PRACTICAL LAB MANUAL

Pharmacology - I

B. Pharm IVth Semester

 CONTENT

Sl. No Content Page No.

1. 2-3
Introduction to experimental pharmacology

2. Commonly used instruments in experimental pharmacology 4-5

3. 6-8
Study of common laboratory animals
4. 9-17
CPCSEA guidelines for laboratory animal facility
5. 18-19
Study of different route of drug administration
6. 20-21
Effect of drugs on ciliary motility of frog oesophagus
7. 22-23
Effect of drugs on rabbit eye

8. 24-25
Effects of skeletal muscle relaxant using rota –rod apparatus
9. 26-27
Study of drugs on locomotor activity using actophotometer

10 28-29
Anticonvulsant effect of drugs by maximal electro-shock induced
convulsions in rats
11. 30-31
Study of stereotype anti catatonic activity of drugs on rat/mice

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 Experiment No: 1

INTRODUCTION TO EXPERIMENTAL PHARMACOLOGY

Pharmacology is the science which deals with the study of drugs. The word ‘pharmacology’ is
derived from the Greek words ‘Pharmakon’ (a drug or poison) and logos (discourse). It broadly
covers the information about the history, source, physiochemical properties, physiological
properties, mechanism of action, absorption, distribution, metabolism and excretion of drugs.
Drugs are chemical agents used for the purpose of diagnosis, prevention relief or cure of a disease
in man or animals. The word drug isderived from the French word ‘drogue’ meaning herb.

Experimental pharmacology is relatively the youngest branch of basic medical sciences. The
advancement in the field of electrophysiology, biochemistry, molecular biology and electronic or
digital recording systems and software’s have enriched and broadened the horizonsof
experimental pharmacology.

The main aims of the experimental pharmacology are to

1. Find out a therapeutic agent suitable for human use

2. Study the toxicity of a drug
3. Study the mechanism of action of drugs
Since experimental pharmacology involves the discovery of new drugs or to study the action of
existing drugs it is done in two main stages

 Preclinical experimental pharmacology which involves the identification and optimization of

novel chemical lead structures and testing them on animals and animal tissues or organs for
their biological actions.
 Clinical pharmacology where testing of drugs is done on human volunteers and patients for
assessing the pharmacokinetics, safety and efficacy in humans.

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 Experiment No: 2

COMMONLY USED INSTRUMENTS IN EXPERIMENTALPHARMACOLOGY

In spite of a tremendous development in electronic devices and recording systems are followed in
institutions and research laboratory. Some common instruments are used in experimental
pharmacology

Organ bath:

The tissue bath used to put the animal tissue for studying the drug actions is called student organ bath.
This was first designed by Rudolph Magnus in 1904. The organ bath essentially consist of

 An outer jacket made of up of steel or glass or Perspex.

 The inner organ or tissue bath made up of glass with a capacity varying from 10 -50 ml
 Thermostatically controlled heating rod
 Stirrer to keep the water in the outer jacket at uniform temperature
 Oxygen or delivery glass tube which also serves as tissue holder
 Glass coil, one end of which is connected having the physiological salt solution.
The student organ bath having two units of inner tissue bath is called double unit organ bath.

Rota rod apparatus:

For the study of muscle relaxant property of diazepam in mice. The loss of muscle grip in an indicator
of muscle relaxation. This effect can be studied in animals using an inclined plane or rotating rods.

Actophotometer:

It is used to study CNS depressant property of chlorpromazine on the loco motor activity of mice.
CNS depressant drugs like alcohol reduces the motor activity with the stimulants like caffeine and
amphetamines increases the activity. The actophotometer operates on photoelectric cell which is
connected in circuit with counter.

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Electro-convulsometer:
It is used to study the anticonvulsant activity of phenytoin against electro-convulsometer induced in
rat. The electric shock is applied through the corneal electrodes; it produces 5 phases such as tonic
flexion, tonic extensor, clonic convulsion, stupor, and recovery/death.

Pole climbing apparatus

It is used to study the anxiolytic activity in rat and mice. The basic principles of pole climbing
apparatus is based on a neuro-chemical mechanism of anxiety disorders drugs like benzodiazepam are
used.

Analgesiometer:

It is used to study the analgesic effects of drugs in mice and rat. The inducer of pain such as heat,
physical compression and chemical inducers. The basal reaction time was noted as an inference of
pain sensation drugs like morphine and other NSAID are used.

Metabolic cage:

It is used to study the metabolic parameters such as fecal and urine for the study of purgative or
laxative, animals such as rat and mice are used.

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 Experiment No: 3

STUDY OF COMMON LABORATORY ANIMALS

1. Guinea pig
Guinea pigs (400-600g) are the commonly used experimental animals. They are very docile and easy
to raise and maintain they are highly sensitive to histamine. They are used in experimental asthma to
study bronchodilators. They are also used to local anaesthetics and as a model in amoebiasis and
cholera as they are sensitive to this microorganism.
2. Albino rat
White rat (200-250g) is the commonest laboratory animal used in experimental pharmacology. Rats
are easy to breed and maintain. Resemble man in several organ function and nutrition and sensitive
to most of the drugs; make them very useful experimental animals. However they do not have
vomiting centre. The various rat tissue used are colon, stomach, uterus, caecum and vas deference.
Besides these organs rat brain tissue is extensively employed in radio receptor ligand studies. The
other strains of rats are Sprague- Dawley and porton.
3. Albino mouse
White mice are the smallest laboratory animals used. Mice are also easy to breed and maintain. They
are small in size (25-30g) and therefore, easy to breed and maintain. They are sensitive to most of the
drugs used in experimental pharmacology. Mice are used extensively in toxicity study, bio assay of
insulin, testing of analgesics, CNS active drugs and chemotherapeutic agents. More recently mouse
brain as well as primary cell culture of mouse spinal cord neurons are used in neuro pharmacology
for studying neurotransmitters receptor functions. The other strains of mice used are Laca and
Balb/C.

4. Rabbit
Domestic rabbits (2-3 kg) are generally used for pyrogen testing. Some of the tissues or
organs from rabbits used are heart, aorta, duodenum and ileum. One peculiar thing about rabbits is
that they are resistant to the actions of atropine as they contain atropine-esterase enzyme, the
presence of which is genetically determined.

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5. Frog

Frogs (150-200g) were one time extensively used in experimental pharmacology. However, recently
the use of wild frogs for experimental purposes has been banned. Earlier, frogs were used for isolated
heart, rectus abdominis muscle preparation, study of muscle nerve and ciliary movements,
respectively. Frogs were also used for the study of nerve block type of local anaesthetics. Frogs are
inexpensive and easily available, and the ban on the use of frogs has been debated.
Other animals
Cats, dogs and monkeys are used for pharmacological investigations of drugs. Cats and dogs were
one time commonly used to study blood pressure experiments. But their use has been now restricted.
However beagle dogs are the only strain approved by regulatory authorities (USFDA) for preclinical
testing of new drugs.
Alternatives to animal experimentation
Because of the growing concern on the use of animals in biomedical research in several countries have
passed legislation to prevent or usage of animal experimentation. These include experiment with tissue
and body fluids of normal animals and human use of micro-organisms, primary cell culture and cell
lines, use of models and computer simulation and software are being used now a days as per the
common laboratory alternate animals

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Zebra fish Danio rerio Developmental assay
Drug discovery
Neurobiology
Toxicity
Worms Habolitis Developmental assay
Neurobiology
Toxicity

Fruit fly Drosphila Cancer biology

Developmental assay
Neuroscience

Tunicates Ciona sp Developmental assay

Neuroscience

Star fish Echinoderms Developmental assay

neuroscience

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 Experiment No: 4

MAINTENANCE OF LABORATORY ANIMALS AS PER CPCSEA GUIDELINES

Good Laboratory Practices (GLP) for animal facilities is intended to assure quality
maintenance and welfare of animals used in laboratory studies while conducting biomedical
and behavioral research and testing of products.
1. GOAL
The goal of these Guidelines is to promote the humane care of animals used in biomedical and
behavioral research and testing with the basic objective of providing specifications that will
enhance animal well-being, quality in the pursuit of advancement of biological knowledge that
is relevant to humans and animals.
2. VETERINARY CARE
a. Adequate veterinary care must be provided and is the responsibility of a veterinarian or a
personwho has training or experience in laboratory animal sciences and medicine.
b. Daily observation of animals can be accomplished by someone other than a veterinarian;
however, a mechanism of direct and frequent communication should be adopted so that timely
and accurate information on problems in animal health, behaviour, and well-being is
conveyed to the attending veterinarian.
c. The veterinarian can also help the establishment in designing appropriate policies and
procedures for ancillary aspects of veterinary care, such as use of appropriate methods to
prevent and control diseases (e.g. vaccination and other prophylaxis, disease monitoring and
surveillance, quarantine and isolation), operative and post-operative care, diagnosis and
treatment of diseases as well as injuries. Reviewing protocols and proposals, animal
husbandry and animal welfare; monitoring occupational health hazards containment, and
zoonosis control programs; and supervising animal nutrition and sanitation. Institutional
requirements will determine the need for full-time or part-time or consultative veterinary
services.
2. ANIMAL PROCUREMENT
All animals (like cattle, buffalo, sheep, goat, pigs, equine etc.) must be acquired lawfully as per
the CPCSEA guidelines. Small animals and dogs can be procured from registered breeders.
Large animals can be procured from farm, farmers or as per guidance of wild life department,
as is done in case of macaques. Cats can be bred for their use. Rodents can be imported from
abroad after necessary license from Director General of Foreign trade (DGFT) is obtained for
import.

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b. A health surveillance program for screening incoming animals should be carried out before
purchaseto assess animal quality. Methods of transportation should also be taken into account
c. Each consignment of animals should be inspected for compliance with procurement
specifications, and the animals should be quarantined and stabilized according to procedures
appropriate for the species and circumstances.
3. QUARANTINE, STABILIZATION AND SEPARATION
a. Quarantine is the separation of newly received animals from those already in the facility until
the health and possibly the microbial status of the newly received animals have been
determined. Aneffective quarantine minimizes the chance for introduction of pathogens into an
established colony. The duration at quarantine in small lab animals is from one week to one
month and large animals allowed up to 6 weeks (cat, dog, monkey, etc.).However, duration
of quarantinecan be increased depending on type of infection /suspected infection noticed in
the animals.
b. Effective quarantine procedures should be used for non-human primates to help limit exposure
of humans to zoonotic infections. The period varies from 2 to 3 months depending on the
reaction of TB testing. Any macaque found positive for TB for at least two times and shows
signs of weight loss or ill health should be euthanized as is practiced internationally to prevent
spreading of TB to workersand other macaques.
c. Regardless of the duration of quarantine, newly received animals should be given a period for
physiological, psychological and nutritional stabilization before their use. The length of time
stabilization will depend on the type and duration of animal transportation, the species involved
andthe intended use of the animals.
d. Physical separation of animals by species is recommended to prevent interspecies disease
transmission and to eliminate anxiety and possible physiological and behavioral changes due to
interspecies conflict.
e. Such separation is usually accomplished by housing different species in separate rooms;
however, cubicles, laminar-flow units, cages that have filtered air or separate ventilation, and
isolators can be used as suitable alternatives.
f. In some instances, it shall be acceptable to house different species in the same room, for
example, if two species have a similar pathogenic status and are behaviorally compatible.

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 Separate set of personnel should be identified for taking care of these infected (sick) animals and
other workers should be restricted from entering in to the facilities unless otherwise required and
after handling these animals they should not be handling any other animals in the facilities
4. SURVEILLANCE, DIAGNOSIS, TREATMENT AND CONTROL OF DISEASE
(a) All animals should be observed for signs of illness, injury, or abnormal behavior by animal
house staff. As a rule, this should occur daily, but more-frequent observations might be
warranted, such as during postoperative recovery or when animals are ill or have a physical deficit.
It is imperativethat appropriate methods be in place for disease surveillance and diagnosis
(b) Post-mortem examination and signs of illness, distress, or other deviations from normal
health condition in animals should be reported promptly to ensure appropriate and timely delivery
of veterinary medical care. Animals that show signs of a contagious disease should be isolated
from healthy animals in the colony. If an entire room of animals is known or believed to be
exposed to an infectious agent (e.g. Mycobacterium tuberculosis in non-human primates), the
group should be kept intact and isolated during the process of diagnosis, treatment, and control.
Animals suffering from contagious diseases like Tuberculosis etc. must be euthanized as is
practiced internationally to preventits spread to other animals and often animal handlers.
(c) The isolation, quarantine and stabilization programs for newly arrived animals are necessary
to provide time to assess their health status, allow them to recover from the stress of shipment and
an opportunity to adapt to their new environment. The extent of these programs depends on
several factors, including species and source of the animals as well as their intended use. For
some animals, such as rodents obtained from reliable sources for which health status is known,
visual inspection on arrival may suffice. For species such as nonhuman primates, farm animals,
wild animals, dogs, cats and non- specific pathogen free rabbits and rodents, appropriate
quarantine and isolation procedures must be employed.
(d) Preventive medicine programs such as vaccinations, ecto- and endo-parasite treatments and
other disease control measures should be initiated according to currently acceptable veterinary
medical practices appropriate to the particular species and source. Only animals of defined health
status shouldbe used in research and testing unless a specific, naturally occurring or

11
induced disease state is being studied. Systems should be established to protect animals within the
institution from exposure to diseases.
(e) Transgenic and mutant animals may be particularly susceptible to diseases and may require
special protection to ensure their health. Systems to prevent spread of disease may include facility
design features, containment/isolation equipment, and use of standard operating procedures.
Training of animal care and research staff is essential to prevent spread of animal diseases.
(f) Disease surveillance is a major responsibility of the veterinarian and should include routine
monitoring of colony animals for the presence of parasitic and microbiological agents that may
causeovert or unapparent disease. Additionally, cells, tissues, fluids, and transplantable tumors that
are to be used in animals should be monitored for infectious or parasitic agents that may cause
disease in animals. The type and intensity of monitoring necessary will depend upon professional
veterinary judgment and the species, source, use and number of animals housed and used in the
facility.
(g) Diagnostic laboratory services must be available and used as appropriate. Laboratory services
should include necropsy, histopathology, microbiology, clinical pathology, serology, and
parasitology as well as other routine or specialized laboratory procedures, as needed. It is not
necessary that all of these services be available within the animal facility (Facilities from other
laboratories with appropriate capabilities may be used).
(h) Animals with infectious / contagious disease must be isolated from others by placin them in
isolation units or separate rooms appropriate for the containment of the agents of concern. In
certain circumstances, when an entire group of animals is known or suspected to be exposed or
infected, it may be appropriate to keep the group intact during the time necessary for diagnosis and
treatment, fortaking other control measures, or for completion of a project.
(i) The veterinarian must have authority to use appropriate treatment or control measures,
including euthanasia in consultation with at least one more additional veterinarian if required,
following diagnosis of an animal disease or injury. If possible, the veterinarian should discuss the
situation with the principal investigator to determine a course of action consistent with
experimental goals. However, if the principal investigator is not available or if agreement cannot
be reached, the veterinarian must have authority to act to protect the health and well- being of the
institutional animalcolony and workers.

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 5. ANIMAL CARE AND TECHNICAL PERSONNEL
(a) Animal care programs require technical and husbandry support. Institutions should employ
people trained in laboratory animal science or provide for both formal and on-the- job training to
ensure effective implementation of the program (Annexure-7).
6. PERSONAL HYGIENE
(a) It is essential that the animal care staff maintain a high standard of personal cleanliness.
Facilities and supplies for meeting this obligation should be provided with appropriate Personnel
ProtectiveEquipment (PPE) e.g. showers, change of uniforms, footwear etc.
(b) Clothing suitable for use in the animal facility should be supplied and laundered by the
institution. A commercial laundering service is acceptable in many situations; however,
institutional facilities should be used to decontaminate clothing exposed to potentially hazardous
microbial agents or toxic substances. It is acceptable to use disposable gloves, masks, head
covers, coats, coveralls andshoe covers. Personnel should change clothing as often as is
necessary to maintain personal hygiene. Outer garments worn in the animal rooms should not
be worn outside the animal facility.
(c) Washing and showering facilities appropriate to the program should be available. Personnel
should not be permitted to eat, drink, smoke or apply cosmetics and perfumes in animal rooms.
They shouldfinish the work with animals as early as possible and sit somewhere else, outside and
not in the animal rooms / areas. A separate area or room should be made available for these
purposes.
7. ANIMAL EXPERIMENTATION INVOLVING HAZARDOUS AGENTS
(a) Institutions should have policies governing experimentation with hazardous agents. Institutional
Bio-safety Committee whose members are knowledgeable about hazardous agents are in place in
most of the higher-level education, research institutes and in many pharmaceutical industries for
taking care of safety issues. This committee shall also examine the proposal on animal experiments
involving hazardous agents in addition to its existing functions
(b) Since the use of animals in such studies requires special considerations, the procedures and the
facilities to be used must be reviewed by both the Institutional Bio-safety committee and
Institutional Animal Ethics Committee (IAEC). Disposing of tissues and fluids from such used
animals must also be appropriately governed as per the laid in practices of the institution / bio-safety
regulation.

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8. MULTIPLE SURGICAL PROCEDURES ON SINGLE ANIMAL
(a) Multiple surgical procedures on a single animal for any testing or experiment are not to be
practiced unless specified in a protocol only approved by the IAEC.
(b) Individual animals should not be used in more than one experiment, either in the same or different
projects, without the express approval of the IAEC. However, it is noted that Appropriate re-use of
animals may reduce the total number of animals used in a project, result in better design of
experiments, and reduce stress or avoid pain to additional animals. Animals that are used in
morethan one experiment should be permitted to recover fully from the first experiment before the
subsequent experiment is performed. Certification of attending veterinarian is however, required
before subjecting animal to the second experiment.
9. DURATIONS OF EXPERIMENTS
No animal should be used for experimentation for more than 3 years unless adequatejustification is
provided.
10. PHYSICAL RESTRAINT
(a) Brief physical restraint of animals for examination, collection of samples, and a variety of other
clinical and experimental manipulations can be accomplished manually or with devices be suitable
in size and design for the animal being held and operated properly to minimize stress and avoid
injuryto the animal.
(b) Prolonged restraint of any animal, including the chairing of non-human primates, should be
avoided unless essential to the research objectives. Less restrictive systems, such as the tether
system or the pole and collar system should be used when compatible with research objectives.
(c) Following points should be considered during handling and restraining animals:-
i. Animals should be handled by competent individuals trained in methods that cause minimal
distress and injury (for example, a person with a publication to his/her credit and post/experience
in relevanttechniques for handling animals is preferable).
ii. The use of restraint devices is sometimes essential for the welfare of the animal and safety of the
handler. Restraint devices should be used to the minimum extent, for the minimum period required
to accomplish the purpose of the experiment and be appropriate for the animal.

iii. Tranquilizers or anesthetics may initially be used to aid restraint but they may prolong recovery
from the procedure. When these agents have been used, recovery of the animals should be closely
monitored.

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(d) The following are important guidelines for the use of restraint equipment:
i. Restraint devices cannot be used simply as a convenience in handling or managing animals. The
period of restraint should be the minimum required to accomplish the research objectives. Animals to
be placed in restraint devices should be given training to adapt to the equipment, prior to initiation of
the experimentation.
ii. Provision should be made for observation of the animal at appropriate intervals. Veterinary care
should be provided if symptoms or illness associated with restraint are observed. The presence of
illness, or severe behavioral change should be dealt with by temporary or permanent removal of the
animal from restraint related protocol.
11. LOCATION OF ANIMAL FACILITIES TO LABORATORIES
 Good animal husbandry and human comfort and health protection require physical separation of
animalfacilities from personnel areas such as offices, break room, training and education room.
 Laboratory animals are very sensitive to their living conditions. It is important that they shall be
housedin an isolated building located as far away from human habitations as possible and not exposed
to dust, smoke, noise, wild rodents, insects and birds. The building, cages and environment of animal
rooms arethe major factors, which affect the quality of animals.
 This separation can be accomplished by having the animal quarters in a separate building, wing,
floor, or room. Careful planning should make it possible to place animal housing areas adjacent to or
near laboratories, but separated from them by barriers such as entry locks, corridors or floors. While
planning an animal facility the space should be well divided for various activities. The animal rooms
should occupy about 50-60% of the total constructed area and the remaining area should be utilized for
servicessuch as stores, washing, office and staff, machine rooms, quarantine and corridors.
 The environment of animal room (Macro- Environment) and animal cage (Microenvironment) are
factors on which the production and experimental efficiency of the animal depends. Since animals are
very sensitive to environmental changes, sharp fluctuations in temperature, humidity, light, sound and
ventilation should be avoided. The recommended space requirements for animal rooms, for different
species

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 12. FUNCTIONAL AREAS
The size and nature of a facility will determine whether areas for separate service functions are
possible or necessary. Sufficient animal area required to:

 Ensure separation of species or isolation of individual projects when necessary;

 Receive, quarantine, and isolate animals
 Provide for animal housing.
(a) In facilities that are small, maintain few animals or maintain animals under special conditions
(e.g., facilities exclusively used for housing germfree colonies or animals in runs and pens) some
functional areas listed below could be unnecessary or included in a multipurpose area. Professional
judgment must be exercised when developing a practical system for animal care.
 Specialized laboratories
 Individual areas contiguous with or near animal housing areas for such activities as surgery, intensive
care, necropsy, radiography, preparation of special diets, experimental manipulation, treatment, and
diagnostic laboratory procedures containment facilities or
 Equipment, if hazardous biological, physical, or chemical agents are to be used Receiving and
storageareas for food, bedding
 Pharmaceuticals and biologics, and supplies
 Space for administration, supervision, and direction of the facility Showers, sinks, lockers and
toiletsfor personnel
 An area for washing and sterilization equipment and supplies,
 An autoclave for equipment
 Food, and bedding and separate areas For holding soiled and cleaned equipment
 An area for repairing cages and equipment

 An area to store wastes prior to incineration or removal

13. PHYSICAL FACILITIES
The physical condition and design of animal facility determine, to a great extent, the efficiency and
economy of this operation. The design and size of an animal facility depend on the scope of
institutional research activities, animals to be housed, physical relationship to the rest of the
institution, and geographic location. A well planned, properly maintained facility is an important
element in good animal care.

Housing facility should be compatible with the needs of the species to be housed.

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 Housing Facilities should be designed and operated to facilitate control of environmental factors to
exclude vermin and limit contamination associated with the housing of animals, delivery of food,
water, bedding, and the entry of people and other animals.

a) Housing Facilities should be maintained in good repair. Walls and floors should be constructed of
durable materials with surfaces that can be cleaned and disinfected readily.
b) Housing Facilities should be kept clean and tidy and operated to achieve maximum possible
hygiene.
c) There should be a pest control programme to monitor and control vermin.
d) There should be adequate and appropriate storage areas for food, bedding and equipment.
e) Deodorants designed to mask animal odour should not be used in Housing Facilities as they may
expose animals to volatile compounds which can alter metabolic processes. In addition, deodorants
mustnot be used as a substitute for good cage and equipment cleaning practices and good ventilation.
f) Cleaning practices should be monitored on a regular basis to ensure effective hygiene and
sanitation. This may include visual inspection, monitoring water temperatures and microbiological
testing of surfaces after cleaning.
g) There should be proper water supply and drainage.
h) There should be adequate contingency plans to cover such emergencies as flooding and fire, or the
breakdown of lighting, heating, cooling or ventilation.
i) In the interest of disease prevention and general animal welfare, access to the Housing Facilities by
unauthorized persons should be restricted

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 Experimental No: 5

STUDY OF DIFFRENT ROUTE OF DRUG ADMINISTRATION

Aim: There are different route of drug administration by using laboratory animal

1. To learn how to handle, treat preparing animals for experiments and ethical guidelines during
thetreatment.
2. Measure the required volume of drugs using aseptic techniques
3. To learn how to give a different types of route of drug administration

How to handle:

The good handling and restrained is most important techniques for correct administration. There strain
methods are 2 types

1. Physical method
2. Manual method
Double handled manual method
Single handled manual method

Route of drug administration

 Enteral route (oral and intregastric)

 Parenteral (I.V, I.M,I.P,S.C,I.D)
 Topical route (surface of the skin membrane)

Enteral route:

The placement of drugs directly into any part of GIT directly it could be oral, sublingual, intragastric.

Oral route:

The swallowing of a drug through mouth by the desired drug along with water (ad libitum) or food. It
is economic and convenient, safe and some animals can be trained to cooperate voluntarily. This route
is not referable since it is in accurate.

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Intra gastric route:

The administration of fluid / drugs directly into oesophagus or stomach it is often used instead of
mixing the substance in water or food. This method is accurate for the dosing of animals.

The gastric lavage tube is a small curved, metallic tube with the ball on the tip, it is often used without
anaesthesia.

Parental route:

Subcutaneous route: the part of the subcutaneous injection and back of neck it doesn’t required large
volume of drugs suitable for suspension.

Intra peritoneal route:

Commonly used in rat and mice, rapid absorption due to large particle surface, mouse may injected
compared with rat (ranges from 2-10ml/kg)

Intravenous route:

It is the most efficient route of drug administration. It should be used with the help of restraining devices
in the appropriate size for the animals to be injected. The tail vein is suitable for drug administration.

Topical route:

Most convenient route for topical drugs, drugs like local anaesthetics, ointments and cream can
be easily applied on the surface of the skin membrane. Eye drops and ointments were also used in this
method. Animals like rabbit, guinea pig and rat are mainly use in this method.

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 Experiment No: 6

EFFECT OF DRUGS ON CILIARY MOTILITY OF FROG OESOPHAGUS

Aim: To study the effect of physostigmine and atropine on ciliary movement in frog buccal cavity.

Principles:

Cilia in the buccal cavity and in the oesophagus helps in the movement of food particles.
Similarly, the importance of mucociliary function has been established in respiratory tract and of
pulmonary diseases such as chronic bronchitis, asthma and in cystic fibrosis. The integrity of
mucociliary function is very important in these air way diseases. Cilia exhibit a great degree of
autonomy in that they are capable of functioning in the absence of nervous innervations. It has also
been demonstrated that the Ach present in the mucous membrane of trachea and buccal cavity helps
in the ciliary movement. Ach serves as a local hormone and the presence of choline acetylase support
the fact that Ach is locally synthesized in the mucous membranes.

Requirements:

Animal: Frog

Drugs: Physostigmine stock solution (1µg/ml), Atropine stock solution (1µg/ml),

Physiological solution: Normal saline

Procedure:

1. Decapitate the frog and pin the frog to the frog board on its back.
2. Pin the lower jaw to the abdomen cutting sufficiently the buccal cavity and exposing the oesophagus
wet by irrigating it with normal saline.
3. To assess the distance travelled by the particles, fix two points. One start from lower jaw to other
beginning of oesophagus. Keep this distance constant to measure the time taken by the particle to
move from a fixed point in the lower jaw to the beginning of the oesophagus.

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4. Place a poppy seed or a small piece of cork at the pre-marked spot in jaw. Turn on the stop watch and
note the time taken by the object to reach the beginning of the oesophagus. Repeat this several times.
5. Put a few drops of physostigmine on the buccal cavity and after 10 min repeat step 4 note the tome.
6. Wash the buccal with normal saline. Put a few drops of atropine on the buccal cavity. After10 min
repeat the step 4. Note the time.
7. Find out the differences in the time taken by the object to move between the pre marked distance
inthe buccal cavity in presence of saline, physostigmine and atropine.

Observation

Treatment Time (seconds)

1st reading 2nd reading 3rd reading

Saline

Atropine

Physostigmine

Inference:

Physostigmine reduces and atropine enhances the time taken by the object to move from the pre
marked point in the lower jaw to reach the oesophagus respectively

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 Experiment No: 7

EFFECT OF DRUGS ON RABBIT EYE

Aim: To study the effects of topically applied drugs on rabbit eye

Principle:

A large number of drugs are used for their local action in the eye as eye drops or eye ointments.
Most of these drugs belongs to anti-microbial, autonomic or local anaesthetic group. The eye is
supplied by both sympathetic and parasympathetic nerves. The superior palpebral muscle and the
dilator papillae of the iris have sympathetic supply. The sphincter pupillae of the iris has
parasympathetic supply which exercises dominant control. The ciliary muscle is also supplied by
parasympathetic nerve and when it contracts, the ciliary body is moved inwards and forwards.
Because of the lens bulges forward and the eye is accommodated for near vision. The opposite
effect is produced by the relaxation of ciliary muscle resulting in paralysis of accommodation. When
the pupil dilates, the iris folds back near the opening of the canal of schlemn and the drainage of
aqueous humour is decreased thereby increasing intraocular pressure. Constriction of the pupil, by
opposite action, will increase drainage and reduce intraocular pressure.
Topically applied drugs can affect the eye by changing conjuctival congestion, papillary
size, light reflex, corneal sensitivity and intraocular pressure. However the effects of drugs on
papillary size, light reflex and corneal reflex can be easily studied by the students. The pupillary
size can be measured by placing a transparent plastic scale in front of the eye but as close as
possible. Light reflex is elicited by directing the light of a torch towards the pupil. The sensitivity of
the cornea is tested by gently touching the cornea with a fine cotton swab stick from the side and
not from the front of the eye. This elicits corneal reflex which manifests as blinking of the crystals.
Requirements:
Animal: Rabbit (2-5kg)
Drugs: Atropine (1%w/v) & Physostigmine (1%w/v)Equipments: rabbit holder, pen torch

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 Procedure:

1. Place the rabbit in the rabbit holder by keeping the head outside
2. Observe size of the pupil in both the eyes
3. Examine the effect of light reflex by holding the torch in front of the eye moving light beam to
and fro
4. Examine the corneal reflex by touching a side of the cornea with a cotton swab.
5. Instil a few drops of atropine solution in the conjunctiva over a period of 8-10 min in the right
eyeof the rabbit would serve as control. Instil normal saline in the left eye.
6. Record the pupillary size, light reflex and corneal reflex after ten minutes of drug initialization and
tabulate the observation.
7. Repeat the experiments with physostigmine and ephedrine

Observation
DRUG PUPILLARY LIGHT REFLEX CORNEAL
SIZE REFLEX
Saline Normal Present Present

Physostigmine Constriction Present Present

Ephedrine Dilation Present Present

Atropine Dilation Absent Present

Inference

Saline: There is no change in the pupil size by instilling saline. The torch and light reflexes present.

Physostigmine: Pupil size decreases and diameter of iris also decreases causing miosis. Light and
touch reflexes are present.

Ephedrine: Increases the pupil size and increases the diameter of iris thereby causing
mydriasis.Touch reflex and light reflex are present but light reflex remains absent.

Atropine: Increases the pupil size and increases the diameter of iris thereby causing mydriasis.
Touchreflex is present but light reflex remains absent.

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 Experiment No: 8

EFFECTS OF SKELTAL MUSCLE RELAXANT USING ROTA-ROD APPARATUS

Aim: To study the muscle relaxant property of diazepam in mice using rota rod apparatus

Principle:

One of the important pharmacological action of anti-anxiety agents of benzodiazepam class of drugs
is muscle relaxing property. The skeletal muscle relaxation together with taming or calming effect,
theseagents reduce anxiety and tension. The loss of muscle grip is an indication of muscle relaxation.
This effect can be easily studied in animals using inclined plane or rotating rods.

The difference in the fall off time from the rotating rod between the control and diazepam treated
animal is taken as an index of muscle relaxation. The angle of the slope of the inclined plane, or the
rate of rotation of the rod should be adjusted such that a normal mouse can stay on the plane or on the
rod foran appreciable period of time.

Requirement:

Animal: Mice (20-25gm)

Drugs: Diazepam of the drug and inject 1ml/100g of body weight of the mouse.

Equipment: Rota rod apparatus

Procedure:

1. Weigh the animals and number them.

2. Turn on the instrument select an appropriate speed (20-25rpm)
3. Place the animal one by one on the rotating rod. One can place more than one mouse. Note down
the fall off time when the mouse falls from the rotating rod. A normal mouse generally falls off
within 3-5 min
4. Inject diazepam to all animals. After 30 min repeat the experiments as done in step 3. Note the fall
off time.
5. Compare the fall off time of animals before and after diazepam treatment.

Observation

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Dose of Diazepam: 4 mg/kg

Sr. No Body weight (g) Dose to be Fall time (seconds) % decrease in fall
administered (mg) time

1 25 0.1 Basel (A) Treatment

(B)
2 30 0.12
3 32 0.128
4 35 0.14
5 33 0.132

Inference:

Mice treated with diazepam spent less time on rota-rod, exhibiting muscle relaxant property.

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 Experiment No: 9

STUDY OF DRUGS ON LOCOMOTOR ACTIVITY USING ACTOPHOTOMETER

Aim: To study the central nervous system depressant property of chlorpromazine on the locomotor
activity of mice using actophotometer.

Principle:

Most of the central nervous system acting drugs influencing the locomotor activities in man and animals.
The CNS depressant drugs such as barbiturates and alcohol reduces the motor activity while the stimulants
such as caffeine, amphetamines increases the activity. In other words, the locomotor activity can be index
of wakefulness of mental activity. The locomotor activity can be easily measured using an actophotometer
which operates on photoelectric cells which are connected in circuit with a counter. When the beam of
light falling on the photocell is cut off by the animal, a count is recorded. An actophotometer could have
either circular or square arena in which the animal moves. Both rats and mice may be used for testing in
this equipment.

Requirement:

Animal: Mice (20-25gm)

Drugs: Chlorpromazine hydrochloride

Equipment: Actophotometer

Procedure:

1. Weigh the animals and number them

2. Turn on the equipment and place individually each mouse in the activity cage for 10 min. Note
thebasal activity score of all the animals.
3. Inject chlorpromazine and after 30 min re –test each mouse for activity scores for 10 min. Note
thedifference in the activity before and after chlorpromazine.
4. Calculate percent decrease in motor activity.

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Dose of chlorpromazine: 3mg/kg

Sr. No B.W Dose to be Basel (A) Treatment (B) Difference % decrease in

administered (A-B) locomotor activity
1 25 0.075
2 30 0.09
3 32 0.096
4 35 0.105
5 34 0.103
6 33 0.099

Inference:

Chlorpromazine was noted to produce CNS depressant property as the locomotor activity score in
chlorpromazine administered animals was found to reduce.

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 Experiment No: 10

ANTICONVULSANT EFFECT OF DRUGS BY MAXIMAL ELECTRO-SHOCKINDUCED

CONVULSIONS IN RATS

Aim: To study the anti convulsant activity of phenytoin against maximal electro shock induced
convulsion rats.

Principle:

Different types of epilepsy i.e., grant mal, petit mal or psychomotor type, can be studied in laboratory
animals. The maximal electroshock (MES) induced convulsions in animals represent grandmal
epilepsy. Similarly chemo- convulsion due to pentylenetetrazol which produce clonic type of
convulsion in man. These are the two procedures used to study convulsion, and to test anti convulsant
drugs in laboratory animals.

In MES- convulsion electroshock is applied through the corneal electrodes. Through optic stimulation
cortical excitation is produced. The MES convulsion are divided into five phases are

1. Tonic flexion
2. Tonic extensor
3. Clonic convulsion
4. Stupor
5. Recovery / death
A substance is known to possess anti-convulsant property if it reduces or abolishes the extensor phase
of MES convulsions both in rats and mice. It is advised the students should have complete
background of the pharmacology of anti-epileptics drugs before performing this experiment.

Requirement:

Animal: Rats (150-200 gm)

Drugs: phenytoin (dose 25 mg/kg); prepare a stock solution
Equipment: Electro-convulsiometer, cornel electrode (150 mA current for 0.2 sec), Stop watch.

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 Procedure:

1. Weigh and number the animals. Divide them into two groups each consisting of 4-5 rats. One
groupis used as control and the other for drugs treatment.
2. Hold the animals properly and place corneal electrodes on the cornea and apply the prescribed
current.Note the difference stages of convulsion
3. Repeat with other animals of control group
4. Inject phenytoin I.P to group of 4-5 rats. Wait for 30 min and subjected to animals to electro
convulsions.
5. Note the reduction in time or abolition of tonic extensor phase of MES convulsions.

Observation

Dose: Phenytoin 25mg/kg

Sr.No Onset time Non-

extensor
Tonic Tonic clonus stupor recovery
seizure
limb extensor
flexor
1
2
3
4
5
6

Inference

Phenytoin demonstrated anticonvulsant effect

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 Experiment No: 11

STUDY OF STEREOTYPE ANTI CATATONIC ACTIVITY OF DRUGS ON RAT/MICE

Aim:

1. To study drug (phenothiazines) induced catatonia (extrapyramidal side effect in rats).

2. To study the anticatatonic (antiparkinsonian) effect of scopolamine.

Principle:

Phenothiazine and butyrophenone type of antipsychotic drugs are known to produce extrapyramidal
side-effects in man .these effects such as akinesia, rigidity and tremors, are called parkinson’s- like
because in parkinson’s disease the major clinical symptoms include difficulty to move and change
posture (akinesia and rigidity) and tremors. These effects of antipsychotic drugs are due to excessive
blockade of dopamine receptors in the extrapyramidal motor system. Therefore, phenothiazines
(choloropromazine or perphenazine) are commonly used to produce parkinson’s-like extrapyramidal
symptoms in laboratory animals and to study anti-parkinsonism drugs. The students are advised to
know the pharmacology of anti-parkinsonism drugs before performing this experiment.

Requirements

Animal: Rats (150-200 g)

Drugs:
 Perphenazine (dose: 5 mg/kg ip; prepare a stock solution containing 1 mg/ml of the drug and
inject 0.5 ml/100g body weight of the animal).
 Scopolqmine (dose: 2 mg/kg, ip prepare a stock solution containing 0.4 mg/ml of the drug and
inject 0.5 ml/100 g body weight of the animal)

Equipment: Two wooden blocks, one being 3 cm high and the other 9 cm high

Procedure

1. Weigh and number the animal. Divide the animals into two groups, one for control (for study of
perphenazine effect) and the other for studying the effects of scopolamine. Each group should consist
of at least 5 animals.

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2. Inject perphenazine to control animals. observe severity of catatonic response (fig 5.14) as follows:
3. Observe the severity of catatonia at 5, 15, 30, 45, 60, 90 and 120 min after perphenazine .
4. To the second group inject scopolamine and after 30 minute inject perphenazine to these animals
already treated with scopolamine. Observe and score the severity of catatonic as instep 2.
5. Compare the onset and severity of catatonic response in the both groups. Plot a graph, time along
thex-axis. Note the difference in the onset and severity of catatonic response in both the groups.
Observation
Sr. Weight Dose Dose to be Mean catatonia after minutes of
No of administered PERP treatment
animal
Perphenazine Perphenazine 15 30 45 60 90 120
(5mg/kg) (mg)
1 180 0.9
2 185 0.92
3 205 1.02
4 210 1.05
5 200 1
Mean

Sr. Weight Dose Dose to be administered Mean catatonia after minutes of

No of PERP treatment
animal
Scopolamine Scopolamine Perphenazine 15 30 45 60 90 120
(2mg/kg)+ (mg) (mg)
1 185 Perphenazine
2 190 (5mg/kg)
3 200
4 205
5 195
Mean

Inference: Scopolamine reduces the mean catatonic score, thus demonstrating anti-catatonic effect.

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