Author: Pankaj Saha

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:

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.

In this post, we will learn about the different types of common laboratory animals which we often use in our Pharmacology Lab.

Guinea pig

Guinea pigs (400-600g) are the commonly used experimental animals. they are very docile and easy to raise and maintain they are highly sensitive to histamine. They are used in experimental asthma to study bronchodilators. they are also used to local anaesthetics and as a model in amoebiasis and cholera as they are sensitive to this microorganism.

Albino rat

The white rat (200-250g) is the commonest laboratory animal used in experimental pharmacology. Rats are easy to breed and maintain. Resemble man in several organ function and nutrition and sensitive to most of the drugs; make them very useful experimental animals. However, they do not have a vomiting centre. The various rat tissue used is colon, stomach, uterus, caecum and vas deference. Besides these organs rat brain tissue is extensively employed in radio receptor-ligand studies. The other strains of rats are Sprague- Dawley and porton.

Albino mouse

White mice are the smallest laboratory animals used. Mice are also easy to breed and maintain. They are small in size (25-30g) and therefore, easy to breed and maintain. They are sensitive to most of the drugs used in experimental pharmacology. Mice are used extensively in toxicity study, bioassay of insulin, testing of analgesics, CNS active drugs and chemotherapeutic agents. More recently mouse brains, as well as primary cell culture ofmouse spinal cord neurons, are used in neuropharmacology for studying neurotransmitters receptor functions the other strains of mice used are laca and balb/C.

Rabbit

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

Frog

Frogs (150-200g) were one time extensively used in experimental pharmacology. However, recently the use of wild frogs for experimental purposes has been banned. Earlier, frogs were used for isolated heart, rectus abdominis muscle preparation, the study of muscle nerve and ciliary movements, respectively. Frogs were also used for the study of nerve block type of local anaesthetics. frogs are inexpensive and easily available, and the ban on the use of frogs has been debated.

Other animals

Cats, dogs and monkeys are used for pharmacological investigations of drugs. Cats and dogs were one time commonly used to study blood pressure experiments. But their use has been now restricted. However, beagle dogs are the only strain approved by regulatory authorities (USFDA) for preclinical testing of new drugs.

Alternatives to animal experimentation

Because of the growing concern about the use of animals in biomedical research in several countries have passed legislation to prevent or usage of animal experimentation. these include experiments with tissue and body fluids of normal animals and human use of microorganisms, primary cell culture and cell lines, use of models and computer simulation and software are being used nowadays. As per the common laboratory alternate animals.

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

Organ bath:

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

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

Rotarod apparatus:

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

Actophotometer:

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

Electro-convulsiometer:

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

Pole climbing apparatus:

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

Analgesiometer:

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

Metabolic cage:

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