Laboratory Manual Final

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Lab Manual phramacology 2nd professional (2014)
Research · April 2015

DOI: 10.13140/RG.2.1.2229.7122
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Laboratory Manual
EXPERIMENTAL PHARMACOLOGY
2nd Year PHARM.D
Dr. Huda Kafeel

Faculty of Pharmacy
Jinnah University for Women
Laboratory Manual 2014
Student name:
_________________________________________
Seat no:
__________________________________________
Course In-charge:
Dr. Huda Kafeel
Lecturer, Faculty of Pharmacy
Jinnah University for women
Dr. Huda Kafeel 2 2nd Professional Pharm.D

This is to certify that
Dr._____________________________________
Roll # ____________________
2nd Professional Pharm.D (Morning) has been
successfully completed the experimental work
assigned in the Course Title Pharmacology &
Therapeutics (408).
Signature of course In-charge:
__________________________________
Dated
________________________________
2nd Professional Pharm.D 3 Dr. Huda Kafeel

CONTENTS
EXP NO. Objective Pg# Date Sign
To study the Basics of Pharmacological

EXP # 1 6-10
experiments
To study handling of different experimental

EXP # 2 11-26
animals.
To study various routes of administration in

EXP # 3 27-31
experimental animals.
To study the physiologic salt solutions used in

EXP # 4 32-34
experimental pharmacology.
Dose calculation and administration guidelines for

EXP # 5 35-39
experimental animals
EXP # 6 Study of Animal ethics for pre-clinical testing. 40-42
To Study various pharmacological experimental

EXP # 7 43-45
instruments.
EXP # 8 study clinical trials of new drugs in human subjects 46-51
Autonomic Nervous System 52-62
EXP # 9 To study the effect of Epinephrine on frog’s heart. 63-64
EXP # 10 To study the effect of Propranolol on frog’s heart. 65-66
EXP # 11 To study the effect of Acetylcholine on frog’s heart. 67-68
EXP # 12 To study the effect of Atropine on frog’s heart. 69-70

CONTENTS
Mydriatic and Miotic effect on rabbit’s eye 71-72
EXP # 13 Evaluation of the effect of Atropine on rabbit’s eye. 73-74
Evaluation of the effect of Phenylephirine on

EXP # 14 75-76
rabbit’s eye.
Evaluation of the effect of Pilocarpine on rabbit’s

EXP # 15 77-78
eye.
Evaluation of the effect of Physostigmine on

EXP # 16 79-80
rabbit’s eye
To study the effect of NSAIDs on bleeding time by

EXP # 17 81-84
Duke method.
To study the effect of NSAIDs on clotting time by

EXP # 18 85-87
Capillary tube method
To study the effect of NSAIDs on clotting time by

EXP # 19 88-90
Modified Lee and White method
To determine anticoagulant effect of Heparin and

EXP # 20 91-96
Warfarin.
Study of Anti-inflammatory effect of NSAIDs on

EXP # 21 97-102
Experimental animal.
Study of Analgesic effect of NSAIDs by Chemical

EXP # 22 103-105
method.
EXP # 23 To study the Diuretic effect of drugs on mice 106-112
EXP # 24 Study of effect of saline purgative on frog intestine. 113-116

BASICS OF
EXPERIMENT # 1
To study the Basics of Pharmacological experiments
Objective: To understand the basic concept and various terms and approaches used in
experimental Pharmacology.
Background: Drugs and their use were well started in pre historic era and even the beneficial
and toxic effects of many plants animal sources were recognized. The concept of pharmacology
began in 17th century when observation and experimentation began to replace traditional drug
use. Many British physicians start to understand the value of experimentation when they
performed experiments to evaluate the effects of some traditional drugs used in their own
practices in the treatment of several diseases. Thus, the science of drug preparation and
therapeutic use of drugs began to develop as part of pharmacology. But, limitation was lack of
facilities regarding methods for obtaining active constituents from different sources.
Purpose of Pharmacological Experiments:

 Discover new therapeutic agents.
 Discover new use of existing therapeutic agents.
 Establish safety profile of new and old therapeutic agents.
Experimental Pharmacology comes under the heads:
1. Pre-Clinical testing- Pre clinical testing is Animal based and serve as basis of clinical
testing. Further sub divided into two types:
 In vivo testing – Experimentation on Whole intact living body.

Animal model based testing and clinical trials are major elements of in vivo research. In

vivo testing is often employed over in vitro because it is better suited for observing the
overall effects of an experiment on a living subject.
 In vitro testing- Experimentation outside the living body. Experimentation performed in

tissue bath, isolated organ, using chamber and test tube based laboratory experiments.
2. Clinical testing- Human Based and further sub divided into different phases.
Approaches for pharmacological experiments:

Approaches that serve as basis for pharmacological experiment can be:
1. Use of ancient /folk remedies, Herbal preparations for evaluation of their possible
pharmacologic activity. For example;
 Evaluation of anti-inflammatory effect of Turmeric (Curcuma longa) on the basis of its use in
regional tribes of India for pain conditions.
 Salicylic acid was isolated from the bark of willow trees after learning that Native Americans
brewed the bark to treat inflammatory ailments.
 South American natives used a tea obtained by brewing Cinchona bark to treat chills and
fever. Further study in Europe led to the isolation of quinine and quinidine, which
subsequently were used to treat malaria and cardiac arrhythmias, respectively.
2. Screening of an old existing drug for their new possible therapeutic effect. For example;
 Atorvastatin (cholesterol-lowering drug) found to be effective in reducing symptoms of
multiple sclerosis (MS) in mice. Leads to the development of drug with new pharmacological
activity.
3. Structural Optimization or modifications in existing drugs for their modified effects in terms of
improved efficacy, safety or new therapeutic effect. And determination of Pharmacophore.
 Structural optimization of lead compound (salicylic acid) by the Bayer Corporation of Germany
resulted in acetylsalicylic acid, or aspirin, the first nonsteroidal anti-inflammatory agent.

 Another example of derivation new chemical entities from biologically unrelated molecules is
illustrated by the development of the potassium channel agonist diazoxide. This molecule was
developed as a result of the observation that the thiazide diuretics, such as chlorothiazide, not
only exhibited diuretic activity, due to inhibition of sodium absorption in the distal convoluted
tubule, but also demonstrated a direct effect on the renal vasculature. Structural modification
to enhance this direct effect led to the development of diazoxide and related potassium
channel agonists for the treatment of hypertension.
Considerations of Pharmacological Experiments:

For any pre-clinical pharmacological experimentation following points must be considered.
 Animal selection
 Dose calculation
 Selection of Vehicle/physiologic solution
 Grouping of Animals
 Development of Experimental Model
1. Animal selection: selection of experimental animals is made in Accordance with the

objective of Study. Different animal related factors like Age, Weight, sex and specie may affect
the results and should be consider during selection.
2. Dose calculation: Dose for experimental subject is appropriately calculated according to

the weight of subject and test dose of new drugs and established human dose of old drugs.
3. Selection of Vehicle/Physiologic solution: Most commonly used vehicle for making

dilutions of different drugs and physiological solution is normal saline. Selection of appropriate
vehicle is important according to the solubility nature of test drug and required route of
administration. Other common vehicle used includes:
 Polysorbate 80 (generic name, Tween 80) is a nonionic surfactant and emulsifier. This
synthetic compound is viscous, water -soluble yellow liquid. It is frequently used to increase
solubility of test drug in water.
 Saline (Normal saline) is an isotonic solution of sodium chloride. (0.90% w/v)
 Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula (CH3)2SO. This
colorless liquid is an important polaraprotic solvent that dissolves both polar and non
polar compounds and is miscible in a wide range of organic solvents as well as water.
 Carboxymethyl cellulose (CMC) or cellulose gum is a cellulose derivative with
Carboxymethyl groups (-CH2-COOH) bound to some of the hydroxyl groups of
the glucopyranose monomers that make up the cellulose backbone. It is often used as
its sodium salt, Sodium Carboxymethyl cellulose.
4. Grouping of Animals: Animals should be divided into two major groups before starting
any pharmacological activity. I-e Test Group (The group of animals in which test dose of test
drug is given) Control Group (The group of animals in which simple saline or any other vehicle
is given and that serve as control for the measurement of pharmacological activity of test
compound) For both the groups all the parameters related to animal housing and food should
be same.
5. Development of Experimental Model: Different models are required for the

experimental purpose according to the objective of study. For example;
 Aged rodents and scopolamine-induced amnesia models can be used for drugs having
memory enhancing effects and it can also be serve as model of Alzheimer’s disease.
 The rodent formalin model is used as an acute and rapid in vivo screening study for
evaluating the potential analgesic effects of novel chemical entities. Injection of formalin
into the rodent’s hind paw induces biphasic nociceptive behavioral responses.
Over view of drug development: Drugs development mainly deals with three stages:
Stage I: Hit and lead compound development phase (Identification of lead compound amongst
the million compounds and selection for further studies)
Stage II: Pre-Clinical Studies (Done in form of In vivo and In vitro animal experiments.
Stage III: Clinical Studies (Experiment in humans) Clinical trials Phase 0, I, II, III, IV and V.
(Figure 1.1)

(Figure 1.1)
Figure 1.1: Developmental Stages of a Drug. Divided into three stages (I) Identification of lead compound and
optimization. (II) Pre-Clinical Studies (III) Clinical Studies.
Additional Notes:

EXPERIMENT # 2
To study handling of different experimental animals
Objective: To study different common Laboratory animals their handling techniques,

Precautions in handling and different terms and basics regarding experimental animals.
LABORTORY ANIMALS:
Animal model selection is one of the most important steps in any of the experimental
pharmacological study. Animal model preferred for the study must be producing similar disease
profile as in human. Hence, suitable animal model should be selected which follow three main
objectives
 Use of an animal phylogenitically closer to men.
 Use of an animal in which the process under investigation is as close as possible to that in man.
 The Anatomy, Physiology and biochemistry and considered to be similar.
Experimental animals can be divided into three categories:

Rodents Non Rodents
Mouse, Rat, Guinea Pig, Gerbil, Hamster etc Rabbit, Monkey, Dog, Cat, Pig etc.
Miscellaneous
Frog, Pigeon, Zebra, Fish, Chicken etc.

Selection criteria for experimental animals:

Since long animal experiments have been a mile stone in advanced medical research. Any or every
animal is not suitable for experimental work. There selection is based on the following criteria.
 Size: Smaller animals are preferred because they are easy to handle & less quantity of drug is
required.
 Availability: Animals which are commonly available should be selected e.g. frogs, rats, rabbits
& dogs.
 Sensitivity: Animals which are sensitive to drugs under trial e.g. guinea pig is sensitive to effect
of histamine.
 Species: In rabbits intra-cerebro ventricular injection of 5-HT induces a lowering of
temperature, but in cats, it induces fever.
-Ethacrynic acid is almost inactive in rats, except at high doses, but quite active in the dog.
-Following the same dose of hexobarbitone per unit body weights, the average sleeping time of
rats is about seven times that of mice, and in the dog its effects lasts for hours.
-Guinea pig and humans are 500 more times sensitive to histamine than are rats & mice. -
Histamine powerfully contracts the uterus of guinea pig while relaxes that of rat. In rodents it
produces stronger arteriolar constriction in cat slight constriction, while in dog, monkey and
man arteriolar dilation.
-The rat heart is known to be very resistant to cardiac glycosides.
-Alloxan produces diabeties mellitus in no. of species but not in guinea pigs.
-Rabbits generally show symptoms following insulin when the blood sugar level is 45 mg
percent while dogs show symptoms only when the level is about 18 mg. percent.

 Housing Requirements- Isolation or group housing.

-Dose of pentoharbitone or phenobarbitone, which produces full hypnosis in isolated mice,
produces marked stimulation when the animals are grouped.
-The CVS becomes more responsive to isoprenaline in solitary housed (6-8 weeks) rats compared
to group housed rats.
Commonly used Experimental animals

MOUSE (Mus musculus):
Swiss albino mice are commonly used. They are smallest cheap & easy to handle.
COMMON BEHAVIOR:
 „ Timid
 „ Social
 „ Territorial
 „ Nocturnal
 „ Rarely aggressive when handled properly
EXPERIMENTAL USE: (Adult weight: 20-25 gm., age suitable for experiment, 2 month)
1. Toxicological studies, especially acute & sub acute toxicities. They are also used in teratogencity
(foetal abnormalities)
2. Bio assay of insulin.
3. Screening of analgesics & anti convulsants
4. Screening of chemotherapeutic agents.
5. Studies related to genetics & cancer research.
6. Study of drugs acts on CNS.
RESTRAINING OF MOUSE:
When attempting to restrain mice, sudden, jerky moves should
be avoided to decrease the likelihood of being bitten.

Approaching mice with gentle confidence is best. It is important to select the appropriate method
of restraint for the procedure you wish to perform and one that will offer the best access to the
area requiring manipulation.
Restraint by the tail or with forceps is only intended for short-
term manipulations, such as transferring animals from one
cage to another.
Tail Restraint: Mice may be picked up by grasping the base

of the tail. Do not grasp the tip of the tail, as this may cause the
skin to be stripped off. This method is only used for brief
restraint; for example transferring animals from cage to cage.
Never suspend the mouse for prolonged periods of time by its tail.
Forceps Restraint: Mice may also be picked up with rubber-

tipped forceps by gently grasping the animal by the scruff of the
neck or the base of the tail. The forceps should be dipped in
disinfectant between cages. This method of restraint should only
be used for short-term procedures such as transferring animals
to a new cage. Never suspend the animal for a prolonged period
of time with the forceps.
Scruff Restraint: Using the scruff or mechanical devices is suggested for procedures requiring
more than momentary restraint, such as injections or blood withdrawal. Restraining the mouse by
the scruff will allow you to perform many technical procedures such as examination, injection and
blood collection. There is a one-hand and a two-hand variation of this technique. The one-hand
method places you at greater risk for being bitten, so beginners should perfect the two-hand
restraint method before attempting the one-hand method. For the two-hand technique, restrain
the mouse by grasping it near the base of the tail and placing it on a toe-gripping surface, such as a
wire bar lid. Pulling back gently on the tail of the mouse causes it to pull forward on the toe-
gripping surface. Caution must be used to avoid injuring the tail or toes of the mouse.
While grasping the tail with one hand, grasp the nape or scruff of
the neck with the other. Position the animal’s body firmly across
your hand by extending your forefinger and thumb back as far as
possible, while maintaining a firm grip on the scruff. Place the tail
between the fingers of this same hand to secure the animal. This
type of restraint will allow the handler complete access to the
ventral side of the mouse. Again, caution must be used. If you do
not grasp enough of the scruff, the animal will be able to turn and
bite. If you grasp too much skin, the airway will become restricted
and the mouse will become cyanotic. Monitor the condition of the
animal the entire time it is restrained, being careful to observe the
breathing rate and color of the ears, nose and oral cavity. The
animal should be released immediately if there are any signs of
gasping or change in coloring from pink to blue.
For the one-hand technique, restrain the mouse by grasping it
near the base of the tail and placing it on a toe-gripping surface. A
good example of an appropriate surface is the wire bar lid. Place
the base of the tail between or underneath your last one or two
fingers. With the thumb and first finger of the same hand, grasp
the nape or scruff of the neck. The same precautions as described
for the two-hand technique must be followed.
INJECTING DUGS IN EXPERIMENTAL MOUSE:

Injections various routes exist for injecting mice.
 It is important that you decide the appropriate route, volume, site and needle selection with
according to nature and type of drug.
 All injections must be performed using sterile needles and syringes. A new needle and syringe
should be used for each cage of mice.

Intramuscular Injections: Regardless of the method used for

intramuscular injections, it must be noted that the sciatic nerve
runs along the length of the femur. It is very important to avoid
injuring this nerve. This is best accomplished by pointing the
needle, caudally rather than cranially, into the caudal thigh
muscles. It is imperative that the mouse be properly restrained.
If the mouse is allowed to kick or struggle, this could cause
injury to the muscles or the nerve.
It is best to swab the area with 70% ethanol before placing the needle
and to aspirate to look for blood before injecting. One of the following
methods may be used:
Method #1 Restrain the mouse by the scruff method. Secure the rear foot
nearest to you beneath your little finger and lower thumb. Swab the area
to be injected with 70% ethanol. Insert the needle, bevel up, into the
caudal thigh at a 45° angle. Aspirate to ensure that you have not entered
a blood vessel. If no blood is seen, slowly inject the material.
Method #2 Restrain the mouse by the scruff method. Swab the area to
be injected with 70% ethanol. Insert the needle into the caudal thigh,
bevel up. Aspirate and inject. Since the foot has not been secured, make
sure the mouse does not kick.
Method #3 A technique using two people can also be used for IM

injections. One person restrains the mouse by the scruff method with
one hand and steadies the leg to be injected with the other. The
second person identifies the caudal thigh muscles, swabs the area
with 70% ethanol, aspirates and injects the material.

Method #4 A tail first restrainer can also be used for IM injections.

Gently pull the foot of the leg to be injected through the restrainer
and locate the caudal thigh muscle. Hold the foot firmly, swab the site
with 70% ethanol, aspirate and inject the material with the needle
bevel up.
Subcutaneous Injections: Restrain the mouse by the scruff method.

Use your thumb and forefinger to make a tent of skin over the scruff.
Prep the area with 70% ethanol. Insert the needle, bevel up, at the base
of the tent. The needle should be inserted parallel to the skin and
should be directed toward the posterior of the animal. Aspirate to
ensure proper placement and inject the material.
Intraperitoneal Injections: Restrain the mouse by the

scruff method. Expose the ventral side of the animal, tilting
the head down at a slight angle. Prep the site with 70%
ethanol. The sterile needle should be placed, bevel up, in the
lower right or left quadrant of the animal’s abdomen. Insert
the needle at a 30° angle. Aspirate to ensure proper
placement and inject the material.
Intradermal Injections: In order to perform intradermal

injections, the mouse should be anesthetized. Shave or pluck an
injection site on the back of the animal to remove the hair. Swab
the site with 70% ethanol. Insert the needle into the skin, bevel
up, holding the needle nearly parallel to the plane of the skin. Do
not aspirate. Inject the material. The volume of the injection
should be small enough to avoid tissue trauma.

Oral Gavage: Gavaging is used to dose an animal with a specified volume

of material directly into its stomach. Only a specialized, commercially
available gavage needle should be used for attempting this procedure. Fill
the syringe with the appropriate volume of material and attach the needle.
Restrain the animal by the scruff. Place the tip or ball of the needle into the
animal’s mouth. Slide the tip gently past the back of the tongue. The needle
should slide easily down the esophagus, if properly placed. DO NOT
FORCE!!! If any resistance is met, remove the needle and reinsert. Do not
aspirate. Once the needle is properly placed, administer the material.
RAT (Rattus norvegicus):

Albino rat is one of the commonest laboratory animals suitable for
experimental work because of its small size & greater sensitivity to most
drugs. It is also the most standardized of all laboratory animals. Wistar
and Sprague-Dawley are also in use in different regions. (Fig 2.2)
It can be used to obtain pure and uniform strains and is found to be very
sturdy to withstand long periods of experimentation under anesthesia.
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 tonsils 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. Pancreas is diffused, therefore difficult to produce pancreactomy. In
stomach, fundus & pyloric parts have clear lining between them. The gastric acid secretion is
continuous.
EXPERIMENTAL USES: (Adult wt. 180-200 gm., age suitable for most of the experiment 1.5 months)
1. Psychopharmacological studies.
2. Study of analysis of anticonvulsants.
3. Bioassay of various hormones, such as insulin oxytocin, vasopressin etc.

4. Study of oestrus cycle, mating behaviour and lactation.

5. Studies on isolated tissue preparation like uterus, stomach, vasdeferens, anococcygeus fundus
strip, heart, etc.
6. Chronic study on blood pressure.
7. Gastric acid secretion studies.
8. Study of hepatotoxic and antihepatotoxic compound.
9. Acute & chronic toxicity studies.
RESTRAINING OF RAT:
Handling and Restraint Rats are typically docile animals, particularly
if they are routinely handled using appropriate techniques. Bites
from rats are uncommon and will typically only occur if the animal is
stressed or in pain. To initially restrain a rat, the handler should gently
grasp it around the shoulders.
The handler’s thumb can then be placed under the rat’s mandible, to
prevent bites, and the rat’s hind limbs can be supported with the other
hand. Restraint should be firm but not too tight as this will impede the
animal’s respiration.
INJECTING DUGS IN EXPERIMENTAL RATS:
Subcutaneous SC injections are performed in rats using the same technique as was described
for mice with the following differences. The volume of material administered can be increased to
approximately 5 ml per site in an adult rat (>300 grams). Syringe size should be increased
proportionately and needles should be 22 gauge or larger gauge.
Intramuscular IM injections may be performed in the rat. Injection volumes are limited to
0.25 ml site because of limited muscle mass. Either the quadriceps muscles located on the cranial
aspect of the femur or the hamstrings on the caudal aspect of the femur can be used. Care must be
taken to avoid depositing material on or near the sciatic nerve which runs along the caudal aspect

of the femur in the thigh. Therefore the needle should be directed cranially if injecting the
quadriceps or caudally when injecting into the hamstrings. A 23 ga 0.5Ó needle or larger gauge
should be used. The needle is directed through the skin into the muscle belly approximately 3-4
mm. Aspiration should be attempted before injecting to determine that accidental penetration of a
blood vessel has not occurred.
Intravenous IV injection technique for the rat is similar to the mouse. However, the vessels are
more difficult to visualize, especially in adult rats. The skin overlying the vessels in adults becomes
quite thick, making vascular access much more difficult. For this reason the preferred site for
vascular access is near the tail base. Injection volumes administered to an adult rat should not
exceed 2 ml and large volumes should be administered slowly to
avoid vascular overload. The technique describing IV administration
and needle size in mice should be followed.
Intra-peritoneal Injection Rats should be restrained with their

abdomen exposed and their head held downward. The injection site,
method and needle size is as described for mice. Because of their
larger size < 5.0 ml of material can be administered to an adult rat.
RABBIT (Oryctolagus cuniculus):

Rabbit are also docile animals with large ears. Usually Newzealand
white rabbits are used. It has huge caccum and large appendix. The
enzyme atropine esterase is present in rabbit liver and plasma, so it
can tolerate large doses of belladona (atropine). Cardioaortic nerve
forms a separate depressive nerve. Vasodilator nerves are absent
and so vasomotor reversal phenomenon cannot be demonstrated.
Histamine causes increase in blood pressure. Ovulation is related to
the release of luteinizing hormone and occurs 10 hours after coitus.

EXPERIMENTAL USE- (Adult weight 1.5-3.0 Kg, Age suitable for experiment 5-6 months)
1. Pyrogen testing
2. Bio assay of antidiabetics, curareform drugs and sex hormones.
3. Screening of agents affecting capillary permeability.
4. Irritancy tests.
5. Screening of antitoxic agents and teratogens.
6. Studies related to reproduction (antifertility agents).
7. Isolated preparation like heart, dudenum, ileum, Finkleman preparation.
8. Study of local anesthetics (surface anaesthesia)
9. Study of miotics & mydriatics
RESTRAINING OF RABBIT:
Rabbits are generally docile and easy to handle.
However, there is still the possibility of getting
scratched or bitten, so be careful. The most important
thing to remember when handling a rabbit is to
support its hind legs. Rabbits have very strong legs and
very little bone mass. When they kick it is very easy for
them to cause serious injury to their spine. To remove
a rabbit from its cage, grasp the loose skin around the
neck and pull the rabbit to the front of the cage. Using
your other hand, support the rabbit’s hind quarters and
lift it out of the cage. After you have the rabbit out of
the cage, tuck the rabbit into the cradle of your arm as
one might carry a football. If at any time the rabbit
begins to struggle, either place it down or hold it close
to your body and re-establish your hold. If the rabbit is
held properly, it will feel secure and will not struggle.
When restraining a rabbit on the tabletop, place one

hand around the rump and rest your other hand across the shoulders.
When placing the rabbit back into the cage, the hind end should go in first. This will prevent the
rabbit from trying to leap into the cage.
INJECTING DUGS IN EXPERIMENTAL RABBIT:

Injection sites should be cleaned with a suitable disinfectant, typically isopropyl alcohol. Sterile
syringes and needles must be used for all injections. The one time use of disposable supplies
insures aseptic techniques and sharp needles. Always select the smallest gauge needle possible to
limit tissue trauma and injection discomfort. A 20-30 gauge needle is recommended for use in a
rabbit. Before injecting, check for correct placement by pulling back on the plunger of the syringe
to create a vacuum. This is known as aspiration.
SUBCUTANEOUS (SQ) INJECTION: The rabbit should

be restrained in the normal manner. With your fingers, lift
the skin to make a “tent”. Disinfect the injection site and
insert needle into the subcutaneous tissue. Aspirate prior to
making the injection. Proper placement should yield no
aspirate. Inject. Most common injection site is the loose skin
around the neck and shoulder area.
INTRAMUSCULAR (IM) INJECTION: Restrain rabbit by either holding the rabbit against your
body (like a football) Disinfect injection site and insert needle into the caudal thigh muscle. You
must first isolate the caudal thigh muscle to prevent injection into the ischiatic nerve. Injection
into the nerve may cause discomfort and lameness. Aspirate and inject. If blood is aspirated, you
must reposition the needle. Another injection site is the lumbar muscles. To administer an IM
injection here, outline the lumbar muscles with your thumb and second finger, using your index
finger locate the vertebral column for orientation. Insert needle lateral to the midline, avoiding the
spine.

INTRADERMAL (ID) INJECTIONS: Restrain the rabbit using either physical or chemical
restraint. Shave or Nair the injection site and disinfect. Isolate the injection site by pinching or
stretching skin. Insert the needle bevel up just under the surface of the skin and inject. A distinct
bleb should form. The recommended needle size for an ID injection is 25 gauge.
INTRAPERITONEAL (IP) INJECTION: Restrain the rabbit by grasping the scruff with one
hand and the hocks with the other. Rotate the rabbit’s body downward and extend the legs
upward. Place the rabbits head and shoulders between your knees and hold firmly. This position
allows the intestines to fall forward and away from the injection site. Disinfect injection site and
insert the needle cranially into the abdomen at a 30-45 degree angle caudal to the umbilicus and
lateral to the midline. Aspirate: * greenish-brown aspirate indicates needle penetration into the
intestines * yellow aspirate indicates needle penetration into the bladder If any fluid is aspirated,
your solution is contaminated and must be discarded and the procedure repeated with a new
syringe and needle. If nothing is aspirated, inject. Once the injection has been given, carefully
reach between your legs and grasp the rabbit’s scruff. Then slowly return the rabbit to the upright
position.
INTRAVENOUS (IV) INJECTION: The most common site for IV

injections is the marginal ear veins. The cephalic, medial, and lateral
saphenous, and lingual are more difficult to use, therefore less
frequently used. Because the rabbits ear veins are so fragile it is
recommended that this procedure be done under anesthesia. Locate a
marginal ear vein and remove the hair. Gentle stroking and tapping of
the ear may make the vein more visible. Disinfect injection site and insert needle into the vein at a
slight angle. You will not be able to aspirate, instead inject slowly and watch for clearing of the
lumen. Incorrect positioning will result in a slight bulge in the ear. If this occurs, remove needle
and repeat process proximal to previous site. Upon completion remove needle and apply pressure
to injection site.

Oral gavage: It is recommended that a 6-8 French feeding tube be used to gavage a rabbit. First,
measure the tube from the tip of the nose to the last rib. This is how far you must insert the tube.
Restrain the rabbit, ideally in a box restraint. Lubricate the tube with sterile lubricant. Stabilize the
rabbit’s head with one hand and with the other insert the tube laterally into the nasal cavity until
the tube begins to pass. At this time straighten out the tube and continue to pass. In the event
resistance is met, DO NOT FORCE as this can cause serious injury. The most common place to meet
restriction is in the back of the throat; the tube should pass freely when the rabbit swallows.
Check for proper insertion by placing the end of the feeding tube in a bowl of water, if air bubbles
are present you may be in the lungs. If you are still in doubt as to the correct positioning, you may
inject a few milliliters of water and aspirate for the presence of greenish-brown stomach contents.
If correct placement is achieved, inject solution. Upon completion remove the tube by pinching the
end of the tube and gently removing the tube. Pinching the end of the tube prevents contents in
the tube from draining into the oral-pharyngeal region. When the procedure is complete, observe
the rabbit for signs of distress such as gasping or frothing at the mouth.
BLOOD WITHDRAWAL FROM EXPERIMENTAL RABBITS:

Withdrawal sites should be cleaned with a suitable disinfectant. Sterile syringes and needles must
be used for all withdrawals. The one time use of
disposable supplies insures aseptic techniques
and sharp needles. Always select the smallest
gauge needle possible to limit tissue trauma and
discomfort. A 22-25 gauge needle is
recommended for use in a rabbit. Check for
correct placement by pulling back on the plunger
of the syringe to create vacuum. This is known as
aspiration.
NOTE: Rabbits have very poor peripheral circulation as a result of a cool environment and
heightened anxiety. In these cases, sedatives or warming methods can be used to dilate the vessels
in the ear.
Marginal ear veins: The marginal ear veins may be used to collect up to 5 milliliters of blood.
Locate a marginal ear vein and remove the hair. Gentle stroking and tapping of the ear may make
the vein more visible. Small quantities of blood may be by disinfecting the site and inserting a
small gauge needle into the ear vein and collecting from the hub of the needle directly into a
microhematicrit tube. For larger quantities of blood a syringe may be used. When using a syringe
it is very important not to apply too much negative pressure, as this will collapse the vein.
Central ear artery: The central ear artery may be used to

collect up to 50 milliliters of blood. Locate the central ear
artery and remove the hair. Gentle stroking and tapping of
the ear may make the artery more visible. It is recommended
that a 1-2 inch needle with the hub broken off be used.
Disinfect the site and insert needle. A rolled up piece of gauze
placed on the underside of the ear will aid in holding. Once
blood starts to flow place receptacle under the needle for
blood collection. If the vessel collapses, gently stroke the ear until the vessel relaxes and blood
begins to flow. A syringe may also be used. When using a syringe it is very important not to apply
too much negative pressure, as this will collapse the artery. Upon completion of either blood
collection technique, ensure proper hemostasis.
Cardiac puncture: This method must be done on an anesthetized rabbit and is only
recommended to be done as a terminal procedure. Repeat survival sampling for small volumes of
blood causes increased morbidity and mortality and is not recommended. It is recommended that
a 20 gauge needle at least 1 ½ inches long be used. Find the xiphoid process as a reference point.
Insert the needle at a 35-40 degree angle just under and to the left of the xiphoid process. As the
needle is inserted into the chest, gently aspirate until blood begins to flow. Overzealous
withdrawal may collapse the heart. If you do not get blood flow on the first try, withdraw the
needle until blood flows or repeat entire process. Probing for the heart is not recommended, this

could cause damage to major vessels and premature death. Upon completion of this procedure the
animal should be euthanized and disposed of properly.
ADDITIONAL NOTES:

EXPERIMENT #3
Various Routes of administration in experimental animals
Objective: Administration of substances to laboratory animals requires careful consideration

and planning to optimize delivery of the agent to the animal while minimizing potential adverse
events. For all species, many different routes are available for administration of substances.
Improper selection of route may result in unintentional adverse effects on experimental animals
and confounded results. Study of all available routes with their limitations is necessary for proper
selection of desired route before planning any study.
Selection of a route: administration of drugs to laboratory animals by a wide variety of

routes. A key factor determining the route selected is whether the agent is being administered for
a local or systemic (either enteral [through the digestive tract] or parenteral [outside the digestive
tract]) effect.
Parenteral administration methods typically produce the highest bioavailability of substances
because these methods avoid the first-pass effect of hepatic metabolism, which occurs commonly
with orally administered chemicals and therapeutics.
Parenteral routes also circumvent some of the unpredictability associated with enteral absorptive
processes.
Regulatory requirements may influence the selection of a particular route, depending on the
purpose of the study (for example, nonclinical safety testing, in which the route of delivery to
animals should closely resemble the projected route of administration to humans).
COMMON ROUTES OF ADMINISTRATION:

 Oral Route - Given into the mouth
 Gastric Gavage- Delivered directly into the stomach
 Intravenous - Delivered into a blood vessel.
 Epicutaneous - Delivered onto skin

 Intradermal - Delivered into skin

 Subcutaneous - Delivered under skin
 Transdermal - Delivered across skin
 Intramuscular - Delivered into a muscle
 Transcorneal - Instilled onto the eye
 Intraocular - Into the eye
 Intracerebral - Into the brain
 Epidural and - Space surrounding the dura mater
 Intrathecal - Spaces that surrounding the distal spinal cord
 Intraperitoneal- Administered into the peritoneal cavity
 Intraosseous - Directly into the marrow cavity
 Intranasal - Sprayed into the nose for absorption across the nasal mucous membranes
 Intratracheal - Delivered into the lungs by direct tracheal instillation
E NTERAL ADMINISTRATION
 Administration of substances directly into the mouth, orogastric or nasogastric gavage is
common in laboratory animal medicine and research.
 Per rectum administration of substances by enema or suppository is less common in animals
than in humans.
 The oral route is economical, convenient, relatively safe, and some animals can be trained to
cooperate voluntarily, depending on the compound being administered.
 Although voluntary consumption of the material being administered is ideal, this dosing
technique may not be reliable in all animals or dose groups or for long-term studies, because of
individual preferences for flavors, palatability issues, and changes in behavior over time.
 Administration of large volumes of substances by orogastric or nasogastric gavage may cause
stress due to gastric distension in species that are unable to vomit, such as rodents. Therefore,
using the smallest volume possible is recommended for the oral route of administration,
optimally 5 ml/kg for all species.
 Limitations of oral dosage may include a slower onset of action
 Oral gavage requires moderate technical skill and confidence.
I NTRAVENOUS ADMINISTRATION .
 The intravenous route of delivery is the most efficient means of delivering substances to
animals because it bypasses the need for solute absorption.
 Substances administered intravenously must be delivered aseptically and should be sterile;
free of particulates.
 The intravenous route of substance delivery, although efficient, can be risky in animals, and
persons conducting this technique require training and practice to ensure competency.
 Careful control of hemostasis must be instituted when the catheter or needle is removed, to
minimize blood loss and painful hematoma formation.
A DMINISTRATION TO SKIN AND MUSCLE .

 Some substances can be administered directly to the skin surface (epicutaneous
administration) for a topical affect. The extent of absorption of materials through the skin and
into the systemic circulation (that is, percutaneous or transdermal delivery) depends on: the
surface area over which the substance is applied; the concentration of the substance
administered; the lipid solubility of the material or vehicle; whether the skin surface is intact;
the skin thickness at the site of application; the length of time that the material is in contact
with the skin surface; and the degree of skin hydration and surface occlusion, in that covered
and well-hydrated skin absorbs substances faster than does uncovered or dry skin.
 Caution must be exercised to avoid applying caustic or irritating material directly onto the
skin, and some substances may induce local sensitization reactions.
 Transdermal or percutaneous delivery represents a similar route of administration except that
materials are applied to the skin surface deliberately, usually by means of a patch, for
absorption across the epithelial barrier into the systemic circulation. Typically, this method
produces very constant blood levels of the substance being administered.
 Percutaneous delivery is an attractive alternative to other parenteral routes, avoiding the need
for repeated animal restraint, painful injections, and sharps hazards. In addition, materials can
readily be removed from the skin surface if dosing needs to be interrupted or if adverse effects
are noted.
 Transdermal delivery of substances may be acute or chronic, and current techniques for
delivering substances by this route have been reviewed recently. Commercially available
human transdermal products can be difficult to use in animals because of the much larger
doses of substances impregnated into products intended for adult human use. Cutting
transdermal patches to scale-down the dose being administered is not recommended;
however, covering a portion of the patch to limit substance administration may be used.
 Nonirritating substances may be given subcutaneously, which represents a rapid, inexpensive,
and simple method of parenteral substance administration. Substances administered
subcutaneously often are absorbed at a slower rate compared with other parenteral routes,
providing a sustained effect.
 Intramuscular administration of substances is a common parenteral route in large animals and
humans but often is avoided in smaller species because of the reduced muscle mass. Generally,
intramuscular injections result in uniform and rapid absorption of substances, because of the
rich vascular supply. The intramuscular technique requires more skill than does subcutaneous
injection and should be conducted only by well-trained personnel.
I NTRAPERITONEAL ADMINISTRATION .
 Injection of substances into the peritoneal cavity is a common technique in laboratory rodents
but rarely is used in larger mammals and humans. Intraperitoneal injection is used for small
species for which intravenous access is challenging and it can be used to administer large
volumes of fluid safely
 Absorption of material delivered intraperitoneally is typically much slower than for
intravenous injection. Although intraperitoneal delivery is considered a parenteral route of
administration, the pharmacokinetics of substances administered intraperitoneally are more
similar to those seen after oral administration, because the substances administered
intraperitoneally may undergo hepatic metabolism before reaching the systemic circulation.
 Materials injected intraperitoneally should be sterile, isotonic, and nonirritating.
ADDITIONAL NOTES:

EXPERIMENT # 4
Physiologic salt solutions used in experimental pharmacology
Objective: To study the physiological salt solutions used in experimental pharmacology &
various drug dilutions.
INTRODUCTION:
As animal experiments have to be done with isolated organs, it is necessary to use a certain no. of
physiological solution of different ionic concentration which almost acts as a substitute to the
tissue fluid Therefore PSS can be defined as artificially prepared solution to keep isolated tissue
alive under experimental conditions. They provide isotonicity, nutrition and acts as a buffer when
drugs are added. It was "Ringer" who first introduced the idea that tissue could be kept alive by
providing proper nutrition, O2 & temperature.
The content of these solutions carries according to tissues & animals taken. These solution provide
food material i.e. energy, O2, electrolytes as in the same proportion as that present in tissue fluid.
They exert same osmotic pressure as that of interstitial fluid i.e. isotonic with body fluids.
Any variant from the principle will lead to shrinkage or blotting depending on hyper tonicity &
loss of physiological function. For these two points should be kept in mind:
 Solution should be prepared carefully with pure material.
 They can be kept for about 24 hr and as they are good media for the growth of micro
organisms they must be refrigerated and should be freshly prepared after 24 hrs.
Following points should be carefully noted at the time of preparation of solution:

1. Balance of cat ions: Absolute quantity of each ion and preparation among each other
especially with ca+2 & K+ must be maintained. The common cations and their significance
are:
o Na+ions: Responsible for maintenance of excitability, contractivility rythimicity of muscles
and nerves.

o K+ions: responsible for increase relaxation of heart increased neuromuscular transmission

and excitability of nerves.
o Ca++ ions: increase force of contraction & tone of heart and decrease excitability of nervous
tissues.
o Mg+2 ions: responsible for contraction of smooth muscle.
2. PH of solution / reaction of solution: pH of various PSS varying from 7.3-7.8 depending
upon organ. At lower pH value tone of preparations tend to decrease & effect of drug is also
altered. pH affects tissue directly and by ionization. At higher pH ionization is less and leads to
alkalinity & thus improves cardiac & smooth muscle activity. During experiment there can be
accumulation of metabolite which may change the pH. Buffering agents like HCO 3 & PO-4
are add in saline solution and solutions are changed frequently.
3. Glucose: Introduced by "Locke" and serves as an energy source, increases contractility of
tissue. It is not essential constituent for amphibians tissue but indispensable for mammalian
tissues.
4. Distilled Water: It acts as a vehicle to dissolve various ingredients.
5. Control of temperature: In order to get consistent effect, it is important to maintain the
temperature of PSS, particularly for mammalian tissue. For instance, when temperature of
solution is below 370C tone of intestine is decreased, increased the contracts become smaller
& contracts and relaxation time increases. Whereas, amphibaian tissues survive for longer
time at room temperature only.
6. Areation: Air, O2 or O2 + 5% CO2 are needed for the proper functioning of the tissues.
Besides providing O2 for the tissues, the stream of gas bubbles also stirs the solutions in the
bath thereby facilitating diffusion of the drugs.
7. The solution in the bath should be changed frequently because prolong areation tends to alter
pH.

Different physiological salt solutions and their uses:

i. Ringer lock's solution: It is used in isolated rabbit heart perfusion.
ii. Frog's Ringer's solution: Used in frog's rectus abdominis muscle and leech dorsalis muscle
preparation.
iii. Tyrode's solution: It is used in experiment of rabbit intestine & guinea pig ileum.
iv. De- jalon's solution: Used in rat uterus, duodenum colon experiment.
v. Kreb's Henseleit solution: Used in guinea pig tracheal chain prep. & rabbits aortic strip
preparation.
Composition of Physiologic Salt Solutions

Ringer Frog's ringer Tyrode's Deialon's Kreb's-Henseleit Mc Ewen's
# Salts in g/l
solution solution solution solution solution solution
1. Nacl 9.00 6.50 8.00 9.00 6.9 7.60
2. KCl 0.42 0.14 0.20 0.42 0.35 0.42
3. CaCl2 0.24 0.12 0.20 0.06 0.28 0.24
4. NaHCO3 0.50 0.20 1.00 0.50 2.10 2.10
5. Mgcl2.6H2O - - 0.10 - - -
6. MgSO4 7H2O - - - - 0.29 -
7. NaH2PO4 - 0.01 0.05 - - 0.14
8. KH2PO4 - - - - 0.16 -
9. Glucose 1.0 1 1 or 2 1.00 0.50 2.00 2.00
10. Areation O2 Air O2 / air O2+5% CO2 O2+5% CO2 O2
ADDITIONAL COMMENTS:

EXPERIMENT # 5
Dose calculation and administration guidelines for
experimental animals
Objective: To study basic concept of drug administration, selection of vehicle (solvent) and
calculation of appropriate dose according to experimental animal variant .
Background:
Characteristics of compound and vehicle to be administered are:
1. pH: Know the pH of the compound AND the vehicle. Aim for pH ~7. If the pH is higher or lower try one
of the following:
 Buffer to pH 7 if possible
 Dilute the solution using sterile normal saline or PBS
 Do not inject a high or low pH preparation IM or SC as this will be painful and cause tissue
necrosis. Recommended alternate routes of administration is IP or IV.
Examples:
 Pentobarbital is a very basic pH (~11). Only administer IV or IP; never IM or SC.
 Ketamine has an acidic pH (~4). In rodents ketamine is always administered IP. IM
administration can cause tissue necrosis.
2. Sterility: The compound and solvent must be rendered sterile either by sterilization or
filtration. Only Pharmaceutical-Grade compounds can be administered into animals.
Investigators are expected to use pharmaceutical grade medications whenever they are
available, even in acute procedures.
3. Odor: An offensive odor would limit voluntary intake of any compound.
4. Taste: Some compounds administered in the drinking water have a bitter taste and are
better accepted if sucrose is added. These include but are not limited to tetracycline,
doxycycline and metronidazole. Adding 2.0-5.0 g sucrose/100 ml water making the solution 2-
5% sucrose enhances palatability. See also IACUC Policy Additives to Drinking Water.

5. Osmolarity: Compounds to be administered parenterally must be ~ isoosmolar (280 milli-

osmoles/l). 5% Dextrose in Lactated Ringer’s Solution (LRS) which are both iso-osmolar by
themselves, may be an exception and can be administered in small amounts SC or IP since the
dextrose it is metabolized to CO2 and water rendering the resultant infusion isoosmotic.
However, caution is advised in a dehydrated animal.
6. Solubility: Some compounds may not be soluble and require administration as a suspension.
Example is sulfamethoxazole-trimethoprim administered in the drinking water. This
suspension must be shaken daily to assure proper dosage.
7. Light sensitivity: Protect against light exposure either by dispensing in a colored glass or
cover clear glass or plastic with foil. Examples include but are not limited to antibiotics such as
Septra, cephalexin and sulfatrim.
8. Toxicity: Detail search of document (literature search) regarding any new compound or
vehicle to be administered to animals in order to determine limitations to use and side effects.
Characteristics of Solvents (vehicle) used are:
1. Water: For enteral administration only. Injectable compounds must be isoosmotic with the
possible exception of 50% dextrose. A small amount of 50% dextrose may be administered IP
or IV in a hypoglycemic crisis.
2. Phosphate Buffered Saline (PBS): Whenever possible use sterile normal bufferd saline (PBS)
as a solvent. If the compound to be administered is not soluble in PBS other possible vehicles
should be considered.
3. DMSO (Dimethyl sulfoxide) 0.5% – 5%: An antioxidant, DMSO [(CH3)2SO] is a highly
reactive, amphipathic molecule with a highly polar domain and two apolar groups, making it
soluble in both aqueous and organic media. Due to its anti-inflammatory properties and the
ability to scavenge reactive oxygen particles, DMSO has been used for the treatment of several
diseases. Therapeutic and toxic agents that are not soluble in water are often soluble in
DMSO.
4. Methylcellulose 2%: is used as a thickener and emulsifier in various food and cosmetic
products, and also as a treatment of constipation. Methylcellulose is a stable sugar with low
toxicity and one of the safest and most widely used vehicles available when administered per
os. PO (gavage) or topical administration appears safe. Other routes of administration must
be scientifically justified. Characteristics of methylcellulose include its viscosity expressed in
centiPoise (cP) and percentage concentration, both of which must be considered when
administered to animals.
5. Corn oil: Gavage administration only. See tables 1 and 2 for recommended volumes.
6. Sesame oil: Gavage administration only. See tables 1 and 2 for recommended volumes.
7. Ethanol: Ethanol can be lethal if injected IV at high
Max. Volume to be given (IM/ IV)
concentration. Therefore, if an experimental compound
Mouse 0.25ml up to 0.4ml
has been purified or dissolved in ethanol, it behooves the
Rat 0.50ml up to 2.0ml
PI to consider concentration of EtOH, possible dilution if
Rabbit 1.00ml up to 10ml
the concentration is high and alternate routes of
administration in order to assure animal safety.
Dose Calculation for Experimental Animal:

For Powdered Drugs: Following are the steps, for calculating the doses and volume of injection
of any given drug, to be administered in the animal.
Step 1. Thinking of “What you have?”

Drug Dose, Weight of animal and volume to be injected.
Suppose;
Drug dose is 25 mg/kg for mice.
Weight of mouse is 20 gm.
Volume to be injected is 0.1 ml.
Step 2.
Hence, 25 mg/kg = 25mg/1000gm.
= 0.025/gm
= 0.025 × 20 (wt of mouse)
= 0.5 mg/ 20 gm wt of mouse.

= 0.5 mg/ 20 gm wt of mouse / 0.1 ml of vehicle.
Step 3. Weighing and dilution of drug.

=0.5 mg/0.1 ml
=5mg/1ml
=50mg/10ml
Hence the solution should be prepared by adding 5mg in 1ml of solvent (vehicle) or 50mg in
10ml of solvent, which will give the dose of 25mg/kg or 0.5 mg/20 gm 0f mouse body weight.
For Injectables: Sometimes pure powder of a drug is not available, so we have to use the
Injectables form or ampule of a drug.
Step 1. So in this case two important things should be kept in mind.

Strength of the injection and its solubility in the solvent.
Suppose;
Diazepam is available in the strength of 15 mg/ml and we have to use this strength in rat at a dose
of 5mg/kg, to show the anxiolytic activity of the drug.
So drug dose = 5mg/kg
=5mg/1000 gm
= 1mg/200 gm (weight of rat)
That means, 1 mg of drug should be administered in a 200gm of body weight rat.
Step 2: Dilution of injection,

Strength 15mg/ml
Put 1ml of drug in test tube and add 2ml of suitable solvent in the test tube.
Now the test tube contains 15mg of drug in 3 ml.
=15mg/3ml
=5mg/1ml
=1mg/0.2ml
SO, we can directly use 0.2 ml of final solution to provide a dose of 1mg in 200 gm of body
weight rat.

Calculate the dose of given drugs for given experimental Animals.

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EXPERIMENT # 6
Study of Animal ethics for pre-clinical testing
Objective: Study of animal ethics for pre-clinical testing and good laboratory practice has
following objectives.
 To realize the importance of using animals for pre-clinical testing.
 To justify the need for adhering to proper standards of maintenance and care in the use of
animals for research and teaching.
EXPERIMENTAL ANIMAL ETHICS:
Animal ethics is a term used in academia to describe human animal

relationships and how animal sought to be treated.
Pharmacological research encompasses a wide range of molecular,
chemical and biological techniques. The use of animals in drug
In 1997 Dr Jay Vacanti and his
discovery is an essential component of this research and is carried out team grew an ear on the back
of a mouse
as part of a broad range of studies in which alternative experimental
approaches are used where possible.
 A harm-benefit analysis in which the potential benefits of a research project are weighed
against the harms likely to be caused to animals - whether done by the competent
authority or by an ethics or animal care and use committee - forms the basis of ethical
frameworks.
 According to different laws established for animal welfare and Different ethics and animal
care and use committees around the world operated under different regulatory systems,
research institutions are required to establish an Institutional Animal Care and Use Committee
(IACUC) “to oversee and evaluate all aspects of the institution’s animal care and use program.”

Acting as “an agent of the research facility,” the IACUC’s responsibilities include:
• Reviewing the facility’s animal care and use program and ensure that animal house follow
GLP guide lines.
• Minimize the pain, stress and discomfort in experimental animal.
• Inspecting the animal labs at least twice a year.
• Reviewing and approving, disapproving, or requiring modifications to research protocols
responding to and, if necessary, investigating facility personnel reports of noncompliance
involving animal care and use.
• Minimize the animal use by peoper design to give the results under 95% of confidence
interval.
• Reporting deficiencies in animal care and use
• Submitting evaluation reports to the institution.
THE THREE RS
The three Rs are a set of principles that scientists are encouraged to
follow in order to reduce the impact of research on animals. The three
Rs are: Reduction, Refinement, and Replacement.
Reduction:
 Reducing the number of animals used in experiments by:
o Improving experimental techniques
o Improving techniques of data analysis
o Sharing information with other researchers
Refinement:
 Refining the experiment or the way the animals are cared for so as to reduce their suffering by:
o Using less invasive techniques
o Better medical care
o Better living conditions
Replacement:
 Replacing experiments on animals with alternative techniques such as:

o Experimenting on cell cultures instead of whole animals

o Using computer models
o Studying human volunteers
o Using epidemiological studies.
ADDITIONAL NOTES:

EXPERIMENT # 7
To Study Basic pharmacological experimental instruments
Objective: For basic understanding of structure, function, SOP and uses of most common
laboratory instruments.
ORGAN BATH (isolated organ bath)

Isolated tissue and organ preparations allow researchers to investigate the physiology and
pharmacology of various tissue samples in a controlled environment without the complications of
an intact animal model. These in vitro experiments can be performed using a variety of tissues and
organs including smooth muscle, skeletal muscle, cardiac muscle, gastrointestinal and urogenital
tissue samples and arterial rings. Typically, these studies are performed in temperature controlled
environments with tissues perfused in an oxygenated physiological buffer solution. Typical
evoked-response protocols can be carried out using pharmacological agents and/or electrical
stimulation.
The apparatus consist of:

 An inner glass tube or organ bath containing PSS and tissue and connected to reservior
through polythene or rubber tube.
 Electrode has its own upright and clamp for mounting to one ledge of the bath giving total
freedom of placement. It has two fixed J-shaped stainless steel wires for stimulation and two
leads for connection to a stimulator.
 Areation cum tissue holder tube to hold tissue and supply O2 / air to perfusion fluid.
 An outer bath made up of glass/perspex filled with water the temperature of which is checked
with the help of thermometers & maintained at 37° for all mammalian experiments.
 A lever for recording the responses of the tissue on a kymograph drum.
 The entire assembly is mounted on tripod stand.

Applications:
The most common application of tissue organ bath is in cardiovascular research, using
isolated heart tissue (papillary muscle, left ventricle) or other muscle strips. For gastro-intestinal
research preparations of ileum and colon are often used, as well as gastric antral muscle and
sphincter. Respiratory physiology and pharmacology can be studied in isolated phrenic diaphragm
preparations, pulmonary arterial smooth muscle and even lung parenchyma. Other smooth muscle
preparations that are used in organ bath research include urinary bladder and penile muscle
strips.
RESEARCH KYMOGRAPH
It is the instrument on which physiological responses such as contraction
and relaxation of muscle are recorded. It consists of a heavy base and a
vertical shaft. Heavy base gives stability to drum. It has (1) base hoofs
(legs): with adjustable leveling screw to keep drum horizontal on the
uneven surface. (2) Side hoofs- to turn the drum on its side so that shaft
becomes horizontal. (3) Gear rods- arrangement with gear & clutch to
obtain desirable speed of drum.
Drum cylinder- is a brass or iron cylinder around which paper is
wrapped and smoked for recording of tracings.
LEVERS- It is the device by virtue of which response of isolated tissue
can be recorded and magnified.
Principle:
1. Fulcrum- the point around which the lever moves on the lever holder is the fulcrum.
2. Stylus- is the writing point which records the tracing on the smoked paper of the drum. It is
either made of celluloid parchuments paper, aluminium foil or thin photographic or x-ray film.
3. Magnification- The fulcrum (F) should be so placed that there is some magnification of the
actual contraction (response). In order to achieve this, the distance between the writing point and
the fulcrum (F) should always be greater than the distance between fulcrum and point of
attchment of tissue (T). By adjustment of these relative distances from the fulcrum any degree of
magnification can be obtained. Therefore lesser the inherent contractility of tissue higher the
magnification needed or vice-versa.
Operating Procedure-(Cardiac activity)
 A graph paper is rolled around the kymograph drum.

 Pith the frog and fix it on the discretions board.
 Expose the heart and transfer it to the myograph board.
 Apply small metallic hook at the apex of the heart.
 Tie a thread on other end of hook and connect it to the sterling heart lever.
 Set the stylus on graph paper mounted on kymograph drum.
 Start the drum and set the speed of kymograph.
 Record normal heart rhythm for one minute.
 Put 1-2 drops of the drug on the heart and record response for 1 minute.
Applications:
It was invented by German physiologist Carl Ludwig in the 1840s and found its first use as a
means to intrusively monitor blood pressure, and has found several applications in the field of
medicine. Its primary use was to measure phenomena such as changes in muscular contractions
or other physiological processes, including speech sounds.
ADDITIONAL NOTES:

EXPERIMENT # 8
To study clinical trials of new drugs in human subjects
Objective: To understand the Purpose, Importance and Phases of clinical trials and also to
familiarize with different terms related with process.
CLINICAL RESEARCH:
 Clinical research is a type of study of clinical or biomedical questions through the use of human
subjects.
 Clinical research studies do not necessarily all involve medical treatments or experimental
therapies. It can also include observational studies, in which people are followed over a period
of time to determine health outcomes.
 Clinical research may also be used to determine the usefulness or safety of a new diagnostic
procedure or drug treatment in the form of a clinical trial.
 Clinical research studies are planned in advance and follow a defined protocol. Epidemiologic
studies examine specific populations to clarify how often a disease occurs or is found in a given
population (the incidence and prevalence), the individual factors that can cause or worsen
disease progression, and the types of health and lifestyle decisions that people make.
 Clinical trials are one important type of clinical research.
CLINICAL TRIALS- (CLINICAL STUDY OF DRUG):
Clinical trials are a form of clinical research that follows a defined protocol that has been carefully
developed to evaluate a clinical question. The U.S. National Institutes of Health (NIH) defines a clinical
trial as: “A prospective biomedical or behavioral research study of human subjects that is designed
to answer specific questions about biomedical or behavioral interventions (such as drugs,
treatments, devices, or new ways of using known drugs, treatments, or devices)”

Although people commonly associate clinical trials with drug trials, in which new medications or
combinations of drugs are tested for their effectiveness against a disease, they may also evaluate
whether interventions such as counseling or lifestyle modifications have an effect on disease
progression. They may be conducted on people who have a disease or on healthy people,
depending upon the purpose of the research.
Goals of clinical trial: include assessing the safety and (relative) effectiveness of a medication
or device:
 On a specific kind of patient (e.g., patients who have been diagnosed with Alzheimer's disease)
 At a different dose (e.g., 10-mg dose instead of 5-mg dose)
 For a new indication
 Is more effective for the patient's condition than the standard therapy
 Relative to two or more already approved/common interventions for that disease (e.g., device
A vs. device B, therapy A vs. therapy B)
Preparations for conducting clinical trials:

Before a clinical trial can be carried out, thorough preparation is necessary, including extensive
reviews of the proposed trial, its methodology, and the goals of the trial. An Institutional Review
Board (IRB) consisting of physicians, statisticians, researchers, patient advocates, and others must
pre-approve every clinical trial. The job of the IRB is to ensure that the trial is ethical, legal, and
that the rights of those participating are fully protected. For example, individual participants'
names are kept secret and not included in the results or publicly available information about a
trial.
Every clinical trial has a strictly defined protocol that is approved by the IRB. A protocol describes
what types of people may participate in the trial; outlines the exact the schedule of tests,
procedures, medications, and/or dosages involved in the trial; and specifies the length of the
study. Generally, doctors check the patient thoroughly at the start of the trial, provide instructions
and directions for participation in the trial, monitor the patient during the actual trial, and remain
in contact, sometimes with further monitoring after the trial is completed.

In many clinical trials, patients will be randomly assigned to a test group or a control group.
(Randomization) The control group receives the standard and accepted treatment for a given
condition, while the test group receives the experimental medication or treatment to be evaluated.
When a trial is "double-blinded," neither the participants nor the treating doctors know if an
individual patient is receiving the standard treatment versus the experimental treatment. Double-
blinded trials offer the advantage of allowing the treating health-care team and the patient to
make unbiased observations about patient progress and the effectiveness of the treatment being
evaluated. A double-blind study is also referred to as a double-masked study. Results obtained
from a randomized, double blind clinical trials are considered the most accurate and reliable types
of results, and help those conducting the trial to draw the most accurate conclusions.
Types of clinical trials:

One way of classifying clinical trials is by the way the researchers behave.
 In an observational study, the investigators observe the subjects and measure their outcomes.
The researchers do not actively manage the study.
 In an interventional study, the investigators give the research subjects a particular medicine
or other intervention. Usually, they compare the treated subjects to subjects who receive no
treatment or standard treatment. Then the researchers measure how the subjects' health
changes.
IMPORTANT TERMS:
 Randomized: Each study subject is randomly assigned to receive either the study treatment or
a placebo.
 Blind: The subjects involved in the study do not know which study treatment they receive. If
the study is double-blind, the researchers also do not know which treatment a subject receives.
This intent is to prevent researchers from treating the two groups differently. A form of
double-blind study called a "double-dummy" design allows additional insurance against bias.
In this kind of study, all patients are given both placebo and active doses in alternating periods.

 Placebo-controlled: The use of a placebo (fake treatment) allows the researchers to isolate the
effect of the study treatment from the placebo effect.
 Informed consent is a legal process (Documentation) in which a recruit is instructed about key
facts before deciding whether to participate. Researchers explain the details of the study in
terms the subject can understand. The information is presented in the subject's native
language. Generally, children cannot autonomously provide informed consent, but depending
on their age and other factors, may be required to provide informed assent.
PHASES OF CLINICAL TRIALS

Clinical trials involving new drugs are commonly classified
into four phases. Each phase of the drug approval process
is treated as a separate clinical trial. The drug-
development process will normally proceed through all
four phases over many years. If the drug successfully
passes through Phases 0, 1, 2, and 3, it will usually be
approved by the national regulatory authority for use in
the general population. An investigational medicine is
often evaluated in tow or more phases simultaneously in
different clinical trials. Also, some clinical trials may
overlap two different phases.
Phase 0 trials: are the first-in-human trials. Single sub-

therapeutic doses of the study drug are given to a small
number of subjects (10 to 15) to gather preliminary data
on the agent's pharmacodynamics and pharmacokinetics.
Phase 1 trial: In which researchers test an experimental

drug or treatment in a small group of people (20–80) for
the first time to evaluate its safety, determine a safe dosage

range, and identify side effects.
Phase IIa trials: Pilot clinical trials to evaluate efficacy (and safety) in selected populations of
patients with the disease or condition to be treated, diagnosed, or prevented. The experimental
treatment is given to a larger group of people (100–300) Objectives may focus on dose-response,
type of patient, frequency of dosing, or numerous other characteristics of safety and efficacy.
Phase IIb: Well controlled trials to evaluate efficacy ( and safety) in patients with the disease or
condition to be treated, diagnosed, or prevented. These clinical trials usually represent the most
rigorous demonstration of a medicine's efficacy. Sometimes referred to as pivotal trials.
Phase IIIa: Trials conducted after efficacy of the medicine is demonstrated, but prior to
regulatory submission of a New Drug Application (NDA). These clinical trials are conducted in
patient populations for which the medicine is eventually intended. Phase IIIa clinical trials
generate additional data on both safety and efficacy in relatively large numbers of patients
(1,000–3,000) in both controlled and uncontrolled trials. Clinical trials are also conducted in
special groups of patients (e.g., renal failure patients), or under special conditions dictated by the
nature of the medicine and disease. These trials often provide much of the information needed for
the package insert and labeling of the medicine.
Phase IIIb: Clinical trials conducted after regulatory submission of an NDA, but prior to the
medicine's approval and launch. These trials may supplement earlier trials, complete earlier trials,
or may be directed toward new types of trials (e.g., quality of life, marketing) or Phase IV
evaluations. This is the period between submission and approval of a regulatory dossier for
marketing authorization. Phase IV: Studies or trials conducted after a medicine is marketed to
provide additional details about the medicine's efficacy or safety profile. Different formulations,
dosages, durations of treatment, medicine interactions, and other medicine comparisons may be
evaluated. New age groups, races, and other types of patients can be studied. Detection and
definition of previously unknown or inadequately quantified adverse reactions and related risk
factors are an important aspect of many Phase IV studies. If a marketed medicine is to be
evaluated for another (i.e., new) indication, then those clinical trials are considered Phase II
clinical trials. The term post- marketing surveillance is frequently used to describe clinical trials.
The term post-marketing surveillance is frequently used to describe those clinical studies in Phase
IV (i.e., the period following marketing) that are primarily observational or non-experimental in
nature, to distinguish them from well controlled Phase IV clinical trials or marketing studies.
ADDITIONAL NOTES:

AUTONOMIC NERVOUS SYSTEM
AUTONOMIC NERVOUS SYSTEM
 Part of the peripheral nervous system that largely acts independent of conscious control
(involuntarily) and consists of nerves in cardiac muscle, smooth muscle, and exocrine and
endocrine glands.
 It is responsible for maintenance functions (metabolism, cardiovascular activity, temperature
regulation, digestion) that have a reputation for being outside of conscious control.
 The other main subdivision of the peripheral nervous system, the somatic nervous system,
consists of cranial and spinal nerves that innervate skeletal muscle tissue and are more under
voluntary control.
DIVISIONS OF AUTONOMIC NERVOUS SYSTEM
Sympathetic nervous Parasympathetic
system nervous system
Deals with the response Deals with sleeping, and
to stress and danger, digesting food, in
releasing epinephrines Enteric nervous system
general, lowers
(adrenaline), in general This subsystem has nerves
metabolic rate, slows
increasing activity and around the intestines,
activity, and restores
metabolic rate. pancreas, and gall bladder.
blood pressure.
FUNCTIONS OF AUTONOMIC NERVOUS SYSTEM

Sympathetic and parasympathetic divisions typically function in opposition to each other. But this
opposition is better termed complementary in nature rather than antagonistic. For an analogy, one
may think of the sympathetic division as the accelerator and the parasympathetic division as the
brake. The sympathetic division typically functions in actions requiring quick responses. The

parasympathetic division functions with actions that do not require immediate reaction. Consider
sympathetic as "fight or flight" and parasympathetic as "rest and digest."
SYMPATHETIC NERVOUS SYSTEM PARASYMPATHETIC NERVOUS SYSTEM
 Diverts blood flow away from the gastro-  Dilates blood vessels leading to the GI tract,
intestinal (GI) tract and skin via increasing blood flow. This is important
vasoconstriction. following the consumption of food, due to
 Blood flow to skeletal muscles and the lung is the greater metabolic demands placed on
not only maintained, but enhanced (by as the body by the gut.
much as 1200 percent, in the case of skeletal  The parasympathetic nervous system can
muscles). also constrict the bronchiolar diameter
 Dilates bronchioles of the lung, which allows when the need for oxygen has diminished.
for greater alveolar oxygen exchange.  During accommodation, the
 Increases heart rate and the contractility of parasympathetic nervous system causes
cardiac cells (myocytes), thereby providing a constriction of the pupil and lens.
mechanism for the enhanced blood flow to  The parasympathetic nervous system
skeletal muscles. stimulates salivary gland secretion, and
 Dilates pupils and relaxes the lens, allowing accelerates peristalsis, so, in keeping with
more light to enter the eye. the rest and digest functions, appropriate
PNS activity mediates digestion of food and
indirectly, the absorption of nutrients.
 Is also involved in erection of genitals, via
the pelvic splanchnic nerves 2–4.
Pharmacology of neurotransmitters: at the effector organs, sympathetic ganglionic neurons

release noradrenaline (norepinephrine) to act on adrenergic receptors, with the exception of the
sweat glands and the adrenal medulla:
 At sweat glands, the neurotransmitter is acetylcholine, which acts on muscarinic receptors.

 At the adrenal cortex, there is no postsynaptic neuron. Instead, the presynaptic neuron
releases acetylcholine to act on nicotinic receptors.
 Stimulation of the adrenal medulla releases adrenaline (epinephrine) into the bloodstream,
which will act on adrenoceptors, producing a widespread increase in sympathetic activity.
In the parasympathetic system, ganglionic neurons use acetylcholine as a neurotransmitter, to
stimulate muscarinic receptors.
Sympathetic Ganglionic neurons

EFFECTOR ORGANS
(A) Releases Norepinephrine
Adrenergic Receptor
Parasympathetic Ganglionic neurons

EFFECTOR ORGANS
(B) Releases Acetylcholine
Muscuranic Receptor
NEUROTRANSMITTORS ASSOCIATED WITH AUTONOMIC NERVOUS SYSTEM
Norepinephrine Synthesis and Release: Norepinephrine (ne) is the primary

neurotransmitter for postganglionic sympathetic adrenergic nerves. it is synthesized inside the
nerve axon, stored within vesicles, and then released by the nerve when an action potential travels
down the nerve. (figure given below)

Amino acid Transported into the sympathetic nerve axon

Tyrosine
An action potential
traveling down the axon
depolarizes the membrane
Tyrosine ⟶ tyrosine hydroxylase ⟶DOPA
and causes calcium to enter
the axon.
DOPA⟶ DOPA decarboxylase ⟶DOPAMINE
DOPAMINE
DOPAMINE
⟶dopamine β
hydroxylase ⟶
NOREPINEPHRINE
Increased intracellular calcium DOPAMINE

⟶dopamine β
hydroxylase ⟶
NOREPINEPHRINE
Norepinephrine Release in synapse and binds to the postjunctional receptor and

stimulates the effector organ response.
EPINEPHRINE SYNTHESIS AND RELEASE:

 Epinephrine is synthesized from norepinephrine within the adrenal medulla, which are small
glands associated with the kidneys.

 Preganglionic fibers of the sympathetic nervous system synapse within the adrenals.
 Activation of these preganglionic fibers releases acetylcholine, which binds to postjunctional
nicotinic receptors in the tissue.
 This leads to stimulation of NE synthesis within adenomedullary cells, but unlike sympathetic
neurons, there is an additional enzyme (phenylethanolamine-N-methyltransferase) that adds a
methyl group to the NE molecule to form epinephrine. The epinephrine is released into the
blood perfusing the glands and carried throughout the body.
NOREPINEPHRINE AND EPINEPHRINE REMOVAL AND METABOLISM:

The binding of NE to its receptor depends on the
concentration of NE in the vicinity of the receptor.
If the nerve stops releasing NE, then the NE
concentration in the junctional cleft will decrease
and NE will leave the receptor. There are several
mechanisms by which the norepinephrine is
removed from the intercellular (junctional) space
and therefore from the postjunctional receptor:
1. Most (~90%) of the NE is transported back into
the nerve terminal by a neuronal reuptake transport system.
2. A small amount of NE (~5%) is taken up by the postjunctional tissue (termed "extraneuronal
uptake") and metabolized.
3. NE (and epinephrine) is metabolized by catechol-O-methytransferase (COMT) and monoamine
oxidase (MAO). The final product of these pathways is vanillylmandelic acid (VMA). This final
product, along with its precursors normetanephrine and metanephrine, is measured in urine
and plasma in the diagnosis of pheochromocytoma, which can cause severe hypertension and
cardiac arrhythmias.

ACETYLCHOLINE SYNTHESIS AND METABOLISM:
Choline Transported into the nerve axon
Pyruvate ⟶⟶Acetyl-CoA
Mitochondria within cholinergic nerves
An action potential
traveling down the axon Choline + Acetyl-CoA ⟶choline acetyltransferase ⟶
depolarizes the membrane ACETYLCHOLINE
and causes calcium to enter

the axon.
ACETYLCHOLINE
Increased intracellular calcium
ACETYLCHOLINE
After ACh is released, it is rapidly degraded within the synapse by

acetylcholineesterase, to form acetate and choline.

RECEPTORS OF SYMPATHETIC AND PARASYMPATHETIC NERVOUS SYSTEM:
Adrenergic receptors:
The two main types of adrenergic receptors are α-receptors & β-receptors. These receptors
further subclassified as α- α1, α2 and β- β1, β2, β3.
α1 & β1 mostly produces excitation & α2 & β2 mostly produces inhibition.
Location:
α1: It is presence at the post junctional on effector organs like radial and sphincter muscles of iris
(eye), heart, some BV (blood vessels), bronchial glands (lungs), liver, gut, skin, sex organs etc.
α2: It is presence on the prejunctional at the nerve ending. On the brain, pancreatic β cells, fat cells,
gut muscles, veins etc.
β1: They are located at heart, salivary glands, juxtaglomerular apparatus of kidney, posterior
pituitary.
β2: Lungs, BV, uterus, liver, eye, gut, urinary bladder, spleen, skeletal muscle, certain veins etc.
β3: Brown adipose tissue, where there function is to generate the heat by thermogenesis. Mostly
presence in to the children.
Cholinergic receptors:
 All sympathetic and parasympathetic neurons are cholinergic and also all parasympathetic
postganglionic neurons are cholinergic.
 There are two types of cholinergic receptors: muscarinic and nicotinic.
- Nicotinic receptors are presence on the dendrites or the cell bodies of postganglionic neurons of
both sympathetic & parasympathetic neurons.
- Muscuranic receptors are present on the all visceral organs. The muscarine, obtain from
mushroom, mimics the action of Ach on these receptors.
Nicotinic receptors
Depending on the location they are classified as NM & NN.
NM: They are presence on the neuromuscular junction mainly on the skeletal muscles. They cause
depolarization at the muscle end plate which leads to contraction of muscle. They are pentameric

having 2α, β, δ and γ or ε subunits and agonist by nicotine and PTMA and antagonist by
tubocurarine.
NN: These are present on autonomic ganglia, adrenal mdulla and CNS. At autonomic ganglia it
causes depolarization of postsynaptic neurons and propogate impulses through it. In the adrenal
medulla releases adr & NA by same mechanism. And at the CNS causes excitation & inhibition
depending up on the neuronal chemical. Nicotine and di methyl phenyl piprizinium are agonist
and hexamethonium is antagonist to them.
Muscuranic receptors
They are subdivided in to M1, M2, M3, M4, and M5.
- M1 : It is presence on the autonomic ganglia, on the gastric gland and at the certain part of the
brain like hippocampus from limbic system and at the corpous straitum. It has role in gastric
secretion. And histamine release. It acts through Gq protein and activates phospholipase C (PLc)
which generate DAG & IP3 as 2 messenger. Some time they also activate PL-A2.
- M2: they are act through Gi protein hich inhibits all the functional activities. Located on the heart
(SA node, AV node, atria, ventricle), on the cholinergic nerve ending and visceral smooth muscle.
They inhibit AC resulting in hyperpolarisation of the neurons and decrease activity of SA node &
conduction through AV node leads to bradycardia.
- M3: it is located on the visceral smooth muscle, iris, ciliary muscle and exocrine glands. They are
also GPCRs acts by Gq protein. Their activity is dominated in smooth muscle the\an M2.
- M4: not abundant in body. They transmit neurotransmitter in certain areas of brain and acts
through Gi protein.
- M5: it acts by Gq protein. Derifinacin is selective antagonist & related to dopamine release.

Effects of sympathetic and parasympathetic stimulation on effector organs
Sympathetic Parasympathetic
Target systems/organs
(adrenergic) (muscarinic)
CIRCULATORY SYSTEM
cardiac output Increases M2: decreases

SA node: heart rate (chronotropic) β1, β2: increases M2: decreases
cardiac muscle: contractility
β1, β2: increases M2: decreases
(inotropic)
conduction at AV node β1: increases M2: decreases
vascular smooth muscle α: contracts; β2: relaxes M3: contracts;
Platelets α2: aggregates ---
renal artery Constricts ---
hepatic artery Dilates ---
mast cells – histamine β2: inhibits ---
RESPIRATORY SYSTEM
β2: relaxes (major

Smooth muscles of bronchioles contribution); α1: contracts M3: contracts
(minor contribution)
NERVOUS SYSTEM
pupil of eye α1: relaxes M3: contracts

ciliary muscle β2: relaxes M3: contracts
DIGESTIVE SYSTEM
β: stimulates viscous, amylase

stimulates watery
salivary glands: secretions secretions; α1 = stimulates
secretions
potassium cation
lacrimal glands (tears) Decreases M3: increases

Effects of sympathetic and parasympathetic stimulation on effector organs

kidney (renin) secretes ---
parietal cells --- M1: secretion
α1, β2: glycogenolysis,
Liver ---
gluconeogenesis
adipose cells β3: stimulates lipolysis ---
GI tract motility Decreases M1, M3: increases
smooth muscles of GI tract α, β2: relaxes M3: contracts
sphincters of GI tract α1: contracts M3: relaxes
glands of GI tract Inhibits M3: secretes
ENDOCRINE SYSTEM
α2: decreases secretion from increases stimulation

pancreas (islets) beta cells, increases secretion from alpha cells and
from alpha cells beta cells
adrenal medulla N: secretes epinephrine ---
URINARY SYSTEM
bladder wall β2: relaxes Contracts

Urethra α1: contracts Relaxes
Sphincter α1: contracts; β2 relaxes Relaxes
REPRODUCTIVE SYSTEM
Uterus α1: contracts; β2: relaxes ---

Genitalia α: contracts M3: erection
OTHERS
M: stimulates (major
sweat gland secretions contribution); α1: stimulates ---
(minor contribution)

Evaluation of drugs acting on autonomic nervous system on

Frog’s heart
The intrinsic properties of the heart are influenced by autonomic nervous system. The
sympathetic discharge is stimulatory while the parasympathetic discharge inhibits the cardiac
contractility. Various drugs acting on specific receptors present in the heart also influence the
cardiac activity. e.g. acetylcholine acts on the muscarinic receptors present in the heart and causes
the decrease in the heart rate and cardiac contractility. Atropine also acts on muscarinic receptors
and is a competitive inhibitor for the acetylcholine. Therefore atropine increases heart rate by
antagonizing the effect of acetylcholine. adrenalin acts on alpha and beta-adrenergic receptors. It
has positive chronotropic (increased heart rate) and positive inotropic (increased force of
contraction)effects. Propranolol is a non-selective beta adrenegic-blocking agent. It acts on beta 1
and beta 2 receptors and inhibits the effects of catecholamines..

EXPERIMENT # 9
Evaluation of Sympathomimitic (Epinephrine) on Frog’s Heart.
Objective: Study the effects of autonomic drug Epinephrine on experimental frog and observe
the pattern of cardiac activity (ionotropic and chronotropic effect) of sympathomimetic drug.
Task: Calculate the following heart rates:

Normal heart rate, heart rate after epinephrine and highest heart rate reached with epinephrine.
Apparatus:
 Frog's dissection instruments
 Normal saline solution
 Adrenalin (concentration= 1:100,000 solution) at a dose of 0.05 ml
Procedure:
 Preparation of frog's heart: A big sized frog is taken. It is pithed and is placed on a frog board
on its dorsal surface. A mid line incision is given and the skin over the anterior abdominal wall
is removed. Heart is exposed by cutting the bones of the girdles with the help of a bone cutter.
 A pin hook is passed through the apex of ventricle and connected to a lever, which writes on a
smoked drum. Adjust the tension transducer, Setup the apparatus for recording the normal
myocardiogram.
 First of all a column of normal tracing is recorded for at least two min. Stop recording and pour
few drops of epinephrine on the frog’s heart and observe the effect on force of contraction and
heart rate. After pouring drug wait, till the heart returns to normal and record a column of
normal tracing before and after testing drug.
 Calculate the heart rate and amplitude of contraction in all recordings and compare with
normal graph.

Observations:
Drug Effect of Drug Inference
Rate Force of Contraction
Normal Saline
Adrenaline (0.05ml)
 Normal heart rate of experimental frog before drug (epinephrine) is ______________beats/minute.

 Heart rate of experimental frog after few drops of epinephrine is__________________beats/minute.
 Highest heart rate reached with epinephrine is_____________________________ beats/minute.
 Percent change in heart rate after epinephrine is________________________%.
Result:__________________________________________________________
________________________________________________________________
________________________________________________________________
Discussion: ______________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

EXPERIMENT # 10
Evaluation of Sympatholytic (Propranolol) on Frog’s Heart
Objective: Study the effects of autonomic drug Propranolol on experimental frog and observe
the pattern of cardiac activity (ionotropic and chronotropic effect) of sympatholytic drug.

Normal heart rate, heart rate after Propranolol and Lowest heart rate reached with propranolol.
Apparatus:
 Propranolol (0.05ml) (1microgm/ml)
Procedure:
myocardiogram.
few drops of Propranolol on the frog’s heart and observe the effect on force of contraction and
normal graph.

Observations:
Normal Saline
Propranolol (0.05ml)
 Normal heart rate of experimental frog before drug (Propranolol) is ______________beats/minute.

 Heart rate of experimental frog after few drops of Propranolol is __________________beats/minute.
 Highest heart rate reached with Propranolol is _____________________________ beats/minute.
 Percent change in heart rate after Propranolol is________________________%.
Result:__________________________________________________________
________________________________________________________________
________________________________________________________________
Discussion: ______________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

EXPERIMENT # 11
Evaluation of Prasympathomimitic (Acetylcholine) on Frog’s
Heart.
Objective: Study the effects of autonomic drug Acetylcholine on experimental frog and observe
the pattern of cardiac activity (ionotropic and chronotropic effect) of Parasympathomimetic drug.

Normal heart rate, heart rate after Acetylcholine and Lowest heart rate reached with
Acetylcholine.
Apparatus:
 Acetylcholine (concentration= 1:100,000 solution) at a dose of 0.05 ml
Procedure:
myocardiogram.
few drops of Acetylcholine on the frog’s heart and observe the effect on force of contraction
and heart rate. After pouring drug wait, till the heart returns to normal and record a column of
normal tracing before and after testing drug. Calculate the heart rate and amplitude of
contraction in all recordings and compare with normal graph.

Observations:
Normal Saline
Acetylcholine (0.05ml)
 Normal heart rate of experimental frog before drug (Acetylcholine) is_____________beats/minute.

 Heart rate of experimental frog after few drops of Acetylcholine is_________________beats/minute.
 Highest heart rate reached with Acetylcholine is_____________________________ beats/minute.
 Percent change in heart rate after Acetylcholine is________________________%.
Result:__________________________________________________________
________________________________________________________________
________________________________________________________________
Discussion: ______________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

EXPERIMENT # 12
Evaluation of Parasympatholytic (Atropine) on Frog’s Heart.
Objective: Study the effects of autonomic drug Atropine on experimental frog and observe the
pattern of cardiac activity (ionotropic and chronotropic effect) of parasympatholytic drug.

Normal heart rate, heart rate after Atropine and highest heart rate reached with Atropine.
Apparatus:
 Atropine (concentration= 1:100,000 solution) at a dose of 0.05 ml
Procedure:
myocardiogram.
few drops of Atropine on the frog’s heart and observe the effect on force of contraction and
normal graph.

Observations:
Normal Saline
Atropine (0.05ml)
 Normal heart rate of experimental frog before drug (Atropine) is ______________beats/minute.

 Heart rate of experimental frog after few drops of Atropine is__________________beats/minute.
 Highest heart rate reached with Atropine is_____________________________ beats/minute.
 Percent change in heart rate after Atropine is________________________%.
Result:__________________________________________________________
________________________________________________________________
________________________________________________________________
Discussion: ______________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

MYDRIATIC & MIOTIC EFFECT OF DRUGS ON RABBIT'S EYE

The eye is supplied both by sympathetic & parasympathetic nerves.
The iris has two types of muscles
1. Dilator pupillae (radial muscle)
2. Sphincter pupillae (circular muscle)
Circular muscle has parasympathetic supply while radial muscle has sympathetic supply. The
cilliary muscle is also supplied by parasympathetic nerves & when it contracts, the cilliary body
move inwards & forwards. Because of this the lens bulges forwards & eye is accommodated for
near vision. The opposite effect is produced by the relaxation of cilliary muscles resulting in
paralysis of accommodation (cycloplegia). When the pupil dilates the iris folds back near the
opening of the canal of schlemn & the drainage of aquous humour is decreased thereby increasing
intraocular pressure and constriction of pupil by opposite action that will increase drainage &
reduce intra ocular tension. Topically applied drugs can affect the eye by changing conjunctival
congestion, pupillary size, corneal sensitivity & intra ocular pressure. However the effect of drugs
on Pupillary size, Light Reflex (L.R.) & Corneal Reflex (C.R) can be studied easily.
1. Pupillary size- Can be measured by placing a transparent plastic scale in front of the eye, as
close as possible.
2. LR- Elicited by directing the light of a torch towards the pupil from back. The positive response
shows constriction of pupil.
3. Corneal Reflex- Sensitivity of cornea is tested by touching cornea gently with a fine cotton
wick/hair aesthesiometer from the side & not from the front of the eye.Positive response show
blinking of the eyelids.
Physostigmine is a reversible cholenestrase inhibitor which increases the endogenous

acetylcholine, this inturn stimulates the circular muscle of iris to produce pupillary constriction
without producing loss of light reflex & corneal reflex.
Ephedrine & Phenylephrine- Stimulate the radial muscle of the iris, this produces dilatation of
pupil without any effect on light reflex & corneal reflex.

Atropine- An antimuscarinic agent, block the effect of endogenously released ACh in the circular
muscle of the iris. This also causes stimulation of ciliary muscle. This produces mydriasis & spasm
of accommodation leading to cycloplegia but without producing loss of corneal reflex.
Pilocarpine- Is a parasympathomimetic drug. It acts directly. It causes contraction of circular
muscle of the iris & leads to decrease in size of pupil (constriction of pupil).
Ciliary muscle is present in the eye mainly supplied by parasympathetic nerves & weak
sympathetic supply. This is responsible for accommodation. Anticholinergic drugs eg. atropine
causes paralysis of ciliary muscle, tightening of suspensory ligaments, the lens is flattened & focal
length increases. Eye is fixed for far vision known as 'paralysis of accomodation' or cycloplegia.
Clinical Importance:
1. Miotic effect of physostigmine is useful in glaucoma as drainage of aquous humor is facilitated
resulting into decrease in I.O.P.
2. Short acting mydriatic effect of phenylepherine is used in fundoscopy, where topical
anticholinergic (mydiatrics) are avoided.
3. Topical effect of xylocaine is used to produce surface anaesthesia for minor local surgery eg.
Foreign body removal, symptomatic relief of corneal/ conjunctival irritation.
4. Atropine is used for fundoscopy & to provide rest to the iris, e.g. in iritis
5. To counteract the mydriatic & cycloplegic effect miotics are used.
6. Miotic effect of phystostigmine is used in glaucoma as the drainage of aquous humor is
facilitated & decrease in I.O.P occurs.
7. Phenylepherine is short acting and not causing cycloplegia. Hence, often preferred over
atropine and its substitutes especially in elderly.

EXPERIMENT # 13
Evaluation of Mydriatic effect of Atropine on Rabbit’s Eye
Objective: Study the effects of autonomic drug Atropine on Rabbit’s eye and observe the
phenomenon of mydriasis. (Dilation of Pupil)
Experimental Animal: Rabbit - (2.5 Kg in wt.)

Experimental Requirement; - Torch, Scale (6”small), Cotton, Scissors, Dropper, Rabbit holder
(Restrainer).
Drugs: Normal saline 0.9%
Atropine 0.5-1%
Procedure:
 Place a rabbit in the rabbit holder (Rabbit Restrainer). Keeping its head outside.
 Observe size of the pupil, LR (Light Reflex) & CR (corneal reflex) in both the eyes [examine the
LR by holding the torch in front of the eye moving light beam to & fro (from back to front),
examine the corneal reflex by touching side of the cornea with cotton wick.
 One eye of the rabbit is used (RE) as 'control' for the experiment.
 Instill few drops of normal saline in right eye & atropine (Drug) in left eye (test eye).
Record the pupillary size, LR & CR after 10 mins of drug instillation & tabulate the observation.
Observations:
Drug Size of Pupil (mm) Light Reflex Corneal Reflex Inference
RE LE RE LE RE LE
B A B A B A B A B A B A
Atropine 1%
B – Before Drug Instillation - P- Present

A – After Drug Instillation - Ab- Absent

Result:__________________________________________________________
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Discussion:
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EXPERIMENT # 14
Evaluation of Mydriatic effect of Phenylephrine on Rabbit’s Eye
Objective: Study the effects of autonomic drug Phenylephrine on Rabbit’s eye and observe the
phenomenon of mydriasis. (Dilation of Pupil)

(Restrainer)
Drugs : Normal saline 0.9%
Phenylepherine 5%
Procedure:
 Instill few drops of normal saline in right eye & Phenylephrine (Drug) in left eye (test eye).
 Record the pupillary size, LR & CR after 10 mins of drug instillation & tabulate the observation.
Observations:
RE LE RE LE RE LE
Phenylephrine B A B A B A B A B A B A
5%


Result:__________________________________________________________
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Discussion:
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EXPERIMENT # 15
Evaluation of Miotic effect of Pilocarpine on Rabbit’s Eye
Objective: Study the effects of autonomic drug Pilocarpine on Rabbit’s eye and observe the
phenomenon of miosis. (Constriction of pupil)

(Restrainer)
Pilocarpine 1-2%
Procedure:
 Instill few drops of normal saline in right eye & Pilocarpine (Drug) in left eye (test eye).
Observations:
RE LE RE LE RE LE
pilocarpine B A B A B A B A B A B A
1-2%


Result:__________________________________________________________
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Discussion:
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EXPERIMENT # 16
Evaluation of Miotic effect of Physostigmine on Rabbit’s Eye
Objective: Study the effects of autonomic drug Physostigmine on Rabbit’s eye and observe the
phenomenon of miosis. (Constriction of pupil)

(Restrainer)
Physostigmine 0.5%
Procedure:
 Instill few drops of normal saline in right eye & Physostigmine (Drug) in left eye (test eye).
Observations:
RE LE RE LE RE LE
Physostigmine
B A B A B A B A B A B A
0.5%


Result:__________________________________________________________
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Discussion:
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COGULATION STUDIES
EXPERIMENT # 17
To study the effect of NSAIDs on bleeding time by Duke Method
Objective: To study the effect of Nonsteroidal anti-inflammatory drugs on coagulation by means
of increase or decrease bleeding time in experimental rabbit by Duke Method.
Background Study:
Nonsteroidal anti-inflammatory drugs, (NSAIDs): Is a class of drugs that
provides analgesic and antipyretic effects, and, in higher doses, anti-inflammatory effects.
The most prominent members of this group of drugs, aspirin, ibuprofen and naproxen, are all
available over the counter in most countries. NSAIDs inhibit the activity of both cyclooxygenase-1
(COX-1) and cyclooxygenase-2 (COX-2), and thereby, the synthesis of prostaglandins and
thromboxanes. It is thought that inhibiting COX-2 leads to the anti-inflammatory, analgesic and
antipyretic effects and that those NSAIDs also inhibiting COX-1, may cause gastrointestinal
bleeding and ulcers. For this reason, the advantages of COX-2 selective inhibitors may be
indicated.
Mechanism of Action:
Most NSAIDs act as nonselective inhibitors of the enzyme cyclooxygenase, inhibiting both
the cyclooxygenase-1 (COX-1) & cyclooxygenase-2(COX-2) isoenzymes. This inhibition is
competitively, as opposed to the mechanism of aspirin, which is irreversible inhibition. COX
catalyzes the formation of prostaglandins and thromboxane from arachidonic acid (itself derived
from the cellular phospholipid bilayer by phospholipase A2).

Prostaglandins act (among other things) as

messenger molecules in the process
of inflammation. COX-1 is a constitutively
expressed enzyme with a "house-keeping"
role in regulating many normal
physiological processes. One of these is in
the stomach lining, where prostaglandins
serve a protective role, preventing the
stomach mucosa from being eroded by its
own acid. COX-2 is an enzyme expressed in
inflammation, and it is inhibition of COX-2 that produces the desirable effects of NSAIDs. When
nonselective COX-1/COX-2 inhibitors (such as aspirin, ibuprofen, and naproxen) lower stomach
prostaglandin levels, ulcers of the stomach or duodenum internal bleeding can result.
Aspirin as Anti platelet Drug:

Aspirin induces a permanent functional defect in platelets, which can be detected clinically as a
prolonged bleeding time. This appears to be primarily, if not exclusively, due to irreversible
inactivation of a key enzyme in platelet arachidonate metabolism through acetylation of a critical
serine residue near its catalytic site. This enzyme, cyclooxygenase (COX)-1, is responsible for the
formation of prostaglandin (PG)H2, the precursor of thromboxane (TX)A2.

Experimental Requirement - Sterile lancet, cotton, rectified spirit, filter paper and stop watch.
Aspirin (75mg /70 kg)
Procedure (Duke Method):

 Weigh the animal and calculate the dose of test drug as per weight.
 Mark the experimental animals to identify control and test groups.

 Administer the test drug and equal volume of normal saline to their respective animals orally.
 After 60 minutes of administration restrain the animal and carefully clip an area of skin where
there are comparatively few hairs. Wash the area with soap and water and dry it thoroughly.
 Using a sterile disposable lancet. Swiftly make two small puncture. Wounds in the skin a short
distance apart (e.g. 2cm) avoiding any major blood vessels start the stopwatch.
 Note the time (start the stop-watch) when bleeding starts.

 At half time intervals gently touch the drop of blood on each wound with a piece of Whatman 1
filter paper. Take care not to touch the edge of the wound because this may dislodge the
platelet plug. This is repeated every 15 seconds; till bleeding stops. I.e. when no spot of blood
appears on the filter paper.

 Note the time (stop the stop-watch). It is seen that the blood stains on the filter paper get
smaller to disappear finally when bleeding stops.
Bleeding time is the interval between the moment when bleeding starts and the moment when
bleeding stops. Normal bleeding time (Duke’s method) is 4 minutes.
Dose Calculation for Experimental animal:

Weight of Experimental animal __________________________
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Observations:
Drug Bleeding Time(Minutes)
Control Normal saline (0.9%)
Test Drug (Aspirin)
Result:___________________________________________________________
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Discussion: ______________________________________________________
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EXPERIMENT # 18
To study the effect of NSAIDs on Whole blood coagulation time
(Clotting Time) by Capillary tube method (Wright’s method)

of change in clotting time in experimental rabbit by Capillary tube method.

Experimental Requirement –
1. Sterile disposable pricking needle or lancet.
2. Stop watch
3. Dry glass capillary tube (narrow diameter 1 top 2 mm, minimum 10 cm long.)
4. Cotton Swab of absorbent cotton.
5. Spirit wetted, cotton swab.
6. 70 % v/v ethyl alcohol
Procedure (Capillary Tube Method):

 After 60 minutes of administration, under sterile precautions make a sufficiently deep prick in
the ear vein of animal. Note the time when bleeding starts (start the stop watch).
 Touch the blood drop at the ear using one end of the dry microhaematocrit capillary tube kept
tilted downwards. The tube gets easily filled by capillary action.
 The tubes should be previously warmed to 37ºC and obviously should not contain
anticoagulant.
 Plug the lower end of each tube and place it in a water bath or beaker of water at 37ºC.
 After about two minute with 15 second interval starts break off a small length e.g. 5mm from
the top of each tube. As this is done, note whether the blood inside separates cleanly.
 As soon as a strand of fibrin is seen to be drawn out between the two broken parts of the tubes
as they are separated, record the time.
 The presence of fibrin indicates that the blood has clotted and the time taken is the whole
blood coagulation time.
Clotting time is the interval between the moment when bleeding starts and the moment when
the fibrin thread is first seen. Normal value is 3to 10 minutes.

Dose of Aspirin 75mg/70kg
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Observations:
Drug Clotting Time(Minutes)
Test Drug (Aspirin)
Result:__________________________________________________________
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Discussion:
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EXPERIMENT # 19
To study the effect of NSAIDs on Whole blood coagulation
time (Clotting Time) by Modified Lee and White method
of change in clotting time in experimental rabbit by Capillary tube method.

1. Stop watch
2. Syringes.
3. Cotton Swab of absorbent cotton.
4. Spirit wetted, cotton swab.
5. 70 % v/v ethyl alcohol
6. Test tubes.
Procedure (Capillary Tube Method):

 After 60 minutes of administration, carefully withdraw at least 1 ml of blood from a vein into a
dry plastic syringe.
 Start the stop clock (or stopwatch) as soon as the blood enters the syringe.
 Immediately remove the needle from the syringe and discharge blood into clean dry glass
tubes (75 mm x 10 mm) which have been previously warmed to 37ºC.
 Immediately replace the tube in the water bath at 37ºC.

 After every one minute from the time the clock was started, take tube and gently tilt it as if to
pour out the blood.
 As soon as one can be tilted past the horizontal without blood being, spilt, record the time.
The time recorded is the whole blood coagulation time.
 Normal value is2 to 7 minutes

Dose of Aspirin 75mg/70kg
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Observations:
Drug Clotting Time(Minutes)
Test Drug (Aspirin)
Result:__________________________________________________________
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Discussion:
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EXPERIMENT # 20
Evaluation of Anticoagulant effect of Warfarin and Heparin
Objective: To study the Anticoagulant effect of common anticoagulant drugs Heparin and
warfarin by means of change in CT and BT in experimental rabbit.
Background:
COAGULATION:
Coagulation (clotting) is the process by which blood changes from a liquid to a gel. It potentially
results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The
mechanism of coagulation involves activation, adhesion, and aggregation of platelets along with
deposition and maturation of fibrin.
Process of Coagulation: Coagulation begins almost instantly after an injury to the blood vessel
has damaged the endothelium lining the vessel.
Exposure of blood to the space under the endothelium initiates two processes:
 Primary hemostasis: Changes in platelets, and the exposure of subendothilial tissue factor to
plasma Factor VII, which ultimately leads to fibrin formation. Platelets immediately form a
plug at the site of injury.
 Secondary hemostasis: occurs simultaneously: Additional coagulation factors or clotting
factors beyond Factor VII, respond in a complex cascade to form fibrin strands, which
strengthen the platelet plug.
Ant platelet and Anticoagulant drugs:

 Anticoagulants and anti-platelet agents are amongst the most commonly used medications.
 Anti-platelet agents include aspirin, dipyridamole, ticlopidine and clopidogrel.
 Among anticoagulants, warfarin and heparin are the most commonly used. Warfarin affects the
vitamin K-dependent clotting factors (II, VII, IX,X) and protein C and protein S, whereas
heparin and related compounds increase the action of antithrombin on thrombin and factor Xa.

HEPARIN, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, It

can also be used to form an inner anticoagulant surface on various experimental and medical
devices such as test tubes and renal dialysis machines.
Heparin is usually stored within the secretory granules of mast cells and released only into
the vasculature at sites of tissue injury Heparin is a naturally occurring anticoagulant produced
by basophils and mast cells. Heparin acts as an anticoagulant, preventing the formation of clots
and extension of existing clots within the blood. While heparin does not break down clots that
have already, it allows the body's natural clot lysis mechanisms to work normally to break down
clots that have formed. Heparin is generally used for anticoagulation for the following conditions:
Acute coronary syndrome, Atrial fibrillation, Deep-vein thrombosis and pulmonary embolism,
Cardiopulmonary bypass for heart surgery, Hemofiltration etc.
HEPARIN
MECHANISM OF ACTION
Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that
result in its activation through an increase in the flexibility of its reactive site loop. The activated
AT then inactivates thrombin and other proteases involved in blood clotting, most notably factor
Xa. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the
binding of heparin.

WARFARIN (also known by the brand name Coumadin) is an anticoagulant normally used in
the prevention of thrombosis and thromboembolism, the formation of blood clots in the
bloodvessels and their migration elsewhere in the body respectively.
 Warfarin inhibits the vitamin K-dependent synthesis of biologically active forms of
the calcium-dependent clotting factors II, VII, IX and X.
 The precursors of these factors require carboxylation of their glutamic acid residues to allow
the coagulation factors to bind to phospholipid surfaces inside blood vessels, on the
vascular endothelium.
 The enzyme that carries out the carboxylation of glutamic acid is gamma-glutamyl carboxylase.
The carboxylation reaction will proceed only if the carboxylase enzyme is able to convert
a reduced form of vitamin K (vitamin K hydroquinone) to vitamin K epoxide at the same time.
WARFARIN
⤬⤬⤬
WARFARIN
Mechanism of Action
 The vitamin K epoxide is in turn recycled back to vitamin K and vitamin K hydroquinone by
another enzyme, the vitamin K epoxide reductase (VKOR).
 Warfarin inhibits epoxide reductase, thereby diminishing available vitamin K and vitamin K
hydroquinone in the tissues, which inhibits the carboxylation activity of the glutamyl
carboxylase. When this occurs, the coagulation factors are no longer carboxylated at
certain glutamic acid residues, and are incapable of binding to the endothelial surface of blood
vessels, and are thus biologically inactive.
 As the body's stores of previously produced active factors degrade (over several days) and are
replaced by inactive factors, the anticoagulation effect becomes apparent.
Some common Pathological terms:

Thrombosis is the pathological development of blood clots. These clots may break free and become
mobile, forming an embolus or grow to such a size that occludes the vessel in which it developed.
An embolism is said to occur when the thrombus (blood clot) becomes a mobile embolus and
migrates to another part of the body, interfering with blood circulation and hence impairing organ
function downstream of the occlusion. This causes ischemia and often leads to
ischemic necrosis of tissue.

FOR CLOTTING TIME MEASURMENT: Stop watch, Syringes, Cotton Swab of absorbent cotton,
Spirit wetted, cotton swab, 70 % v/v ethyl alcohol, Test tubes and Dry glass capillary tube (narrow
diameter 1 top 2 mm, minimum 10 cm long.)
FOR BLEEDING TIME EVALUATION: Sterile lancet, cotton, rectified spirit, filter paper and stop
watch.
Sodium Warfarin -0.5mg/kg
Heparin-70 Units/kg
Procedure:
 Mark the experimental animals to identify control and test groups of both drugs.

 After 60 minutes of administration, perform the procedure for both BT (Duke method) and CT
(capillary tube method) evaluation as described previous.
Bleeding time and clotting time are not the same. BT depends on the integrity of platelets and
vessel walls, whereas CT depends on the availability of coagulation factors.
Marking of animal Weight of Animal (kg) Drug to be given Dose of drug
Warfarin
Heparin
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Observations:
Drug Bleeding Time(Minutes) Clotting Time(Minutes)
Control Normal saline
Test Drug (Heparin)
Test Drug (Warfarin)
Result:__________________________________________________________
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Discussion:
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ANTIINFLAMATORY & ANALGESIC STUDIES
EXPERIMENT # 21
Study of Anti-inflammatory effect of NSAIDs on
Experimental animal
Objective: NSAIDs are classic anti inflammatory agents and thus the aim of this experiment is to
observe the induction of inflammation in the form of paw edema in experimental animal and thus
evaluation of anti-inflammatory effect of indomethacin.
Background:
Inflammation:
• Response of immune system to foreign organism or antigenic substance
• Characterized by edema, erythema, swelling, and pain or tenderness.
Phases of inflammation:
Acute: vasodilation & increase capillary permeability Delayed: infiltration of
leukocytes and phagocyte cells Chronic proliferative: tissue degeneration and
fibrosis.
Process of Inflammation:
 The process of acute inflammation is initiated by cells already present in all tissues (mainly
resident macrophages and mastocytes).
 These cells present on their surfaces certain receptors named pattern recognition
receptors(PRRs), which recognize molecules that are broadly shared by pathogens but
distinguishable from host molecules, collectively referred to as pathogen-associated molecular
patterns (PAMPs).

 At the onset of an infection, burn, or other injuries, these cells undergo activation (one of their
PRRs recognizes a PAMP) and release inflammatory mediators responsible for the clinical
signs of inflammation.
 Vasodilation and its resulting increased blood flow causes the redness (rubor) and increased
heat (calor).
 Increased permeability of the blood vessels results in an exudation (leakage)
of plasma proteins and fluid into the tissue (edema), which manifests itself as swelling (tumor).
 Some of the released mediators such as bradykinin increase the sensitivity to pain
(hyperalgesia, dolor). The mediator molecules also alter the blood vessels to permit the
migration of leukocytes mainly neutrophils, outside of the blood vessels (extravasation) into
the tissue.

The neutrophils migrate along a chemotactic gradient created by the local cells to reach the
site of injury.
 The loss of function (functio laesa) is probably the result of a neurological reflex in response to
pain.
 In addition to cell-derived mediators, several a-cellular biochemical cascade systems
consisting of preformed plasma proteins act in parallel to initiate and propagate the
inflammatory response.
 These include the complement system activated by bacteria and the
coagulation and fibrinolysis systems activated by necrosis, e.g. a burn or a trauma.
 The acute inflammatory response requires constant stimulation to be sustained.
 Inflammatory mediators have short half lives and are quickly degraded in the tissue. Hence,
acute inflammation ceases once the stimulus has been removed.
Mediators of inflammation:
 Bradykinin
 leukocytes
 Lysosome granules
 Histamine
 T-cells, NK cells

 Leukotriene
 Intraleukins
 Prostaglandins
Anti-inflammatory Drugs:
• Steroidal: example: Corticosteroids
• Non-steroidal Anti-inflammatory Drugs (NSAID) Examples: ASA, indomethacin, ibuprofen.
INDOMETHACIN or indometacin is a non-steroidal anti-inflammatory drug (NSAID) commonly

used as a prescription medication to reduce fever, pain, stiffness, and swelling. It works by
inhibiting the production of prostaglandins, molecules known to cause these symptoms.
Experimental Animal: Rat/Mice/Rabbit.

Requirements: Vernier calipers. Syringes, oral feeding tube
Drugs and Solution: Carragenin (2%, 0.1 ml SC.)
Indomethacin 10 mg/kg
Normal saline (0.9%)
Procedure:
 Weigh the animal and calculate the dose of test drug (Indomethacin) as per weight.
 Experimental Mice were fasted for 12 h before the experiment with free access to water.
 50 µlit 2.0% carrageenan suspension in saline was injected into the plantar side of right hind
paws of the mice.
 While Indomethacin at dose of 10mg/kg was administered orally 30 min. before the
carrageenan injection in sub plantar region of mouse paws of test group and equal volume of
saline for control group.
 The paw thickness was measured before injecting the carrageenan and after 60, 120, 180, 240
min. using vernier caliper.
 The anti-inflammatory activity was calculated as percentage inhibition of oedema in the
animals treated with indomethacin in comparison to the carrageenan control group.
 The percentage (%) inhibition of edema is calculated using the formula:
Where T t is the thickness of paw of mouse given indomethacin T o is

the paw thickness of mouse of control group at the same time.
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Observations:
Change in Paw thickness (mm)

Groups
0 min 60 min 120 min 180min 240min
Control (Normal saline)
Test (Indomethacin)
Diclofenac (5mg/kg, i.p.).
Ibuprofen 20mg/kg (p.o.)
Aspirin (300mg/kg.p.o.)
Calculations:
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Result:__________________________________________________________
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Discussion: ______________________________________________________
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EXPERIMENT # 22
Study of Analgesic effect of NSAIDs by Chemical method
Objective: NSAIDs are potent analgesic agents and thus the aim of this experiment is to evaluate
the analgesic effect of analgesic effect of NSAIDs by chemical method. (Acetic acid induced
abdominal constrictions)
Experimental Animals: 2 Mice (if female)

Requirements: Syringe.
Drug & Solutions: Acetyl salicylic acid 1mg/ml
Acetic acid 0.6%
Normal Saline (0.9%)
Procedure:
 Observe the response of mice to application of a clamp near the base of the tail, while holding
the tip of the tail, place the mouse on the table and observe for biting behavior. Select the mice
which show a good pain response and discard those mice which do not exhibit an aggressive
biting response to clamp.
 Weigh 2 mice (in gm) and mark them. Pre treat one mouse with Aspirin 6 mg/Kg sc and the
other with saline same volume sc. Note time of injection. 30 minutes after injection of
drug/saline, inject 0.3ml of 0.6% acetic acid (I.P) to each mouse.
 Place the mouse on the top of a stool for better observation.
 Observe the no. of abdominal constrictions (stretching syndrome) occurring in the next 15
minutes.
 Record each observation and then total.
 One abdominal constriction is taken as the complete movement from side to side (both sides).
Observe only one mouse at a time and note the position of the tail in the mouse treated with
Aspirin.

 Number of abdominal constrictions to be noted for 15 minutes from the time of administration
of acetic acid.
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Observations:
Groups Number of Abdominal Contractions
Control (Normal saline)
Test (Aspirin)
Result:___________________________________________________________
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Discussion: ______________________________________________________
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Diuretics
EXPERIMENT # 23
Evaluation of the diuretic activity of the Loop Diuretic
(Furosamide) in mice
Objective: The aim of this experiment is to observe or evaluate the diuretic potential of the
common diuretic drugs like loop diuretic or Thiazide diuretic in mice after acute administration.
Background:
DIURTICS:
A diuretic is any substance that promotes the production of urine. This includes forced diuresis.
There are several categories of diuretics. All diuretics increase the excretion of water from bodies,
although each class does so in a distinct way.
Diuretics are used to treat heart failure, liver cirrhosis, hypertension, water poisoning, and
certain kidney diseases.
CLASSIFICATION:
Loop diuretics, such asfurosemide, inhibit the body's ability to reabsorb sodium at the
ascending loop in the nephron, which leads to an excretion of water in the urine, whereas water
normally follows sodium back into the extracellular fluid. Other examples of high ceiling loop
diuretics include ethacrynic acid and torsemide.
Thiazide-type diuretics such as hydrochlorothiazide act on the distal convoluted tubule and
inhibit the sodium-chloride symporter leading to retention of water in the urine, as water
normally follows penetrating solutes. Frequent urination is due to the increased loss of water that
has not been retained from the body as a result of a concomitant relationship with sodium loss
from the convoluted tubule.
Carbonic anhydrase inhibitors inhibit the enzyme carbonic anhydrase which is found in the
proximal convoluted tubule. This results in several effects including bicarbonate retention in the
urine, potassium retention in urine and decreased sodium absorption. Drugs in this class
include acetazolamide and methazolamide.
Potassium Spearing Diuretics: These are diuretics which do not promote the secretion
of potassium into the urine; thus, potassium is retained and not lost as much as with other
diuretics. The term "potassium-sparing" refers to an effect rather than a mechanism or location;
nonetheless, the term almost always refers to two specific classes that have their effect at similar
locations:
 Aldosterone antagonists: (spironolactone) which is a competitive antagonist of aldosterone.
Aldosterone normally adds sodium channels in the principal cells of the collecting duct and
late distal tubule of the nephron. Spironolactone prevents aldosterone from entering the
principal cells, preventing sodium reabsorption. Similar agents areeplerenoneand potassium
canreonate.
 Epithelial sodium channel blockers: amiloride and triamterene.
Osmotic Diuretics: Compounds such as mannitol are filtered in the glomerulus, but cannot be
reabsorbed. Their presence leads to an increase in the osmolarity of the filtrate. To maintain
osmotic balance, water is retained in the urine.
LOOP DIURETICS (FUROSAMINDE)
Loop diuretics are diuretics that act at the ascending loop of Henle in the kidney. They are
primarily used in medicine to treat hypertension and edema often due to congestive heart failure
or renal insufficiency.
Loop diuretics act on the Na+-K+-2Cl- symporter (cotransporter) in the thick ascending limb of
the loop of Henle to inhibit sodium and chloride reabsorption. This is achieved by competing for
the Cl- binding site. Because magnesium and calcium reabsorption in the thick ascending limb is
dependent on the positive lumen voltage gradient set up by potassium recycling through renal

outer medullary potassium channel, loop diuretics also inhibit their reabsorption. By disrupting
the reabsorption of these ions, loop diuretics prevent the generation of a hypertonic renal
medulla.[2] Without such a concentrated medulla, water has less of an osmotic driving force to
leave the collecting duct system, ultimately resulting in increased urine production. Loop diuretics
cause a decrease in the renal blood flow by this mechanism. This diuresis leaves less water to be
reabsorbed into the blood, resulting in a decrease in blood volume.
The collective effects of decreased blood volume and vasodilation decrease blood pressure and
improve edema.
Clinical Indication:
Loop diuretics are principally used in the following indications:
 edema associated with heart failure, hepatic cirrhosis, renal impairment, nephrotic syndrome
 hypertension
 adjunct in cerebral/pulmonary edema where rapid diuresis is required (IV injection)
They are also sometimes used in the management of severe hypercalcemia in combination with
adequate rehydration.
Adverse Effects:
ADRs include: hyponatremia, hypokalemia, hypomagnesemia, dehydration, hyperuricemia, gout,
dizziness, postural hypotension, syncope.[3] The loss of magnesium as a result of loop diuretics
also been suggested as a possible cause of pseudogout (chondrocalcinosis) Infrequent ADRs
include: dyslipidemia, increased serum creatinine concentration, hypocalcemia, rash and
Ototoxicity (damage to the ear) is a serious, but rare ADR associated with use of loop diuretics.
This may be limited to tinnitus and vertigo, but may result in deafness in serious cases.
Experimental Animal: Mice - (20-25gm in wt.)

Requirements: Metabolic cage for mice, Measuring cylinder and Oral dosing tube.
Furosamide 10mg/kg

Procedure:
 Each mouse was placed in an individual metabolic cage 24 h prior to commencement of the
experiment for adaptation and then fasted overnight with free access to water.
 The animals were pretreated with physiological saline (0.9% NaCl) at an oral dose of 0.15
ml/10 g body weight, to impose a uniform water and salt load.
 Administer the test drug to the animals orally.
 Immediately after administration, the mice were individually placed in a metabolic cage. Urine
was then collected and measured 1, 2, 3, 4, and 5 h after dosing.
 The following parameters were calculated in order to compare the effects of the extracts with
vehicle and standard on urine excretion.
 The urinary excretion independent of the animal weight was calculated as total urinary output
divided by total liquid administered (Formula−1).
 The ratio of urinary excretion in test group to urinary excretion in the control group was used
as a measure of diuretic action of a given dose of a drug (Formula −2).
 A parameter known as diuretic activity was also calculated. To obtain diuretic activity, the
diuretic action of the extract was compared to that of the standard drug in the test group
(Formula – 3)

Marking Weight of Animal (gm) Amount (mg) Volume (ml)
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Observations:
Effect of the loop diuretic (Furosamide) on 5 hour urine volume in mice
Volume of Urine (ml) Diuretic Diuretic

Mice
1hr 2hr 3hr 4hr 5hr Action Activity
T1
T2
T3
C1
C2
C3

Calculations:______________________________________________________
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Result:__________________________________________________________
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Discussion:
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Gastrointestinal Pharmacology
EXPERIMENT # 24
Effect of Saline Purgative on frog intestine
Objective: Evaluation of effect of saline purgative by the use of hypertonic, hypotonic and
isotonic solutions of saline, magnesium sulphate and Frog’s ringer.
Background:
LAXATIVE: any drug used in the treatment of constipation to promote the evacuation of feces. Laxatives produce
their effect by several mechanisms. The four main types of laxatives include: saline purgatives, fecal softeners, contact
purgatives, and Bulk Laxatives.
Saline purgatives: are salts containing highly charged ions that do not readily cross cell
membranes and therefore remain inside the lumen, or passageway, of the bowel. By retaining
water through osmotic forces, saline purgatives increase the volume of the contents of the bowel,
stretching the colon and producing a normal stimulus for contraction of the muscle, which leads
to defecation. Some commonly used salts are magnesium sulfate (Epsom salts), magnesium
hydroxide (Milk of Magnesia), sodium sulfate (Glauber salt), and potassium sodium tartrate.
Fecal softeners: are not absorbed from the gastrointestinal tract and act to increase the bulk of
the feces. Liquid paraffin (mineral oil) can be used either as the oil itself or as a white emulsion.
Other fecal softeners have a detergent action that increases the penetration of the stool by water.
Contact purgatives: act directly on the muscles of the intestine, stimulating the wave like
contractions (peristalsis) that result in defecation. This type of laxative includes cascara, senna,
castor oil, and phenolphthalein. After regular use, their effect tends to lessen, so larger and

more frequent doses are necessary until finally they cease to be effective. They are useful,
however, when short-term purging is required (e.g., before surgery or after an illness).
Bulk laxatives act by increasing the size of the feces, in part because of their capacity to attract
water. This group includes methylcellulose and carboxymethylcellulose, the gums agar and
tragacanth, psyllium (plantago) seed, and dietary fibre.
Experimental Animals: Frog

Requirements: Frog's board, dissecting instruments, pithing needle, needle with thread, syringe
with needle.
Drug & Solutions: Magnesium Sulphate 27% (Hypertonic)
Saline 0.9% made upto 0.45% (Hypotonic)
Frog's Ringer (Isotonic)
Procedure:
 Pith a frog.
 Expose the abdominal cavity.
 Trace the small intestine and make three compartments by tying threads at equal distance.
 Secure the threads tightly so that no fluid can seep through from one compartment to the
other.
 In the first compartment, inject 0.2ml of hypotonic saline, 0.2ml of magnesium sulphate in the
second compartment, and 0.2ml of Frog's Ringer in the third compartment.
 Wait for 20 minutes and record the observation.

Observation:
Compartment Observation Inference
First Compartment :
0.2ml of saline (hypotonic)
Second Compartment :
0.2ml of Magnesium sulphate
(Hypertonic)
Third Compartment:
0.2ml of frog's ringer (Isotonic)
Result:___________________________________________________________
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Discussion: ______________________________________________________
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MARK DISTRIBUTION FOR FINAL LAB PHARMACOLOGY

Total Mark: 50
SECTION MARKS
Quiz 1 10
Quiz 2 10
Discussion in the manual 05
Spot Viva 05
Result in the final lab 05
Discussion over results in the final lab 05
Methodology 05
Lab Performance throughout semester 05
-------------------------------------WISH YOU BEST OF LUCK----------------------------------
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Lab Manual phramacology 2nd professional (2014)

Research · April 2015

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Dr. Huda Kafeel

Dr. Huda Kafeel 2 2nd Professional Pharm.D

This is to certify that

Signature of course In-charge:

2nd Professional Pharm.D 3 Dr. Huda Kafeel

To study the Basics of Pharmacological

To study handling of different experimental

To study various routes of administration in

To study the physiologic salt solutions used in

Dose calculation and administration guidelines for

EXP # 6 Study of Animal ethics for pre-clinical testing. 40-42

To Study various pharmacological experimental

EXP # 8 study clinical trials of new drugs in human subjects 46-51

Autonomic Nervous System 52-62

EXP # 9 To study the effect of Epinephrine on frog’s heart. 63-64

EXP # 10 To study the effect of Propranolol on frog’s heart. 65-66

EXP # 11 To study the effect of Acetylcholine on frog’s heart. 67-68

EXP # 12 To study the effect of Atropine on frog’s heart. 69-70

Dr. Huda Kafeel 4 2nd Professional Pharm.D

EXP # 13 Evaluation of the effect of Atropine on rabbit’s eye. 73-74

Evaluation of the effect of Phenylephirine on

Evaluation of the effect of Pilocarpine on rabbit’s

Evaluation of the effect of Physostigmine on

To study the effect of NSAIDs on bleeding time by

To study the effect of NSAIDs on clotting time by

To study the effect of NSAIDs on clotting time by

To determine anticoagulant effect of Heparin and

Study of Anti-inflammatory effect of NSAIDs on

Study of Analgesic effect of NSAIDs by Chemical

EXP # 23 To study the Diuretic effect of drugs on mice 106-112

EXP # 24 Study of effect of saline purgative on frog intestine. 113-116

2nd Professional Pharm.D 5 Dr. Huda Kafeel

Purpose of Pharmacological Experiments:

Experimental Pharmacology comes under the heads:

 In vivo testing – Experimentation on Whole intact living body.

Dr. Huda Kafeel 6 2nd Professional Pharm.D

 In vitro testing- Experimentation outside the living body. Experimentation performed in

Approaches for pharmacological experiments:

2nd Professional Pharm.D 7 Dr. Huda Kafeel

Considerations of Pharmacological Experiments:

1. Animal selection: selection of experimental animals is made in Accordance with the

2. Dose calculation: Dose for experimental subject is appropriately calculated according to

3. Selection of Vehicle/Physiologic solution: Most commonly used vehicle for making

5. Development of Experimental Model: Different models are required for the

2nd Professional Pharm.D 9 Dr. Huda Kafeel

Dr. Huda Kafeel 10 2nd Professional Pharm.D

Objective: To study different common Laboratory animals their handling techniques,

Experimental animals can be divided into three categories:

2nd Professional Pharm.D 11 Dr. Huda Kafeel

Selection criteria for experimental animals:

Dr. Huda Kafeel 12 2nd Professional Pharm.D

 Housing Requirements- Isolation or group housing.

Commonly used Experimental animals

2nd Professional Pharm.D 13 Dr. Huda Kafeel

Tail Restraint: Mice may be picked up by grasping the base

Forceps Restraint: Mice may also be picked up with rubber-

INJECTING DUGS IN EXPERIMENTAL MOUSE:

2nd Professional Pharm.D 15 Dr. Huda Kafeel

Intramuscular Injections: Regardless of the method used for

Method #3 A technique using two people can also be used for IM