LABORATORY MANUAL OFisca.co.in/ANI_VAT_AND_FISHRY/lab_manual/Laborato… · ·...

LABORATORY MANUAL OF

NEUROPHARMACOLOGY

(For Students of Pharmacology of Veterinary, Medical, Ayurvedic and

Pharmacy Disciplines)

By

DR. GOVIND PANDEY BVSc & AH, MVSc & AH, PhD Hon. (Pharmacol.), DSc (std.), LLB, LLM, MBA,

MA (Soc.), MA (Hin.), MA (Eng.), MA (Pol.), Acharya (Jyotish), PGDPA & LSG,

PGDCA, AvR, MDEH, SR, DNHE, AIT, PGPHT, FSLSc, FASAW, FISCA

Professor / Principal Scientist & Sectional Head, Department of Pharmacology & Toxicology, College of Veterinary Science

& Animal Husbandry, Rewa (The Nanaji Deshmukh Veterinary Science

University, Jabalpur), MP, India

2013

International E – Publication www.isca.co.in

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Phone: 91-731-2616100, Mobile: 91-8057083382, E-mail: [email protected],

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© Copyright Reserved

2013 All rights reserved. No part of this publication may be reproduced, stored, in

a retrieval system or transmitted, in any form or by any means, electronic,

mechanical, photocopying, reordering or otherwise, without the prior

permission of the publisher.

ISBN: 978-81-927544-4-4

PREFACE

“Nervous system” is the system of cells, tissues and organs regulating the body’s

responses to internal and external stimuli. In vertebrates, it consists of the brain, spinal

cord, nerves, ganglia and parts of the receptor and effector organs. It is the system of

neurons and tissues regulating the actions and responses of vertebrates and many

invertebrates. It receives chemical information from hormones in the circulating blood,

and can also regulate the secretions of endocrine system by the action of neurohormones.

Therefore, this system is of great importance.

With regard to “Neuropharmacology”, the nervous system is very sensitive to

various drugs. The drugs acting on brain (CNS) may be either stimulants or depressants

that block or enhance certain neurotransmitters. Dopamine is involved with all forms of

pleasure. Cocaine interferes with the uptake of dopamine from the synaptic cleft. Alcohol

causes a euphoric ‘high’, followed by a depression. Heroin is a derivative of morphine

which causes ‘euphoria’. Similarly, several drug act on the peripheral nervous system.

Here, some drugs or neurotransmitters like acetylcholine, norepinephrine, serotonin and

dopamine may be excitory. Some of these are associated with relaxation, e.g., dopamine

and serotonin. Dopamine release seems related to sensations of pleasure. Endorphins are

natural opioids that produce elation and reduction of pain, as do artificial chemicals such

as opium and heroin. The neurological diseases, e.g., Parkinson’s disease and

Huntington’s disease, are due to imbalances of neurotransmitters. Parkinson’s disease is

due to the dopamine deficiency. Huntington’s disease is thought to be cause by

malfunctioning of an inhibitory neurotransmitter. Alzheimer’s disease is associated with

the protein plaques in the brain. In this view, therefore, the actions of

neuropharmacological agents must be studied experimentally on different tissues/organs

of the animals. Hence with this aim, the “Laboratory Manual of Neuropharmacology”

has been put forward. All 26 chapters/experiments included in this manual are the

practical tools of the “Neuropharmacology”. Therefore, this manual is a fruitful and

resourceful material for the students of Pharmacology of Veterinary, Medical, Ayurvedic

and Pharmacy disciplines, as well as to the teachers and scientists of Pharmacology.

In this context, I would like to pay my sincere thanks and deep regards to Dr. S.P.

Shukla, Dean, College of Veterinary Science & Animal Husbandry, Rewa (NDVSU,

Jabalpur), MP for the “Foreword” he has provided for this manual. I am also thankful to

all the authors/publishers/books/websites, especially to the Google Website, Purves et al.

(Life: The Science of Biology, 4th

edition by Sinauer Associates- www.sinauer.com and

WH Freeman- www.whfreeman.com), Dennis Kunkel (www.DennisKunkel.com) and

HowToMedia, Inc. (Innerbody.com), from where the matters and photographs have been

extracted and incorporated in this manual; I gratefully acknowledge to all those. Last but

not the least, I am cordially thankful to Dr. Nitish Kumar, Dr. Swatantra Singh and Dr.

Anjana Panicker (all teachers of Veterinary Pharmacology and Toxicology in the College

of Veterinary Science & AH, Rewa).

26th

June, 2013 Dr. Govind Pandey

iv

CONTENTS

S. No. Description Page

- Foreword iii

- Preface iv

- Contents v

1 General Study of Nervous System

Objective

Nervous System

Functions of Nervous System

Divisions of Nervous System

Central Nervous System

Peripheral Nervous System

Nervous Tissues

Reflexes

Sense Organs

Action Potential

Brain and Drugs

Important Questions

1

1

1

1

2

3

6

10

17

17

19

20

21

2 Alternatives of Animals as Models for Experimentation

Objective

Principle

Common Laboratory Animals

Alternatives of Animal

Problems Concerned with Animal Alternatives

Important Questions

22

22

22

22

23

24

24

3 Study of Analgesic Effects of Drugs

Objective

Principle

Methods

Important Questions

25

25

25

25

26

4 Study of Analgesic Effects of Drugs by Hot Plate Method

Objective

Requirements

Procedure

Observations and Conclusion

Important Questions

27

27

27

27

28

28

5 Study of Analgesic Effects of Drugs by Tail Flick Method

Objective

Requirements

Procedure


Important Questions

29

29

29

29

30

30

6 Study of Effects of Local Anesthetics

Objective 31

31

Definition and Types of Local Anaesthesia

Requirements

Principle

Procedure


Important Questions

31

31

31

32

32

32

7 Study of Effects of Central Nervous System Depressants

Objective

Requirements

Procedure


Important Questions

33

33

33

33

34

34

8 Study of Effects of Pentobarbitone and Chlorpromazine

Objective

Requirements

Principle

Procedure


Important Questions

35

35

35

36

36

37

37

9 Study of Effects of Central Nervous System Stimulants

Objective

Requirements

Procedure


Important Questions

38

38

38

38

39

39

10 Study of Effects of Muscle Relaxants

Objective

Requirements

Principle

Procedure


Important Questions

40

40

40

40

41

41

41

11 Study of Effects of Drugs on Motor Activity by Actophotometer

Objective

Requirements

Principle

Procedure


Important Questions

42

42

42

42

43

43

43

12 Study of Effects of Drugs on Conditioned Avoidance Response

Objective

Requirements

Principle

Procedure


44

44

44

44

45

45

Important Questions 45

13 Study of Effects of Anticonvulsants by Electro-Convulsometer

Objective

Requirements

Principle

Procedure


Important Questions

46

46

46

46

47

47

47

14 Study of Effects of Anticonvulsants by Leptazole Test

Objective

Requirements

Principle

Procedure


Important Questions

48

48

48

48

48

49

49

15 Study of Equipments and Preparation of Physiological Salt Solutions

for Experiments on Isolated Tissues

Objective

Principle of Physiological Salt Solution and Isolated Tissue Preparation

Student’s Organ Bath (Isolated Organ Bath)

Preparation of Different Materials

Commonly used Physiological Salt Solutions

Sacrificing of Animals

Preparation of Tissues

Important Questions

50

50

50

51

53

54

55

57

58

16 Study of Effects of Sympathetic and Parasympathetic Drugs on

Duodenum

Objective

Requirements

Principle

Procedure and Observations

Important Questions

59

59

59

59

59

61

17 Study of Effects of Histaminic and Antihistaminic Drugs on Ileum

Objective

Requirements

Principle

Procedure

Observations and Inference

Important Questions

62

62

62

62

63

64

64

18 Study of Effects of Sympathetic and Parasympathetic Drugs on Fundus

Objective

Requirements


Important Questions

65

65

65

65

67

19 Study of Effects of Some Drugs on Gastrointestinal Motility 68

Objective

Requirements

Principle

Procedure


Important Questions

68

68

68

68

69

69

20 Study of Effects of Some Drugs on Uterus

Objective

Requirements

Principle

Procedure


Important Questions

70

70

70

70

70

71

71

21 Study of Effects of Cardiac Glycosides on Heart

Objective

Requirements

Principle

Procedure


Important Questions

72

72

72

72

73

73

73

22 Study of Effects of Parasympathetic Drugs on Eye

Objective

Requirements

Principle

Procedure


Important Questions

74

74

74

74

74

75

75

23 Study of Effects of Some Drugs on Blood Coagulation

Objective

Requirements

Principle

Procedure


Important Questions

76

76

76

76

76

77

77

24 Study of Effects of Some Drugs on Blood Pressure During Anaesthesia

Objective

Requirements

Principle

Procedure


Important Questions

78

78

78

78

79

79

80

25 Study of Effects of Ganglionic Stimulants and Blockers

Objective

Requirements

Principle

81

81

81

81

Procedure


Important Questions

81

82

82

26 Study of Effects of Some Drugs on Autonomic Nervous System

Objective

Requirements

Principle


Important Questions

83

83

83

84

84

85

- About the Author

86

ix

1

GENERAL STUDY OF NERVOUS SYSTEM

OBJECTIVE

To study about the nervous system in general.

NERVOUS SYSTEM

“Nervous system is defined as all the cells, tissues and nerves that coordinate

responses to internal and external stimuli”. For example, what brain tells to react when

we cut our finger while chopping the vegetables? Therefore, according to the medical

science, the nervous system is the system of cells, tissues and organs regulating the

body’s responses to internal and external stimuli. In vertebrates, it consists of the brain,

spinal cord, nerves, ganglia and parts of the receptor and effector organs.

According to science, nervous system is the system of neurons and tissues regulating

the actions and responses of vertebrates and many invertebrates. Nervous system of

vertebrates is a complex information-processing system that consists mainly of brain,

spinal cord, and peripheral and autonomic nerves. It receives chemical information from

hormones in circulating blood and can also regulate the secretions of endocrine system by

the action of neurohormones. The nervous systems of invertebrates vary from a simple

network of nerves to a complex nerve network under the control of a primitive brain.

All the nerve cells and nervous tissues in an organism, and the brain, spinal cord,

ganglia, nerves and nerve centres in vertebrates, coordinate and control the responses to

stimuli and conditions behaviour and consciousness.

FUNCTIONS OF NERVOUS SYSTEM

Multicellular animals must monitor and maintain a constant internal environment, as

well as monitor and respond to an external environment. In many animals, these two

1

functions are coordinated by two integrated and coordinated organ systems: the “nervous

system” and the “endocrine system”.

The nervous system monitors and controls almost every organ system through a

series of positive and negative feedback loops. Hence, the nervous system has 3 main

functions: sensory, integration and motor.

1. Sensory function: The sensory function involves collecting the information from

sensory receptors that monitor the body’s internal and external conditions. These

signals are then passed on to the “central nervous system” (CNS) for further

processing by afferent neurons (and nerves). It means that the nervous system

receives the sensory input from internal and external environments. The sensory input

can be in many forms, including pressure, taste, sound, light, blood pH, or hormone

levels which are converted to a signal and sent to the brain or spinal cord.

2. Integration function: Integration process is processing of many sensory signals that

are passed into CNS at given time. These signals are evaluated, compared, used for

decision making, discarded or committed to memory. Integration takes place in gray

matter of brain and spinal cord, and is performed by interneurons. Many interneurons

work together to form complex networks that provide this processing power.

3. Motor output function: Once the networks of interneurons in the CNS evaluate

sensory information and decide on an action, they stimulate efferent neurons. Efferent

neurons (also called ‘motor neurons’) carry signals from gray matter of CNS through

the nerves of peripheral nervous system (PNS) to effector cells. Effector may be

smooth, cardiac, or skeletal/glandular tissue. The effector then releases a hormone or

moves a part of body to respond to the stimulus. In the sensory centers of brain, or in

spinal cord, barrage of input is integrated and a response is generated. The response, a

motor output, is a signal transmitted to organs than can convert the signal into some

form of action, such as movement, changes in heart rate, release of hormones, etc.

DIVISIONS OF NERVOUS SYSTEM

All animals necessarily do not have highly specialized nervous systems. Those with

2

simple systems tend to be either small and very mobile or large and immobile. Large,

mobile animals have highly developed nervous systems: the evolution of nervous systems

must have been an important adaptation in the evolution of body size and mobility.

Coelenterates, cnidarians and echinoderms have their neurons organized into a nerve net.

These creatures have radial symmetry and lack a head. Although lacking a brain or either

nervous system (CNS or PNS) nerve nets are capable of some complex behaviour.

Bilaterally symmetrical animals have a body plan that includes a defined head and a tail

region. Development of bilateral symmetry is associated with cephalization, the

development of a head with the accumulation of sensory organs at the front end of the

organism. Flatworms have neurons associated into clusters known as ganglia, which in

turn form a small brain. Vertebrates have a spinal cord in addition to a more developed

brain. Chordates have a dorsal rather than ventral nervous system. Several evolutionary

trends occur in chordates: spinal cord, continuation of cephalization in the form of larger

and more complex brains, and development of a more elaborate nervous system.

The vertebrate nervous system is divided into the following main parts (Fig. 1 & 2):

1. Central Nervous System (CNS)- Brain and spinal cord.

2. Peripheral Nervous System (PNS)- Somatic nervous system (SNS) and autonomic

nervous system (ANS- sympathetic and parasympathetic systems).

CENTRAL NERVOUS SYSTEM

CNS is composed of the “brain” and “spinal cord”. The CNS is surrounded by bone-

skull and vertebrae. Fluid and tissue also insulate the brain and spinal cord.

Brain:

During embryonic development, the “brain” first forms as a tube, the anterior end of

which enlarges into three hollow swellings that form the brain and the posterior of which

develops into the spinal cord. Some parts of the brain have changed little during the

vertebrate evolutionary history. The vertebrate evolutionary trends include:

a. Increase in brain size relative to body size.

3

Fig. 1: Divisions of Nervous System

Fig. 2: Nervous System

(Source of figures: Different websites/authors/publishers that are gratefully acknowledged)

b. Subdivision and increasing specialization of forebrain, midbrain and hindbrain.

c. Growth in relative size of the forebrain, especially cerebrum, which is associated

with increasingly complex behaviour in mammals.

Brain is composed of 3 parts: cerebrum (seat of consciousness), cerebellum and

medulla oblongata (these latter two are the ‘part of unconscious brain’) (Fig. 3 & 4).

4

I. Forebrain, Diencephalon and Cerebrum-

“Forebrain” consists of the ‘diencephalon’ and ‘cerebrum’. The diencephalon is

divided into ‘thalamus’ and ‘hypothalamus’. Thalamus acts as a switching centre for

nerve messages. Hypothalamus is a major homeostatic centre having both nervous and

endocrine functions. Hypothalamus has regulatory areas for thirst, hunger, body

temperature, water balance and blood pressure (BP), and links the nervous system to the

endocrine system. Thalamus serves as central relay point for incoming nervous messages.

Cerebrum, the largest part of the human brain, is divided into left and right ‘hemispheres’

connected to each other by the ‘corpus callosum’. Thus, the conscious brain includes the

‘cerebral hemispheres’, which are separated by the corpus callosum. The hemispheres

are covered by a thin layer of gray matter known as the ‘cerebral cortex’, the most

recently evolved region of the vertebrate brain. Fish have no cerebral cortex; while

amphibians and reptiles have only rudiments of this area. In reptiles, birds and mammals,

the cerebrum coordinates sensory data and motor functions. The cerebrum governs

intelligence and reasoning, learning and memory.

Cortex in each hemisphere of cerebrum is between 1 and 4 mm thick. Folds divide

the cortex into 4 lobes: occipital, temporal, parietal and frontal. No region of brain

functions alone, although major functions of various parts of lobes have been determined.

The ‘occipital lobe’ (back of the head) receives and processes visual information. The

‘temporal lobe’ receives auditory signals, processing language and the meaning of words.

The ‘parietal lobe’ is associated with the sensory cortex and processes information about

touch, taste, pressure, pain, heat and cold. The ‘frontal lobe’ conducts motor activity and

integration of muscle activity; speech; and thought processes.

Most people have their language and speech areas on the left hemisphere of their

brain. Language comprehension is found in the Wernicke’s area. Speaking ability is in

the Broca’s area. Damage to Broca’s area causes speech impairment but not impairment

of language comprehension. Lesions in Wernicke’s area impair the ability to comprehend

written and spoken words but not speech. Remaining parts of the cortex are associated

with higher thought processes, planning, memory, personality and other human activities.

5

II. Midbrain, Brain Stem and Medulla Oblongata-

“Midbrain” consists of connections between hindbrain and forebrain. Mammals use

this part of brain only for eye reflexes. Midbrain and pons are also the part of

unconscious brain. “Brain stem” is the smallest, oldest and most primitive part of the

brain. It is continuous with the spinal cord, and is composed of the parts of hindbrain and

midbrain. ‘Medulla oblongata’ and ‘pons’ control the heart rate, constriction of blood

vessels, digestion and respiration. The medulla oblongata is closest to the spinal cord and

is involved with the regulation of heart beat, breathing, vasoconstriction (blood pressure),

and reflex centres for vomiting, coughing, sneezing, swallowing and hiccupping.

III. Hindbrain and Cerebellum-

The third part of “hindbrain” is ‘cerebellum’, but it is not considered part of the brain

stem. Cerebellum is the second largest part of brain after the cerebrum. Its functions

include fine motor/muscle coordination and body movement, posture and balance. This

region of the brain is enlarged in birds and controls muscle action needed for flight.

Spinal Cord:

“Spinal cord” runs along the dorsal side of the body and links the brain to the rest of

the body. Vertebrates have their spinal cords encased in a series of (usually) bony

vertebrae that comprise the vertebral column. The ‘gray matter’ of the spinal cord

consists mostly of ‘cell bodies’ and ‘dendrites’. The surrounding ‘white matter’ is made

up of the bundles of ‘interneuronal axons’ (tracts). Some tracts are ascending (carrying

messages to the brain) and others are descending (carrying messages from the brain). The

spinal cord is also involved in reflexes that do not immediately involve the brain.

PERIPHERAL NERVOUS SYSTEM

The PNS (Fig. 5) connects the CNS to the rest of body, and is composed of nerves

(‘bundles of neurons’). On the other hand, the PNS consists of all body nerves.

The ‘axons’ and ‘dendrites’ are surrounded by a white myelin sheath. The cell bodies

6

Fig. 3: Parts of Brain

Fig. 4: Brain’s Functional Areas

Fig. 5: Functions of Peripheral Nervous System

(Source of figures: Different websites/authors/publishers that are gratefully acknowledged)

7

are in the CNS or ganglia. The ‘ganglia’ are collections of nerve cell bodies. The cranial

nerves in the PNS take impulses to and from the brain (CNS). The spinal nerves take

impulses to and away from the spinal cord (CNS). The main components of the PNS are:

1. Sensory (afferent) pathways- They provide input from the body into the CNS.

2. Motor (efferent) pathways- They carry signals to muscles and glands (effectors).

Most of the sensory inputs carried in the PNS remains below the level of conscious

awareness. The input that does reach the conscious level contributes to the perception of

our external environment. There are two major subdivisions of the PNS motor pathways:

‘somatic’ (skeletal) and ‘autonomic’ (smooth muscle, cardiac muscle, and glands).

I. Somatic Nervous System:

SNS includes all nerves controlling the muscular system and external sensory

receptors. The external sense organs (including skin) are ‘receptors’. The muscle fibers

and gland cells are ‘effectors’. The ‘reflex arc’ is an automatic, involuntary reaction to a

‘stimulus’. The reaction to the stimulus is involuntary, with the CNS being informed but

not consciously controlling the response. The examples of reflex arcs are balance,

blinking reflex and stretch reflex. The sensory input from the PNS is processed by the

CNS and responses are sent by the PNS from the CNS to the organs of the body. The

‘motor neurons’ of the SNS are distinct from those of the ANS. The inhibitory signals

can not be sent through the motor neurons of the SNS.

II. Autonomic Nervous System:

ANS is a part of PNS consisting of motor neurons which control the internal organs.

The ANS controls muscles in the heart, smooth muscles in the internal organs like

intestine, bladder and uterus. The motor neurons in the ANS do not reach their targets

directly (as do those in the SNS), but connect to a secondary motor neuron which in turn

innervates the target organ.

The ANS is mainly subdivided into two systems (Fig. 6): the “sympathetic” and the

“parasympathetic”. According to some, one more subdivision of ANS is the “enteric”.

8

Fig. 6: Functions of Sympathetic and Parasympathetic Nervous Systems (Source of figure: Website/author/publisher that is gratefully acknowledged)

A. Sympathetic Nervous System-

This is involved in the ‘fight’ (flight) response to stress, danger, excitement, exercise,

emotions and embarrassment. This increases respiration and heart rate, releases

adrenaline and other stress hormones, and decreases digestion to cope with the situations.

B. Parasympathetic Nervous System-

This system forms the body’s ‘rest’ and ‘digest’ response when the body is relaxed,

resting or feeding. This system works to undo the work of sympathetic system after a

stressful situation. Other functions of this are: to decrease respiration and heart rate,

increase digestion, and permit the elimination of wastes. Each of these subsystems

operates in the reverse of other (antagonism).

Both above systems innervate the same organs and act in opposition to maintain

homeostasis. For example, when we are scared, the sympathetic system causes our heart

to beat faster; while the parasympathetic system reverses this effect.

C. Enteric Nervous System-

“Enteric nervous system” (ENS) is responsible for regulating digestion and function

9

of the digestive organs. ENS receives signals from CNS through both sympathetic and

parasympathetic divisions of ANS to regulate its functions. However, the ENS mostly

works independently of CNS and continues to function without any outside input. For

this reason, ENS is often called as “brain of the gut” or body’s “second brain”. The ENS

is an immense system- almost as many neurons exist in the ENS as in the spinal cord.

NERVOUS TISSUES

Most of the portion of the nervous system is “tissue” made up of two types of cells:

the “neuron” and the “neuroglia” (glial cell). The neurons transmit nerve messages, while

the glial cells are in direct contact with the neurons and often surround them.

Neuron:

“Neuron” (or nerve cell) is the functional unit of nervous system. Possibly, humans

have about 100 billion neurons in their brain alone. The neurons communicate within the

body by transmitting electrochemical signals. They look quite different from other cells

in body due to many long cellular processes which extend from their central cell body.

While variable in size and shape, all neurons have 3 parts (Fig. 7):

1. Dendrites-

They are ‘small tree-like structures’ extend from cell body to pick up stimuli from

the environment, other neurons, or sensory receptor cells. Thus, the dendrites receive

information from another cell and transmit message to cell body.

2. Cell Body-

It is the roughly round part of a neuron which contains the nucleus, mitochondria and

most of the cellular organelles typical of eukaryotic cells.

3. Axon-

The long transmitting processes called axons extend from the cell body to send the

10

Fig. 7: Structure of Neuron and Direction of Nerve Message Transmission

Fig. 8: Structure of Nerve Bundle

(Source of figures: Websites/authors/publishers that are gratefully acknowledged)

signals onward to other neurons or effector cells in the body (Fig. 8).

There are 3 basic classes of neurons:

A. Afferent neurons (Sensory neurons)- They have a long dendrite and short axon.

B. Efferent neurons (Motor neurons)- They have a long axon and short dendrites.

They transmit signals from CNS to effectors (e.g., muscles and glands) in body.

C. Interneurons- They are found only in CNS, where they connect neuron to neuron.

They form complex networks within CNS to integrate the information received

from afferent neurons and to direct the function of body through efferent neurons.

11

Some axons are wrapped in a myelin sheath formed from plasma membranes of

specialized glial cells called ‘Schwann cells’, which serve as supportive, nutritive and

service facilities for neurons. The gap between Schwann cells is called ‘node of Ranvier’,

which serves as points along the neuron for generating a signal. The signals jumping

from node to node travel 100s of time faster than the signals traveling along the surface

of axon. This allows our brain to communicate with our toes in a few 1000th

of a second.

Neuroglia (Glial Cell):

“Neuroglia” acts as ‘helper cells’ of nervous system. Neuron in body is surrounded

by anywhere from 6 to 60 neuralgias that protect, feed and insulate the neuron. Because

neurons are extremely specialized cells that are essential to the body function and almost

never reproduce, neuroglias are vital to maintain a functional nervous system.

Nerves:

“Nerves” are the bundles of axons in the PNS that act as information highways to

carry signals between the brain and spinal cord and the rest of the body. Each axon is

wrapped in a connective tissue sheath called the ‘endoneurium’. Individual axons of the

nerve are bundled into groups of axons called ‘fascicles’, wrapped in a sheath of

connective tissue called the ‘perineurium’. Finally, many fascicles are wrapped together

in another layer of connective tissue called the ‘epineurium’ to form a whole nerve. The

wrapping of nerves with connective tissue helps to protect the axons and to increase the

speed of their communication within the body. Different types of nerves in the body are:

a. Afferent, Efferent and Mixed Nerves-

Some of the nerves in the body are specialized for carrying information in only one

direction, similar to a one-way street. The nerves which carry information from sensory

receptors to CNS only are called ‘afferent nerves’. Other neurons called ‘efferent nerves’

carry signals only from the CNS to the effectors such as muscles and glands. Some

nerves are ‘mixed nerves’ which contain both afferent and efferent axons. The mixed

12

nerves function like two-way streets where afferent axons act as lanes heading toward the

CNS, and efferent axons act as lanes heading away from the CNS.

b. Cranial Nerves-

Extending from inferior side of the brain are 12 pairs of ‘cranial nerves’. Each

cranial nerve pair is identified by a ‘Roman numeral’- 1 to 12 based upon its location

along the anterior-posterior axis of the brain. Each nerve also has a descriptive name

(e.g., olfactory, optic, etc.) which identifies its function or location. The cranial nerves

provide a direct connection to brain for the special sense organs, muscles of head, neck

and shoulders, heart, and GIT.

c. Spinal Nerves-

Extending from the left and right sides of the spinal cord are 31 pairs of ‘spinal

nerves’. The spinal nerves are mixed nerves which carry both sensory and motor signals

(messages) between the spinal cord and specific regions of the body. The 31 pairs of the

spinal nerves are split into 5 groups named for the 5 regions of the vertebral column.

Thus, there are 8 pairs of cervical nerves, 12 pairs of thoracic nerves, 5 pairs of lumbar

nerves, 5 pairs of sacral nerves and 1 pair of coccygeal nerves. Each spinal nerve exits

from the spinal cord through the ‘intervertebral foramen’ between a pair of vertebrae or

between the first cervical (C1) vertebra and the occipital bone of the skull.

Synapses:

Junction between a nerve cell and another nerve cell is called a “synapse” (Fig. 9).

The synapses may form between two neurons or between a neuron and an effector cell.

There are two types of synapses found in the body:

1. Chemical Synapses-

At the end of a neuron’s axon is an enlarged region of axon, the ‘axon terminal’, is

separated from the next cell by a small gap called ‘synaptic cleft’ (space between two

13

Fig. 9: Structure of Synapse

(Source of figure: Website/author/publisher that is gratefully acknowledged)

cells). When an ‘action potential’ (AP) reaches the axon terminal, it opens voltage-gated

calcium ion channels. To cross the synaptic cleft requires the actions of

‘neurotransmitters’ (NTs). Calcium ions cause synaptic vesicles containing chemicals

NTs to release their contents by exocytosis into synaptic cleft. So, the NTs are stored in

small synaptic vesicles clustered at the tip of axon. The NT molecules cross the synaptic

cleft and bind to receptor molecules on cell, forming a synapse with neuron. These

receptor molecules open ion channels that may either stimulate the receptor cell to form a

new AP or may inhibit the cell from forming an AP when stimulated by another neuron.

2. Electrical Synapses-

The ‘electrical synapses’ are formed when 2 neurons are connected by small holes

called ‘gap junctions’. The gap junctions allow electric current to pass from one neuron

to the other, so that an AP in one cell is passed directly on to the other cell through the

synapse. The messages travel within the neuron as an ‘electrical action potential’.

Some NTs cause an AP, others are inhibitory. The NTs tend to be small molecules,

some are even hormones. The time for NT action is between 0.5 and 1 millisecond. The

NTs are either destroyed by specific enzymes in the synaptic cleft, diffuse out of the cleft,

or are reabsorbed by the cell. More than 30 organic molecules are thought to act as NTs.

The NTs cross the cleft, binding to receptor molecules on the next cell, prompting

transmission of the message along that cell’s membrane. Acetylcholine (Ach) is an

14

example of a NT, as is norepinephrine, although each acts in different responses. Once in

the cleft, the NTs are active for only a short time. Enzymes in the cleft inactivate the NTs.

The inactivated NTs are taken back into the axon and recycled.

Meninges:

“Meninges” are the protective coverings of the CNS. They consist of three layers:

1. Dura Mater-

The ‘dura mater’ (‘tough mother’) is the thickest, toughest and most superficial layer

of meninges. Made of dense irregular connective tissue, it contains many tough collagen

fibers and blood vessels. It protects CNS from external damage, contains cerebrospinal

fluid that surrounds the CNS, and provides blood to the nervous tissue of the CNS.

2. Arachnoid Mater-

The ‘arachnoid mater’ (‘spider-like mother’) is much thinner and more delicate than

the dura mater. It lines the inside of dura mater and contains many thin fibers which

connect it to the underlying pia mater. These fibers cross a fluid-filled space called

‘subarachnoid space’ between the arachnoid mater and pia mater.

3. Pia Mater-

The ‘pia mater’ (‘tender mother’) is a thin and delicate layer of tissue that rests on

the outside of the brain and spinal cord. Containing many blood vessels that feed the

nervous tissue of the CNS, the pia mater penetrates into the valleys of the sulci and

fissures of the brain as it covers the entire surface of the CNS.

Cerebrospinal Fluid:

The space surrounding the organs of the CNS is filled with a clear fluid called as the

“cerebrospinal fluid” (CSF). The CSF is formed from blood plasma by special structures

epithelial tissue which filters blood plasma and allows the filtered fluid to enter the space

15

around the brain. The newly created CSF flows through the inside of the brain in hollow

spaces called ‘ventricles’ and through a small cavity in the middle of the spinal cord

called ‘central canal’. The CSF also flows through the subarachnoid space around the

outside of brain and spinal cord. The CSF is constantly produced at the choroid plexuses,

and is reabsorbed into the bloodstream at structures called ‘arachnoid villi’.

CSF provides the following vital functions to the CNS:

(1) The CSF absorbs shocks between the brain and skull, and between the spinal cord

and vertebrae. This shock absorption protects the CNS from blows or sudden

changes in velocity, such as during a car accident.

(2) The brain and spinal cord float within the CSF, reducing their apparent weight

through buoyancy. The brain is a very large but soft organ that requires a high

volume of blood to function effectively. The reduced weight in the CSF allows

the blood vessels of brain to remain open and helps protect the nervous tissue

from becoming crushed under its own weight.

(3) The CSF helps to maintain chemical homeostasis within the CNS. It contains

ions, nutrients, oxygen and albumins which support the chemical and osmotic

balance of the nervous tissue. The CSF also removes waste products which form

as byproducts of cellular metabolism within the nervous tissue.

Myelination:

Axons of many neurons are covered by a coating of insulation known as ‘myelin’ to

increase the speed of nerve conduction throughout the body. Myelin is formed by 2 types

of glial cells: ‘Schwann cells’ (in PNS) and ‘oligodendrocytes’ (in CNS). In both cases,

the glial cells wrap their plasma membrane around the axon many times to form a thick

covering of lipids. The development of these ‘myelin sheaths’ is called “myelination”.

The myelination speeds up the movement of APs in the axon by reducing the number

of APs that must form for a signal to reach the end of an axon. The myelination process

begins speeding up nerve conduction in the foetal development and continues into early

adulthood. The myelinated axons appear white due to the presence of lipids and form the

16

‘white matter’ of the inner brain and outer spinal cord. The white matter is specialized for

carrying information quickly through brain and spinal cord. The ‘gray matter’ of brain

and spinal cord are unmyelinated integration centres where information is processed.

REFLEXES

The “reflexes” are fast, involuntary responses to stimuli. Most well known reflex is

‘patellar reflex’, which is checked when a physicians taps on a patient’s knee during

physical examination. Reflexes are integrated in gray matter of spinal cord or in brain

stem. They allow body to respond to stimuli quickly by sending responses to effectors

before nerve signals reach the conscious parts of brain. This explains why people will

often pull their hands away from a hot object before they realize that they are in pain.

SENSE ORGANS

“Sensory input” to the nervous system is in the form of five ‘special senses’: vision,

hearing, balance, smell and taste. Structurally, there are three classes of ‘sensory

receptors’- free nerve endings, encapsulated nerve endings and specialized cells. Free

nerve endings are simply free dendrites at the end of a neuron that extend into a tissue.

Pain, heat and cold are all sensed through free nerve endings. Encapsulated nerve ending

is a free nerve ending wrapped in a round capsule of connective tissue. When the capsule

is deformed by touch or pressure, the neuron is stimulated to send signals to CNS.

Specialized cells detect stimuli from the above five senses, each of which has its own

sensory cells, e.g., ‘rods’ and ‘cones’ in the retina to detect the light for sense of vision.

Sensory Receptors:

The “sensory receptors” are classified according to the type of energy they can detect

and respond to. Functionally, there are following 7 major categories of sensory receptors:

i. ‘Mechanoreceptors’ are responsible for hearing, balance and stretching. They are

sensitive to mechanical stimuli like touch, pressure, vibration and blood pressure.

ii. ‘Photoreceptors’ detect light in the retina to provide the sense of vision.

17

iii. ‘Chemoreceptors’ detect chemicals in bloodstream and provide the senses of taste

and smell, as well as internal sensors in the digestive and circulatory systems.

iv. ‘Nociceptors’ respond to stimuli, such as extreme heat, cold or tissue damage by

sending the pain signals to the CNS.

v. ‘Osmoreceptors’ monitor blood osmolarity to determine body’s hydration levels.

vi. ‘Thermoreceptors’ detect temperatures inside the body and in its surroundings.

vii. ‘Electroreceptors’ detect electrical currents in the surrounding environment.

The ‘mechanoreceptors’ vary greatly in the specific type of stimulus and duration of

stimulus/action potentials. The most adaptable vertebrate mechanoreceptor is the hair

cell. The hair cells are present in the lateral line of fish. In humans and mammals, the hair

cells are involved with the detection of sound and gravity, and providing balance.

Hearing:

“Hearing” involves the actions of the external ear, eardrum, ossicles and cochlea. In

hearing, the sound waves in air are converted into vibrations of a liquid, then into

movement of hair cells in the cochlea and finally, they are converted into action

potentials in a sensory dendrite connected to the auditory nerve. Very loud sounds can

cause violent vibrations in the membrane under hair cells, causing a shearing or

permanent distortion to the cells, resulting in permanent hearing loss.

Orientation and Gravity:

“Orientation and gravity” are detected at the semicircular canals. Hair cells along 3

planes respond to shifts of liquid within the cochlea, providing a three-dimensional sense

of equilibrium. Calcium carbonate crystals can shift in response to gravity, providing

sensory information about gravity and acceleration.

Photoreceptors:

The “photoreceptors” detect vision and light sensitivity. The human eye can detect

light in the 400 to 700 nanometers (nm) range. The light with wavelengths shorter than

18

400 nm is termed ‘ultraviolet’ (UV) light. The light with wavelengths longer than 700 nm

is termed ‘infrared’ (IR) light.

In the “eye”, two types of photoreceptor cells (viz., ‘rods’ and ‘cones’) are clustered

on the retina, or back portion of the eye. These receptors, rods and cones, apparently

evolved from the hair cells. The rods detect differences in the light intensity; the cones

detect colour. The rods are more common in a circular zone near the edge of the eye. The

cones occur in the centre (or ‘fovea centralis’) of the retina. The light reaching a

photoreceptor causes the breakdown of the chemical ‘rhodopsin’, which in turn causes a

membrane potential that is transmitted to an action potential. The action potential

transfers to ‘synapsed neurons’ which connect to the optic nerve. The optic nerve

connects to the occipital lobe of the brain. The humans have 3 types of cones, each

sensitive to a different colour of light: red, blue and green. The ‘opsins’ are chemicals

which bind to cone cells and make those cells sensitive to the light of a particular

wavelength (or colour). The humans have three different forms of opsins coded for by

three genes on the ‘x chromosome’. The defects in one or more of these opsin genes can

cause colour blindness, usually in the males.

ACTION POTENTIAL

Neurons function through the generation and propagation of electrochemical signals

known as “action potential” (AP). An AP is created by the movement of sodium and

potassium ions through the membrane of neurons. The following steps happen in the AP:

1. Resting Potential:

At rest, the neurons maintain a concentration of sodium (Na) ions outside of the cell

and potassium (K) ions inside of the cell. This concentration is maintained by the

‘sodium-potassium pump’ of the cell membrane which pumps three sodium ions out of

the cell for every two potassium ions that are pumped into the cell. The ion concentration

results in a resting electrical potential of -70 millivolts (mV), which means that the inside

of the cell has a negative charge compared to its surroundings.

19

2. Threshold Potential:

If a stimulus permits enough positive ions to enter a region of the cell to cause it to

reach -55 mV, that region of the cell will open its voltage-gated sodium channels and

allow sodium ions to diffuse into the cell. The -55 mV is the threshold potential for

neurons as this is the ‘trigger’ voltage that they must reach to cross the threshold into

forming an AP.

3. Depolarization:

Sodium carries a positive charge that causes cell to become depolarized (positively

charged) compared to its normal negative charge. The voltage for depolarization of all

neurons is +30 mV. The depolarization of the cell is the AP that is transmitted by the

neuron as a nerve signal. The positive ions spread into neighboring regions of the cell,

initiating a new AP in those regions as they reach -55 mV. The AP continues to spread

down the cell membrane of the neuron until it reaches the end of an axon.

4. Repolarization:

After the depolarization the voltage of +30 mV is reached, the voltage-gated

potassium ion channels open, allowing the positive potassium ions to diffuse out of the

cell. The loss of potassium along with the pumping of sodium ions back out of the cell,

though the sodium-potassium pump restores the cell to the -55 mV resting potential. At

this point, the neuron is ready to start a new AP.

BRAIN AND DRUGS

Some NTs are excitory, such as acetylcholine (Ach), norepinephrine, serotonin and

dopamine. Some are associated with relaxation, such as dopamine and serotonin.

Dopamine release seems related to sensations of pleasure. Endorphins are natural opioids

that produce elation and reduction of pain, as do artificial chemicals such as opium and

heroin. Neurological diseases, e.g., Parkinson’s disease and Huntington’s disease, are

due to imbalances of NTs. Parkinson’s disease is due to the dopamine deficiency.

20

Huntington’s disease is thought to be cause by malfunctioning of an inhibitory NT.

Alzheimer’s disease is associated with the protein plaques in the brain.

The brain acting drugs may be either stimulants or depressants that block or enhance

certain NTs. Dopamine is involved with all forms of pleasure. Cocaine interferes with the

uptake of dopamine from the synaptic cleft. Alcohol causes a euphoric ‘high’, followed

by a depression. Marijuana, material from the Indian hemp plant (Cannabis sativa), has a

potent chemical tetrahydracannibinol (THC) that in low concentrations, causes a euphoric

‘high’ (if inhaled, the most common form of action is smoke inhalation). High dosages

may cause severe effects such as hallucinations, anxiety, depression and psychotic

symptoms. Cocaine is derives from the plant Erthoxylon coca. It is inhaled, smoked or

injected. The cocaine users report a ‘rush’ of euphoria after its use. After the ‘rush’, there

is a short (5-30 minutes) period of ‘arousal’, followed by a ‘depression’. Repeated cycle

of use terminate in a ‘crash’ when the cocaine is gone. Its prolonged use causes

production of less dopamine, causing the user to need more of the drug. Heroin is a

derivative of morphine, which in turn is obtained from opium, the milky secretions

obtained from the opium poppy, Papaver somniferum. Heroin is usually injected

intravenously, although snorting and smoking serve as alternative delivery methods.

Heroin binds to opioid receptors in the brain, where the natural chemical endorphins are

involved in the cessation of pain. Heroin is physically addictive, and its prolonged use

causes less endorphin production. Once this happens, the euphoria is no longer felt, only

dependence and delay of withdrawal symptoms are seen in humans.

IMPORTANT QUESTIONS

1. Describe the role of nervous system in animals.

2. Differentiate between the sympathetic and parasympathetic nervous systems.

3. Enumerate five drugs acting on the central nervous system.

21

2

ALTERNATIVES OF ANIMALS AS MODELS FOR

EXPERIMENTATION

OBJECTIVE

To study the alternatives of animals as models for experimentation.

PRINCIPLE

The ‘modern laboratory animal science’ is built up on the principle of three “R”, i.e.,

replace, reduce and refine.

“Replace” denotes that ‘replace the animal experimentation wherever possible with

the alternatives’. “Reduce” means ‘to minimize the number of experiments or animals in

each experiment’. “Refine” indicates that ‘refine the experiments which are to be carried

out so that the animals undergo the minimum of discomfort, and the scientific quality of

the experiments should be as high as possible’.

The laboratory animal unit is basically to ensure that the three “R” are followed in

practice. Students, animal welfare organization, teachers and others have frequently

questioned the use of animals for the experimental purposes. The use of animals in

teaching and research is often opposed on moral grounds, and from educational and

practical standpoints. Due to these considerations, more attention is now paid to the

approaches that reduce, refine or replace the use of animals in education.

COMMON LABORATORY ANIMALS

Mice (singular- mouse), rats, guinea pigs, rabbits, cats, dogs and frogs (Fig. 10-16)

are the common laboratory animals that are used for different biological (or

pharmacological) experiments.

22

Fig. 10: Mouse Fig. 11: Rat Fig. 12: Guinea Pig

Fig. 13: Rabbit Fig. 14: Frog

Fig. 15: Cats Fig. 16: Dog


ALTERNATIVES OF ANIMAL

Followings are the important alternatives of animal which can be used in place of

animals for the experiments:

1. Models, mannequins and mechanical stimulation;

2. Film and interactive videos;

3. Computer simulation and virtual reality systems;

4. Self experiments and human studies;

5. Plant experiments;

23

6. Observation and field studies;

7. Waste materials from slaughter houses and fisheries;

8. In vitro studies on cell lines;

9. Dead animals from humane and ethical sources;

10. Clinical practices; and

11. Extension practices.

Many alternatives have been developed for educational purposes. However, their

impact on animal use can only be determined when reliable data are available which

report the numbers and species of animals being used, and the purpose of their use. This

information would permit more effective targeting of resources for developing

alternatives where they will have greatest impact.

PROBLEMS CONCERNED WITH ANIMAL ALTERNATIVES

Although animal alternatives are now widely available but the number of animals

being used in the education seems to be decreasing slowly. For many reasons, the

introduction and subsequent use of alternatives in education is not straight forward. Some

of the reasons/problems in this concern may be:

1. Information about potential animal alternatives is not widely available.

2. Some teachers are resistant to change, so there is need to be convinced about the

benefits of using alternatives.

3. Integration of an alternative into a course usually involves initial investment of

time and money.

4. There are financial, technical and other factors which restrict the use of animal

alternatives.

IMPORTANT QUESTIONS

1. Enumerate ten laboratory animals used in different experiments.

2. Why there is a need of animal alternatives?

3. Which are the alternatives developed for educational purpose?

24

3

STUDY OF ANALGESIC EFFECTS OF DRUGS

OBJECTIVE

To study the analgesic effects of drugs.

PRINCIPLE

The basic principle involved in this experiment is to elicit the reaction to

standardized painful stimuli before and after the administration of any analgesic drug. A

significant increase in reaction time (prolongation) after the administration of drug

indicates the analgesic effect of a test drug.

METHODS

There are different methods (Table 1) on the basis of noxious stimuli used to produce

pain sensation. These are:

1. Chemical irritant method

2. Physical pressure method

3. Pinching mechanical stimuli method

4. Electric shock method

5. Thermal and radiant heat method

Table 1: Different Methods for Induction of Pain Sensation

S.

No.

Method Stimulus Nature of

Analgesic Drug

Laboratory

Animal Used

1 Physical By clipping the tail Central Mouse

2 Thermal and radiant heat

a. Hot plate

On hot plate surface at

55oC temperature

Peripheral,

central

Mouse, rat

b. Radiant heat or tail flick Red hot nichrome wire

touching with tail

Central Mouse, rat

3 Chemical writhing Intraperitoneal Peripheral Mouse

administration

4 Electric Shock- Finger

electrode

Electric current Central Dog

IMPORTANT QUESTIONS

1. Define and classify the analgesics with examples.

2. Discuss the general mechanism of action of analgesic drugs.

3. Describe the procedure of evaluating the analgesic effect of a test drug.

26

4


BY HOT PLATE METHOD

OBJECTIVE

To study the analgesic activity of test drugs by ‘Hot Plate Analgesiometer’.

REQUIREMENTS

Instruments and other materials- Hot plate analgesiometer (Fig. 17), tuberculin

syringe with needle (Fig. 18), distilled water and normal saline.

Drugs- Pentazocine, paracetamol and aspirin.

Animals- Mice.

Fig. 17: Hot Plate Analgesiometer Fig. 18: Tuberculin Syringe with Needle


PROCEDURE (To be described by Teacher, or written by Student)

27

OBSERVATIONS AND CONCLUSION

The observations and conclusion should be written as per the Table 2.

Table 2: Analgesic Activity of Drugs by Hot Plate Analgesiometer

Treatment Dose (mg/kg)

and Route of

Administration

Reaction Time

(sec.) Before

Treatment

Reaction Time (sec.)

After Treatment

Percent

Analgesia

Score

Normal Saline

(Control)

Pentazocine

Paracetamol

Aspirin

IMPORTANT QUESTIONS

1. Differentiate between the narcotic and non-narcotic analgesics.

2. Name different methods for induction of pain in laboratory animals.

3. Discus the mechanism of action of drugs used in the experiment.

28

5


BY TAIL FLICK METHOD

OBJECTIVE

To study the analgesic activity of a test drug by ‘Radiant Heat Analgesiometer’ (Tail

Flick Method).

REQUIREMENTS

Instruments and other materials- Radiant heat analgesiometer (Fig. 19), tuberculin

syringe with needle, distilled water and normal saline.

Drugs- Pentazocine, paracetamol and aspirin.

Animals- Rats.

Fig. 19: Radiant Heat Analgesiometer



29



Table 3: Analgesic Activity of Drugs by Tail Flick Method

Treatment Dose (mg/kg)

and Route of

Administration

Reaction Time

(sec.) before

Treatment

Reaction Time

(sec.) after

Treatment

Percent

Analgesia

Score

Normal Saline

(Control)

Pentazocine

Paracetamol

Aspirin

IMPORTANT QUESTIONS

1. What do you mean by the ‘Tail flick’?

2. How radiant heat analgesiometer works?

3. Name other drugs which can be used in the experiment.

30

6

STUDY OF EFFECTS OF LOCAL ANESTHETICS

OBJECTIVE

To study the onset and duration of anaesthetic effect of local anaesthesia.

DEFINITION AND TYPES OF LOCAL ANAESTHESIA

“Local anaesthesia” is a state in which a drug causes a reversible loss of sensation in

a localized area of the body. In general, there are following five types of local

anaesthesia:

1. Surface or topical anesthesia

2. Infiltration anesthesia

3. Conduction or nerve blocking anesthesia

4. Epidural anaesthesia

5. Intrathecal or spinal anaesthesia

REQUIREMENTS

Instruments and other materials- Tuberculin syringe with needle, distilled water

and normal saline.

Drugs- Lignocaine (0.5%).

Animals- Rabbits or guinea pigs.

PRINCIPLE

Local anaesthesia blocks the prolongation and conduction of nerve impulses when

applied locally. They desensitize the nerve/nerve endings. The onset and duration of this

desensitization is checked by the response of painful stimuli. The absence of pain

sensation indicates the local anaesthetic effect.

31

PROCEDURE

1. Secure the animal and clip its hair from an area of about 4 cm in diameter on the

back on either side of the vertebral column, and shave it. This is done a day prior

to conducting the experiment.

2. Check the area with pin pricks for the presence of twitching reflex, i.e., on

pricking the animal, contraction of its muscle occurs due to pain.

3. On one side, inject 0.5% of lignocaine, intradermally. Due to this, the formation

of wheel occurs. Mark this area with a marker.

4. Do pricking at 6 different sites on the patch at interval of 5 minutes and note the

progressive disappearance and reappearance of twitching reflex.

5. Compare the results by noting the time (min.) of the onset of local anaesthetic

effect and its total duration (min.).



Table 4: Effect of Lignocaine (Local Anaesthesia)

Group/Patch Administered Drug Twitching Reflex at Interval (Time) of

0 min. 5 min. 10 min. 15 min. 20 min.

I (Control) Normal saline

II Lignocaine (0.5%)

IMPORTANT QUESTIONS

1. Discus the mechanism of action of lignocaine.

2. Give the examples of some other local anaesthetics.

3. Differentiate between the local and systematic anaesthetics.

32

7

STUDY OF EFFECTS OF

CENTRAL NERVOUS SYSTEM DEPRESSANTS

OBJECTIVE

To study the effects of hypnotics and their potentiation by sedatives.

REQUIREMENTS

Instruments and other materials- Glass jar (3 L capacity), absorbent cotton, cones

and cloth towel.

Drugs- CNS depressants, viz., anaesthetic ether, chloroform, alcohol and vaseline.

Animals- Rats or mice.


33



Table 5: Effects of Hypnotics/Sedatives (CNS Depressants)

Observation Time

(min.) of

Anaesthetic

Amount

of anaes-

thetic

Duration

of anaes-

thesia

Body

Tempe-

rature

Pulse

Rate/

min.

Respi-

ration

Rate

Reflexes Other

Symp-

toms

Normal (Control)

Ether

0 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

Chloro-

form

0 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

Alcohol

(Ethanol)

0 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

IMPORTANT QUESTIONS

1. Name the different reflexes to be observed during anaesthesia.

2. Describe the merits and demerits of ether and chloroform anaesthetics.

3. Write the significance of atropine administration before chloroform anaesthesia?

34

8

STUDY OF EFFECTS OF PENTOBARBITONE

AND CHLORPROMAZINE

OBJECTIVE

To study the effects of pentobarbitone and chlorpromazine.

REQUIREMENTS

Instruments and other materials- Tuberculin syringe with needle, stopwatch (Fig.

20), weighing balance (Fig. 21), distilled water and normal saline (0.89% or 0.9%

NaCl solution).

Drugs- 1% pentobarbitone (25 mg/kg) and 0.1% chlorpromazine (3 mg/kg).

Animals- Mice.

Fig. 20: Stopwatch Fig. 21: Weighing Balance


PRINCIPLE

Pentobarbitone sodium induces sleep by depressing the CNS. This is indicated by

loss of rightening reflexes, i.e., when animal is placed on its back, it fails to correct its

posture. Chlorpromazine (a tranquilizer) has the hypnotic CNS depressant effect, which

increases the sleeping time.

35




Table 6: Effects of Pentobarbitone and Chlorpromazine (CNS Depressants)

Observation Time

(min.) of CNS

Depressant

Heart

Rate/

min.

Respira-

tion Rate/

min.

Spontaneous

Movement

Reflexes Other

Symptoms

Normal (Control)

Pentobar-

bitone

0 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

Chlorpro-

mazine

0 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

5 min.

10 min.

15 min.

20 min.

25 min.

30 min.

IMPORTANT QUESTIONS

1. Classify CNS depressants with suitable examples.

2. Enumerate the mechanism of action of test drugs.

3. Mention the indications of test drugs.

37

9

STUDY OF EFFECTS OF

CENTRAL NERVOUS SYSTEM STIMULANTS

OBJECTIVE

To study the actions of strychnine.

REQUIREMENTS

Instruments and other materials- Glass syringe (Fig. 22), needle, scalpel (Fig. 23),

probe (Fig. 24), distilled water and normal saline.

Drugs- Strychnine sulphate solution (0.1%) and mephenesin (10%).

Animals- Frogs.

Fig. 22: Glass Syringes Fig. 23: Surgical Scalpels Fig. 24: Surgical Probes



38



Table 7: Effects of Strychnine (CNS Stimulant)

Treatment Observation Remark

Control (Normal)

Strychnine + external stimuli

Strychnine + mephenesin

IMPORTANT QUESTIONS

1. Enumerate the examples of different respiratory stimulants.

2. Describe the mechanism of action of strychnine.

3. What is the role of mephenesin?

39

10

STUDY OF EFFECTS OF MUSCLE RELAXANTS

OBJECTIVE

To determine the effects of drugs on muscle tone and body balance by ‘Rota-Rod’.

REQUIREMENTS

Instruments and other materials- Rota-Rod (Fig. 25), tuberculin syringe with

needle, distilled water and normal saline.

Drugs- Chlorpromazine, chlordiazepoxide and diazepam.


Fig. 25: Rota-Rod Apparatus


PRINCIPLE

Antianxiety drugs have important action on muscle relaxation. Besides, these drugs

have taming or calming effect which reduces the anxiety and tension. Loss of muscle grip

indicates the muscle relaxation. This effect can be studied by using inclined plane or

rotating rods. Difference in fall of time from rotating rod between control and drug

treated animals is taken as an index of muscle relaxation. Animals rotating over the rod

should be adjusted such as that the normal animal can stay on the rod for 3 to 5 minutes.

40




Table 8: Effects of Muscle Relaxants

Drug Dose (mg/kg) and

Route of

Administration

Number of

Animals

Treated

Number of

Animals

Fallen

Percent

Reduction in

Activity

Control

(Normal Saline)

Chlorpromazine

Chlordiazepoxide

Diazepam

IMPORTANT QUESTIONS

1. Describe different characteristics of the skeletal muscle.

2. What is meant by the term ‘muscle tone’?

3. Mention the mechanism of action of drugs used in the experiment.

41

11

STUDY OF EFFECTS OF DRUGS ON

MOTOR ACTIVITY BY ACTOPHOTOMETER

OBJECTIVE

To study the effects of drugs on spontaneous motor activity by ‘Actophotometer’.

REQUIREMENTS

Instruments and other materials- Actophotometer (Fig. 26), tuberculin syringe with


Drugs- Chlorpromazine, meprobamate and diazepam.


Fig. 26: Actophotometer


PRINCIPLE

Actophotometer consists of photocells. These photocells are activated when the rays

of light falling on the photocells are obstructed/cut off due to movement of animal

crossing the path of light beam. This cut off quantity of light beam is measured

electronically. This is proportional to the movement of animal in cage. This instrument

42

measures the active exploratory movement of animal. This equipment is used to measure

the effect of drug on motor activity of the rat/mice, and useful in screening and evaluation

of drug for pharmacological and toxicological experiments.




Table 9: Effects of Drugs on Spontaneous Motor Activity

Drug Dose (mg/kg) and Route of

Administration

Cumulative Count Percent Reduction in

SMA

Control

(Normal Saline)

Chlorpromazine

Meprobamate

Diazepam

IMPORTANT QUESTIONS

1. What do you mean by the ‘spontaneous motor activity’ (SMA)?

2. What is the working principle of actophotometer?

3. Mention the mechanism of action of meprobamate.

43

12

STUDY OF EFFECTS OF DRUGS ON

CONDITIONED AVOIDANCE RESPONSE

OBJECTIVE

To establish the conditioned response and study the effects of drugs on conditioned

avoidance response (CAR).

REQUIREMENTS

Instruments and other materials- Cook’s pole-climbing apparatus (Fig. 27),

tuberculin syringe with needle, distilled water and normal saline.

Drugs- Chlorpromazine, chlordiazepoxide and diazepam.

Animals- Rats.

Fig. 27: Cook’s Pole-Climbing Apparatus


PRINCIPLE

Cook’s pole-climbing apparatus is used to study the effect of drug on CAR in

experimental animals. This apparatus consist of pole in the lid of box, electrified grid on

floor and buzzer arrangement (Fig. 27). During training, the buzzer and electric shock are

simultaneously applied through devises. If needed, shocks are applied intermittently than

44

variable length of time, but each time not more than 30 seconds. The rats gradually learn

to climb the pole. Initially, training is given 3 times a day for a week only. Thereafter,

only buzzer is tried. Shock is applied only when rats do not climb. Thus, the rats develop

conditioned reflex (climbing on hearing the buzzer in 2 weeks). The drug under study is

administered to the rats, and the rats are subjected to test as determined. Conditioned

reflex (CR) blocking is exhibited to climb the pole within 30 seconds. This is considered

as the main parameter in the evaluation of drug effect on CAR.




Table 10: Effects of Drugs on Spontaneous Motor Activity

Drug Dose (mg/kg)

and Route of

Administration

Number

of

Animals

Treated

Number of

Animals with

Conditioned

Response

Number of

Animals with

Conditioned

Reflex Blocking

Percent

Activity

Control

(Normal Saline)

Chlorpromazine

Chlordiazepoxide

Diazepam

IMPORTANT QUESTIONS

1. Define the conditioned reflex (CR) blocking.

2. What do you mean by the conditioned and unconditioned responses?

3. Discuss the mechanism of action of tranquillizers.

45

13

STUDY OF EFFECTS OF ANTICONVULSANTS

BY ELECTRO-CONVULSOMETER

OBJECTIVE

To study the anticonvulsant activity by ‘Electro-Convulsion’ method (MES test).

REQUIREMENTS

Instruments and other materials- Electro-Convulsometer (convulsometer, Fig. 28),

tuberculin syringe with needle, distilled water and normal saline.

Drugs- Dilantin sodium and phenobarbital sodium.

Animals- Mice.

Fig. 28: Electro-Convulsometer


PRINCIPLE

Seizures are produced by electrical stimulation and their phases are then antagonized

by systemic administration of an anticonvulsant. Different types of epilepsies can be

studied in the laboratory animals. The maximal electro shock (MES) induced convulsion

in animals represents the ‘grand mal’ type of epilepsy. There are two procedures used to

study the convulsion and anticonvulsion drugs in the laboratory animals. In MES, the

convulsion electro-shock is applied through the corneal electrodes; while through the

46

optic stimulations, cortical excitation is produced. The MES convulsions are divided into

five phases, viz., tonic flexion, tonic extensor, clonic convulsion, stupor and recovery or

death. A substance is known for its anticonvulsant property if it reduces or abolishes the

extensor phase of MES convulsion.




Table 11: Anticonvulsant Activity of Drugs by Electro-Convulsion Method

Drug/Treatment Dose (mg/kg)

and Route of

Administration

Number

of

Animals

Treated

Time (min.)

for Onset of

Convulsion

Time (min.)

for Survival of

Rat

Percent

Anticon-

vulsant

Activity

Control

(Normal Saline)

Dilantin sodium

Phenobarbital

sodium

IMPORTANT QUESTIONS

1. How the anticonvulsant activity can be seen in convulsometer?

2. Give the mechanism of action of dilantin and phenobarbitone.

3. What are therapeutic uses of test drugs?

47

14

STUDY OF EFFECTS OF ANTICONVULSANTS

BY LEPTAZOLE TEST

OBJECTIVE

To study the anticonvulsant activity (‘Leptazole test’) against leptazole

(pentylenetetrazole) induced convulsion.

REQUIREMENTS

Instruments and other materials- Convulsometer (agressometer), tuberculin syringe

with needle, distilled water and normal saline.

Drugs- Dilantin sodium, phenobarbital sodium and pentylenetetrazole.

Animals- Mice.

PRINCIPLE

Pentylenetetrazole (leptazole) is a CNS stimulant. It produces jerky type of ‘clonic’

convulsion in rats and mice. The convulsive effect of pentylenetetrazole is similar to

‘petit mal’ convulsion seen in humans.


48



Table 12: Anticonvulsant Activity of Drugs by Electro-Convulsion Method

Drug/Treatment Dose (mg/kg)

and Route of

Administration

Number

of

Animals

Treated

Time (min.)

for Onset of

Convulsion

Time (min.)

for Survival of

Rat

Percent

Anticon-

vulsant

Activity

Control

(Normal Saline)

Dilantin sodium

Phenobarbital

sodium

Leptazole

IMPORTANT QUESTIONS

1. How the anticonvulsant activity can be seen in agressometer?

2. Differentiate between the ‘tonic’ and ‘clonic’ convulsions. Also describe the

different methods for inducing convulsions in laboratory animals.

3. Give the mechanism of action of pentylenetetrazole.

49

15

STUDY OF EQUIPMENTS AND PREPARATION OF

PHYSIOLOGICAL SALT SOLUTIONS FOR

EXPERIMENTS ON ISOLATED TISSUES

OBJECTIVE

To study the equipments and preparation of physiological salt solutions for

experiments on isolated tissues.

PRINCIPLE OF PHYSIOLOGICAL SALT SOLUTION AND ISOLATED TISSUE

PREPARATION

‘Physiological salt solution’ is required to keep the isolated tissue/organ preparation,

surviving as long as the experiment goes on by providing ionic requirement and

nutritional supply to tissues. Composition of physiological salt solution is such that it

provides artificial media resembling inorganic composition of blood plasma with a buffer

pH of 7.0, glucose to supply energy and aeration with oxygen (mixture of 95% O2 and

5% CO2). It should be clear and no turbidity should be seen. By using ‘isolated tissues’

against the intact animals, many test preparations can be tested from a single animal.

Likewise, relatively small amount of the test material is required; and the effect of drug is

tested directly without the factors of absorption, metabolism, excretion or interference

due to nerve reflexes. On the other hand, results obtained on isolated experiments are not

always reproducible when tested on the whole animals. Guinea pigs, rabbits and rats are

usually the common sources of isolated tissues. Mice are also sometimes used, while cats

and dogs are too big to be sacrificed for just a piece of tissues. In the latter case, if at all,

the preparations are generally obtained from anaesthetized animals used for other tests.

Isolated strips of intestine are most commonly used smooth muscle preparations as:

50

a. Isolated strips of intestine are abundant.

b. They are more resistant to handling.

c. Relatively the set up of intestinal smooth muscle preparations is easy.

d. Various smooth muscle preparations have variable degree of spontaneous activity.

e. They permit the study of different types of pharmacological actions.

STUDENT’S ORGAN BATH (ISOLATED ORGAN BATH)

Besides tremendous development in electronic devices, gazetteries and recording

system, conventional equipments (e.g., “Student’s organ bath”) are routinely used in the

experimental pharmacology. The complete assembly of “Student’s organ bath” (Fig. 29)

consists of many parts, including organ bath, aerator, Brodie’s heart lever (Fig. 30),

frontal writing lever (Fig. 31) and Sherrington rotating drum. “Organ bath” (isolated

organ bath) is used to study the effects of drugs on isolated tissues. It was first designed

by Rudolph Magnus in 1904. This equipment essentially consists of the following parts:

1. Water Bath or Outer Jacket:

The water bath is made up of steel, glass or ‘perspex’ (transparent thermoplastic

resin). It holds water and other parts of organ bath. It consists of a glass vessel called

‘organ tube’ surrounded by a perspex or glass tank filled with water maintained at a fixed

temperature by an electric heater and thermostat. The organ tube is connected through

polythene or rubber tube to the reservoir which contains physiological saline solution

(Ringer’s or Tyrode’s solution). In between the reservoir and organ tube, there is a glass

preheating coil. The tissue is suspended in organ tube by means of a tissue holder that

also serves the purpose of aeration (hence known as ‘aeration tube cum tissue holder’).

One end of the tissue is tied to this and the other end is connected to lever, the recording

device (kymograph, Fig. 32; or physiograph, Fig. 33).

2. Recording Device:

The commonly used recording devices are ‘levers’. For recording of isometric

51

Fig. 29: Student’s Organ Bath Fig. 30: Brodie’s Heart Lever Fig. 31: Frontal Writing lever

Fig. 32: Kymograph Fig. 33: Physiograph


contraction, ‘spring lever’ is used; while for recording of isotonic contraction, ‘isotonic

frontal writing lever’ is commonly used. Isotonic contractions indicate the change in

length at uniform tension. Isometric recordings are records of change in tension (force)

developed irrespective of the length. There is another type of recording called ‘auxotonic’

in which the change in force of contraction is recorded with respect to change in length.

3. Heart Lever:

Commonly used ‘heart levers’ are spring levers (“Starling’s heart lever” or “Brodie’s

heart lever”). In this type of lever, fulcrum lies at one end beyond the point of attachment.

It consists of a horizontal arm suspended by a fine spring. To the horizontal arm, the

thread is attached which connects it to the heart. At its free end, the ‘sty let arm’ is fixed.

52

4. Isotonic Frontal Writing Lever:

It is made up of the light aluminium or stainless steel rod, passing through a wheel

which rotates freely about its axle (commonly known as ‘lever holder’). At one end, the

frontal writing point is fixed. The advantage of ‘frontal writing lever’ is that it gives a

record with the uniform friction, and the writing point falls vertically against the paper.

The traces thus obtained are linear and not curves. The lever should be adjusted such that

it gives a fair and fixed magnification of the response and should exert suitable tension.

PREPARATION OF DIFFERENT MATERIALS

1. Adjustment of Magnification:

In order to achieve 10 times magnification, the distance from the fulcrum to the point

of recording (frontal point) should be 10 times more than the distance from fulcrum to the

point of tissue attachment. Therefore,

Magnification = distance from fulcrum to the writing point ÷

distance from fulcrum to the point of attachment of tissue

2. Adjustment of Tension:

The lever is made horizontal by applying the plasticin (modeling wax) at the shorter

end. A small amount of plasticin is also placed on the point of attachment of tissue. One

gramme (g) weight or 500 mg weight is placed over this plasticin and the lever is again

balanced (i.e., made horizontal) by putting plasticin on other side and adjusting distance

from fulcrum. While taking responses, weight should be taken off. When the tissue is tied

and lever is adjusted in horizontal position, it will exert the tension of 500 to 1000 mg.

3. Smoking of Drum:

Tissues responses are recorded on ‘smoked drum’ which is prepared as under-

Glazed paper is laid down on the table, keeping glazed surface downwards. One end

of the paper is gummed. Drum cylinder is placed in the middle of the paper. Proximal

53

ungummed end is rolled around the drum and held tightly between the thumbs. Other end

is also rolled on the other side and the gummed end is pasted on the proximal ungummed

end. Cylinder with paper is passed over a rod fixed in a smoking rack. A flame is

obtained by passing the gas through benzene or using a mixture of benzene and kerosene

in the ratio of 1:9. Burner is brought nearer to the drum that is rolled uniformly at the

maximum possible speed. Outer orange zone of flame should touch the paper. Uniform

rolling of drum should be continued so as to set the uniform deposit of soot.

4. Fixing of Graph (Varnishing of Graph):

The paper is cut after obtaining the recordings and then it is dipped in a solution of

resin in methylated spirit. This solution is prepared by dissolving 150 g of resin in 2 L of

spirit. After passing the paper through the solution, it is drained and then allowed to dry.

5. Physiological Salt Solution or Ringer’s Solution:

Ionic requirement and nutritional supply can be provided by ‘physiological salt

solution’ or “Ringer’s solution”. Composition of Ringer provides artificial media,

resembling the inorganic composition of blood plasma together with buffer mechanism to

maintain optimal pH at about 7.0 to 7.2 and glucose to facilitate the tissue metabolism.

6. Aeration:

Physiological salt solution in organ bath should be bubbled with air with sufficient

oxygen or carbogen (95% O2 + 5% CO2). This is important for regular supply of oxygen,

at the maintained pH. Bubbling of air gives the uniform distribution of drug.

COMMONLY USED PHYSIOLOGICAL SALT SOLUTIONS

1. Ringer-Locke

2. Frog’s Ringer

3. Tyrode

4. De Jalon

54

5. Krebs-Henseleit (Krebs)

Chemical compositions of some physiological salt solutions are shown in Table 13.

Table 13: Chemical Compositions of Some Physiological Salt Solutions

Chemical

Compound

(g/L)

Physiological Salt Solution

Ringer-

Locke

Locke Frog’s

Ringer

Tyrode De Jalon Mammal’s

Ringer

Krebs

NaCl 9.15 9.00 6.50 8.00 9.00 9.00 6.90

KCl 0.42 0.42 0.14 0.20 0.42 0.42 0.35

CaCl2 0.24 0.24 0.12 0.20 0.06 0.24 0.28

MgCl2 - - - 0.10 - 0.10 0.10

MgSO4 - - - - - 1.28

NaHCO3 0.15 0.50 0.20-0.40 1.00 0.50-1.00 0.50 2.10

NaH2PO4 - - 0.01 0.05 0.05 0.11 0.11

KH2PO4 - - - - - 0.16

Glucose 1.00 1.00 1.50-2.00 1.00-2.00 0.50 1.00 1.00-2.00

The functions of chemical compounds of physiological salt solutions are as under:

1. Sodium chloride (NaCl)- It maintains isomolarity, isotonicity, excitability and

contractility of the tissue preparation.

2. Potassium chloride (KCl)- It maintains ionic balance of the tissue preparation.

3. Calcium chloride (CaCl2)- It maintains contractility of the tissue preparation.

4. Magnesium chloride (MgCl2)- It stabilizes the tissue preparation, and hence

reduce the spontaneous activity.

5. Magnesium sulphate (MgSO4)- It stabilizes the tissue preparation, and hence

reduce the spontaneous activity.

6. Sodium bicarbonate (NaHCO3)- It provides alkaline pH.

7. Sodium dihydrogen phosphate (NaH2PO4)- It acts as a buffer.

8. Potassium dihydrogen phosphate (KH2PO4)- It acts as a buffer.

9. Glucose- It provides the energy.

SACRIFICING OF ANIMALS

The animals should be sacrificed painlessly (euthanasia method). Small animals can

55

be made unconscious by head blow (stunning), pithing, decapitation or anaesthesia.

I. Pithing:

“Pithing” means the destruction of brain and spinal cord. Under this condition, the

animal (e.g., frog) as a whole does not remain alive but the individual organ continues to

maintain the vitality for some time. If the same tissues are placed in suitable isotonic

solution, they may survive even for hours. Frog is slippery to hold and it can easily slip

away due to excessive mucous secretion. The animal is held from the waist region and as

the first step of pithing, the dorsal part of head is struck with iron rod or against edge of a

table. As a result of this, the animal gets sudden shock and temporarily becomes

unconscious. The animal should be stunned by a single hard stroke only. If the stroke is

light, the frog will make violent movements to escape. The head is pressed down to

locate a groove indicating position of foramen magnum. This is at the posterior border of

tympanic membrane crossing the midline. A stout needle is pierced through the

depression into the skull. This will destroy the brain. If destruction is proper, the eye

reflexes will be abolished. The probe or the needle is withdrawn up to the level of

foramen magnum and then passed into the canal of vertebral column. This will destroy a

part of the spinal cord. When the spinal cord is destroyed, limbs of the animal are out

stretched that go into the contractile convulsions.

II. Decapitation:

Another method used for sacrificing the animals (e.g., mice, rats, rabbits and guinea

pigs) is stunning them, followed by cutting the blood vessels of neck region, or cutting

the head (i.e., “decapitation”).

III. Use of Anaesthesia and Other Methods:

Different anaesthetics are used to make the animal unconscious. To kill the animal,

intravenous saturated magnesium sulphate solution (15 g/kg) can also be used. Another

method is to inject a large volume of air to cause air embolism. The dosages of different

56

anaesthetic agents in different species of animals are described in Table 14.

Table 14: Dosages of Different Anaesthetic Agents in Different Species of Animals

Animal

Species

Anaesthetic Agent

(Anaesthetic)

Dose

(mg/kg)

Route of

administration

Mouse Pentobarbitone sodium 30-50 Intraperitoneal (ip)

Rat Pentobarbitone sodium 30-50 ip

Urethane 1250 ip

Urethane 1250-1750 Intramuscular (im)

Guinea pig Pentobarbitone sodium 30-50 ip

Urethane 1500 ip

Rabbit Pentobarbitone sodium 30-40 Intravenous (iv)

Urethane 500-1500 im, or iv

Cat and

dog

Chloralose 80-120 iv

Pentobarbitone sodium 180 ip

Pentobarbitone sodium 30-40 iv

PREPARATION OF TISSUES

The body organs to be studied should be removed rapidly and immersed in Ringer

solution in a Petri dish. After mounting the tissue, give rest for about 30 to 60 minutes.

The tissue should relax during this period. However, due to increase tones, the tissue may

fail to relax until the tension on the lever is increased by readjustment of weights. The

organ bath is emptied at regular intervals and the fluid is replaced by fresh solution

during the experimental period to avoid alteration of pH due to prolonged aeration, which

in turn changes the tone of the muscle.

The drum/chart movement is started at a slow speed. After measuring the volume of

the drug solution accurately (0.1-0.5 ml) with the help of a 1 ml graduated pipette of a

tuberculin syringe fitted with needle and then dipping its tip into the bath fluid, the drug

is added with a uniform speed. Simultaneously, with the addition of the drug, a stopwatch

is started. Every time, the drug is added into the bath. A mark is put on the drum near the

base line and labeled appropriately, indicating the drug and its dose or concentration.

The drug gets mixed up quickly due to bubbling of the gas, and is allowed to act till

the response reaches a steady level or up to a fixed time (30-60 sec.) depending on the

nature of the experiment after which the bath fluid containing the drug is washed out and

57

fresh solution is allowed to fill the bath. The washing is repeated 2 or 3 times, if

necessary, in order to allow the lever to come back to the original base line. This can,

however, be avoided by emptying the organ bath by overflow method, that is by

displacing the fluid by fresh solution from the bottom without actually emptying the bath.

After some definite interval depending on the tissue as well as the drug, another dose of

drug is added and the stopwatch is restarted, thus completing a cycle. Sensitivity of the

preparations may sometimes be improved by raising the temperature or by increasing the

dose intervals. Testing should start with a very low concentration producing slight or no

response and then repeating after each washing a dose 10 times greater than the preceding

concentration until the activity range is found or until total ineffectiveness is proved by

giving sufficiently large concentrations.

IMPORTANT QUESTIONS

1. Name the basic equipments used for autonomic drugs experiments on isolated

tissues.

2. Describe various types of levers.

3. Write down the working of student’s organ bath.

58

16

STUDY OF EFFECTS OF SYMPATHETIC AND

PARASYMPATHETIC DRUGS ON DUODENUM

OBJECTIVE

To study the actions of sympathetic and parasympathetic drugs on the duodenum

(intestine).

REQUIREMENTS

Instruments and other materials- Isolated organ bath, kymographic monodrum/

physiograph, dissecting instruments, tuberculin syringe with needle, distilled water

and normal saline.

Drugs- Adrenaline, acetylcholine (Ach), ephedrine, histamine, pilocarpine, arecoline,

atropine and barium chloride (BaCl2).

Animals- Rabbits.

PRINCIPLE

Rabbit intestine is more resistant to trauma and is easier to set up, and has larger

contractions. The functional components of the isolated intestine are terminal

sympathetic and parasympathetic ganglionic synapses. The rabbit intestine preparation is

particularly suitable for adrenaline and noradrenaline which produce relaxation.

PROCEDURE AND OBSERVATIONS

1. Pour 50 ml of Ringer’s solution in an isolated organ bath and maintain it at a

temperature of about 370C (rabbit belongs to mammalian species which is a warm

blooded animal, so it is necessary to maintain the body temperature at 370C).

59

2. Remove about 3 to 4 cm piece of duodenum from a freshly killed rabbit.

3. Remove faecal matter from intestine, and cut the adhering vessels and mesentery.

4. Ligate the loop of intestine on both the sides.

5. Tie one end of intestinal strip to the oxygen tube. Suspend it in Ringer’s solution.

6. Attach another end of intestinal strip to writing lever or transducer of

physiograph.

7. The lever should be weighed with plasticin, so that the muscle is kept stretched to

a moderate extent.

8. Pass oxygen through the tube.

9. Inject test drugs by tuberculin syringe as follows and note the observations:

(a) Add 2 mg of adrenaline to the bath. After 2 minutes, change the Ringer’s

solution- ……..

(b) Add 2 mg of Ach to the bath. After one minute, change the bath fluid- ……..

(c) Add 1 mg of ephedrine to the bath. Do not wash out. After 5 minutes, add 2

mg adrenaline to the bath and after 2 minutes, wash out. Add again 2 mg

adrenaline and after 2 minutes, wash out- ……..

(d) Add 1 mg of histamine acid phosphate. After one minute, change the Ringer’s

solution- ……..

(e) Add 0.25 mg of pilocarpine. Wash after the height of its action- ……..

60

(f) Add 0.5 mg of arecoline hydrobromide. At the height of its effect, add 0.5 mg

of atropine- ……..

(g) Repeat pilocarpine and wash- ……..

(h) Add 20 mg of BaCl2. At the height of its effect, repeat atropine- ……..

IMPORTANT QUESTIONS

1. Classify the sympathetic and parasympathetic drugs.

2. Describe the mechanism of action of sympathetic and parasympathetic drugs.

3. Enumerate the antagonists of certain sympathetic and parasympathetic drugs.

61

17

STUDY OF EFFECTS OF HISTAMINIC AND

ANTIHISTAMINIC DRUGS ON ILEUM

OBJECTIVE

To study the actions of histaminic and antihistaminic drug on the ileum (intestine).

REQUIREMENTS

Instruments and other materials- Tyrode’s solution, isotonic frontal writing lever

(500 mg tension and 8-10 folds magnification), organ bath, kymograph/physiograph,

dissecting instruments, tuberculin syringe with needle, distilled water and normal

saline (0.89% or 0.90% NaCl solution).

Drugs- (i) Spasmogenics/histaminics- acetylcholine (Ach- 10 mg/ml), carbachol (10

mg/ml), histamine (10 mg/ml) and barium chloride (BaCl2- 10 mg/ml);

(ii) Spasmolytics/antihistaminics- atropine (1 mg/ml), mepyramine (1 mg/ml)

and papaverine (100 mg/ml).

Animals- Guinea pigs.

PRINCIPLE

Histamine is an autacoids drug. Beside the “triple” response, it acts on H1 receptors

of the intestinal smooth muscles and causes the contraction of these muscles. The guinea

pig ileum is highly sensitive to histamine. Ach, carbachol, histamine and BaCl2 produce

contractile effect. These drugs are ‘spasmogenics’. However, atropine, mepyramine and

papaverine (they are ‘spasmolytics’) do not produce any effect of their own but inhibit

the responses to Ach, histamine and BaCl2, respectively. The antagonism by atropine and

mepyramine to Ach and histamine, respectively is specific; while that produced by

papaverine is non-specific. It is possible that Ach and histamine act through specific

62

receptors, i.e., muscarinic and H2 receptors. Atropine and mepyramine specifically block

these receptors, respectively. BaCl2 produces effect by direct action, not involving

receptors, and papaverine inhibits this action and, hence it is non-specific.

PROCEDURE

1. Set up the organ bath assembly and other experimental arrangements should be

made.

2. Guinea pig kept for overnight fasting is stunned by a sharp blow on the head and

carotid bleeding. The abdominal cavity is quickly opened and ilio-caecal junction

is traced and cut. Remove (isolate) a few cm long ileum portion, and immediately

place it in the Petri dish having Tyrode’s solution maintained at 370C.

3. Trim the mesentery and clean the contents of ileum (handle the muscles with

fingers rather than forceps) by pushing Tyrode’s solution into the lumen of ileum.

4. Take 2 to 3 cm long piece of ileum and tie the thread to top and bottom ends in

the tissue organ bath with one end to the tissue holder and other end to the

recording drum (isotonic frontal writing lever).

5. Fill the tissue bath with the Tyrode’s solution and allow the tissue to stabilize for

30 minutes. During this period, washing is given at every 8 to 10 minutes.

6. Add 0.3 ml of Ach, carbachol, histamine and BaCl2 with the help of tuberculin

syringe in the organ bath in the increasing dose concentration. Record the effect

till the drugs show their maximum effects.

7. After recording each observation, give proper washing to the tissue.

8. Add 0.1 ml of atropine into the tissue bath, incubate for 15 to 20 minutes and then

add 0.3 ml of each Ach, carbachol, histamine and BaCl2. Record the spasmolytic

effect of atropine against the contraction of Ach, carbachol, histamine and BaCl2.

9. Further, add 0.1 ml of mepyramine into the tissue bath, incubate for 10 to 15

minutes and then add 0.3 ml of each Ach, carbachol, histamine and BaCl2. Record

the spasmolytic effect of mepyramine against the contraction of Ach, carbachol,

histamine and BaCl2.

63

10. Similarly, add 0.1 ml of papaverine into the tissue bath. Incubate for 10 to 15

minutes and then add 0.3 ml of each Ach, carbachol, histamine and BaCl2. Record

the spasmolytic effect of papaverine against the contraction caused by the Ach,

carbachol, histamine and BaCl2.

OBSERVATIONS AND INFERENCE

The tracing on the black paper of kymograph monodrum shows that histamine, Ach,

carbachol and BaCl2 produce contraction of guinea pig ileum, and pretreatment with

antihistaminics (viz., atropine, mepyramine and papaverine) blocks the action of

histamine and other agonist test drugs. This ‘antagonism’ of antihistaminic test drugs is

reversible by increasing the concentration of histamine or histamine agonist test drugs

(viz., Ach, carbachol and BaCl2).

IMPORTANT QUESTIONS

1. What is the effect of antagonists on agonists?

2. What is the use of H2 receptor blockers in the treatment of peptic ulcer?

3. What is meant by the non-specific action of papaverine?

64

18

STUDY OF EFFECTS OF SYMPATHETIC AND

PARASYMPATHETIC DRUGS ON FUNDUS

OBJECTIVE

To study the actions of sympathetic and parasympathetic drugs on the fundus strip

(stomach).

REQUIREMENTS

Instruments and other materials- Krebs’s solution, organ bath assembly,

kymograph/physiograph, writing lever/transducer, dissection set, tuberculin syringe

with needle, distilled water and normal saline.

Drugs- 5-Hydroxytryptamine (5-HT), acetylcholine (Ach), nicotine, histamine,

tryptamine and atropine.

Animals- Rats.


1. Rat is sacrificed by cutting the jugular vein and the abdomen is opened. The

fundus part of stomach can easily be identified by its grey colour, whereas the

pyloric part is pink. It is cut away, opened out longitudinally, placed in a dish

containing Krebs’s solution and made into a strip about 4 or 5 cm long by suitable

transverse cuts.

2. A thread is attached at each end, and the preparation is mounted in Krebs’s

solution at 370C, aerated with oxygen (95%) and carbon dioxide (5%). One end of

the strip is attached to oxygen tube in the bath and the other end to a lever writing

frontally on a smoked drum.

65

3. Load in horizontal position is usually about 1 g. Muscle does not relax

spontaneously after it has been caused to contract, so it must be stretched to assist

its recovery by adding extra 1 g load. Preparation should be left for 30 minutes to

stretch before use.

4. Because the tissue contracts slowly, a long time-cycle is needed, similar to one

used with frog rectus preparation. At 0 minute, start the kymograph; at 1 minute,

add the test drug; at 2.5 minutes, stop the kymograph, wash the preparation and

add the extra load; and at 6 minutes, remove extra load and start the kymograph,

so the interval between doses is 6 minutes. Sometimes, the tissue only recovers

slowly after stretching, making it very difficult to obtain a steady base-line. If this

happens, do not stretch the preparation for as long as 3.5 minutes but allow a

longer period for recovery with stretching. Stretch the preparation for 2 minutes

and allow 5.5 minutes for recovery, so doses are added once at every 10 minutes.

5. Observe the sensitivity of the test drugs in doses of 0.1 or 0.2 ml as follows:

(a) 5-HT (10-7

M)- ……..

(b) Ach (10-6 M)- ……..

(c) Nicotine (10-3

M)- ……..

(d) Histamine (10-3

M)- ……..

(e) Tryptamine (10-4

M)- ……..

66

6. Replace the Krebs’s solution by fresh Krebs’s solution containing atropine (2 x

10-6

M) and repeat the testing of doses of these agonists, which were previously

effective. Note the observations as observed: ……..

IMPORTANT QUESTIONS

1. Why fundus strip has been selected in this experiment?

2. Summarize the tissue responses produced by various test drugs.

3. What happens with the tissue responses when atropine is added to the Ringer’s

solution?

67

19

STUDY OF EFFECTS OF SOME DRUGS ON

GASTROINTESTINAL MOTILITY

OBJECTIVE

To study the effects of various drugs on the gastrointestinal (GIT) motility.

REQUIREMENTS

Instruments and other materials- 1% activated charcoal suspension, animal cages,

tuberculin syringe, blunt and hypodermic needles, distilled water, and normal saline.

Drugs- Carbachol, metochlopromide and atropine.

Animals- Mice.

PRINCIPLE

Metochlopromide, carbachol and neostigmine increases the GIT motility.

Metochlopromide inhibits D2 receptors in central nervous system (CNS), increases

gastric emptying and motility in upper duodenum and inhibit 5-HT receptors.

PROCEDURE

1. The mice are fasted overnight.

2. The mice are divided into 4 groups of 3 animals each.

3. Weigh each mouse separately and put the identification marks, and keep each of

them in separate cages.

4. Administer the test drugs, viz., carbachol (1 mg/mouse), metochlopromide (2

mg/mouse) and atropine sulphate (1 mg/mouse) intraperitoneally (ip) to the

animals of groups 2, 3 and 4, respectively. However, in group 1 (control), 3 ml of

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1% activated charcoal suspension is administered.

5. After 15 minutes of administration of carbachol, metochlopromide and atropine,

administer 3 ml of 1% activated charcoal suspension to rats of groups 2, 3 and 4.

6. After 30 minutes, sacrifice all the mice humanely, and remove the intestine and

clean the mesentery. Place it on the white sheet in straight position.

7. Measure the distance traveled by the charcoal with the help of a scale and record

separately.

8. Calculate mean difference in the rate of movement of charcoal suspension in GIT.


Observe the effects of test drugs in the following groups of mice:

a) Group 1 (Control- Charcoal meal at 3.15 PM)- ……..

b) Group 2 (1.5 ml carbachol, ip at 3.20 PM + charcoal meal at 3.35 PM)- ……..

c) Group 3 (1.5 ml metochlopromide, ip at 3.25 PM + charcoal meal at 3.40

PM)- ……..

d) Group 4 (1.5 ml atropine sulphate, ip at 3.30 PM + charcoal meal at 3.45 PM)-

……..

After the post mortem (P.M.) examination, compare the intestine of the rat of each

group to know the extent of charcoal meal movement. The observations revealed that the

charcoal meal travels more in groups 2 and 3, least in group 4.

IMPORTANT QUESTIONS

1. Name any four drugs that increase the GIT motility.

2. Describe the action of charcoal.

3. Discuss the mechanism of action of carbachol, metochlopromide and atropine.

69

20

STUDY OF EFFECTS OF

SOME DRUGS ON UTERUS

OBJECTIVE

To study the effects of different drugs on the uterus.

REQUIREMENTS

Instruments and other materials- De Jalon’s modified Ringer solution, frontal

writing lever, organ bath, kymograph/physiograph, dissecting instruments, tuberculin

syringe with needle, distilled water and normal saline.

Drugs- Oxytocin, ergometrine, 5-hydroxytryptamine (5-HT) and adrenaline.

Animals- Female rats.

PRINCIPLE

Uterus is mainly used for assay of oxytocin, ergometrine, 5-HT and adrenaline. The

uterus is very sensitive to agonist action of adrenaline. The contractions of uterine smooth

muscles are produced by addition of Ach or carbachol to bath. The suitable animals are

female rats not pregnant for 4 weeks or more and not in oestrus stage. The spontaneous

rhythmic contractions of uterine smooth muscles are abolished by De Jalon’s modified

Ringer solution containing less calcium and less glucose with temperature of 30oC to

32oC. If rat is in oestrus, or stilbesterol is given, the spontaneous contraction is reduced.

The ‘tocolytic’ drugs cause relaxation of uterus or inhibit the uterine contraction.

PROCEDURE

1. For estimation of the effects of test drugs, the uterine the horns of female rat are

70

dissected and transferred to a dish containing the De Jalon’s Ringer solution.

2. Each horn is cut into two bits longitudinally, thus four pieces of uterine

preparation can be obtained from a rat. One piece is selected and tied at both ends.

3. The isolated uterine smooth muscle preparation is mounted in organ bath, by

tying one end to the bent tip of the oxygen tube and the other end to the simple

lever with frontal writing point. The writing point is adjusted so as to write

smoothly on the smoked paper of a slow moving drum.

4. Air can be bubbled through organ bath by using a respiratory pump. The bath

temperature is kept at 30oC to 32

oC. About

30 minutes is taken to get it stabilized.

5. The spontaneous movements produced by the test drugs in the uterine smooth

muscle (tissue) preparation are recorded for 1 minute. 1 ml of oxytocin (posterior

pituitary extract) is added in the preparation and its effect is recorded for 1

minute. The tissue is then washed twice and allowed to restabilize for 5 minutes.

6. Then, 1 ml of ergometrine is added in the preparation and the effect is recorded

for 1 minute. Again, the tissue is washed twice and restabilized for 5 minutes.

7. Then, 1 ml of 5-HT is added in the preparation and the effect is recorded for 1

minute. Again, the tissue is washed twice and restabilized for 5 minutes.

8. Then, 1 ml of adrenaline is added in the preparation and the effect is recorded for

1 minute. Again, the tissue is washed twice and restabilized for 5 minutes.


Oxytocin and ergometrine have produced increased contraction of uterus preparation,

suggesting that they are stimulants of uterine smooth muscles. However, the 5-HT and

adrenaline have caused inhibition of spontaneous contractions of uterine smooth muscles.

IMPORTANT QUESTIONS

1. Name two drugs which produce stimulatory/excitatory effect on the uterus.

2. Define the ‘tocolytics’.

3. Give the mechanism of action of oxytocin and ergometrine.

71

21

STUDY OF EFFECTS OF

CARDIAC GLYCOSIDES ON HEART

OBJECTIVE

To study the effects of cardiac glycosides (CGs) on the heart.

REQUIREMENTS

Instruments and other materials- Frog’s Ringer solution (containing 6.5 g of

sodium chloride, 0.14 g of potassium chloride, 0.03 g of calcium chloride called ‘low

calcium Ringer’ and other chemicals of Frog’s Ringer solution), kymograph,

Starling’s or Brodie’s heart lever, perfusion tube holder, Marriott flask connected to

Symme’s cannula, frog board, threads, dissecting instruments, tuberculin syringe with

needle, aneurysm needles, bent pin, distilled water and normal saline.

Drugs- Cardiac glycosides (CGs), adrenaline, acetylcholine (Ach) and isoprenaline.

Animals- Frogs.

PRINCIPLE

“Cardiac glycosides or cardio-active glycosides” (CGs) are linked by an O2 atom to

sugar molecule. They are group of steroidal glycosides, which act as cardiotonic agent.

They are synthesized from plant carbohydrate by hydrogen. CGs were firstly used by

William Withering to treat oedema due to heart failure. CGs increase tone, excitability

and contractibility of cardiac muscles. Normally, the CGs cause tachycardia (increased

heart rate) but in failing heart, they cause bradycardia (decreased heart rate). They have

no significant effect on BP. Adrenaline increases heart rate and BP. There is biphasic

response, first there is rise in BP; then the BP is fallen and heart rate decreases; while for

72

respiration, there is broncho-dilation. Ach causes decreased BP and broncho-constriction.

Isoprenaline causes decreased heart rate and BP, and broncho-constriction.

PROCEDURE

1. Heart and great blood vessels of frog are exposed and pericardium is removed.

2. Through the apex of the heart, a bent pin is passed and connected to the Starling’s

heart lever with a thread.

3. The sinus venosus is exposed. The Symm’s cannula is introduced in the sinus

venosus and ligature is applied with the help of aneurysm needle.

4. Cannula is directed towards heart and ligature is tightened at the neck of cannula.

5. Cannula is connected to Mariotte bottle by a rubber tube. Perfusion tube holder is

used to hold the rubber tube. The Marriott bottle is then filled with frog’s Ringer

solution, and adrenaline, Ach and isoprenaline are added separately in solution.

6. Both the right and left aorta are cut.

7. Writing point of Starling’s heart lever is adjusted to write smoothly on the drum.

8. Finally, the perfusion is started and recorded on the drum.


Perfusion with low calcium Ringer causes decreased heart rate, decreased force of

contraction and reduced tone. When any CG (e.g., isosorbide mononitrate) is dosed, the

force of cardiac muscle contraction is increased. Heart failure is produced experimentally

by low calcium Ringer. Adrenaline has increased the heart rate, force of contraction and

tone even in the normal heart. It also increased the oxygen demand and tachycardia. Ach

and isoprenaline have caused decrease in heart rate and BP, and broncho-constriction.

IMPORTANT QUESTIONS

1. Define CGs.

2. Discuss the mechanisms of action of CGs.

3. Write the effects of CGs on heart rate, blood pressure and respiratory rate.

73

22

STUDY OF EFFECTS OF

PARASYMPATHETIC DRUGS ON EYE

OBJECTIVE

To study the effects of parasympathetic drugs on the eye.

REQUIREMENTS

Instruments and other materials- Observation cages, rabbit’s holders, sterile

droppers and distilled water.

Drugs- Physostigmine (Neostigmine, 1-2%), pilocarpine (2-4%) and atropine (1-2%).

Animals- Rabbits.

PRINCIPLE

Pupil of the eye is dilated by paralysis of parasympathetic stimulation of the

sympathetic nervous system. Due to parasympathetic stimulation, the circular muscle

fibres of the iris contract, while the sympathetic action contracts the radial muscle fibres.

“Myosis” occurs due to stimulation of the parasympathetic nerves. Rabbit is resistant to

atropine because the rabbit produces an enzyme ‘atropinase’ which metabolises atropine.

Atropine is a ‘mydriatic drug’, where as physostigmine and pilocarpine are ‘miotic

drugs’. The physostigmine is particularly used to reverse the skeletal muscle paralysis.

PROCEDURE

1. Hold the rabbit in hand or keep it in the rabbit holder, so that its head will remain

protruding.

2. Put the test drugs, viz., physostigmine (neostigmine, 1-2%), pilocarpine (2-4%)

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and atropine (1-2%) in one eye of the rabbit carefully till the corneal surface is

wetted by the drug solution. Keep the other eye as control for that particular drug.

One rabbit should be used for each test drug.

3. Observe the effects of each drug after 5 minutes and note the size of pupil, and

response to light and touch.


The observations are described in Table 15.

Table 15: Effects of Parasympathetic Drugs on Eye

Test Drug Size (Diameter)

of Pupil

Light

Reflex

Corneal

Reflex

Physostgmine Decreased Present Present

Pilocarpine Decreased Present Present

Atropine Increased Absent Present

The diameter of pupil in the tested eye due to atropine is increased, indicating that

the atropine has caused papillary dilatation. The light reflex due to atropine is absent

when compared with the control eye. Due to physostigmine and pilocarpine, the size of

pupil is decreased and the light reflexes are present. In all the cases, the conjunctiva is red

and the corneal and conjunctival reflexes are normal (present).

IMPORTANT QUESTIONS

1. Why the rabbit is resistant to the effect of atropine?

2. Write at least one use of atropine, physostigmine and pilocarpine.

3. Differentiate between the ‘mydriatics’ and ‘miotics’.

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23

STUDY OF EFFECTS OF

SOME DRUGS ON BLOOD COAGULATION

OBJECTIVE

To study the effects of some drugs on the blood coagulation.

REQUIREMENTS

Instruments and other materials- Test tubes, hypodermic syringe with needle and

distilled water.

Drugs- Sodium chloride (NaCl) solution (0.89% or 0.90%, i.e., normal saline),

heparin (0.5%) and disodium ethylene diamine tetra acetate (EDTA- 0.5%).

Animals- Dogs or cats.

PRINCIPLE

Anticoagulants (e.g., heparin and EDTA) prolong the clotting time of blood. They

exert the action by 3 means: by ant thrombin activity, by inhibiting formation of

prothrombin and factor VII, and by removing the free ionized calcium from the blood.

PROCEDURE

1. Take a healthy dog or cat for the experiment and shave the hairs over the jugular

vein region by a shaving razor.

2. Collect 2 ml of blood from the jugular vein of a dog or cat with the help of a

hypodermic syringe (with needle).

3. Transfer 0.5 ml of blood in a test tube containing 0.1 ml of 0.89% or 0.9% NaCl

solution (normal saline).

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4. Transfer 0.5 ml of blood in a test tube containing 0.1 ml of heparin solution.

5. Transfer 0.5 ml of blood in a test tube containing 0.1 ml of disodium EDTA

solution.

6. Transfer 0.5 ml of blood in a test tube containing 0.1 ml of distilled water.

7. Cork all the test tubes tightly and mix the sample gently.

8. Observe the time required for clotting.


Heparin and EDTA have clotted the blood as compared to the control distilled water

and normal saline, which indicate that the heparin and EDTA have anticoagulant effect.

IMPORTANT QUESTIONS

1. Describe coagulation occurs by the two clotting pathways.

2. What is the role of prothrombin and factor VII in the clotting of blood?

3. Describe the use of heparin and EDTA.

77

24


BLOOD PRESSURE DURING ANAESTHESIA

OBJECTIVE

To study the effects of various drugs on blood pressure (BP).

REQUIREMENTS

Instruments and other materials- Condon mercury manometer (Fig. 34),

kymograph, surgical equipments, heparin (1000 IU/ml), tuberculin syringe with


Drugs- Adrenaline (10 µg/ml), isoprenaline (10 µg/ml), acetylcholine (Ach solution

of graded strength), atropine (100 µg/ml), phenoxybenzamine (100 µg/ml) and

propanolol (100 µg/ml).

Animals- Rats.

Fig. 34: Condon Mercury Manometer


PRINCIPLE

The modulation of BP by various drugs and their antagonists are easily elicitable

78

experimentally, and their drug receptor interaction can be studied. The drugs acting on

the cardiovascular system act in different mode as they either reduce or increase the BP.

PROCEDURE

1. Take a healthy adult rat and anesthetize it with chloroform or ether.

2. Place the anesthetized rat on a small animal operating table and secure it by tying

its all limbs.

3. Give midline incision on the neck and extend the skin laterally.

4. Expose the trachea and give a transverse cut between two rings. Insert a small

tube or cannula in between two rings pointing towards lungs and secure it

properly with the help of twin ligature to ensure a free airway.

5. Expose and clean common carotid artery on one side and ligate it at superior end,

and put a clamp at inferior end. Give a small cut on carotid artery, and insert a

polythene cannula already attached to a 23- gauge needle and connected via a

three way stop-cock to a Condon mercury manometer filled with normal saline

containing heparin (1000 IU/ml). Flush system with saline after each injection.

6. Record the baseline mean arterial BP.

7. Inject various test drugs one by one in the rubber tubing. Always keep at least 5 to

10 minutes gap between the two injections. Record the response of each test drug.

8. Label and fix the tracing with fixing solution. Interpret the findings of test drugs.



Table 16: Effects of Some Drugs on Blood Pressure

Drug/Treatment Dose Response (mm Hg) Remark

Normal -

Adrenaline 2 µg/kg

Isoprenaline 2 µg/kg

Acetylcholine 2 µg/kg

Atropine 1 mg/kg

Phenoxybenzamine 1 mg/kg

Propanolol 1 mg/kg

IMPORTANT QUESTIONS

1. Classify the drugs acting on the cardiovascular system.

2. Write the functions of Condon mercury manometer.

3. How much is the arterial BP in normal humans and animals.

80

25

STUDY OF EFFECTS OF GANGLIONIC

STIMULANTS AND BLOCKERS

OBJECTIVE

To study the effects of ganglionic stimulants and blockers on blood pressure (BP).

REQUIREMENTS

Instruments and other materials- Heparin (1000 IU/ml) or sodium citrate (100

mg/ml) as anticoagulant, pentobarbitone sodium (30 mg/ml), mercury manometer,

kymograph, surgical equipments, dog operating table, kymograph (big), arterial

cannula, venous cannula, surgical equipments, intubation tube, syringe with needle,

distilled water and normal saline.

Drugs- Adrenaline (10 µg/ml), 1,1- dimethyl-4-phenylpierazinium (DMPP- 1 mg/ml)

and pentolinium tartrate (20 mg/ml).

Animals- Dogs.

PRINCIPLE

Autonomic ganglionic stimulant drugs cause the increase in BP due to stimulation of

sympathetic nervous system (predominant tone on blood vessels). The administration of

ganglionic blocker abolishes the predominant tone of sympathetic nervous system,

resulting in the fall of BP.

PROCEDURE

1. Take a healthy dog or cat for the experiment.

2. Anaesthetize the dog with 30 mg/kg of pentobarbital sodium intravenously. If the

81

respiratory arrest occurs, institute the artificial respiration immediately and

administer the metrazol or picrotoxin.

3. The dog is fixed on a table in supine position.

4. The neck of dog is dissected to expose carotid arteries and vagus nerves. Isolate

both common carotid arteries, so that they can readily be occluded.

5. Cannulate the right common carotid artery and connect it to the mercury

manometer or pressure transducer for the recording of BP.

6. Record the base line mean arterial BP.

7. Record pressure responses to adrenaline and DMPP by injecting drugs

individually in the rubber tubing close to the venous cannula.

8. Label and fix the tracing with fixing solution. Interpret the findings of test drugs.



Table 17: Effects of Ganglionic Stimulants and Blockers on Blood Pressure

Drug/Treatment Dose Response

(mm Hg)

Remark

Normal saline -

Adrenaline 2 µg/kg

DMPP 50 µg/kg

Pentolinium 2 mg/kg

IMPORTANT QUESTIONS

1. Give the example of autonomic ganglionic stimulants and blockers.

2. Describe the mechanism of action of DMPP and pentolinium tartrate.

3. Why the carotid artery is applied to measure the BP.

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26


AUTONOMIC NERVOUS SYSTEM

OBJECTIVE

To study the actions of some drugs on the autonomic nervous system.

REQUIREMENTS

Instruments and other materials- Heparin (1000 IU/ml) or sodium citrate (100

mg/ml), pentobarbitone sodium (30 mg/ml) as anticoagulant, mammalian blood

pressure assembly with mercury manometer, electronic stimulator (Fig. 35),

kymograph, surgical equipments, dog operating table, kymograph (big), arterial

cannula, venous cannula, surgical equipments, intubation tube, glass syringe with


Drugs- Acetylcholine chloride, carbachol, adrenaline, ephedrine hydrochloride,

histamine phosphate, posterior pituitary extract (oxytocin), barium chloride (BaCl2),

neostigmine methyl sulphate and atropine sulphate.

Animals- Dogs.

Fig. 35: Electronic Stimulator


83

PRINCIPLE

The actions of many drugs are not confined to a single system of the body. A drug

may act on single organ or multiple organs of the same or different systems. The present

experiment needs a live anaesthetized dog to demonstrate the effects of some drugs on

the cardiovascular and respiratory systems. These drugs act on different receptors in an

organ to produce similar or opposite effects.


1. Take a healthy dog or cat for the experiment.

2. Anaesthetize the dog with pentobarbital sodium (30 mg/kg) or chloralose (100

mg/kg) intravenously. If the respiratory arrest occurs, institute the artificial

respiration immediately and administer the metrazol or picrotoxin.

3. Dog is fixed on a table in supine position and neck of dog is dissected to expose

carotid arteries and vagus nerves. Isolate both common carotid arteries, so that

they can readily be occluded. Cannulate the right common carotid artery and

connect it to mercury manometer or pressure transducer for the recording of BP.

4. Left side vagus is cut into the central and peripheral end for applying electrical

stimulation. Right femoral vein is cannulated with a catheter to inject test drugs.

5. A chart recorder or kymograph is set up to receive the signals from the

physiograph to produce graphical representation of changes in the BP.

6. Open abdomen and expose ileum. Put a purse string suture in the ileum. Introduce

balloon attached to glass tubing and tight the purse string. Close the abdomen.

7. Connect the open end of the glass tube at tambour for recording of the

movements. Canulate a femoral vein for intravenous injections.

8. All injections are to be made intravenously. The rate of injection should be slow

and regular. The data should be taken before the dose and at the height of action.

(a) Occlude both common carotid arteries until response is obtained (for 30 sec.;

be careful not to stimulate the vagus). Then, note the observations:

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(b) For stimulation of peripheral end of the vagus nerve, determine the threshold

electrical stimulus, applied for 5 seconds to the right vagus, which produces a

detectable effect on the BP. Further, give a stronger stimulus for 5 seconds to

produce a detectable effect on the intestinal motility- ……..

(c) Effect of Ach chloride (0.0001 mg/kg and 0.001 mg/kg)- ……..

(d) Effect of carbachol (0.002 mg/kg)- ……..

(e) Effect of adrenaline (0.001 mg/kg)- ……..

(f) Effect of ephedrine hydrochloride (0.3 mg/kg)- ……..

(g) Effect of histamine phosphate (0.01 mg/kg)- ……..

(h) Effect of posterior pituitary extract (oxytocin- 0.1 unit/kg)- ……..

(i) Effect of BaCl2 (1.5 mg/kg)- ……..

(j) Effect (potentiation) of neostigmine (inject neostigmine methyl sulphate @

0.03 mg/kg; wait for 5 minutes; and repeat above b and c)- ……..

(k) Effect of atropine (give very slowly @ 2 mg/kg; and repeat above b, c, d, g

and h)- ……..

IMPORTANT QUESTIONS

1. Discuss the occlusion of both carotid arteries.

2. Describe the effect of vagal stimulation on body systems.

3. Conclude and interpret the observations/results of the experiment.

85

ABOUT THE AUTHOR

Dr. Govind Pandey, “Professor/Principal Scientist” of Pharmacology

& Toxicology, is having the experience of about 33 years in ‘Research/

Teaching/Extension/Administration’. He is an able academician, scientist,

veterinarian and administrator; a Hindi literalist and eloquent speaker

endowed with strong writing flair. Dr. Pandey is probably the “Only person

in Madhya Pradesh and alone veterinarian in India with maximum academic

qualifications” (20 Degrees/Diplomas/Certificates). He obtained PhD

Honours degree in Veterinary Pharmacology & Toxicology from the Jawaharlal Nehru

Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, MP in the year 1990. Presently, he is

doing DSc. He has been ‘awarded/honoured’ by eminent persons; and also

‘published/broadcasted’ in different media for great contribution in education, science,

research, Hindi literature and culture, public, governmental and social works. His

“Biography” is included in the famous book of the world, ‘Who’s Who in the World

2011’ (28th

edition, America). Dr. Pandey has been honoured with 3 prestigious national

and international ‘Fellowship Titles’, viz., “FASAW”, “FSLSc” and “FISCA”.

Dr. Pandey started his career as “Veterinary Assistant Surgeon/Lecturer” on 7th

August, 1980 at Artificial Insemination Training Institute, Mandla, Animal Husbandry

(AH) Department, Government of MP. Under this Department, he also worked as

“Veterinary Surgeon” and “Senior Veterinary Surgeon” in different offices at Jabalpur,

including “Officer-In-Charge cum Drawing Disbursing Officer (DDO)” of Rinder Pest,

Jabalpur Division, Jabalpur till 19th

April, 2012. During these periods, he also served as

“Chief Executive Officer/Block Development Officer cum DDO” of some Janapad

Panchayats under the Panchayat & Rural Development Department, Govt. of MP; and as

“Assistant Professor & Head” and “Professor/Principal Scientist & Head” of

Pharmacology in Pharmacy colleges. On 20th April, 2012, he joined as “Deputy Director

of Research/Associate Professor/Senior Scientist” at the Directorate of Research

Services, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur. On 26th

November, 2012, he has resumed the post of “Professor/Principal Scientist & Sectional

Head”, Department of Pharmacology & Toxicology, College of Veterinary Science &

AH, Rewa (NDVSU, Jabalpur).

Dr. Pandey is working in different areas of Life Science, with specialization in

Pharmacology & Toxicology. He has investigated some “Antihepatotoxic and anticancer

herbal drugs, and experimental hepatotoxic and cancer models’ in animals. He has also

made a good contribution in Fishery Science, Hindi literature, Human Resource

Management, Political Science, Sociology, Public Administration, Law and Astrology.

He has published more than 225 scientific papers and delivered many speeches in a

number of platforms. He has supervised/guided/co-guided some PhD/MVSc & AH

‘Research Theses’; and guided many M Pharm/B Pharm ‘Academic Project Reports’. He

has also carried out some ‘Research Projects’. His 1 scientific ‘e-Book’, “Recent

Research on the Anticancer Herbal Drugs and Cancer Causing Agents” (International E

- Publication, ISCA; 2013) has been published; while and 6 ‘Books’ are under

publication. Dr. Pandey is the recipient 30 “Awards/Fellowships/Sponsorships/Honours/

86

Recognitions” (including “ICAR Senior Research Fellowship” and “Sri Ram Lal

Agrawal National Award”) in science, research and Hindi literature. In Hindi literature,

he has published 5 books, released 2 audiocassettes of own lyrics and edited 1 book. His

several poems, lyrics, dramas, stories or speeches have been published/broadcasted

through various media. He is the “Life Member” of 25 scientific, professional, literary

and cultural associations/societies/journals. He has acted as the “Chairperson/Chief

Guest/Judge/Expert” in many ‘Conferences/Seminars/Projects/Committees/Programmes’.

He also acted as the “Editorial Board Member/Editor/Reviewer” of some journals or

magazines. He has successfully organized many academic, official, literary, cultural and

social programmes in an exemplary manner. Dr. Pandey had been the “Captain of

Badminton”, “Sergeant of NCC”, “Literary Secretary” and “Hostel Prefect” in college.

He has passed “C Certificate of NCC”; and completed two years’ course of “National

Service Scheme” (NSS).

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