Mechanisms of reduced effectiveness in a rat model of neuropathic pain

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Authors Nichols, Michael Lorne, 1967-

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Mechanisms of Reduced Opioid Effectiveness in a Rat Model of Neuropathic Pain

by Michael Lome Nichols

A Dissertation Submitted to the Faculty of the COMMITTEE ON AND TOXICOLOGY (Graduate) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1997 UMI Nximber: 9729518

UMI Microform 9729518 Copyright 1997, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arirar, MI 48103 2

THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Michael L. Nichols entitled Mechanisms of Reduced Opioid Effectiveness in a Rat

Model of Neuropathic Pain

and recommend that it be accepted as fulfilling the dissertation requirement for tlie Degree of Doctor of Philosophy

OA.KSLCS. 4-- FV^k Porr^a,^h.D. Date ^9-^7 Edward D. , Ph.D. Date

Lai, Ph.D. Date

r. PhUlilsCMa^./M.D'./,In,/;M.D^, Ph.D. Date H-lih7 D. Palmer, M.D., Ph.D. Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and re^mmend that it be accepted as fulfilling the dissertation requirement.

hAiAlt. f ~/0 -^1 Usfeertation Director Date 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under mles of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the m ^ * • ^ ' ' hip. In all other instances, however, permission must be 4

ACKNOWLEDGEMENTS

I thank Dr. Frank Porreca for giving me the opportunity to be successful when no one else would. Frank has provided me with an excellent setting to develop and excel as a young scientist. Further, Frank has given me both the guidance, motivation and example of a true mentor. I also thank my conunittee members, Drs. Ed French, Josephine Lai. Phil Malan and John Palmer for their time and effort to help me complete my Ph.D. I also thank Dr. Larry Reid everything he has given me along the way. Di (Chuck) Bian and Michael (Rash) Ossipov deserve special recognition. Chuck has worked with me on nearly every project in the laboratory, and his surgical skills have saved me countless hours of frustration. Mike O. has helped me through my confusion with his expertise and guidance. On the intellectual side of things he has given his all to help me understand. I thank them both very much. I have shared many things with Ed (Dr. Dork) Bilsky, including office space, for the greater part of nearly twelve years. Ed has been a great friend who has kept the lab from feeling like a job. I will dearly miss Ed and his antics and I wish him and his lovely wife, Jill, all the best. Other members of the Porreca tribe that deserve recognition are Joe Tumminia, C.J. (Squeegee) Kovelowski, Dr. Todd (Squirrel) Vanderah, Dr. Robert (Bob) Horvath, Sandy (Flower) Wegert, Eddie Mata, Dr. Ken (Vem) Wild and Jason Lashbrook for always lending a hand or an ear to me along the way. I consider all of you my friends. I also thank all of the friends I have made in Arizona including Ray-ray, Steve Stratton, Lungs of a Smoker (Jill, Kent, AI, Mr. Potatohead and EFang), softball teamates and camping crews. Russ (Crusty) Ingersoll is my dear friend, whom I'll miss very much. Lastly, I thank my best friend, Wanda, who has not only been my friend, but has managed to survive living with me for nearly four years. I love you very much, Wanda. 5

DEDICATION

This disseration is dedicated to my parents, Robert and Marie Nichols. Their sacrifice gave me the opportunity to begin my academic career. Their support helped to propel me through my bachelor's, master's, and now, my doctoral degree. I would never have been given these opportunities without them. 6

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS 13

ABSTRACT 20

INTRODUCTION 22

Nociception 23

The neurotransmission of pain 25

Hyperalgesia 27

Opiates and and pain perception 30

Brief history 30

Opioid receptors and endogenous opioids 31

Opioids and the inhibition of pain 36

Endogenous pain control 40

Abnormal pain states: peripheral neuropathy 41

Animal models of peripheral neuropathy 43

Opiates in neuropathic pain 44

Excitatory amino acids and neuropathic pain 46 7

TABLE OF CONTENTS-ConfmueJ

Page

The non-opioid effects of and neuropathic pain 49

Afferent fibers and neuropathic pain 50

Modulation of the effectiveness of mu opiates () 52

HYPOTHESIS OF THE DISSERTATION 57

Hypothesis 1 57

Hypothesis 2 57

Hypothesis 3 58

METHODS 59

Animals 59

Chemicals 59

Surgical implantation of the intrathecal catheter 60

Drug administration 61

L5/L6 nerve ligation and sham surgery 62

Neonatal treatment 63

Behavioral Testing 63

von Frey assay (including normalized von Frey fibers) 63 8

TABLE OF CONTENTS- Continued

Page

Warm-water tail flick assay 64

Radiant heat foot flick assay 65

Pin prick assay 65

Statistics 65

RESULTS OF PRELIMINARY EXPERIMENTS 67

Baseline testing 67

Testing with normalized von Frey fllaments 68

Effect of anesthetic on the development of tactile allodynia 71

SUMMARY OF PRELIMINARY EXPERIMENTS 77

RESULTS OF STUDIES INVOLVING OPIATES AND OPIOIDS 79

Lack of effect of intrathecal morphine to probing with von Frey

monofilaments 79

Reduced antinociceptive effectiveness of morphine in rats with

nerve-ligation injury 83

Studies on the effect of intrathecal [D-Ala^, NMePhe^, Gly-

oljenkephalin (DAMGO) to probing with von Frey monofilaments. .83 9

TABLE OF CONTENTS- Continued

Page

Studies on the antiallodynic effect of intrathecal SNC 80 in rats with

nerve injury 88

Studies on the antiallodynic effect of intrathecal [D-Ala^,

Glu^ldeltorphin in rats with nerve injury 90

Studies on the antiallodynic effect of intrathecal in rats with

nerve injury 93

Studies on the antiallodynic effect of intrathecally administered

kappa in rats with nerve injury 97

SUMMARY OF STUDIES INVOLVING OPIATES AND OPIOIDS 100

RESULTS OF EXPERIMENTS INVOLVING NEONATAL CAPSAICIN

TREATMENT 106

Effects of capsaicin treatment on nociceptive thresholds 106

Warm-water Tail Flick {48'C) 106

Warm-water Tail Flick (52"C) 107

Radiant Heat Paw Withdrawal 107

Pin Prick 110 10

TABLE OF CONTENTS- Continued

Page

Von Frey probing 110

Effects of morphine in capsaicin treated rats 114

Morphine in the warm-water tail flick (52" C) 114

Morphine in the von Frey assay 120

Effects of DAMGO in capsaicin treated rats 123

DAM GO in the warm-water tail flick (52" C) 123

DAMGO in the von Frey assay 129

SUMMARY OF EXPERIMENTS INVOLVING NEONATAL CAPSAICIN

TREATMENT 134

RESULTS OF EXPERIMENTS INVOLVING EXCITATORY

ANTAGONISTS AND (1-13) ANTISERA 140

Studies on the antiallodynic effects of excitatory amino acid

antagonists 140

MK 801 versus tactile allodynia 140

CNQX versus tactile allodynia 142

L-AP3 versus tactile allodynia 142 11

TABLE OF CONTENTS- Continued

Page

Studies on the effects of morphine in the presence of MK 801 148

Tactile allodynia 148

Warm-water tail flick (55°C) 151

Studies on the effect of morphine following pretreatment with

antisera to dynorphin A (1-13) 154

Tactile allodynia 154

Warm-water tail flick 55"C 154

SUMMARY OF EXPERIMENTS INVOLVING EXCITATORY AMINO

ACID ANTAGONISTS AND DYNORPHIN A (1-13) ANTISERA 159

RESULTS OF EXPERIMENTS INVOLVING DYNORPHIN A (1-17) 163

SUMMARY OF EXPERIMENTS INVOLVING DYNORPHIN A (1-17)... 166

RESULTS OF EXPERIMENTS INVOLVING CCKg AND DELTA

MODULATION OF MORPHINE AND 169

Studies on the modulation of the effect of morphine by a CCK^

antagonist or a delta opioid 169 12

TABLE OF CONTENTS- Continued

Page

Co administration of morphine and L 365, 260 169

Co administration of morphine and [D-Alcr, Glu^] 171

Studies on the modulation of the effects of thiorphan by a CCK„

antagonist 171

SUMMARY OF EXPERIMENTS INVOLVING CCKb AND DELTA

MODULATION OF MORPHINE AND THIORPHAN 178

GENERAL CONCLUSION 180

APPENDIX A: Animal use forms 187

REFERENCES 188 13

LIST OF FIGURES

Page

1 Baseline paw withdrawal thresholds of nerve-ligated and sham-operated rats 69

2 Baselines of standard and normalized von Frey filiments in nerve-ligated rats 70

3 Development of tactile allodynia following nerve ligation surgery performed under

/xylazine anesthetic 72

4 Development of tactile allodynia following nerve ligation surgery performed under

halothane anesthetic 73

5 Development of tactile allodynia following nerve ligation surgery performed under

pentobarbital anesthetic 74

6 Development of tactile allodynia following nerve ligation surgery performed under

Innovar-vet anesthetic 75

7 Development of tactile allodynia following nerve ligation surgery performed under

chloral hydrate anesthetic 76

8 Lack of antiallodynic effect of Lth. morphine in nerve-ligated rats 80

9 Antiallodynic dose response relationship of morphine in nerve-ligated rats 81

10 Lack of effect of Lth. morphine in sham-operated rats 82

11 Antinociceptive effect of Lth. morphine in nerve-ligated and sham-operated rats... .84 14

LIST OF FIGURES-Co/irmae^/

Antiallodynic effect of i.th. [D-AIa^ NMePhe\ GIy-ol] in nerve-ligated

rats 85

13 Antiallodynic dose response relationship of [D-AIa\ NMePhe'*, Gly-ol]enkephalin in

nerve-ligated rats 86

14 Lack of effect of i.th. [D-Ala^ NMePhe"*, Gly-oI]enkephalin in sham-operated rats. 87

15 Antiallodynic effect of i.th. SNC 80 in nerve-ligated rats 89

16 Antiallodynic effect of i.th. [D-Ala% Glu"']deltorphin in nerve-ligated rats 91

17 Antiallodynic dose response relationship of [D-Ala", Glu"*]deltorphin in nerve-ligated

rats 92

18 Antiallodynic effect of i.th. biphalin in nerve-ligated rats 94

19 Antiallodynic dose response relationship of biphalin in nerve-ligated rats 95

20 Dose response comparison of morphine, [D-Ala', NMePhe"*, Gly-ol]enkephalin and

biphalin 96

21 Antiallodynic effect of i.th. U 69,593 in nerve-ligated rats 98

22 Antiallodynic effect of i.th. fedotazine in nerve-ligated rats 99

23 Effect of neonatal capsaicin treatment of nerve-ligated and sham-operated rats in the

48''C warm water tail flick assay 108 15

LIST OF FIGURES-Confi/2Me

24 Effect of neonatal capsaicin treatment of nerve-ligated and sham-operated rats in the

52°C warm water tail flick assay 109

25 Effect of neonatal capsaicin treatment of nerve-ligated and sham-operated rats in the

radiant heat foot flick assay Ill

26 Effect of neonatal capsaicin treatment of nerve-ligated and sham-operated rats in the

pin prick assay 112

27 Effect of neonatal capsaicin treatment of nerve-ligated and sham-operated rats on von

Frey filiment probing 113

28 Effect of ith. morphine on neonatally vehicle treated, sham-operated rats in the 52"C

warm water tail flick assay 115

29 Effect of i.th. morphine on neonatally capsaicin treated, sham-operated rats in the

52°C warm water tail flick assay 116

30 Effect of i.th. morphine on neonatally vehicle treated, nerve-ligated rats in the 52^

warm water tail flick assay 117

31 Effect of i.th. morphine on neonatally capsaicin treated, nerve-ligated rats in the 52°C

warm water tail flick assay 118

32 Dose response relationship of i.th. morphine on capsaicin treated rats in the 52"C

warm water tail flick assay 119 16

LIST OF FIGURES-Conri/zMeJ

33 Effect of Uh. morphine in neonatal capsaicin or vehicle treated, sham-operated rats to

probing with von Frey filaments 121

34 Antiallodynic effect of Lth. morphine in neonatal capsaicin or vehicle treated, nerve-

ligated rats 122

35 Antinociceptive effect of Lth. [D-Ala^ NMePhe'', Gly-ol]enkephalin on sham-

operated rats, neonatally treated with vehicle 124

36 Antinociceptive effect of Lth. [D-AIa^, NMePhe\ GIy-ol]enkephalin on nerve-ligated

rats, neonatally treated with vehicle 125

37 Antinociceptive effect of Lth. [D-Ala\ NMePhe^ Gly-ol]enlcephalin on sham-

operated rats, neonatally treated with capsaicin 126

38 Antinociceptive effect of Lth. [D-Ala^ NMePhe"*, Gly-ol]enkephalin on nerve-ligated

rats, neonatally treated with capsaicin 127

39 Antinociceptive dose response comparison of [D-Ala", NMePhe"*, Gly-ol]enkephalin

in capsaicin and vehicle treated sham-operated and nerve-ligated rats 129

40 Effect of Lth. [D-Ala", NMePhe"*, Gly-ol]enkephaIin in neonatal capsaicin or vehicle

treated, sham-operated rats to probing with von Frey filaments 130

41 Andallodynice effect of Lth. P-Ala", NMePhe"*, Gly-ol]enkephalin on nerve-ligated

rats, neonatally treated with vehicle 131 17

LIST OF FIGURES-ConrinMe^/

42 Antiallodynic effect of i.th. [D-Ala% NMePhe'*, Gly-ol]enkephalin on nerve-ligated

rats, neonatally treated with capsaicin 132

43 Antiallodynic dose response comparison of [D-Ala^, NMePhe"*, Gly-ol]enkephalin in

nerve-ligated rats neonatally treated with capsaicin or vehicle 133

44 Lack of antiallodynic effect of i.th. MK 801 141

45 Antiallodynic effect of i.th. CNQX 143

46 Antiallodynic effect of i.th. CNQX in rats pretreated with MK 801 144

47 Dose response relationship of i.th. CNQX alone and in the presence of

MK 801 145

48 Lack of antiallodynic effect of i.th. L-AP 3 146

49 Lack of antiallodynic effect of i.th. L-AP 3 in rats pretreated with MK 801 147

50 Antiallodynic effect of i.th. MK 801 administered prior to 2 doses of i.th.

morphine 149

51 Antiallodynic effect of 3 doses of i.th. MK 801 administered prior to

morphine 150

52 Antinociceptive effect of i.th. morphine in the presence of i.th. MK 801 in nerve-

ligated rats 152

53 Antinociceptive effect of i.th. morphine in the presence of i.th. MK 801 in sham-

operated rats 153 LIST OF FIGURES-Con/mMet/

54 Antiallodynic effect of Lth. morphine in the presence of antisera to dynorphin A

(1-13) 156

55 Antinociceptive effect of i.th. morphine in the presence of antisera to dynorphin A (1-

13) in nerve-ligated rats 157

56Antinociceptive effect of i.th. morphine in the presence of antisera to dynorphin A (1-13)

in sham-operated rats 158

57 Paw withdrawal thresholds following i.th. administration of dynorphin A (1-17) to

normal rats 164

58 Dose and time response of paw withdrawal thresholds following i.th. administration

of dynorphin A (1-17) to normal rats 165

59 Antiallodynic effect of i.th. co-administration of morphine and L 365,260 in the

absence and presence of 170

60 Antiallodynic effect of i.th. co-administration of morphine and [D-Ala^

Glu"*]deltorphin in the absence and presence of naltrindole 172

61 Antiallodynic effect of i.th. co-administration of thiorphan and L 365,260 in the

absence and presence of naltrindole 174

62 Antiallodynic effect of i.th. co-administration of thiorphan and L 365,260 in the

absence and presence of antisera to [Leu^]enkephalin 175 19

LIST OF FIGVRES-Continued

63 Antiallodynic effect of i.th. co-administration of thiorphan and L 365,260 in the

absence and presence of antisera to [Met^]enkephalin 176

64 Antiallodynic effect of i.th. co-administration of thiorphan and L 365,260 in the

absence and presence of control sera 177 20

ABSTRACT

Peripheral nerve injury can result in long-lasting, abnormal pain states referred to as

neuropathic pains. These pains can result in increased sensitivity to both noxious

(hyperalgesia) and non-noxious stimuli (allodynia) and are often characterized as resistant

to alleviation by opioids. Neuropathies are accompanied by various neuropathological

changes, including: (1) alterations in spinal neurotransmitter levels (including

(CCK), enkephalin and dynorphin); (2) degeneration of primary afferents;

(3) formation of ectopic foci and dorsal horn sensitization; (4) abnormal sympathetic

innervation and (5) sprouting of Ap fibers to form novel synapses. The hypotheses of this

dissertation are (1) information of an allodynic nature is transmitted by Ap fibers, which do

not contain opioid receptors, therefore only the post-synaptic pool of receptors is available

for opioid interaction, resulting in a loss of opioid efficacy; (2) the maintenance of the

neuropathic pain state is mediated by tonic activity at excitatory amino acid receptors

(possibly by dynorphin) which may contribute to the loss of opioid effectiveness: and (3)

increases in spinal CCK attenuate opioid effectiveness by inhibition of the activity of

endogenous .

The initial hypothesis is supported by results showing that low efficacy opioids

(morphine and SNC 80) are ineffective at alleviating nerve injury-induced allodynia while

high efficacy opioids (DAMGO, [D-Ala", Glu"*]deltorphin and biphalin) produced a significant antiallodynic action. This strengthens the suggestion that a reduced opioid 21 receptor pool exists for the treatment of allodynia. Additionally, selective destruction of C- fibers by capsaicin alters responses to thermal but not mechanical stimuli. These results suggests that allodynia from nerve injury is mediated by AP fibers, which do not possess opioid receptors. The second hypothesis is supported by the result that MK 801 and dynorphin A (1-13) antisera restore the effectiveness of morphine suggesting that increased dynorphin levels affect opioid efficacy, possibly through an NMDA mediated mechanism.

Finally, blockade of CCKg receptors can also enhance the efficacy of opioids in a naltrindole reversible fashion, suggesting that CCKg blockade may increase morphine's effectiveness by increasing the availability of an endogenous 5-opioid, possibly [Leu-

^]enkephalin. These findings may lead to a better understanding of the role of opioids in neuropathic pain and the development of better treatments for these conditions. 22

INTRODUCTION

The perception of pain, also referred to as nociception (Perl, 1980), is a necessary function of the central nervous system. Pain is the body's warning sign for the potential of injury as well as a signal to avoid further injury and protect an injured area in order to facilitate the healing process. Tissue damage can arise from numerous external sources, such as scalding bath water or a bowling ball dropped on the foot, as well as internal sources, such as numerous disease states. Therefore, the mechanisms for nociception must be responsive to the various types of stimuli which may result in tissue damage, specifically, to stimuli of a thermal, mechanical and/or chemical nature. Further, the system must provide information associated with the location of the painful event as well as the intensity of such an event. Additionally, in order for pain to serve as a protective mechanism, there are systems involved which result in continuing nociception even after the source of the initial insult may have been withdrawn. These systems also provide increased sensitivity to various stimuli to prevent further damage.

Pain is also classified into two categories, acute pain and chronic pain. Individuals commonly suffer from acute pain, the cause of which is tissue damage of a brief nature

(Bonica, 1953). Chronic pain typically refers to pain caused by ongoing disease states or massive tis.sue damage which result in difficult to treat, long-term pain states. The focus of this dissertation is primarily to try to better understand the chronic pain states which arise following peripheral nerve injury. A better understanding of these types of pain states will 23

aid in the development of better as well as further our understanding of the

physiology associated with nerve injury.

Nociception

The "pain receptors" or nociceptors are generally thought to be free nerve endings

which respond to noxious stimuli. The exact mechanism by which the interaction between

the noxious stimuli and the nociceptors take place, a process known as transduction, is not

yet fully understood (Fields, 1987). Nociceptors have generally been classified into two different groups of pain receptors based on the size and transmission velocity of the neuron itself and the types of stimuli which result in nociceptor activation. The first type of

nociceptor is referred to as the C-polymodal nociceptor. These nociceptors are called

"polymodal" because the have been shown to respond to a number of different kinds of noxious stimuli, including mechanical, thermal or chemical stimuli (Bessou and Perl, 1969.

Georgopouious, 1974, Torebjork, 1974). C-polymodal nociceptors are found on C-fibers.

C-fibers are unmyelinated, small diameter (0.3 - 3 |im, Ochoa and Mair, 1969) afferents having slow conduction velocities (0.5 - 2 m/sec, LaMotte and Campbell, 1978). C-fibers comprise the largest proportion of afferent axons found in peripheral nerves (Fields, 1987).

The second group of nociceptors, A6-high threshold mechonoreceptors are found on thinly myelinated, larger diameter (2-5 |im, Ochoa and Mair, 1969) afferents and have more rapid conduction velocities (about 20 m/sec, Adriaensen, et al.. 1983). While all A5- 24

nociceptors are thought to respond to mechanical stimuli, some also respond to thermal

stimuli, referred to as A5-mechanothermal receptors (Adriaensen, et al., 1983).

Nociceptive neurons terminate primarily in the superficial layer of the dorsal horn of

the spinal cord (Lamina I and II) as well as in deeper lamina. These neurons form synapses

on second order neurons in the spinal cord which ascend to the thalamus after crossing the

midline. Larger (typically non-nociceptive) afferent fibers can directly asccnd along the

midline without synapsing in the dorsal horn. There are a wide variety of neurons found in

the dorsal horn of the spinal cord including inhibitory and excitatory intemeurons and

projection neurons. It is thought that nociceptive afferents form synapses with all these

types of neurons. Intemeurons can transmit inhibitory or excitatory information to

projection neurons, which then send pain signals to higher brain areas, motor neurons (to mediate spinal reflex action from painful stimuli), other intemeurons involved in pain regulation, and back onto the spinal afferent as a possible control mechanism. In addition, primary afferents may synapse directly on projection neurons to transmit nociceptive information to supraspinal sites. The projection neurons can generally be classified into two classes: nociception specific neurons and wide dynamic range (WDR) neurons. As the name implies, the synapses to nociception specific projection neurons arise solely from nociceptive C- and A5-fibers. Several nociceptive afferents may synapse on a single projection neuron, which results in relatively larger receptive fields for these second-order neurons. Nociceptive afferents also synapse on WDR neurons in the dorsal horn. WDR neurons respond to non-noxious stimuli, but also fire (and to a greater degree) when

stimuli is of a noxious nature (Fields, 1987).

The neurotransmission of pain

There are a number of substances which are found in primary afferent neurons

which may be responsible for the transmission of pain including various and

amino acid neurotransmitters. The peptides include -gene-related

(CGRP), somatostatin, vasoactive intestinal peptide (VIP) and substance P and other

neurokinins. Glutamate and aspartate are the primary excitatory amino acid

neurotransmitters. While the are found in high concentrations only in small diameter myelinated and unmyelinated afferents, the excitatory amino acids are also found

in these fibers as well as large, heavily-myelinated fibers. (LaMotte, 1986; Fields. 1987;

Rang, et al. 1994). These neurotransmitters have been localized in the dorsal horn as well as in the dorsal root ganglia, where the afferent cell bodies are located (LaMotte, 1986).

There is strong evidence to suggest that substance P may be a major contributor to pain neurotransmission. Substance P release can be measured in the dorsal horn following noxious thermal or mechanical stimuli (Duggan, et al., 1988) and can result in the slow depolarization of second order neurons (Urban and Randic, 1984). Substance P acts at the neurokinin family of G- coupled receptors which is divided into three subtypes,

NK-1, NK-2 and NK-3. Substance P is more selective for the NK-1 subtype, while Neurokinin A and B are more selective for the other subtypes (Maggio, 1988; Regoli, et

al., 1994).

Glutamate is also thought to be involved in the neurotransmission of pain. It is co-

localized in small-diameter afferents with the neurokinins and has been shown to be

released following noxious stimulation (Battaglia and Rustioni, 1988) and can result in the

rapid depolarization of second order neurons (Gerber and Randic, 1989 a,b). Glutamate

has been shown to act on three potential post-synaptic sites: The N-methyl-D-aspartate

(NMDA) receptor complex, the 2-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid

(AMPA) receptors and the metabotropic glutamate receptors. Additionally, glutamate acts

on presynaptic kainate receptors. The NMDA receptor complex is a ligand-gated Ca"^-

channel (also Na^ and K"*") which has a number of important binding sites (MacDermott, et

al., 1986). Glutamate interacts with a glutamate binding site on the complex but appears to

be unable to result in channel conductance without concomitant occupation of a strychnine-

insensitive glycine site (Kleckner and Dingledine, 1988). Further, the activation of AMPA

receptors on the postsynaptic neuron may also be required for the activation of the NMDA channel. Partial depolarization of the membrane by AMPA activation can free a Mg"^ ion trapped in ihe channel due to the inward potential of the cell. The Mg"^ ion cannot pass through the channel due to the presence of a large hydration sphere surrounding the molecule and therefore blocks other ions from passing. Additionally, the NMDA receptor complex has a site which binds channel blockers. This site is often referred to as the PCP 27

() site or channel occlusion site. The AMPA receptor is also a ligand gated ion channel, although it is thought to be primarily a Na"^ /K^ channel. Activation of the AMPA receptor can result in the direct depolarization of the neuron by Na^ flux and subsequent activation of voltage-gated Ca^-channels (Murphy and Miller, 1989). Unlike the NMDA and AMPA receptors, the metabotropic glutamate receptor family are G-protein coupled receptors which may be linked to the release of intracellular Ca^ when activated (Berridge andGalionne, 1988).

In addition to the peripheral and spinal mechanisms, there are supraspinal components of pain perception. As mentioned, nociceptive afferents can synapse directly on projection neurons or on excitatory intemeurons which subsequently synapse on projection neurons. Many of these projection neurons subsequently cross the midline of the cord and ascend primarily via the spinothalamic (Dubner, et al., 1989; Simone, et al..

1991) and spinoreticularthalmic (Guillbaud, et al., 1994) tracts. The neurons of these ascending tracts subsequently terminate in the thalamus, medulla, pons, midbrain and limbic structures (Albe-Fessard, et al, 1974, Brodal, 1981, Villaneuva, et al., 1988). These projection neurons innervate specific regions of the thalamus which are responsible for sending information to selected cortical regions for processing (Price and Dubner, 1977).

Hyperalgesia 28

Interestingly, nociceptors do not adapt to repeated activation, as has been seen in typical receptors. In fact, the sensitivity of nociceptors has been shown to increase, rather than decrease, following repeated stimulation (Adriaensen, et al., 1983, Campbell, et al..

1979, Fitzgerald and Lynn, 1977), a characteristic which seems essential to reduce the occurrence of further tissue damage. This heightened responsiveness of nociceptors, also known as peripheral sensitization, may be mediated by the local release of a number of different chemicals many of which are associated with the inflammatory process. The sources of these chemicals include release from damaged cells, release from recruited

(mast) cells, local enzymatic synthesis and direct release from the affected afferent. These substances include protons (Steen, et al., 1992), potassium (Fock and Mense, 1976), (Rochae Silva and Rosenthal, 1961, Fock and Mense, 1976), histamine (Fock and Mense. 1976, Lembeck, 1983), serotonin (Sicuteri, et al, 1965; Fock and Mense.

1976) substance P (see Fields, 1987), as well as certain arachidonic acid metabolites

(eicosanoids), such as and leukoUienes (Ferriera, et al, 1974). These compounds may be responsible for sensitization of nociceptors by altering the membrane permeability of neurons either by direct effects on membrane potential, direct action on ion channels or through second messenger mediated cascades which may result in facilitation of cellular depolarization. Peripheral sensitization leads to a decrease of the intensity level of noxious stimuli required to produce pain. This decreased pain threshold is evident at the site of the initial injury and is known as primary hyperalgesia. 29

In addition to peripheral sensitization, additional events take place at the level of the

spinal cord which also lead to increased pain sensitivity. Central sensitivity occurs

following continued afferent barrage caused by severe peripheral tissue damage. This

increased afferent activity form the periphery and DRG can result in the hyperexcitability of

the neurons in the dorsal horn of the spinal cord. This hyperexcitability is manifest by the

spreading of the area of hyperalgesia outward from the site of injury or secondary

hyperalgesia. In addition, central sensitization results in the direct sensitization of the

second order neuron, resulting in a reduced requirement of afferent input for activation of

the neurons of the DRG following repeated input from the afferents. This process is

referred to as "wind up" (Mendell, 1966). It is known that secondary hyperalgesia is

produced by a central mechanism, rather than peripheral, because the administration of

nerve block prior to the injury, will attenuate the spreading of hyperalgesia away from the

site of the injury without altering primary hyperalgesia. Taken together, primary and

secondary hyperalgesia result in an increased sensitivity to painful stimuli at and surrounding the injury to serve, in general, as a protective mechanism. Sometimes massive

tissue damage will result in the development of allodynia. Allodynia is the perception of

pain from a typically innocuous stimuli. Occasionally, if the peripheral and central sensitization is great enough, even stimuli of such low intensity as light touch, a cool

breeze or the very presence of one's own clothing can be perceived as being painfial

(Payne, 1986). 30

Opiates and opioids and pain perception

Brief histoiy

Crude extracts from , the poppy, have been in medicinal

(as well as psychotropic) use for thousands of years. It was found that slicing the fruit of

the unripened plant resulted in the secretion of a tar-like sap that could be harvested from

the plant. This sap, raw opium, contains over twenty-five -alkaloids including

and morphine. These products have been shown to produce analgesia, as well as

conistipative and psychoactive effects.

It wasn't until 1806 that the German chemist, Friedrich Sertiimer is credited with

the first isolation an active opiate alkaloid, morphium (named after the Greek god of dreams, Morpheus and later renamed morphine) from the juice of the opium poppy.

Sertiimer's morphium proved to be a much more potent, pure alkaloid than the raw opium.

Sixteen years later, Robiquet isolated codeine from opium. These pure substances were then utilized as analgesics instead of the cmde opium extracts, because of their increased potency and control of dosing. The advent of the hypodermic syringe, followed shortly by the Americiin civil war, led to the finding that direct injection of morphine into the blood produced rapid and accurate administration of morphine. Unfortunately, this also exposed the abuse potential of morphine to such an extent that morphine dependence was dubbed

"soldier's disease" due to the large number of civil war veterans who exhibited the 31

symptoms of dependence. Subsequently, a number of other opiates have been synthesized

in the hopes that analgesics with lower addiction liability might be found. was

marketed by the German company, Bayer, as an in the late 1800s, but was

proven to possess even greater addiction liability than morphine. and

meperidine were later synthesized as morphine substitutes. To this day, morphine, codeine,

methadone and meperidine are still used in the United States as analgesics, antitussives and

treatment for opiate withdrawal. Besides abuse liability, there are other side effects

associated with the use of currently available opioids, including respiratory depression,

constipation and the production of tolerance. For these reasons, the study of the chemistry

and pharmacology of opiates continues. Many additional opiates have been derived and

synthesized in the hopes of reducing the unwanted effects of the classic opiate compounds.

Additionally, a greater understanding of the physiological mechanisms by which opiates

produce their effects has been achieved.

Opioid receptors and endogenous opioids

Many opiate-like compounds have been synthesized in the second half of the 20th

century including the development of partial agonists and antagonists ( being the

classic , McClane and Martin, 1967). The in vitro testing of these

compounds in isolated tissue preparations has led to the development of strucUire-activity

relationships for these compounds which suggested that opiates could be acting at receptors either in the periphery or in the central nervous system (CNS). 32

Radioligand binding techniques finally provided evidence for the existence of opioid receptors. Opioids were labeled with radioactive markers and the amount and rates of specific binding to receptor , could be measured by quantification of the radioactive emmissions after the tissue preparations had been washed. While the initial studies by Goldstein and colleagues (1971), using competition of radiolabeled against unlabeled and levorphanol, were not particularly fruitful, they were instrumental in the development of the radioligand binding technique. These experiments were followed up with a number of studies utilizing better techniques to show that the opioids were binding to specific receptors. Pert and Snyder (1973) demonstrated specific and saturable binding of radiolabeled naloxone. High affinity specific binding was also demonstrated using radiolabeled , a potent opioid agonist (Simon, et al., 1973) and radiolabeled (Terenius, 1973). The criteria for binding includes evidence of stereoselectivity and saturability as well as evidence for high concentrations of opioid receptors in brain areas diought to be involved in pain control

(Kuharetal., 1973).

The finding that opioid receptors existed strongly implied the existence of endogenous ligands with which they could interact. Various brain extracts were isolated and purified. Some extracts were shown to produce opioid-like effects in isolated tissue preparations (Hughes, 1975; Kraulis et al., 1975) as well as attenuate opioid binding in inhibition studies (Pasternak et al., 1975; Terenius and Wahlstrom, 1975). These peptides. 33

[Leu^]enkephalin and [Met^jenkephalin were later isolated and sequenced from pig brains

(Hughes et al., 1975b). The term enkephalin is derived firom the Greek word for "in the

head". These compounds were shown to possess many of the characteristics of morphine,

but were considerably less potent. They produced analgesia (Belluzzi et al., 1976; Buscher et al., 1976; Loh et al., 1976; Pert et al., 1977) and physical dependence (Wei and Loh,

1976) that was reversed by the administration of the opioid antagonist, naloxone.

Following these discoveries, an explosion of activity took place in the field of opioid pharmacology. Along with the enkephalins, two additional categories of endogenous peptides were discovered, the endorphins and the . First it was discovered that the polypeptide P-lipotrophin (j3-LPH) contained a copy of the

[Met^]enkephalin sequence and that a portion of the P-LPH peptide had opioid activity (Li and Chung. 1976). This portion was named P-endorphin, a contraction of "endogenous morphine". Additionally, dynorphin 1-13 (meaning powerful morphine, 13 amino acids in length) was isolated from pituitary tissue (Goldstein, et al., 1979). After it was discovered diat a 32 amino acid peptide contained dynorphin 1-17 (dynorphin 1-13 plus 4 additional amino acids) in the amino portion and was enzymatically cleaved to form two shorter peptides, the amino portion of the peptide was renamed dynorphin A, while the carboxy portion was named (Fischli, et al., 1982). All three classes of endogenous opioids were discovered to be cleavage products of much larger precursor polypeptides.

Proenkephalin contained six copies of [Met^]enkephalin and one copy of [Leu^]enkephalin 34

(Noda et al., 1982). Proopioimelanocortin (POMC) contained P-endorphin as well as copies of P-LPH, corticotropin and melanocyte stimulating peptides and a copy of

[Met5]enkephalin (Mains, et al., 1977, Rubenstein, et al., 1978, Chretien, 1979).

Prodynorphin contained the 32-amino acid dynorphin A and B precursor (Rossier, 1982).

In addition to the discovery of the endogenous opioid peptides, it had been suggested, based on slightly different pharmacological characteristics, that different opioids may be acting at different types of opioid receptors. It has subsequently been shown that there are three distinct classes of opioid receptors: mu (ji), delta (5) and kappa (ic). The naming of the |x and k receptors was based upon the prototype opioid agonists found to act upon them, morphine and ketocyclazocine, respectively (Martin, et al., 1976, Gilbert and

Martin, 1976). The 5-opioid receptor was named based on the isolated tissue preparation in which it was found, the mouse vas deferens (5 referring to deferens. Lord et al., 1977).

Since these revelations, a number of fi, 6 and K receptor selective agonists and antagonists have been developed, some of which will be discussed later. Additionally, the three types of opioid receptors have been cloned. Two groups of researchers (Evans, et al.,

1992 and Keiffer, et al., 1992) independently cloned the 6-opioid receptor using cDNA from mouse NG108 neuroblastoma cells. Subsequently, the 5-opioid receptor in rat

(Fukada, et al., 1993) and human (Knapp, et al., 1994) have been cloned showing high homology to the mouse. Additionally sequence homology screening techniques have been used to clone the rat ^-opioid receptor (Chen, et al., 1993a, Fukada, et al., 1993, Wang, et 35

al., 1993) and the mouse K-opioid receptor (Yasuda, et al., 1993). Subsequendy the human |i-opioid receptor (Wang, et al., 1994b) and the rat, guinea pig and human K-opioid receptor (Meng et al., 1993, Chen, et al, 1993b, Nishi et al, 1993, Minami, et al, 1993,

Xie, et al, 1994, Mansson, et al., 1994, Zhu, et al, 1995), have all been idennfied in various cDNA libraries. All of these opioid receptors were of a seven trans-membrane, G- protein coupled nature of similar length (372 - 400 amino acids) and have a high degree of homology especially within the transmembrane spanning regions.

Opioid receptors can be found throughout the central nervous system. Using autoradiography, radioligand binding and, more recently, in situ hybridization techniques to measure mRNA, the location and density of opioid receptors throughout the CNS has been observed. All three types of opioid receptors have been localized in numerous brain, brain stem and spinal cord regions. Here, the focus will be on opioid receptors found in the spinal cord and the DRG. All three types of opioid receptors are distributed in the superficial layer (Lamina I and H) of the dorsal horn, as well as at presynaptic sites on the terminals of C- and A5-fibers (Arvidsson et al., 1995; Besse et al., 1990; Dado et al..

1993; Ji et al., 1996). Besse and coworkers (1990), using autoradiographic techniques with radiolabeled DAMGO (a |i opioid), DTLET (a 6 opioid ligand) and ethylketocyclazocine (a K opioid), demonstrated that of the total opioid receptor population found in spinal cord preparations 69 % of the opioid receptors were |i opioid receptors, 24

% were 5 opioid receptors and the remaining 7 % were K opioid receptors. Furthermore, 36

they were able to estimate the ratio of pre- and post-synaptic opioid receptors by performing dorsal rhizotomy (the sectioning of the dorsal roots from the spinal cord) on some animals and repeating the autoradiographic procedures. The results demonstrated that

76, 61 and 53 % of the [i, 6 and k opioid receptors, respectively, were found on presynaptic sites. These findings suggest that the majority of opioid receptors (primarily p. and 5) are found on the primary afferents responsible for the transmission of signals associated with noxious stimuli, while the remainder of the opioid receptors are found on second order neurons (or possibly intemeurons) found the dorsal horn of the spinal cord.

Opioids and the inhibition of pain

It is thought that opioids produce their analgesic effects at the level of the spinal cord by interacting with receptors on C- and A6-fibers to inhibit the release of pain neurotransmitters (i.e., neurokinins and excitatory amino acids) and thus prevent these neurotransmitters from activation of the second order neurons (Mudge, et al., 1979).

Additionally, opioids are thought to produce their effects by interacting with opioid receptors on second order neurons, thus producing further inhibition of pain pathways and that both the pre- and post-synaptic effects of opioids play a role in producing analgesia

(Lombard and Besson, 1989).

Opioids also act at numerous supraspinal sites to prevent the depolarization of neurons associated with pain. Opioid receptors are found in the thalamus and cortical regions as well as in the midbrain regions, where they are thought to disinhibit descending 37

inhibitory control mechanisms (discussed below). There is sufficient evidence to suggest that opioids produce a significantly greater degree of analgesia when both spinal and supraspinal sites are activated. In fact, this effect has been shown to not just be additive but synergistic or "super-additive" (Yeung and Rudy, 1980).

The renaissance in opioid pharmacology since the 1970s has led to the development of numerous potent and selective compounds, many of which have been shown to produce antinociception in the laboratory and in clinical studies. In addition many specific antagonists have been developed. These compounds have proven useful as tools with which to further elucidate opioid pharmacology. The development of more selective opioid agonists is important because it helps us better understand the pharmacology of the opioid system which can result in the development of better analgesic compounds which do not possess many of the side effects associated with the current regimen of clinically available opioids.

Many of these compounds have been developed by modification of the endogenous opioid peptides and are, therefore, rapidly broken down by enzymatic degradation following systemic administration. These peptides can, however, be administered direcdy into the spinal cord or into specific regions of the brain, which, although possible in laboratory animals, is not recommended in humans. [D-Ala\ NMePhe"*, Gly-ol]enk:ephalin

(DAMGO) is a highly selective and potent peptidic |i-opioid agonist synthesized by Handa and colleagues (1981) which has been shown to produce antinociception when 38

administered into the lateral ventricle of the brain or into the lumbar enlargement of the

spinal cord (Miaskowski, et al. 1991; Dickenson and Sullivan, 1987; Mattia, et al., 1991).

Evidence suggests that DAMGO is of higher efficacy than the prototypic |i-opioid agonist,

morphine (Galligan, 1993; Mjanger and Yaksh, 1991;Seali and Yaksh, 1993). In addition

to |i-opioid agonists, a number of peptidic S-selective opioid agonist have been discovered.

Erspamer and colleagues have isolated a family of peptides called , from

the skin of a south African frog {Phyllomedusa sauvagei) (Erspamer et al., 1989; Kreil et

al., 1989). These compounds were shown to by much more potent in isolated tissue

preparation than any other previously discovered 5-selective compounds. One particular

compound, commonly referred to as deltorphin n ([D-Ala\ Glu'']deltorphin) is highly selective for the 6-opioid receptor. [D-Ala", Glu'*]deltorphin has been shown to produce 5- selective analgesia when administered at either spinal (Mattia, et al., 1992; Stewart and

Hammond, 1993) or supraspinal sites (Jaing, et al, 1990).

Extremely potent non-selective opioid peptides have also been developed.

Lipkowski and colleagues (1982) synthesized the dimeric enkephalin analogue, biphalin, which binds to both (i and 5 sites. Horan and colleagues (1993) demonstrated that both spinal as well as supraspinal administration of biphalin produced potent antinociception in mice. Furthermore, biphalin produced andnociception when administered systemically.

This suggested that although biphalin was peptidic in nature, its structure was stable enough to resist enzymatic degradation. 39

More recently, the first advances in the development of non-peptidic 5-opioid agonists have occurred. BW 373U86, developed by Burroughs-Wellcome (Chang et al.,

1993), showed some selectivity for 5 receptors (Wild et al., 1993) but was of limited use in vivo and produced severe behavioral toxicity manifest by convulsions when administered (Comer, et al., 1993). Calderon and colleagues began making modifications to the structure of the enantiomers of BW 373U86. One result was the 5-selective agonist

SNC 80 (Calderon, et al., 1994). This compound was shown to produce antinociception in mice when administered centrally, spinally and systemically. The finding of a systemically active, non-peptidic, 5-selective opioid is encouraging towards the development of better analgesics (Bilsky, et al., 1995).

Non-peptidic K-selective opioids have been more readily available. U 69,593

(Lahti, et al., 1985), a K-selective agonist, has been shown to produce antinociception when administered at either spinal or supraspinal sites (Millan, et al., 1989). has primarily been tested against gastrointestinal transit and motility (Reviere, et al., 1993) but may prove useful as an analgesic.

The endogenous enkephalins, [Leu^]enkephalin and [Met^]enkephalin. are not particularly good choices as potential analgesics because they are rapidly degraded by enzymatic processes. Thiorphan inhibits the degradation of endogenous enkephalins by inhibition of the enzyme responsible, neutral 24.11. Thiorphan has been shown to produce antinociception in some animal models (Chaillet, et al., 1983, Evans and 40

Mathre, 1985, Roques, et al., 1980) and serves as a useful instrument in the understanding of the effects of the endogenous enkephalins.

Endogenous pain control

The existence of opioid receptors and endogenous opioids suggests that some form of endogenous opioid pain control system exists. This can be seen in that various stressors can induce analgesia by the release of endogenous opioids (Moskowitz, et al., 1985;

Marek, et al., 1987; Przewlocki, 1993). In addition to control at die level of the spinal cord, where opioidergic inhibitory intemeurons can inhibit the pain signal, descending pain control systems originating from midbrain regions also influence pain control. Electrical stimulation of the periaquaductal gray (PAG) can produce analgesia in humans and in lab animals (Hosobuchi, et al., 1977, Mayer and Liebeskind, 1974) and that this is caused by inhibition of cells in the dorsal horn (Guillbaud, et al., 1973). Lesions of the dorsolateral funiculus (u spinal cord structure which includes a bundle of descending neurons) can block brainstem induced analgesia (Basbaum, et al., 1976). The PAG neurons project to the rostral ventral medulla (RVM, Basbaum and Fields, 1984) and subsequently to die spinal cord. These descending systems are thought to mediate antinociception by the release of serotonin (5-HT) and norepinephrine (NE). Yaksh and colleagues (1979) have shown that the antinociceptive effects of PAG morphine are blocked by spinal administration of 5-

HT and NE antagonists. Both n and 5 opioid receptors are found in the PAG (Kalyuzhny 41

et al., 1996) and opioid administration in the PAG results in disinhibition of these

descending amine control systems. Disinhibition by morphine refers to the suggestion that

these descending system are tonically inhibited by y-amino butyric acid (GABA)

intemeurons. The inhibition of these GABA intemeurons results in the activation of the descending control systems (Mason, et al., 1990). Although this descending inhibitory

pain control system plays an important role in endogenous pain control, this dissertation focuses primarily on the role of opioids at the spinal level.

Abnormal pain states: peripheral neuropathy

Peripheral nerve damage can lead to abnormal pain states characterized by a sensitivity to nociceptive input, indicative of a lowered nociceptive threshold (hyperalgesia) and the perception of nociceptive input by typically innocuous stimuli (allodynia). These abnormal pain states are often referred to as neuropathic pain. Although sensitization to stimuli, in general, is not abnormal, but rather a protective mechanism, these neuropathic pains often last for very long periods of time and are difficult to treat. The causes of nerve injury which result in neuropathic pain include disease states, such as diabetes, cancer, and herpes zoster, among others, to direct nerve damage caused by compression or traumatic tissue damage.

Although the mechanisms underlying neuropathic hyperesthesias are not yet fully understood, they are often associated with various neuroanatomical changes occurring in. 42 and associated with, the spinal cord. These changes include a partial degeneration of both myelinated and unmyelinated afferents in the periphery (Nuytten, et al. 1992), which may suggest the involvement of these fibers in abnormal pain perception. Additionally, severely damaged or severed nerves may form ectopic foci. Following damage, nerves can form terminal bulbs which may subsequently sprout, presumably in an attempt to re-innervate the peripheral tissue, however, should the growth of these fine sprouts be inhibited, they can stop growing or occasionally grow back onto themselves forming a tangled network of nerve fiber called a neuroma (reviewed in Fawcett and Keynes, 1990). These neuromas can produce massive spontaneous discharges (Wall and Gutnick, 1974) which can lead to the perception of pain (Kajander and Bennett, 1992). As mentioned above, massive afferent discharge can lead to central sensitization of dorsal horn neurons. Furthermore, abnormal sympathetic innervation of the dorsal root ganglia and spinal nerves (Chung, et al., 1993,

McLachlan. et al., 1993, Xie et al., 1995) can lead to efferent maintenance of pain

(Roberts, 1986). In addition, peripheral nerve damage can result in the sprouting of large diameter, low-threshold AP-fibers (mechanoceptors) to form novel synapses with second order neurons associated with the transmission of nociceptive input (Woolf et al, 1992;

Lekan, et al., 1995). A-P-fibers are large, heavily myelinated afferents with rapid conduction velocities which are typically involved in the transmission of low threshold mechanical nature, such as light touch. Because these fibers, normally associated with innocuous stimuli, now with second order nociceptive neurons, it has been proposed that 43 this mechanism may be responsible, in part, for the production of allodynia caused by nerve injury (Woolf et al, 1992, Woolf and Doubell, 1994). In addition to these neuroanatomical changes, increases in spinal levels of cholecystokinin (Stanfa, et al,

1994), enkephalins and dynorphin (Dubner, 1991) have been observed following nerve injury.

Animal models of peripheral neuropathy

Several animal models of peripheral neuropathy have been developed which demonstrate many of the characteristics of neuropathic pain syndromes in man. These models include the loose ligation of the sciatic nerve, often referred to as the chronic constriction injury model (CCI, Bennett and Xie, 1992), the tight ligation of one third to one half of the sciatic nerve, the partial nerve ligation model (PNL, Seltzer, et al., 1990) and the tight ligation two of the three spinal nerves which form the sciatic nerve (Kim and

Chung, 1992). All of these models result in the development of mechanical and thermal hyperalgesia, as well as the development of tactile allodynia (see Bennett, 1994). The CCI model has also been shown to result in autonomy as well as exaggerated deformation of the paw, while the both the CCI and PNL model are difficult to reproduce exactly from animal to animal due to inconsistencies with the strength of the ligation (CCI) or amount of the nerve ligated when only a portion of the nerve is used (PNL). For these reasons, his 44 dissertation focuses on the highly reproducible spinal nerve ligation model of peripheral neuropathy.

Opiates in neuropathic pain

Interestingly, neuropathic pains often demonstrate a resistance to treatment with opioids. Amer and Meyerson (1988) showed that intravenous (/.v.) morphine was ineffective at altering the mean responses to a visual analogue pain assessment scale in patients exhibiting various types of neuropathic pain. Portenoy and colleagues (1990) demonstrated that while i.v. morphine was able to produce adequate analgesia in neuropathic patients, that higher doses of morphine and , than those normally used, were required to alleviate the pain. Likewise results from patients with cancer pain of a neuropathic nature, showed reduced opioid effectiveness (Twycross.

1988). However, some studies were able to demonstrate that opioids were able to elicit pain relief in neuropathic patients (Rowbotham, et al, 1991; Urban, et al., 1986). These results suggest that while neuropathic pains may be resistant to treatment with opioids, that perhaps these pains could be alleviated by administration of higher doses or perhaps alternative (i.e., other than morphine) opioids in the alleviation of neuropathic pain syndromes (Portenoy, 1990).

Studies in animal models of neuropathic pain showed similar results. Xu and colleagues 11993) were unable to reduce autonomy produced by sciatic nerve section with 45

10 [Lg of i.th. morphine. Yaksh and colleagues (1995) showed that 30 jig of i.th. morphine was unable to attenuate tactile allodynia in the spinal nerve ligation model of neuropathic pain. Mao and collaborators (1995) demonstrated that i.th. morphine was able to produce antihyperalgesic and antinociceptive effects (using a radiant thermal stimulus) in the CCI model of nerve injury, but that the efficacy of morphine was greatly reduced.

Others have shown that i.th. morphine is effective in reducing the autonomy associated with sciatic nerve section (Wiesenfeid-Hallin, 1984).

Altliough many of these results point toward the idea that opioids are less than effective in treating neuropathic pains, systematic studies have not been performed. Most of these studies have been performed primarily using only morphine and further, the testing has been done in different models of neuropathy having different endpoints (i.e., autonomy, tactile allodynia and thermal nociception). The clinical studies are not much different. Again morphine is the primary agent tested in patients with neuropathic pain of various nature and often by recording pain index using a generalized pain scale. In order to better understand the role of opioids in animal models of neuropathic pain, we must look at the effectiveness of many opioids, by multiple routes of administration, in the various model of neuropathy, and using numerous endpoints (e.g., thermal and mechanical stimuli in tests of nociception, hyperalgesia and allodynia). Then by making comparisons across all these groups we can better understand the effectiveness of these . This dissertation 46

attempts to examine a small portion of this story: primarily i.th. opioids in the spinal nerve ligation model of neuropathy, against tactile allodynia.

Excitatory amino acids and neuropathic pain

Previous studies suggest that a portion of the mechanisms of neuropathic pains include collateral sprouting of AP-fibers to form novel synapses in superficial lamina of the spinal dorsal horn and the development of ectopic foci which can result in central sensitization (Woolf et al, 1992, Woolf and Doubell, 1994). In addition to these neuroanatoiTiical changes, increases in spinal levels of cholecystokinin (Stanfa, et al.

1994), enkephalins and dynorphin (Dubner, 1991) have been observed following nerve injury. It has been suggested that excitatory amino acids (EAAs, Wilcox, 1991; Woolf and

Thompson, 1991, Ma and Woolf, 1995) or dynorphin (Vanderah, et al, 1996) interacting at the NMDA receptor contribute to the development of central sensitization associated with increased spontaneous activity. Activity at the NMDA receptor has also been demonstrated to be responsible, in part, for the reduced potency of morphine (Hoffman and Weisenfeld-

Hallin, 1996; Advokat and Rhein, 1995). Activity at non-NMDA EAA receptors has also been shown to be involved in the development and maintenance of abnormal pain states.

Selective antagonists for the EAA receptors have been developed. MK 801

(dizocilipine), developed by Merck (Christy, et al., 1979), is a non-competitive inhibitor of the NMDA channel (Wong, et al., 1986). MK 801 is thought to produce its effects by 47

occlusion of the ion channel via allosteric mechanisms and not by inhibition at the putative

glutamate of glycine sites found on the NMDA receptor complex. 6-cyano-7-

nitroquinoxaline-2,3-dione (CNQX) is an antagonist which blocks both the AMPA and

kainate sites (Honore, et al., 1988). L-2-amino-3-phosphonopropionic acid (L-AP3) is general metabotropic glutamate (Collingridge, et al., 1989). Both the

AMPA/kainate antagonist, CNQX and the metabotropic receptor antagonist, L-AP3 have

been demonstrated to inhibit the allodynia associated with intrathecal administration (Ferriera and Lorenzetti, 1994; Minami, et al., 1994). Further, CNQX has

been shown to attenuate the development of thermal hyperalgesia associated with the CCI

model of neuropathy (Mao, et al., 1992) as well as inhibit the hyperalgesia produced by the intraperitoneal injection of lippopolysaccharide (Watkins, et al, 1994) or hindpaw injection of carageenan (Ren, et al., 1992). Conversely, both of these compounds have been shown to be ineffective in altering the hyperalgesia associated with other models of chronic pain such as the formalin test (Codere and Van Empel, 1994). In addition, CNQX has been shown to be ineffective in inhibiting thermal hyperalgesia in the CCI model, when administered after the production of nerve injury (Mao, et al., 1992).

There is compelling evidence to suggest that activity at the NMDA receptor is involved in the maintenance and production of abnormal pain states. It is known that the development of neuropathies involves an increase in the spontaneous activity of injured nerves and subsequent central sensitization, which is mediated, in part, by the release of 48

excitatory amino acids (EAAs) acting at NMDA receptors in the dorsal hom of the spinal cord (Wilcox, 1991; Woolf and Thompson, 1991). Although activation of NMDA receptor does not result directly in the depolarization of dorsal hom neurons in the resting state, it does produce prolongation of action potentials initiated by neurokinins or non-NMDA

EAAs (Ma and Woolf, 1995). The release of glutamate, acting at NMDA receptors, yields central sensitization during repeated C-fiber stimulation (Davies and Lodge, 1987) and, following nerve injury, the subsequent development of allodynia and hyperalgesia (Mao, et al., 1992). Administration of NMDA produces hyperalgesia (Aanonsen and Wilcox, 1986) and enhances die firing of dorsal hom neurons in response to noxious stimuli (Aanonsen, et al., 1987). Further, the development of central sensitization can be blocked by administration of NMDA antagonists (Ma and Woolf, 1995). In addition. Seltzer and colleagues (1991) found that i.th. administration of NMDA antagonists suppressed the autonomy associated with nerve transection and suggested that the intensity of neuropathic pain is mediated by an NMDA-mediated spinal disinhibition. Smidi and colleagues (1995) have shown that the systemic administration of MK801 was able to delay, but not prevent, the onset of mechanical hyperalgesia in the chronic constriction injury model of neuropathic pain. These studies suggest that the activity of excitatory amino acids may be responsible, in part, for the development and maintenance of the neuropathic pain state. For these reasons, the role of antagonists to the NMDA and non-NMDA receptors in nerve injury induced allodynia will be explored. Further, the effect of tonic activity at the NMDA 49

receptor on the efficacy and potency of morphine in nerve-injured animals will also be

discussed.

The non-opioid effects of dynorphin and neuropathic pain

Elevated levels of endogenous dynorphin have been associated with spinal cord

injury. The behavioral effects of spinal cord trauma have been shown to be reduced by the

intrathecal {i.th.) administration of antisera to dynorphin suggesting that dynorphin may

play a role in the pathology of spinal cord injury (Shukla and LeMaire. 1994; Faden,

1990).

It is well accepted that i.th. administration of dynorphin produces neurological dysfunctions in rats (Long, et al., 1988). NMDA antagonists have been shown to block the paralytic and nociceptive effects of dynorphin (Caudle and Isaac, 1988; Shukla and

Lemaire, 1994). The sequelae of nerve injury involves significant plastic changes in the dorsal horn of the spinal cord which result in central sensitization (Woolf et al, 1992;

Woolf and Doubell, 1994; Xie et al., 1995) and that this central sensitization is dependent on NMDA receptor activation (Woolf and Thompson, 1991). Further, the development of central sensitization can be blocked by administration of NMDA antagonists (Ma and

Woolf, 1995). In some animal models, NMDA antagonists have been shown to attenuate the behavioral manifestations associated with nerve injury (Seltzer et al., 1991; Lee and

Yaksh, 1995). 50

These findings, along with the observations that spinal dynorphin levels are

elevated following peripheral nerve injury and that dynorphin may act at NMDA receptors

in the spina! cord to produce its "non-opioid" effects, lead to the present investigation of the effects of dynorphin A (1-13) antisera alone, and in combination with morphine, in a nerve

injury model of neuropathic pain. Further, since elevated dynorphin levels may be involved

in the activity at the NMDA receptor and NMDA activation is involved in the inidal development of the neuropathic state, it is possible that the initial afferent barrage which results in neuropathic syndromes may also be mediated by dynorphin. For this reason, the effects of a single administration of subparalytic doses of dynorphin A (1-17) to normal animals was assessed.

Afferent fibers and neuropathic pain

Treatment with capsaicin (8-methyl-A/-vanillyl-6-nonenamide). the pungent component of peppers of the Capsicum family, on the second post-natal day has been shown to induce a selective, permanent degeneration of up to 95% of unmyelinated primary afferent C-fibers (Jansco, et al., , 1977, Nagy et al, 1980, Nagy, et al., 1983).

Further, neonatal capsaicin treatment results in the attenuation thermal nociceptive responses (Jansco and Jansco-Gabor, 1980, Lorez, et al., 1983, Doucette, et al., 1987,

Meller, et al., 1992, Shir and Seltzer, 1990), while its effects on mechanical nociceptive 51

have been inconsistent (Faulkner and Growcott, 1980, Saumet and Duclaux, 1982, Shir and Seltzer. 1990, Ren, et al., 1994, Kinnman and Levine, 1995, Kim, et al., 1995).

Keep in mind that the sprouting of large diameter, low-threshold AP fibers to form novel synapses with second order neurons typically associated with nociception (Woolf et al, 1992) results following nerve injury and that they may be responsible for the transduction of the allodynic signal. In addition, studies involving conduction velocity have suggested that AP fibers, not C-fibers, are involved in the transmission of nociceptive information in animals with nerve injury (Treede, et al., 1991).

Capsaicin-sensitive afferents have been demonstrated to be involved in the development of thermal hyperalgesia in the chronic sciatic nerve constriction model of neuropathic pain (Meller, et al., 1992). Further, Shir and Seltzer (1990), suggest that the thermal hyperalgesia associated with a partial ligadon of the sciatic nerve is mediated by C- fibers, while the mechanical hyperesthesias were mediated by A-fibers. Little is known about the involvement of capsaicin-sensitive fibers in the development of mechanical allodynia. Conversely, Kinnman and Levine (1995), demonstrated a slower onset and reduction in sensitivity to probing with von Frey filament in neonatally capsaicin treated rats with L5 spinal nerve resection. These results contradict with the hypothesis that mechanical allodynia is mediated by large, heavily myelinated AP fibers.

The present studies were undertaken to determine die involvement of capsaicin- sensitive afferents in behavioral responses to high intensity thermal, as well as high and 52 low intensity mechanical stimuli in a spinal nerve ligation model of neuropathic pain.

Further the effectiveness of morphine and DAMGO will be assessed against thermal and low-threshold tactile stimuli in neonatally capsaicin treated rats with nerve injury.

Modulation of the effectiveness of mu opiates (morphine)

CCK is found throughout the CNS including the dorsal horn of the spinal cord

(Suberg and Watkins, 1987). Further, a considerable proportion of CCK and CCK receptors have been shown to be distributed in the same regions of the CNS as the endogenous opioid peptides (Stengaard-Pederson and Larson, 1981) and opioid receptors

(Ghilardi et al., 1992). The antiopioid effect of CCK has been well documented. The systemic or intrathecal administration of CCK produces physiological antagonism of morphine-induced antinociception (Paris, et al., 1983). In addition, studies have shown that the non-specific CCK antagonist, enhanced the antinociceptive effects of morphine without producing any antinociceptive effects alone (Suh and Tseng, 1990,

Watkins, et al, 1985a). Electrophysiological studies by Kellstein and colleagues (1991) showed that CCK attenuates and the non-selective CCK antagonists, , enhances the inhibitory role of morphine on nociceptive neuronal activity in the dorsal horn of the spinal cord. Later experiments demonstrated that the CCKg specific antagonists L365,260

(Dourish, et al., 1990) and CI-988 (PD134308) (Hughes, et al., 1991) enhanced the antinociception of morphine without producing any antinociceptive activity alone. Recent 53 evidence suggests that the blockade of CCKg may result in alterations in the activity of endogenous 5-opioid compounds to result in the reduced activity of morphine. In agreement with die previously mentioned studies, the antinociceptive activity of systemic and intrathecal morphine to thermal nociceptive stimuli has been shown to be enhanced by administration of the CCKg specific antagonist, L365,260. Interestingly, this effect was inhibited by the 5-opioid selective antagonist, naltrindole (NTI) (Ossipov, et al., 1994).

NTI did not, however, inhibit the antinociceptive effects of morphine alone. Vanderah and colleagues (1996) demonstrated similar findings in the mouse tail flick test. Furthermore, this study demonstrated that the intracerebroventricular {i.e.v.) administration of morphine was enhanced by L 365, 260 and that this effect was blocked by NTI. The study also showed that the enhancement of morphine's antinociceptive effects was inhibited by i.e. v. pretreatment of mice with antisera raised against [Leu^]enkephalin, but not

[Met^]enkephalin.

A number of studies have been performed to suggest that there is a significant interaction between ji-opioid receptor induced analgesia and 5-opioid receptor activation.

Vaught and Takemori (1979) have shown that administration of [Leu^Jenkephalin. at below antinociceptive doses, enhances the antinociceptive effects of morphine. Additionally, the interaction of systemic morphine and [Leu^]enkephalin has been shown to be a synergistic effect in the mouse tail flick test (Porreca, et al, 1990). The exogenous administration of other 5-opioid agonists, including [D-Ala*, Glu'']deltorphin have also been shown to result 54

in a significant enhancement of the antinociceptive effects of morphine (Vaught and

Takemori, 1979, Vaught, etal, 1982, Porreca, et al., 1992, Heyman, et al, 1986, 1989

a,b). These findings further strengthen the hypothesis that CCK may tonically inhibit the

release and/or availability of an endogenous 5-opioid, possibly [Leu^jenkephalin, which

may result in the reduced effectiveness of morphine.

There is compelling evidence to suggest that there is upregulation of spinal CCK in

response to nerve injury (Stanfa, et al., 1994, Xu , et al., 1993). In situ hybridization

studies have demonstrated increased CCK mRNA levels in the rat dorsal root ganglia and

monkey dorsal horn following nerve transection (Verge, et al., 1993), further suggesting a

role for CCK in the abnormal spinal pharmacology following nerve injury. Xu and

colleagues (1993) demonstrated that the antinociceptive effects of morphine were attenuated

following sciatic nerve section and that this phenomenon was coincident with increases in

CCK mRNA in the spinal cord. Further, this decreased potency of morphine was restored

by the administration of the CCKg antagonist CI-988. These studies, taken together,

suggest that nerve injury may result in increased spinal levels of CCK and which may

inhibit the effectiveness of endogenous 6-opioids, such as [Leu']enkephalin, and

subsequently be responsible, in part, for the reduced effectiveness of morphine against

neuropathic pain. The experiments performed here attempt to determine whether blockade

of CCKg receptors or administration of exogenous 5-opioid agonists, can restore the efficacy and potency of morphine against the production of tactile allodynia in a model of 55

peripheral neuropathy. Further, these experiments aim to determine if these effects are dependent upon activation of 6-opioid receptors.

The degradation of endogenous enkephalins can be attenuated by thiorphan, a neutral endopeptidase (24.11) inhibitor, which produces antinociception in some models

(Chaillet, etal., 1983, Evans and Mathre, 1985, Roques, et al., 1980). Given that CCKg antagonists can increase the activity of endogenous enkephalins and the short duration of action of tliese compounds, Vanderah and colleagues (1996) administered thiorphan, in combination with L365,260, at doses which were ineffective alone. Coadministraion of thiorphan and L 365,260 produced an NTI reversible antinociception in mice. Since blockade of CCKQ receptors may increase levels of endogenous enkephalins.

These results suggested that the enhancement of the activity of endogenous enkephalins, along with the prevention of their degradation can result in sufficient antinociceptive activity. In order to further examine the role of endogenous enkephalins in a model of peripheral neuropathy, experiments involving the coadministration of morphine or thiorphan with L 365,260 were undertaken. An additional aim of these studies was to identify the endogenous 5-opioid, which may be affected by increased spinal levels of

CCK. 56

HYPOTHESIS OF THE DISSERTATION

Hypothesis 1

The attenuation of the antiallodynic efficacy (and potency) of opioids following

nerve injury, may result, in part, because such pain signals are transmitted by AP-fibers. rather than C- or A8-fibers. Since Ap-fibers do not possess opioid receptors, the pool of opioid receptors available to inhibit nerve injury-induced allodynia is found only on the second order neuron. Thus, the opioid receptor pool, and available spare receptors, are considerably smaller to inhibit nerve injury-induced allodynia, than seen against "acute" pain.

Hypothesis 2

The neuropathic pain state is maintained by increased spinal afferent drive which is manifest by heightened activity at excitatory amino acid receptors, specifically at NMDA and AMPA sites. Further, NMDA receptor activation, due possibly to increased levels of spinal dynorphin, results in a reduction in morphine efficacy and potency in a rat model of neuropathic pain.

Hypothesis 3 57

Opioid efficacy and potency are affected by alterations in spinal levels of other neurotransmitters following nerve injury. Specifically, increases in spinal levels of the endogenous anti-opioid, cholecystokinin, which subsequently attenuates the availability of

[Leu^]-enkephalin. This may result in the removal of a naturally occurring synergistic effect occurring following endogenous activation of 5 opioid receptors (by [Leu^]-enkephalin) in combination with exogenous activation of |i opioid receptors (by morphine), resulting in a loss of morphine's potency and efficacy. 58

METHODS

Animals

Male Sprague-Dawley rats (175-350 g) were used for all experiments. In the case of experiments involving neonatal capsaicin treatment. Female Sprague-Dawley rats were obtained 15-17 days into gestation. Following birth, all rats were treated as described below. Rats were purchased from Harlan Sprague-Dawley, inc., Indianapolis, Indiana.

Chemicals

Ketamine, pentobarbital, chloral hydrate, capsaicin, and thiorphan were purchased from Sigma Chemical Co. (St. Louis, Mo.). Capsaicin was dissolved in 10% ethanol,

10% Tween-80, and 80% saline. Xylazine was purchased from Lloyd Laboratories

(Shenandoah, lA). Halothane was purchased from Halocarbon Laboratories (River Edge,

NJ). Morphine, [D-Ala% NMePhe"*,Gly-ol]enkephalin (DAMGO), U 69,593 and

Dynorphin A (1-17) were obtained through the National Instimte of Abuse drug supply program. SNC 80 was synthesized as previously described (Calderon, et al..

1994). Fedotozine was a generous gift of Institut de Recherche Jouveinal (Fresnes,

France). Deltorphin was synthesized by methods previously described (Mosberg, et al.,

1983) and dissolved in 75 % DMSO. Biphalin was synthesized as previously described

(Lipkowski, et al, 1982). MK801, CNQX, L-AP3 and naltrindole (NTI) were purchased from Research Biochemicals Inc. (Natick, MA). L365,260 was a generous gift of Merck, 59

Sharpe and Dohme Research Laboratories (Rathway, NJ) and was dissolved in 20 %

DMSO/80 % propylene glycol. Dynorphin A (1-13) antisera, [Leu^]enk;ephalin antisera.

[Met'jenkephalin antisera and control sera were a generous gift of Dr. Leon Tseng.

Antisera were produced by repeated injection of rabbits with peptide-coupled bovine thyroglobulin. Control sera was taken from uninjected rabbits. [Met^]enkephalin antisera was shown to lack (i.e., < 2 %) cross-reactivity with [Met']enkephalin-Arg-Phe,

[Met^]enkephalin-Arg-Gly-Leu, [Met^]enkephalin-Leu, dynorphin A (1-13), dynorphin A

(1-17) and P-endorphin. It did possess some cross-reactivity with [Leu"'']enkephalin antisera (29.4 %). [Leu^Jenkephalin antisera was shown to lack (i.e., < 2 %) cross- reactivity witii dynorphin A (1-13), dynorphin A (1-17) and P-endorphin. It showed some cross-reactivity for [Met^]enkephalin (14 %). Dynorphin A (1-13) antisera lacked (i.e., < 2

%) cross-reactivity with [Leu^]enkephalin, [Met^Jenkephalin, P-endorphin and dynorphin

1-8. It was 100% cross-reactive with dynorphin A (1-17). Prior to use, all compounds were dissolved in distilled water unless noted otherwise.

Surgical implantation of the intrathecal catheter

Rats were anesthetized by halothane induction as described below. While under anesthesia, rats were implanted with catheters for intrathecal administration of drugs into the region of the lumbar spinal cord according to the method described by Yaksh and Rudy

(1976). An 8 cm length of PE-IO polyethylene tubing was inserted into the vertebral canal 60

through an incision made in the atlanto-occipital membrane such that it terminated at the

level of the lumbar enlargement. The catheter was secured to the musculamre at the

incision, which was then closed. The rats received 4.4 mg/kg, i.m. of gentamycin and

were allowed to recover for a period of at least 5 days prior to testing. Rats exhibiting

motor deficiency were discarded from testing.

Drug administration

Ketiunine, xylazine, pentobarbital, chloral hydrate and Innovar-vet were administered via the intraperitoneal (/./?.) route by direct injection into the peritoneal cavity.

Capsaicin was administered by subcutaneous {s.c.) injection into the subcutaneous space of the central back. I.p. and s.c. injections were made in unanesthatized rats in a volume of 1 ml per kg of body weight. I.p. and s.c. injections were given with a 1 ml tuberculin syringe through a 26, 27 28 or 30-gauge needle. Halothane was administered by inhalation. The nose and mouth of the rat were placed in a plastic "mask" attached to a

Fluomatic \ aporizer (Foregger Industries, Smithtown, NY) and anesthesia was induced by administration of 4% halothane in O,. A maintenance concentration of 1.5% halothane was utilized for continuous anesthesia. All additional injections were administered by intrathecal {i.th.) administration. I.th. injections were given through the chronically indwelling catheter (described above) at a volume of 5 fil, followed by a 9 |il flush with vehicle. 61

L5/L6 nerve ligation and sham surgery

Nerve ligation was performed according to the method of Kim and Chung (1992).

Rats were anesthetized with halothane "to effect". The dorsal pelvic area was clipped using oscillating surgical shears and prepared with a surgical scrub. An incision (3-4 cm) was made along the left side of the vertebral column using the level of the posterior iliac crests as the midpoint. Fascia and muscle tissue were cleared to expose the dorsal vertebral column and the vertebral posterior articular process just anterior to the sacrum was removed using a fine rongeur. The L4 and L5 spinal nerves were exposed and delicately separated using a smooth glass hook to avoid damaging the nerves. The L5 nerve was tightly ligated with 4-0 silk suture distal to the dorsal root ganglia. The L6 spinal nerve was then drawn from beneath the sacrum using the glass hook and ligated as above. The incision was sutured closed with 3-0 Vicryl and the animals were allowed to recover for 7 days. Sham- operated controls were prepared in an identical manner, with the exception that the nerves were not ligated. All rats received 4.4 mg/kg, i.m. of gentamycin. Rats exhibiting motor deficiency were discarded from testing.

Neonatal capsaicin treatment

Capsaicin treated animals were prepared according to the method of Jansco and colleagues (1977). On the second post-natal day, rat pups were subcutaneously injected 62

with 50 mg/kg capsaicin or vehicle. Subsequent to injection, pups were placed in a high O,

(95%) environment for approximately 10 min to reduce the incidence of death due to respiratory depression. Rat pups receiving capsaicin were given an ear punch in order to determine treatment regimen upon adulthood. Capsaicin vehicle was 10% ethanol. 10%

Tween-80, and 80% saline. On the 21st post-natal day, rats were weaned from die mothers and sex was determined. Only male rat pups were utilized in the subsequent procedures. Nerve-ligation and sham surgeries took place approximately 6-7 weeks after birth when the rats weighed 175-200 g.

Behavioral Testing von Frey assay (including normalized von Frey fibers)

Rats were placed in a clear acrylic chamber having a 1/4 inch wire mesh floor and were allowed to habituate for a minimum of 30 min. Tactile allodynia was determined by measuring the paw withdrawal in response to probing the plantar surface of the left hind paw with von Frey monofilaments to determine baseline withdrawal threshold. Withdrawal threshold was determined by increasing and decreasing stimulus intensity between 0.4 and

15.1 gram equivalents of force and estimating using a Dixon non-parametric test (Chaplan. et al. 1994). Nerve-ligated rats having baseline thresholds of greater than 4 g were discarded from experimental groups except in the case of studies of anesthetic and capsaicin treatments. In those studies, all rats were used, regardless of baseline threshold. An 63

example is provided below: The initial fiber applied to all animals is marked 4.31. This fiber produces force equivalent to approximately 2 g. If the subject withdraws the paw

(represented by an X in the testing table), the next smaller fiber (4.08, equivalent to 1.2 g) is applied, [f the rat does not withdraw from the 4.08 fiber (represented by an O in the testing table) the next larger fiber is applied (4.31). A negative response results in an O and increasing the force to the next higher fiber (4.56, equivalent to 3.6 g).This is continued until a pattern of at least 6 positive or negative responses is achieved. In the case of the example, the patem is XOOXXO. This pattern 3.61 3 3.84 4.08 02 m 4.31 X m X 4.56 m 4.74 4.93

5.18 >

Following the determination of baseline responses, some rats received doses of agonists, antagonists, antisera, enzyme inhibitors or some combination of these compounds. The effect of drug treatments on paw withdrawal thresholds were then determined at distinct time intervals following drug administration.

Warm-water tail flick assay

To determine spinal reflex behavior to thermal nociceptive stimuli, the warm water tail-flick assay was performed. Animals were tested by gently holding the rat in a vertical position and immersing the tail in a water bath maintained at either 48''C, 52°C or 55°C.

The endpoint was determined by measuring the latency to the first sign of an attempt at rapid removal of the tail from the water bath. As above, baseline latencies were measured, followed by drug treatment (in some cases) and subsequent post-treatment testing. A maximum latency of 15 sec was assigned in order to avoid tissue damage.

Radiant heat foot flick assay

The presence of thermal hyperalgesia was measured utilizing the radiant heat paw withdrawal assay as described by Hargreaves and colleagues (1988). Rats were placed in clear plastic chambers on a glass surface maintained at 30°C and allowed to habituate for at least 30 min. A focused light source was then directed at the plantar surface of the left hind paw and the latency to paw withdrawal was measured. Again, baseline latencies were measured followed by drug treatment, where applicable. A maximum latency of 40 sec was assigned in order to avoid tissue damage.

Pin prick assay

In order to determine the presence of mechanical hyperalgesia the paw pin prick assay (Tal and Bennett, 1994) was performed. The subjects were placed in the same clear acrylic chamber used in the von Frey assay and allowed to habituate for at least 30 min. A blunt metal pin was then rapidly applied to the plantar surface of the left hind paw such that 65

it was forceful enough to raise the paw from the wire mesh surface, but without penetrating or physically damaging the skin. The latency of the resultant paw lift is measured.

Statistics

Subjects were randomly assigned to test and treatment groups. Experiments involving drug treatment immediately prior to testing were performed using a between- groups paradigm. Dose response relationships that involved linear regression and the calculation of A50 values and 95% confidence limits (C.L.) and relative potencies were performed using the computer program designed by Talarida and Murray (1986) as modified by Michael H. Ossipov (unpublished). Only the linear portion of the dose- response curves were used for the fitting of the linear regression and calculation of the A50 values. All data were analyzed using two-way or one-way analysis of variance (ANOVA) where applicable. Post-hoc tests were Student's t-test and least squared difference test

(LSD). All data points represent the mean plus or minus the standard error of the mean for groups having a minimum of 4 subjects. 66

RESULTS OF PRELIMINARY EXPERIMENTS

The objectives of this section are to (1) determine whether the nerve-Iigation surgery was a successful and valid experimental procedure for causing the development of

nerve-injury induced tactile allodynia in rats, (2) To verify that the different diameters of the

various von Frey filaments used in testing would not be a factor in the interpretation of

withdrawal thresholds, and (3) to determine whether the use of different anesthetics during

the nerve ligation surgery might affect the development of allodynia in this model.

Baseline testing

Following either sham or nerve ligation surgery, rats were allowed to recover for a minimum of seven days upon which time all subjects were tested by probing with von Frey filaments. The paw withdrawal thresholds of typical groups of sham-operated and nerve- ligated rats are shown in Figure 1. The baseline withdrawal threshold for sham-operated rats was 13.5 ± 1.2 g. The baseline withdrawal threshold for nerve ligated rats was 2.3 ±

0.5 g. These results are consistent with previous work (Chaplan, et al., 1995). Throughout these experiments (except the neonatal capsaicin procedures) any nerve-ligated animal not exhibiting tactile allodynia (i.e., baseline withdrawal latency > 4 g) was discarded from testing. Further, animals exhibiting hind-limb flacidity or motor deficit were also discarded. Although exact statistics were not kept, 10 to 15% of nerve ligated subjects were discarded for these reasons. 67

Testing with normalized von Frey fliaments

Von Frey filaments are calibrated to produce a specific amount of force when bent.

This is achieved by producing monofilaments of different diameters. The net result is that the amount of pressure (defined as force/unit area) applied by each monofilament is not directly proportional to the force applied by each filament. In addition, the different size of the contact area of each monofilament may activate different numbers of mechanoreceptors, thereby confounding the accuracy of the withdrawal threshold. To address these potential problems, a set of "normalized" von Frey monofilaments were manufactured. The diameter of the tip of each of the "normalized" monofilaments was made a constant 0.61 mm by gluing a small ( about 1 mm) piece of PE-10 tubing to the tip of each filament. A group of nerve-ligated rats was subsequently tested with both the standard and the "normalized" monofilaments. The paw withdrawal threshold of the nerve-ligated rats when tested with the standard von Frey filaments was 1.8 ± 0.2 g. The paw withdrawal threshold of the same group of rats tested with the "normalized" monofilaments was 2.1 ± 0.3 g. A within groups t-test revealed no difference in paw withdrawal threshold (Figure 2). 68

15

E u t/5 12 +1 -25 2 o ® Nerve ligated -0~Sham operated

C3

St o.C3

BASE 10 20 30 45 60 Time (min)

Figure 1. Mean paw withdrawal thresholds of nerve-ligated and sham-operated rats at baseline (BASE) and 10, 20, 30,45 and 60 min after i.th. injection of distilled water

(n=10/group) 69

Q standard von Frey filaments

I von Frey filaments with 0.61 mm dia. P 4

o 3

X

Figure 2. .Mean paw withdrawal threshold of nerve-ligated rats tested with both standard

and "normalized" von Frey filaments. 70

E^ect of anesthetic on the development of tactile allodynia.

A comparison of a five anesthetics was made to determine whether their different

mechanisms of action might affect the development of allodynia in the model of nerve

injury. Ketamine/xylazine (lOOmg/kg and 10 mg/kg), halothane ("to effect"), pentobarbital

(50 mg/kg). chloral hydrate (100 mg/kg), and Innovar-vet (20 mg/kg, a /droperidol

cocktail) were administered at the doses in parentheses and maintained "to effect" .

Subsequently, the rats underwent the nerve ligation surgery and were tested for the development of allodynia each day for 10 days. In all cases, at least 4 of the 5 animals in each group met the paw withdrawal threshold < 4 g criteria on day 7 following surgery

(Figures 3-7). 71

15 Ketaminc/xylazinc

o x: 10 V5P

f3

I'

5 6 7 10 Time (day)

Figure 3. Mean paw withdrawal thresholds following nerve ligation surgery performed under ketainine/xylazine anesthetic. The ratio above each data point indicate the number of

rais per group having paw withdrawal thresholds > 4 g on the given day. 72

Halothane

Figure 4. Mean paw withdrawal thresholds following nerve ligation surgery performed under halothane anesthetic. The ratio above each data point indicate the number of rats per

group having paw withdrawal thresholds > 4 g on the given day. Pentobarbital

Figure 5. Mean paw withdrawal thresholds following nerve ligation surgery performed under pentobarbital anesthetic. The ratio above each data point indicate the number of rats

per group having paw withdrawal thresholds > 4 g on the given day. Innovar

Figure 6. Mean paw withdrawal thresholds following nerve ligation surgery performed under Innovar-vet anesthetic. The ratio above each data point indicate the number of rats

per group having paw withdrawal thresholds > 4 g on the given day. 75

Chloral hydrate

Figure 7. Mean paw withdrawal thresholds following nerve ligation surgery performed under chloral hydrate anesthetic. The ratio above each data point indicate the number of

rats per group having paw withdrawal thresholds > 4 g on the given day. SUMMARY OF PRELIMINARY EXPERIMENTS

The initial experiments demonstrate that the nerve ligation surgery results in the

development of tactile allodynia of the ipsilateral hind paw of the rat and that this allodynia

is produced by the nerve ligation itself and is not an artifact of the surgical procedures. The

results coincide with previous reports (Chaplan, et al., 1995).

Since no significant difference was seen between paw withdrawal thresholds tested

the standard von Frey filaments and tested with the "normalized" monofilaments, the concern that the different diameters of the various von Frey filaments might be confounding

the logarithmic relationship between the force of the monofilaments and the resultant

behavioral effect was demonstrated to be invalid. This concern was important because the

Dixon test, the non-parametric statistic used to estimate paw withdrawal, requires a linear

(or in this case a logarithmic transformation to linear) relationship between the different treatment levels in order to accurately estimate the threshold (Dixon, 1980). The von Frey filaments utilized in the testing of tactile allodynia are separated by approximately 0.225 log units of force.

The use of different anesthetics was not shown to result in dramatic differences in the development of tactile allodynia. Halothane was selected as the anesthetic of choice because it demonstrated ease of use, rapid recovery time and reduced potential for animal loss due to overdose because, in most cases, rats could be resuscitated by removal from the maintenance dose and aspirated. Although the ketamine/xylazine cocktail was not shown to 77

alter the development of tactile allodynia, it was discontinued because previous studies have

shown that the development of hyperesthesia from nerve injury can be retarded by

administration of other NMDA channel blockers (Mao, et al., 1992). Pentobarbital resulted

in the least desirable outcome. It is unknown if the inconsistent results to probing with von

Frey filaments were caused by the pharmacological effects of pentobarbital or if there was an abnormal number of surgical failures in that particular group. Innovar-vet did not alter the development of allodynia in nerve-ligated rats, but was not used in these studies to avoid any potential effect the opioid component of the anesthetic might have on the future use of opioids in these experiments. Lastly, chloral hydrate also did not alter the development of allodynia. Chloral hydrate, however, is not recommended as an anesthetic by the University Animal Care and Use Committee of the university of Arizona for "non­ terminal" surgeries. 78

RESULTS OF STUDIES INVOLVING OPIATES AND OPIOIDS

The objectives of this section are to (1) compare the antaillodynic effects of i.th. administered mu (morphine, DAMGO), delta (SNC 80, [D-Ala^ Glu'']deItorphin), kappa

(U 69,593, fedotozine) and a mixed mu/deita (biphalin) opioid agonists, (2) address the effects of i.th. morphine and DAMGO in sham-operated animals and (3) discuss the antinociceptive effect of morphine in the tail-flick assay in both nerve-ligated and sham- operated rats.

Lack of effect of intrathecal morphine to probing with von Frey monofilaments

I.th. administration of morphine, a mu opioid agonist, at doses of 30, 60 and 100

|ig (90, 180 and 300 nmol) did not produce any significant increase in paw withdrawal threshold in L5/L6 nerve-ligated rats up to 60 min after drug injection (maximum % antiallodynia =13.1 ± 18.8 %, Figures 8 and 9). Similarly, morphine (30 |ig, i.th.) did not significantly alter the paw withdrawal threshold to probing with von Frey monofilaments in sham-operated rats at any time point up to 60 min following administration (Figure 10). 79

—O—Vehicle "•"SOjig ~*~60ng -^lOOfig

30 40 Time (min)

Figure 8. Lack of antiallodynic actions of i.th. morphine (30, 60 and 100 |ig) in groups of

5 to 7 rats with nerve ligation injury. 80

100

I I f •t

10 30 100 300 Morphine dose (p. g)

Figure 9. Antiallodynic dose response relationship of morphine in nerve-ligated rats 30 min

following i.th. injection. 81

Vehicle

Figure 10. Lack of effect of i.th. morphine (30 ^ig) in sham-operated rats on probing with

von Frey filaments. 82

Reduced antinociceptive effectiveness of morphine in rats with nerve- ligation injury

I.th. administration of morphine (30 fig) was able to produce thermal antinociception in sham-operated rats (maximum % antinociception = 83.3 ± 10.5 at 30 min), however, the same dose of morphine in rats with nerve injury was unable to produce a maximal antinociceptive effect at any time point (maximum % antinociception =61.0 ±

13.5) (Figure 11.)

Studies on the effect of intrathecal [D-Aia^, NMePhe^, GIy-oI]enkephaiin

(DAMGO) to probing with von Frey monoHlaments

In contrast to the effects of morphine, intrathecal administration of the high efficacy

opioid, D-Ala% NMePhe'*, Gly-ol]enkephalin (DAMGO) at doses of 0.15, 0.5 and 1.5

[Lg (0.3, 1 and 3 nmol) was able to produce a full antiallodynic action having a maximum

% antiallodynia of 83.7 ± 8.9 % at 30 min following DAMGO administration (Figure 12).

Figure 13 shows the dose-effect relationship for DAMGO at 30 min after drug injection.

The Ajo and 95 % C.L. for DAMGO at 30 min was 0.6 (0.4 - 0.9) fxg. 83

—O— Morphine (30(Xg)-Sham # Morphine (30fig)-L5/L6 ligated

10 20 30 40 50 60 70 Time (min)

Figure 11. Antinociceptive effect of i.th. morphine (30 jig) in nerve-ligated and sham-

operated rats in the 55''C warm-water tail flick test. (n=4-5 rats/group) 84

100 Vehicle 0.15 fig 0.5 fig O 80

0 10 20 30 40 50 60 70 Time (min)

Figure 12. Time response lines for the antiallodynic actions of graded doses (0.15. 0.5 and

1.5 ^g) of i.th. [D-Ala*, NMePhe"*, Gly-ol]enkephalin or vehicle in nerve-ligated rats (n=5

to 8/group). Asterisk indicates a significant difference from pretreatment withdrawal

thresholds. DAMGO (Hg)

Figure 13. Antiallodynic dose response relationship of [D-Ala", NMePhe"*, Gly- ol]enkephalin in rats with nerve ligation injury, 30 min following administration. Vehicle-Control DAMGO (0.15 ng) DAMGO (0.5 (ig) DAMGO (1.5 ng)

BASE 10 20 30 45 60 Time (min)

Figure 14. Time response lines for the effects of 1.5 |ig of i.th. [D-Ala\ NMePhe'*.

ol]enkephalin or vehicle in sham-operated rats (n=5/group). 87

Again, unlike moq)hine, i.th. DAMGO produced a small effect in sham-operated rats. The highest dose of DAMGO (1.5 jig) resulted in all rats having a maximum withdrawal threshold of 15 grams at the 20, 30 and 45 min time points. This effect, however, was not statistically significant (Figure 14). No differences were seen in the paw wididrawal thresholds of sham-operated rats i.th. administered 0.5 of 0.15 |ig of DAMGO

(Figure 14).

Studies on the antiallodynic effect of intrathecal SNC 80 in rats with nerve injury

A single dose of SNC 80, a non-peptide, delta opioid-selective agonist was administered (100 nmol, i.th.) to rats with nerve ligation injury. SNC 80 was unable to produce a statistically significant antiallodynic effect having a maximum % antiallodynia =

30.4 ± 17.9 at 20 min after injection (Figure 15). This dose of SNC 80 has been shown to produce approximately a 60% reduction in flinching behavior in the rat formalin test

(Nichols and Porreca, unpublished observation) suggesting that SNC 80 is not a good candidate for the treatment of neuropathy induced allodynia. 100 Vehicle

SNC 80C100nmol)

0 10 20 30 40 50 60 70 Time (min)

Figure 15. Lack of antiallodynic effect of 100 nmol i.th. SNC 80 in nerve-ligated rats. 89

Studies on the antiallodynic effect of intrathecal [D-Ala^, GIu^]deltorphin in rats with nerve injury

In contrast to the effects of SNC 80, i.th. administration of the high efficacy, peptidic, 5 opioid [D-AIa", Glu'*]deitorphin at doses of 9, 27, 90 and 269 jig (10, 30, 100 and 300 nmol )was able to produce a statistically significant antiallodynic action having a maximum % antiallodynia of 68.2 ± 11.0 % at 30 min following [D-Ala\ Glu'*]deltorphin administration (Figure 16). Figure 17 shows the dose-effect relationship for [D-Ala^

Glu'*]deltorphin at 30 min after drug injection. The Aj^ and 95 % C.L. for [D-Ala',

GIu"^]deltorphin at 30 min was 100 (82 - 122) |ig. In addition, in order to control for the possibility that [D-Ala", Glu"']deltorphin might be having its action systemically, this compound was given by the intravenous (/.v.) at the highest dose tested (i.e.. 269 fig)-

This dose of /.v. [D-Ala", Glu'*]deltorphin had no effect on paw withdrawal threshold (data not shown). 0 10 20 30 40 50 60 70 Time (min)

Figure 16. Time response lines for the effects of graded doses (9, 27, 90 and 269 |J.g) of

i.th. [D-Ala", Glu"*]deltorphin or vehicle in nerve-ligated rats (n=5/group). Asterisk

indicates a significant difference from pretreatment withdrawal thresholds. 91

[D-Ala-, Glu-*]deltorphin (nmol)

Figure 17. Antiallodynic dose response relationship of [D-Ala*, Glu"']deltorphin in rats

with nerve ligation injury, 30 min following administration. 92

Studies on the antiallodynic effect of intrathecal biphalin in rats with nerve

injury

Lth. administration of biphalin (0.03, 0.1, 0.3, 1.0 and 3.0 jig), a highly

efficacious, dimeric enkephalin analogue that acts at both p. and 5 opioid receptors, to rats

with nerve ligation injury resulted in a robust antiallodynic action. I.th. biphalin

administration resulted in a maximum % antiallodynia = 94.7 ± 5.3 at 30 min after injection

(Figure 18). Figure 19 shows the dose-effect relationship for biphalin at 30 min after drug

injection. The and 95 % C.L. for biphalin at 30 min was 0.1 (0.03 - 0.4) |j.g.

Figure 20 shows the comparison of the dose effect curves for the low efficacy |i opioid, morphine, the high efficacy |i opioid DAMGO and the mixed [i and 5 opioid

biphalin. Morphine is completely ineffective while biphalin is approximately 6 fold more potent than DAMGO. 93

—O— Vehicle —•—0.03 iig -•-0.1 ng -*-0.3 ng 100

B o C/3 +1

40 C3 Wc <

•==o 0 10 20 30 40 50 60 70 Time (min)

Figure 18. Time response lines for the antiallodynic actions of graded doses (0.15, 0.5 and

1.5 fig) of i.th. biphalin or vehicle in nerve-ligated rats (n=4 to 5/group). Asterisk indicates

a significant difference from pretreatment withdrawal thresholds. Biphalin (|ig)

Figure 19. Antiallodynic dose response relationship of biphalin in rats with nerve ligation

injury, 30 min following administration. 95

9 Biphalin

-•-DAMGO

~0—Morphine

0.01 0.1 10 100 Dose (|ig)

Figure 20. Dose response comparison of morphine, [D-Ala", NMePhe"*, Gly-ol]enkephalin

and biphalin at 30 min after injection. 96

Studies on the antiailodynic effect of intrathecaily administered kappa agonists in rats with nerve injury

A single dose of U 69,593, a kappa opioid-selective agonist was administered (250

[ig, i.th.) to rats with nerve ligation injury. This high dose of U69,593 was able to produce a small, but statistically significant antiailodynic effect having a maximum % antiallodynia = 60.6 ± 12.6 at 30 min after injection.

In addition to U 69,593, two doses of fedotozine, a kappa opioid-selective agonist were tested (300 and 600 |j.g, i.th.) to rats with nerve ligation injury. Again, these were very high doses of fedotozine, which produced a small, but statistically significant antiailodynic effect having a maximum % antiallodynia = 45.3 ± 22.4 at 10 min after injection. 97

"~0~ Vehicle

Figure 21. Time response lines for the antiallodynic actions of a single dose (250 fig) of i.th. U69,593 or vehicle in nerve-Iigated rats (n=5/group). Asterisk indicates a significant

difference from pretreatment withdrawal thresholds. 98

100 Vehicle 300 ^g 600

•a

I -10 1 0 10 20 30 40 50 60 70 Time (min)

Figure 22. Time response lines for the antiallodynic actions of a two doses (300 and 600

|j.g) of Ltli. fedotozine or vehicle in nerve-ligated rats (n=5/group). Asterisk indicates a

significant difference from pretreatment withdrawal thresholds. 99

SUMMARY OF STUDIES INVOLVING OPIATES AND OPIOIDS

Yaksh and colleagues (1995) demonstrated that 30 |xg of i.th. morphine failed to prevent tactile allodynia using the L5/L6 nerve ligation model of neuropathy. In the present studies, dose up to 100 (ig of i.th. morphine did not produce any observable change in sensitivity to paw withdrawal. Previous studies have shown that 10 p.g of i.th. morphine were able to produce antinociception in the tail flick or hot plate tests (Ossipov, et al.,

1994). These data therefore support the hypothesis that there is a marked reduction in the antiallodynic potency and efficacy of morphine following nerve injury. In addition, the finding that the 30 |j,g dose of morphine was unable to produce a full antinociceptive effect in the 55°C tail flick test suggest that along with a reduction in the antiallodynic effectiveness of morphine there is a marked reduction of the ability of morphine to modulate acute nociception. This is effect has been further characterized by the constmction of dose effect relationships which suggest an approximate 4 fold decrease in potency of morphine in nerve injured animals when compared to sham controls (Ossipov, et al, 1995). Although these data suggest the reduced efficacy of i.th. morphine in this model, it should be noted that a complete antiallodynic effect of morphine is produced when administered by other routes of administration. Bian and colleagues (1995) have shown that subcutaneous morphine is able to produce a full antiallodynic activity in this model, although at higher doses than those seen for antinociceptive activity. Further, intracerebroventricular administration of morphine was also fully active in the suppression 100

of allodynia. These results suggest that the reduced efficacy of i.th. morphine may be

related to our understanding of the presumed underlying cause of allodynia.

It has been suggested that morphine acts in large part through presynaptic opioid

receptors localized on the central terminals of primary afferent C-fibers to alleviate acute

pain (Yaksh, et al., 1995). Low threshold mechanical stimulation, however, is mediated

primarily by AP fiber input and the mechanical allodynia associated with nerve injury is

thought to be mediated through aberrant responses to AP fiber inputs involving novel

synaptic connections between the AP fibers and ascending nociceptive pathways (Woolf

and Doubeil, 1994). Because AP fibers are not known to contain any opioid receptors, any

observed opioid mediated activity must occur at post-synaptic sites. Besse and coworkers

(1990) have recently shown that dorsal rhizotomy produces a marked loss of |i and 5 opioid receptors to 30 and 40% of control, respectively. These observations show that the

majority of spinal opioid |j. (70%) and 6 (60%) receptors are found at presynaptic sites.

Other evidence suggests that opioid 5 receptors are found at presynaptic sites on terminals of C-fibers or on descending modulatory fibers (Arviddson, et al., 1995). Given that allodynia is likely mediated by AP fibers suggests that presynaptic opioid receptor (found on C-fibers) are not involved in the alleviation of allodynia. This hypothesis is further supported by the finding that low threshold mechanical stimulation in sham-operated animals is also not affected by the administration of spinal morphine suggesting that in the normal state, AP fiber inputs are not affected by i.th. morphine. The implication of this 101

view is that the receptor pool available for the spinal antiallodynic action of morphine are

those receptors found on post-synaptic sites. This smaller "available" receptor pool for antiallodynic activity would thus require high-efficacy opioid agonists or large doses of lower efficacy opioids to produce a full effect.

Altliough Uh. morphine was unable to produce any antiallodynic action, the higher efficacy opioid DAMGO (Galligan, 1993, Mjanger and Yaksh, 1991, Saeki and Yaksh,

1993) was able to produce a fiill antiallodynic action when administered by the same route.

It is possible that DAMGO possesses sufficient efficacy to produce its antiallodynic action by activity solely at postsynaptic opioid receptors, whereas a partial ^ agonist, such as morphine, would not have sufficient efficacy to achieve a measurable antiallodynic action.

It is important to note that electrophysiological studies have shown that DAMGO can produce effects at a post-synaptic site in spinal cord slice preparations (Glaum, et al, 1994).

The highest dose of DAMGO tested resulted in the maximum paw withdrawal threshold being achieved when administered to sham-operated animals. Although this effect was not statistically significant, this dose appears to produce some behavioral toxicity expressed in the form of rigidity and ataxia. Unfortunately the nature of testing with von Frey filaments does not allow for higher stimuli to be applied. The next higher monofilament in the progression (nearly 30 g) is of sufficient strength to occasionally raise the hind paw from the surface of the observation chamber without bending, if the subject has not placed sufficient weight on the limb. The signs of behavioral toxicity with DAMGO were not 102

evident in nerve-ligated rats. A possible explanation for the lack of toxicity is that the spinal dorsal horn plasticity, such as upregulation of antiopioid substances (e.g., CCK and dynorphin) (Woolf and Doubell, 1994; Dubner, 1991), along with a number of other changes, may be sufficient enough following nerve injury to result in altered spinal pharmacology including a subsequent loss of behaviorally toxic effects.

Similar results as seen with the opioid |X agonists can be demonstrated with the opioid 8 agonists. The finding that SNC 80 (Bilsky, et al., 1995) was unable to produce any antiallodynic action at a dose that had previously been shown to produce antinociception in rats in a tonic model of inflammatory pain (Nichols, unpublished observations) suggests that there is also a marked reduction in the antiallodynic effectiveness of this compound. Administration of i.th. [D-Ala\ Glu'']deltorphin was able to produce a significant antiallodynic effect. In this case, however, even the doses of ID-

Ala^, Glu"^]deltorphin required to produce the antiallodynic effect are much higher than those required to elicit an antinociceptive effect. Stewart and Hammond (1993, 1994) have shown that i.th. [D-Ala", Glu"*]deltorphin produced 6-antagonists sensitive antinociceptive effects in the tail-flick and paw-withdrawal assays at doses up to 100 |ig. The dose of ID-

Ala^, Glu"']deltorphin required to produce any statistically significant, and at that just less than a 70%, antiallodynic action shown here was 269 |j,g, nearly 3-fold higher than that required to produce a full antinociceptive effect. 103

In agreement with the above results, the high efficacy dimeric enkephalin analogue, biphalin, w:is also able to produce a full antiallodynic action when administered via the i.th. route. Biphalin acts at both |i, and 6 opioid receptors to produce its antinociceptive activity

(Horan, et al., 1993). There is significant evidence to suggest that activation of both .u and

5 opioid receptors may result in a synergistic interaction, thereby greatly potentiating antinociceptive effects (Vaught and Takemori, 1979, Lee, et al, 1980, Rothman and

Westfall, 1982). Although this is one potential explanation for the high efficacy if biphalin,

5 opioid agonists have not been shown to potentiate the effects of |i opioid agonists

(DAMGO and PL 17, Heyman et al 1989b) other than morphine.

The administration of the K-opioid agonists U 69,593 and fedotozine were also able to produce some limited antiallodynic effect, however the massive doses required for these compounds to produce an effect do not substantiate there usefulness as potential therapies for the treatment of allodynia produced by nerve injury. Previous studies have shown that i.th. administration of 200 |xg of fedotozine in mice produced about a 50% antinociceptive effect in the warm-water tail flick. (Bilsky, personal communication). Further the i.th. doses of U 69,593 required to produce antinociception in rats against pressure and thermal stimulation of the tail were 20 |ig to double the baseline tail withdrawal latencies (Millan, et al., 1989). These doses of are clearly much greater than the doses which typically produce antinociception in rats. 104

The results of these experiments suggest that there is a substantial reduction in the effectiveness of many opioids against tactile allodynia. It should be noted that other investigators have shown that opioids may be effective at alleviating neuropathic pain

(Jadad, et al., 1992, Portenoy, et al, 1990, Portenoy and Foley, 1986) but at higher than normal doses. Many studies, however have focused on the use of morphine or opioids of lesser efficacy (Amer and Meyerson, 1988) to conclude that opioids are ineffective. It seems possible that opioids of higher efficacy might prove useful in the treatment of neuropathic pain. 105

RESULTS OF EXPERIMENTS INVOLVING NEONATAL CAPSAICIN

TREATMENT

The objectives of this section are to (I) examine the effects of selective C-Fiber destruction by neonatal administration of capsaicin on nociceptive thresholds in both nerve- ligated and sham-operated rats. Specifically, the purpose is to observe the effects of capsaicin treatment on the warm water-tail flick assay, a test of thermal nociception, the radiant heat foot flick assay, a test of thermal hyperalgesia, the pin prick assay, a test of mechanical hyperalgesia, and in the von Frey assay, a test for the development of tactile allodynia. (2) To determine the effect of C-fiber destruction on the effectiveness of the low efficacy |i-opioid, morphine, and the high efficacy |X-opioid, DAMGO, against thermal nociception and tactile allodynia in nerve-ligated rats.

Effects of capsaicin treatment on nociceptive thresholds

Warm-water Tail Flick (48'C)

Sham-operated rats which were neonatally administered vehicle (Sham-Vehicle) had a mean tail flick latency at 48°C of 4.4 ± 0.2 s (mean ± s.e.m., n = 13) which was significantly lower than the tail flick latency of sham-operated, capsaicin-treated (Sham-

Capsaicin) animals tested at the same temperature (8.2 ± 0.9 s, n = 20). Nerve-ligated, vehicle-treated (Ligated-Vehicle) rats also demonstrated a significantly lower (3.6 ±0.2 s, n = 17) tail flick latency than their capsaicin treated counterparts (Ligated-Capsaicin. 7.2 ± 106

0.9 s, n = 18). The Ligated-Vehicle group also showed a small, but significant, reduction in tail flick latency when compared to the Sham-Vehicle group (Figure 23).

Warm-water Tail Flick {52'C)

The mean tail-flick latency at 52°C for the Sham-Vehicle group was 2.6 ± 0.1 s (n

= 22) which was, again, significantly lower than the tail-flick latency of the Sham-

Capsaicin :inimals (4.0 ± 0.4 s, n = 25). Similarly, the Ligated-Vehicle rats also demonstrated a significantly lower (2.3 ± 0.1 s, n = 25) tail-flick latency than the Ligated-

Capsaicin group (3.1 ± 0.2 s, n = 25) at 52°C. Both the Ligated-Vehicle group and the

Ligated-Capsaicin group had small, but significantly lower tail flick latencies at 52°C when compared to the respective sham groups (Figure 24).

Radiant Heat Paw Withdrawal

The mean paw withdrawal latency from a focused radiant heat source for the Sham-

Vehicle group was 9.9 ± 1.0 s (n = 22). Similar to the tail flick results, this was significantly lower than the paw withdrawal latency of Sham-Capsaicin treated animals

(15.92 ±0.9 s, n = 25). In addition, Ligated-Vehicle latencies (11.71 ± 1.0, n = 25) were significantly lower than Ligated-Capsaicin group latencies (19.4 ± 2.3, n = 25). 107

10 r • Sham/control

Q Sham/capsaicin

* Ligated/control

' LigaCed/capsaicin

Figure 23.NeonataI capsaicin treatment attenuates the withdrawal reflex of the tail immersed

in a 48°C water bath in both sham-operated and L5/L6 spinal nerve ligated rats. Open bar

represents sham-operated, vehicle treated rats (n = 13). Hatched bar indicates sham-

operated, capsaicin treated rats (n = 20). Gray bar indicates L5/L6 nerve ligated, vehicle

treated rats (n = 17). Closed bar represents nerve ligated, capsaicin treated rats (n = 18).

Asterisks indicate significant difference (p < 0.05) within surgery groups. Cross indicates

significant difference within dmg treatment groups. 108

• Sham/control

• Sham/capsaicin

• Ligated/control

• Ligated/capsaicin

Figure 24. Neonatal capsaicin treatment attenuates the withdrawal reflex of the tail immersed in a 52°C water bath in both sham-operated and L5/L6 spinal nerve ligated rats.

Open bar represents sham-operated, vehicle treated rats (n = 22). Hatched bar indicates

sham-operated, capsaicin treated rats (n = 25). Gray bar indicates L5/L6 nerve ligated, vehicle treated rats (n = 25). Closed bar represents nerve ligated, capsaicin treated rats (n =

25). Asterisks indicate significant difference (p < 0.05) within surgery groups. Cross

indicates significant difference within drug treatment groups. 109

Comparisons within drug treatment groups (i.e., Sham-Vehicle versus Ligated-Vehicle and

Sham-Capsaicin versus Ligated-Capsaicin) revealed no differences in paw withdrawal

latency (Figure 25).

Pin Prick

The mean latencies of sham-operated rats to hold the paw aloft following rapid application of a blunt pin to the plantar surface of the paw were nearly indiscernible from zero (0.1 ± 0.06 s. n=22; 0.03 ± 0.03 s, n=25 for vehicle and capsaicin treated groups, respectively). However, the mean pin prick latency for each ligated group was significantly higher than their sham-operated counterparts (2.861 ± 0.6, n = 25; 1.9 ± 0.6. n = 25 for vehicle and capsaicin treated groups, respectively). There were no differences within drug treatment groups (Figure 26).

Von Frey probing

The mean paw withdrawal threshold determined by von Frey filament probing for

Sham-Vehicle animals was 13.5 ± 0.4 g (n = 22). Capsaicin treatment did not produce any difference in paw withdrawal threshold (13.9 ± 0.4 g, n = 25). Nerve ligation resulted in significantly lower paw withdrawal threshold in both the vehicle treated group (4.0 ± 0.7, n = 25) and the capsaicin treated group (2.7 ± 0.3, n = 25). Capsaicin treatment in nerve 110

ligated animals did not produce a significant difference in paw withdrawal threshold

(Figure 27).

25 r

•Sham/control

20 •Sham/capsaicin

B Li gated/control

•Ligated/capsaicin 15

10

Figure 25. Neonatal capsaicin treatment attenuates the withdrawal latency of the paw following application of radiant heat to the plantar surface in both sham-operated and L5/L6

spinal nerv e ligated rats. Open bar represents sham-operated, vehicle treated rats (n = 22).

Hatched bar indicates sham-operated, capsaicin treated rats (n = 25). Gray bar indicates

L5/L6 nerve ligated, vehicle treated rats (n = 25). Closed bar represents nerve ligated,

capsaicin treated rats (n = 25). Asterisks indicate significant difference (p < 0.05) within

surgery groups. Ill

• Sham/control

0 Sham/capsaicin

• Ligated/control

1 Ligated/capsaicin

Figure 26. Neonatal capsaicin treatment does not alter the paw lift latency following rapid application of a blunt pin to the plantar surface of the paw in either sham-operated or L5/L6 spinal nerve ligated rats. Open bar represents sham-operated, vehicle treated rats (n = 22).

Hatched bar indicates sham-operated, capsaicin treated rats (n = 25). Gray bar indicates

L5/L6 nerve ligated, vehicle treated rats (n = 25). Closed bar represents nerve ligated,

capsaicin treated rats (n = 25). Cross indicates significant difference (p < 0.05) within

drug treatment groups. 112

• Sham/control

ZI Sham/capsaicin

B Ligated/control

' Ligated/capsaicin

Figure 27. Neonatal capsaicin treatment does not alter the paw withdrawal threshold determined by systematic application of calibrated von Frey filaments to the plantar surface

in either sham-operated or L5/L6 spinal nerve ligated rats. Open bar represents sham-

operated. vehicle treated rats (n = 22). Hatched bar indicates sham-operated, capsaicin

treated rats (n = 25). Gray bar indicates L5/L6 nerve ligated, vehicle treated rats (n = 25).

Closed bar represents nerve ligated, capsaicin treated rats (n = 25). Cross indicates

significant difference (p < 0.05) within drug treatment groups. 113

Effects of morphine in capsaicin treated rats

Morphine in the warm-water tail flick (52"C)

The i.th. administration of morphine (3, 10 and 30 p.g) produced dose-related

antinociception in all groups of rats. Morphine given to sham-operated rats which were

neonatally treated with vehicle resulted in an (95 % C.L.) = 7.8 (2.6 - 22.9) |ig and a

maximum % antinociception = 87.5 ± 12.5 at 45 min after administration (Figure 28). In

nerve-Iigated rats which were neonatally treated with vehicle, morphine produced a

maximum % antinociception = 61.9 ± 17.2 at 30 min after administration with an Ajq (95

% C.L.) = 16.7 (5.8 - 48.1) |ig (Figure 29). Further, morphine produced a maximum %

antinociception = 80.0 ± 20.0 in capsaicin-treated, sham-operated rats, A50 (95 % C.L.) =

11.3 (6.1 - 20.6) |ig. Figure 30) while eliciting a similar response in capsaicin-treated.

nerve-ligated rats (maximum % antinociception = 84.1 ± 17.2 at 45 min, A50 (95 % C.L.)

= 21.9 (8.4 - 56.6) |ig. Figure 31). Figure 32 shows the dose response comparison of

morphine in these four groups of rats. Contrary to what was expected, neither capsaicin

treatment nor nerve ligadon surgery significandy altered the potency of morphine, although

there is a trend suggesting the reduction of the potency of morphine caused by both capsaicin treatment and nerve ligation. 114

100 Vehicle

3 lig 80 10 tig 30 tig

60 o.

40

20

0 0 10 60 Time (min)

Figure 28. Antinociceptive effect of i.th. morphine on sham-operated rats, neonatally treated with vehicle in the 52°C warm water tail flick assay (n=5-7 rats/group). Asterisks

indicate statistical significance (p < 0.05) versus baseline. 115

100 Vehicle

3 Jig 80 10 ng 30 Ug

60 o.

40

20

0 0 10 20 30 40 50 60 70 Time (min)

Figure 29. Antinociceptive effect of i.th. morphine on nerve-ligated rats, neonatally treated with vehicle in the 52°C warm water tail flick assay (n=5-6 rats/group). Asterisks indicate

statistical significance (p < 0.05) versus baseline. 116

100

a.

-<>-

0 10 20 30 40 50 60 70 Time (min)

Figure 30. Antinociceptive effect of Lth. morphine on sham-operated rats, neonatally treated with capsaicin in the 52''C warm water tail fliclc assay (n=4-6 rats/group). Asterisks

indicate statistical significance (p < 0.05) versus baseline. 117

Vehic e

E 80

30 40 Time (min)

Figure 31. Antinociceptive effect of i.th. morphine on nerve-ligated rats, neonatally treated

with capsaicin in the 52°C warm water tail flick assay (n=4-10 rats/group). Asterisks

indicate statistical significance (p < 0.05) versus baseline. 118

100 -o- Sham/Control 90 ' -a- Ligated/Control ? 80 : Sham/Capsaicin o 70 Ligated/Capsaicin

c 60 o 50 (Uo. o o 40 c S 30 < 20

10

10 100 Dose Morphine (jig)

Figure 32. Dose response comparison of morphine in capsaicin and vehicle treated sham-

operated and nerve-ligated rats in the 52°C warm water tail flick assay 30 min after

injection. 119

Morphine in the von Frey assay

The i.th. administration of 30 |ig morphine, a dose which produced antinociception in all groups of rats did not alter paw withdrawal thresholds to von Frey probing in either sham-operated or nerve-ligated rats pretreated with capsaicin or vehicle. Results of von

Frey probing in nerve-ligated rats are expressed as % antiallodynia while results in sham- operated rats are expressed in paw withdrawal threshold. This is due to the presence of a

"ceiling effect", i.e., the baseline values in sham-operated rats (approximately 12-14 g) are very close to the maximum possible effect (15 g), resulting in very small changes of withdrawal threshold translating into the appearance of dramatic changes if the data were expressed as % maximum possible effect. Morphine (30 jxg, i.th.) did not alter the paw withdrawal threshold of sham-operated rats which were neonatally treated with vehicle of capsaicin (Figure 33). Similarly, the same dose of morphine failed to be effective against tactile allodynia in rats with nerve-injury, regardless of neonatal vehicle (maximum % antiallodynia = 7.2 ± 4.2) or capsaicin treatment (maximum % antiallodynia = 13.6 ±8.1)

(Figure 34). ""O— Vehicle-Conlrol # Morphine (30 ng)/ControI HZ}~ Vehicle-Capsaicin I Moqjhine (30 ng)/Capsaicin

BASE 10 20 30 45 60 Time (min)

Figure 33. Effect of i.th. morphine in neonatal capsaicin or vehicle treated, sham-(

rats to probing with von Frey filaments (n-4-5 rats/group) 121

100 r —O— Vehicle-Control

9 Morphine (30 ^lg)-Control

HZH Vehicle-Capsaicin

H Morphine (30 ^g)-Capsaicin

10 20 30 40 50 60 70 Time (min)

Figure 34. Antiallodynic effect of i.th. morphine in neonatal capsaicin or vehicle treated.

nerve-ligated rats (n=4-5 rats/group). 122

Effects of DAMGO in capsaicin treated rats

DAMGO in the warm-water tail flick (52" C)

The i.th. administxation of the high-efficacy |j.-opioid, DAMGO (0.05, 0.15, 0.5 and 1.5 |ig) produced dose and time-related antinociception in all groups having a maximum effectiveness of 100% antinociception in all groups except the nerve-Iigated, vehicle treated group (maximum % antinociception = 84.3 ± 16.7). DAMGO had an Ajg

(95 % C.L.) = 0.1 (0.06 - 0.2) |ig in sham-operated, vehicle-treated rats (Figure 35), an

Ajo (95 % C.L.) = 0.1 (0.08 - 0.2) jig in nerve-ligated, vehicle-treated rats (Figure 36), an

A50 (95 % C.L.) = 0.1 (0.1 - 0.2) [Lg in sham-operated, capsaicin-treated rats (Figure 37) and an A50 (95 % C.L.) = O.I (0.06 - 0.2) |ig in nerve-ligated, capsaicin-treated rats

(Figure 38). Figure 39 shows the antinociceptive dose response comparison of DAMGO in these four groups of rats. Neither nerve injury nor capsaicin treatment significantly altered the potency or efficacy of DAMGO in the warm-water tail flick assay. 123

Vehicle 0.05 ug 0.15 ug 0.5 ug 1-5 ^g

Figure 35. Antinociceptive effect of i.th. [D-Ala^, NMePhe'*, Gly-ol]enkephalin on sham- operated rats, neonatally treated with vehicle in the 52°C warm water tail flick assay (n=4-7

rats/group). Asterisks indicate statistical significance (p < 0.05) versus baseline. 124

100 —O— Vehicle —•—0.05 ng -•-0.15 (ig 80 -^0.5 ng 1.5 ng

B 60 Q.

= 40

20

0 0 Time (min)

Figure 36. Antinociceptive effect of i.th. [D-Ala", NMePhe"*, Gly-ol]enkephalin on nerve-

ligated rats, neonatally treated with vehicle in the 52°C warm water tail flick assay (n=4-6

rats/group). Asterisks indicate statistical significance (p < 0.05) versus baseline. 125

—O— Vehicle —0.05 p.g -•—0.15 fig 100

E u t/5 +1 c o a. ou o o c c <

I) 10 20 30 40 50 60 70 Time (min)

Figure 37. Antinociceptive effect of i.th. [D-Ala", NMePhe'*, Gly-ol]enkephalin on sham-

operated rats, neonatally treated with capsaicin in the 52°C warm water tail flick assay

(n=4-6 rats/group). Asterisks indicate statistical significance (p < 0.05) versus baseline. 126

Vehicle 0.05 ng 0.15 ng

100 r 1-5 Jig

30 40 Time (min)

Figure 38. Antinociceptive effect of i.th. P-Ala% NMePhe'', Gly-oI]enkephalin on nerve- ligated rats, neonatally treated with capsaicin in the 52°C warm water tail flick assay (n=4-5

rats/group). Asterisks indicate statistical significance (p < 0.05) versus baseline. 127

100 —O— Sham/Control 90 —Q— Ligated/Control 80 B # Sham/Capsaicin iS C/3 70 B Ligated/Capsaicin

a 60 _o wo. 50 o u • ow* 40 o c •ac 30 < 20

10

0.03 0.1 0.3 I.O Dose DAMGO (|i g)

Figure 39. Dose response comparison of [D-Ala*, NMePhe^, GIy-ol]enkephalin in capsaicin and vehicle treated sham-operated and nerve-ligated rats in the 52°C warm water

tail flick assay 30 min after injection. 128

DAMGO in the von Frey assay

Unlike the results seen with morphine, i.th. administration of DAMGO (0.15, 0.5 and 1.5 |ig), was able alter paw withdrawal thresholds to von Frey probing in either sham- operated or nerve-ligated rats pretreated with capsaicin or vehicle. Again, results of von

Frey probing in nerve-ligated rats are expressed as % antiallodynia while results in sham- operated rats are expressed in paw withdrawal threshold. The highest dose of DAMGO produced a maximum possible effect (i.e., 15 g) in both vehicle and capsaicin treated sham- operated rats (Figure 40), these results were not statistically significant, however. DAMGO was also able to produce dose and time-related antiallodynic action in nerve-ligated rats treated with either vehicle or capsaicin. DAMGO had an Ajq (95 % C.L.) = C.5 (0.3 - 0.9)

|j.g in nerve-ligated, vehicle-treated rats (Figure 41) and an Ajg (95 % C.L.) = 0.5 (0.2 -

1.4) |j.g in nerve-ligated, capsaicin-treated rats (Figure 42). Figure 43 shows the antiallodynic dose response comparisons of DAMGO capsaicin and vehicle treated rats.

Neither capsaicin treatment did not significantly alter die antiallodynic potency or efficacy of DAMGO. 129

•~0— Vehicle-Control DAMGO (1.5 (ig)/Control ~D~ Vehicle-Capsaicin H DAMGO (1.5 (X g)/Capsaicin

BASE 10 20 30 45 60 Time (min)

Figure 40. Effect of i.th. [D-Ala*, NMePhe"', GIy-ol]enkephaIin in neonatal capsaicin or vehicle treated, sham-operated rats to probing with von Frey filaments (n-4-5 rats/group) 130

Vehicle 0.05 tig 0.15 ng 100 r 0.5 \ig 1.5 ng

•a

0 10 20 30 40 50 60 70 Time (min)

Figure 41. Antiallodynic effect of i.th. [D-Ala% NMePhe"*, Gly-ol]enkephaIin on nerve- ligated rats, neonatally treated with vehicle (n=4-5 rats/group). Asterisks indicate statistical

significance (p < 0.05) versus baseline. 131

—0~ Vehicle A 0.05 ng

100

80

60

40

20

0 0 10 0 40 Time (min)

Figure 42. Antiallodynic effect of Uh. [D-AIa^ NMePhe"*, Gly-ol]enkephalin on nerve-

ligated rats, neonatally treated with capsaicin (n=4-5 rats/group). Asterisks indicate

statistical significance (p < 0.05) versus baseline. 132

Ligated/Control

Ligated/Capsaicin

.2 c < ^ 20

0.03 0.1 0.3 Dose DAMGO (|i g)

Figure 43. Antiallodynic dose response comparison of [D-Ala", NMePhe"*, Gly- ol]enkephaIin in nerve-Iigated rats neonatally treated with capsaicin or vehicle treated 30

min after injection. 133

SUMMARY OF EXPERIMENTS INVOLVING NEONATAL CAPSAICIN

TREATMENT

These present results help further our understanding the involvement of capsaicin-

sensitive afferents in the development of the behavioral sequalae associated with

neuropathy pain in rats.

Neonatal capsaicin treatment is thought to produce its antinociception through

neurotoxicity of C-fibers (Jansco, et al.,, 1977, Nagy et al, 1980). This effect is C-fiber specific at doses of 50 mg/kg or less (Nagy, et al., 1983). The neurotoxicity results from

two mechanisms, increased Ca"^ influx and subsequent mitochondrial damage, in combination with capsaicin-receptor linked Na* influx and passive CI" conductance, which

results in a net increase in internal NaCI. An influx of water follows, resulting in swelling and osmotic lysis (Bevan and Solcsanyi, 1990).

A number of studies have been performed showing that neonatal capsaicin treatment can attenuate the nociceptive responses to thermal stimuli (Jansco and Jansco-Gabor. 1980,

Lorez, et al., 1983, Doucette, et al., 1987). In neuropathic rats, neonatal capsaicin treatment h;is been shown to be effective in reducing thermal hyperalgesia associated with nerve injury (Shir and Seltzer, 1990, Meller, et al., 1992). In normal rats, capsaicin sensitive afferents appear to play a role in mediation of high intensity mechanical stimuli. It has been shown that neonatal capsaicin treatment attenuates tail or paw withdrawal and vocalizations following either pressure or pricking with a needle (Faulkner and Growcott. 134

1980, Saunietand Duclaux, 1982). However, the involvement of capsaicin-sensitive fibers

in the mechanical hyperesthesias associated with either inflammation or nerve injury is less

clear. Ren and colleagues (1994) were unable to demonstrate an effect of neonatal capsaicin

in alleviating the mechanical hyperalgesia, as determined by von Frey probing, resulting

from complete Freund's adjuvant-induced inflammation in the paw. Further, neonatal capsaicin did not attenuate the mechanical allodynia in the paw following partial ligation of

the sciatic nerve (Shir and Seltzer, 1990). Conversely, neonatal capsaicin did attenuate the

increased sensitivity to pressure applied to the yeast injected paw (Faulkner and Growcott.

1980) and, in rats with L5 nerve resection, neonatal capsaicin treatment was shown to

reduce the frequency of paw withdrawal following repeated application of selected von

Frey filaments (BCinnman and Levine, 1995). In addition, mechanical allodynia in the tail following transection of the inferior caudal trunk between the S3 and S4 spinal nerves was inhibited by neonatal capsaicin (Kim, et al., 1995).

The present study demonstrates that L5/L6 spinal nerve ligation leads to a small decrease in tail-flick latencies at both 48 and 52°C. Although this difference was statistically significant, the magnitude of the difference was less than 0.9 sec at 48''C and less than 0.4 sec at 52°C. Further, nerve ligation results in the development of distinct mechanical hyperalgesia and allodynia. Although previous studies suggest that spinal nerve ligation results in thermal hyperalgesia of the paw (Wegert, et al., unpublished observation) the current study was unable to reveal such effects. The previous study showed 135

differences in paw withdrawal latency from a radiant heat source of about 4 sec. In those experiments, only rats demonstrating marked allodynia (i.e., less than 4 g paw withdrawal threshold) were used as subjects thus insuring that only successful surgery were utilized.

In the current study, 5 of the 25 nerve-ligated, vehicle treated subjects did not meet this requirement. Due to the nature of the study, these rats could not be excluded, and therefore

20% of the nerve ligated rats were "failed" surgeries. Due to the relatively small difference in radiant heat paw withdrawal latency between ligated and sham animals compared to large differences in von Frey threshold and pin prick latency, the inclusion of borderline and non-neuropathic subjects potentially accounts for the lack of thermal hyperalgesia in the paw.

Further, neonatal capsaicin treatment results in a marked attenuation of thermal nociceptive responses in both sham-operated and nerve-injured animals. This finding is consistent with previous studies. Although neonatal capsaicin treatment resulted in a modest reduction of paw lift latency following pin prick, the differences were not found to be statistically significant and in no way was capsaicin treatment able to alleviate the behavioral signs of mechanical hyperalgesia in this model. Although there may be some C- fiber involvement it is clear that non-capsaicin-sensitive afferents, possibly A5 or AP fibers, play a role in the mediation of mechanical hyperalgesia. Similarly, the development of mechanical allodynia was not altered by neonatal capsaicin treatment. This finding strengthens the theory that aberrant synapses formed at typically nociceptive second order 136

neurons by normally non-nociceptive, low threshold AP fibers following peripheral nerve

injury are important the development of mechanical allodynia (Woolf and Doubell, 1994).

Yaksh and colleagues (1995) have suggested that the ineffectiveness of morphine in

models of neuropathic pain is due to a reduction in presynaptic opioid receptors as a result

of the degeneration of primary afferent C-fibers subsequent to nerve damage. The current

study suggest that although capsaicin-sensitive C-fibers are involved in the mediation of

thermal nociceptive input, they are not involved in the mediation of mechanical allodynia.

It is more likely that the reduced efficacy of opioids versus allodynia is due to transmission

of such information by AP fibers, leaving only the post-synaptic opioid receptors, an

effective reduction in the receptor pool, available for interaction with drugs such as

morphine.

These studies also attempted to determine the effect of selective C-fiber destruction on the ability of morphine and DAMGO to elicit antinociceptive effects in nerve-injured

rats. Since neonatal capsaicin administration results in C-fiber destruction, and the majority of opioid receptors are found on presynaptic sites (Besse, et al., 1990) a resultant decrease

in the number of presynaptic opioid receptors should occur. This reduction in the available opioid receptor pool should result in the reduction of antinociceptive potency of opioids. C-

fiber destruction should not alter the effectiveness of opioids against mechanical allodynia,

however, since it is likely that allodynia is mediated by AP-fibers. The results of the present experiments somewhat support this theory. The effectiveness of morphine and 137

DAMGO were not altered by neonatal capsaicin treatment in the von Frey probing assay.

This result further strengthens the argument that information from low-threshold stimuli is transmitted by non C-fibers in both normal animals and in neuropathic animals. The antinociceptive potency and efficacy of DAMGO were also not altered due to capsaicin treatment or nerve-ligation. These results further suggest that DAMGO is of sufficient efficacy that the partial loss of presynaptic opioid receptor produced by capsaicin ueatment is not dramatic enough to alter the antinocicepdve effectiveness of DAMGO. Again,

DAMGO may be efficacious enough to produce its actions at post-synaptic opioid receptors, just as was suggested to explain DAMGO's antiallodynic activity. It should be noted, however that previous results (Ossipov, et al., 1997) have shown that the antinociceptive potency of DAMGO is significantly reduced (form 0.2 |ig to 1.0 jig) in rats with L5/L6 nerve ligation injury. These experiments were performed in the 55''C tail flick test and it would be expected that the potency of analgesic compound would be reduced in a test of higher stimulus intensity. Further, although in a lower stimulus intensity test, like the 52°C tail flick test, there is no difference in the potency of DAMGO in either sham- operated or nerve-ligated rats, a higher stimulus intensity might be able to reveal changes in the potency of DAMGO. One would expect, however, to see a dramatic loss of the antinociceptive effectiveness of morphine following capsaicin treatment. Although these data show a rightward trend in the dose response curves for both sham-operated and nerve- ligated animals receiving neonatal capsaicin treatment, the effect is not statistically 138

significant. Again, perhaps testing these morphine in capsaicin treated animals under higher stimulus intensity might widen the shifts of the dose response curves. Neonatal capsaicin treatment has been shown to result in the destruction of a large portion, but not all of

(Jansco, et al., 1977, Nagy et al, 1980, Nagy, et al., 1983). Preliminary results in these experiment indicate that there is approximately a 20% reduction in the immunoreactivity for substance P in spinal cord slices. Although this small reduction in substance P levels does not directly translate into equivalent reduction of C-fibers, they do suggest that the amount of C-fiber loss is not as dramatic as has been seen previously (Jansco, et al., 1977, Nagy et al, 1980, Nagy, et al., 1983). This C-fiber loss may be of a great enough magnitude to result in alterations of nociceptive thresholds, while it may not be sufficient to significantly alter the effectiveness of morphine. 139

RESULTS OF EXPERIMENTS INVOLVING EXCITATORY AMINO ACID

ANTAGONISTS AND DYNORPHIN A (1-13) ANTISERA

The objectives of this section are to (1) detennine the antiallodynic activity of the selective excitatory amino acid antagonists, MK 801, an NMDA antagonist, CNQX, an

AMPA antagonist, and L-AP3, a glutamate metabotropic-site antagonist alone and CNQX and L-AP3 in combination with MK 801, (2) observe the effect of NMDA channel

blockade by MK 801 on the antiallodynic and antinociceptive efficacy of morphine and (3) compare the effects of inhibition of endogenous dynorphin (by administration of selective antisera), which may play a role in the activation of the NMDA complex, to those seen by direct blockade of the NMDA channel with MK 801.

Studies on the antiallodynic effects of excitatory amino acid antagonists

MK 801 versus tactile allodynia

The i.th. administration of MK 801 (3.4 fig), which occludes the Ca++ channel associated with the NMDA receptor complex, did not produce any antiallodynic activity alone (Figure 44). The maximum % antiallodynia produced by MK 801 alone was 8.9 ±

4.3. Higher doses of MK 801 could not be tested due to the development of behavioral toxicity. 100

Vehicle MK801 (3.4 Hg)

Figure 44. Lack of antiallodynic effect of i.th. MK 801. 141

CNQX versus tactile allodynia

Unlike the NMDA antagonist, MK 801, CNQX (0.2, 0.6 and 2 (ig, i.th.), an

AMPA antagonist, produced dose and time related antiallodynic effects in animals with nerve injury ( maximum % antiallodynia = 92.6 ± 7.4, Figure 45). The Aj^ (95 % C.L.) for CNQX at 30 min was 1.0 (0.4 - 2.5) |ig.

Administration of CNQX (O.I, 0.2, 0.6 and 1 |ig, i.th.) to rats pretreated with MK

801 (3.4 |ig, i.th.) also produced significant dose and time related antiallodynic effects

(maximum % antiallodynia = 91.7 ± 6.1, Figure 46).. The A50 (95 % C.L.) for CNQX in the presence of MK 801 at 30 min was 0.3 (0.2 - 0.5) fig. Pretreatment with MK 801 produced a 3.5 fold (1.2 - 10.3, 95 % C.L.), statistically significant leftward shift of the dose-response curve of CNQX (Figure 47).

L-AP3 versus tactile allodynia

Administration of L-AP3 (0.01, 0.1, 1, 10 |ig, i.th.), the glutamate metabotropic antagonist, to rats with nerve injury was unable to alter paw withdrawal thresholds

(maximum % antiallodynia = 27.4 ± 24.3 at 10 min. Figure 48). Further, unlike the results seen with CNQX, MK 801 was unable to potentiate any antiallodynic action of 10 |ig of i.th. L-AP3 (maximum % antiallodynia = 7.1 ± 3.7, Figure 49). -Q-Vehicle •"^0.2 ng ® 0.6 ng ^ 2 ng

Time (min)

Figure 45. Antiallodynic effect of i.th. CNQX in rats with nerve ligation injury. Asterisks

indicate statistical significance (p < 0.05) versus baseline. 143

~0— Vehicle -D-MKSOI (3.4 ng) CNQX (0.1 ^g) + MK801 CNQX (0.2ng) + MK801 CNQX (0.6ng) + MK801 100 CNQXd ng) + MK801

0 10 20 30 40 50 60 70 Time (min)

Figure 46. Antiallodynic effect of Lth. CNQX in rats with nerve ligation injury pretreated

with Lth. MK80I (-10 min). Asterisks indicate statistical significance (p < 0.05) versus

baseline. 144

100

—O— CNQX alone

CNQX + MK801 (3.4 tig) ^ 80 C (O C/3

60 2 'S >» -oo 40 c <

20

0.01 0.1 1 10 Dose CNQX (|i g)

Figure 47. Dose response relationships of i.th. CNQX alone and in rats pretreated with i.th. MK80I (-10 min). MK801 significantly shifted the dose response curve of CNQX

3.5-fold to the left. 145

100 r Vehicle 0.01 Jig 0.1 Jig

10 20 30 40 50 60 70 Time (min)

Figure 48. Lack of antiallodynic effect of i.th. L-AP3 in rats with nerve ligation injury. 100 r

-O- Vehicle -•-MK801 (3.4 ng) 80 -•~L-AP3 (10 ^g) -•-L-AP3 + MK801

60

40

20

10 20 30 40 50 60 70 Time (min)

Figure 49. Lack of antiallodynic effect of i.th. L-AP3 in rats with nerve ligation injury

pretreated with i.th. MK80I (-10 min). 147

Studies on the effects of morphine in the presence of MK 801

Tactile allodynia

The intrathecal {i.th.) administration of morphine, at a dose of 30 jig did not produce any significant increase in paw withdrawal threshold in the L5/L6 nerve ligated rats (maximum % antiallodynia = 9.4 ± 6.9). Similarly, MK801 (3.4 |j,g, i.th.) did not produce any increase in paw withdrawal threshold (maximum % antiallodynia 8.9 ± 4.3).

Co-administration of these same doses of morphine and MK801 was able to produce a significant antiallodynic effect, achieving a maximal effect of 86.0 ± 14.0 % MPE.

Likewise, co-administration of MK801 (3.4 fig, i.th.) with 10 ^ig morphine was also able to produce a significant antiallodynic effect (maximum % antiallodynia = 52.7± 19.7)

(Figure 50). Figure 51 shows the antiallodynic effect of a fixed dose of i.th. morphine (30

(ig) in the presence of varied doses of i.th. MK 801 (0.34, 1.02 and 3.4 |ig). ~0 Vehicle HZH Morphine (60 |ig) -A- MK80I (3.4 Hg) — Mor (10 ng) + MK801 -B- Mor (30 ng) + MK801

Figure 50. Antiallodynic effect of 2 doses of i.th. morphine alone and in rats pretreated with i.th. MK801 (-10 min). Asterisks indicate statistical significance (p < 0.05) versus

baseline. 149

—O—Vehicle —Q—Morphine (60 ng) -:^r-MK801 (3.4 ng) -•-Mor+ MK801 (0.34 ng) -•-Mor+ MK801 (1.02 jig) A Mor + MK80I (3.4 (ig)

30 40 Time (min)

Figure 51. Antiallodynic effect of i.th. morphine alone and in rats pretreated with 3 doses

of i.th. MK801 (-10 min). Asterisks indicate statistical significance (p < 0.05) versus

baseline. 150

Warm-water tail flick (55"C)

As mentioned, i.th. morphine has also been demonstrated to have reduced efficacy in the warm-water tail flick test in rats with nerve injury. Further, the antiallodynic efficacy of i.th. morphine can be restored by pretreatment with the NMDA antagonist, MK 801.

Similarly, the antinociceptive efficacy of i.th. morphine can be restored by pretreatment with i.th. MK 801 (Figure 52). In nerve-ligated rats, morphine (30 |ig, i.th.) produced a maximum % antinociception = 57.2 ± 9.9 at 60 min after injection. The same dose of morphine administered to nerve-ligated rats which had been pretreated 15 min with MK

801 (3.4 [ig, i.th.), produced a maximum % antinociception = 87.0 ± 13.0. In both cases the drug effects were shown to be opioid-mediated by reversal with intraperitoneal {i.p.) naloxone (5 mg/kg, administered at 61 min) (Figure 52).

MK 801 pretreatment did not increase the antinociceptive efficacy of morphine in sham-operated rats. Morphine (30 |ig, i.th.) produced a maximum % antinociception =

83.3 ± 10.5 (30 min), while administration of morphine plus MK 801 (3.4 |ig, i.th.) produced a maximum % antinociception = 67.0 ± 13.0 (60 min). Again, the antinociceptive effects of morphine were reversed by i.p. administration of naloxone (Figure 53). 151

Morphine (30 (ig) Mor+ MK-801 (3.4 ng)

Time (min)

Figure 52. Antinociceptive effect of i.th. morphine alone and in nerve-ligated rats pretreated

with i.th. MKSOl (-10 min). Asterisks indicate statistical significance (p < 0.05) versus

baseline. Morphine (30 |J. g) 100 Mor +MK-801 (3.4 |ig)

c/3 MK-801 M.S.

CX

NLX

-20 -10 0 10 20 30 40 50 60 70 80 Time (min)

Figure 53. Antinociceptive effect of i.th. morphine alone and in sham operated rats pretreated with i.th. MK801 (-10 min). Asterisks indicate statistical significance (p < O.i

versus baseline. 153

Studies on the effect of morphine following pretreatment with antisera to dynorphin A (1-13)

Tactile allodynia

Dynorphin A (1-13) antisera (400 mg, i.th.) was able to produce a significant increase in paw withdrawal threshold, when compared to baseline values, but only at the

10 min time point (maximum % Antiallodynia =29.1 ± 18.6). At all other time points, dynorphin A (1-13) antisera was not significantly different than baseline. Co­ administration of dynorphin A (1-13) antisera (200 and 400 mg, i.th.) with morphine (30 mg, i.th.) was able to produce a significant effect (maximum % antiallodynia = 78.5 ± 19.7 and 100 ± 0, respectively) (Figure 54).

Warm-water tail flick 55"C

Again, the efficacy of morphine (30 mg, i.th.) was greatly reduced in animals with the L5/L6 nerve injury, only achieving a maximal % antinociception of 61.0 ± 13.5 at the

45 min time point. Similar to the effects with MK 801, dynorphin A (1-13) antisera (200 mg, i.th.) with morphine (30 mg, i.th.) was able to restore the efficacy of morphine in animals with nerve-injury (maximum % antinociception = 85.9 ± 12.3), without altering tail-flick latency, alone (maximum % antinociception = 7.7 ± 1.0) (Figure 55).

Morphine (30 mg, i.th.) was able to significantly increase the tail-withdrawal latency m sham-operated rats, achieving a maximum % antinociception of 83.3 ± 10.5. 154

Again, like MK 801, Pretreatment with dynorphin A (I-13) antisera (200 mg, i.th.) and subsequent administration of morphine (30 mg, i.th.) did not produce any alteration in the tail-withdrawal latency of sham-operated rats when compared with animals receiving morphine alone (maximum % antinociception = 80.8 ± 12.0). In addition, the tail-flick latencies of animals pretreated with dynorphin A (1-13) antisera were determined 5 min prior to morphine administration. The tail-flick latency of these animals was not significantly different than baseline responses (maximum % antinociception = 0.1 ±3.0)

(Figure 56). 155

~0— Vehicle HZ1~ Morphine (30 ng) —£s— Dyn A antisera (400 |ig) 100 Mor + Dyn A/S (60 (i g) Mor + Dyn A/S (200 ^lg) Mor + Dyn A/S (400 n g) B d Dyn A M.S. HH A/S C3 C T3 O

C <

-20 -10 0 20 30 40 50 60 70 Time (min)

Figure 54. Antiallodynic effect of Uh. morphine in the presence of antisera to dynorphin A

(1-13). Asterisks indicate statistical significance (p < 0.05) versus baseline. 156

—O— Morphine (30 ng) 100 • Mor + Dyn AJS (200 p. g)

Dyn A M.S. A/S

-20 -10 0 10 20 30 40 50 60 70 Time (min)

Figure 55. Antinociceptive effect of i.th. morphine alone and in the presence of antisera to

dynorphin A (1-13) in nerve-ligated rats. Asterisks indicate statistical significance (p <

0.05) versus baseline. 157

100 ~0— Morphine (30 jig) # Mor + Dyn A/S (200 |ig)

Dyn A M.S. A/S

-20 -10 0 10 20 30 40 50 60 70 Time (min)

Figure 56. Antinociceptive effect of i.th. morphine alone and in the presence of antisera to dynorphin A (1-13) in sham-operated rats. Asterisks indicate statistical significance (p <

0.05) versus baseline. 158

SUMMARY OF EXPERIMENTS INVOLVING EXCITATORY AMINO

ACID ANTAGONISTS AND DYNORPHIN A (1-13) ANTISERA

Tonic activation of NMDA receptors in the dorsal horn may occur following nerve injury (Wilcox, 1991; Woolf and Thompson, 1991). Further, glutamate release, and its activity at NMDA receptors can yield central sensitization (Davies and Lodge, 1987) and allodynia and hyperalgesia in a model of nerve injury (Mao, et al., 1992). Further, central sensitization is blocked by administration of NMDA antagonists (Ma and Woolf, 1995).

Seltzer and colleagues (1991) found that i.th. NMDA antagonists were suppressed autonomy in nerve injured rats. Smith and colleagues (1995) were able to delay the onset of mechanical hyperalgesia in the CGI model. The present findings that MK 801 was not able to produce any antiallodynic activity, suggests that blockade of NMDA receptor activation is not sufficient to inhibit allodynia. CNQX, however, did produce a significant antiallodynic action. This suggests that activation of the AMPA/kainate site is directly involved the mediation of allodynia in nerve injured animals. Further, the result that MK

801 was able to potentiate the antiallodynic effect of CNQX suggests that following nerve injury, there may be tonic activation of the NMDA receptor following nerve injury, but that this tonic activity may play a role in the maintenance of the neuropathic pain state, rather than the direct transfer of information from the presynaptic site to the second-order neuron.

In other words, it is possible that low threshold stimuli in rats with nerve injury results in the release of excitatory amino acids or possibly some other neurotransmitter which could 159 subsequently activate the post-synaptic AMPA receptor. Sufficient AMPA activation may result in the depolarization of the second order neuron. In the neuropathic state, this process is potentiated by tonic activation of the NMDA receptor. Therefore, blockade of the

AMPA receptor by CNQX would inhibit cell firing, while blockade of the NMDA site by

MK 801 would inhibit the long term potentiation of the action potential, it would not prevent the second order neuron from firing. Further, the effects of AMPA blockade should be enhanced by the inhibition of the NMDA site, which is what has been demonstrated here. The metabotropic glutamate site does not appear to be involved in the mediation of tactile allodynia in this model of nerve injury.

The result that MK801 was able to restore the both the antinociceptive and antiallodynic efficacy of morphine suggests that central sensitization, due to tonic NMDA receptor activation may account, in part, for the loss activity of spinally administered morphine, [njured nerves may develop spontaneously firing ectopic foci which may be responsible for this tonic NMDA receptor activation and potentially for the subsequent reduction in efficacy of spinal morphine.

Evidence exist that spinal dynorphin levels are elevated following nerve injury

(Dubner, 1991) and that the intrathecal administration of antisera to dynorphin can attenuate neurological impairment associated with such injuries (Cox, et al., 1985; Faden. 1990) suggesting the involvement of dynorphin in the development of some neuropathology.

Here, i.th. administration of dynorphin A (1-13) antisera produced only a very modest 160

antiallodynic action and did not alter tail-flick latency. However, dynorphin A (1-13) antisera, similar to MK801, did enhance the effectiveness of morphine against nerve-injury induced tactile allodynia. Additionally, dynorphin A (1-13) antisera restored the antinociceptive efficacy of morphine in nerve-injured, but not sham-operated rats, in a similar manner to that seen with MK 801.

Evidence exists to suggest dynorphin may interact with N-Methyl-D-aspartate

(NMDA) receptors and produce "non-opioid" effects including neurological deficits, motor dysfunctions and flaccid hind-limb paralysis (Walker, et al, 1982). Further, the paralytic and nociceptive effects of dynorphin can by inhibited by NMDA receptor antagonists

(Caudle and Isaac, 1988; Shukra and Lemaire, 1994). Binding experiments have shown that radiolabeled L-glutamate is displaced by dynorphin A (1-13) (Massardier and Hunt.

1989) and that, in trigeminal neurons, dynorphin binds to the dithiothreitol-sensitive site of the NMDA receptor complex (Chen, et al., 1995).

It has been suggested that the ineffectiveness of morphine in models of neuropathic pain is due to a reduction in presynaptic opioid receptors as a result of the degeneration of primary afferent neurons subsequent to nerve damage (Yaksh, et al, 1995). The current study, showing that the NMDA antagonist, MK801, while not producing any antiallodynic effect alone, can enhance the antiallodynic effectiveness of morphine. Similarly, dynorphin

A (1-13) antisera, produced little effect alone, but was able to restore the antinociceptive and antiallodynic efficacy of morphine in nerve-injured rats without altering morphine's 161 action in sham-operated rats. These results suggest that tonic NMDA receptor activation is responsible, in part, for the maintenance of the neuropathic pain state and that endogenous dynorphin, levels of which are elevated in the spinal cord following nerve injury, could be involved in this maintenance, either through the release of EAAs or through direct agonist action of dj norphin on the NMDA receptor complex. 162

RESULTS OF EXPERIMENTS INVOLVING DYNORPHIN A (1-17)

The objectives of this section are to (1) determine if a administration of a single,

low dose of exogenously administered dynorphin A (1-17) into the spinal cord will result

in the development of tactile allodynia and (2) to determine if this effect is dose related.

Normal rats with i.th. catheters were administered a single dose (15 nmol, Uh.) of dynorphin A (1-17). A marked reduction in paw withdrawal threshold developed, as determined by von Frey filament probing. The effect was shown to be bilateral and was significantly lower than baseline in either paw from 4 days after drug administration through 14 days (except in the right paw on day 5). In that time span paw withdrawal threshold ranged from 4.0 ± 0.4 g (day 4) to 2.0 ± 0.6 g (day 13) in the left paw and 5.2 ±

1.6 g (day 5) to 1.8 ± 0.1 g (day 12) in the right paw. Animals receiving vehicle had paw withdrawal thresholds ranging from 9.0 ± 1.7 to 14.2 ± 0.6 over the course of testing.

(Figure 57). Subsequently, additional doses of 1.5 and 5 nmol of dynorphin A (1-7) were tested (using the left paw only). Although neither dose resulted in statistically lower paw withdrawal thresholds, the effect appears to be dose related (Figure 58). 163

Left paw-Vehicle Right paw-Vehicle Left paw-15 nmol Dynorphin A. i.th. Right paw-15nmol Dynorphin A, i.th.

B 30m 2h 6h Id 2d 3d 4d 5d 6d 7d 8d 9d lOd lid 12d 13d 14d Time

Figure 57. Time-response relationship of paw withdrawal thresholds following i.th.

administration of a single dose (15 nmol) of dynorphin A (1-17) to normal rats. 164

Vehicle 1.5 nmol Dynorphin A, i.th. 5.0 nmol Dynorphin A, i.th. 15.0 nmol Dynorphin A, i.th.

1 I 0 I X Time

Figure 58. Dose and time response of the development of tactile allodynia following i.th.

administration of graded doses of dynorphin A (1-17) to normal rats. 165

SUMMARY OF EXPERIMENTS INVOLVING DYNORPHIN A (1-17)

Spinal dynorphin levels are increased following nerve injury (Dubner, 1991).

Exogenous dynorphin has been shown to produce dose-dependent paralytic effects in rats

(Long, et 111., 1988) through an NMDA receptor mediated process. Further, antisera to

dynorphin can reduce the magnitude of impairment from spinal cord injury (Cox, et al..

1985, Faden, et al., 1990) suggesting that dynorphin plays a role in some spinal

neuropathology. Dynorphin may produce these neuropathological effects by the destruction

of spinal neurons, possibly due to severe Ca"^-efflux (Long, et al., 1988). Dynorphin has

also been siiown to produce local ischemia which can subsequently result in cell death and

damage to of the blood-brain barrier (Thomhill, et al., 1987; Long and Tortella, 1991).

This ischemia is a result of decreased spinal cord perfusion which can also result in

neurological damage following removal of dynorphin, when reperfusion injury may occur

(Long, et al., 1987, 1994; Burkley, 1987; Schmeltzer, et al., 1989). The finding here, that subparalytic doses of exogenously administered (/.r/i.) dynorphin results in the development of a long lasting tactile allodynia suggests that the pathophysiological role of dynorphin is not limited to paralytic effects. These studies also show that the development of tactile allodynia is a dose-related effect similar to that seen with the other pathophisiology of dynorphin. Subparalytic doses of dynorphin produce sufficient pathophysiology to result in the development of tactile allodynia similar to that seen in rats with spinal nerve injury. The previous results demonstrate that tonically effective dynorphin following nerve 166 injury is involved in the maintenance of the neuropathic pain state. This effect is possibly mediated via NMDA receptors. The present finding suggest that elevated dynorphin levels may be involved in the development of the hyperesthesia associated with nerve injury.

There is further evidence to suggest that exogenous dynorphin acts through similar mechanisms to that seen in both neuropathic pain and spinal cord trauma. Vanderah and colleagues (1995) have shown that dynorphin-induced allodynia can be prevented by pretreatment with MK 801, but not by naloxone. Further,

Dynorphin may produce its non-opioid effect at NMDA receptors by increasing the release of excitatory amino acids (Skilling, et al., 1992) or through a direct action on the

NMDA receptor complex (Caudle, et al., 1994). As mentioned earlier, dynorphin has been shown to bind to sites on the NMDA receptor in trigeminal neurons (Chen, et al., 1995).

The current findings suggest that dynorphin plays an important role in the development of allodynia, but unlike nerve-injury induced allodynia, the components of neuropathic pain related to disruption of afferent flow are not present. Therefore, i.th. injection of low doses of dynorphin may prove to be a useful tool in the understanding of 167 one aspect of the pathophysiology of nerve injury. The physiology and pharmacology of these effects warrant further investigation, however. 168

RESULTS OF EXPERIMENTS INVOLVING CCKb AND DELTA

MODULATION OF MORPHINE AND THIORPHAN

Studies on the modulation of the effect of morphine by a CCKq antagonist or a delta opioid agonist

Co administration of morphine and L 365, 260

Again, morphine (30 (ig, i.th.) did not produce any antiallodynic action alone.

Similarly, neither NTI (30 (ig, maximum % antiailodynia = 4.5 ± 3.3), L 365,260, the

selective CCKg antagonists (2.5 ng, maximum % antiailodynia = 5.3 ± 5.7) nor vehicle,

produced a significant change in paw withdrawal threshold. This dose of NTI has been shown to selectively block 5-opioid receptors (Stewart and Hammond, 1994). The dose of

L 365,260 was considered optimal because a biphasic dose-response relationship has been demonstrated with regard to morphine antinociception (Zhou, et al., 1993). The co­ administration of morphine with L 365,260 produced a significant and long-acting antiallodynic action having a maximum % antiailodynia = 63.7 ± 13.8 at 30 min after administration. This effect was blocked by pretreatment with the 5-selective dose of NTI

(maximum % antiailodynia = 5.6 ± 4.0 at 20 min) (Figure 59). 169

—0~ Vehicle 100 HZ]~ Morphine -A- L 365,260 -O- NTI Mor + L 365.260 E 80 Mor + L365.260+ NTI ai +1 .2 60 c >. •a _o •2 40 c <

20

10 20 30 40 50 60 70 Time (min)

Figure 59. Time-response curves for the antiallodynic actions of i.th. vehicle (distilled water), morphine alone (30 jig), L 365,260 alone (2,5 ng), naltrindole alone (30 fig), or co-administration of morphine and L 365,260 in the absence and presence of naltrindole.

Asterisks indicate statistical significance (p < 0.05) versus baseline. 170

Co administration of morphine and [D-Alcr, GlW ]deltorphin

The concurrent administration of [D-Ala^, GIu'*]deItorphin (9 |ig, i.th.-, a dose which does not produce any antiallodynic action alone. Figure 16) with morphine (30 ^lg, i.th.) produced a significant antiallodynic action (maximum % antiallodynia = 80.2 ± 16.6 at 20 min). The increase in paw withdrawal threshold seen with the co-administration of

[D-Ala^, GIu'']deItorphin and morphine was completely abolished by pretreatment with the

5-selective dose of NTl (maximum % antiallodynia = 7.5 ± 3.8 at 20 min) (Figure 60)

Studies on the modulation of the effects of thiorphan by a CCKq antagonist

Thiorphan (300 |ig, i.th.) did not produce any significant increase in paw withdrawal threshold (maximum % antiallodynia = 31.4 ± 23.4). Similarly, neither NTI

(30 fig, maximum % antiallodynia = 4.5 ± 3.3), L365,260 (2.5 ng, maximum % antiallodynia = 5.3 ± 5.7) nor vehicle, produced a significant change in paw withdrawal threshold. In contrast, co-administration of the same doses of thiorphan and L365,260 did produce a significant antiallodynic effect (maximum % antiallodynia = 80 ± 19.8). This effect was blocked (maximum % MPE = 36 ± 19.3) by the 5-seIective dose of NTI (Figure

61). [Leu^]enkephalin antisera (200 |ig, maximum % antiallodynia = 9.4 ± 4.3). 171

100 ~0~ Vehicle HZf" Moqjhine -A-DELT -O-NTI Mor + DELT Mor + DELT + NTI

•a

I) 10 20 30 40 50 60 70 Time (min)

Figure 60. Time-response curves for the antiallodynic actions of i.th. vehicle (distilled

water), morphine alone (30 ^ig), [D-Ala*, Glu"*]deltorphin alone (9 ng), naltrindole alone

(30 |ig), or co-administration of morphine and [D-Ala', Glu'']deltorphin in the absence and

presence of naltrindole. Asterisks indicate statistical significance (p < 0.05) versus

baseline. 172

[Met^]enkephalin antisera (200 p.g, maximum % antiallodynia = 10.8 ± 5.5) or control sera

(200 (ig, maximum % antiallodynia = 2.7 ± 1.7) were unable to produce any antiallodynic effect alone. [Leu^]enkephalin antisera antagonized die antiallodynic effect of thiorphan plus L365.260 (maximum % antiallodynia = 10.5 ± 6.2) (Figure 62), while neither

[Met^]enkephalin antisera (maximum % antiallodynia = 61.9 ± 8.6) (Figure 63) nor control sera (maximum % antiallodynia = 64.9 ± 14.4) (Figure 64) altered the effect of thiorphan plus L365,260. 173

—Q— Vehicle ~r~l~ Thiorphan -A- L 365.260 -O- NTI 9 Thior + L 365 Thior + L 365 + NTI

^ 20

30 40 Time (min)

Figure 61. Antiallodynic effect of i.th. vehicle (open circles), thiorphan (3CX) |ig, open

squares). L365,260 (2.5 ng, open triangles), naltrindole (30 ng, open diamonds), co administration of thiorphan and L365,260 (closed circles) and thiorphan plus L365,260

following pretreatment (3 min) with naltrindole (closed squares) (n=4-6 rats/group).

Asterisks indicate a significant difference (p < 0.05) from baseline, crosses indicate a

significant difference from thiorphan plus L365,260. 174

~0— Vehicle —Q—Thiorphan -A-L 365.260 —0~ LeuEnk A/S ~^~Thior + L 365 B Thior + L 365 + LeuEnk A/'. Thior/ L365

20 30 Time (min)

Figure 62. Antiallodynic effect of Uh. vehicle (open circles), thiorphan (300 [ig, open squares), L365,260 (2.5 ng, open triangles), antisera to [Leu^]enkephalin (200 |ig, open

diamonds), co administration of thiorphan and L365,260 (closed circles) and thiorphan plus L365.260 following pretreatment (15 min) with antisera to [Leu"'']enkephalin (closed

squares) (n=4-6 rats/group). Asterisks indicate a significant difference (p < 0.05) from

baseline, crosses indicate a significant difference from thiorphan plus L365,260. 175

—O— Vehicle I0() —I I— Thiorphan -A- L 365.260 —0~ MetEnk A/S 9 Thior + L 365 Thior + L 365 + MetEnk A

V3 Met Thior/ A/S L365

40

-20 -10 0 10 20 30 40 50 60 70 Time (min)

Figure 63. Antiallodynic effect of Uh. vehicle (open circles), thiorphan (300 |ig, open squares), L365,260 (2.5 ng, open triangles), antisera to [Met^]enkephalin (200 p.g, open

diamond.s), co administration of thiorphan and L365,260 (closed circles) and thiorphan plus L365.260 following pretreatment (15 min) with antisera to [Mer'lenkephalin (closed squares) (n=4-13 rats/group). Asterisks indicate a significant difference (p < 0.05) from

baseline. 176

100 ~0~ Vehicle —Q-Tliiorphan -z^r- L 365.260 —O— Control Sera Thior + L 365 H Thior + L 365 + Control AJS +1 Con Thior/ A/S L365 2 "c •a o C3 C <

-20 -10 0 10 20 30 40 50 60 70 Time (min)

Figure 64. Antiallodynic effect of i.th. vehicle (open circles), thiorphan (300 jig, open squares), L365,260 (2.5 ng, open triangles), antisera to [Met^]enlcephalin (200 jig, open diamonds), co administration of thiorphan and L365,260 (closed circles) and thiorphan plus L365.260 following pretreatment (15 min) with antisera to [Met^]enkephalin (closed

squares) (n=4-8 rats/group). Asterisks indicate a significant difference (p < 0.05) from

baseline. 177

SUMMARY OF EXPERIMENTS INVOLVING CCKb AND DELTA

MODULATION OF MORPHINE AND THIORPHAN

These findings extend our understanding of the role of elevated spinal CCK levels following peripheral nerve damage. Spinal CCK levels increase in response to peripheral nerve injury (Stanfa, et al., 1994; Xu, et al., 1993) and CCK mRNA levels are elevated in rat dorsal root ganglia and monkey dorsal horn following nerve transection (Verge, et al..

1993) The antiopioid effects of CCK are well documented (Paris, et al., 1983; Suh and

Tseng, 1990, Watkins, et al, 1985a, Kellstein et al., 1991, Dourish, et al., 1990; Hughes, et al., 1990).Recent evidence suggests that the antiopioid effects of endogenous CCK may have a modulatory effect on the release of endogenous 5 opioid ligands. Down regulation by i.th. antisense to CCKg receptors produced an increase in the antinociceptive potency of morphine in mice which was blocked by the i.th. NTI or [Leu^]enkephalin antisera

(Vanderah, et al., 1994). In addition NTI blocked the enhancement of morphine's antinociceptive effect by administration of-L365,260 (Ossipov, et al, 1995).

The present experiments show that the antiallodynic efficacy of morphine is restored by blockade of CCKq receptors and that the effect is 5 opioid receptor mediated.

Further, co administration of exogenous 5 opioid agonists also result in an NTI reversible antiallodynic action. Lastly that the co-administration of L365,260 with thiorphan produce antiallodynic effects, apparently by the combination of an attenuation of CCK-mediated tonic inhibition of [Leu^Jenkephalin, but not [Met^Jenkephalin, activity and the reduction 178 of enkephalin degradation by thiorphan. The argument that the antiallodynic action is mediated by 5-opioid receptors is strengthened by the observation of the blockade with

NTI.

These observations, along with the coincident distribution of CCK with enkephalins in the CNS (Stengaard-Pederson and Larson, 1981) suggest that activation of

CCKb receptors may attenuate opioid activity, possibly by inhibiting the release of enkephalins or increasing the metabolism of enkephalin. The current results along with the observation that the exogenously administered 6 opioid agonist, [D-Ala^, Glu^Jdeltorphin is also partially effective at alleviating allodynia suggest that 6 opioid receptors modulate allodynia in this model of neuropathic pain.

In summary, the hypothesis that increased spinal CCK following nerve injury may act at CCKq receptors in the rat to produce an inhibitory effect on endogenous opioid activity. The blockade of CCK^ receptors along with morphine or inhibition of enkephalin degradation results in increased availability of enkephalin. The increase of

[Leu5]enkephalin, but not [Met^Jenkephalin, can result in sufficient activation of 5 opioid receptors, leading to the amelioration of the mechanical allodynia associated with nerve ligation injury. The data suggest that opioid 6 receptors can play a significant role in diminishing the abnormal sensations associated with injuries to peripheral nerves. 179

GENERAL CONCLUSION

Neuropathic pains are often considered to be opioid resistant both in the laboratory

and the clinical setting. However, this conclusion is clouded by a number of factors

including a limited number of opioids that have been tested (primarily morphine) and often

the tests are performed in different models of neuropathy using different endpoints.

Further, clinicians often administer only "normally" effective doses of opioids (Portenoy,

at al., 1990) to neuropathic patients. A more systematic approach to the assessment of the

effectiveness of opioids in neuropathic pains is required.

The objective of this dissertation was to determine if opioid efficacy, with a focus

on antiallodynic efficacy, is reduced in a spinal nerve ligation model of neuropathic pain.

Although this model exhibits signs of reduced opioid efficacy versus tactile allodynia and acute thermal nociception, a number of opioid compounds have been demonstrated to be fully effective against nerve-injury induced allodynia. The administration of spinal morphine, the classic opioid |i-agonist, at doses which far exceed those typically shown to produce antinociception in normal animals, was ineffective at alleviating nerve injury- induced allodynia. In contrast, DAMGO, a high efficacy opioid |i-agonist, was able to produce a significant dose related antiallodynic action.

Similarly, the newly developed, non-peptidic opioid 6-agonist, SNC 80. was unable to produce any significant antiallodynic effect. However, the peptidic opioid 5- agonist, [D-Ala", Glu"*]deltorphin, was able to significantly attenuate the allodynia 180

produced by nerve injury, but at much higher doses than those which typically produce

antinociception. Additionally spinal administration of biphalin, a very high efficacy peptidic

opioid which is thought to act at both |i,- and 5-opioid receptors, produced a full

antiallodynic action in this model.

A pair of K-selective opioid agonists were also tested against allodynia. While both

U 69,593 and fedotozine were able to significantly raise the paw withdrawal threshold in

nerve injured rats, the effect was achieved only at doses which were extraordinarily higher

than those shown to produce antinociception in normal animals.

Funher, morphine was tested against a thermal nociceptive stimulus in both nerve-

injured and sham-operated rats. The data demonstrate that the effectiveness of morphine in

the warm-water tail flick test is also greatly reduced in nerve injured animals when compared to controls, although not to the extent of that which was seen against tactile allodynia.

These results suggest that the effectiveness of low-efficacy opioids, like morphine and SNC 80, is greatly reduced in nerve-injured animals, while high-efficacy opioids, like

DAMGO, [D-Ala\ Glu'*]deltorphin and biphalin, are able to produce significant antiallodynic action. Although the K-opioids also produced antiallodynia, they were also considerably less effective than when tested against nociceptive stimuli. One possible reason why low-efficacy opioids are ineffective, while high efficacy opioids are effective against allodynia is that the transduction of afferent information from low-intensity stimuli. 181

even in rats with nerve injury, is thought to occur via Ap-fibers (Treede, et al., 1992). AP-

fibers have not been shown to express opioid receptors. This would cause the apparent

reduction in the opioid receptor pool to include only those opioid receptors found at post­

synaptic sites, thereby allowing only high-efficacy opioids to be effective.

Subsequently, the goal of this dissertation was to determine the possible

mechanisms of reduced opioid effectiveness in this model. To determine the role of C-

Fibers in pain transmission in nerve-injured rats, studies with capsaicin were performed. In

these studies capsaicin was administered to rat pups, which results in selective C-fiber

destruction and subsequent thermal antinociception. The effects of capsaicin on mechanical

nociception is less clear, however. At adulthood the subjects underwent the nerve-ligation

surgery and were subsequently tested against various nociceptive stimuli. Behavioral

measures of thermal nociception were attenuated in both sham-operated and nerve-injured

rats. Neither the mechanical hyperalgesia nor the mechanical allodynia associated with

nerve injury were altered by neonatal capsaicin treatment. These results suggest that in a

nerve ligation model of neuropathic pain in rats, noxious thermal information is mediated

via C-fibers, while high intensity and low intensity mechanical information is mediated by

A6 or AP fibers.

To extend these findings, spinal morphine and DAMGO were tested in these capsaicin-treated groups of rats. Contrary to what was expected, neither DAMGO nor

morphine exhibited any significant reduction in efficacy against either low-threshold 182

mechanical nor high-threshold thermal stimuli. Because a trend appeared, suggesting the

possibility that morphine's potency may be reduced in capsaicin treated animals, these

finding should be extended by increasing group sizes and altering stimulus intensities to elucidate the effects of capsaicin treatment on opioid effectiveness.

Additionally, an upregulation of compounds which may produce the physiological antagonism of morphine occurs at the level of the spinal cord following nerve injury, specifically, dynorphin and CCK. The maintenance of neuropathic pain states appears to be mediated, in part, by excitatory amino acid receptor activation. Administration of the

AMPA selective antagonist, CNQX was able to produce dose-related antiallodynic activity, while neither MK801, an NMDA antagonist, nor L-AP3, a metabotropic glutamate antagonist, was effective. Further, pretreatment with MK801 significantly enhanced the potency of CNQX, but not L-AP3. These findings suggest that the direct transduction of allodynic information is mediated via non-NMDA excitatory amino acid receptor (although not metabotropic receptors) and that the maintenance of the neuropathic pain state is mediated via NMDA receptors. Since evidence exists that the "non-opioid" effects of dynorphin, levels of which are elevated following nerve injury, are also mediated via the

NMDA receptor (see Shukla and LeMaire), additional saidies were undertaken.

Pretreatment with MK801 was able to restore the potency and efficacy of morphine against tactile allodynia and acute thermal nociception in rats with nerve injury. Similarly, i.th. treatment with antisera raised to dynorphin A (1-13) produced little antiallodynic effect 183

alone but did result in a fiill antiallodynic action when given 15 min prior to morphine.

Further, the antinociceptive efficacy of Lth. morphine was greatly reduced in animals with

nerve-ligation injury when compared to sham-operated controls. Antisera to dynorphin A

(1-13) restored the attenuation of die tail-flick reflex by morphine in nerve-injured animals,

without altering morphine's effect in sham-operated controls. These results suggest that

tonic activation of NMDA receptors, following peripheral nerve injury, is involved, in part,

with the attenuation of the effectiveness of morphine in a model of neuropathic pain.

Additionally, this tonic NMDA activity may be mediated by elevated levels of endogenous dynorphin associated with peripheral nerve injury. The hypothesis that increased dynorphin levels play an important role in the development of neuropathies is supported by the finding that administration of low doses of exogenously administered dynorphin A (I-17) produces stable, long-lasting tactile allodynia.

CCK may act as an endogenous anu-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid 5-receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish die release and/or availability of endogenous enkephalins. The present studies have further evaluated the role of elevated CCK levels in rats with nerve injury. Co-administration of a CCKg antagonist

(L365, 260) or sub-effective doses of [D-Ala\ Glu'*]deltorphin significantly enhanced the antiallodynic efficacy of morphine. Further, this effect was antagonized by the 5-receptor antagonist, naltrindole. This suggests that CCKg blockade may increase morphine's 184 effectiveness by increasing the availability of an endogenous 5-opioid, thus enhancing a ji-

6 receptor interaction. These studies were also extended by administration of thiorphan, a neutral , in combination with L 365, 260. Co-administration of these compounds produced a significant antiallodynic action which was again antagonized by naltrindole. In addition, antisera to [Leu^Jenkephalin, but not to [Met^]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCKg receptors may enhance the actions or availability of endogenous

[Leu^Jenkephalin or a like substance which can elicit a significant antiallodynic action via opioid 5 receptors (when its degradation is retarded by thiorphan) or can enhance the effectiveness of morphine.

The central goal of pharmacological sciences is to find effective and selective treatments for the numerous ailments which afflict human beings. The goal of this disseratation is to gain a better understanding of neuropathic pains and the use of opioids in the treatment of these pains. Taken together, the findings of this disseration suggest that use of higher doses of the currently available opioids or use of higher efficacy opioids, such a fentanyl, may result in effective treatment of neuropathic pains. Additionally, CCK antagonists or 8-opioid agonists could be useful adjuncts to treatment with morphine.

However, because of distinct differences in the role of CCK receptors in primates in comparison with rodents (Ghilardi, et al., 1992) perhaps the use of non-selective CCK antagonists would be a better choice in man. Finally, control of the tonic activity at NMDA 185 receptors, possibly by dynorphin, is an effective method of enhancing the efficacy and potency of morphine. Using higher efficacy and more selective drugs may reduce the side effects associated with regular opioid use. A similar effect may be seen with drug combinations. Therefore, the continued understanding of the physiology and pharmacology associated with neuropathic pains is warranted. 186

APPENDIX A: Animal care forms

THtUNivERsmrof

lasliluWrnlAninuJOrH iARIZONA. VIVI ^ / V ' luorni. Aruon.. 55721 jnd Uic Commrtiw TUCSON ARIZONA

Verification of Rcv-iinv BY lltt InslituliMiMl Anitnol Care nod Us« Cnmmilicc(lACUC) Final Approval Granled

nlS Awunmce No. A-3248-01 - IISDA No 86-3

TITIP.. PROTOCOL. CONTROL «f9-»-03S

"Drug Modulation of a Model of Neuropathic Piiin"

PRINCIPAL INVF5rnGA10R/r)FJ>ARTM!:NT:

Fniuk Porrcca & Mike Nichols - Pharmacology

SUBMISSION DATE; M«rch 9,19« APPROVAL UATE; M«j 26,1994

C5RANTING AGENCY-

Nin Funded • Dissertatiuu Research

The Univ'ersilyof Aii/jxa Institutional Animal Care and Use Committee reviews all section* of prupos^U relaling to anmial care and use. The above nanicd proposal has Uxn granted Rnal Approval acconlin)! to tlx review- policies of the lACUC.

NOTES;

Full approval of tliif conlrol number i$ valid IhriKigih*: May 25,1997

* When pnyecu or gr«nt rvhodr r\l«Tid Uc sbotT notcO cxfnraiiAn dxtc, Ifac PriiMpjl Invx&Usator «iU Hiboiit a new protocol prOfosAl (or fuU rrvw Folkminf lACUC Nvmv. a ncu- Pn4o,-el Control Kumhcr and ExpiniMin I)«te will be aMipMd.

••• Coutinucd appnival for thi* projcct was eonfinncd: April 11,1 y!>7 »•* Revisions (if any), arc lifted lielow:

Michael A. Cusanovich, Hh.D. Vice President for Research

OATC Ann! II. 1997 187

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