Forced-Exercise Alleviates Neuropathic Pain in Experimental

133 Pages·2016·1.51 MB·English

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Dissertations Theses and Dissertations
2010
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Sahadev A. Shankarappa
Loyola University Chicago
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Shankarappa, Sahadev A., "Forced-Exercise Alleviates Neuropathic Pain in Experimental Diabetes: Effects
on Voltage-Gated Calcium Channels" (2010). Dissertations. 187.
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Copyright © 2010 Sahadev A. Shankarappa
LOYOLA UNIVERSITY CHICAGO

FORCED-EXERCISE ALLEVIATES NEUROPATHIC PAIN IN

EXPERIMENTAL DIABETES: EFFECTS ON VOLTAGE-GATED CALCIUM

CHANNELS

A DISSERTATION SUBMITTED TO

THE FACULTY OF THE GRADUATE SCHOOL

IN CANDIDACY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

PROGRAM IN NEUROSCIENCE

BY

SAHADEV A SHANKARAPPA

CHICAGO, ILLINOIS

MAY 2010
Copyright by SAHADEV SHANKARAPPA, 2010
All rights reserved
ACKNOWLEDGEMENTS
Firstly, I would like to acknowledge Dr. Evan B. Stubbs, Jr., who has been my
graduate advisor, mentor, and racquetball teacher, for his guidance in helping me to
understand, critically think, and do science in an organized and logical manner. I
sincerely thank Dr. Erika S. Piedras-Rentería, who has been my second mentor, for all
her help, suggestions, and guidance for the electrophysiology component of this
dissertation. I also thank the members of my graduate committee, Dr. Edward J. Neafsey,
Dr. Morris Fisher and Dr. Gwendolyn Kartje, for their valuable suggestions and
encouragement.
I acknowledge the Arthur J Schmitt foundation for awarding me the Schmitt
fellowship, and the Veteran Affairs department of rehabilitation services for the financial
assistance, that made this study possible.
I would like to thank my colleagues, Dr. Jason Sarkey, Dr. Cynthia Von ee and
Mr. Michael Richards, for their assistance, advice and encouragement during my
graduate career.
I thank my mother, Gowthami; my father, Shankar; and my sister, Parvathi; for
their unrelenting emotional support throughout my career. I thank my wife Shivanee, and
my daughter Aarika without whom I would not have come this far.

iii
“To my lovely wife Shivanee and my beautiful daughter Aarika
TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS v
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xii
ABSTRACT xiv
CHAPTER
1. INTRODUCTION 16
STATEMENT OF PROBLEM 16
HYPOTHESIS 19
SPECIFIC AIMS 19
LITERATURE REVIEW 21
DIABETES MELLITUS 21
Epidemiology of DM 23
Complications of DM 23
DIABETIC NEUROPATHY 25
Pathogenic mechanisms of diabetes-associated nerve injury 25
Neuropathic pain in diabetic neuropathy 28
Experimental animal models of diabetic neuropathy 29
Streptozotocin (STZ)-induced DM 30
STZ-induced diabetic polyneuropathy 32
v
Treatment options in diabetic neuropathy 32
Exercise as a therapeutic strategy 33
Exercise and voltage-gated calcium channels 34
VOLTAGE GATED CALCIUM CHANNELS 36
High-voltage activated Ca2+ channel isoforms 39
Low-voltage activated Ca2+ channels 41
Role of voltage-gated Ca2+ channels in neuropathic pain 42
2. FORCED-EXERCISE ALLEVIATES NEUROPATHIC PAIN IN
EXPERIMENTAL DIABETES: EFFECTS ON VOLTAGE-GATED
CALCIUM CHANNELS 46
ABSTRACT 46
INTRODUCTION 48
MATERIALS AND METHODS 51
Animal care 51
Induction of experimental diabetes mellitus 51
Experimental exercise regimens 52
Tactile-responsiveness to Von-Frey filaments 53
Thermal responsiveness to infra-red heat stimulus 54
Peripheral nerve conduction studies 55
Dissociation of dorsal root ganglia neurons 57
Voltage clamp electrophysiology 57
Statistical analysis 59

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RESULTS 60
Streptozotocin-induced diabetes and voluntary-exercise 60
Streptozotocin-induced diabetes and forced-exercise 60
Forced-exercise protects against STZ-induced diabetes-associated
nerve conduction deficits 61
Forced-exercise delays onset of diabetes-associated hyperalgesia 62
Responsiveness to thermal stimulus is not altered by forced-exercise 63
Naloxone reverses forced-exercise induced analgesia 64
Forced-exercise prevents diabetes associated alteration in
Ca2+ current densities 64
Diabetes-associated changes in LVA Ca2+ current
steady-state inactivation 66
DISCUSSION 79
3. D-GLUCOSE DEPENDENT MODULATION OF Ca 3.2 α CHANNEL
V 1H
FUNCTION 85
ABSTRACT 85
INTRODUCTION 87
MATERIALS AND METHODS 89
Cell culture 89
Voltage Clamp Electrophysiology 89
Statistical Analysis 91
RESULTS 92
D-Glucose induced enhancement of T-type currents in HEK-293 cells 92
D-glucose induced alteration of Ca 3.2 channel gating properties 92
V
Effect of D-glucose on Ca 3.2 channel steady state properties 93
V
vii
Aldose reductase inhibition does not alter D-glucose induced
enhancement of Ca 3.2 currents 93
V
DISCUSSION 104
SUMMARY 107
REFERENCES 111
VITA 132
viii
LIST OF TABLES
Table Page
1. Classification of Diabetes Mellitus 22

2. Clinical classification of glucose tolerance based on plasma glucose levels. 23

3. Types, symptoms, and signs of diabetic neuropathy. 26

4. Mechanisms of diabetes induced nerve injury. 28

5. Animal models of DM and its subtypes 30

6. Pharmacology and function of the α1 Ca2+ channel subunit. 37

7. Evoked compound muscle action potential response from the sciatic
nerve of STZ-diabetic rats subjected to sedentary or forced-exercise. 71

8. Conduction velocity calculated from the tail nerve in diabetic rats
subjected to forced-exercise. 72

9. Passive membrane properties of dissociated small diameter DRG neurons. 78

10. Comparison of in vitro and in vivo glucose concentrations. 95

11. Osmolarity measurements of normal and high-glucose containing media. ......... 95
ix