EXPERIMENTAL PHARMACOLOGY

2nd D-PHARM
EXPERIMENT NO :1

INTRODUCTION TO EXPERIMENTAL PHARMACOLOGY

Pharmacology is the branch of medical science which deals with the study
of both beneficial and harmful effect of drugs on biological systems.

The word “pharmacology” is derived from Greek words pharmakon (an active
principle) and logos (discourse or treatise).

Experimental pharmacology is one of the sub-branches of medical science

which play a crucial role in the identification of new drug molecule with
characteristic pharmacological activities along with elucidation of its mechanism
of action.

It provides basis for new drug development and includes different

molecular and biochemical aspects.

The study also covers it employs use of various equipment’s for

the evaluation of various parameters like behavioral, biochemical,
histopathological etc. With respect to the different therapeutic agents.

Thus, the main aims of experimental pharmacology are:

❖ To determine the suitability of therapeutic agents for their use in

human beings.
❖ To determine the probable mechanism(s) and site(s) of action of
drugs.
❖ To study the toxicity consequences of drugs.

The pharmacological experiments can be performed either in intact animal

(anaesthetized or non-anaesthetized called whole animal experiments) or in
isolated tissues/ organs of various laboratory animals.
 The experiments conducted on whole animals are known as in vivo
experiments, while those which are conducted on isolated organs/ tissues (e.g.
heart, intestine, fundus, stomach, uterus, etc.) are known as in vitro experiments.

The effect of drugs in whole animals will be altogether different from

studying them on isolated tissues or organs and have individual advantages and
limitations.

While performing the experiments on whole animal, the effects of drugs

on any organ or parts of the body is influenced by several physiological factors
like metabolic state of the body, influence of hormones and neurotransmitters,
blood supply to that part, diseased state of the body, other bio-availability factors
etc.

These factors alter the pharmacological actions of the drugs and it is

difficult task to conduct the influence of such diverse factors.

Furthermore, the alteration of various pharmacological effects will produce

remarkable difficulties in the elucidation of exact mechanism of action of drug.

However, the whole animal experiments play an important role in studying

the effects of various drugs, their interactions with other simultaneously
administered drugs and their ultimate effect on various physiological parameters.

On the other hand, in vitro experiments are specifically used to investigate the
mode of action of drug and they provide distinct advantage, since the effect of
drug can be studied without the influence of above mentioned parameters and
other systems thereby provide exact mechanism of drug action.
 EXPERIMENT NO :2

DIFFERENT EXPERIMENTAL ANIMALS AND THEIR

APPLICATION IN EXPERIMENTAL PHARMACOLOGY

AIM:
To study the different laboratory animals andtheir application in experimental
pharmacology. Laboratory animals can be breed and also be reared in the laboratory
under suitable conditions.The common laboratory animals are

Rat, Mice, Rabbit, Frog, Guinea Pig, Hamster, Cat, Dog

RAT:

Rats are very robust and devoid of vomiting centers. There is no gall bladder or tonsil.
Albino rat- white rat (Wistar strain) are commonly used. Another strain is Sprague-
Dawlely.

Advantages:

1) It resembles a man in several functions, nutrition, and sensitivity towards most

of the drugs.
2) It is small in size compared to other animals. So drugs are required in small
quantities.
3) It is devoid of a vomiting center so any noxious substance can be administered.
4) The stomach, funds, and pyloric parts have clear linings between them.
Experimental Use:

• Normal adult rat weighs about 200-250gm and the age suitable for most of the
experiments is 1-5 months.
• Psycho-pharmacological studies.
• Study of analgesic and anti-convulsion.
• Bio-assays of various Hormones
• Study of hepatotoxic and anti-hepatotoxic compounds.
GUINEA PIG:

• It is a very docile animal.

• It is highly sensitive to Histamine and Penicillin.
• It is highly suitable for tuberculosis and anaphylaxis.

Experimental Use:

• Normal adult weighs about 400-600gms and the age suitable for the experiment
is 30 months.
• Evaluation of the broncho dilatory activities.
• Anaphylactic and Immunological studies.
• Evaluation of local anesthetics study of digitalis.
MICE:

• Zoological name- Mus musculus

• Albino mice – white mice (Swiss strain) are commonly used. It is very cheap,
smallest and easy to handle.
• Other strains are Laca and Bulb/C
Experimental Use:

Normal adult weighs about 20-25gms and agesuitable for experiment isone
month

• Toxicological studies - Especially acute and sub acute toxicity studies.

• Employed in teratogenicity.
• Bio-assay of drugs(insulin).
• Screening of analgesics and anti-convulsions.
• Studies related to genetic and cancer research
RABBIT:

UsuallyNew Zealand, white rabbits are used. It is a docile animal with large ears. A
peculiar thing about rabbits is that they contain an atropine esterase enzyme and hence
resistant to the action of atropine.
Experimental Use:

A normal adult weighs about 1.5 to 3kg and the age suitable for the experiment is 5-
6months.

• Pyrogen testing
• Bio-assay of anti-diabetic drug and sex hormone.
• Studies related to reproduction
• Isolated preparation like heart, aorta, duodenum, and ileum.

HAMSTER:

• They have short bodies with legs and tail

• The skin is loose and soft.
• The chest pouches are prominent and extend up to shoulder regions.
Experimental Use:

A normal adult weighs about 80-90gms and the age suitable for the experiment
is one month.

• It is used for psychological investigation, cell genetics, and radiation therapy.

• Research on diabetic mellitus
• Bio-assay of prostaglandin.
FROGS:

• 150-200gms are extensively used in experimental pharmacology.

• Isolated frog heart, rectus, abdomen is muscle preparation, muscle nerve
preparation, ciliary movement and gastrointestinal tract are used in experimental
pharmacology.
Experimental Use:

• It is used for the study of nerve block type of local anesthesia.

• Inexpensive and easily available.
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,

several countries have passed legislation to prevent or usage of animal
experimentation. These include experiments 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
nowadays as per the common laboratory alternate animals.
EXPERIMENT NO :3

COMMONLY USED INSTRUMENTS USED IN EXPERIMENTAL

PHARMACOLOGY

AIM: To study the commonly used instruments in experimental pharmacology

ACTOPHOTOMETER:

Actophotometer consists of photocells. These photocells are activated when the

rays of light falling on the photocells are obstructed to the movement of animals
crossing the path of the light beam. This cut-off quantity of light beam is
electrical. This is proportional to the movement of animals in a cage. This
instrument measures the active exploratory movement of animals. This
equipment is used to measure the effect of drugs on the motor activity of the
rat/mice, and is useful in screening and evaluation of drugs for pharmacological
and toxicological experiments.

ROTAROD APPARATUS:

The Rota rod performance test is a performance test based on a rotating rod with
forced motor activity being applied, usually by a rodent. The best measures
parameters such as riding time or endurance. Some of the functions of tests
include evaluating the balance, grip strength, and motor coordination of the
subjects, especially in testing the effect of experimental drugs or after traumatic
brain injury. In the test, a rodent is placed on a horizontally oriented, rotating
cylinder(rod) suspended above a cage floor, which is low enough not to injure the
animal, but high enough to induce avoidance of fall. Rodents naturally try to stay
on the rotating cylinder, or rotarod, and avoid falling to the ground, the length of
time that a given animal stays on the rotating rod is a measure of their balance,
coordination, and motor planning. The speed of the Rota rod is mechanically
driven, and may either be held constant or accelerated. A human analog to the
Rota rod test might be tread mill running. Hamster, gerbils, and mouse, owners
can observe the principle in action when an animal climbs on the outside of its
wheel.

ELECTRO-CONVULSOMETER:

Seizures are produced by electrical stimulation and their phases are then
antagonized by systemic administration of anticonvulsants, Different types of
epilepsy can be studied in laboratory animals. The maximal electroshock-induced
convulsion drug in the laboratory animals. The MES convulsions are divided into
fire phases tonic fission, tonic extensor, clonic convulsion.

EDDY’S HOTPLATE:

In the hotplate, we use it as the Min stimulant or source of pain. Mice are the
choice of animal for this experiment hotplate is a device having a boundary or
outer side and a plate that gets heated by a heating coil from inside. Mice are
placed one by one on this apparatus which temperature is kept constant by a
regulator, analgesic increases the time. Eddy and Leimbach is also known as
Eddy’s hot plate. Eddy’s Hot plate consists of 30x30 cm. Heating surface with
perspex enclosure and solid-state temperature controller with micro-controller
based digital temperature indicator controller to set surface temp. A constant
temperature (550C) was maintained and the reaction of animals, such as paw
licking/jumping response was taken as endpoint. Eddy's hot plate test is used to
assess the analgesic activity of drugs.
ELEVATED PLUS MAZE

The test set consists of a plus-shaped apparatus with two open and two enclosed
arms, each with an open roof, elevated 40–70 cm from the floor. The model is
based on rodents' aversion to open spaces. This aversion leads to the behavior
termed thigmotaxic, which involves avoidance of open areas by confining
movements to enclosed spaces or to the edges of a bounded space. In EPM this
translates into a restriction of movement to the enclosed arms

Anxiety reduction in the plus-maze is indicated by an increase in the proportion

of time spent in the open arms (time in open arms/total time in open or closed
arms), and an increase in the proportion of entries into the open arms (entries into
open arms/total entries into open or closed arms). A total number of arm entries
and a number of closed-arm entries are usually employed as measures of general
activity.
EXPERIMENT NO :4

DIFFERENT ROUTES OF DRUG ADMINISTRATION FOR VARIOUS

ANIMAL MODELS

AIM: To study the different routes of drug administration on mice/rats

INTRAPERITONEAL INJECTION:

• First the point of entry of the needle was located. An imaginary line was
drawn across the abdomen first above the knees.
• The needle was inserted along this line on the animal’s right side and
close to the midline.
• In the case of females, the point of entry is cranial to and slightly medial
to the last nipple.
• To perform an I.P. injection, the mouse/rat must be well restrained so that
it cannot move during the procedure.
• The mouse/rat was restrained and titled so that the head is facing
downward and the abdomen is exposed. The needle was inserted into the
abdomen at about 30 degrees after disinfecting the injection site.
• The shaft of the needle should enter to a depth of about half of a
centimeter. The needle was aspirated to be sure that the needle has not
penetrated a blood vessel, the intestines or the urinary bladder.
• Recommended needle size I.P. is 25-27 G

SUBCUTANEOUS INJECTION:

• The mouse/rat was restrained in the normal manner and the skin was
lifted to make a tent. The injection site was disinfected and the needle
was introduced into the subcutaneous tissue.
 • The loose skin around the neck and shoulder area was selected for the
injection site. Subcutaneous injections were mostly used to administer
fluids for hydration and to inject anesthetics.
• Typical volumes injected area of range 1ml or less
• The needle was inserted at the base of the tent, the needle was held
parallel to the animal’s body to avoid puncturing underlying structures.
• The needle was aspirated to ensure that the needle had not entered a
blood vessel.
• The full volume was injected at a moderate rate.
• The needle was withdrawn, the skin was pressed, and the Animal was
checked for any bleeding.
• Fluid was deposited in subcutaneous space and the bubble of fluid
Recommended needle size S.C. is 23-25 G.

INTRAMUSCULAR INJECTION:

• Intramuscular infections are not recommended due to lack of muscle

mass in mice/rat.
• Injection may cause discomfort and local tissue irritation.

INTRAVENOUS INJECTION:

• The tail vein of the mouse/rat was used.

• The mouse was restrained with physical or chemical restraint. The tail
was rotated slightly to visualize the vein.
• The injection site was disinfected and a needle of 27-30 G was
inserted into the vein at a slight aspiration was not possible inserted
injection was given slowly and cleaning of the lumen was watched.
• Incorrect positioning resulted in a slight bulge in the tail, the needle
was removed and the process was repeated proximal to the previous
 site, after completion the needle was removed and pressure was
applied at injection site. Recommended needle size I.V. is 27-30 G.

INTRADERMAL INJECTION:

• Intradermal injection was done only under anesthesia.

• The hair on the back was clipped and prepared with alcohol swab.
• The needle was inserted in between the layers of skin on back at 30
degree.
• The syringe was aspirated to ensure proper placement
• Any sign of blood or other fluid indicated improper placement
• The drug was administered slowly with a maximum volume of
100μl for the injection site to avoid tissue trauma.
• Successful injection resulted in a small circular melt.

ORAL FEEDING / GAVAGING IN MOUSE:

• Biomedical needles are used with 1-3ml syringe

• The distance from the tip of the nose to the first rib was
measured. The needle of measured length was used
• The syringe was filled with the appropriate amount of drug.
• Mouse was restrained.
• The tip was slid down the back of the mouth moving the lip
forward in one felt motion Any substance felt indicated
improper placement
• The drug was administered once the needle was properly
placed.
EXPERIMENT NO :5

EUTHANASIA AND VARIOUS BLOOD COLLECTION TECHNIQUES

FOR EXPERIMENTAL ANIMALS

AIM: To study about the laboratory animal euthanasia

DESCRIPTION:

• The killing of animals used for scientific purposes is a very sensitive issue
and requires special consideration to ensure that animal anxiety and fear is
reduced to a minimum.
• Euthanasia is defined as killing painlessly
• When stock is not required for certain reasons, such as sex preference
utilization.

OBJECTIVES OF EUTHANASIA:

• Avoid distress and produce rapid loss of consciousness until death occurs.
• Be reliable, reproducible and irreversible.
• Be appropriate for age, species, and health of the animal.
• Require minimum restraint.
• Be compatible with the objectives of the study.
• Be simple to administer.
• Be safe for the operator.
• Be aesthetically acceptable to the operator, where at all possible.

TECHNIQUES OF EUTHANASIA

The techniques listed here fall within two general categories:

• Those methods that are recommended.

 • Those methods that is acceptable with reservations.
The reservations may be on aesthetic grounds, the need for special
equipment, or pose some possible human safety hazard. These might be
used where, for example, the recommended method may impact negatively
on the science, or as a second method to ensure that death has occurred.
Techniques can be further divided into:
• Chemical, which is further subdivided into: Inhalant, and injectable
• Physical

RECOMMENDED TECHNIQUES CARBON DIOXIDE:

• Carbon dioxide, passed through a reduction valve, can be piped into plastic
bags or deep containers at an optimal flow rate that displaces 20% of the
chamber volume per minute.

Considerable debate has occurred relative to three methods of administration

of carbon dioxide for euthanasia of rats and mice:

• Placing them into a container pre-filled with carbon dioxide Placing them
in air and then rapidly filling the container with carbon dioxide Using a
carbon dioxide/oxygen mixture e.g. 70%CO2/30%O2.

LABORATORY ANIMAL BLEEDING TECHNIQUES PROCEDURE:

(Rat and mouse )

TAIL VEIN PUNCTURE

• Animal was restrained.

• Lateral or dorsal veins were dilated by dipping the tail into water at 40 to
50 C or by rubbing with xylol and then cleaning the part with some
disinfectant.
• The tail was grasped between the thumb and index finger.
• The needle (25-27 G fitted with 1 ml syringe) was introduced near the distal
portion of the tail.
• It was aspirated for confirmation and to avoid the collapse of the wall of
the vein obliterating the needle opening.
• Another way of collecting blood repeatedly is by cutting the tip of the tail
with some sharp instrument.

ORBITAL SINUS VENIPUNCTURE

• Animal was anesthetized with ether.

• The capillary tube was inserted in the medial canthus with gentle
rotation while directing the tube caudally and towards the midline.
• Pressure was applied after blood collection to prevent hematomas.
• 0.5 ml of blood can be obtained weekly using this method.

RABBIT

• The rabbit was restrained by using a rabbit holder.

• The lateral margin of the ear was shaved and swabbed with a
disinfectant.
• The ear was grasped between the thumb and the index finger.
• The lateral margin vein was punctured at a site immediately
proximal to the thumb by using a 20-G needle.
• Very gentle aspiration was applied in order to avoid collapse of the
vein.
• 0.5-1 ml of blood sample was collected.
EXPERIMENT NO :6

EFFECT OF TOPICAL APPLICATION OF DRUG ON RABBIT EYE

AIM: To study the effects of drugs on rabbit eye.

PRINCIPLE:

• Local action of large number of drugs in an eye can be achieved without

systemic effect by the application of drugs belonging to antimicrobial,
autonomic or local anesthetic groups, the eye is supplied with both
sympathetic and parasympathetic nerves.
• Ciliary muscles are supplied by parasympathetic nerves, when it contracts
the ciliary body is mooned 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 muscles
resulting in paralysis of accommodation.
• Topically applied drugs can affect the eye by changing conjunctival
conjunction, papillary size, light reflux, corneal sensitivity, and
intraocular pressure.
• The pupillary size can be measured by placing a transparent plastic scale
in front of the eyes as closely as possible.
• Atropine an anti-muscarinic agent blocks the effects of endogenously
released acetylcholine on the circular muscles of the iris and muscles
ciliary produces Mydriatics and spasm of accommodation leading to
cyclopedia but without producing loss of corneal reflux.

REQUIREMENTS: (write in left side)

▪ Animals- rabbit (2 - 5kgs)

▪ Drugs- Normal saline, Atropine, Physostigmine, Ephedrine, Nor
adrenaline and Lignocaine
 ▪ Equipment- rabbit holder, torch light, and ExPharm software

PROCEDURE:

▪ Rabbits were placed in the rabbit holder, and the heads were kept outside.
▪ The right eye was served as test The pupil size was observed in both the
eyes
▪ The effect of light reflux was examined by holding the torch near the eye
and moving the light beam to and for both the animals
▪ The corneal reflex was examined by touching a side of the cornea with a
cotton swab
▪ The left eye served as control, and saline was instilled. The pupillary size,
light reflux, and corneal reflex action were recorded 10 min after drug
instillation. Observations were tabulated for a period of 60 minutes.