DEPARTMENT OF VETERINARY PHARMACOLOGY AND TOXICOLOGY

Department of veterinary Pharmacology and Toxicology offers undergraduate courses to students in third professional year preparing for B.V.Sc.&A.H. degree. The courses of study envisage study of actions, mechanism of action and therapeutic and side or adverse effects of drugs in animals. Emphasis is given so that the students gain enough knowledge to select and prescribe a drug of choice for the treatment of an animal.

Students are given practice in pharmacy practical class to prepare some commonly used drug formulations. As per mandate of CPCSEA guidelines, practical classes in experimental pharmacology are conducted using software, simulation, video clips for demonstration of observation of effects of various classes of drugs in both intact laboratory animals and isolated organs/tissues as an alternate to use of animals.

The Department has following sanctioned strength of Faculty:

Professor - 1

Associate Professor – 1

Assistant Professors – 1

COURSES and SYLLABI

THEORY

UNIT-1 (GENERAL PHARMACOLOGY)

Introduction, historical development, branches and scope of Pharmacology. Sources and nature of drugs. Pharmacological terms and definitions, nomenclature of drugs. Principles of drug activity: Pharmacokinetics - Routes of drug administration, absorption, distribution, biotransformation and excretion of drugs. Pharmacodynamics - Concept of drug and receptor, dose-response relationship, terms related to drug activity and factors modifying the drug effect and dosage. Adverse drug reactions, drug interactions.

UNIT-2 (DRUGS ACTING ON AUTONOMIC NERVOUS SYSTEM)

Neurohumoral transmission, Pharmacology of neurotransmitters. Adrenoceptors agonists and antagonists, adrenergic neuron blockers, cholinoceptor agonists and antagonists. Autacoids: Histamine, histamine analogues and antihistaminic agents, 5-Hydroxytryptamine and its agonists and antagonists, eicosanoids, platelet activating factors, angiotensin, bradykinin and kallidin.

UNIT-3 (DRUGS ACTING ON CENTRAL NERVOUS SYSTEM)

Classification of drugs acting on CNS. History, mechanism and stages of general anaesthesia. Inhalant, intravenous and dissociative anaesthetics. Hypnotics and sedatives; psychotropic drugs, anticonvulsants, opioid analgesics, non-steroidal anti-inflammatory drugs, analeptics and other CNS stimulants. Drugs acting on somaticnervous system: Local anaesthetics, muscle relaxants. Euthanizing agents.

UNIT-4 (DRUGS ACTING ON DIFFERENT BODY SYSTEMS)

Drugs acting on digestive system: Stomachics, antacids and antiulcers, prokinetics, carminatives, antizymotics, emetics ,antiemetics, purgatives, antidiarrhoeals, choleretics and cholagogues. Rumen pharmacology. Drugs acting on cardiovascular system: Cardiotonics and cardiac stimulants, antiarrhythmic drugs, vasodilators and antihypertensive agents, haematopoietic drugs, coagulants and anticoagulants, Drugs acting on respiratory system: Expectorants and antitussives, respiratory stimulants, bronchodilators and mucolytics. Drugs acting on urogenital system: Diuretics, drugs affecting urinary pH and tubular transport of drugs, ecbolics and tocolytics. Pharmacological basis of fluid therapy. Pharmacotherapeutics of hormones. Drugs acting on skin and mucous membranes: Emollients, demulcents and counter irritants

UNIT-5 (VETERINARY CHEMOTHERAPY)

Introduction and historical developments of chemotherapy. Antimicrobial agents: Classification, general principles inantimicrobial chemotherapy, antimicrobial resistance, combined antimicrobial therapy. Sulphonamides and theircombination with diaminopyrimidines. Penicillins, cephalopsorins, cephamycins and other beta lactams, beta lactamaseinhibitors. Aminoglycosides and aminocyclitols, tetracyclines, amphenicols (chloramphenicol, thiamphenicol, florfenicol), macrolides, quinolones and fluoroquinolones, polypeptides (polymixins, bacitracin) and glycopeptide antibiotics, miscellaneous agents: Lincosamides, novobiocin, virginiamycin, tiamulin, nitrofurans and methenamine, Antitubercular drugs. Antifungal agents: Topical and systemic agents including anti-fungal antibiotics. Antiviral and anticancer agents. Anthelmintics: Drugs used against nematodes, cestodes, trematodes. Antiprotozoal agents: Drugs usedin trypanosomosis, theileriosis, babesiosis, coccidiosis, amoebiosis, giardiosis and trichomoniasis. Ectoparasiticides, Antiseptics and disinfectants. Pharmacology of drugs of abuse in animals Pharmacology of indigenous medicinal plants: Scientific name, common name, active principles, pharmacological actions and therapeutic uses of Ginger, ocimum, neem, piper longum, withania, leptadenia, tinospora, embilica,eucalyptus, glycerrhiza, trichospermum, curcuma, adhantoda, butea, aloes, sena, rheubarb, catechu etc.

UNIT-6 (VETERINARY TOXICOLOGY)

General Toxicology: Definitions, history of toxicology, fundamentals and scope of toxicology. Sources and classification of toxicants, factors modifying toxicity, general approaches to diagnosis and treatment of poisoning. Toxicity caused by metals and non-metals: Arsenic, lead, mercury, copper, molybdenum, selenium, phosphorus, fluoride, nitrates or nitrites, chlorate, common salt and urea. Poisonous plants: Cyanogenetic plants, abrus, ipomoea, datura, nuxvomica, castor, oxalate producing plants, plants causing thiamine deficiency, plants causing photosensitization and lathyrism, oleander, and cotton. Toxicity caused by Agrochemicals: Insecticides - Chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids, newer insecticides. Herbicides, fungicides and rodenticides. Fungal and bacterial toxins: Aflatoxins, rubratoxin,  ochratoxin, sporidesmin, citrinin, F-2 toxin, trichothecenes, ergot, fescue, botulinum toxin and tetanus toxin Venomous bites and stings: Snake, scorpion, spider, bees and wasp, toad and fishes (puffer fish, shellfish). Toxicity caused by food additives and preservatives. Drug and pesticide residue toxicology. Environmental pollutants: Air and water pollutants. Concept of radiation hazards.

PRACTICAL

UNIT-1 (GENERAL PHARMACOLOGY)

Handling and washing of laboratory wares. Handling and operation of commonly used laboratory instruments. Concept of good laboratory practices (GLP). Pharmacy appliances. Principles of compounding and dispensing. Metrology, systems of weights and measures, pharmacy calculations. Pharmaceutical processes. Pharmaceutical dosage forms .Prescription writing, incompatibilities. Drug standards and regulations, custody of poisons. Compounding and dispensing of powders, ointments, mixtures, liniments, lotions, liquors, tinctures, emulsions, and electuaries.

UNIT-2 (ANS PHARMACOLOGY)

Demonstration of the action of autonomic agonists and antagonists on intact or isolated preparations of the laboratory animals. Simulated animal experiments should be preferred over use of live animals. The lab for simulated experiments should be established within a span of one year.

UNIT-3 (CNS PHARMACOLOGY)

Handling of lab animals. Regulatory guidelines for use of lab animals. Demonstration of the effect of CNS active drugs and local anaesthetics in laboratory animals. The lab for simulated experiments should be established within a span of one year.

UNIT-4 (VETERINARY CHEMOTHERAPY)

Demonstration of various chemotherapeutic agents and their dosage forms. Demonstration of antibiotic sensitivity test and its interpretation.

UNIT-5 (VETERINARY TOXICOLOGY)

Collection, preservation and dispatch of material for toxicological analysis. General principles for toxicological analysis. Detection of heavy metals or non-metals or plant poisons. Demonstration of agrochemical toxicity and its antidotal therapy via simulation methods. Demonstration of toxic weeds and plants of local area. Methods of calculation of median lethal dose (LD50) or maximum tolerated dose (MTD).

                                                      ANNUAL EXAMINATION

PAPERS              U N I T S     MAXIMUM MARKS                   WEIGHTAGE

THEORY

Paper-I                         1, 2, 3 and 4                              100                                           20

Paper-II                        5 and 6                                     100                                           20

PRACTICAL

Paper-I                         1 and 2                                      60                                            20

Paper - II                      3, 4 and 5                                  60                                            20

 INTERNAL ASSESSMENT.                                                                          20

       

 

 NEUROHARMACOLOGY PRACTICAL MANUAL

NEUROPHARMACOLOGY

 

CHAPTER - 10

 

Gross observable effects of cns active drugs

The gross observable effects of drugs can be demonstrated in mice by using multidimensional screening procedure devised by Irwin (1963). The screening procedure is simply based on observational techniques following administration of the drug. The scoring of different signs or effects or characters is done as under:

a. Normal signs: Normal score is 4 for all the normal signs. For stimulation or enhancement of intensity of signs, scoring is done from 4 to 8, whereas the decrease of intensity of effects or sign is scored from 4 to 0.

b. Abnormal signs: Normal scores is 0. If any abnormal sign or some effects appear, scoring is done between 0 and 8 depending on the intensity of the abnormal signs.

The gross observable effects or activity profiles have been placed into four main groups.

a. Behavioural profiles

b. Neurological profiles

c. Autonomic profiles

d. Miscellaneous.

Behavioural profiles:

These effects are divided into three sub-groups. (i) awareness, (ii) mood and (iii) motor activity.

(I)  Awareness:

The effects on awareness can be judged from the following parameters:

1. Alertness (4): indicates stimulation of CNS

2. Stupor (0): indicates depression of CNS

3. Visual placing (4): measures the animals’ response and its ability to orient itself without bumps or falls when placed in a difficult position, like on a rotating rod or a fine rope. Subnormal ability may indicate motor in-coordination.

4. Stereotypy (0): is frequent mechanical repetition of only one kind of movement, e.g. searching movements of head with amphetamine, circling or circular movement with morphine, self-biting with apomorphine and tail-lashing with spinal stimulants.

 

5. Passivity (0) measures the struggle behaviour of the animal when placed in un-accustomed or unusual position.

                                                 

Unaffected (normal) mouse struggles (moves head and limbs) to escape the unusual position and orients itself back to normal position. The scoring is done to indicate abolition of struggle behaviour. The scheme of scoring is as under:

 i). Score 1 - when mouse is suspended vertically by loose fold of skin over the neck,

ii). Score 2 – when mouse is rotated horizontally on its back,

iii). Score 3 – when suspended by fore limb, and

iv). Score 4 – when mouse does not struggle to escape when suspended by hind limb.

Passivity indicates tranquillization, CNS depression, myorelaxation, paralysis or anaesthesia.

  1. Catalepsy/Catatonia indicates the tone of limb muscles of the animals. If one of the fore limbs is placed  on a piece of wood at height of 4 cm in mice and 8 cm in rats and the animal is unable to remove the limb for more than one minute, the presence of  catalepsy is indicated.

                                          Fig. shows catalepsy in rabbit

(II)  Mood:

Following activities determine the mood of the animals.

1. Grooming (4): Generally grooming behaviour is prevalent in unaffected, normal mice. But excessive grooming indicates central stimulation or sympathetic stimulation

2. Vocalization (0): Mice generally do not utter any sound. Vocalization such as ‘squeaks’ indicates discomfort or noxious stimuli.

3. Restlessness, irritability, aggressiveness and fearfulness are normally absent in mice. Their presence indicates CNS stimulation or discomfort.

 

 

 

(III)  Motor activity:

1. Spontaneous motor activity (4): Normally animals exhibit spontaneous motor activity (SMA) to explore its surroundings i.e. exploratory behaviour. The drugs may increase or decrease the SMA.

Reduction in SMA is scored as under:                       The increase in SMA is scored as under:

b. Slight reduction- 3                                                  a. Constant walking - 6

c. Occasional spontaneous movements – 2                b. Walking with running - 7

d. Very rare spontaneous movements – 1                   c. Agitated and vigorous running – 8

e. Absence of spontaneous activity – 0

Decrease in SMA indicates CNS depression, blockade of ganglia or neuromuscular junctions, whereas increased SMA indicates stimulation of CNS, ganglia or neuromuscular junctions.

2. Reactivity (4): Reactivity is scored on the basis of intensity of locomotor response and spontaneous activity following snapping of fingers and gentle manipulation of animals. An increase or decrease in reactivity indicates stimulation or depression of CNS, ganglia or neuromuscular junctions.

3. Touch response (4): The touch response is scored on the basis of intensity of locomotor activity by touching the animals with a pencil or forceps at various parts of the body e.g. on the side of the neck or abdomen or on the groin. No response to touch indicates CNS depression.

4. Pain response (4): The response to pain is recorded by applying a small bulldog clamp to the base of the tail. The absence of any response to pain measures analgesia, CNS sedation or depression.

Neurological profiles

(I) CNS excitation:

1. Startle response (0): Shaking of the animal body in response to a sudden loud sound is called startle response. It can be assessed by striking the outside of the observation tray or cage with a metal object. It is rated as below.

a. No response – 0

b. The animal visibly jerks – 2

c. Jerks and jumps – 4

d. Jerks and frantic escape attempts – 6

e. Clonic convulsions – 8

2. Straub tail (0): Generally the mice keep their tail on the floor of cage. The degree of elevation of the animal’s tail is recorded as straub tail response. Spinal stimulant drugs e.g. morphine, strychnine etc. produce such responses.

 

3. Tremors (0):

4. Twitches (0):

5. Convulsions (0): The animals are observed for pre-convulsive seizures and clonic or tonic convulsions.

(II)  Posture (4):

Any deviation in normal posture of the animals is observed, e.g. relaxed, hunched, lying, or stretched. The deviation in body or the limb position from the normal may indicate neuromuscular blockade or central depression.

(III)  Motor in-coordination (0):

Motor in-coordination indicates CNS depression and muscular relaxation, synaptic blockade or anesthetic activity.

1. Ataxia (0): Ataxia, staggering or abnormal gait can be scored as under:

a. Score – 2, when animal’s movements show occasionally detectable in-coordination,

b. Score – 3, constant in-coordination, but animal walks straight,

c. Score – 4, animal does not walk straight stumbles and the course is zigzag.

2. Righting reflex (4): When an animal is placed on its back, it orients itself to correct posture. This reflex is called righting reflex and indicates normal mid brain activity. The scoring is done as under:

a. Score – 4 when corrects immediately

b. Score – 2 sluggish but corrects within five seconds (partial loss of righting reflex).

c. Score – 0 when does not correct within 15 seconds (complete loss of righting reflex). The time elapsed between loss and regain of righting reflex is measured as ‘sleeping time’.

3. Somersault test (0): This is also like righting reflex but procedure of observation is different. In this test mouse is picked up by tail and tossed in the air five times. The scoring is done according to the posture adopted by mouse when it touches the rubber-padded floor.

a. Standing on four feet: score 0, if every time it stands on four legs.

b. Lying on sides: score 1, if 1-2 times, score 2, if 3-4 times and score 3, if 5 times.

c. Lying on the back: score 4, if 1-2 times; score 5, if 3-4 times and score 6 if 5 times.

d. Sluggish regaining of posture from supine or side position: score 7.

e. Remains on the back:  score 8.

(IV)  Muscle tone:

Loss of muscle tone indicates myorelaxation, neuromuscular blockade or CNS depression.

  1. Limb tone (4): Limb tone may be assessed by grasping a foreleg of the mouse and feeling

the resistance on the extension of the leg.

b. Grip strength (4): It can be assessed in two ways:

i. Allow the mouse to grasp a pencil in the horizontal position and then lift the animal and note the ease with which the animal drops the pencil.

ii. Inclined plane test: In this test the mouse is placed on top of an inclined plane, usually a wooden board having rough surface and kept at 45o angle. Normal animals walk down the board while affected animals slide down.

c. Abdominal tone or body tone (4): Body tone can be estimated by noting the muscle tone in comparison to that in normal animals. The body tone can also be estimated by performing Raubichaud test: To perform this test, pick up the mouse by holding between thumb and fore-finger the loose fold of skin on the back of the animal and then suddenly drop the animal on the floor. Now observe the perpendicular skin folds for adopting the body contours. The scoring is done as under:

  1. Score 0, if the perpendicular fold of skin adopts to body contours immediately.
  2. Score 1, if the fold persists for 3-5- seconds.
  3. Score 2, if the fold persists for more than 5 seconds.

(V)  Reflexes:

a. Pinnal reflex (4): Pinnal reflex is observed by touching the external auditory meatus with a hair brush, feather or other soft fine object. Normally mouse withdraws its head from the offending objects and shows pinnal movements.

b. Corneal reflex (4): This reflex is observed by touching cornea with stiff hair. Normally the mouse closes its eye lids or withdraws itself.

Pinnal and corneal reflexes are scored as follows.

i. Normal response - score 4

ii. Sluggish response - score 2

iii. No response - score 0

Loss of these reflexes indicates blockade of sensory nerves, motor nerves or the spinal synapses.

 

 

 

autonomic profiles

(I)  Optical sign:

1. Pupil size (4): Make observations for increase or decrease in the size of the pupil. Enlargement (mydriasis) indicates sympathomimetic or parasympatholytic activity.

Constriction (miosis) indicates parasympathetic stimulation or morphine induced (pin-point pupil) activity.

2. Palpebral opening (4): Observation is made for the degree of opening or closure of eye lids. It is scored as under:

i. Normal opening of eye lids – score 4

ii. Slight closure of eye lids – score 3

iii. Half closure of eye lids – score 2

iv. Three fourth closure of eye lids – score 1

v. Complete closure of upper eye lids (ptosis) – score 0

Wide palpebral openings are signs of sympathomimetic activity and narrow palpebral opening are signs of tranquillization and CNS depression.

3. Exopthalamus (0): Bulging of eye balls indicates sympathetic stimulation.

(III)  Secretory signs: normal scores are as under.

1. Urination – score 0

2. Salivation – score 0

3. Lachrymation – score 0

4. Sweating – score 0

5. Defecation – score 0

Increase in all these secretary signs/scores indicates parasympathetic (musacrinic) stimulation.

IV)  General signs:

1. Writhing (0): Movements with abdomen stretched or rubbing of abdomen with floor indicates irritation of peritoneum or stimulation of sensory receptors.

2.  Piloerection (0): indicates sympathetic stimulation, hypothermia or tranquillizing activity.

3. Colour of skin and mucus membrane:

Red color - indicates vasodilatation or sympatholytic activity.

White color - indicates vasoconstriction or sympathomimetic activity.

Cyanosis - indicates decreased oxygenation.

4. Heart rate:

Tachycardia – cardiotonic or sympathetic stimulation.

Bradycardia –cardiac depression or sympatholytic or parasympathetic stimulation.

  1. Respiratory rate:

Increase - indicates analeptic activity or CNS stimulation.

Decrease - indicates CNS depression.

MISCELLANEOUS PROFILES

1. Snout withdrawal: The scoring is based on the time taken by the animals to withdraw

head when its snout is held between thumb and index finger. Score 4, 3, 2 and 1 is given when time taken by the animal is 1, 2, 6 and 8 seconds, respectively.

2. Head drop: It is produced when a drug has peripheral muscle relaxant action.

3. Hind drop: It is produced when a drug has central muscle relaxant action.

4. Other profiles are: analgesia, anaesthesia, blood pressure, pulse rate, biting, fighting, vision, cage climbing and mortality.

                         

Fig: Intraperitoneal and oral injections in mouse.

 

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EXERCISE - 10

Objective: To study the gross observable effects of pentobarbitone.

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic             needle, pentobarbitone solution.

Pentobarbitone is a short acting general anaesthetic / hypnotic.  The barbiturates are derived from barbituric acid which is a conjugate of malonic acid and urea (malonyl urea). The mechanism of action of barbiturates is not clear. These may be affecting the synaptic transmission by increasing the Cl ions conductance in the excitatory and inhibitory postsynaptic neurons. Barbiturates also increase the binding of GABA with its receptors. Thus the transmission of impulses is inhibited.

Procedures:

1. Prepare the solution of pentobarbitone sodium (2.5 mg/ml and 5.0 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF PENTOBARBITONE

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

EXERCISE  - 11

Objective: To study the gross observable effects of diazepam.

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic                  needle, diazepam solution.

Diazepam is a benzodiazepine compound. The first compound of this group, chlordiazepoxide, was synthesized accidentally in 1961. Diazepam acts at all levels of neuroaxis and potentiates the neural inhibition mediated by GABA. Primarily, it is a CNS active agent and causes reduction of anxiety, CNS sedation, induction of sleep, reduction of muscle tone and coordination, and anticonvulsant effect. Among the peripheral effects coronary vasodilation is produced after intravenous administration and neuromuscular blockade at a very high dose.

Procedure:

1. Prepare the solution of diazepam (0.25 mg/ml and 0.5 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF DIAZEPAM

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

EXERCISE  - 12

Objective: To study the gross observable effects of chlorpromazine.

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic                  needle, chlorpromazine solution.

Chlorpromazine is a phenothiazine derivative. It has been used as chemical restraint for animals in veterinary practice for various diagnostic and clinical procedures. It has been used to quieten and calm-down unfriendly animals and thus helps in preparation of animals for neuroleptanalgesia and general anaesthesia. It acts by blocking dopaminergic, adrenergic, serotonergic, histaminergic and cholinergic receptors. Blockade of central adrenergic transmission produces tranquillizing action and blockade of dopaminergic receptors in chemo-sensitive trigger zone in medulla produces antiemetic action.  

Procedure:

1. Prepare the solution of chlorpromazine (0.25 mg/ml and 0.5 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF CHLORPROMAZINE

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:

                                                                                                            Date:

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

 

EXERCISE - 13

           

Objective: To study the gross observable effects of amphetamine.

 

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic              needle, chlorpromazine solutions.

Amphetamine is a non-catecholamine sympathomimetic drug. d-isomer of amphetamine produces more CNS effects than l-isomer. Amphetamine is an indirectly acting sympathomimetic amine. It crosses blood brain barrier and produces powerful CNS stimulant action. Its CNS actions are mostly due to the release of biogenic amines from their storage sites in the nerve terminals. It has peripheral actions mediated through α and β receptors. Various CNS actions mediated by nor-adrenaline are alerting effect, anorectic effect and locomotor stimulating action (partially mediated). Whereas dopamine mediated activities which are seen at a higher dose are locomotor activity and stereotyped behaviour. Still higher doses produce disturbances of perception and overt psychotic behaviour, probably mediated by 5-HT and dopamine.

Procedure:

1. Prepare the solution of amphetamine (0.25 mg/ml and 0.5 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF AMPHETAMINE

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:

                                                                                                            Date:

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

 

EXERCISE - 14

           

Objective: To study the gross observable effects of metrazol.

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic

 needle, metrazol solution.

It is an analeptic and convulsant drug. The word analeptic means “reviver”, therefore, it was previously used to stimulate the vital centers in medulla to overcome the depression of the nervous system produced by an overdosage of drugs such as morphine or barbiturates. All analeptics are convulsant in higher doses, but all convulsants are not necessarily analeptics. It seems to enhance CNS excitability by reducing GABA-ergic inhibition. All levels of cerebrospinal axis are stimulated; therefore, various centers controlling the specific physiologic functions may also be stimulated. Convulsions results due to the stimulation of cortex. 

Procedure:

1. Prepare the solution of metrazol (4.0 mg/ml and 8.0 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF METRAZOL

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

PROFORMA FOR GROSS OBSERVABLE EFFECTS OF DRUGS

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration  (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

  1. Type/class of drug __________________________________________
  2. Name of known / unknown drug ________________________________

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

 

 

EXERCISE  - 15

           

Objective: To study the gross observable effects of strychnine.

Requirements: Mice, animal cage, marker solution, tuberculin syringe, hypodermic

needle, strychnine solution.

Strychnine is an alkaloid obtained from the seeds of the Indian plant Strychnos nuxvomica. Nuxvomica mean emetic nut. Strychnine antagonizes the action of inhibitory neurotransmitter glycine in the spinal cord which is released from Renshaw cells. The strychnine sensitive postsynaptic inhibition in higher centers of CNS is also mediated by glycine. This causes inhibition of the inhibitory effect over the motoneurons resulting into their over excitation. Therefore, a sensory stimulus produces exaggerated reflex effects and even convulsions. It does not produce selective analeptic action. The convulsions produced by strychnine are essentially reflex in nature and are not accompanied by loss of consciousness. In early stage there is a coordinated extensor thrust and at the later stage it may be followed by tonic convulsions and opisthotonus condition. Death occurs because of respiratory failure due to paralysis of respiratory muscles.

Procedure:   

1. Prepare the solution of strychnine (0.1 mg/ml and 0.2 mg/ml) in distilled water.

2.  Apply the identification marks to the mice and then record the body weight.

3.  Administer the drug solution at the rate of 1 ml/100g body weight.

4.  The control mice are administered normal saline solution.

 

 

 

 

 

 

 

 

 

 

 

 

 

GROSS OBSERVABLE EFFECTS OF STRYCHNINE

 

Species of animal_____________ Sex_____________ Body Weight _________________

Name of Drug ________________________________ Dose & Route_________________   

     Activity profiles

Normal

Score

 

                Time after drug administration (min.)

   5            10           15          20          30          60

SMA

4

 

 

 

 

 

 

Ataxia

0

 

 

 

 

 

 

Passivity

0

 

 

 

 

 

 

Stereotypy

0

 

 

 

 

 

 

Reactivity

4

 

 

 

 

 

 

Startle response

0

 

 

 

 

 

 

Straub tail

0

 

 

 

 

 

 

Convulsions (tonic/clonic)

0

 

 

 

 

 

 

Righting reflex

4

 

 

 

 

 

 

Pinal reflex

4

 

 

 

 

 

 

Corneal reflex

4

 

 

 

 

 

 

Pupil size

4

 

 

 

 

 

 

Palpebral opening

4

 

 

 

 

 

 

Urination

0

 

 

 

 

 

 

Salivation

0

 

 

 

 

 

 

Defaecation

0

 

 

 

 

 

 

Lachrymation

0

 

 

 

 

 

 

Piloerection

0

 

 

 

 

 

 

Snout withdrawal

4

 

 

 

 

 

 

Robichaud test

0

 

 

 

 

 

 

Body posture

4

 

 

 

 

 

 

Limb tone

4

 

 

 

 

 

 

Exophthalmia

0

 

 

 

 

 

 

Any other

0/4

 

 

 

 

 

 

 

Interpretation of results:

1. Type/class of drug __________________________________________

2. Name of known/unknown drug ________________________________

 

 

 

 

Signature of instructor                                                                        Signature of student

Date:                                                                                                   Date:

 

CHAPTER - 11

 

Pharmacological Evaluation of Specific Activities of drugs

 

1. Evaluation of general anaesthetic activity:

            General anaesthetics are the agents that produce reversible loss of whole body sensations and consciousness. These drugs first depress the reticular activating systems of mid brain and produce analgesia and sleep. The cortex is then depressed, followed by progressive depression of the mid brain and spinal cord, and the production of surgical anaesthesia. The medullary structures are the last to be depressed in deeper planes of anaesthesia, but once  they are affected, respiration and circulation may fail.

Screening procedure in rats and mice:  A 1 gallon (3800 ml) wide-mouth screw-cap glass jar is flushed with oxygen for 1 min and a measured amount of volatile substance is placed on the bottom through a suitable syringe or cotton swab. The amount is calculated to give 1.25 vol % concentration of the vapours in the jar (or a logarithmic multiple of 1.25% i.e. 0.63, 2.5, 5.0, 10.0). The volume % concentration of the anaesthetic mixture can be calculated by the density and molecular weight of the liquid (assuming 1 mol. of vapour = 22.4 L).

            The jar is closed and evaporation of the anesthetic is facilitated by gentle rotation of the jar. One rat or five mice are quickly poured from a beaker into the jar and the jar is immediately closed. Every 15 sec. the jar is gently rotated and the time noted for each animal to become anaesthetized (i.e. loss of righting reflexes). This procedure is repeated until all animals are anaesthetized. Induction should occur no sooner then 0.5 min and no later then 5 min. The animals are allowed to remain in anaesthesia jar for 10 min., with testing of righting reflexes every min. until they are removed. They are then ‘poured’ out into room air and the time of recovery (to righting and walking) is noted for each animal. The results are reported as mean induction time and mean recovery time. If the induction time is less than 0.5 min. or longer than 10 min., the conccentration of anaesthetic agent is increased or decreased until proper concentration is found. The animals are watched for 24 hour survival rate to reveal any latent toxic effect of the agent.

2. Evaluation of hypnotic activity:

            Hypnotics (somnifacients, soporifics) are the agents that produce CNS depression equivalent to natural sleep and the individual can be easily aroused. Pharmacologically the term ‘hypnosis’ and ‘sleeping time’ has been applied to the state of drug-induced unconsciousness associated with the loss of muscle tone and static righting reflexes. The animals are in a state of general anaesthesia or coma and consequently aren’t arousable. Pharmacologic hypnosis is thus a much deeper state of CNS depression than clinical hypnosis. In clinical hypnosis, the purpose is to obtain a normal nights sleep from which the subject can be aroused without any subsequent ‘hangover’. Therefore, the induction of sleep by hypnosis may be regarded as night time ‘sedation’. The day time sedation is considered as the therapeutic equivalent of the state of somnolence (pre-sleep) and is accepted as the basis for hypnotic activity evaluation in animals. The duration of drug-induced sleep/hypnosis can be computed by noting the time elapsing between the loss and recovery of   righting reflex.

3. Evaluation of CNS sedative Activity:

            The CNS sedatives are the agents that reduce the level of brain perception to external stimuli. The CNS sedative activity of a drug can be evaluated in mice/rats by observing its effect on followings:

(a). Pentobarbitone-induced hypnosis: Potentiation indicates CNS sedative activity.

(b) Spontaneous Motor Activity: Reduction in SMA indicates CNS sedative activity. Actophotometer cage can be used to quantitate SMA.

(c) Rota-rod (Tread-mill) Test: The animals trained to walk on the rotating rod fall within 2 minutes under sedative effect.

                                               

                                          Fig. Rota-Rod Tread Mill

(d)  Amphetamine-induced Hyperactivity:  antagonism  indicates CNS sedative activity.

4. Evaluation of tranquillizing activity

                                                  

                                 Fig.  Cooks Pole-Climbing Apparatus     

            Among the various methods, conditioning methods are very frequently used for the evaluation of tranquillizing activity. One of the conditioning methods employed for this purpose was devised by Cook and Weidley (1957). This involves the use of an instrument

called the Cook’s pole climbing apparatus, which has an electrified grid and a buzzer arrangement with a pole attached to the removable lid. Rats are conditioned to buzzer/sound to escape/avoid electric shock. Buzzer is a conditioned stimulus and shock is an unconditioned stimulus. During the training, the buzzer and shock (80V, 5pulse/second) are applied simultaneously and rats are guided to climb the pole to escape shock. 

            The conditioned avoidance response (CAR) considered to be established when the rats climb on the pole within 3 seconds in response to buzzer only. The escape response to electric shock is known as unconditioned avoidance response (UCAR). The conditioned avoidance response is considered blocked when rats do not climb the pole within 30 seconds with buzzer. The presence of unconditioned avoidance response (UCAR) is seen by applying the shock to the rats. Major tranquillizers block the CAR without blocking UCAR, but hypnotics block both.

5. Evaluation of analgesic activity:

            Analgesics are the drugs that increase the threshold of pain perception. Pain is a subjective sensation expressed as a result of stimulation of nerve fibers by noxious stimuli. The pain threshold is the intensity of the stimulus required to cause a just perceptible sensation of pain. Thus the analgesic activity of drugs is evaluated against pain response produced by some noxious stimuli such as heat, pressure or electric shock. Analgesic drugs raise the pain threshold and are evaluated on quantal (all or none) basis.

Methods:

A. Thermal method: The most commonly employed method is the thermal method where heat is used as noxious stimulus to evoke pain. Following are the two routinely used thermal methods:

                                                 

                                    Fig. Hot-Plate                                      Fig. Tail Flick Unit 

 

 

(i) Conduction Heat Method (Hot plate method): 

The heat stimulus comes in direct contact with the animal body. This method is very popular and applicable to mice. It uses an electrically heated plate maintained at 55±0.5º C. Individual mouse is placed on hot plate and the reaction time is recorded. Reaction time is defined as the “time elapsed between placing of mouse on hot plate and occurrence of the signs of discomfort such as licking of forepaws, kicking by hind legs or jumping to escape out of the chamber”. This method is used to assess centrally acting analgesic drugs. A three fold increase in reaction time is considered to be the analgesic effect. In any case mouse should not be allowed to remain on the hot plate beyond 15 seconds to avoid tissue damage.

(ii) Radiation Heat Method (Tale-flick method): The heat stimulus does not come in direct contact with the animal body. A red hot nichrome wire about 1/8 inch below the tail plateform is the source of heat. A constant amperage is supplied to the nichrome wire throughout the galvanometer of the instrument. The tail of the rat is placed on the tail plateform and reaction time in the form of flicking of the tail is recorded. A three fold increase in reaction time is indicative of analgesia.         

B. Other methods:

(i) Mechanical method: One of the most common methods of applying stimulus is the application of pressure on the tail of a rat. A constant pressure is applied either by a motor, gas from a cylinder or a bull dog clamp on the tail or toe of the rat. Untreated animals cry or attempt to remove clip by biting it. Centrally acting analgesic drugs increase reaction time. Drugs which inhibit release of the substances of pain (anti-inflammatory) do not increase reaction time.

(ii) Electrical method: Mice are placed on floor made of metallic rods that can be electrified. When specified voltage is applied across the rods, the animal squeaks and struggles to escape. The increase in reaction time indicates analgesic activity. Both centrally and peripherally acting rugs increase the reaction time in this test.               

(iii) Chemical method: Production of pain by chemical method is most suitable procedure for screening of peripherally acting analgesic drugs. Benzoquinon, bradykinin or acetic acid when given intraperitoneally produce a characteristic irritation symptom “writhing”. Numbers of ‘writhes’ are counted before and after the administration of drugs under test. Decrease in number of writhes is considered analgesic effect.

 

 

 

6. Evaluation of Anticonvulsant Activity:

            Anticonvulsant (antiepileptic) action of drugs is studied on convulsions produced by electric shock or chemo-shock in mice or rats. The protection of experimentally-induced convulsions is the criteria for anticonvulsant activity.

Methods of producing convulsions (seizures): There are two commonly employed methods.

A. Electroshock-induced seizures: This includes Supramaximal or Maximal Electroshock Seizures (SMES or MES). An electric stimulus about 5 times greater than the threshold (48 mA, 0.2 sec) is applied via pinnal or corneal electrodes with help of a convulsiometer. 

                                                                                                                                                                                                        

                  Fig. ECT Unit.                             Fig. Mouse showing tonic convulsions (Extensor phase)

            The resultant seizure in normal mice shows tonic flexor followed by tonic extensor and few clonic jerks thereafter. The blockade of the ‘tonic phase’ of convulsions is taken as criteria for anticonvulsant effect and such drugs are effective in grand mal epilepsy. Phenylhydantoin and phenobarbitone are most effective whereas trimethadione is least effective.

B. Chemoshock-induced seizures: This includes Metrazol Seizure Threshold Test (MST). Metrazol is given sub-cutaneously at the rate of 80-85 mg/kg in mice. It produces primarily clonic convulsions, but tonic convulsions may occur before death. Protection of clonic convulsions is the criteria for anticonvulsant effect. Such drugs are effective in petitmal epilepsy. Phenobarbitone is most potent whereas diphenylhydantoin is ineffective.

7. Evaluation of anti-inflammatory activity:

                                                  

                                                              Fig.  Plethysmometer   

            Inflammation is a defensive response of the  body to an infection, irritation or foreign substances. Though, it is a defensive response, but when it becomes too great it may be fatal, therefore, it is counteracted. The drugs which counteract inflammation are called anti-inflammatory agents. The anti-inflammatory activity is tested against experimentally induced inflammation by some phlogestic (inflammatory) agent. Following are different models used for evaluating anti-inflammatory activity of a compound:

A. Acute models: Rat hind-paw oedema model is the most commonly used model. Oedema is induced mostly by subcutaneous injection of either carrageenin (0.1 ml of 1% in corboxymethyl cellulose) or formaline (0.05-0.1 ml of 4%) in the hind-paw of rats. Oedema is measured by Plethysmograph by dipping the paw in mercury. Protection of the oedema formation indicates anti-inflammatory activity.

B. Sub-acute models: Cotton pellet test and granuloma pouch test are the commonly used sub-acute models. Two autoclaved pellets of cotton or sponge weighing 50±1 mg are implanted aseptically, one on each side of the mid line incision. On 8th day, the rats are killed by an overdose of ether and the pellets surrounded by granuloma tissue are dissected out carefully and dried in an oven at 60º C till a constant weight is obtained. The per cent inhibition is calculated by comparing with control group.

C. Chronic models: Adjuvant-induced arthritis and collagen-induced arthritis are commonly used chronic models. For collagen-induced arthritis a Native type II collagen is prepared (dissolved in 0.1M acetic acid at a concentration of 2 mg/ml and emulsify at 4º C with equal volume of complete Freund’s adjuvant) and 0.5 mg of the collagen in the cold emulsion is injected intra-dermally on the back of each rat. Sometimes an intraperitoneal booster dose of collagen alone is required on day 21. The disease symptoms start from day 10 to 29 and persist for 4 to 8 weeks. Rats of 4 to 5 weeks of age give good results. The development of the disease can be graded by scoring from 0 to 4 each paw every day. Simple erythema is given the lowest score and complete immobility of the joints get the highest score. Volume of both the hind paws can be measured with plethysmograph. Only 40–60% of animals develop polyarthritis.

8. Evaluation of Local-anaesthetic Activity:

            The drugs which block conduction of impulses and block sensation (pain) from relatively restricted area of the body are called ‘local anesthetics’. Any drug which blocks sensory nerve is most likely to block motor nerve also, if given in higher concentrations. Local anesthetics prevent generation and propagation of action potential along the nerve. Thin nerve fibers conducting pain sensation are more sensitive to nerve block than thick fibers conducting sensation of pressure or touch and motor nerves. The effects of drugs causing blockade of conduction can be detected (i) electrically, (ii) by observing disappearance of a reflex and (iii) by observing disappearance of sensation. Isolated preparations like frog-sciatic gastrocnemius muscle preparation can be used to demonstrate the nerve block effect of local anesthetics. The nerve is exposed to the solution of drug under test and muscle contraction to nerve stimulation is recorded.  Electric impulses are given by a nerve stimulator. To study disappearance of a reflex, a drug is directly applied in different concentrations on the cornea. The disappearance of the blink response, when cornea is touched with hair, determines the onset of local anesthetic action.

            The disappearance of sensation is observed with twitch response test in guinea pig skin. The duration and intensity of effect of drugs can be assessed with this technique. For wheal preparation the skin on lower back of guinea pig is shaved by clipping or using depilatory cream 24 hours before the experiment. The upper part of the back is more sensitive than the lower part. The skin on the back is stretched and 0.25 ml of the drug is injected intradermally using 26 G needle. The raised wheals over the skin are marked with a felt tipped or a ball point pen (see fig.).                                    

                                                         

                               Fig. Raised wheals marked on back of guinea pig.

            Five minutes after drug injection, the sensitivity of wheal area is compared with skin of some distant point by pricking with needle. Appearance of twitch with each prick shows negative response while absence of twitch on pricking gives positive response. Six twitches are recorded at 5 minutes intervals till 30 minutes after drug injection. Failure of twitch i.e. negative response is indication of anesthesia. The result is expressed as the total number of negative responses out of maximum of 36 possible. The graphs of log dose-effect are compared and the activity is compared by standard assay methods.

Equipment:

              i. Electrical clipper or safety razor or depilatory cream.

             ii. 26Gx3/8 inch hypodermic needles.

            iii. Tuberculin syringe (2).

             iv. Time clock

Drugs: Procaine hydrochloride (3.3x10-3 M and 3.3x10-2M) or lignocaine (1x10-3M)

 

9. Evaluation of skeletal muscle relaxant activity:

            Drugs which cause relaxation of skeletal muscles are called skeletal muscle relaxants. These are of two types depending upon the site of action.

  1. Central skeletal muscle relaxants: Such drugs act on central nervous system and relax the skeletal muscles.
  2. (ii) Peripheral skeletal muscle relaxants: Such drugs act on peripheral nervous system at the motor end-plate.

These are further of two types:

            (a) Competitive or non-depolarizing type- cause flaccid paralysis e.g. d-tubocurarine, gallamine, and

            (b) Non-competitive or depolarizing type- cause spastic paralysis e.g. decamethonium, suxemethonium.

Screening Procedures:

A. Central skeletal muscle relaxants.

(i) Primary screening tests:

(a) Gross observational techniques: These are mostly employed in mice. A decrease in tone of the abdominal muscle, selective depression of the pinnal reflex, relative absence of pawing movements, ascending reversible hind limb weakness, hind drop, paraplegia, loss of righting reflex, good respiratory exchange and absence of stupor  provide the evidence for the potential central muscle relaxant activity.

(b) Anticonvulsant activity: Central muscle relaxants block the tonic phase of maximal electroshock convulsions, strychnine and high dose of metrazole- induced convulsions.

(c) Anti-tremor activity: Tremors produced by nicotine, veratrum or oxotremorine are blocked by central muscle relaxants.

(d) Analgesic activity: central muscle relaxants possess good analgesic activity and potentiate morphine analgesia.

(e) Potentiation of barbiturate sleeping time.

(f) Local anaesthetic activity: Central muscle relaxants possess local anaesthetic activity comparable to procaine.

(ii) Secondary screening tests:

(a) Effect on decerebrate rigidity: The decerebrate rigidity of skeletal muscles is produced mostly in cats by intercollicular prepontine transaction or by causing ischemia of brain by ligating both common carotid and the basilar artery. The central muscle relaxants block the decerebrate rigidity.

(b) Spasticity produced by temporary spinal cord asphyxia (by compression of the descending aorta) is antagonized by central muscle relaxants.

(c)  Muscle spasm caused by tetanus toxin and strychnine are antagonized by central muscle relaxants.

(d) Monosynaptic reflexes are not affected by central muscle relaxants e.g. patellar reflex produced by taping the tendon of quadriceps femoris muscle at the patella.

(e) Polysynaptic reflexes are blocked by central muscle relaxants e. g. linguomandibular reflex, flexor reflex, cross extensor reflex etc.

B. Peripheral muscle Relaxants:

(a) Frog rectus abdominis muscle preparation– acetylcholine-induced contractions are blocked by  peripheral muscle relaxants.

(b) Sciatic nerve-tibialis anterior muscle preparation–The contractions caused by stimulating the nerve are blocked by peripheral muscle relaxants.

(c) Sciatic nerve-gastrocnemius muscle preparation– Contractions caused by stimulating the nerve are blocked by peripheral muscle relaxants.

(d) Rat phrenic nerve diaphragm preparation– Contractions caused by stimulating the nerve are blocked by peripheral muscle relaxants.   

(e) Gross observation–Head drop is noticed   (see fig.)    

                        

                                                             Fig. Head drop in rabbit  

 

 

 

 

 

 

 

 

EXERCISE – 16

Objective:       To demonstrate the effect of drugs on neuromuscular coordination in

                        mice (Rota-rod Test)

Procedure:

  1. Select adult mice of either sex.
  2. Train the mice to walk on a rota-rod (2 cm in diameter), rotated at by an electric motor at 12 rpm, till the mice can balance on rotating rod for at least 2 minutes (2 to 4 trials are given per day for two days).
  3. Select the mice which successfully pass three complete trials per day for two days.
  4. Administer any of the following test compounds, intraperitoneally. 

Chlorpromazine: 1,2,4 and 8 mg/kg.

Chlordiazepoxide: 5, 10, 20, 40 and 80 mg/kg,

Meprobamate: 50, 100, 150, 200 and 400 mg/kg,

  1. Mice in control group are to be treated with normal saline. Use 10 mice per group per dose.
  2. Place the mice on rotating rod at 5, 10,15, 30, 60, 90, 120, 180 and 240 min after the drug treatment.
  3. Note the number of mice in each group falling off within 120 seconds, at each time interval.
  4. Plot the percentage of mice falling against time interval and determine
    1. The time of peak effect at any dose level.
    2. Note the onset of action and recovery from drug effect.
    3.  Determine the ED50 of drug from the percentage fall with different doses at the time of peak effect.

Various drugs can be compared for their potency / efficacy.

 

Table: Neuromuscular incoordination in mice

Drug

                          No. of mice falling off (Total mice =10)

5 min

10 min

15 min

30 min

60 min

90 min

120 min

180 min

240 min

Control

 

 

 

 

 

 

 

 

 

CPZ

1 mg/kg

 

 

 

 

 

 

 

 

 

2 mg/kg

 

 

 

 

 

 

 

 

 

4 mg/kg

 

 

 

 

 

 

 

 

 

8 mg/kg

 

 

 

 

 

 

 

 

 

 

Determine the time of peak drug effect at any dose level.

 

 

 

Note the onset of action and recovery from drug effect.

 

 

 

Determine the ED50 of drug from the percentage fall with different doses at the time of peak effect.

 

 

 

Various drugs can be compared for their potency.

 

 

 

 

 

 

 

Signature of instructor                                                                  Signature of student
EXERCISE – 17

Objective:       To demonstrate the effect of drugs on spontaneous motor activity in mice using photoactometer.

Procedure:

  1. Divide mice into groups of 6 mice each.
  2. Mice in control group are treated with normal saline.
  3. The mice in rest of the groups are injected different doses of chlorpromazine e.g. 0.5, 1, 2 and 4 mg/kg, intraperitoneally.
  4. Measure the activity of each group by placing them in an photoactometer for 5 min, at an intervals of ½, 1, 1½, 2 and 3 hr after drug treatment.
  5. Calculate the percentage decrease in activity in comparison to saline treated animals, considering saline control activity as 100 % activity.
  6. Determine the time at which each drug shows maximum effect.
  7. To calculate ED50 value, take the motor activity of each group for 5 min at the time of peak drug effect. Calculate the ED50 from the results obtained. For this purpose at least six drug treated groups are taken.

 

Table: Spontaneous motor activity in mice.

 

 Drugs

                            Counts on photoactometer

½ hr

1 hr

1½ hr

2 hr

3 hr

Saline

 

 

 

 

 

CPZ (0.5)

 

 

 

 

 

CPZ (1.0)

 

 

 

 

 

CPZ (2.0)

 

 

 

 

 

CPZ (4.0)

 

 

 

 

 

 

 

                                                    Counts of drug treated group × 100

 Per cent decrease in activity =   ––––––––––––––––––––––––––––

                                                     Counts of control group

 

EXERCISE-18

Objective:      To demonstrate the effect of drugs on conditioned avoidance response                         (CAR) in rats using Cook’s Pole climbing Apparatus.

Procedure:    

  1. Select some rats of either sex weighing 80 to 100 g.
  2. Place a single rat in the chamber of Cook’s pole climbing apparatus.
  3. Deliver a series of foot shocks (80 volts, 5 pulse/sec) through the grid floor, concurrently the buzzer is switched on.
  4. The rats learn to climb on hanging wooden rod to avoid foot shock (UCAR) and climb on buzzer sound only even when foot shock is not delivered (CAR).
  5.  Administer appropriate doses of chlorpromazine and test for inhibition of CAR and UCAR at various time intervals after drug administration.
  6. Administer appropriate doses of diazepam and test for inhibition of CAR and UCAR at various time intervals after drug administration
  7. Tabulate your observations.

 

Table: Inhbition or blockade of CAR / UCAR.

 

Drugs

                                    Time after drug administration

½ hr

1 hr

1½ hr

2 hr

3 hr

Saline

 

 

 

 

 

CPZ

 

 

 

 

 

Diazepam

 

 

 

 

 

 

 

 

 

 

 

 

 

EXERCISE – 19

Objective:       To demonstrate the analgesic effect of drugs in mice using Hotplate analgesiometer.

Procedure:    

  1. Take albio mice of either sex weighing 20-30 g.
  2. Select the mice which show reaction when put on hot plate. 
  3. Take ten mice, weigh and mark them and divide into two groups of five mice each.
  4. Prepare the solution of pentazocin.
  5. Put the individual mouse on the hot plate maintained at 55.5±0.5°C temperature. Note down the reaction time of all the mice before administering the drug.
  6. Administer two different doses of pentazocin to mice in both the groups and note down the reaction time of individual mouse at various time intervals.
  7. Record the increase in reaction time.   

 

Table: Analgesic effect of pentazocin (Group I)

 

Time (min)

Animal No./Reaction time (min)

Mean

1

2

3

4

5

0

 

 

 

 

 

 

30

 

 

 

 

 

 

60

 

 

 

 

 

 

90

 

 

 

 

 

 

 

Table: Analgesic effect of pentazocin (Group II)

 

Time (min)

Animal No./Reaction time (min)

Mean

1

2

3

4

5

0

 

 

 

 

 

 

30

 

 

 

 

 

 

60

 

 

 

 

 

 

90

 

 

 

 

 

 

 

EXERCISE – 20

Objective:       To study the anticonvulsant effect of drugs in mice using electroshock method.

 

Procedure:

  1. Prepare the solution of phenobarbital sodium.
  2. Select mice of either sex weighing 20-30 g.
  3. Screen the mice on previous day by giving electrical shock through corneal/pinnal electrodes (48 mA, 0.2 sec.). Select the mice which show extension of hind limbs.
  4. Make two groups of five mice each.
  5. Keep one group as control and another as test group.
  6. Administer normal saline or vehicle of the drug to the control group and the drug solution to the test group.
  7. Compare the response of the mice in each group when subjected to electrical shock after one hour of drug administration.
  8. Note percent of mice which do not show hind limb extension.

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Notes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

99999

 

 

 

 

 

 

 

 

 

 

 

 

 

Notes

 

 

 

 

 

Facilities