or subcutaneous administration of the test compound

18
OBSERVATIONS TABLE:
Reaction time in second

Rat No. Control Minutes after drug administration

15 30 45 60

1.

2.

3.

4.

5.

6.

EVALUATION:

 Shows the prolongation of latency time by comparing value before and after
administration of test compound.
 Comparison done by statistical analysis using t-test.
 The value which is exceeding the value before administration for 50% or 100%
can be regarded as positive response.
RESULTS:

RESOURCES AND HINTS FOR TEACHERS:

1. Check work books after students calculate the volume of drug to be injected. Check
volumes in syringes prior to injection. Restrain the animal for the student to inject.
Timings of injections should be noted. Only one animal to be observed at a time.
2. The experiment can be completed in 60 minutes. This can be followed by a small
group discussion on evaluation of analgesics in humans.

19
 LAB 8: SCREENING OF DIURETICS

AIM: To study the Effect of different Diuretics on the Production of Urine in Laboratory
Animals
ANIMALS: Wistar albino Rats
APPARATUS: Oral feeding needle, metabolic cages, measuring cylinder, funnel
DRUG & SOLUTIONS:
Furosemide (5 mg/kg; oral)
Urea (900 mg/kg; oral)
Normal Saline (2 ml/kg; oral)
BACKGROUND INFORMATION:

 Some of pathological conditions associated with retention of sodium and water in

the body e.g. Congestive Heart failure, pulmonary edema, renal edema and
hypertension.

 Diuretics are the drugs that cause a net loss of Na+ and water in urine.

 The first important site with regard to renal function is the glomerulus itself. Drugs
which increase glomerular filtration rate (Xanthines, cardiac glycosides, saline)
produces diuresis.

THE FORMATION OF URINE:

 In summary, three processes occurring in

successive portions of the nephron
accomplish the function of urine
formation:

 Filtration of water and dissolved

substances out of the blood in the
glomeruli and into Bowman's
capsule;

 Reabsorption of water and

dissolved substances out of the
kidney tubules back into the blood

20
 (note that this process prevents substances needed by the body from being
lost in the urine);

 Secretion of hydrogen ions (H+), potassium ions (K+), ammonia (NH3),

and certain drugs out of the blood and into the kidney tubules, where they
are eventually eliminated in the urine.

PRINCIPLE: Diuretics are the compounds which increase the flow of urine. Normal
urine output in rats is very small (1-2 ml/rat/day). Hence, to get the measurable
quantity the animals are first hydrated. The urine output is increased after
administration of diuretics like urea and furosemide. Increase in volume of urine is
measured with the help of measuring cylinder and compared with the normal urine
output.

PROCEDURE: Albino rats of either sex weighing between 150-200 g are fasted
(deprived of food & water) overnight and saline is administered orally. These rats are
divided into three groups as follows:

1. Only normal saline

2. Saline + Urea

3. Saline + Furosemide

After administration of drugs, rats are placed in the three different metabolic cages.
Urine is collected in measuring cylinder. Time when the first drop of urine is collected
in a cylinder for each group is noted and the volume is recorded at intervals of 15 min
for 3-4 Hours. The difference in the volume collected at different time interval and total
volume can be compared with various diuretics.

OBSERVATIONS TABLE:

Groups I II III
Amount of
After 15 Min
urine collected
After 30 Min
(ml)
After 1 Hr.
Total Volume

EVALUATION: Calculate percentage increase in volume of urine taking control value

as 100%.

RESULTS:

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 LAB 9: SCREENING OF ANTI-INFLAMMATORY DRUGS USING HIND PAW
METHOD

AIM: To Evaluate the Anti-inflammatory Activity of the Drug (acetyl salicylic acid)
Using Hind Paw Method
MODEL: Acute inflammatory
METHOD: Rat hind paw method
ANIMALS: Wistar albino Rats
APPARATUS: Oral feeding needle, Plethysmometer,
DRUG & SOLUTIONS:
Aspirin (100 mg/kg; oral)
BACKGROUND INFORMATION:
Inflammation is a protective response to injury. It occurs in three phases:
(a) The first phase being oedema and swelling with accompanying pain. These
effects are produced as a result of the dilation and increased permeability of the
blood vessels (veins) due to the release of certain mediators such as histamine,
serotonin and kinins etc.
(b) In the second phase, leukocytes migrate to this area and mopping up operations
starts.
(c) Second phase is followed by repair, which is ushered in by the
proliferation of fibroblasts and synthesis of connective tissue.
The ability of a compound to reduce the local oedema induced in rat paw by various
irritants is the most widely used test to screen new non-steroidal anti-inflammatory
drugs. Many compounds like formalin, carrageenan, Kaolin, yeast and dextran have
been used as irritants to produce oedema.
PROCEDURE: Twelve healthy male albino rats weighing 100-200 gms will be selected
and made into two groups of six animals each. All the animals will be kept on fasting
for 18 hours. The hind paw of the rats will be marked at the level of tibio tarsal junction
of hind leg, so that while measuring the volume, the dipping will be done to the same

22
level. One group serve as control, 0.3 ml of Normal
saline will be given orally. Another group will receive
the test drug, Acetyl salicylic acid 300 mg/Kg. After
30 minutes of the administration of drug, 0.1 ml of 1%
Carrageenan will be administered to the rats into the
plantar surface of the right hind limb to induce paw
oedema. The volume will be measured immediately
and after 3 hours using plethysmometer.
The change in the paw volume was compared with
the control animals. The percentage of oedema
compared to the control by the test drug was
calculated using the formula

CALCULATION: Vc - Vt
% oedema inhibition = --------------- X 100
Vc
Where:
Vt- means increase in paw volume in rats treated with test drug;
Vc-means increase in paw volume in control group of rats.
OBSERVATION TABLE:

Initial Reading 3 hr after Difference in Percentage

Group
Reading carrageenan volume oedema

Control

Test

RESULT: The % oedema inhibition was found to be ______________

23
Laboratory Manual
Pharmacology II
(PHL 322)

Dr/ Majid A Ganaie

Department of Pharmacology
College of Pharmacy
Salman bin Abdulaziz University

1435-1436 / 2014 – 2015

24
 Content Index:

Exp.
Experiment Page no. Date Remarks
No.

To study various terms, definitions and

1. principles used in experimental 26-29
neuropharmacology.
To learn how to assessing appearance and
2. 30-32
behavior.

To learn the laboratory animal (mouse)

3. 33-37
handling technique.
To determine the time required for the
4. induction and recovery from anesthesia for 38-39
various volatile general anesthetics.
To study the effect of drugs on Spontaneous
5. Motor Activity (SMA) and to evaluate their 40-42
nature as CNS stimulants.
To study the effect of drugs on Spontaneous
6. Motor Activity (SMA) and to evaluate their 43-45
nature as CNS depressants.
To study the effect of various analgesics on
7. pain sensitivity to thermal stimulus using hot 46-47
plate.
To study the effect of drugs on anxiety
8. 48-49
behavior using Elevated Plus Maze.

To study the effect of drugs on depression

9. 50-51
using Forced swim test.
To study the effect of various tranquilizers
10. and sedatives on motor co-ordination by Rota 51-53
Rod test in rodents.
To evaluate antiepileptic activity of drug
using maximum electroconvulsive shock
11. 54-56
seizures (M. E. S.) and chemical induced
convulsions methods.
To study the antiparkinsonian activity of
12. 57-58
drugs by phenothiazine induced catatonia.

25
 LAB-1: INTRODUCTION TO EXPERIMENTS IN CNS PHARMACOLOGY

(Tutorial)

AIM: To study various terms, definitions and principles used in experimental

neuropharmacology

NEUROPHARMACOLOGY: The study of drugs specifically employed to affect the

nervous system. Several drugs used for the treatment of extra-neural pathology may

have an effect on the Nervous system

EXPERIMENTAL PHARMACOLOGY: deals with effect of various pharmacological

agents studied on different animal species. The effects of a drug can be considered at
different levels:

• Molecular
• Cellular
• Behavioral
As the complexity of the system increases it becomes

more difficult to predict the effects of a drug

Chemical Signals (Synapse): One neuron will

transmit info to another neuron or to a muscle or

gland cell by releasing chemicals called

neurotransmitters. The site of this chemical

interplay is known as the synapse. An axon

terminal (synaptic knob) will abut another cell, a

neuron, muscle fiber, or gland cell. This is the site of

26
transduction – the conversion of an electrical signal into a chemical signal.

Synaptic Transmission: An action potential reaches the axon terminal of the

presynaptic cell and causes Voltage-gated Ca2+ channels to open. Ca2+ rushes in, binds

to regulatory proteins & initiates NT exocytosis. NTs diffuse across the synaptic cleft

and then bind to receptors on the postsynaptic membrane and initiate some sort of

response on the postsynaptic cell.

How does a drug reach the brain?

A drug stays in the blood for some time after administration, before reaching the CNS,

where it has to cross the Blood Brain Barrier BBB. The BBB barrier prevents that all the

substances reach the brain. Also the placental barrier has to be taken into account. Once

the drug reaches the brain it can go through the cell membrane or binds to a receptor on

the cell surface.

TOLERANCE: A diminished response to drug administration after repeated exposures

of that drug (metabolism or compensatory changes in the nerve cell.

27
SENSITIZATION (reverse tolerance): when repeated drug administration causes an

enhancements of the drug effectsUp-or down-regulation of the receptors (1/2 weeks)

reflect compensatory changes in the number of receptors after prolonged use of the

drugs. Increase in the number of receptors is called up-regulation and decrease in the

number of receptors is called down regulation.

Basic properties of the nerve cells:

 Ability to conduct electric signals

 Specific intercellular connections with other nervous cells or with other tissues

Drugs can affect either one of these properties

28
Discovery of new drugs or to study the actions of existing drugs

Preclinical Two ways: Intact animal study – invivo

isolated organ study – invitro

Clinical Phase -1,2,3,4

Examples of the tests

 General Behavior

 Stimulant and depressant activity tests

 Memory Impairment tests

 Learning & Memory tests

 Anxiety & Fear tests

 Psychomotor Behavior & Coordination

 Anti-depressant Activity Tests

29
 LAB-2: ASSESSING APPEARANCE AND BEHAVIOR

AIM: To learn how to assessing appearance and behavior.

PROCEDURE: A common approach to assessing animal appearance and behavior is

through observation of the following:

 Activity Level

For example: Hypoactivity (hunched, huddled, lethargic), hyperactivity,

restlessness, lack of inquisitiveness

 Attitude

For example: Arousal, depression, awareness of surroundings

 Behavior, Spontaneous

For example: vocalization, self-trauma, isolation from cage mates. These

observations are made without disturbing the animal.

 Behavior, Provoked

e.g., vocalization, hiding, aggressiveness, minimal response. These observations

are made when the animal is disturbed or even prodded.

 Body Condition

e.g., emaciation, missing anatomy

 Food and Fluid Intake

e.g., elimination of feces and urine

 Fur and Skin

e.g., unkempt or greasy or dull fur; porphyrin staining around eyes and nostrils;
cyanotic, pale, or congested mucous membranes or skin (ears, feet, tail); skin
lesions; soiled anogenital area

30
 Eyes

e.g., clarity/condition of lens, cornea; position of globe (e.g., sunken in orbit or

protruding); condition of eyelids, encrustation

 Posture

e.g., hunched back, tucked abdomen; prostrate; head tucked down

 Locomotion

e.g., gait, ataxia, lameness, action of each limb, position of tail when ambulating

 Neurological

e.g., tremor, convulsion, circling, paralysis, head tilt, coma

 Vital Signs

e.g., respiratory distress (open mouth breathing, pronounced chest movement)

 Other clinical parameters that are relevant to your study

e.g., presence and status of tumors, infection, or surgical wounds

31
OBSERVATION TABLE:

Score (0-5)
0 1 2 3 4 5
Activity Level
Attitude
Behavior, Spontaneous

Behavior, Provoked
Body Condition
Food and Fluid Intake
Fur and Skin
Eyes
Posture
Locomotion
Neurological

Vital Signs
Other clinical parameters
that are relevant to your
study

Results:

32
 LAB-3: LABORATORY ANIMAL (MOUSE) HANDLING TECHNIQUE

AIM: To learn the laboratory animal (mouse) handling technique.

A. Blood collection from tail vein

B. Blood collection from orbital sinus

C. Blood collection from cardiac puncture

D. Blood collection from saphenous vein

E. Determining Sex and Age

F. Acclimation

A. Blood collection from tail vein

For collection of small amount of blood

(Approximate 0.1 ml)

Tools for Blood Collection from Tail

 75% alcohol cotton ball for surface

disinfection
 Small plastic bottle with 1/2 cm diameter
holes in both ends as mouse restrainer
 Scissors
 Pipetteman and tips
 A vial for blood collection

1. Placing a mouse on a cage lid and grasping the

loose skin behind the ears by the thumb and
forefinger

2. Push the mouse into the restrainer

3. Leave the tail of the mouse outside the cover of the

restrainer

4. Amputate the tip of the mouse tail by scissors

5. Massage the tail and collect blood by pipetteman

33
B. Blood collection from orbital sinus

Use anesthesia before blood withdraw and blood

collection amount up to 0.5 ml

Tools for Blood Collection from Orbital Sinus in

Mouse

 75% alcohol cotton ball for surface disinfection

 Hypnorm for general anesthetic

 27 G needle with 1 ml syringe for injection

 Glass capillary tube and vial for blood collection

1. Anesthetize a mouse by intraperitoneal injection

2. Use a sharp end glass capillary tube to penetrate the

orbital conjunctiva and rupture the orbital sinus

3. Collect blood with a vial

C. Blood collection from cardiac puncture

It is used for collection of blood up to 1 ml within a

short period of time and must be performed under
general anesthetic
Tools for Cardiac puncture in Mouse

 75% alcohol cotton ball for surface disinfection

 Hypnorm used as anesthetic
 27G needle with 1 ml syringe for injection
 24G needle with 3 ml syringe for blood withdraw

34
1. Anesthetize a mouse

2. Disinfect the thorax area with 75% alcohol cotton ball

3. Search for the maximum heart palpitation with your finger

4. Insert a 24G 1” needle through the thoracic wall at the point of maximum heart
palpitation

5. Withdraw blood slowly by your right hand

D. Blood collection from saphenous vein

This method is used of multiple samples are taken in the course of a day and it can also
be applied on rats, hamsters, gerbils and guinea-pigs

Tools for blood collection from Saphenous vein in

mice

 75% alcohol cotton ball for surface disinfection

 50 ml syringe tube with small holes at the end as
restrainer
 a scalpel and shaver for remove of hair
 24 G 1 “ needle for release of blood
 tips and pipetteman for blood collection

1. Place the mouse in the restainer

2. Pull out the leg and removed the hair by a assistant

35
3. The saphenous vein is seen on the surface of the thigh

4. Apply vaseline after disinfect the surface area to reduce

clotting and coagulation during blood collection.

5. Use a 24 G 1” needle to puncture the vein and release blood from the saphenous vein

6. Use a Micropipette with tip to collect blood from the saphenous vein, Approximate

7. Flex the foot of the mouse to reduce the flow of blood back to the puncture site

8. A cotton ball is applied to the puncture site to stop further bleeding

E. Determining Sex and Age

(Refer to the image) the top two mice are neonates

and note that the anogenital distance is larger in
the male than in the female neonates, the penis and
vulva cannot be easily differentiated and so are
referred to as a genital papilla.

36
The bottom two animals are adults; genitalia are differentiated. Also, nipples become
evident in females at about 10 days of age.

F. Acclimation

This period of time allows animals to adapt to a new environment. Upon arrival to your
facility, your mice should have an acclimation period before they are used.

The period of time necessary for biological stabilization will depend on the parameters
to be studied.

37
 LAB-4: GENERAL ANESTHETICS

AIM: To determine the time required for the induction and recovery from anesthesia

for various volatile general anesthetics.

REQUIREMENTS: Transparent box or Glass bell jars, Cotton, Stop watch, Rats

DRUGS: Chloroform, Ether

PRINCIPLE:

General anesthetics produce a reversible loss of consciousness throughout the body and

it is usually accompanied by inhibition of sensory autonomic reflexes and skeletal

muscle relaxation. When anesthetic is given to the rat, slowly it loses its righting reflex

(when animal is placed on its back, quickly retain its position, i.e. initial posture). Loss

of righting reflex is considered as a parameter of induction of anesthesia and

reappearance of righting reflex is considered as a parameter for recovery from

anesthesia).

PROCEDURE:

1. Rats weighing 150-200 g are selected. They are placed separately under glass bell

jars.

2. A swab of cotton soaked with 5 ml of chloroform is placed in each jar.

3. The stop watch is started and time required for the induction of anesthesia is

recorded.

38
 4. The bell jars and cotton swabs are removed and the time of recovery from

anesthesia is recorded.

5. The mean induction and recovery time for chloform is calculated.

6. The same procedure is repeated for ether.

OBSRVATION TABLE

a. Diethyl ether

Time required for

Time required for
induction of anesthesia
recovery (Sec.)
(Sec.)

Animal 1

Animal 2

Animal 3

Mean

b. Chloroform

Time required for

Time required for
induction of anesthesia
recovery (Sec.)
(Sec.)

Animal 1

Animal 2

Animal 3

Mean

RESULT:

39
 LAB-5: CNS STIMULANTS

AIM: To study the effect of drugs on Spontaneous Motor Activity (SMA) and to
evaluate their nature as CNS stimulants.

REQUIREMENTS: Activity cage, Cotton, Stop watch, rats/mice

DRUGS: CNS Stimulants

PRINCIPLE:

A drug increases or decreases CNS activity will also produce increase or decrease in

spontaneous motor activity (SMA) in the animals. The Activity cage is designed on this

principle. The Activity Cage is a reliable research instrument for recording spontaneous

coordinate activity in rats and mice and is designed as mentioned below:

1. The I.R. Beam Cage consists of: - an animal cage of clear Perspex

1.1.1 Set of emitter/sensor arrays for horizontal activity

1.1.2 Set of emitter/sensor arrays for vertical activity

2. The Cage consists of a cubicle, dimensioned 41 x 41 x 33 (h) cm, entirely made of

clear Perspex, upper lid and bottom catch pan, detachable for cleaning.

3. The cubicle rests on a sturdy base made of black Perspex, provided with four
vertical notched bars of stainless steel to which the horizontal/vertical detecting
systems can be fastened.

4. The animal cage is designed for one rat or up to 3-4 mice.

5. The transparent cubicle can be easily removed for cleaning purposes.

6. The activity detection of Activity Cage relies on horizontal and/or vertical sensors.
The movements the animal makes inside the cage interrupt one or more I.R. beam/s.
The beam interruptions, counted and recorded by the electronic unit, enable the user
to assess and analyze the animal activity.

40
7. Data related to activity, either horizontal and/or vertical, are printed in a convenient
format

PROCEDURE:

1. Insert the mains lead into the socket

2. Switch the instrument "ON" with the help of switch marked mains near the socket
(Warm for 2-3 min)

3. It shows four functional keys F1, F2, F3 and F4 from the top row of the keyboard.
The function of these keys is according to what the corresponding display shows.

4. Habituation: Move animals in their home cages into the laboratory, 45 – 60 minutes
prior to testing so that the animals can acclimate to the testing room.

5. After the acclimation period, weigh the first set of mice to be tested, and place them
into individual, bedding-lined, holding cages within 10 minutes of testing.

6. Prepare drug vehicle (usually saline), administer and immediately place the animal
into the activity cage.

7. Record activity for 5 minute periods, depending upon the expected duration of
action of the drug to be tested.

8. Remove animals from activity cage and place into home cage.

9. Clean activity monitors by removing fecal boli and urine with a wipe or paper towel
and then with 10% isopropyl alcohol.

10. The same procedure is repeated for other set of animals.

41
 OBSRVATION TABLE

Horizontal activity Vertical activity

(5 min) (5 min)
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Mean
Results:

Conclusion: Since the number of counts is increased by Amphetamine, it indicates that

spontaneous motor activity is increased by this drug. Hence it is CNS stimulant drug.

Discussion:

CNS Stimulants: Atropine, Amphetamine, strychnine, Picrotoxin, caffeine

42
 LAB-6: DEPRESSANTS

AIM: To study the effect of drugs on Spontaneous Motor Activity (SMA) and to
evaluate their nature as CNS depressants.

REQUIREMENTS: Activity cage, Cotton, Stop watch, rats/mice

DRUGS: CNS depressant

PRINCIPLE:

A drug increases or decreases CNS activity will also produce increase or decrease in

spontaneous motor activity (SMA) in the animals. The Activity cage is designed on this

principle. The Activity Cage is a reliable research instrument for recording spontaneous

coordinate activity in rats and mice and is designed as mentioned below:

1. The I.R. Beam Cage consists of: - an animal cage of clear Perspex

1.1.1 Set of emitter/sensor arrays for horizontal activity

1.1.2 Set of emitter/sensor arrays for vertical activity

2. The Cage consists of a cubicle, dimensioned 41 x 41 x 33 (h) cm, entirely made of

clear Perspex, upper lid and bottom catch pan, detachable for cleaning.

3. The cubicle rests on a sturdy base made of black Perspex, provided with four
vertical notched bars of stainless steel to which the horizontal/vertical detecting
systems can be fastened.

4. The animal cage is designed for one rat or up to 3-4 mice.

5. The transparent cubicle can be easily removed for cleaning purposes.

6. The activity detection of Activity Cage relies on horizontal and/or vertical sensors.
The movements the animal makes inside the cage interrupt one or more I.R. beam/s.
The beam interruptions, counted and recorded by the electronic unit, enable the user
to assess and analyze the animal activity.

43
7. Data related to activity, either horizontal and/or vertical, are printed in a convenient
format

PROCEDURE:

1. Insert the mains lead into the socket

2. Switch the instrument "ON" with the help of switch marked mains near the socket
(Warm for 2-3 min)

3. It shows four functional keys F1, F2, F3 and F4 from the top row of the keyboard.
The function of these keys is according to what the corresponding display shows.

4. Habituation: Move animals in their home cages into the laboratory, 45 – 60 minutes
prior to testing so that the animals can acclimate to the testing room.

5. After the acclimation period, weigh the first set of mice to be tested, and place them
into individual, bedding-lined, holding cages within 10 minutes of testing.

6. Prepare drug vehicle (usually saline), administer and immediately place the animal
into the activity cage.

7. Record activity for 5 minute periods, depending upon the expected duration of
action of the drug to be tested.

8. Remove animals from activity cage and place into home cage.

9. Clean activity monitors by removing fecal boli and urine with a wipe or paper towel
and then with 10% isopropyl alcohol.

10. The same procedure is repeated for other set of animals.

44
 OBSRVATION TABLE

Horizontal activity Vertical activity

(5 min) (5 min)

Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Mean

Results:

Conclusion: Since the number of counts is decreased by Phenobarbitone, it indicates

that spontaneous motor activity is decreased by this drug. Hence it is CNS depressant
drug.

Discussion:

CNS Stimulants: Atropine, Amphetamine, strychnine, Picrotoxin, caffeine

CNS depressants: Anesthetics, alcohols, sedatives, hypnotics and narcotics.

45
 LAB-7: ANALGESICS

AIM: To study the effect of various analgesics on pain sensitivity to thermal stimulus
using hot plate.

REQUIREMENTS: Hot plate analgesiometer, Cotton, Stop watch, rats/mice, 70%

alcohol.

DRUGS: Morphine, Indomethacin, Saline

PRINCIPLE:

Pain is induced to a suitable animal and the response of the animal to the painful

stimuli is recorded before and after administration. One of the established methods of

inducing painful stimulus is bu thermal method using hot plate analgesiometer. Drugs

that selectively inhibit the perception (sensation) of the pain can be used as analgesic

drugs.

PROCEDURE:

1. Insert the mains lead into the socket

2. Switch ON the hot plate apparatus and wait until the plate reaches the defined

temperature (55°C, adjust if necessary(.

3. Clean the metal surface and the plastic cover with disinfectant (e.g. 70% ethanol).

Wait 1 minute to re-establish the surface temperature before commencing with the

test.

4. Put the first mouse on the plate and simultaneously start the stopwatch to measure

the withdrawal latency.

46
5. Stop the stopwatch after the mouse displays any reaction to heat (paw shaking,

licking or jumping). If the mouse does not react to heat after 30 seconds, it is

removed from the hot plate and 30 seconds is considered as latency by default.

6. The hot plate and the jar are cleaned with water and disinfectant (e.g. 70% ethanol)

before testing another animal, wait 1 minute to re-establish the surface temperature.

The transparent cubicle can be easily removed for cleaning purposes.

7. At the end of testing, return the mice to the housing rooms and thoroughly clean the

equipment.

OBSRVATION TABLE

Reaction time Reaction time

(before treatment) (After treatment)

Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
Mean

RESULTS:

47
 LAB-8: ANXIOLYTICS

AIM: To study the effect of drugs on anxiety behavior using Elevated Plus Maze.

REQUIREMENTS: Elevated Plus Maze, Cotton, Stop watch, rats/mice

DRUGS: Diazepam, Saline (0.9% NaCl)

PRINCIPLE:

The standard elevated plus-maze is commonly used to assess anxiety-like behavior in

laboratory animals (rats/mice). The maze is usually a cross shaped maze with two open
arms and two closed arms, which is elevated above the floor.

This task exploits the conflict between the innate fear that rodents have of open areas
versus their desire to explore novel environments. Security is provided by the closed
arms whereas the open arms offer exploratory value. When anxious, the natural
tendency of rodents is to prefer enclosed dark spaces to opened brightly lit spaces. In
this context, anxiety-related behavior is measured by the degree to which the rodent
avoids the unenclosed arms of the maze.

PROCEDURE:

1. Habituation: Move animals in their home cages into the laboratory, 45 – 60 minutes
prior to testing so that the animals can acclimate to the testing room.

2. After the acclimation period, weigh the first set of mice to be tested, and place them
into individual, bedding-lined, holding cages within 10 minutes of testing.

3. Prepare drug vehicle (usually saline), administer and immediately place the animal
into the Plus Maze.

4. The plus-maze apparatus, consisting of two open arms (16 x 5 cm) and two closed
arms (16 x 5 x 12 cm) having an open roof, with the plus-maze elevated (25 cm) from
the floor used to observe anxiolytic behavior in mice.
5. Each mouse is placed at the center of the elevated plus maze with its head facing the
open arm.

48
6. The behavior of the mouse is recorded for 5 min.
7. The parameters observed are
7.1 The number of entries into the open arms
7.2 Average time spent by the mouse in open arms (average time = total time spent
in open arms/number of entries in arms).
8. Clean the plus maze by removing fecal boli and urine with a wipe or paper towel
and then with 10% isopropyl alcohol.

OBSRVATION TABLE

Number of entries into open Average time spent in open

Animal
arms arms
number
(5 min) (5 min)
1
2
3
4
5
Mean

RESULTS:

Conclusion:

Since the number of open arm entries and average time spent in open arms are
increased, it indicates that the drug has anxiolytic effect.

Discussion:

Anti-anxiety drugs: Diazepam, Alprazolam, Buspirone

49
 LAB-9: ANTI-DEPRESSANTS

AIM: To study the effect of drugs on depression using Forced swim test.

REQUIREMENTS: Glass chamber, Cotton, Stop watch, mice, Towel,

DRUGS: Flouxetine, Saline (0.9% NaCl)

PRINCIPLE:

The
forced
swim
test( also
called
the
Porsolt
test
or
behavioral
despair
test
)
is
regarded
as
a
physiological
measure
of
despair
exhibited
by
the
mouse
from

being
in
an
environment
from
which
it
cannot
escape.

This
is
exhibited
through
immobility
behavior.

This
test
is
often
used
whentesting
various
antidepressants
and
models
of
mood

disorders.

Mice are subjected to two trials during which they are forced to swim in an acrylic
glass cylinder filled with water, and from which they cannot escape. The first trial (if
rats are used) lasts 15 minutes. Then, after 24-hours, a second trial is performed that
lasts 5 minutes. For mice no pretest trial is needed. The time that the test animal spends
without moving in the second trial is measured. This immobility time is decreased by
antidepressants.

PROCEDURE:

1. Bring
your
animals
to
the
Behavioral
Suite
for
acclimation.


2. Set
up
the
beaker with
water
on
a
stable
tabletop
surface. These should be tall

enough to fill to a depth of 30 cm, leaving space at the top so that the rat cannot
escape, and at least 20 cm in diameter.

3. Fill the swim cylinders to 30 cm with water of 23–25 °C, which should be checked
with a thermometer. If the temperature is warmer (i.e., 30 °C) the animals merely

50
 float, whereas lower temperatures (i.e., 15–20 °C) lead to hypothermia and cause the
animal to be more active.

4. Prepare drug vehicle (usually saline), administer and place the animal into the
beaker with water.

5. Gently place the animal in the water and record the immobility for 5 min.
Immobility consists of the animal floating in the water without struggling and only
making movements necessary to keep its head above water
6. There are three predominant behaviors in the modified FST: immobility, swimming
and climbing. Swimming consists of the rat making horizontal movements
throughout the swim cylinder, which also includes crossing into another quadrant.
7. Climbing comprises upward-directed movement of the forepaws, usually against
the side of the swim cylinder
OBSRVATION TABLE

Animal Immobility
number (6 min)

1
2
3
4
5
Mean

Results:

Anti-depressant drugs: Imipramine, Amitriptyline, Fluoxetine.

51
 LAB-10: Motor Co-ordination

AIM: To study the effect of various tranquilizers and sedatives on motor co-
ordination by Rota Rod test in rodents.

REQUIREMENTS: Rota rod apparatus, Cotton, Stop watch, rats/mice

DRUGS: Diazepam, Chlorpromazine, Pentobarbitone.

PRINCIPLE:

Minor tranquilizers or anxiety agents like benzodiazepines produce specifically the

skeletal muscle relaxation. The site of this activity is the central nervous system.

Disturbances in the maintenance of tone and posture, is the first sign of centrally

mediated skeletal muscle relaxation. A mouse/rat when allowed to stay on a slow

rotating rod fails to stay on the rod maintaining its posture, when a skeletal muscle

relaxant is given. This property is utilized in the rota rod test.

EQUIPMENT & PROCEDURE:

1. Commercially available Rota Rod apparatus (IITC Life Science) has the capabilities
of having up to five mice or rats tested at any given time. The user can select from
one to five lanes to be included in an experiment.
2. For mice experiments 1¼ inch diameter drums are supplied and for rat
experiments 3¾ inch diameter drums are supplied. All that is needed is a standard
Phillips screwdriver to change the drums, no other tool required.
3. The LED display shows all test results for each animal position; they are: Stopping
RPM, length of test and distance traveled.
4. Animals drop is sensed by accurate magnetic switches and vertically sliding
acrylic front panels prevent escape of animals.
5. The electronic gear is built into the base of the unit.

52
 6. All parameters are digitally controlled and entered via the keypad on the front
panel. Start, stop and reset buttons are also on the front of the unit. An LED
display allows the user to view all parameters and test results.

OBSRVATION TABLE

Animal No. Animal Latency

(Seconds)

1
2
3
4
5
Mean

PRECAUTIONS:

1. To keep the instrument clean and dry.

2. Do not handle the instrument with wet hands.
3. Never poke the instrument with the nail, pencil, spatula etc
4. Avoid scratches on the chambers and panels.
5. After experimentation clean the chamber with distilled water.

RESULT:

53
 LAB-11: Anti-Epileptic Drugs

AIM: To evaluate antiepileptic activity of drug using maximum electroconvulsive

shock seizures (M. E. S.) and chemical induced convulsions methods.

REQUIREMENTS: Electro-convulsiometer, electrodes, stopwatch.

Drugs: Pentyleneterazol (Leptazole, 80 mg/kg); Phenytoin (100 mgkg).

PRINCIPLE

The convulsions in rat or mice can be induced by giving high voltage current near the
brain or by suitable CNS stimulants (e.g. pentylenetetrazal). The screening of
antiepileptic agents can be done by experimentally induced convulsions (seizures) and
their prevention by drugs under test.

PROCEDURE

1. Maximum electro-convulsive seizure (MES)

The rats (weighing 150-250g) or mice (weighing 20-40g) are used in the experiments.
The animals are first tested by giving maximum current of 150mA (in rat) or 80mA (for
mice) for 0.2 sec. duration. Those animals which show characteristic course of
convulsions are selected. This course has a short latent period (4-8 sec.) followed by a
tonic convulsion (up to about 15 sec) and then a phase of clonic convulsions (upto about
30 sec.). The selected animals are divided into two groups of 4 animals. One group
receives the test drug while the other receives saline as control. Shock is given to all
animals after 2 hours and the time taken by each phase of convulsion is noted.

2. Chemical method

The animals are injected with pentylenetetrazole (80mg/kg) given intraperitoneally.

Those animals which show convulsions are selected for the experiment. The animals are
divided into two groups. One group receives test drug while other receives saline as

54
control. The pentylenetetrazole is again given in the same dose and the time taken for
convulsions to start is noted. Picrotoxin (6-7 mg) may also be used instead of
pentylenetetrazole to produce convulsions.

OBSERVATIONS:

1. Maximum electro-convulsive seizure (MES)

Effect of MES
Groups Extensor Recovery
Latent period
tonus

2. Chemical method

Chemical method
Groups
Time of recovery Total time

RESULTS:

55
DISCUSSION
Epilepsy is synchronous discharge of impulses from brain characterized by ora (noice),
cry, tonic and clonic convulsions. There is spontaneous occurrence of brief episodes
associated with disturbance in consciousness and excessive EEG pikes.
It is characteristic that a drug showing prevention against electrically induced
convulsions are effective in Grand mal epilespy in human beings and those drugs
which prevent only chemically induced convulsions are effective therapeutically in Petit
mal epilepsy.

Various drugs used

1. Barbiturates, e.g. Phenobarbitone, Methylphenobarbitone.
2. Hydantoins, e.g. Phenytoin, Ethotoin, Methoin
3. oxazolidinediones, e.g. Trimethadione, Paramethadione.
4. Succinimides, e.g. Phensuximide Thiosuximide.
First groups are effective in Grand mal epilepsy while other two groups two are
effective in Petit mal epilepsy.

56
 LAB-12: ANTIPARKINSONIAN ACTIVITY

AIM: To study the antiparkinsonian activity of drugs by phenothiazine induced

catatonia.

REQUIREMENTS Rats (150-200g), wooden blocks (3cm high or 9 cm high).

DRUGS: Haloperidol, Perphenazine and Scopolamine

PRINCIPLE:

Antipsychotics like phenothiazines, reserpine etc. produce extrapyramidal side-effects

in man and in animals. These effects may simulate catatonia in rats and mice. The
extrapyramidal effects are mediated through dopamine antagonistic action of
antisychotic drugs. Anti-parkinsonian drugs produce the beneficial effects through
dopaminegic action or anticholinergic etc.

PROCEDURE

1. Animals are weighed and numbered. They are divided into two groups. one which
receives perphenazine and other receiving both perphenazine and scopolamine or
levodopa.

2. Catatonic effects are observed and scored as follows:

 Score 0: Rat moves normally when placed on table

 Score 1: Rat moves only when touched or pushed

 Score 2: Rat fails to correct the posture when the front paws set on 3 cm
high block.

 Score 3: Rat fails to remove the front paws when placed on 9 cm block.

The catalonia is observed at 10, 30, and 60min after drug administration.

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In treated groups antiparkinsonian drug is given 30 min. before the administration of
anti-parkinsonian drug.

OBSERVATION:

treatment Animal Body Degree of catatonia

no. weight

10 min 30 min 60 min Total

RESULTS:

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