Pharmacology-I_sem-4_RDP lab manual

Pharmacology -1 B.Pharm Semester IV
INDUKAKA IPCOWALA COLLEGE OF PHARMACY
B.Pharm Semester 4
PHARMACOLOGY-I
Practical Manual

INDEX
SR.
NO.
PRACTICAL
Date
PAGE. No. SIGN
1.
(A)Introduction to experimental pharmacology,
commonly used instruments in experimental
pharmacology.
(B) Study of experimental conditions required for
isolated tissue
2 Study of common laboratory animals.
3 Maintenance of laboratory animals as per CPCSEA
guidelines.
4
Standard techniques for drug administration
(intravenous injection, intra gastric administration) and
collection of blood samples, anesthetics and euthanasia
used for animal studies.
5
To study the effect of hepatic microsomal enzyme
inhibitors and inducers on pentobarbitone sleeping time
in mice.
6
To study the effect of physostigmine and atropine on the
ciliary movement of frog’s oesophagus.
7 To study the effects of Autonomic drugs on rabbit’s eye.
8
To study the effect of various tranquillizers and sedatives on
motor Co-ordination by Rota rod test in mice.
9
To study the effects of drugs on spontaneous motor activity
and to evaluate their nature as CNS stimulants or
depressants.

10
(A) Aim: To study anticonvulsant property of phenytion
against maximal electro-shock induced convulsion in
rat.
(B)Aim: To study anticonvulsant property of Diazepam
against Pentylenetetrazole Induced convulsion
in mice.
11
To study the taming effect of chlorpromazine in rats and
mice or to study the effect of chlorpromazine on
apomorphine induced compulsive behavior (stereotypy) in
rats and mice.
12
To Study the anxiolytic (anti-anxiety) effect of diazepam in
mice using elevated zero-maze.
13
To evaluate local anesthetic agent by various method:
A) Surface anaesthesia B) Intradermal method
C) Plexus Anaesthesia D) Conduction Anaesthesia.

DATE: / /2018
PRACTICAL: 1 (A)
Aim: Introduction to experimental pharmacology, commonly used instruments in
experimental pharmacology
General considerations
Pharmacology is the study of the effects of drugs on living systems. It attempts to characterize
the biological action of chemicals and to define the mechanisms by which these actions are
evoked. A pharmacologist seeks to desire and quantify both the undesirable effects in laboratory
animals and to establish the condition under which such observation can be extrapolated to
human population. Experimental pharmacology thus helps in understanding the nature of drug
action and the vulnerability of the living system to the alteration by chemicals that serves as the
basis of which
(a) New therapeutic agents are developed, and
(b) Toxic consequence of chemical may be alleviated.
The experiments can be carried out in whole animal or in isolated organs. In whole animals
experiments (e.g. dog blood pressure), the effect of drug on any organ is influenced by many
factors such as metabolic state, blood supply, nervous control, hormonal influence, factors of
bioavaibility etc. It is difficult to estimate & control the influence of such a diversity of factor.
Elucidation of action of drug may be hindered in such cases. The use of isolated (in-vitro)
preparation provides distinct advantages to facilitate the experimental investigations concerning
mode of action of drug. The isolated preparation is commonly used in experiments because of
these advantages. The intact experiments (whole animal experiments) are also of importance to
study the effects of drugs, the interaction between two drugs & between the body factors and
drugs.
INSTRUMENTATION
The in vitro tissue preparation represents isolated preparation or a piece of living tissue which
has been taken from freshly killed animal. The function of the preparation within the body is
maintained by certain provision of certain basic requirement like 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.
ISOLATED ORGAN BATH
The apparatus providing the basic requirements of life to the tissue and facilitating the record of
response was first designed by Rudolph Magnus in 1904. It is commonly known as the isolated

organ bath. It consist the glass tube called organ tube fixed in Perspex or glass tank filled with
water maintained at fixed temperature by an electrical heater and a thermostat. The organ tube
is connected through polyethylene or rubber tube or the reservoir, which contains Physiological
salt solutions (PSS). In between the reservoir and the organ tube is the glass preheating coil. The
tissue is suspended in the organ tube by means of tissue holder which also serves the purpose of
aeration (hence also known as aeration tube cum tissue holder). One end of the tissue is tied to
this and the other end connected to the lever, the recording device.
RECORDING DEVICE
Sherrington Recording Drum and Drum Cylinder
It is the instrument on which physiological responses such as contraction and relaxation of the
muscle are recorded. It consists of heavy base and a vertical shaft. Heavy base give s stability to
drum. It has (1) base hoofs (legs) with adjustable leveling screws to keep the drum horizontal if
the surface of the table is uneven. (2) Side hoof to turn the drum on its side so the shaft becomes
horizontal. (3) Gear rod arrangement with fast, slow and neutral gears and clutch (starter). The
gear rod is attached to a cone wheel which has 4 pulley grooves. Desirable speed of drum can be
obtained by changing the gear position and shaft drum pulley connections. (4) Contact screw on
the surface. A wire can be fixed from main plug to convey the current through base and (5)
Contact foil with a contacted screw mounted on an insulated material on the superior surface of
the base. Second wire can be connected here.

Drum cylinder is a brass or iron cylinder around which a paper is wrapped and smoked. Drum
with smoked paper is fitted on the vertical shaft. At the base of vertical shaft, there two
projecting striker which can be drawn apart to set any desired angle between them. When the
striker makes the contact with foil, the make in the circuit occurs.
These days electrical drum is more commonly used. This is similar to Sherrington Recording
Drum but speed is controlled electrically with the help of gears.
LEVERS
For recording of isometric contractions, spring lever is used while for the recording of isotonic
contractions; isotonic frontal writing lever is commonly used. Isotonic contraction indicates
change in length at uniform tension. (Force) develop irrespective of length. There is another
type of recording called auxotonic in which the change in the force of contraction is recorded
with respect to change in length.
Heart lever
Commonly used heart lever is spring lever (Starling heart lever or brodies heart lever). It is of
type class II lever, i.e. fulcrum lies at one end beyond the point of attachment. It consists of a
horizontal arm suspended by a fine spring. To the horizontal arm, a thread is attached which
connect it to heart. At the end of this arm, a stylet is fixed.

Isotonic frontal writing lever
It is made of light aluminum or stainless steel passing through a wheel which rotates freely
about axis (commonly known lever holder). At one end the frontal writing lever is fixed.
The advantage of frontal writing point is that it gives the recording with a uniform friction and its
writing point falls vertically against the paper. The trace thus obtained is liner as straight lines
and not the areas.
The lever should be adjusted such that it gives a fair and fixed magnification of the response and
should exert suitable tension.
Recording of responses on the drum-cylinder without smoking (sketch-pen tip)
The responses with the help of frontal writing lever or simple levers can be recorded on
the drum cylinder using unsmoked paper. Simple sketch-pen tip can be tied with the help of
cotton thread with a very small amount of wool and drop of ink (or eosin) can be placed before
starting the recording. This avoids the trouble of smoking as well as varnishing of graph.

Self-study Questions
1. Write functions of Sherrington Recording drum and frontal writing lever.
2. Enlist parts of lever. Write difference between frontal writing lever and heart lever.
3. Write functions of thermostat and heater.
4. What is invivo experiment and invitro experiment?

DATE: / /2018
PRACTICAL: 1(B)
EXPERIMENTAL CONDTIONS FOR ISOLTED TISSUE EXPERIMENTS
Aim: Study of experimental conditions required for isolated tissue.
The in vitro tissue preparation represents isolated preparation or a piece of living tissue which
has been taken from freshly killed animal. The function of the preparation within the body is
maintained by provision of certain basic requirement like Physiological Salt Solution,
Temperature, Aeration, Magnification, Tension, Stabilization Period, Contact time of drug.
PHYSIOLOGICAL SALT SOLUTION (PSS)
ingredients Concentration in gms/l
Frog ringer Tyrode Kreb’s
bicarbonate
de Jalon
NaCl
KCl
CaCl2
NaH2PO4
KH2PO4
MgSO4
MgCl2
Glucose
NaHCO3
6.5
0.14
0.12
0.05
-
-
-
1.5
0.4
8.0
0.2
0.18
0.1
-
-
0.1
1.0
1.0
5.9
0.35
0.28
-
0.16
0.11
-
2.0
2.1
9.0
0.350
0.003
-
-
-
-
0.5
0.5
The ionic requirements and the nutritional supply can be provided by using thr suitable solution,
commonly known as physiological salt solution. Its composition is such that it provides an
artificial media resembling the inorganic composition of blood plasma together with buffer

mechanism to maintain the optimum pH about 7.0 to 7.2 and glucose to facilitate tissue
metabolism. Commonly used PSS are frog ringer (for heart rectus abdominis and other
preparation of frog) Tyrode (for guinea pig ileum, rat ileum, rabbit ileum etc), de Jalon (for rat
uterus), Kreb’s solution (for rat fundus strip, tracheal preparations, vas deferens) etc.
PURPOSE OF EACH INGREDIENT:
Sodium Chloride (NaCl) To maintain iso-osmolarity, isotonicity,
excitability and contractility of the preparation.
Potassium Chloride (KCl) To maintain ionic balance.
Calcium chloride (CaCl2) To maintain the contractility of the preparation.
Sodium Bicarbonate (NaHCO3) To provide alkaline pH.
Glucose To Provide energy.
Sodium or Potassium dihydrogen Act as buffer.
Phosphate (NaH2PO4 or KH2PO4)
Magnesium Chloride or sulphate To stabilize the preparation and
(MgCl2 or MgSO4) and hence to reduce the spontaneous activity.
TEMPERATURE
Temperature of the physiological salt solution should be kept at 370
C (plus or minus 0.50
C).
AERATION
To supply oxygen, the solution is bubbled with air, oxygen or carbogen (i.e., 5% CO2 + 95%
Oxygen). The purpose of the aeration are (1) supply of oxygen, (2) uniform distribution of the
drug due to bubbling, (3) maintenance of the pH specially in kreb’s solution where amount of
sodium bicarbonate is more.
The gas cylinder can be identified by standard colour. Oxygen cylinder has black body with
white top whereas the carbon dioxide cylinder has grey body. Carbogen cylinder has black with
grey and white top. Compressed air has grey body with black and white top.

TENSION
Tone is required for working of tissue. For fast contractile tissue like ileum preparations tension
adjustment required is 500 mg. For slow contractile tissue like frog rectus abdominus muscle, rat
fundus strip and rat anococcygeus muscle tension adjustment required is 1 gm.
ADJUSTMENT OF MAGNIFICATION
In order to achieve 10 times magnification, the distance from the fulcrum to the point of
recording (frontal point) should be 10 times more than the distance from the fulcrum to the point
of tissue attachment.
ADJUSTMENT OF TENSION
The lever is made horizontal by applying plasticine (modeling wax) at the shorter end. A small
amount of plasticine is also placed at the point of attachment of tissue. 1 gm or 500 mg weight is
placed over this plasticine and the lever is again balanced (i.e., made horizontal) by putting the
plasticine on the other side and adjusting the distance from fulcrum. While taking the responses
the weight should be taken off. Thus, when the tissue is tied and the lever is adjusted in
horizontal position, it will exert the tension of 1 gm or 500 mg.
STABILIZATION PERIOD
Tissue must be allowed to stabilize for atleast 30 minutes.
CONTACT TIME
In isolated tissue experiments, one should clearly express the time for which the tissue
preparation remains in contact with drug (contact time). The contact time depends on the type of
tissue preparation and the nature of drug. For slowly contracting tissue preparation, e.g. rectus
abdominis muscle it is 60 to 90 sec. whereas, for fast contracting tissue preparation like guinea
pig ileum it is 30 sec.

1. Write formula for magnification of lever.
2. Write experimental conditions for isolated tissue experiments.
3. Write uses of each ingredients used in preparation of physiological salt solution.
4. Enlist different PSS tissue preparation for which they are used.

DATE: / /2018
PRACTICAL: 2
ANIMALS AND LEGAL REGULATION OF EXPERIMENTAL ANIMALS
Aim: Common laboratory animals.
(A) COMMON LABORATORY ANIMALS
Laboratory animals are those animals which can be bred and reared (maintained) in the
laboratory under suitable conditions. The common laboratory animals are: rat, mice, guinea pig,
rabbit and hamster. The other animals used for experimental purpose are cat, dog, frog, monkey,
pigeon etc.
All animals as a rule should be handled in humane method. In no case animal should suffer from
pain. The health of animal should be maintained. When they are sacrificed the approach should
be euthanasia (painless death).
RAT (Rattus norvegicus: Norway rat) (Adult weight 200-250g; Age suitable for most of the
experiments: 1.5 months)
Albino rats of Wistar strain are commonly used. Other strains used are Sprague-Dawley and
Wistar Kyoto.
Advantages and characteristics:
It is small in size compared to other animals so drugs are required in small quantity. Vomiting
centre is absent and so drug can be administered orally. Gall bladder and tonsil are absent.
Because of the absence of gall bladder in rat there is continuous flow of bile into the intestine.
This facilitates the study of drugs acting on bile, cholesterol reabsorption etc. Pancreas is
diffused, therefore, difficult to produce pancreactomy. In stomach, fundus and pyloric parts have
clear lining between them. The gastric acid secretion is continuous.
Estrus cycle lasts for four to five days and can be divided into four stages as follows.
Estrus: It is characterized by increased running activity, quivering of the ears and lordosis in the
presence of another rat. The vaginal smear shows cornified epithelial cells only. It lasts form 9-
15 hrs and ends with ovulation. Experimentally it can be induced by administration of
diethylstilbestrol (100 µg/kg; i.p.).
Metaestrus: It occurs shortly after ovulation. Leucocytes start appearing in this stage and
predominate over cornified epithelial cells. It lasts for about 15-18 hrs.

Diestrus: It is the longest stage (60-70 hrs). Vaginal smear shows only leucocytes.
Proestrus: It is of about 12 hrs. characterized by nucleated epithelial cells either singly or in
groups.
Experimental uses
1. Psychopharmacological studies.
2. Study of analgesics and anticonvulsants.
3. Bioassay of various hormones such as insulin, oxytocin, vasopressin etc.
4. Study of estrus cycle, mating behavior and lactation.
5. Studies on isolated tissue preparations like uterus, stomach, vasdeferens, anococcygeus
muscle, fundus strip, aortic strip, heart etc.
6. Chronic study on blood pressure.
7. Gastric acid secretion studies.
8. Study of hepatotoxic and antihepatotoxic compound.
9. Acute and chronic toxicity studies.
10. Study on mast cells using peritoneal fluid and mesenteric attachments.
GUINEA PIG (Cavia porcellus) (Adult weight 400-600g; Age suitable for experiments: 3
months)
It is a docile animal. It is highly susceptible to tuberculosis and anaphylaxis. It is also highly
sensitive to histamine and penicillin. It requires exogenous ascorbic acid in diet.
Experimental uses
1. Evaluation of bronchodilators.
2. Anaphylatic and immunological studies
3. Study of histamine and antihistamines.
4. Bioassay of digitalis.
5. Evaluation of local anaesthetics.
6. Hearing experiments because of sensitive cochlea.

7. Studies on isolated tissues specially, ileum, tracheal chain, vas-deferens, teania coli, heart
etc.
8. Study of tuberculosis and ascorbic acid metabolism.
MOUSE (Mus muculus) (Adult weight: 20-25g; Age suitable for experiments 1 month)
Swiss albino mice are commonly used. They are the smallest, cheap and easy to handle.
Experimental uses
1. Toxicological studies, specifically acute and subacute toxicity. They are also used in
teratogenicity (foetal abnormalities).
2. Bioassay of insulin.
3. Screening of analgesic and anticonvulsants.
4. Screening of chemotherapeutic agents.
5. Studies related to genetics and cancer research.
6. Study of drugs acting on central nervous system.
RABBIT (Lupas or Oryctolagus cuniculus) (Adult weight: 1.5-3.0kg; Age suitable for
experiments: 5-6 months)
It is also a docile animal with large ears. Usually New Zealand white rabbits are used. It has huge
caecum and long appendix. The enzyme atropine esterase is present in rabbit liver and plasma, so
it can tolerate large dose of belladonna (atropine). Cardioaortic nerve forms a separate depressor
nerve. Vasodilator nerves are absent and so Dale’s vasomotor reversal phenomenon cannot be
demonstrated. Histamine causes rise in blood pressure. Ovulation is coitus induced and occurs
10th
day after coitus.
Experimental uses
1. Pyrogen testing.
2. Bioassay of anti-diabetics, curareform drugs and sex-hormones.
3. Screening of agents affecting capillary permeability.
4. Irritancy tests.
5. Study of drugs used in glaucoma.
6. Pharmacokinetic studies.
7. Screening of embryotoxic agents and teratogens.
8. Studies related to reproduction (antifertility agents).
9. Isolation preparations like heart, duodenum, ileum, Finkleman preparation.
10. Study of local anaesthetics (surface anaesthesia).
11. Study of miotics and mydratics.

HAMSTER (Mesocriceiuz auratus and Cricetulus griseus) (Adult weight: 80-90 g; Av. age
suitable for experiments: 1 month)
They have short body with short legs and tail. The skin is loose and covered with dense short soft
fur. The cheeks pouches are prominent and extend upto the shoulder region.
Experimental uses
1. Chinese hamsters have low chromosome number making it useful for cytological
investigations, genetics, tissue culture and radiation research.
2. Research on diabetes mellitus.
3. Research related to virology, immunology and implantation studies.
4. Bioassay of prostaglandins.
FROG (Rana tigrina)
This is one of the most commonly used experimental animals used in physiology, pharmacology
and toxicology. It has been used in the experiments for more then 200 years. It is easily available
during rainy season. It is an amphibian animal and safe to handle. It cannot be bred in laboratory.
Adrenaline is neurotransmitter in the sympathetic system.
Experimental uses:
1. Study of isolated tissues such as rectus abdominis muscle and heart preparation.
2. Study of drugs acting on central nervous system.
3. Study of retinal toxicity of drugs, light bleaches rhodopsin in eye within one hour and is
regenerated within one hour in dark.
4. Study of drugs acting on neuromuscular junctions. (using gastrocnemius sciatic muscle
nerve preparation)
CAT (Felis catus, Felis sylvestris)
It is a carnivore, relatively easy to obtain and to use for experimental purpose. The physiology of
circulatory and neuromuscular system is very much similar to that of man. It has a highly
developed nictitating membrane (which is contracted by sympathetic nerves). Morphine
produces excitation of central nervous system in cat.
Experimental uses:
1. Acute experiments for drugs affecting blood pressure.
2. Bioassay of noradrenaline (using spinal cat )
3. Studies on ganglion blockers (using nictitating membrane in vivo).
4. Studies on neuromuscular system (using gastrocnemius-sciatic muscle nerve
preparation.)

5. Toxicity studies of compounds like acetanilide.
DOG (Canis familaris)
Commonly Mongrel or Beagle dogs are used. It is easily available and large sized animal. Dogs
can be easily tamed as well as trained. It has a small stomach and short intestinal tract resembling
those of human beings. It can be conditioned to carry a stomach cannula. The cervical
sympathetic and vagus nerves run together inseparably in the same trunk (vagosympathetic
nerve).
Experimental uses:
1. Gastric acid secretion studies (Pavlov’s pouch).
2. Acute experiment for drugs affecting blood pressure, intestinal movements etc.
3. Studies on anti-diabetic agents.
4. Pharmacokinetic study
MONKEY
Monkey and apes are the primates belonging to the highest order of mammals. The anatomy and
physiology of monkeys and apes closely resemble that of man. The studies done in monkeys may
directly be translated to human beings. Considering the humane aspects, tests in primates should
be done only in the last stage of the evaluation of drugs before clinical trials. They are used in the
fields of psychopharmacology, virology, immunology, nutrition, reproduction, parasitology etc.
(1) Enlist animal which are used in common lab experiments and write down experiment
uses.

DATE: / /2018
PRACTICAL: 3
LEGAL REGULATIONS FOR THE USE OF EXPERIMENTAL ANIMALS
Aim: Maintenance of laboratory animals as per CPCSEA guidelines.
Russel and burch in his book “The principles of humane experimental technique” published in
1959 gave the concept of the 3 Rs… and now 4 Rs concept immerge.
Reduction- in use of animal
Refinement- in experimental techniques
Replacement- of various non animals methods for certain experimental procedures
Rehabilitation- care of animals after experimental uses.
This concept has been gradually accepted all over the world.The prevention of cruelty to
Animals Act of 1960 provided for constitution of a committee to oversee the conduct of
experiment on animals.
CPCSEA- Committee for purpose of control and supervision of experiment on animals
It makes rules for conduct of experiment on animals authorizes its officers to inspect any place
and prohibit a person or institution from carrying out experiments on animals.
Mrs. Maneka Gandhi was chair person of CPCSEA.
CPCSA guidelines state that all lads doing work on animals have to be registered with CPCSEA
and follow rules and guidelines laid down.
All institute level an IAEC- Institutional Animal Ethics Committee has to be constituted to
oversee experiments on animals.
CPCSEA guidelines for laboratory animal facility
• Provide adequate veterinary care – through full time or part time or consultative
veterinary services.
• All animals should acquire lawfully as per CPCSEA guidelines and newly received
animals should be kept separate from animals all ready in the animal house, for at least a week.
• Different species of animals should be kept in separate rooms to prevent interspecies
diseases transmission and prevent behavioral changes due to interspecies conflict.
• Lab animals should be housed in an isolated building located as far away from human
habitation as possible and not exposed to dust, smoke, noise, rodents, insects, and birds.
• Separate rooms for store, washing, office and staff quarantine and corridors should be
provided .
• Sharp fluctuations in temperature, humidity, light, sound, and ventilation should be
avoided.
• Corridors should be wide enough to facilitate movement of personnel as well as
equipments and should be kept clean.
• Animal room doors should be rust and dust proof and should be provided with an
observation window.
• Floor should be smooth, moisture proof, nonabsorbent, skid proof, and resistant to acid,
solvents and adverse effects of detergent and disinfectants.

• Drainage must be adequate to allow for removal of water and drying of surfaces.
• Walls should be free from cracks.
• Storage area should be provided for feed, bedding cages and other material not in use.
• Separate area should be provided for experiments.
• Temperature-18-29 c. relative humidity-30-70%.
• AC should be provided to regulate environmental parameter for lab animals or proper air
circulation should be provided by use of fans. Exhaust fans should be provided to remove foul
odor.
• Proper lighting and ventilation should be provided.
• 0Environment should be noise free.
• Polypropylene, polycarbonate and stainless steel cages should be used to house small lab
animals.
• Feeding and water devices should be provided, with cages.
• Animals should be fed palatable, non contaminated and nutritionally adequate food daily.
• Feeder should be allow easy access to food and avoid contamination by urine, and faces.
• Food should contain moisture, fiber, protein, vitamins, minerals, fat, carbohydrate, for
providing appropriate nutrition.
• Bedding should be absorbent, free of toxic chemicals or ether substances that could injure
animals or personnel. It should be used in amount sufficient to keep animals dry. It should be
removed and replaced with fresh material at least twice a week to keep animals clean and dry.
• Generally tissue paper, cotton, rice husk, is used as bedding.
• Animals should have continuous access of fresh potable drinking water.
• Proper sanitation should be maintained-animal rooms, corridors, storage space, and other
areas should be cleaned with appropriate detergent and disinfectants to keep them free of dirt and
contamination.
• Cages should be sanitized before placing animals in them. Animal cages, racks, accessory
equipments such as feeder and water bottles should be sanitized and washed frequently to keep
them clean and contamination free.
• Transport of animals from one place to another should be undertaken with care. They can
be transported by roads, rails, air. Transport stress should be avoided and cages should be
appropriate size to enable these animals to have comfortable move and protect from injury. Food
and water should be provided during transport.
• Since lab animals are subjected to an environment in captivity, unnatural to their natural
environment, it is essential that specialized care and handling should be taken, since the
psychology of animals play an important role in their health, breeding, well being, and
experiment.
1. Write 4 Rs.
2. What is full form of CPCSEA and IAEC.

DATE: / /2018
PRACTICAL: 4
STANDARD TECHNIQUES FOR DRUG ADMINISTRATION AND COLLECTION OF
BLOOD SAMPLE
Aim: Some common and standard techniques for drug administration (intravenous
injection, intra gastric administration) and collection of blood samples, anesthetics and
euthanasia used for animal studies.
HANDLING OF ANIMALS
Rabbit and Guinea pigs
Hold in firm grip of both hands.
Place in the box in such a way that head projects out from box hole with with two ears free.
Figure 1. Handling of rabbit
Mice and rats
Mouse and rat is gripped at neck firmly with index finger and thumb finger and rest of body
gripped in palm of hand with remaining fingers. Then animal is introduced in restrainer by
keeping tail projecting outside.

Figure 2. Handling of animals.
MODE OF ADMINISTRATION OF DRUG
Intraperitoneal
In this technique animals is held as discussed before and skin is picked up by fingers. Needle is
inserted in the skin first, just by piercing skin; drug is injected by ensuring needle is not in
muscle layers or near nerves.
Oral
Hold animal as shown in finger.
Introduce needle in mouth and push position to force down orally.

Figure 3: Intraperitoneal injection Oral administration
COLLECTION OF BLOOD SAMPLE
The smaller animals may be stunned and blood may be collected from the neck blood vessels.
However, blood samples can also be collected from the animals without sacrificing the animals
by following methods.
Figure 4.Collection of blood from rat/mice tail vain.

Figure 5.Collection of blood from rat/mice retro orbital plexus.
Figure 6. Collection of blood from rabbit marginal ear vein.

Table 1: Standard techniques for injuction of drug and collection of blood sample in
experimental animals.
Animals Intravenous Injections Collection of blood
Mice and Rat Tail vein Heart, puncture of tail vein, by cutting the
tip of tail, retro orbital sinus.
Guinea Pig Penile vein in males Small quantity from marginal ear vein, large
heart puncture, penile vein in males
Rabbit Marginal ear vein Marginal ear vein, cutting the marginal vein
or central artery of the ear at its tip, heart.
Dog and Cat Cephalic vein or Saphaneous
vein
Cephalic vein or Saphaneous vein
ANAESTHESIA AND EUTHENESIA
The anesthesia should be given for 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 experiments or he has inflicted irreparable injury,
the animal should be sacrificed. Neuromuscular blocking agents must not be used without
adequate general anesthesia.
In event of a decision to sacrifice an animal on termination of an experiment or otherwise 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.
Anaesthesia:
Unless contrary to the achievement of the results of study, 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 anesthetics the animal is prepared for anesthesia by overnight 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
anesthesia 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 anesthesia.
A number of general anesthetic agents are used in the form of inhalants. General anaesthetics
are also used in the form of intravenous or intramuscular injection such as barbiturates. Species
characteristics and variation must be kept in mind while using an anesthetic. Side effects such as
excessive salivation, convulsion, excitement and disorientation should be suitably prevented and
controlled. The animal should remain under veterinary care till it completely recovers from
anesthesia and post operative stress.
Table 1. Doses of Anaesthetic agents
Species Anaesthetic agent Dose (mg/kg) Route
Mouse Petobarbitone Sodium 30-50 i.p.
Rat Petobarbitone Sodium
Urethane
30-50
150
125-175
i.p.
i.p.
i.m.
Guinea Pig Petobarbitone Sodium
Urethane
30-50
500-1500
i.p.
i.v.
Rabbit Petobarbitone Sodium
Urethane
30-40
500-1500
i.v.
i.v.
Cat Chloralose
Phenobarbitone Sodium
Petobarbitone Sodium
80-120
180
30-40
i.v.
i.p
i.v.
Dog Chloralose
Phenobarbitone Sodium
Petobarbitone Sodium
Thiopentone Sodium
80-120
180
30-40
12-16
i.v.
i.p.
i.v.
i.v.

Euthanasia
The animals as a rule should be killed in ‘euthanasia’ that is ‘painless or mercy killing’
Euthanasia is resorted to events where animal is required to be sacrificed on termination of an
experiment or otherwise for ethical reason. The procedure should be carried out quickly and
painlessly in an atmosphere free form 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 choice of method will depend on the nature of study, the species of
animal to be killed.
Physical Methods
Decompression
Stunning
The quickest and commonest method for killing mice, rats, guinea pigs and rabbits is preliminary
crushing of head (stunnig) followed by cutting the neck blood vessels to death. Stunning is carrid
out by striking the dorsal part of head against the edge of table or sink. This leads to stiffiening
of all the muscles followed by a series of convulsions and then gradual relaxation of the limbs
and body. As a result fo stunning animal gets sudden shock and temporarily becomes
unconscious. One should have the practice to stun by a single hard stroke only.
INHALATION OF GASES
Nitrogen flushing
Argon flushing
DRUG ADMINISRATION
Curariform drugs, Nicotine sulphate, Magnesium sulphate, Potassium chloride, Strychnine,
Paraquat, Dichlorvos, Air embolism.
The Method of euthanasia should in all cases meet the following requirements:
• Death, without anxiety, pain or distress with minimum time lag phase.
• Minimum physiological and psychological disturbance.
• Compatibility with the purpose of study and minimum emotional effect on the operator.
• Location should be separate from animal rooms and free environmental contaminants.
• Tranquilizers have to be administered to larger species such as monkeys, dogs and cats
before the euthanasia procedure.

(1) Write vein is used in Rat, Mice, G. Pig, Rabbit and Cat for administration of drugs?
(2) What are the way for colletion of small amout of blood and large amout of blood in
Rat, G.pig and Rabbit?
(3) What is Euthenesia? Enlist methods of euthenesia.
(4) What is anaesthesia? Enlist anaesthetic agents.

DATE: / /2018
PRACTICAL: 5
EFFECT OF MICROSOMAL ENZYME INHIBITORS AND INDUCERS
Aim: To study the effect of hepatic microsomal enzyme inhibitors and inducers on
pentobarbitone sleeping time in mice.
Requirements: Mice (20-25 gm), Syringe, Needle (22-24), stopwatch.
Drugs: Pentobarbitone sodium (40 mg/kg), Phenylbutazone (10mg/kg), Ethanol (1mg/kg),
Saline (0.9% NaCl)
Procedure:
1. Weigh the mice and make four groups of 3 mice each.
2. Inject saline (0.1 ml) in first group of mice.
3. In second group inject pentobarbitone sodium and in third and fourth group, in addition
to pentobarbitone sodium, inject ethanol and phenylbutazone respectively. Volume of drug
injected should not exceed 0.5 ml in mice.
4. The injections are given by intraperitoneal route.
5. The time of onset of action is loss of righting reflex in mice, i.e. animal fails to uphold its
normal position or it falls asleep (hypnosis) is recorded for each animal.
6. The animals are placed on their back leaving sufficient space in between two animals.
7. The time of recovery from sleep is recorded. Sleeping time is the time from the loss of
righting reflex and the time when animals turns to recover its normal posture.

Tabulate your observations as follows:
Observation table:
Group
No.
Drug Dose Time of onset Time of recovery
1 Normal saline (0.9 %
NaCl)
1 ml/kg
2 Pentobarbitone 40 mg/kg
3 Pentobarbitone +
Ethanol
40 mg/kg + 1 ml/kg
4 Pentobarbitone +
Phenylbutazone
40 mg/kg + 10mg/kg
Result: Ethanol is a enzyme inducer, which decreased Pentobarbitone induced sleep. Whereas,
Phenylbutazone is a enzyme inhibitor that increased pentobarbitone induced sleep.
Self study questions
1. What is enzyme induction?
2. What is enzyme inhibition?
3. Give examples of enzyme inducers and enzyme inhibitors.

DATE: / /2018
PRACTICAL: 6
Aim: To study the effect of physostigmine and atropine on the ciliary movement of frog’s
Oesophagus.
Requirement: Frog, dissection tray, glass plate, poppy seeds, frog’s ringer solution and drug
solutions.
Principle:
The ciliary movement of esophagus depends on the stimulating action of Ach. It is normally
secreted in the trachea and causes onward contraction of the cilia.
Procedure:
Frog is decelerated in such a way that the lower jaw and the buccal cavity remain intact. The
frog is put on the wooden board with its vertical surface facing the board. Dissection is carried
out from above downwards to half way of the frog’s back. The posterior wall of the abdominal
cavity is opened to about 2.5 to 3.5 cm.
Blood is removed by cotton swabs wetted with frog’s ringer solution. The esophagus is opened
from the buccal cavity (side up) right up to the stomach. The frog is put in a shallow chamber in
which air is kept moist by swabbing with hot water cotton pads. Poppy seeds of uniform size are
used for the experiment. For studying the rest of movement, two lines are marked on the glass
plate at a distance of about 2.5cm.
In the beginning of the experiment, esophageal membrane is wetted with frog’s ringer solution
and the time taken for 10 particles of poppy seeds is first recorded and mean time is calculated.
Membrane is again wetted with frog’s ringer solution after half an hour to see whether similar
results are obtained.
For studying the action of physostigmine, oesophagus membrane is treated with physostigmine
solution, prepared in the frog’s ringer. 10 poppy seeds are used and the mean time for the transist
is noted. This experiment is again carried out after half an hour to confirm the results. If two
consecutive results vary then their mean should be taken as the final, provided the variation is
not very large.

Remove the effects of physostigmine by repeatedly washing the membrane of the oesophagus.
Result:
Physostigmine increases the rate and atropine decreases the rate of ciliary movements.

DATE: / /2018
PRACTICAL: 7
EFFECT OF AUTONOMIC DRUGS ON RABBIT EYE
Aim: To study the effects of Autonomic drugs on rabbit’s eye.
Requirements: Rabbits, Dropper, drug solutions. (Adrenaline 0.1 %, Physostigmine 2 %,
Atropine 1%, Homatropine 0.5%, Ephedrine 5%, Pilocarpine 0.5%, Cocaine 1%, Guanthedine
5%)
Procedure:
1. Put the rabbits in holders in such a way that the head will be protruding outside.
2. Instill the drug solution in one of the eyes carefully.
3. Rabbits will try to remove the solution by blinking eyes (corneal reflex).
4. Instill the solution again till the corneal surface is wetted by the drug solution.
5. Then, study the effect after five minutes.
6. Note the size of pupil, response to light (light reflex) and touch reflex (corneal reflex).
Mechanism of Action: Pupil of the eye is dilated (Mydriasis) by paralysis of the
parasympathetic or stimulation, the circular muscle fibers of the iris contract and due to
sympathetic contracts the radial muscle fibers. Miosis (constriction of pupil) can also occur due
to stimulation of the parasympathetic nerve, occulomotor nerve (3rd
cranial nerve) or inhibition
of the sympathetic nerves.
Discussion:
Pilocarpine is a directly acting parasympathomimetic agent.
Physostigmine is an anticholinesterase agent (directly acting parasympathomimetic agent). Both
the drugs can be absorbed locally and produce miosis.
Morphine is not absorbed by locally and hence effect is not observed. However, if given
intravenously it stimulates Edinger Westphall nucleus in the brain and results in pinpoint pupils.
Ephedrine is an indirectly acting sympathomimetic agent. It produces mydriasis. The light
reflex and corneal reflex are not affected by ephedrine.
Cocaine is a local anaesthetic agent hence, corneal reflexes are lost if cocaine is instilled. The
mydriatic action of cocaine is due to its noradrenaline uptake block activity. Since uptake of
noradrenaline is available at the site to produce mydriasis.

Atropine blocks the muscarinic receptors hence; the sympathetic system predominates, resulting
in mydriasis. Since parasympathomimetic responses are abolished cycloplegia (loss of
accommodation) is seen.
Guanethidine is an adrenergic neuron blocker. It produces slight mydiatic action due to
noradrenaline uptake blocking activity. Because it is adrenergic neuron blocker, ephedrine fails
to produce mydriatic effect in the presence of guanethidine.
Observations:
Drug Size of Pupil Light reflex Corneal reflex
Pilocarpine Decreased Present Present
Physostigmine Decreased Present Present
Morphine No change Present Present
Ephedrine Increased Present Present
Cocaine Increased Present Absent
Atropine Increased Absent Present
Guanethidine No change Present Present
Decreased: Miosis, Increased: Mydriasis
Result: Miotic drugs increased pupil size and Mydriatic drugs decreased pupil size of rabbit.
Atropine showed absence of light reflex whereas, local anaesthetics (cocaine, xylocaine) showed
absence of corneal reflex.

Self study questions
1. Enlist miotics and mydriatic.
2. Write mechanism of miosis and mydriasis.
3. Explain effect of sympathetic and parasympathetic stimulation of rabbit eye.
4. What is light reflex and corneal reflex.

DATE: / /2018
PRACTICAL: 8
RECORDING OF SKELETAL MUSCLE RELAXANT ACTIVITY
Aim: To study the effect of various tranquillizers and sedatives on motor Co-ordination
by Rota rod test in mice.
Principle:
Minor tranquilizers or antianxiety agents like benzodiazepam produce specifically the skeletal
muscle relaxation. The site of this activity is the central nervous system (CNS). Disturbance in
the maintenance of tone and moisture is the first sign of centrally mediated skeletal muscle
relaxation. A mouse 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. The rate of rotation of the rod should be adjusted such that a normal mouse
can stay on the rod for an appreciable period (3-5 min) of time.
Requirements: Rota rod apparatus, mice, syringe and needle.
Drugs: Diazepam, Chlorpromazine, Pentobarbitone.
Apparatus:
Rota rod apparatus consists of a metal axle, with a horizontal wooden or iron rod 2.5 cm in
diameter and 5.3 cm in length. Rod is rotating at variable speed (15-25 r.p.m). Rod is divided
into 4-5 sections with the help of wheels or card board disc. Timer and counter are at the base
of instrument.
Rota rod apparatus

Procedure:
1. Mice weighing 20 to 25 g are selected. They are placed on a rod rotating at 20 r.p.m.
More than one mouse can be placed at a time on the rod which is divided into many
sections.
2. “Fall off time” is being recorded when the mouse falls the rod for each mouse. A normal
(untreated) mouse generally falls off within 3-5 minutes.
3. Various CNS depressants are injected and after half an hour of injection, the “fall off
time” is recorded.
4. Compare the fall off time of animals before and after drug treatment.
Observation
After drug treatment, reduction in fall off time indicating skeletal muscle relaxant property.
Study Questions:
1. Classify skeletal muscle relaxants.
2. Explain the principle of working of rota-rod apparatus.
3. Which parameter is observed for evaluation of skeletal muscle relaxant activity?

DATE: / /2018
PRACTICAL: 9
RECORDING OF SPONTANEOUS MOTOR ACTIVITY USING PHOTOACTOMETER
Aim: To study the effects of drugs on spontaneous motor activity and to evaluate their
nature as CNS stimulants or depressants.
Principle:
A drug increasing or decreasing CNS activity will also produce increase or decrease in
spontaneous motor activity (SMA) in the animals. Photoactometer is designed on this
principle. The instrument consisits of a cage which is 30cm long and 30cm deep. It has a wire
mesh at the bottom. Six lights and six photocells placed in the outer periphery in such a way
that single mice can block only one beam. Technically, it’s principle is that a photocell is
activated when the rays of light falling on photocells are cut-off by animals crossing the beam
of light. The photocells are connected to an electronic automatic counting device which counts
the number of “cut-offs”.
Instrument: Photoactometer (Actophotometer, Techno.)
Drugs: Amphetamine, Phenobarbitone, Saline (0.9% NaCl).
Procedure:
Mice weighing 20-25g are divided into 3 groups of 4 each. One group received saline. Other
two groups receive amphetamine (1 mg/kg) and phenobarbitone (30 mg/kg). The drugs are
administered through i.p. route.
Each group of animals is placed in the photoactometer separately and number of ‘cut offs’ is
recorded for 10 min. duration at the interval of 30 minutes, till the maximum effect of drug is
recorded.
Conclusions:
1. Since the number of counts are increased by amphetamine, it indicates that spontaneous
motor activity is increased by this drug. Hence, this is a CNS stimulant drug.
2. Since the numbe of counts are decreased by phenobarbitone, it indicate spontaneous motor
activity is decreased by this drug. Hence, this is a CNS depressant drug.
CNS stimulants: Atropine, Amphetamine, Strychnine, Picrotoxin, Pentylenetetrazole,
Doxapam, Ethamivan, Nikethemide, Methylphenidate, Caffiene, Theophylline, Theobromine

CNS depressants: Anaesthetics, alcohols, sedatives, hypnotics, narcotics
Study Questions:
1. Which instrument is used for spontaneous motor activity evaluation?
2. Explain principle of working of Photoactometer.
3. Explain effect of CNS stimulants and CNS depressants on spontaneous motor activity.

DATE: / /2018
PRACTICAL: 10
RECORDING OF ANTICONVULSANT ACTIVITY
(B) Aim: To study anticonvulsant property of phenytion against maximal electro-shock
induced convulsion in rat.
Principle:
Different types of epilepsies - grand mal, petit mal or psychomotor type can be studied in
laboratory animals. The maximal electro-shock (MES) induced convulsions in animals
represent grand mal type of epilepsy. Similarly, chemo-convulsions due to pentylenetetrazol
which produce clonic type of convulsions resemble petit mal type of convulsions in man.
These are the two procedures used to study convulsions and to test anticonvulsant drugs in
laboratory animals.
In MES-convulsions electroshock is applied through the corneal electrodes. Through optic
stimulation cortical excitation is produced. The MES-convulsions are divided into five phases
such as (a) tonic flexion (b) tonic extensor (c) clonic convulsions (d) stupor and (e) recovery or
death. A substance is known to possess anticonvulsant property if it reduces or abolishes the
extensor phase of MES convulsions. This procedure may be used in rats and mice.
Requirements:
Drugs: Phenytoin (100 mg/kg)
Equipment: Elecro-convulsometer, corneal electrode (apply 150 mA current for 0.2 sec), stop-
watch.
Procedure:
1. Weigh and number the animals. Divide them into two groups each consisting of 4-5 rats.
One group is used as control and the other for drug (phenytoin) treatment.
2. Hold the animal properly, place corneal electrodes on the cornea and apply the prescribed
current. Note different stages of conclusions i.e. (a) tonic flexion, (b) tonic extensor
phase, (c) clonic convulsions, (d) stupor, and (e) recovery or death. Note the time (sec)
spent by the animal in each phase of the convulsions. Repeat with other animals of
control group.
3. Inject phenytoin intraperitoneally to a group of 4-5 rats. Wait for 30 min and subject the
animals to elecroconvulsions as described in step-2.
4. Note the reduction in time or abolition of tonic extensor phase of MES-convulsions.

Observation: Reduction in time or abolition of tonic extensor phase of MES-convulsions
indicative of protective action of drug.

(C) Aim: To study anticonvulsant property of Diazepam against Pentylenetetrazole
Induced convulsion in mice.
Requirements:
Drugs: Pentylenetetrazol (80 mg/kg, i.p.), Diazepam (Dose 4 mg/kg, i.p.)
(Strychnine 4 mg/kg, i.p or picrotoxin 8 mg/kg, i.p. can also use for induction of convulsion in
rats.)
Procedure:
1. Weigh and number the animals. Divide the animals into two groups each comprising of 5
animals. One group is used for studying the effects of pentylenetetrazol alone (control) and the
other for studying the protective effect of diazepam.
2. Inject pentylenetetrazol to control animals and note the onset of action (indicated by
Straub’s tail, jerky movements of whole body and convulsions) and severity of convulsions
due to the drug.
3. Inject diazepam to second group. After 30 min inject pentylenetetrazol to these animals
which have received diazepam. Note onset and severity of convulsions (step 2).
4. Note either delay or complete abolition of convulsions in mice treated with diazepam.
Observation: reduction in time or abolition of clonic convulsions indicating protective action
of drug.
Discussion
Epilepsy is synchrous discharge of impulses from brain characterized by aura, cry, tonic and
clonic convulsions. There is spontaneous occurrence of brief episodes associated with
disturbance in consciousness and excessive EEG discharges.
Drugs showing prevention against MES- induced convulsions are effective in grand mal
epilepsy in human beings and drugs which prevent chemically induced convulsions are
effective therapeutically in petit mal epilepsy.
Antiepileptics
1. Barbiturates: Phenobarbitone, Methylphenobarbitone
2. Hydantoins: Phenytoin, Ethotoin
3. Oxazolidinediones: Trimethadione, paramethadione
4. Succinimides: Phensuximide, Thiosuximide
First two groups are effective in grand mal epilepsy while other two groups effective in petit
mal epilepsy.

Study questions:
1. What is Epilepsy? Describe different types of Epilepsy also Give recommended treatment
for each type of Epilepsy.
2. Name chemicals used for induction of convulsion in experimental animals.
3. Name experimental method used for grandmal and petitmal epilepsy.

DATE: / /2018
PRACTICAL: 11
RECORDING OF APOMORPHINE INDUCED STEREOTYPY
Aim: To study the taming effect of chlorpromazine in rats and mice or to study the effect
of chlorpromazine on apomorphine induced compulsive behavior (stereotypy) in rats
and mice.
Principle:
Compulsive behavior is defined as a purposeless activity exhibited by the animal. This
purposeless activity is supposed to be identical to the behavioural disorder seen in
schizophrenic (psychotic) patients who also show repetitive purposeless activity. This
behavioural abnormality in schizophrenia is due to the excessive neuronal activity of dopamine
in the limbic system. Apomorphine, a dopamine receptor agonist, through its dopaminergic
activity induces compulsive stereotyped behavior in rats and mice. The stereotyped behavior
includes repetitive standing (rearing), continuous sniffing (touching the nose to the wall of the
container) and licking of the wall of the container. These behaviours can be easily observed
and subjectively scored also.
Chlorpromazine, an antipsychotic agent inhibits apomorphine induced compulsive behavior by
blocking the dopamine receptors.
Requirements: Rats (150-200g), mice (15-25g), syringe and needle, clean beakers (250 ml for
mice and 1000 ml for rats).
Drugs: Apomorphine (2.5 mg/kg, i. p.), Chlorpromazine (3 mg/kg, i. p.)
Procedure:
1. Weigh the animal and divide them into two groups of three animals.
2. Inject saline into one group and chlorpromazine (3 mg/kg) into another group of animals.
3. After thirty minutes, inject apomorphine (2.5 mg/kg) into all the animals and place them
individually into separate beakers and observe the intensity of compulsive behavior, i.e.
rearing, sniffing and licking. Note the onset of these responses after 15-30 and 60 min
after apomorphine injection. Depending on the severity of responses scores like 1.
Presence of response, 2. Moderate response, 3. Severe response. The actions are
compared in two groups of animals.
Observation: Apomorphine induces compulsive rearing, sniffing and licking behavior in mice.
This effect is blocked by pretreatment with chlorpromazine.

Study Question:
1. Explain mechanism of Apomorphine induced Compulsive behavior and chlorpromazine in
rats and mice.
2. Classify Antipsychotic drugs.
3. What is compulsive behavior?

DATE: / /2018
PRACTICAL: 12
Aim: To Study the anxiolytic (anti-anxiety) effect of diazepam in mice using elevated zero-
maze.
Principle: The elevated zero-maze test is a behavioural test of anxiety based on the naturalistic
tendency of rodents to avoid open and elevated areas. It is similar to more widely used elevated
plus maze, accept that the open and closed arms are arranged circularly, thus eliminating the
center area which removes ambiguity in interpretation of time spend in the central square of the
traditional design. This maze is an elevated (40cm), white or black, annular having outer
diameter of 45cm and inner diameter of 30cm. The runway ring where the mouse can explore is
of 6cm width, which is divided into 4 quadrants, 2 opposing “open” quadrants without walls and
2 opposing “closed” quadrants having 12cm high walls. The open quadrants have a ridge of 2-
3mm to prevent the mouse to fall off. The walls have thickness of 0.75cm (Fig.5.9.). The model
is widely used for the evaluation of anxiolytic agents belonging to chemically different class of
drugs.
Requirements:
Animal Mice (20-25g)
Drugs Diazepam (Dose 2 mg/kg I.p.). Prepare a stock solution containing 0.2 mg/ml of the
drug in 1% w/v gum acacia or carboxymethylcellulose and inject 1ml/100g body weight of the
mouse.
Equipment the Zero-maze with the dimensions described as above.
Procedure:
1. Weigh and number the animals. Divide them into two groups each consisting of
minimum of 6 mice. One group is used as control and other for drug (Diazepam)
treatment.
2. Place the animal individually in the open arm facing towards the closed arm. Start the
stopwatch and note the following parameters for a period of 6 minute:
(a) Latency to enter the open arm.
(b) Average time each animals spends in the open/closed arm.
(c) Total number of entries in the open arm (an open arm entry is defined as the entry
when all the four paws of the animal are in the arm).
(d) Stretchings (elongated body posture) into open quadrant.
3. Clean the maze thoroughly using tissue paper after each trial.

4. Inject diazepam to the test group. After 30 minutes place the animals individually as
described above and note all parameters as in step 2.
5. Compare the time spend in open arm, latency to enter the open arm, number of entries in
open arm as well as number of stretchings .
Observations:
Sr.no Treatment
(mg/kg)
Animal id Open arm
Time spent
(sec)
Latency
(sec)
Number
of entries
Number of
stretchings
1. Control 1
2
3
4
5
6
2.8
3.5
3.9
256
262
246
1
1
1
10
9
9
Mean
SEM

2 Diazepam (2) 1
2
3
4
5
6
41.16
45.00
39.25
48
47
51
12
13
16
3
1
2
Mean
SEM
Results: Diazepam increases the time spent in open arm, decreases the latency to enter the open
arm, increases the number of entries in the open arm and decreases the number of stretchings.
Inference: Diazepam shows anti-anxiety effect in mice zero-maze model of anxiety.

DATE: / /2018
PRACTICAL: 13
STUDY OF LOCAL ANESTHETICS BY DIFFERENT METHOD.
AIM:-To evaluate local anesthetic agent by various method:
A) Surface anaesthesia B) Intradermal method
C) Plexus Anaesthesia D) Conduction Anaesthesia.
A) SURFACE ANAESTHESIA
Apparatus: Animal holder, cotton, stopwatch
Drugs: Procaine hydrochloride
Principle:
Cornea is sensitive part of eye which respond to a very light touch. When local
anaesthetic is applied or instilled in the eye, it will abolish the neuronal activity in that area. So
when a plug of cotton is touched to cornea the blinking(corneal reflex)is not observed.
Procedure:
1. An adult is restrained in animal holder and ther corneal reflex is checked by
touching cotton plug to the cornea from the side of animal.
2. The local anaesthetic is instilled into the eye.
3. After a few seconds,the corneal reflux is agained checked. This is repeated ath
every one minute for five minutes.the number of failures are noted down.
For different drugs and concentration different animals are used, keeping one eye as the
control.

Observation:
Drug Conc. Eye Corneal reflex(minutes) Total
number
of failure
per no. of
test
1 2 3 4 5
Right + - - - + 3/5
Left
B) INTRADERMAL METHOD
Apparatus: Scissors, Pricking needle
Principle:
Pricking on the sin produces a twitch or movement indicating that the pain is produced.
Intradermal injection of local anaesthetic in the skin inhibits this response in the area where the
injection is given.
Procedure:
Adult guinea pig of either sex is used. The hairs on the back are removed from two areas
of 4.5cm.
Wheals are produced in the shaved area by intradermal injection of drug. Size of the
wheal is marked by ink. Time of injection is noted. After 5 minutes 6 prickes are given to the
area at the interval of 30 sec. and no of pricks that fail to produce twitching are noted.
This is repeated for 6 times every 5 minutes. This method can be used in human using
skin. However, if a drug is vaso constrictor, the local anaesthetic value cannot be evaluated by
this method

Observations:
Drug Conc. NO.of failure to responses(min)
6 12 18 24 30
Total no. of
failure
Percentage
response
(out of 36)
C) PLEXUS ANAESTHESIA
Apparatus:- scalpel, forceps, scissors, stand
Principle:-
Local anaesthesia produces blockade of all type of nerves. Thus, stimulation to these
nerves will fail to produce any response (eg-sensory or motor reflux)if the drug under test is
applied between the stimulating point and muscle.
Procedure:
Frogs of either sex are used. Animal is decapitated (head is cut down) and spinal cord is
destroyed upto the third vertebrae just below the sternum a transverse incision is made in
abdomen and viscera are removed through this opening.
The frog is the mounted vertically on a stand.
The solution of local anaesthetic is filled in thepouch of lower abdomen.this will
submerge the lumbar plexus. Sensory impulse is then applied by immersing the feet in dil.HCl
for 30 sec. and the response is observer(i.e.withdrawal of limb from the solution).after this limbs
are washed with water. This is done after every one min. and time required to abolish withdrawal
response is noted.
For quantitative work, atleast four frogs are used.
(This method is not accepted by CPCSEA).

D) CONDUCTION ANAESTHESIA
Apparatus: Artery clamp, Tuberculine syringe, mice
Principle: Same as that of plexus anaesthesia
Procedure:
Mice of either sex weighing 18-25 g are used and tail is pressed by artery clamp. Those
animal which try to remove the clip are selected.
For each dose of drug , a group of 10 mice is used . each animal is injected with 0.1 ml of
drug subcuteneously into the tail above the point where the clip is applied. After 15 minutes of
injection the clip is applied to each animal and observed for reflex action( removal of clip).
Degree of conduction anaesthesia is expressed as number of mice in group producing for
response vs. number of mice used in group(x/10).

Pharmacology-I_sem-4_RDP lab manual

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Pharmacology-I_sem-4_RDP lab manual