Category: Pharmacology

Aim: To study the local anaesthetic property of procaine HCL using nerve block anaesthesia by
frog foot withdrawal method.

Principle: Local anaesthetics reversibly block impulse conduction along nerve axons and excitable membranes. Their action is used to produce local or regional anaesthesia and for blocking pain sensation. the local anaesthetic property can be easily studied by using

• Nerve block anaesthesia
• Surface anaesthesia
• Infiltration anaesthesia

Requirements:

Animal: Frog
Drugs: Procaine HCL (1%w/v)
Equipment: Frog board surgical instruments

Procedure:

1. Decereb the frog and Place the frog (100-150 gm) and destroy the upper part of the spinal
   cord with the help of pithing needle.
2. Cut open the abdomen and remove all the abdominal organs , so that a pouch is made of
   abdominal walls.
3. Expose the spinal nerves in the cavity
4. Put the frog on the frog board with two of its hind legs hanging free from the board.
   Alternatively the frog may be pinned to the board placed vertically so that its hind legs
   hang free
5. Immense the right hind leg in the beaker containing 0.1N HCL.
6. Note the brisk reflex withdraw of the leg
7. Wash the immerse leg with flowing water or normal saline.
8. Repeat the same with the left hind leg.
9. Wash the immersed left.
10. Place about 10 ml of 1% w/v procaine Hcl in the abdominal pouch. The sciatic nerve flex is exposed to the local anaesthetic actions of the drugs. allow the drug to act for 5 min.

Aim: To study the anxiolytic activity of diazepam in mice using elevated zero mazes.

Principle: The elevated zero maze test is a behavioural test of anxiety based on the naturalistic
the tendency of rodents to avoid open and elevated areas. It is similar to the more widely used elevated plus maze, except that the open and closed arms are arranged circularly, thus eliminating the centre area which removes ambiguity in the interpretation of time spent in the central square of the traditional design. The maze is an elevated, white or black, annual having an outer diameter of 45 cm and an inner diameter of 30 cm. The runway ring where the mouse can explore 6cm width, which is divided into 4 quadrants, 2 opposing “open” quadrants without walls and 2 opposing closed quadrants. The model is widely used for the elevation of anxiolytic agents belonging to a chemically different class of drugs.

Requirements:
Animal: Mice (20-25gm)
Drugs: Diazepam (dose 2 mg/kg. i.p)
Equipment: Zero mazes with the dimension

Procedure:

1. Weigh and number the animals . divide them into two groups each consisting of
   minimum 6 mice. One group is used as control and other drugs treatment.
2. Place the animal individually in the open arm facing towards the closed arm. Start the
   stop watch and note the following parameters for a period of six minutes.
   • Latency to enter the open arm
   • Average time each animals spends in the open arm
   • Total number of entries in the open arm
   • Stretching into open quadrants
3. Clean the maze thoroughly using tissue paper after each trail.
4. Inject diazepam to the test group. After 30 min place the animals individually as described.

Aim:

• To study drug (phenothiazines)- induced catatonia (extrapyramidal side effect in rats.
• to study the anticatatonic (antiparkinsonian) effect of scopolamine.

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

Requirements
Animal: Rats (150-200 g)
Drugs: Perphenazine (dose: 5 mg/kg IP; prepare a stock solution containing 1 mg/ml of the drug and inject 0.5 ml/100g bodyweight of the animal). Scopolamine (dose: 2mg/kg, IP prepare a stock solution containing 0.4 mg/ml of the drug and inject 0.5 ml/100 g body weight of the animal)
Equipment: two wooden blocks, one being 3 cm high and the other 9 cm high

Procedure:

1. Weigh and number the animal. Divide the animals into two groups, one for control (for
   study of perphenazine effect) and the other for studying the effects of scopolamine. Each
   group should consist of at least 5 animals.
2. Inject perphenazine to control animals. observe severity of catatonic response (fig 5.14) as follows:

3. Observe the severity of catatonia at 5,15,30,45,60,90 and 120 min after perphenazine .
4. To the second group inject scopolamine and after 30 minute inject perphenazine to these
   animals already treated with scopolamine. Observe and score the severity of catatonic as in
   step 2.
5. Compare the onset and severity of catatonic response in the both groups. Plot a graph, time
   along the x-axis. Note the difference in the onset and severity of catatonic response in both
   the groups.

Aim: To study the effect of hepatic microsomal enzyme induction on the duration of action of
pentobarbital sodium.

Principle: The drugs induce hepatic microsomal oxidative enzyme systems to enhance the metabolism of other drugs. as a result, in the presence of an enzyme-inducer, the duration of action of the second drug will be reduced. This has significant clinical relevance because when more than one drug is administered at a time one drug may modify the action of another through the microsomal enzyme-inducing property.
The common drugs which induce hepatic microsomal enzyme systems are phenobarbitone and meprobamate. Co-administration of any drug with either of these drugs may affect the disposition of the second drug and therefore, the desired pharmacological effects. The students are advised to study various processes of drug metabolism before doing this experiment.

Requirement:
Animal: Mice(20-25g)
Drugs: Phenobarbitone sodium (dose: 50mg\kg IP, once a day for 5 days. Prepare a stock containing 5mg\ml of the drug and inject 1ml\100g of body weight of the animal. Prepare fresh solution every day). Pentobarbital sodium (dose45 mg\ml of the drug and inject 1 ml\100 g of body weight of the animal).

Procedure:

1. Weigh and number the animals. divide them into two groups, each comprising of at least 6
   mice.
2. To the first group inject phenobarbitone once daily for 5 days. To the second group inject
   distilled water, similarly, for 5 days.
3. One hour after the last dose of phenobarbitone on the 5th day, inject pentobarbital to both the groups.
4. Note the onset and duration of sleepdue to pentobarbital in both the groups as done in the
   earlier experiment.

The inference the animals pretreated with phenobarbital sleep for a shorter duration of the time as
compared to animals treated with distilled water.

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

Principle: Different types of epilepsy i.e., grand mal, petit mal or psychomotor type, can be studied in
laboratory animals. The maximal electroshock (MES) induced convulsions in animals represent
grand mal epilepsy. Similarly chemo- convulsion due to pentylenetetrazole which produce the clonic
type of convulsion in man. These are the two procedures used to study convulsion and to test anticonvulsant drugs in laboratory animals.
In MES- convulsion electroshock is applied through the corneal electrodes. Through optic
stimulation, cortical excitation is produced. The MES convulsion is divided into five phases are

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

Requirement:
Animal: Rats (150-200 gm)
Drugs: phenytoin (dose 25 mg/kg); prepare a stock solution
Equipment : electro- convulsiometer, cornel electrode (150 mA current for 0.2 sec) Stop watch

Procedure:

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

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

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

Requirement:

Animal: Mice (20-25gm)
Drugs: Chlorpromazine hydrochloride
Equipment : Actophotometer

Procedure:

• Weigh the animals and number them.
• Turn on the equipment and place individually each mouse in the activity cage for 10 min. Note the basal activity score of all the animals.
• Inject chlorpromazine and after 30 min re-test each mouse for activity scores for 10 min. Note the difference in the activity before and after chlorpromazine.
• Calculate percent decrease in motor activity.

Aim: To study the muscle relaxant property of diazepam in mice using rotarod apparatus.

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

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

Requirement:

Animal : Mice (20-25gm)
Drugs: Diazepam of the drug and inject 1ml/100g of body weight of the mouse.
Equipment: Rotarod apparatus

Procedure:

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

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

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

Requirements:

Animal: Frog
Drugs: Physostigmine stock solution (1µg/ml), Atropine stock solution (1µg/ml)
Physiological solution: Normal saline

Procedure:

• Decapitate the frog and pin the frog to the frog board on its back.
• Pin the lower jaw to the abdomen cutting sufficiently the buccal cavity and exposing the oesophagus wet by irrigating it with normal saline.
• To assess the distance travelled by the particles, fix two points. One starts from the lower jaw to the other beginning of the oesophagus. Keep this distance constant to measure the time taken by the particle to move from a fixed point in the lower jaw to the beginning of the oesophagus.
• Place a poppy seed or a small piece of cork at the premarked spot in the jaw. Turn on the stop watch and note the time taken by the object to reach the beginning of the oesophagus repeat this several times.
• Put a few drops of physostigmine on the buccal cavity and after 10 min repeat step 4 note the tome.
• Wash the buccal with normal saline. Put a few drops o atropine on the buccal cavity. After 10 min repeat step 4. Note the time.
• Find out the differences in the time taken by the object to move between the premarked distance in the buccal cavity in presence of saline, physostigmine and atropine.

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

Aim: To study the Mydriatic effect of topically applied atropine on rabbit eyes.

Principle: A large number of drugs are used for their local action in the eye as eye drops or eye ointments. Most of these drugs belong to an antimicrobial, autonomic or local anaesthetic group. The eye is supplied by both sympathetic and parasympathetic nerves. The superior palpebral muscle and the dilator papillae of the iris have sympathetic supply. The sphincter pupillae of the iris has a parasympathetic supply that exercises dominant control. The ciliary muscle is also supplied by the parasympathetic nerve and when it contracts, the ciliary body is moved inwards and forwards. Because the lens bulges forward and the eye is accommodated for near vision. The opposite effect is produced by the relaxation of ciliary muscle resulting in paralysis of accommodation. When the pupil dilates, the iris folds back near the opening of the canal of schlemn and the drainage of aqueous humour is decreased thereby increasing intraocular pressure. constriction of the pupil, by opposite action, will increase drainage and reduce intraocular pressure.

Topically applied drugs can affect the eye by changing conjunctival congestion, papillary size, light reflex, corneal sensitivity and intraocular pressure. however, the effects of drugs on papillary size, light reflex and corneal reflex can be easily studied by the students. the pupillary size can be measured by placing a transparent plastic scale in front of the eye but as close as possible. Light reflex is elicited by directing the light of a torch towards the pupil. The sensitivity of the cornea is tested by gently touching the cornea with a fine cotton swab stick from the side and not from the front of the eye. This elicits corneal reflex which manifests as the blinking of the crystals.

Requirements:

Animal : Rabbit(2-5kg)

Drugs: Atropine (1%w/v) & Physostigmine (1%w/v)

Equipment: rabbit holder, pen torch

Procedure:

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

Observation:

DRUG	PUPILLARY SIZE	LIGHT REFLEX	CORNEAL REFLEX
Saline	Normal	Present	Present
Physostigmine	Constriction	Present	Present
Ephedrine	Dilation	Present	Present
Atropine	Dilation	Present	Present

There are different routes of drug administration by using laboratory animals.

Aim:

• To learn how to handle, treat preparing animals for experiments and ethical guidelines during the treatment.
• Measure the required volume of drugs using aspectic techniques.
• To learn how to give a different types of route of drug administration.

How to handle: Good handling and restraint are the most important techniques for correct administration. The restrain methods are 2 types.

• Physical method
• Manual method
  • Double handled manual method
  • Single handled manual method

Route of drug administration:

• Enternal route (oral and intregastric)
• Parentral (I.V, I.M,I.P,S.C,I.D)
• Topical route (surface of the skin membrane)

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

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

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

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

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

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

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

Topical route: The most convenient route for topical drugs, drugs like local anaesthetics, ointments and cream can be easily applied on the surface of the skin membrane. Eye drops and ointments were also used in this method. Animals like rabbits, guinea pigs and rats are mainly used in this method.