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Handbook of Experimental Pharmacology

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ISBN 93-85046-42-X

9 789385 046421
 Dedicated to
“My Parents, Teachers and Students”
 Handbook of
Experimental Pharmacology

Shivsharan B. Dhadde
Department of Pharmacology
D.S.T.S. Mandal’s College of Pharmacy
Solapur, Maharashtra – 413 004

2019

Studium Press (India) Pvt. Ltd.

 Handbook of
Experimental Pharmacology

© 2019

This book contains information obtained from authentic and highly regarded
sources. Reprinted material is quoted with permission, and sources are indicated.
A wide variety of references are listed. Reasonable efforts have been made to
publish reliable data and information, but the author and the publisher cannot
assume responsibility for the validity of all materials or for the consequences of
their use.

All rights are reserved under International and Pan-American Copyright

Conventions. Apart from any fair dealing for the purpose of private study,
research, criticism or review, as permitted under the Copyright Act, 1956, no
part of this publication may be reproduced, stored in a retrieval system or
transmitted, in any form or by any means–electronic, electrical, chemical,
mechanical, optical, photocopying, recording or otherwise–without the prior
permission of the copyright owner.

ISBN: 978-93-85046-34-XXXXXXXXXX

Published by:

Studium Press (India) Pvt. Ltd.

4735/22, 2nd Floor, Prakash Deep Building
(Near Delhi Medical Association),
Ansari Road, Darya Ganj, New Delhi-110 002
Tel.: + 91-11-43240200-15 (15 lines); Fax: 91-11-43240215
E-mail: pubdir@studiumpress.in

Printed at India
 About the Author

Mr Shivsharan B. Dhadde is serving as Assistant

Professor at D.S.T.S. Mandal’s College of
Pharmacy, Solapur. He completed his graduation
in Pharmacy from B.L.D.E.A’s S.S.M. College of
Pharmacy and Research Center, Vijaypur,
Karnataka and post graduation in Pharmacology
from Sree Siddaganga College of Pharmacy,
Tumkur, Karnataka under Rajiv Gandhi
University of Health Sciences, Karnataka,
Bangalore. Mr Dhadde, qualified GATE (PY)-2009
and received several scholarships and awards during his studies. His
research interests are Meta-Analysis of natural and synthetic
pharmacological agents and evaluation of drugs for neurological and
metabolic disorders. He is serving as “Executive Editor for SPER Journal
of Advances in Novel Drug Delivery” and Editorial Board Member and
Reviewer for many reputed national and international journals. Mr
Dhadde has more than 6 years of teaching and research experience.
He worked as a resource person in more than six national and
international workshops/conferences. Mr Dhadde has presented scientific
posters in four national and six international workshops/conferences.
There are more than 20 publications in national and international
journals in his credit with a cumulative impact factor of ~ 30 (Thomason
Reuter). He also contributed three book chapters in reputed reference
books in the area of diabetes mellitus, nanotechnology and polymer
science. Mr Dhadde gained a good amount of knowledge in experimental
pharmacology and he has commendable skills in typical pharmacological
experiments like invasive blood pressure measurement in rats, recording
the response of phrenic nerve-diaphragm preparation, recording the
response of drug in rat thoracic aorta ring preparation, ischemia (bifocal)
and reperfusion-induced neuronal damage in rats, middle cerebral artery
occlusion induced brain injury in rats, ischemia (bifocal) and reperfusion-
induced renal damage in rats and mice, Langendorff’s isolated perfused
heart preparation, nerve conduction recording, 3-nitropropionic acid-
induced experimental Huntington’s disease in rats etc. Mr Dhadde
gained good citation index with H-Index of 09 and i10-index of 08.
31 March 2018
 Acknowledgements

I would like to thank the Management, Principal and all the staff
members of D.S.T.S. Mandal’s College of Pharmacy, Solapur for their
constant support and suggestions during the preparation of this
manuscript. I wish to place on record my deep gratitude to my wife
Sakshi S. Dhadde for her relentless help while preparing this book. I
wish to express my deep appreciation and warmest thanks to J N Govil,
Studium Press (India) Pvt. Ltd, New Delhi, India for his continued
support and exemplary job coordination; I also extend my
acknowledgement to all the staff of Studium Press Pvt. Ltd, New Delhi
(India) for their commendable efforts in handling the manuscript with
professional skill and accuracy.

Shivsharan B. Dhadde
 Preface

The purpose of the present book is to provide fundamental knowledge

of practical aspects of the subject right from laboratory animals handling
and tissue mounting to practical implications of various important
complex experimental procedures. Today the experimental
pharmacology has tremendously drifted from the conventional approach
to molecular and biochemical aspects. The advancements in the field of
electrophysiology, biochemistry, molecular biology, electronic or digital
recording systems and software have been enriched and broadened the
horizons of experimental pharmacology. Due to the stringent regulations
laid down by the government (CPCSEA), so getting animals for
experiments is not an easy task. Thus, with the changing scenario,
alternative methods of teaching pharmacology experiments for
undergraduate students have been developed along with the advances
in computer technology. Experiments performed by using animals can
be substituted by demonstrations using computer-simulated learning
programs. Exact simulation of real animal experiments on a computer
is not easy because the biological responses are very complex. Moreover,
imagination of experimental outcome and acquiring the skill of hands-
on experiment is not satisfactorily learned by observing the effect of the
drug in simulated experiments. This book helps to reduce the gap
between hands-on experiments in animal and simulated experiments.
Fundamental principles of experimental pharmacology deal with
essential aspects of pharmacology, animal experimental methodology
and interpretation of obtained results. In this book, the reader could
find some of the useful aspects, e.g. number of worked out examples
which will help to translate theory into practice. Made a sincere attempt
to include as much relevant information as possible with illustrated
points and suitable examples to make this book comprehensive. Topics
covered in this book have been carefully selected based on most of the
improvised problems as per curriculum designed for pharmacology. I
hope that the present book will be helpful for all the graduates and
postgraduates related to pharmacology, trainees, research workers
during their day-to-day activities including allied health discipline and
scientists in industrial drug discovery set-up and CRO. Several simple
and newer experimental models have been incorporated which will help
viii Handbook of Experimental Pharmacology

the students to engage in drug discovery in future. Besides this, several

important points have been discussed in this book, e.g. ethics of animal
experimentation, care and handling of experimental animals,
preparation of solutions, tissue mounting for in vitro studies.

Every care has been taken to ensure that the book is free from errors
but absolute perfection is usually unattainable. Hence, errors if any
may please be communicated to me.

Solapur Shivsharan B. Dhadde

31 March 2018
Table of Contents ix

Table of Contents

About the Author v

Acknowledgements vi
Preface vii
Basics of Experimental Pharmacology –
1. Introduction to experimental pharmacology –
2. Commonly used instruments in experimental pharmacology –
3. Commonly used laboratory animals in experimental –
pharmacology
4. CPCSEA guidelines for laboratory animal facility –
5. Handling and restraint of small laboratory animals –
6. Routes of drug administration in laboratory animals –
7. Techniques of blood collection in laboratory animals –
8. Anaesthetics used in experimental pharmacology –
9. Techniques of euthanasia used in experimental –
pharmacology
Experiments on Isolated Preparations (Ex-Vivo) –
10. Physiological salt solutions (PSS)used in isolated tissue –
preparation
11. Procedure for recording isolated tissue responses –
12. Dose-response curve (DRC) of acetylcholine using frog –
rectus abdominis muscle.
13. Effect of physostigmine on DRC of acetylcholine using frog –
rectus abdominis muscle
14. Effect of atropine on DRC of acetylcholine using rat ileum –
15. Effect of spasmogens and spasmolytics on rabbit jejunum/ –
Effect of drugs on gastrointestinal motility
16. Effect of agonist and antagonists on guinea pig ileum –
17. Effect of saline purgative on frog intestine –
18. Determination of PA2 value of prazosin using rat –
anococcygeus muscle
19. Determination of PD2 value using guinea pig ileum –
preparation
20. Effect of drugs on isolated frog heart preparation –
x Handbook of Experimental Pharmacology

Bioassays –
21. Introduction to bioassay –
22. Bioassay of histamine using guinea pig ileum by matching –
method
23. Bioassay of oxytocin using rat uterine horn by interpolation –
method
24. Bioassay of serotonin using rat fundus strip by three-point –
method
25. Bioassay of acetylcholine using rat ileum by four-point –
method
26. Bioassay of atropine using guinea pig ileum by –
interpolation method
27. Bioassay of d-tubocurarine by rabbit head-drop method –
28. Bioassay of digitalis by guinea pig cardiac arrest method –
29. Bioassay of insulin by rabbit hypoglycemic effect method –
30. Bioassay of heparin sodium by sheep blood coagulation –
method
In-Vivo Experiments in Animals –
31. Effect of drugs (miotics, mydriatics and local anaesthetics) –
on rabbit eye
32. Effect of drugs on ciliary motility of frog oesophagus –
33. Skeletal muscle relaxant property of drug using rota-rod –
apparatus
34. Skeletal muscle relaxant property of drug using hanging –
wire test
35. Local anaesthetic activity of drugs by different methods –
36. Effect of drugs on locomotor activity in mice using –
actophotometer
37. Analgesic activity of drugs using central and peripheral –
methods
38. Anti-inflammatory activity of drugs using carrageenan- –
induced paw-oedema model
39. Anti-ulcer activity of a drug using pylorus ligated (SHAY) –
rat model
40. Anti-ulcer activity of a drug using NSAIDS induced ulcer –
model
41. Effect of sulfasalazin on indomethacin-induced –
inflammatory bowel disease in rats
42. Anti-allergic activity by mast cell stabilization assay –
43. Effect of finasteride on testosterone-induced prostatic –
hyperplasia in rats
44. Hypoglycemic effect of insulin in rabbits –
Table of Contents xi

45. Effect of drugs on blood pressure and heart rate of dog –

46. Diuretic activity of drugs using rats/mice –
47. Effect of hepatic microsomal enzyme inducers on the –
phenobarbitone sleeping time in mice
48. Anticonvulsant effect of drug by MES induced convulsion –
method
49. Anticonvulsant effect of drug by PTZ induced convulsion –
method
50. Antistereotypy activity of haloperidol in rats (stereotyped –
behaviour)
51. Anti-catatonic activity of drugs on rats/mice –
52. Anxiolytic activity of drugs in mice using Light-dark box –
apparatus
53. Anxiolytic activity of drugs in mice using elevated plus –
maze apparatus
54. Pyrogen test of pharmaceutical preparation by rabbit –
method
55. Determination of acute skin irritation/corrosion of a test –
substance
56. Determination of acute eye irritation/corrosion of a test –
substance
57. Determination of acute oral toxicity of drug –
Introduction to Cell-Based Assays –
Bibliography –
Subject Index –
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