Peripheral and spinal mechanisms of neuropathic pain in the rat
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PERIPHERAL AND SPINAL MECHANISMS OF NEUROPATHIC PAIN IN THE
RAT
by
Di Bian
A Dissertation Submitted to the Faculty of the
COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2000 UMI Number 9965862
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THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE
As members of the Final Examination Commitcee. we certify that we have
read the dissertation prepared by ni Bian
entitled PERIPHERAL AND SPINAL MECHANISMS NEUROPAmCC PAIN IN THE RAT
and recommend that it be accepted as fulfilling Che dissertation requirement for the Degree of Doctor of Phd-losophy
J
Porrecaj, Fn.D. Date
.IT
- ¥-6-00 Edward French, PnTo. Date
T. Philip_^Maian»^MD, PH.D. . Dace /T //i ^ 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 recommend that it be accepted as fulfilling the dissertation requirement.
ertation ector Frank Porreca, Ph.D. 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfilbnent of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special pemiission, 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 material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
r SIGNED: 4
ACKNOWLEDGMENTS
I would like to especially acknowledge my primary advisor Dr. Frank Porreca for his guidance and support. He has been an inspiration to me in numerous ways and sets an example of an excellent scientist. The most impressing memories during my graduate study with him are his critical thinking and commitment to science. His brilliance in science is something I will enjoy throughout my career. A sincere thanks is in order for my other committee members, Dr. Edward French, Dr. Josephine Lai, Dr. Phil Malan and Dr. Jay Gandolfi, for their input and assistance in helping me achieve my doctorate degree.
Lastly, I would like to thank numerous co-workers, previous graduate students and friends for helping me in my graduate studies, experiments and for general support. First, I would like to thank Dr. Chengmin Zhong, Dr. Michael Ossipov, Dr. Todd Vanderah, Dr. Mike Nichols, and Dr. Edward Bilsky for their help in my experiments for this dissertation. I would also like to thank several of my fellow co-workers and friends that have helped make life bearable during graduate school including Terri Vorholzer, Sandra Wegert, Luis Gardell, Reggie Wang, Hong Sun, Mohab Ibrahim, Kun Ren, Terry Namkoong, Robert Smith, Lisa Sebastien and Carl Kovelowski.
Very special thanks are in order for those who have helped me in preparing, proofi-eading and supporting this dissertation including Dr. Frank Porreca, Dr. Ken Wild, Dr. James Treanor and Dr. Michael Ossipov. In addition, I would like to express my gratitude to the entire pharmacology and toxicology department for having an excellent graduate program. 5
DEDICATION
I would like to dedicate this dissertation to my wonderful wife, Xiao Ding and my beautiful daughter Shelly. My wife and daughter have continually supported and encouraged me throughout my graduate study career here in the University of Arizona.
Although there have been times were I have been extremely busy at experiment, my wife and family understood and never discouraged me from completing my degree.
Over the past six years my family has done more for me than I can ask for in a lifetime.
Therefore, it is only proper that I dedicate this dissertation to them. 6
TABLE OF CONTENTS Page LIST OFRGURES 8 LIST OF TABLES 12 ABSTRACT 13 INTRODUCTION 15 Nociception 17 Pain transmission 20 Hyperalgesia and allodynia 23 Neuropathic pain 25 Animal models of peripheral neuropathy 27 Possible mechanisms of neuropathic pain 30 Opioids and pain perception 42 Applications of antisense technology in pain research 51 HYPOTHESES OF THIS DISSERTATION 60 Hypothesis 1 60 Hypothesis 2 60 Hypothesis 3 61 Hypothesis 4 61 METHODS 62 Animals 62 Surgical introcerebroventricular (i.e.v.) cannulation and drug administrations 62 Surgical implantation of intrathecal catheter (i.th.) and drug administrations 63 Spinal cord transection 63 Chung and sham surgery 64 Behavioral Testing 65 Warm- water tail flick test 65 Hot plate test 65 Tactile allodynia test and calculations 65 Radiant heat paw-withdrawal test (thermal hyperalgesic test) 67 Immunohistochemistry Staining 68 Statistics 69 EXPERIMENTS 71 Study 1. Loss of morphine antinociceptive and antiallodynic spinal/supraspinal synergy in Chung model rats; Restoration by intrathecal MK-80I or dynorphin antiserum 71 Experimental procedure 73 Results 75 Summary 1 93 Study 2. Tactile allodynia, but not thermal hyperalgesia, of the hindpaw is blocked by spinal transection in Chung model rats 99 Experimental procedure 101 Results 102 Tactile allodynia and/or mechanical hyperreflexia 102 7
TABLE OF CONTENTS - Continued Page Thermal Hyperalgesia 103 Summary 2 110 Study 3. Lack of involvement of capsaicin-sensitive primary afferents in tactile allodynia in Chung model rats 114 Experimental procedure 118 Results 119 Summary 3 137 Study 4. Antisense oligodeoxynucleotides against a TTX-resistant sodium channel, PN3, prevent and reverse chronic, inflammatory and neuropathic pain in the rat 148 Experimental procedure 158 Chung and other inflammatory model rat preparations 158 Antisense and mismatch oligodeoxynucleotide sequences 158 Immunohistochemistry procedure 159 Results 159 Antisense (ASl), not mismatch (MMl), ODN to PN3 prevents the development of allodynia and thermal hyperalgesia in Chung rats 159 AS 1, not mismatch MMl, ODN to PN3, reverses tactile allodynia and thermal hyperalgesia in Chung rats 160 AS 1 or MMl ODN to PN3 does not affect nociception in acute pain models 161 AS2, not MM2 ODN to PN3, prevents and reverses tactile allodynia and thermal hyperalgesia in Chung rats 162 ASl ODN, not MMl ODN to PN3 inhibits chronically, but not acutely developed neuropathic pain in inflammatory pain models 162 ASl, not MMl ODN to PN3, reverses tactile allodynia and thermal hyperalgesia in diabetic neuropathic pain model rats 163 Effects of AS or MM ODN to PN3 on immunohistochemical studies 164 Summary 4 193 CONCLUSIONS 197 REFERENCES 200 8
LIST OF RGURES Page Figure 1-1, Concurrent i.th./i.c.v. morphine produces a synergistic antinociceptive interaction in the tail-flick test in normal rats 79
Figure 1-2, Concurrent i.th./i.c.v. morphine produces an additive antiallodynic effect in Chung rats 80
Figure 1-3, In the presence of i.th. antiserum to dynorphin, concurrent i.thVi.c.v. morphine produces a synergistic anti-allodynic effect in Chung rats 81
Figure 1-4, In the presence of i.th. MK-801, concurrent i.th./i.c.v. morphine produces a synergistic anti-thermal hyperalgesic effect in Chung rats 82
Figure 1-5, Dynorphin content increases in the spinal cord in Chung rats 83
Figure 1-6, Concurrent i.th./ i.e.v. morphine produces a synergistic antinociceptive effect in the tail-flick test in sham-operated rats 84
Figure 1-7, Antiserum to djmorphin i.th. does not alter the antinociceptive potency of morphine given i.th., i.c.v. or in combination in sham-operated rats 85
Figure 1-8, MK-801 i.th. does not alter the antinociceptive potency of morphine given i.th., i.c.v. or in combination in sham-operated rats 86
Figure 1-9, Concurrent i.th./i.c.v. morphine produces an additive antinociceptive effect in Chung rats in the tail-flick test 87
Figure 1-10, In the presence of antiserum to dynorphin, concurrent i.th./i.c.v. morphine produces a synergistic antinociceptive effect in the tail-flick test in Chung rats 88
Figure 1-11, In the presence of i.th. MK-801, concurrent i.th./i.c.v. morphine produces a synergistic antinociceptive effect in the tail-flick test in Chung rats 89
Figure 2-1, Allodynia in Chung rats is abolished by spinal transection 104
Figure 2-2, Spinal transection produces a tail hyperreflexia to mechanical stimuli in Chung and sham-operated rats 105
Figure 2-3, Spinal transection increases ipsilateral paw withdrawal latencies of sham- operated rats to thermal stimuli 106 9
LIST OF FIGURES - Continued page Figure 2-4, Spinal transection increases ipsilateral paw withdrawal latencies of Chung rats to thermal stimuli 107
Figure 2-5, There is no significant difference between ipsilateral and contralateral paws withdrawal latencies to thermal stimuli in Chung and sham-operated rats before spinal transection 108
Figure 2-6, There is no significant difference of paw withdrawal latencies to thermal stimuli between ipsilateral and contralateral paws in Chung and sham-operated rats after spinal transection 109
Figure 3-1, AUodynia time course in Chung rats 122
Figure 3-2, Thermal hyperalgesia time course in Chung rats 123
Figure 3-3, Thermal nociceptive response time course in Chung and sham-operated rats in the tail-flick test 124
Figure 3-4, Thermal nociceptive response time course in Chung and sham-operated rats in the hot plate test 125
Figure 3-5, RTX (0.1 mg/kg i.p.) does not reverse allodynia in Chung rats 126
Figure 3-6, RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the radiant heat paw withdrawal test in Chung and sham-operated rats 127
Figure 3-7, RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the tail flick test in Chung and sham-operated rats 128
Figure 3-8, RTX (O.l mg/kg i.p.) produce a thermal antinociceptive effect in the hot plate test in Chung and sham-operated rats 129
Figure 3-9, RTX (0.3 mg/kg s.c.) does not reverse allodynia in Chung rats 130
Figure 3-10, RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effect in the radiant heat paw withdrawal test in Chung and sham-operated rats 131
Figure 3-11, RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effect in the tail flick test in Chung and sham-operated rats 132
Figure 4-12, AS2 ODN to PN3 reverses thermal hyperalgesia in Chung rats 178 10
LIST OF FIGURES — Continued page Figure 3-12, RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effect in the hot plate test in Chung and sham-operated rats 133
Figure 3-13, RTX (0.1 mg/kg i.p.) does not prevent the development of allodynia in Chung rats 134
Figure 3-14, RTX (0.1 mg/kg i.p.) produces thermal antinociceptive effects in the tail flick test in Chung and sham rats 135
Figure 3-15, Systemic RXT treatment dose not change tactile allodynia in Chung rats 136
Figure 4-1, Pretreatment with AS 1 ODN to PN3 prevents the development of tactile allodynia by Chung surgery 167
Figure 4-2, Pretreatment with AS 1 ODN to PN3 prevents the development of thermal hyperalgesia by Chung surgery 168
Figure 4-3, ASl ODN to PN3 reverses tactile allodynia in Chung rats 169
Figure 4-4, ASl ODN to PN3 reverses thermal hyperalgesia in Chung rats 170
Figure 4-5, AS 1 and MMl ODNs to PN3 do not prevent allodynia of the affected paw by carrageenan 171
Figure 4-6, AS 1 and MMl ODNs to PN3 do not prevent thermal hyperalgesia of the affected paw by carrageenan 172
Figure 4-7, ASl and MMl ODNs to PN3 do not change the tail flick latency in carrageenan pain model rats 173
Figure 4-8, ASl and MMl ODNs to PN3 do not prevent mechanical hyperalgesia of the affected paw in carrageenan pain model rats 174
Figure 4-9, AS 1 and MMl ODNs to PN3 do not change the tail flick latency in CFA pain model rats 175
Figure 4-10, ASl and MMl ODNs to PN3 do not prevent mechanical hyperalgesia in the affected paw in CFA pain model rats 176
Figure 4-11, AS2 ODN to PN3 reverses tactile allodynia in Chung rats 177 II
LIST OF FIGURES - Continued Page Figure 4-13, AS I ODN to PN3 prevents tactile allodynia in CFA pain model rats .... 179
Figure 4-14, AS 1 ODN to PN3 prevents thermal hyperalgesia in CFA pain model rats 180
Figure 4-15, Allodynia time course in streptozotocin-induced diabetic model rats .... 181
Figure 4-16, Thermal hyperalgesia time course in streptozotocin-induced diabetic model rats 182
Figure 4-17, A single dose of streptozotocin causes a body weight loss in streptozotocin-induced diabetic model rats 183
Figure 4-18, ASl ODN to PN3 reverses tactile allodynia in streptozotocin-induced diabetic model rats 184
Figure 4-19, AS 1 ODN to PN3 reverses thermal hyperalgesia in streptozotocin-induced diabetic model rats 185
Figure 4-20, AS 1 ODN to PN3 knocks down PN3 protein in the L4 DRG of Chung model rats 186
Figure 4-21, ASl ODN to PN3 knocks down PN3 protein in the L5 DRG of Chung model rats 187
Figure 4-22, ASl ODN to PN3 does not alter PNl or PN4 protein in normal rats 188
Figure 4-23, AS 1 ODN to PN3 reverses increased dynorphin levels back to normal in Chung model rats 189
Figure 4-24, AS 1 ODN to PN3 knocks down PN3 protein in the L4 DRG of streptozotocin-induced diabetic model rats 190
Figure 4-25, AS 1 ODN to PN3 knocks down PN3 protein in the L4, L5 and L6 DRGs in streptozotocin-induced diabetic model rats 191 12
LIST OF TABLES page Table I, Summary of the antiallodynic effect of morphine given alone and in the presence of i.th. antiserum to dynorphin or MK-801 in Chung rats 90
Table 2, Summary of the antinociceptive effect of morphine in the tail-flick test given aione and in the presence of i.th. antisemm to dynorphin or MK-801 in sham-operated rats 91
Table 3, Summary of the antinociceptive effect of morphine given alone and in the presence of i.th. antiserum to dynorphin or MK-801 in Chung rats 92
Table 4, Summary of PN3 staining in large and small diameter neurons in the L4 DRG in Chung model rats 192 13
ABSTRACT
The Chung peripheral nerve injury model shows consistent allodynia and
thermal hyperalgesia, which represent the most common clinical neuropathic pain
symptoms. Also clinically relevant, in the Chung model spinal morphine was inactive
against tactile allodynia and diminished in potency in acute nociception without
spinal/supraspinal antinociceptive synergy. Further, there are increased levels of
dynorphin in multiple segmental regions of the spinal cord. This loss of
spinal/supraspinal synergy and the spinal antiallodynic effect of morphine is restored by
spinal MK-801 or antiserum to dynorphin.
It is shown here that spinal transection blocks tactile allodynia, but not thermal
hyperalgesia in Chung model rats, suggesting that thermal hyperalgesia involves both
spinal and supraspinal circuits, whereas tactile allodynia depends on a supraspinal loop.
The c-fiber specific neurotoxin resiniferatoxin before or after Chung surgery
abolishes thermal nociception in Chung and sham-operated rats, but not allodynia in
Chung model rats. These data suggest that tactile allodynia may be mediated by Ap-
fibers rather than c-fibers, offering a mechanistic basis for the observed insensitivity of
allodynia to spinal morphine in Chung model rats.
The data also show that PN3 sodium channel protein expression is increased in
medium to large diameter neurons in the L4 ipsilateral DRG of Chung rats, and that selective knockdown of PN3 protein in the DRG with specific antisense prevents and
reverses allodynia and hyperalgesia in Chung model rats without affecting normal 14 nociceptive functions. Meanwhile, the increased dynorphin level in the spinal cord of
Chung model rats returns to normal following spinal PN3 antisense. This suggests that increased PN3 protein in the DRG of Chung model rats may underlie an important mechanism for central sensitization and peripheral ectopic firing after nerve injury.
Increased expression of PN3 is also found in the DRGs of diabetic and CFA model rats; knockdown of PN3 reverses allodynia and thermal hyperalgesia in these models.
Together, these data suggest that relief from peripheral nerve injury, chronic inflammation, or diabetic neuropathy might be achieved by selective blockade of PN3.
In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability. 15
INTRODUCTION
The sensation of pain is produced by stimulation that is potentially injurious to the body. The major physiological function of pain is to protect against bodily injury and to act as a warning sign for the potential of injury as well as a signal to avoid further injury and protect an injured area in order to speed up the healing process. If individuals have the conditions of congenital deficits in neural apparatus for detection of noxious stimuli, the lack of pain sensation would result in failing to avoid harmful stimuli and unawareness of diseases.
The perception of pain is also referred to as nociception (Perl, 1980). Many external noxious stimuli such as thermal, mechanical, chemical and numerous internal diseases can produce pain sensation. Therefore, the mechanisms of nociception must vary with the various natures of noxious stimuli. The relationship between the noxious stimulus to the tissue and nociception is a series of complex electrical and chemical events; the system must provide information associated with the location of the painful stimulation as well as the intensity of such stimulation.
Additionally, in order for pain sensation to serve as a protective mechanism, there are systems involved that result in continuing nociception even after the source of the initial noxious stimulation may have been eliminated. These systems also provide increased sensitivity to various stimuli to prevent further damage. 16
There are four distinct processes involved from the detection of noxious stimulation in the periphery to the perception of pain in the brain. They are transduction
(or nociception), transmission, modulation and perception.
Transduction is the process by which noxious stimuli act at the nociceptors to produce electrical activity in the appropriate sensory nerve endings.
Transmission is the neuronal event subsequent to transduction; once the noxious stimulus has been transduced into impulses in the peripheral sensory nerves, pain sensations are determined by neurons of the pain transmission system. The transmission system includes three major neuronal comfjonents: the peripheral sensory nerves, secondary neurons ascending from the spinal cord to the brain stem and the thalamus, and the neurons which project from the brain stem and thalamus to the sensory cortex.
Modulation is the neuronal activity leading to control of the pain transmission neurons. The neuronal activity of pain modulation is a complex process involved in both the peripheral and central nervous system (CNS) mechanisms. Stress, emotional states and arousal attention, which clearly involve CNS mechanisms, profoundly alter responses to noxious stimuli. For example, traumatic injuries sustained during athletic competitions or combat are often initially reported as being relatively painless. In other circumstances, these same injuries are extremely painful (Fields et al., 1994).
Peripheral factors can modulate pain perception by peripheral and CNS mechanisms.
For example, pain thresholds to mechanical stimulation can be dramatically lowered in an area of inflammation by the sensitization of primary afferent fibers. Long-lasting 17
increases in excitability of noci-responsive dorsal horn neurons by continuous input
from primary afferent fibers can also lower pain thresholds.
Perception is the least understood process so far. We do not yet fully understand
how the neuronal activity of pain transmission neurons produces a subjective correlate and how the objectively observable neuronal events produce subjective experiences of
pain.
Pain is often classified into two categories, acute pain and chronic pain. The common cause of acute pain is tissue damage of a brief nature (Bonica, 1980). Chronic pain typically refers to pain caused by ongoing disease states or massive tissue damage that is difficult to heal, leading to long-term pain states. One of the most conmion chronic pain states is neuropathic pain. It is also referred as painful neuropathy, which is caused by peripheral nerve injury.
The focus of this dissertation is primarily to try to better understand this chronic pain state. A better understanding of neuropathic pain will aid in the development of better analgesics as well as further our understanding of the physiology and pharmacology associated with nerve injury.
Nociception
Nociceptors are also called "pain receptors". C. S. Sherrington first introduced this concept in 1906. Nociceptors are generally thought to be free nerve endings of neurons responding to noxious stimuli. The physiological mechanisms by which 18 noxious stimuli stimulate nociceptors and produce pain perception are not yet fiilly understood (Heinricher et al., 1987).
Primary nociceptive neurons (nociceptors) can be classified into two different groups based on their size and transmission velocity and their activation by noxious stimulation. The first type of nociceptors is referred to as the c-polymodal nociceptor.
These nociceptors are called "polymodal" nociceptors because they have been shown to respond to a number of different kinds of noxious stimuli, including mechanical, thermal or chemical stimuli (Bessou and Perl, 1969; Georgopoulous, 1974; Torebjork,
1974).
C-fibers are unmyelinated axons of small diameter neurons (0.3-3 |im, Ochoa and Mair, 1969) with 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 is A5-high threshold mechanoreceptors. AS- high threshold mechanoreceptors are found on thinly myelinated, large diameter (2-5
^im, Ochoa and Mair, 1969) afferents and conduct neuronal signaling at a rapid conduction velocity (approximately 20 m/sec, Adriaensen, et al., 1983). While all A6- nociceptors are thought to respond to high threshold mechanical stimulation, a proportion of A&-nociceptors also respond to thermal stimuli and are accordingly referred to as A5-mechanothermal receptors (Adriaensen, et al., 1983).
Nociceptors have their cell bodies located in the sensory ganglia such as dorsal root ganglia (DRG). They are generally pseudo unilateral axon neurons with peripheral 19
nerve endings distributed in the peripheral tissues to sense external and internal noxious
stimuli. They have shorter central axons terminating primarily in the superficial layers
of the dorsal horn of the spinal cord (Lamina I and U) as well as in deeper lamina
(lamina V). These neurons form synapses on second order neurons (also known as
projection neurons) in the spinal cord which ascend to the thalamus after crossing the
midline of the spinal cord. Larger (typically non-nociceptive fibers such as AP) afferent
fibers can directly ascend along the midline of the spinal cord without synapsing in the
dorsal horn.
There are a wide variety of other neurons found in the dorsal horn of the spinal cord including inhibitory and excitatory intemeurons as well as motor neurons. It is
thought that the nociceptive afferent fiber forms synapses with all these types of
neurons. Intemeurons can transmit inhibitory or excitatory information to projection
neurons. Motor neurons mediate spinal reflexes to painful stimuli.
Once a projection neuron receives input from a nociceptor, it then sends pain signals to supraspinal sites in the brain. Projection neurons can generally be classified into two classes: nociception specific neurons and wide dynamic range (WDR) neurons.
As their names imply, the synapses to nociception specific projection neurons arise solely from nociceptive c- and A6-fibers. Several nociceptive afferent fibers may synapse on a single projection neuron, which results in relatively larger receptive fields for these second-order neurons. Nociceptive afferent fibers also synapse on WDR neurons in the dorsal horn. WDR neurons respond to non-noxious stimuli, but also fire to a greater degree when stimulation is of a noxious nature (Fields, 1987). 20
In addition to the peripheral and spinal mechanisms, there are supraspinal components of pain perception. As mentioned above, nociceptive afferents can synapse directly on projection neurons and/or on excitatory intemeurons that subsequently synapse on projection neurons. Most of these projection neurons subsequently cross the
midline of the spinal cord and ascend primarily via the spinothalamic (Dubner et al.,
1989; Simone et al., 1991) and spinoreticularthalamic (Guillbaud et al., 1994) tracts.
The neurons of these ascending tracts terminate in the thalamus, medulla, pons, midbrain and limbic system (Brodal, 1980; Villaneuva et al., 1988). Specific areas of the thalamus are responsible for sending information to selected cortical regions for processing (Price and Dubner, 1977).
Pain transmission
There are a number of substances that are found in primary afferent neurons, which may be responsible for the transmission of pain. These substances include various peptides and amino acid neurotransmitters such as calcitonin-gene-related peptide (CGRP), somatostatin, vasoactive intestinal peptide (VIP), substance P and other neurokinins as well as glutamate and aspartate. While the neuropeptides are found in high concentrations only in small diameter myelinated and unmyelinated afferent fibers, the excitatory amino acids are found in these fibers as well as large, thick- myelinated fibers (LaMotte, 1986; Fields, 1987; Rang, et al., 1994). These neurotransmitters have been localized in the dorsal horn of the spinal cord as well as in the DRG, where the afferent neurons are located (LaMotte, 1986). 21
There is strong evidence to suggest that substance P may be a major
neurotransmitter of pain transmission. Substance P release can be released in the dorsal
horn following noxious thermal or mechanical stimuli of the periphery (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-protein 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). The research work from
transgenic mice over-expressing substance P has shown that the mice exhibit allodynia
and hyperalgesia and these nociceptive responses are reversed by substance P and
NMD A antagonists ( McLeod et al., 1999).
Glutamate is also thought to be involved in the neurotransmission of pain. It is
co-localized in small-diameter afferent fibers with the neurokinins, 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).
Glutamate has been shown to act on three potential post-synaptic sites: The N-methyl-
D-aspartate (NMDA) receptor complex, 2-amino-3-hydroxy-5-methyl-4-isoxazole-
proprionic acid (AMPA) receptors and metabotropic glutamate receptors. Additionally,
glutamate acts presynaptically on kainate receptors leading to further release of
glutamate from primary neurons. The NMDA receptor complex is a ligand-gated Ca^- channel (also some Na^ and composed of two different protein subunits named
NMDARl and NMDAR2. At present, it is not clear how many NMDARl and 22
NMDAR2 subunits are present in each functional NMDA receptor complex. The
NMDA receptor complex plays a pivotal role in long-term depression, long-term
potentiation, and developmental plasticity in the nervous system. Overactivation or
prolonged stimulation of NMDA receptors on the target neurons can damage and
eventually kill these neurons via a process referred to as excitotoxicity (MacDermott et
al., 1986). At present, there are at least six pharmacologically distinct sites through
which compounds can alter the activity of this receptor. Glutamate and related
compounds interact with a glutamate binding site on the complex to promote the
opening of the ion channels for Ca"^ and Na"*". However, this activity is unable to result
in channel conductance unless a strychnine-insensitive glycine site is also occupied
(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 release a Mg^ ion
trapped in the NMDA 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 phencyclidine and related noncompetitive antagonists such as ketamine and MK-801. This site is referred to as the PCP (phencyclidine) 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^ influx and subsequent activation of voltage-gated Ca^ channels (Murphy and 23
Miller, 1989). Unlike the NMDA and AMP A receptors, the metabotropic glutamate receptor family is a G-protein coupled receptor that may be linked to the release of intracellular Ca^ when activated (Berridge and Galione, 1988).
Hyperalgesia and Allodynia
Generally, nociceptors do not adapt to repeated activation, as has been seen in typical sensory receptors. Actually, 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). This characteristic of nociceptors is essential to prevent injury from the occurrence of further tissue damage and is also known as peripheral sensitization. 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 can be production by enzymatic synthesis by damaged local tissues, release from recruited (mast) cells, and direct release from affected afferent nerve fibers. The chemicals released following noxious stimulation could be the consequences of tissue injury or of the neurogenic inflammatory reactions in which nociceptive afferent fibers release infianunatory mediators in response to noxious stimulation. These substances include protons (Steen et al., 1992), potassium ions (Fock and Mense, 1976), bradykinin (Rocha and Rosenthal, 1961; Pock and
Mense, 1976), histamine (Fock and Mense, 1976; Lembeck, 1983), serotonin (Sicuteri et al., 1965; Fock and Mense, 1976) substance P and glutamate (Fields, 1987), and 24 certain arachidonic acid metabolites (eicosanoids). such as prostaglandins and
leukotrienes (Ferriera et al., 1994).
Some of these compounds are responsible for peripheral sensitization of nociceptors by altering the membrane permeability of neurons either by direct effects on membrane potential or through second messenger mediated cascades that may result in facilitation of cellular depolarization. Peripheral sensitization leads to a decrease of the threshold of noxious stimuli required to produce pain. This decreased pain threshold is evident at the site of the initial injury and is also known as primary hyperalgesia. In addition to peripheral sensitization, additional events occur at the level of the spinal cord, which also lead to increased pain sensitivity. Some of the compounds from continuous peripheral tissue damage result in hyperexcitability of the neurons in the dorsal horn of the spinal cord. This hyperexcitability is manifested by the spreading of the area of hyperalgesia outward from the site of injury and is also called secondary hyperalgesia.
In addition, central sensitization results in the direct sensitization of the second order neurons, resulting in a reduced requirement of afferent input for activation of the neurons of the DRG following repeated input from the afferent fibers. 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 around 25 the injury to serve 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 stimulus. 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 painful (Payne, 1986).
Neuropathic pain
One of the most important health problems in the world is the inefficient treatment of pain. The impact of pain to the daily life of human beings is not only in economic terms (approximately SICX) billion annually, NIH Guide, Vol. 24, 1995) but also in terms of human suffering. By far the most common pain symptoms are seen in patients who have suffered peripheral or central nerve damage. Amputees, peripheral nerve injury caused by trauma, herpes infections (post-herpetic neuralgia or shingles), the various other neuropathies caused by diabetes mellitus and chemical therapy for cancers make up the largest numbers. It has been estimated that as many as one-third of
Americans suffer from some form of chronic neuropathic pain, and that one-third of these patients have chronic neuropathic pain which is resistant to the commonly used medical and pharmacological interventions.
Neuropathic pain is one of the most difficult chronic pains to treat because our understanding of the mechanism is limited. Neuropathic pain refers to abnormal pain states caused by damage to peripheral nerves. Causes of neuropathic pain include diabetic neuropathy, herpes zoster, nerve contraction or compression, radiation or 26 chemotherapy for cancer, causalgia and complications from AIDS. Individuals afflicted with neuropathic pain often show exaggerated sensitivity to nociceptive stimuli
(hyperalgesia) and or perceive non-painful stimuli as painful, i.e. allodynia (Payne,
1986.). Patients with neuropathic pain often have spontaneous buming or shooting pains along with tingling, crawling sensations. Neuropathic pain may continue for long periods of time even after the initial nerve injury has healed.
Painful neuropathies follow peripheral nerve injury due to trauma or diseases.
The neuropathic pain symptoms include thermal or mechanical hyperalgesia, allodynia, spontaneous pain and reflex sympathetic dystrophy (RSD). RSD is included among these neuropathies because of the similarity to symptoms of causalgia and the suspicion of a similar underlying neurologic dysfunction. Causalgia refers to buming pain in association with nerve injury, in which the pain syndrome is attributed to sympathetic maintained pain (SMP). RSD is a more obscure term than causalgia to describe the disorder in patients who present with chronic pain in a limb in which there is edema and
/or a temperature change (the affected side is usually colder than the normal side). The manifestations of chronic neuropathic pain symptoms are different clinically when the peripheral injuries are different. For example, post-herpetic neuralgia and diabetic neuropathy usually cause burning and itching sensations; RSD and peripheral nerve damage or partial nerve damage causes causalgia; and nerve amputation and RSD cause phantom pain. Although it is true that some of these different disease statuses have the unique features of neuropathic pain, all have several different neuropathic pain manifestations. The many different neuropathic pain sensations and their occurrence in 27 many different combinations suggest that there are several different and independent mechanisms producing neuropathic pain symptoms. Up to now, our understanding of neuropathic pain has been limited, as have been the available treatments.
Animal models of peripheral neuropathy
Several animal models of peripheral neuropathy have been developed, especially in rodents. These neuropathic pain models have demonstrated many characteristics of neuropathic pain symptoms in various disease states of human beings. These animal models have greatly facilitated neuropathic pain research ranging from behavioral endpoints to molecular analysis (Fields et al., 1994).
Partial nerve injury is the main cause of causalgia in humans. Seltzer et al. described the first nerve injury model by tight ligation of one third to one half of the sciatic nerve, the partial nerve ligation model (PNL, Seltzer, et al., 1990). The Seltzer rats developed guarding behavior of the ipsilateral hind paw and licked it often, suggesting the possibility of spontaneous pain. The affected paw was evenly hyperesthetic to non-noxious and noxious stimuli including thermal and mechanical hyperalgesia as well as allodynia.
The second neuropathic pain model was created by loose ligations of the sciatic nerve, often referred to as the chronic constriction injury model (CCI, Bennett and Xie,
1988). This neuropathic pain model was induced by the placement of 4 loose ligatures around the entire sciatic nerve. The CCI model gives rise to a painful peripheral neuropathy that is characterized by allodynia, hyperalgesia, and possible spontaneous 28 pain. To compare the difference between neuropathic pain behaviors of the CCI model with those of the PNL model on the time course and magnitude of hyperalgesia, the CCI model has a faster time frame than that the PNL model. In addition, the PNL model shows a reduction in both the duration and magnitude of behavioral hyperalgesia obtained for those animals in which local anesthetic (lidocaine) was applied to the sciatic nerve, while the CCI model does not show this sensitivity.
Kim and Chung described the third neuropathic pain model that mimics peripheral nerve injury right after the CCI model, also known as the Chung model. This model was produced by tight ligation of L5 and L6 spinal nerve roots that form the sciatic nerve (Kim and Chung, 1992). The Chung model rats have a long-lasting thermal hyperalgesia and mechanical allodynia of the affected paw for at least 10 weeks. In addition, there were behavioral signs of the presence of spontaneous pain in the affected paw. The Chung model manifests the symptoms of human patients with causalgia and is compatible with previously developed neuropathic models. The Chung model has four unique features. First, the surgical procedure is stereotyped and thus highly reproducible. Second, Injured and uninjured nerves in the origin of the sciatic nerve is clearly presented, which makes nociceptive afferent pathway from the paw and ectopic foci from the site of injury easy to be identified. Third, the levels of injured and intact spinal segments are completely separated, allowing independent experimental manipulations of the injured and intact spinal segments in future experiments to answer questions regarding mechanisms underlying causalgia. Fourth, there is little if any. 29 motor function deficient manifestations in the affected paw with neuropathic pain symptoms.
Importantly, all three of these models result in the development of mechanical and thermal hyperalgesia, as well as the development of tactile allodynia (Bennett,
1994). The CCI and PNL models have been shown to result in a high incidence of autonomy as well as exaggerated deformation of affected paw, while both models are also difficult to reproduce exactly from animal to animal due to inconsistencies with the strength of the ligation (CCI) or amount of the nerve being ligated when only a portion of the nerve is used in the PNL. In addition, the behavioral neuropathic pain symptoms become less evident over time with the development of the affected peripheral nerve degeneration. For these reasons, this dissertation focuses on the highly reproducible spinal nerve ligation model of the Chung peripheral neuropathy.
In addition to above animal models of neuropathic pain produced by ligation injury on the peripheral nerves. The other animal models of neuropathic pain have been described extensively in the literature, which produce neuropathic pain symptoms most commonly seen in clinic. For Example, models of diabetic neuropathy and vincristine neuropathy as well as chronic inflammatory pain in rats that produce similar neuropathic pain symptoms following the progress of diabetes mellitus, long term vincristine cancer chemotherapy as well as chronic inflammatory diseases such as arthritis clinically (Fields, 1994). Some of these neuropathic pain models in rats will be used to investigate possible mechanisms of neuropathic pain in this dissertation. 30
Possible mechanisms of neuropathic pain
The most common phenomenon in the clinic is that patients with neuropathic pain often demonstrate a resistance to treatment with opioids. Amer and Meyerson
(1988) showed that intravenous (i.v.) morphine was ineffective at altering the mean responses to a visual pain assessment scale in the patient with various types of neuropathic pain. Portenoy et al., (1990) reported that i.v. morphine produced analgesic effects in neuropathic patients with higher doses than normally used to relieve pain.
Likewise, results from patients with cancer pain of a neuropathic nature showed reduced opioid effectiveness (Twycross, 1982). 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 for the alleviation of neuropathic pain symptoms (Portenoy,
1990).
Research work in animal models of neuropathic pain has shown similar results to clinical findings. Xu and colleagues (1993) showed that i.th. morphine did not reduce the incidence of paw autonomy produced by sciatic nerve section. Yaksh and colleagues (1995) showed that 30 Hg of i.th. morphine, an effective dose for acute pain models, was unable to attenuate tactile allodynia in the Chung model of neuropathic pain. Mao and collaborators (1995) demonstrated that i.th. morphine was able to produce antihyperalgesic and antinociceptive effects in the CCI model of nerve injury, but that the efficacy of morphine was greatly reduced. 31
A number of neuroanatomical and neurochemical changes occurring after peripheral nerve injury may underlie the dysesthesias associated with neuropathic pain states. These changes include a partial degeneration of both myelinated and unmyelinated afferents in the periphery (Nuytten, et al., 1992). This may suggest the involvement of these fibers in abnormal pain perception. Additionally, severely damaged or severed nerves may form ectopic foci. After peripheral nerve damage, injured nerves can form terminal bulbs that may subsequently sprout, presumably in an attempt to re-innervate the peripheral tissue. However, if the growth of these fine sprouts is inhibited, they can stop growing or occasionally grow back onto themselves, forming a tangled network of nerve fibers called a neuroma (Fawcett and Keynes,
1990). These neuromas can produce massive spontaneous discharges (Wall and
Gutnick, 1974) which can lead to neuropathic pain (Kajander and Bennett, 1992). This massive afferent discharge can lead to central sensitization of dorsal horn neurons.
Also, abnormal sympathetic innervation of the DRG and spinal nerves (Chung, et al.,
1993, McLachlan, et al.; 1993, Xie et al., 1995) can lead to the sympathetic maintenance of pain or RSD (Roberts, 1986).
In addition to neuroma formation, peripheral nerve damage can result in the sprouting of large diameter, low-threshold A|3-fibers (mechanoceptors) to form novel synapses with second order neurons of the dorsal horn associated with the transmission of nociceptive input (Woolf et al., 1992; Lekan, et al., 1995). AP-fibers are large, heavily myelinated afferents with rapid conduction velocities that are typically involved in the transmission of low threshold mechanical stimuli, such as light touch. It is 32 suggested that the iarge-diameter myelinated neurons undergo phenotypic changes in response to peripheral nerve injury, resulting in inappropriate synaptic connections.
Because these fibers, normally associated with innocuous stimuli, now form synapses with second order nociceptive neurons, this mechanism may be responsible for the production of allodynia caused by nerve injury (Woolf et al., 1992; Woolf and Doubell,
1994). In the Chung model there is a significant sprouting of A^-fibers into the superficial laminae of the dorsal horn of the spinal cord, presumably forming anomalous synapses with rostrally projecting dorsal horn neurons, which normally transmit nociceptive information. By forming such pathological synapses, Ap-fibers normally transmitting touch sensation might cause the same to be interpreted as tactile allodynia (Woolf et al., 1992; Woolf and E>oubell, 1994).
Injured peripheral nerves may form peripheral sprouts and ectopic foci, resulting in spontaneously consistent discharges that are perceived as nociceptive (Kajander and
Bennett, 1992). There is a correlation between ectopic discharge and behavioral signs of neuropathic pain (Kim et al., 1995). Continuous afferent barrage from ectopic foci may elicit hypersensitivity of wide dynamic range neurons, which respond to both low-
(innocuous) and high-threshold (nociceptive) stimuli so that normally innocuous mechanical touch or thermal stimulus may excessively activate these neurons to produce spontaneous pain, allodynia and hyperalgesia.
The consistent firings from ectopic foci of injured peripheral nerves can cause the excess release of excitatory amino acids such as glutamate in the central terminal of afferent neurons. Substantial amounts of evidence have demonstrated that the 33 activation of giutamate release from afferent fibers in the dorsal horn of the spinal cord and subsequent activation of NMDA receptors mediate central sensitization. This central sensitization may also be responsible for allodynia and hyperalgesia (Yamamoto et al., 1993).
A sympathetic component of neuropathic pain has been investigated.
Sympathetic efferent fibers may stimulate peripheral mechano- or thermal nerve endings, resulting in a continuous feedback loop of sympathetically maintained pain.
Likewise, peripheral nerve injury could result in sprouting of postganglionic sympathetic fibers into the dorsal root ganglion; such innervation is virtually absent in normal rats (Chung et al., 1993; McLanchian et al., 1993). In Chung neuropathic pain rats, clonidine, an tti-adrenoceptor agonist, administered i.th. produces an antiallodynic effect and surgical sympathectomy prevents mechanic allodynia and thermal hyperalgesia (Yaksh et al., 1995; Chung et al., 1993). A sympathetic component of neuropathic pain is also suggested by the observations that chemical symathectomy therapy such as administration of clonidine or phentolamine, another a-adrenoceptor agonist effectively alleviates some types of neuropathic pain clinically (Byas et al.,
1995; Shiretal., 1993).
Treatment of rats with capsaicin (8-methyl-N-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 of thermal 34 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 responses 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).
Capsaicin-sensitive afferent fibers have been demonstrated to be involved in the development of thermal hyperalgesia in the CCI model of neuropathic pain (Meller, et al., 1992). Further, it was suggested that the thermal hyperalgesia associated with a partial ligation of the sciatic nerve was mediated by c-fibers, while the mechanical hyperesthesias were mediated by A-fibers (Shir and Seltzer, 1990). 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 filaments in neonatally capsaicin treated rats with L5 spinal nerve transection. These results contradict with the hypothesis that mechanical allodynia is mediated by large, heavily myelinated Ap fibers. One of the present studies will determine the involvement of capsaicin-sensitive afferents in behavioral responses to thermal, as well as mechanical stimuli in the Chung model of neuropathic pain.
Since the sprouting of large diameter, low-threshold AP fibers to form novel synapses with second order neurons is typically associated with nociception following nerve injury, AP fibers may be responsible for the transduction of the allodynic signal
(Woolf et al., 1992). In addition, studies involving conduction velocity have suggested 35
that Ap fibers, not c-fibers, are involved in the transmission of nociceptive information
in animals with nerve injury (Treede et al., 1992).
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 and opioid receptors (Stengaard-Pederson and Larson,
1981). The antiopioid effect of CCK has been well-documented (Ghilardi et al., 1992).
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, proglumide enhances the antinociceptive effects of morphine without producing any antinociceptive effects alone (Sun and
Tseng, 1990; Watkins et al., 1985). Electrophysiological studies have shown that CCK attenuates and the non-selective CCK antagonist, lorglumide, enhances the inhibitory role of morphine on nociceptive neuronal activity in the dorsal horn of the spinal cord
(Kellstein et al., 1991). Later experiments demonstrated that the CCKB 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.
A number of studies have been performed to suggest that there is a signiHcant interaction between ^-opioid receptor induced analgesia and 5-opioid receptor activation. Vaught and Takemori (1979) have shown that administration of
[Leu^]enkephalin, at below antinociceptive doses, enhances the antinociceptive effects 36 of morphine. Additionally, the interaction of systemic morphine and [Leu^]enkephalin has been shown to produce 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'^Jdeltorphin have also been shown to result in a significant enhancement of the antinociceptive effects of morphine (Vaught and Takemori, 1979; Vaught, et al., 1982:
Porreca et al., 1992; Heyman et al., 1989). These findings suggest that CCK may tonically inhibit the release and/or availability of an endogenous 5-opioid, possibly
[Leu^]enkephalin, 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 DRG and monkey dorsal horn following nerve transection (Verge et al., 1993). Administration of CCK has been shown to antagonize the antinociceptive effects of morphine (Itoh et al., 1982;
Wiesenfeld-Hallin and Duranti, 1987), while antagonists of CCK receptors have been consistently shown to enhance morphine antinociceptive potency (Hill and Woodruff,
1990). Xu and colleagues (1993) demonstrated that the antinociceptive effects of morphine were attenuated following sciatic nerve transection and that this phenomenon was coincident with increases in the CCK mRNA in the spinal cord after the sciatic nerve injury. Further, the decreased potency of morphine in rats with sciatic nerve transection was restored by the administration of the CCKB 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 5-opioids, such as 37
[Leu^]enkephalin, and subsequently be responsible, in part, for the reduced
effectiveness of morphine against neuropathic pain. In addition to these findings,
increases in spinal levels of cholecystokinin (Stanfa et al., 1994), enkephalins and
dynorphin (Dubner, 1991) have been observed following nerve injury. The evidence
shows that there are increased levels of CCK in the spinal cord of neuropathic pain rats
with spinal nerve transection, suggesting a role for CCK in augmenting sensory input
subsequent to peripheral nerve injury in neuropathic pain. The importance of elevated
spinal CCK levels also suggests the role of increased CCK levels in neuropathic pain
states in decreasing morphine efficacy in neuropathic pain states.
The degradation of endogenous enkephalins can be attenuated by thiorphan, an
endopeptidase inhibitor, which produces antinociceptive effects by increase in the
activity of endogenous enkephalins (Chaillet et al., 1983; Scott et al., 1985). I.th.
administration of thiorphan, in combination with L365,260, at low dose, which is
ineffective alone, has shown to produce antinociceptive effects. Coadministration of
thiorphan and L365,260 produces a naltrindole (NTI, a 5-seIective antagonist)
reversible antinociception in mice (Vanderah et al., 1996).
Peripheral nerve injury has also been associated with elevated spinal quantities of endogenous opioid peptides, including enkephalins and dynorphin (Ruda, 1992).
Although enkephalins may produce some antinociceptive activity, the higher levels of endogenous dynorphin in the spinal cord seem to play a pathological role after peripheral nerve injury; dynorphin antiserum administered intrathecally reduces neuropathic pain symptoms (Cox et al., 1985; Faden et al., 1990). Long et al., 1988, 38 demonstrated dose-dependent neurological impairments by i.th. dynorphin, which depletes populations of neurons in the spinal cord. In addition, a single subparalytical dose of dynorphin A (2-17) (5 jig i.th.) produces a long-lasting neuropathic pain in rats.
This neuropathic pain can be prevented by i.th. pretreatment with dynorphin antiserum
(Vanderah et al., 1996).
In addition to these neuroanatomical changes, 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 (Celerier et al., 1999; Advokat and Rhein, 1995). Activity at non-NMDA EAA receptors has also been shown to be involved in the development and maintenance of neuropathic pain states (Faden et al.,
1990).
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 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).
It is well accepted that i.th. administration of dynorphin produces neurological dysfunctions in rats (Lx)ng 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 consequence of nerve injury involves significant plastic changes 39 in the dorsaJ hom of the spinal cord which result in central sensitization (Woolf et al.,
1992; Woolf and Doubell, 1994; Xie et al., 1995) and 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).
Selective antagonists for the EAA receptors have been developed, such as MK
801 (Dizocilpine), which is a non-competitive inhibitor of the NMDA channel (Wong et al., 1986). MK 801 produces its effects by occlusion of the ion channel via allosteric mechanisms 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 a metabotropic glutamate receptor antagonist (Collingridge et al., 1989). Both the AMPA/kainate antagonist,
CNQX and the metabotropic receptor antagonist, L-AP3 have been demonstrated to inhibit the allodynia produced by i.th. prostaglandin administration (Ferriera and
Lorenzetti, 1994; Minami et al., 1994). CNQX has also been shown to attenuate the development of thermal hyperalgesia in 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 carrageenan (Ren et al., 1992). 40
Although activation of NMDA receptors does not result directly in the
depolarization of dorsal horn 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, produces 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 the flring of dorsal horn 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 disinhibitory
process. Smith and colleagues (1995) have shown that the systemic administration of
MK801 delays, but does not prevent, the onset of mechanical hyperalgesia in the CCI
model of neuropathic pain. These studies suggest that the activity of excitatory amino acids is responsible for the development and maintenance of neuropathic pain.
Increased concentrations of sodium channels at the neuronal fibers of the site of
injury as well as at the cell bodies of the DRG of injured nerves have been suggested because of the therapeutic effectiveness of many sodium channels blockers on neuropathic pain. (Tanelian et al., 1991; Jett et al., 1997; Rizzo, 1997). The generation of spontaneous, ectopic discharges after nerve injury also strongly implicates voltage- 41 gated sodium channels in neuropathic pain (Sangameswaran et al., 1996). These channels are located in the plasma membrane and jjermit entry of sodium ions into the cell causing depolarization and generation of the action potential (Catterall, 1992;
Kallen et al., 1993). A number of voltage-gated sodium channel subtypes have been identified based on biophysical properties and sensitivity to tetrodotoxin (TTX).
Recently, a novel TTX-resistant sodium channel has been identified and cloned firom rat DRG. (Akopian et al., 1996). This TTX-resistant sodium channel is identified as either peripheral nerve 3 (PN3; Sangameswaran et al., 1996) or sensory nerve specific
(SNS; Akopian et al., 1996). Targeting individual voltage-gated sodium channel subtypes that may be either specific to sensory nociceptive neurons or selectively regulated (Waxman et al., 1994) in response to peripheral nerve injury may serve as a unique and novel approach to the treatment of neuropathic pain.
Opioid therapy for neuropathic pain in clinic has been a controversy issue. A substantial amount of clinical evidence has been shown that neuropathic pain is resistant to amelioration by opioids. For example, neuropathic pain symptoms of several origins were shown to be resistant to treatment with morphine (Amer et al., 1988). Several other clinical studies have demonstrated that potency of opioids in controlling neuropathic pain is decreased dramatically (Portenoy et al., 1986; Urban et al., 1986;
Galer et al., 1992; Zenz et al., 1992). It has been reported that intrathecal administration of morphine produces dose-dependent antihyperalgesic and antinociceptive effects in the CCI model of neuropathic pain in rats, but with six-fold less potency compared to normal rats (Mao et al., 1995). It has been known that the spinal action of morphine in 42
acute pain is largely mediated through opioid receptors located on central terminals of
small diameter afferent fibers (Lombard et al., 1989; Yaksh et al., 1995). It has been
shown that the CCI model of neuropathic pain is associated with decreased levels of
substance P and calcitonin gene-related peptide in the cells of the DRG and in laminae I
and II of the dorsal hom. This implies proximal degeneration of small-diameter
neurons in the DRG after peripheral nerve injury (Bennett et al., 1989). It has been shown that unilateral dorsal rhizotomy of T13 to S2 decreases n- and 5-opioid binding sites about 60% and 70%, respectively (Besse et al., 1990). In the CCI model of
neuropathic pain, the |X-opioid binding sites increased transiently and then decreased dramatically at 2 weeks after nerve injury (Besse et al., 1992). Evidence has shown that morphine produces a significant antinociceptive synergistic interaction at supraspinal and spinal sites. Morphine administered intrathecally and intracerebraventricularlly in a
1:1 fixed ratio produced an approximately 30-fold increase in potency compared to introcerebroventricular morphine alone when evaluated in the tail-flick test (Yeung et al., 1980).
Opioids and pain perception
Crude extracts from Papaver Somniferum, the opium poppy, have been used in medicine for thousands of years. The slicing of the unripe plant fruit makes the secretion of juice. This sap, raw opium, contains over twenty-five opiate-alkaloids including codeine and morphine. These products have been shown to produce analgesic, antitussive as well as psychoactive effects. The German chemist, Friedrich 43
Sertiimer first isolated an active opiate alkaloid, morphium (named after the Greek god
of dreams. Morpheus and later renamed morphine) from the sap of the opium plants.
Sertiimer's morphium proved to be a much more p>otent, pure alkaloid than the raw
opium. Subsequently, in the hopes of opioid analgesics with lower psychoactive
addictive liability a number of other opioid compounds have been synthesized.
Heroin was developed by the German pharmaceutical company, Bayer, as an
analgesic in the late 1800s, but was proven to possess even greater addiction liability
than morphine. Methadone and meperidine were later developed as sjmthesized analgesics and treatment for opiate withdrawal. Besides abuse liability, there are similar side effects as morphine with the use of the opioids. For these reasons, the study of the chemistry and pharmacology of opiates has been continued to nowadays in the
hopes of reducing the unwanted effects of the classic ones. Additionally, a further understanding of the pharmacology of opioids has been achieved. For example, the development of agonists and antagonists and testing of these compounds in isolated tissue preparations have led to the development of structure-activity relationships.
Radioligand binding techniques gready advanced opioid pharmacology and provided evidence for the existence of opioid receptors.
The criteria for opioid receptor binding includes evidence of stereoselectivity and saturability as well as evidence for high concentrations of opioid receptors in brain areas thought to be involved in pain control (Kuhar et al., 1973). The finding that opioid receptors exist from the periphery to the CNS strongly implied the existence of pain control pathway and endogenous opioid ligands. Various brain extracts were 44 isolated and purified and shown to produce antinociceptive effects (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 include [Leu^]enkephalin and [Met^jenkephalin which were isolated and sequenced from pig brains (Hughes et al., 1975). The term enkephalin is derived from the Greek word for "in the head".
These compounds 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) but considerably less potent comparing to morphine. These effects were reversed by the administration of the opioid antagonist, naloxone.
Along with the enkephalins, two additional categories of endogenous peptides were discovered, the endorphins and the dynorphins. 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 (Noda et al., 1982). Proopioimelanocortin (POMC) contained jJ- endorphin as well as copies of P-LPH, corticotropin and melanocyte stimulating peptides and a copy of [Met^jenkephalin (Mains et al., 1977; Rubenstein et al., 1978;
Herbert, 1980). Prodynorphin contained the 32-amino acid dynorphin A and B precursor (Berman et al., 1994).
In addition to the discovery of the endogenous opioid peptides, based on slightly different pharmacological characteristics, it had been suggested that different opioids act at different types of opioid receptors. It has subsequently been shown that there are three distinct classes of opioid receptors: mu (^i), delta (S), and kappa (k). The naming 45 of the and K receptors was based upon the prototype opioid agonists found to act upon them, morphine and ketocyciazocine, 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 |i, 5, and K receptor selective agonists and antagonists have been developed. Additionally, the three types of opioid receptors have been cloned. All of these opioid receptors belong to a seven trans membrane, G-protein coupled receptor family with similar length (372 - 400 amino acids) and a high degree of homology especially within the transmembrane spanning regions.
Opioid receptors can be found throughout the CNS. Using autoradiography, radioligand binding and in situ hybridization techniques, the location and density of opioid receptors throughout the CNS has been observed. 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 II) 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 (|i agonist), DTLET
(5 agonist) and ethylketocyclazocine (K agonist), demonstrated that of the total opioid receptor population found in spinal cord preparations 69 % of the receptors were (i, 24
% were 5 and the remaining 7% were k receptors. Furthermore, they were able to estimate the ratio of pre- and post-synaptic opioid receptors by performing dorsal 46 rhizotomy (the sectioning of the dorsal roots from the spinal cord) 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 observations indicate that the majority of opioid receptors (primarily n and 5) is on the primary afferents responsible for the transmission of signals associated with noxious stimuli, while the remainder of the opioid receptors is on second order neurons in the dorsal horn of the spinal cord. It is possible that opioids produce their analgesic effects at the level of the spinal cord by interacting with receptors on c- and A5-fibers to inhibit the release of pain neurotransmitters and thus prevent the release of these neurotransmitters (Mudge et al., 1979). Additionally, opioids are also thought to produce their effects by interacting with opioid receptors on second order neurons, thus producing further inhibition of pain signals through 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 produce analgesic effects by disinhibiting descending inhibitory control mechanisms. Numerous studies have shown that opioids produce a significandy greater degree of analgesia when both spinal and supraspinal sites are activated (Yeung and Rudy, 1980).
The great progress in opioid pharmacology has led to the development of numerous potent and selective opioids, many of which have been shown to produce 47 antinociception in the laboratory and in clinical studies. In addition, many speciflc antagonists have been developed. These compounds have proven useful as tools to further elucidate opioid pharmacology. Further understanding the pharmacology of the opioid system would be helpful for the development of better analgesic compounds which do not have many of the side effects associated with the current regimen of clinically available opioids.
Many of these peptides have been developed by modification of the endogenous opioids. Because of their peptidic natures, they can be quickly broken down by enzymatic degradation following administration. For example, [D-Ala", NMePhe'*,
GIy-ol]enkephalin (DAMGO) is a highly selective and potent peptide ^-opioid, which has been shown to produce antinociception when administered into the cerebral ventricle of the brain or into the lumbar region of the spinal cord (Miaskowski et al.,
1991; Dickenson and Sullivan, 1987; Mattia et al., 1991). Evidence has also shown that
DAMGO is of higher efficacy than the prototypic ^.-opioid agonist morphine (Galligan,
1993; Mjanger and Yaksh, 1991; Seali and Yaksh, 1993). In addition to ^.-opioid agonists, a number of peptidic S-selective opioid agonists have been developed.
Erspamer and colleagues have isolated a family of peptides called deltorphins, from the skin of a South African frog (Phyllomedusa sauvagei) (Erspamer et al., 1989; Kreil et al., 1989). These peptides are shown to by much more potent in isolated tissue preparations than any other previously discovered 5-selective compounds. One particular compound, commonly referred to as deltorphin U ([D-Ala", Glu^]deltorphin) is highly selective for the 6-opioid receptor. [D-Ala~, Glu'*]deltorphin has been shown to produce 5-seleciive analgesia when administered at either spinal (Mattia et al., 1992) or supraspinal sites (Jiang 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 and 5 receptors. Horan and colleagues (1993) demonstrated that both spinal and supraspinal administration of biphalin produced potent antinociception in mice. Furthermore, biphalin produced antinociception when administered systemically. This suggested that although biphalin is peptidic in nature, its structure is stable enough to resist enzymatic degradation.
Advances in the development of non-peptidic S-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 due to severe behavioral toxicity manifested as convulsions when administered (Comer et al.,
1993). The 5-selective agonist SNC 80 was developed by modifications to the structure of the enantiomers of BW 373U86 (Calderon et al., 1994). This compound was shown to produce antinociception in experimental animals. The finding of a systemically active, non-peptidic, 5-selective opioid is optimistic to the development of better analgesics (Bilsky et al., 1995). Non-jjeptidic K-selective opioid agonist U69,593 (Lahti et al., 1985), has been shown to produce antinociception when administered at either spinal or supraspinal sites (Millan et al., 1989).
Because endogenous opioids are rapidly degraded by enzymatic processes they are not good choices as analgesics. Attempts to stabilize [Met^l- or [Leu^] enkephalin by incorporating D-amino acids into the peptide sequence resulted in compounds such 49
as [D-Ala"]-Met-enkephaIinamide (DAME) (Pert et al., 1976) and [D-Ala". D-
Leu^Jenkephalin (DADLE). Although such compounds were stable and suitable for studies in vivo, they maintained only very limited selectivity for the S opioid receptor
(Mosberg et al., 1983). With the progress in protein chemistry and opioid pharmacology they could become good potential analgesics. For example, Thiorphan produces antinociception in some animal models by inhibiting the degradation of endogenous enkephalins (Chaillet et al., 1983; Evans and Mathre, 1985; Roques et al.,
1980) and serves as a useful tool in the understanding of the effects of the endogenous enkephalins.
The first examples of 6 selective molecules were DPDPE (Mosberg et al., 1983) and ICI 174,864 (Cotton et al., 1984), a selective 5 opioid agonist and antagonist, respectively. DPDPE, and naturally occurring 6 opioid receptor agonist [D-Ala",
Glu'^Jdeltorphin (Erspamer et al., 1989; Kreil et al., 1989), have been shown to be capable of eliciting antinociception in rats and mice at both supraspinal (Porreca et al.,
1984; 1987; Galligan et al., 1984; Jensen and Yaksh, 1986; Heyman et al., 1987; 1988;
Takemori and Portoghese, 1987; Jiang et al., 1990, 1991; Mattia et al., 1991) and spinal
(Tung and Yaksh, 1982; Porreca et al., 1984, 1987; Heyman et al., 1987; Drower et al.,
1991; Mattia etal., 1992) sites.
The existence of opioid receptors and endogenous opioids suggests that there is an endogenous opioid pain control system, since stressors induce analgesia by the release of endogenous opioids (Moskowitz et al., 1985; Marek et al., 1987; Przewlocki,
1993). In addition to control at the level of the spinal cord, descending pain control 50 systems originating from midbrain regions also influence pain control. Electrical stimulation of the periaqueductal gray (PAG) can produce analgesia (Hosobuchi et al.,
1977, Mayer and Liebeskind, 1974) and this is caused by inhibition of cells in the dorsal horn (Guillbaud, et al., 1973). Lesions of the dorsolateral funiculus 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 the spinal cord.
These descending pathways mediate antinociception by the release of serotonin (5-HT) and norepinephrine (NE). Reddy and colleagues (1979) have shown that the antinociceptive effects of PAG morphine are blocked by spinal administration of 5-HT and NB antagonists. Both |i and 5 opioid receptors are found in the PAG (Kalyuzhny et al., 1996) and opioid administration in the PAG results in disinhibition of these descending amine control systems. Disinhibition produced by morphine suggests that these descending systems are tonically inhibited by y-amino butyric acid (GABA) intemeurons. The disinhibition of these GABA intemeurons results in the activation of the descending control systems (Reichling et al., 1990). This descending inhibitory pain control system plays an important role in endogenous pain control (Fields et al.,
1997).
It has been determined that the three endogenous opioid peptides, P-endorphin, enkephalin and dynorphin are derived from the precursor protein products of three separate genes. These genes are referred to as pro-opiomelanocortin (POMC) (Mains et al., 1977), pro-enkephalin A (Hoellt et al., 1982) and pro-enkephalin B (pro-dynorphin)
(Kakidani et al., 1982). Interestingly, the products of these three genes are all similar in 51 size and structure. For example, each precursor contains a hydrophobic signal peptide followed by a cysteine residue in the amino terminus, allowing the peptides to be transported across the endoplasmic reticulum (Hollt, 1985). Also, there are paired basic amino acid residues at the border of most cleavage sites in the precursor protein, thought to serve as processing signals. Lastly, the sequence for either [Met^]enkephalin or [Leu^]enkephalin can be found at the N-terminus of each of the opioid peptides derived from these three precursor proteins (Hoellt et al., 1982). The anatomical distributions of the opioid peptides P-endorphin, enkephalin and dynorphin are consistent with their probable involvement in antinociceptive processes (Przewlocki et al., 1983; Finley et al., 1981; Watson et al., 1982; Millan, 1986; Khachaturian et al.,
1982; Cruz and Basbaum, 1985).
Applications of antisense technology in pain research
Antisense RNA and DNA techniques have been developed as a relatively recent approach to the specific modulation of gene expression in vitro and in vivo. Interest in developing antisense technology and exploiting it for research and therapeutic purposes has become intense in recent years. Although the progress has been rapid, the technology still remains empirical and questions remain to be answered (Marcusson,
1999; Weintraub, 1990).
First of all, it is important to understand the antisense terminology. DNA has two strands, one bears the same sequence as the mRNA, except with thymine instead of uracil. The other strand is used as a template and u-anscribed into mRNA, therefore 52
being the antiparailel complement of the mRNA. In some molecular biology textbooks,
the template strand of DNA was referred to as "sense" (Gardner, 1991; Mathews, 1990)
and in some other literature, the template strand of DNA was referred to as "antisense"
(Baertschi. 1994; Forsdyke, 1995). Since there is confusion of the use of antisense
terminology in the literature (Forsdyke, 1995; Lewin, 1994; Gardner, 1991), it has been
suggested that with respect to mRNA, the term antisense means a DNA or RNA
oligonucleotide complementary to the mRNA expressed from a given gene (Hengen,
1996). I would like to use this reconmiended antisense terminology in this dissertation.
The antisense concept comes from an understanding of basic nucleic acid
structure and function and depends on Watson-Crick hybridization theory (Watson and
Crick, 1953). The demonstrations that nucleic acid hybridization is successful and the
advances in in situ hybridization have built the foundation of the antisense concept. A
profusion of reports have described the modulation of various oncogenes by both DNA
and RNA antisense technology (Yu et al., 1989; Yokoyama et al., 1987). Such
manipulations offer the possibility of combining the specificity of genetic approaches
with the reversibility and temporal control of more pharmacological approaches.
DNA antisense has been shown to block the translation of mRNA in a sequence- specific manner in vitro (Gibson, 1994). DNA oligomers present distinct advantages and disadvantages compared to RNA antisense in vivo and in vitro. In the in vivo system, although DNA antisense is susceptible to be digested by nucleases, unmodified
DNA oligomers are far more stable than RNAs in biological fluids. In the in vitro 53 system, DNA antisense can be applied to cells via microinjection, bulk addition to culture medium, or by the use of uptake facilitating vehicles such as liposomes.
Comparing to vector-generated RNA antisense systems, DNA oligomer modulation of gene expression is transient. Degradation of the applied DNA antisense oligomer either intracellularly or extracellularly results in rapid return to baseline expression. Although serum free cultme conditions (i.e. nuclease free) can enhance extracellular stability of DNA antisense oligomers, repeated addition may significantly modulate the expression of proteins with half-lives in excess of several hours.
However, such repeated DNA antisense application with the breakdown of large quantities of extracellular exogenous DNA may have significant impact on intracellular nucleotide pools and unpredictable potential toxicities. With the concern of endpoint interference by exogenous DNA oligomer breakdown and alteration of nucleotide pools, control DNA mismatch oligomer sequences are necessary to be selected to contain the same base composition as the antisense sequence, especially if the target
DNA sequence is particularly purine or pyridine rich. For convincing demonstration of gene-specific effects of DNA antisense, adequate control oligo sequences should be used as well as appropriate biological endpoints. A sensitive technique for determining specific antisense down-regulation of the target gene expression either at the RNA level
(not always evident) or protein level is also required (Jaskulski et al., 1988; Nekers et al., 1993).
In order to get relatively stable antisense oligonucleotides and facilitate the uptake of DNA antisense oligos many modified oligomers such as phosphorothioates 54 and methylphosphonates have been developed (Nekers et al., 1992). For application of modified oligos, two important points should be considered carefully. First, the kinetics of uptake and subcellular localization of a particular modification may be quite different from those of a normal phosphodiester oligomer of the same sequence. Consequently, both the biological effects and the toxicities may be peculiar to the specific modification and not generalizabie to other modifications or even to the natural phosphodiester. For example, phosphorothioates, although more resistant to nuclease degradation, are also more slowly accumulated within the cells and appears to possess significantly more nonspecific toxicity in a variety of cell culture systems (Ghosh et al., 1993). A second important point is the incorporation of the modified bases into cellular DNA after a modified oligo is broken down. Modified bases have shown significant potential to induce mutagenesis or interfere with normal DNA repair mechanisms (Wickstrom et al.,
1989). It has been found that in a number of oncogenes systems the DNA antisense oligomer inhibits gene expression by acting at the 5' cap region or translational site
(Daaka et al., 1990). The other mechanisms of DNA antisense include triple helix formation at the transcriptional level and transcriptional factor inactivation (Moser et al., 1993).
RNase H is a ubiquitous enzyme that degrades the RNA strand of an RNA-DNA duplex in various organisms from virus to human being (Crouch, 1985). It plays an important role in the down regulation of gene expression using antisense technology.
At least two classes of RNase H have been identified in eukaryotic cells (Gerasimov et al., 1985). It has been shown that the backbone modifications of nucleotide have great 55 influences on the activation of RNase H. For example, methyl phosphonates do not activate RNase H (Shaw et al., 2000). In contrast, phosphorothioates have great ability to activate RNase H (Mirabelli et al., 1991). When DNA antisense binds to its target
RNA to form a hybrid complex, the complex in turn obtains an ability to activate RNase
H right away (Quartin et al., 1989). Furthermore, a single ribonucleotide in a sequence of deoxyribonucleotides is shown to activate RNase H when bound to its complementary deoxyribonucleotide (Eder et al., 1991). The evidence also shows that the efficacy of RNase H is considerably less against an RNA target duplexed with a
RNA type of antisense oligo than a full DNA type of antisense oligo.
RNA antisense oligonucleotides are designed to bind to RNA targets via hybridization. A RNA antisense oligo can bind a specific gene sequence of the target
RNA and inhibit the interaction of RNA with proteins, other nucleic acids, or other factors required for basic steps in the intermediary metabolism of the RNA and its utilization of the cell.
A most important step in the intermediary metabolism of most mRNA molecules is the excition of introns. These "splicing" reactions are sequence specific and require the concerted action of splicesomes. Consequently, RNA oligonucleotides that bind to sequences required for splicing may prevent binding of necessary factors or physically prevent the required cleavage reactions. This would result in inhibition of
RNA to become mature. Although there are several examples of oligonucleotides directed to splice junctions, none of the studies present data showing inhibition of RNA processing, accumulation of splicing intermediates, or a reduction in mature mRNA. 56
Nor are there published data in which the structure of the RNA at the splice junction
was probed and the oligonucleotides demonstrated to hybridize to the sequences for
which they were designed (McManaway et al., 1990).
Translational arrest represents another important mechanism of action of RNA
antisense. However, detailed events leading to translational arrest by RNA antisense
are little understood. Target RNAs that have been reported to be inhibited by a
translational arrest mechanism include HIV (Agrawal et al., 1988), vesicular stomatitis
virus (Lemaitre et al., 1987), N-myc (Rosolen et al., 1990), and a number of normal cellular genes (Vasanthakumar et al., 1989; Sburlati et al., 1991).
RNA adopts a variety of three-dimensional structures induced by intra
molecular hybridization, in which the most common form is the stem-loop. These structures play crucial roles in a variety of functions. For example, they can provide additional stability for RNA and as recognition motifs for a number of proteins, nucleic acids and ribonucleoproteins. Thus, disruption of three-dimensional structures of RNA could be a mechanism of antisense (Ecker et al., 1992).
The first clear elucidation of the concept of using antisense oligonucleotides as therapeutic agents is in the work of Zamecnik and Stephenson of 1978. The authors reported the synthesis of an oligonucleotide with 13 nucleotides long that was complementary to a sequence in the Rous sarcoma virus genome. They suggested that this oligonucleotide could be stabilized by 3'- and 5'-terminal modifications and showed evidence of antiviral activity. More importantly, they discussed possible sites for binding in RNA and mechanisms of action of oligonucleotides. Similar to DNA 57 antisense oligomer, a number of reports have been shown for the activities of anti-c- myc and anti-viral RNA oligonucleotides with phosphodiester, methyl phosphonate, and phosphorothioate backbones (Ghosh et al., 1993; Deanet al., 1996; Crooke et al.,
1993).
Two basic approaches have been used in the application of RNA antisense to cells. The first involves the synthesis of the desired molecule in vitro followed by its application to cells either via microinjection or bulk addition to culture medium.
Because single stranded RNA is extremely sensitive to degradation in biological fluids, techniques have been explored to protect the RNA. For example, liposome encapsulation and DNA/RNA hybrid molecules have been developed (Renneisen et al.,
1990; Milhaud et al., 1989). The second approach to RNA antisense involves utilizing eukaryotic expression vectors, which can transcribe a desired RNA antisense sequence within transfected target cells. The ultimate biological effect of the nucleotide would be influenced by many factors. For example, the local concentration of the oligonucleotide at the target RNA, the concentration of the RNA, the rates of synthesis and degradation of the RNA, the type of terminating mechanism, and the rates of the events that result in termination of the activity of the RNA. So far the understandings of these factors that may greatly affect experimental interpretations are very limited. Evidence has shown that different synthetic and purification procedures produced oligonucleotides that varied in cellular to.xicity and that potency varied from batch to batch (Crook et al.,
1991). 58
Antisense oligonucleotides are generally designed to be single stranded. Some
evidence showed that certain sequences, e.g. stretches of guanosine residues, are prone
to adopt more complex structures such as secondary and tertiary structure. The structure of target RNA has been shown to profoundly influence the affinity of the oligonucleotide to its RNA target (Egholm et al., 1993; Ecker et al., 1993). Moreover,
RNA structure produces asymmetrical binding sites that then result in divergent affinity constants depending on the position of the oligonucleotide in that structure (Ecker et al.,
1993). This in turn affects the optimal length of an oligonucleotide needed to achieve maximal affinity. Up to now it is not known how RNA structure and RNA-protein interactions influence antisense action. Several studies have shown that cells in tissue culture may actively take up phosophorothioate oligonucleotides and the uptake is highly variable (Crooke et al., 1991; Crooke et al., 1994). Cell type has a dramatic effect on total uptake, kinetics of uptake, and pattern of subcellular distribution. Uptake varies as a function of sequence, and stability in cells is also influenced by sequence
(Crooke et al., 1994; Crooke et al., 1995). Furthermore, a report showed that receptor- mediated endocytosis is a mechanism of uptake for phosphorothioate oligonucleotides
(Loke et al., 1989). Therefore, it is not clear that phosphorothioates taken-up by cells in vitro or in vivo primarily is simply a receptor-mediated endocytosis. Therefore, extrapolations from in vitro uptake studies to predictions about in vivo pharmacological behavior are entirely inappropriate. There are several lines of evidence from animal and human studies showing that, even after careful consideration of all in vitro uptake data. 59 one can not predict in vivo pharmacokinectics of the oligos (Cossum et al., 1993, 1994;
Crooke et al., 1994; Sands et al., 1994).
Although the antisense field is still in its infancy, it has generated considerable enthusiasm about potential applications (Crooke, 1996). Since the target for antisense oligos is a site in a specific mRNA species, affinity and specificity of binding derive from hybridization interactions which are theoretically much larger that can be achieved with small molecules. In addition to substantial theoretical advantages in specificity, the rational design of a specific antisense is easier than the design of small molecules interacting with target proteins. The use of oligonucleotides in neuropathic pain research represents a new paradigm for the mechanism of discovery. For example, as the sequences of the n, 5 and k opioid clones became available, the antisense approach was used to address aspects of jt, 5 and k opioid receptor pharmacology (Bilsky et al.,
1995; Rossi et al., 1994; Lai et al., 1994; Adams et al., 1994). This dissertation will further explore mechanisms of neuropathic pain caused by peripheral nerve injury using antisense technology. 60
HYPOTHESES OF THIS DISSERTATION
The main objective of this dissertation is to study the mechanisms of
neuropathic pain from the periphery to the spinal cord level due to peripheral nerve
injury or inflammation. The central hypothesis will be composed of the following 4 parts:
Hypothesis 1
Peripheral nerve injury may attenuate the normally present spinal/supraspinal synergistic antinociceptive effects of morphine. Further, the attenuation of the activity of morphine in neuropathic pain states may be due to a pathological action of elevated levels of spinal dynorphin. Elevated levels of spinal dynorphin after peripheral nerve injury might in turn produce a state of spinal sensitization through actions at NMDA receptors.
Hypothesis 2
Neuropathic pain due to peripheral nerve injury may involve supraspinal, as well as spinal modulatory pathways and these central modulatory pathways may differentially affect (facilitate or inhibit) neuropathic pain symptoms such as allodynia and thermal hyperalgesia. 61
Hypothesis 3
The formation of ectopic foci due to peripheral nerve injury is responsible for
central sensitization, which consequently promotes neuropathic symptoms such as
allodynia and thermal hyperalgesia. These neuropathic pain symptoms may involve
different afferent fiber types.
Hypothesis 4
The generation of spontaneous discharges from ectopic foci caused by different
peripheral neuropathies such as peripheral nerve injury, inflanmiation or diabetic
neuropathy may be due to TTX-resistant voltage gated sodium channels, which are responsible for neuropathic pain symptoms due to peripheral neuropathies. Knocking- down the expression of such sodium channels may diminish these spontaneous discharges and therefore, eliminate neuropathic pain symptoms. 62
METHODS
Animals
Male Sprague-Dawley rats (150-300 g) were used for all experiments. Rats were purchased from Harlan Sprague-Dawley, Inc., Indianapolis, Indiana. Rats were maintained in a climate-controlled room on a 12-h light/dark cycle (lights on at 06:00 h) and food and water were available ad libitum. All tests, surgery and drug delivery procedures were performed in accordance with the policies and reconunendations of the
International Association for the Study of Pain (lASP) and the National Institutes of
Health (NIK) guidelines for the handling and use of laboratory animals and received approval from the Animal Care and Use Committee (ACUC) of the University of
Arizona. Groups of 6 to 10 rats were used in all experiments.
Surgical intracerebroventricular (i.e.v.) cannulation and drug administration
Rats were anesthetized for i.c.v. cannulation surgeries with anesthesia of
Vetamine (Mallinckrodt Veterinary, ketamine/xylazine, 10/1; 100 mg/kg, i.p.). A 22-ga guide cannula (Plastics One Inc., Roanoke, VA) was directed towards the right lateral ventricle 2mm caudal to bregma and 1.5 mm lateral to the sagittal suture and 3.5 mm ventral from the skull surface using stereotaxic coordinates obtained from Paxinos and
Watson (1986). The guide cannula was cemented in place and secured to the skull by small stainless steel machine screws. The animals were allowed to recover five to seven days post-surgery before any pharmacological manipulations were made. Drug 63
administration was performed by slowly expelling 5 |jJ of drug solution through a 28-
gage injection cannula followed by 10 ^1 saline flush. Pontamine blue was injected
into the i.e.v. site through cannula to verify correct i.c.v. cannulation and drug delivery.
Surgical implantation of intrathecal catheter (i.th.) and drug administration
Rats were anesthetized by Vetamine 100 mg/kg i.p.. I.th. catheter indwelling surgery
was performed in a stereotaxic headholder. An 8 cm length of PE-10 polyethylene
tubing was inserted into the vertebral canal of the spinal column through an incision
made in the atlanto-occipital membrane such that it terminated at the level of the lumbar
enlargement according to the method described by Yaksh and Rudy (1976). All rats
were allowed to recover for a period of at least five days prior to any pharmacological
manipulations. Rats exhibiting motor deficiency from the surgery were discarded.
Drugs were injected to the lumbar region of the spinal cord in a volume of 5 and
followed by 9 jiL saline flush with a 1 nL airgap in between to separate drug and saline.
Spinal cord transection
A laminectomy exposed the spinal cord in the thorathic region. The spinal
transection surgery was made at vertebra Tg- The dura was reflected and the spinal cord
was transected with spring scissors with removal of an 1mm piece of spinal cord to
ensure a complete spinal transection. Sheun operated animals received laminectomy at
vertebra Tg and dural reflection only. A piece of gelfoam was placed over the
laminectomy of the lesion to stop bleeding and the muscle and skin were sutured. The surgical animals were individually housed in clean cages. The rats were monitored for signs of spinal shock after recovery and the bladder was periodically expressed. 64
Transected rats were used twenty-four hours after spinal transection. At the termination of the experiment, the spinal cord was removed and the transection was verified histologically.
Chung and sham surgery
Chung L5/L6 nerve ligation was performed according to the method of Kim and
Chung (1992). Anesthesia was induced with 4% halothane in air at 2.5 L/min and maintained with 2.5% halothane in air. The dorsal pelvic area was clipped using oscillating surgical shears and prepared with a surgical scrub. An incision (1cm) 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 (finger tight) with 4-0 silk suture distal to the dorsal root ganglia. The L6 spinal nerve was then drawn from beneath the edge of the sacmm using the glass hook and ligated as above. The incision was sutured closed with 3-0 silk thread and the animals were allowed to recover for at lease five days prior to use.
Sham-operated controls were prepared in an identical manner, with the exception that the nerves were not ligated. Rats that exhibited motor deficiency (such as paw-dragging or dropping) or failure to exhibit subsequent tactile allodynia were excluded from further testing (less than 5% of the animals were excluded). 65
All rats that underwent surgical procedures received 4 mg/kg i.m. of gentamicin immediately after surgical procedures to prevent infections.
Behavioral Testing
Warm-water tail flick test
The tail flick test (TF) was performed by gently holding the rat in a vertical position and immersing the tail in a water bath maintained at 52 or 55°C. The latency to withdrawal of the tail from the warm water bath was determined both before and after drug administration. A cut-off latency of 10 sec for those tests at 52°C or 55°C was employed to prevent tissue damage.
Hot plate test
The hot plate assay (HP) was performed by placing the rats in a plexiglass enclosure on a thermostatically controlled metal plate maintained at 55° C. The latency to licking of a hindpaw as well as jumping from the plate was taken as the nociceptive endpoint and determined both before and after drug administration. A cut-off time of
40 sec was employed to prevent tissue damage.
Tactile allodynia test and calculations
Rats were placed in a clear plastic chamber having a 1/4 inch wire mesh floor and were allowed to habituate for a minimum of 15 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 0.2 and 15.1 gram equivalents of force and estimating using a Dixon 66 non-parametric test (Chaplan et al., 1994). Chung rats having baseline thresholds of greater than 4 g were discarded from the experiment. In all studies, all rats were used, regardless of baseline threshold. An example is provided below.
The initial fiber applied to all animals is marked 4.31. This fiber produces a 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. If 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 pattern is XOOXXO. This pattern gives a threshold of 3.134 g.
3.61 3.84 4.08 O o 4.31 X o X 4.56 X 4.74 4.93 5.18
Following the determination of baseline responses, some rats received doses of agonists, antagonists, antisera, enzjnne 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. In this behavioral test, the critical point is to distinguish between "annoyance-avoidance" paw withdrawal responses and allodynic responses. "Annoyance-avoidance" paw withdrawal response is usually a slow response to avoid probing stimulation. Habituating the rats to be 67
tested long enough to the climate of the testing environment usually stops escape from
stimulation behavior. Allodynic response is a dramatic paw-lifting response to avoid
painful stimulation. The rat with allodynia usually has a quick and reflex-like response
to avoid stimulation. Careful observation and parallel normal or sham control standards
are the two best ways to avoid pseudo-positive results.
The assessment of tactile allodynia (i.e., decreased threshold to paw-withdrawal
following probing with non-noxious mechanical stimuli) consisted of measuring the
withdrawal threshold of the paw ipsilateral to the site of nerve injury or local
inflanunation in response to probing with a series of eight calibrated von Frey filaments
(0.40, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.1 g). Each filament was applied
perpendicularly to the plantar surface of the affected paw of rats kept in suspended
wire-mesh cages. Measurements were taken both before and after administration of
drug or vehicle. Withdrawal threshold was determined by sequentially increasing and
decreasing the stimulus strength ("up and down" method), analyzed using a Dixon non-
parametric test and expressed as the mean withdrawal threshold in gram force values
(Chaplan et al., 1994).
Radiant heat paw withdrawal test (thermal hyperalgesic test)
The presence of thermal hyperalgesia was measured utilizing the radiant heat
paw withdrawal test 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 15 min. A radiant heat source (i.e., high intensity projector lamp) was activated with a timer and focused onto the plantar surface of the left hindpaw of a 68
rat and the latency to paw withdrawal was measured. Paw-withdrawal latency was
determined by a photocell that halted both lamp and timer when the paw was
withdrawal. Again, baseline latencies were measured followed by drug treatment,
where applicable. A maximum cut-off of 40 sec was assigned in order to avoid tissue damage.
Immunohistochemistry Staining
Rats were anesthetized with Nembutal (Abbott Laboratories, 25 mg/kg, i.p.).
The blood was flushed out from rats with an ice-cold flush solution (PBS + 0.05%
Heparin). Each rat was then perfused with about 350 ml ice-cold fixative solution (4% paraformaldehyde + 0.1 glutaraldehyde + 0.36mM CaCh)- DRGs were taken out and then post-fixed with the same fixative solution without glutaraldehyde overnight.
DRGs were equilibrated with 30% sucrose/PBS at least over night to until DRGs sink to the bottom of the sucrose. DRGs were sliced to 10 ^un sections in a cryostat. The DAB staining procedure was then followed;
1. Wash sections in PBS for 10 minutes twice.
2. Treat sections with 1% H2O2 for 30 minutes.
3. Wash sections in PBS for 10 minutes twice.
4. Treat sections with 10% normal goat serum for 1 hour.
5. Incubate sections in 5% normal serum (goat serum) + PBS + 1:2,000-50,000
primary antibody (A-IgG) + 0.1% triton l(X)x + 0.02% sodium azide overnight.
6. Wash sections in PBS 20 min twice. 69
7. Incubate sections in secondary antibody (B-IgG) 1:1,000-2,000 in 5% normal serum
+ 0.1% Triton lOOX 0.5 for 1 hour.
8. Wash sections in PBS for 10 minutes twice.
9. Incubate sections in A+B mixtures for 30 minutes.
10. Wash sections in PBS for 10 minutes twice.
11. Incubate sections in DAB buffer solution for 5 minutes.
12. Mount sections to glass slides and dry them at room temperature.
13. Dehydrate each slide in alcohol with serial concentrations from 20% to 100% for 15
minutes at each concentration.
14. Treat each slide with xylene for 5 minutes twice.
15. Add mounting medium to slides and cover them with cover slips.
16. Examine slides under light microscope.
Note: PBS solution used in the above protocol is 0.1 M.
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 Prizm (GraphPad, San Diego, CA). Only the linear portion of the dose-response curve was 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 70 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 5 subjects. The
A50 with 95% CL and relative potencies with 95% CL of dose response curves were calculated by linear regression of log dose response curves. 71
EXPERIMENTS
Study 1 Loss of morphine antinociceptive and antiallodynic spinal/supraspinal synergy in Chung model rats: Restoration by intrathecal MK-801 or dynorphin antiserum
The Chung model of peripheral nerve ligation injury produces behavioral signs that are suggestive of those seen in clinical conditions of neuropathic pain, such as tactile allodynia and thermal hyperalgesia (Bian et al., 1995; Chaplan et al., 1994; Kim et al., 1992). A number of pathological events are known to occur which may promote the development of neuropathic dysesthesias, including the development of ectopic foci
(Devor et al., 1990), sprouting of AP primary afferent fibers into the superficial laminae of the dorsal horn (Lekan et al., 1996; Woolf et al., 1992), and changes in spinal neurotransmitter levels, which include increases in spinal dynorphin (Dubner et al.,
1992) and possibly cholecystokinin (Stanfa et al., 1994). There is also strong evidence to indicate that excitatory amino acids may be released from tonically active afferent neurons and that this abnormal spontaneous, persistent afferent drive is mediated through an action at the NMDA (N-methyl-D-aspartate) receptor complex (Davies et al., 1987; Dickenson et al., 1987; Wilcox et al., 1991).
It has been demonstrated that a spinal/supraspinal synergistic antinociceptive interaction is produced by morphine against acute noxious stimuli (Fig. 1-1; Yeung et al., 1980). It is because of this site-site synergy that systemic morphine is sufHciently 72
potent for clinical analgesic utility. The loss of spinal efficacy of morphine, demonstrated in Chung model rats, might contribute to a loss of this spinal/supraspinal synergy (Bian et al., 1995). Evidence has shown that morphine does not reduce nociceptive response in several acute nociceptive tests in homozygous ^-receptor knockout mice, and displays rightward and downward shifts in morphine analgesic dose-response relationships in heterozygous |i-receptor knockout mice, supporting |i- receptor-mediation of morphine-induced supraspinal and spinal analgesia (Sora et al.,
1997). Previous studies have demonstrated that one consequence of Chung nerve- ligation injury in rats is a significant reduction in the antinociceptive potency and efficacy of i.th. morphine and a complete loss of i.th. antiallodynic efficacy (Bian et al.,
1995; Ossipov et al., 1995). These losses are restored by an i.th. injection of antiserum to dynorphin A Therefore, we hypothesized that the normally present spinal/supraspinal synergy of morphine for analgesia known to exist with ^-opioid receptors, might be lost, or substantially diminished, probably as a consequence of the loss of antiallodynic activity of i.th. morphine in neuropathic pain states caused by peripheral nerve injury. Further, the loss of antiallodynic activity of morphine in neuropathic pain states may be due to a pathological action of elevated levels of spinal dynorphin. Elevated levels of spinal dynorphin after peripheral nerve injury might in turn promote a state of spinal 73 sensitization through direct or indirect actions at the NMDA receptor. Study 1 was undertaken in order to determine the effect of experimental nerve injury on the spinal/supraspinal antinociceptive and antiallodynic activity of morphine which may involve different nociceptive processing mechanisms (Bian et al., 1998;). Additionally, the possible involvement of spinal dynorphin was studied using antiserum to this peptide and the NMDA antagonist, MK-801. Experimental Procedure Several doses of morphine were administered in order to generate dose-response curves. In the experiments where antisemm to dynorphin A (3.4 jig or 10 nmol) was used, it was given by i.th. injection 20 min prior to morphine. These doses and times have been established based on previous work (Nichols et al., 1997; Ossipov et al., 1995). This dose of MK-801 did not produce adverse behavioral effects. The combinations of the i.th. and i.c.v. injections of morphine were given concomitantly, as the times of peak effect (30 min) by either route of administration coincided. The warm-water tail-flick test at 52°C was performed before and after drug treatment. Rats were tested between 10 and 15 days after Chung ligation once allodynia was firmly established. Measurements were taken before and 10, 20, 30, 45 and 60 min after administration of drug or vehicle. 74 The assessment of tactile allodynia was taken before and 10, 20, 30, 45 and 60 min after administration of drug or vehicle. Dose-response curves were obtained for individual compounds and for combinations of compounds. Rats were deeply anesthetized with ether and decapitated 10 days after Chung or sham surgery. Incisions were made at the S] vertebral level and the spinal cord was ejected by injection of ice-cold saline. The spinal cord was placed on an iced glass dish and the spinal segments of lumbar region were rapidly dissected. The tissue was divided into left and right segments by a sagittal cut and the dorsal and ventral quadrants by a cut in a plane slightly dorsal to the central canal. Tissue samples obtained from ipsilateral as well as contralateral dorsal quadrants of the lumbar spinal cord were weighed, immediately frozen on dry ice and stored at -70°C. Thawed tissues were placed in IN acetic acid, disrupted using a Polytron homogenizer and incubated at 95°C for 20 min. After centrifugation at 10,000 x g for 20 minutes (4°C) the supernatant was lyophilized and stored at -70°C. Protein concentrations were determined using the bichinchoninic acid method with bovine serum albumin as a standard. Immunoassay was performed using a commercial enzyme immunoassay kit using an antibody specific for dynorphin Ad-i?) (Peninsula Laboratories, Belmont, CA). Standard curves were constructed and dynorphin content of each sample was determined. The behavioral data were converted to percent of maximal possible effect (% MPE) using the formula: % MPE = (WT - CT)/(CO-CT) x 100, where WT = the withdrawal threshold or latency obtained experimentally, CT = the baseline control 75 value before drug administration and CO = the cut-off value (i.e.; 15 g for the tactile allodynia test, 10 sec for the tail-flick test). Dose-response curves were generated where possible and the A50 value (dose estimated to produce 50% MPE) and 95% confidence intervals were determined for additional statistical calculations and isobolographic analyses (Tallarida et al., 1989). An additive or synergistic interaction between the two drug treatments was determined by isobolographic analysis when both routes of administration produced a dose-response curve, or by a modified isobolographic analysis when only one route of administration produced a dose- dependent effect (Porreca et al., 1990; Tallarida et al., 1989). Significant differences in drug potencies were determined by the relative potency assay and a t-test as described by Tallarida et al., (1989). For the radioimmunoassay, pair-wise comparisons between treatments were detected using Student's t-test. Significance was determined at P<0.05 level. Results Morphine was inactive against allodynia after i.th. injection up to the highest tested dose of 100 pg, but produced a dose-dependent antiallodynic effect after i.e.v. injection to Chung rats (A50 = 1.7 ± I.l |ig). In a 1:1 fixed ratio, concurrent i.th. and i.c.v. administration of morphine also produced a dose-dependent antiallodynic effect (A50 = 1.6 ± 2.7 ^ig as total dose). This interaction was found to be additive and not synergistic after isobolographic analysis (P > 0.05) (Fig. 1-2). 16 In the presence of i.th. antiserum to dynorphin A(|.i7). morphine given i.th. produced a dose-dependent antiallodynic effect in Chung rats (Fig. 1-3). The A50 (± 95% C.L.) value was 11.6 ± 9.42 pg. Likewise, i.c.v. morphine also produced dose- dependent antiallodynia in the presence of i.th. dynorphin A(i.i7) antiserum and produced an A50 value of 2.7 ± 0.89 pg, a value which was not significantly different (p > 0.05) from the potency of i.c.v. morphine alone. The concurrent injection of morphine i.th. and i.c.v. produced a remarkably robust synergistic effect in the presence of i.th. dynorphin antiserum (Fig. 1-3) with an A50 of 0.24 ± 0.072 ng expressed as total dose. Similar results to those seen with dynorphin antiserum were seen with i.th. MK- 801. After i.th. MK 801, morphine produced a dose-dependent antiallodynic effect after both i.c.v. (A50 = 1.4 ± 0.42 pg) and i.th. (A50 = 6.0 ± 3.9 pg) administration (Fig. 1-4); the i.c.v. morphine effect in the presence of i.th. MK-801 did not differ significantly (p > 0.05) from the effect of i.c.v. morphine alone. The concurrent injection of morphine i.th. and i.c.v. also produced a remarkably robust synergistic effect in the presence of i.th. MK-801 (Fig. 1-4) with an A50 of 0.14 ± 0.03 pg expressed as a total dose. The A50 values are summarized in Table 1. Neither dynorphin antiserum nor MK-801 given alone was active against tactile allodynia. Morphine alone produced a dose-dependent antinociception in the tail-flick test after either i.c.v. or i.th. injection to sham-operated rats (A50 = 3.9 ± 0.92 pg and 1.4 ± 0.42 pg, respectively). A 1:1 fixed i.th./i.c.v. ratio of morphine produced a synergistic antinociceptive effect as demonstrated by the A50 of 0.33 ± 0.13 pg (total dose)(Fig. 1-6 77 and Table 2). The i.th. injection of antiserum to dynorphin A In the presence of i.th. dynorphin Ad-i?) antiserum, the A50 values for the i.th., i.c.v. or combined routes of administration are 2.85 ± 1.20 pg, 2.07 ± 1.02 and 0.11 ± 0.037 ng, respectively. Similar results were obtained with the i.th. injection of MK- 801 (Fig.1-8). The A50 (± 95% C.L.) values are summarized in Table 2. The injection of either i.th. or i.c.v. morphine alone to Chung rats also produced a dose-dependent antinociception with A50 values of 2.3 ± 0.77 ng and 1.3 ± 0.56 ^ig, respectively. A 1:1 fixed ratio of i.th. and i.c.v. morphine also produced dose- dependent antinociception, but isobolographic analysis revealed the interaction to be additive in nerve-ligated rats (Fig. 1-9); the A50 value in total dose was 1.35 ± 0.46 ng (Table 3). The antinociceptive dose-response curve for i.th. morphine in the presence of antisemm to dynorphin A (Table 3). The combination of i.thVi.c.v. morphine produced a robust synergistic antinociceptive interaction and an A50 value of 0.072 ± 0.014 jig (Fig. I-10 and Table 3). 78 Similar results were seen in the presence of i.th. MK-80i(Fig. 1-11). The A50 (± 95% C.L.) values are summarized in Table 3. Neither dynorphin antiserum nor MK- 801 given alone was active against acute nociception in Chung rats. Dynorphin immunoreactivity was significantly (p < 0.05) elevated in the ipsilateral lumbar dorsal quadrant in Chung rats 10 days after Chung surgery (Figure 1- 5). compared to sham-operated and untreated animals. The dynorphin content of the ipsilateral dorsal lumbar quadrant was 1321 ± 189 pg/mg protein. The dynorphin content of sham-operated rats was 595 ±61 pg/mg protein, which was not significantly different (p > 0.05) from that of naive rats (593 ± 95 pg/mg protein). Additionally, dynorphin content was significantly (p < 0.05) increased in the sacral segment of the spinal cord of Chung rats compared to sham-operated rats. The dynorphin content of the sacral cord of Chung rats with was 943 ± 92 pg/mg protein and that of sham- operated rats was 746 ± 90 pg/mg protein. 79 Potency Ratio = 29. Adapted from: Yeung and Rudy JPET2I5 633-642, 1980 Total Morphine Dose ((ag) Figure 1-1. Concurrent i.th7i.c.v. administration of morphine in a 1:1 ratio produces a synergistic antinociceptive interaction in the 52°C tail-flick test in normal rats. 80 100 I.thVi.c.v 80 l.C.V. e •T3 O 60 •3 40 20 0 0.1 .3-^1 3 10 30 100 Total Morphine Dose (|ig) Figure 1-2. I.th. injection of morphine does not produce an antiallodynic effect up to 100 ng and the concurrent i.thVi.c.v. injection of morphine produces an additive antiallodynic effect in Chung rats (p < 0.05). 81 100 i.th. i.c.v. 80 i.thVi.c.v. u ^ 60 ^ 40 20 %3 .1 .3 1 3 10 30 Total Morphine Dose (fig) Figure 1-3. In the presence of i.th. antiserum (200 jig) to dynorphin A(l-17), i.th. injection of morphine produces a does-dependent antiallodynic effect and the concurrent i.th./i.c.v. injection of morphine pr(^uces a synergistic antiallodynic effect in Chung rats (p < 0.01). 82 100 .2 80 *T3 O 60 •3 I 40 20 c.v. 0 L 0.1 .3 1 10 30 Total Morphine Dose ((ig) Figure 1-4. In the presence of i.lh. MK-801 (3.4 ^g), i.th. injection of morphine produces a does-dependent antiallodynic effect and the concurrent i.th./i.c.v. injection of morphine produces a synergistic antiallodynic effect in Chung rats (p < 0.01). 83 1500 500 •>? Figure 1-5. Dynorphin content increases significantly in the ipsilateral dorsal quadrant of the spinal cord in both lumbar and sacral regions in Chung rats 10 days after Chung surgery (n = 8 - 20, *p <0.05). (Shown with permission of Dr. T. Philip Malan and Mr. Mohab Ibrahim at the University of Arizona, Department of Anesthesiology). 84 100 80 u a- 60 40 20 • i.th./i.c.v. 0 .03 .3 1 10 30 Morphine Dose (jig) Figure 1-6. Either i.th. or i.c.v. injection of morphine produces an antinociceptive effect in the 52°C tail-flick test and the concurrent i.thV i.c.v. injection of morphine produces a synergistic antinociceptive effect in the 52°C tail-flick test in sham-operated rats (p < 0.05). 85 100 80 ft- 60 40 l.C.V. i.th. 20 .03 30 Total Morphine Dose (fig) Figure 1-7. In the presence of i.th. antiserum (200 jig) to dynorphin A(l-17), i.th. injection of morphine produces a dose-dependent antinociceptive effect and the concurrent i.th./i.c.v. injection of morphine produces a synergistic antinociceptive effect in the 52°C tail-flick test in sham-operated rats (p < 0.01). The i.th. antiserum to dynorphin A(l-17) did not alter the antinociceptive potency of morphine given i.th., i.c.v. or in combination in sham-operated rats (p < 0.01). 86 100 80 u a. 60 40 l.C.V. 20 1.thy l.C.V 0_ .03 .3 I 3 10 30 Morphine Dose (fig) Figure 1-8. In the presence of i.th. MK-801 (3.4 ^g), i.th. injection of morphine produces an antinociceptive effect and the concurrent i.th./i.c.v. injection of morphine produces a synergistic antinociceptive effect in the 52°C tail-flick test in sham-operated rats (p < 0.01). The i.th. MK-801 did not alter the antinociceptive potency of morphine given i.th., i.e.v. or in combination in sham-operated rats (p < 0.01). 87 100 80 UJ O. 60 40 i.c.v. 20 i.thJi.c.v. Q .03 .1 .3 1 3 10 30 Total Morphine Dose (^g) Figure 1-9. Either i.th. or i.c.v. injection of morphine produces an antinociceptive effect in the 52°C tail-flick test and the concurrent i.th./i.c.v. injection of morphine produces only an additive antinociceptive effect in the 52°C tail-flick test in Chung rats (p < 0.01). 88 100 80 u I 60 i.th. ^ 40 20 0.01 .03 30 Total Morphine Dose (ng) Figure 1-10. In the presence of antiserum (200 (ig) to dynorphin (1-17), either i.th. or i.c.v. injection of morphine produces an antinociceptive effect and the concurrent i.th. /i.c.v. injection of morphine produces a synergistic antinociceptive effect in the 52°C tail-flick test in Chung rats (p < 0.01). 89 100 80 u I i.th. ^ 40i l.C.V. 20 .03 30 Total Morphine Dose (jig) Figure 1-11. In the presence of i.th. MK-801 (3.4 p,g), either i.th. or i.e.v. injection of morphine produces an antinociceptive effect and the concurrent i.th./i.c.v. injection of morphine produces a synergistic antinociceptive effect in the 52°C tail-flick test in Chung rats (p < 0.01). 90 Morphine alone + Dynorphin A1.17 + MK-801 (3.4 Mg, A50 (± 95% CL) Antiserum (200 MS' i.th.) i.th.) Aso (± 95% CL) A5o(±95%CL) Morphine i.th. Not Active (up to 11.6 (± 9.42) Mg 6.0 (± 3.9) Mg 100 Mg) Morphine i.c.v. 1.7 (± 1.1) Mg 2.7 (±0.89)Mg 1.41 (±0.42) Mg Morphine 1.6 (±2.7)Mg 0.24 (± 0.072) Mg 0.14 (± 0.03) Mg i.th./i.c.v. (Additive) (Synergistic) (Synergistic) (Total dose) Calculated 3.4 (± 2.1)Mg 4.36 (± 1.32) Mg 2.28 (± 0.63) Mg Additive Dose Table 1. Antiallodynic effect of morphine given alone and in the presence of i.th. antiserum to dynorphin Am? or MK-801 in Chung rats. Values are the A50 ± 95% C.L. derived from isobolographic analysis of the dose-response curves. A synergistic interaction is indicated when the experimental A50 for the i.th./i.c.v. combination is significantly less than the calculated theoretical additive value for the combination. N = 6 to 10 rats per group. 91 Morphine alone + Dynorphin A1.17 + MK-801 (3.4 Mg, A5O(±95%CL) Antiserum (200 pg, i.th.) i.th.) A5o(±95% CL) A5O(±95%CL) Morphine i.th. 1.45 (±0.42) ng 2.85 (± 1.20) ng 1.7 (±) 0.37 Mg Morphine i.e.v. 3.92 (±0.92) ng 2.07 (± 1.02) Mg 2.3 (±)0.48 Mg Morphine 0.33 (±0.13) Mg 0.11 (± 0.037) Mg 0.08 (±) 0.013 Mg i.th./i.c.v. (Synergistic) (Synergistic) (Synergistic) (Total dose) Calculated 2.12 (±0.46) ng 2.40 (± 0.76) Mg 1.9 (±)0.30 Mg Additive Dose Table 2. Antinociceptive effect of morphine in the tail-flick test when given alone and in the presence of i.th. antiserum to dynorphin Ai-n or MK-801 in sham-operated rats. Values are the A50 ± 95% C.L. derived from isobolographic analysis of the dose- response curves. A synergistic interaction is indicated when the experimental A50 for the i.th./i.c.v. combination is significantly less than the calculated theoretical additive value for the combination. N = 6 to 10 rats per group. 92 Morphine alone + Dynorphin Ai.i? + MK-801 (3.4 A5O(±95% CL) Antiserum (200 pg, Mg, i.th.) i.th.)A5o (± 95% CL) A5O(±95%CL) Morphine i.th. 2.3 (±0.77) ^g 1.27 (± 0.28) Mg 0.21 (±0.05) Mg Morphine i.e.v. 1.3 (±0.56) ng 2.26 (± 0.58) ^ig 1.7 (± 0.4) ng Morphine 1.35 (± 0.46) ^lg 0.072 (± 0.014) ng 0.068 (± 0.02) ng i.th./i.c.v. (Additive) (Synergistic) (Synergistic) (Total dose) Calculated 1.67 (±0.45) Mg 1.62 (± 0.27) fig 0.37 (±0.08) ng Additive Dose Table 3. Antinociceptive effect of morphine in the tail-flick test when given alone and in the presence of i.th. antiserum to dynorphin Am? or MK-801 in Chung rats. Values are the A50 ± 95% C.L. derived from isobolographic analysis of the dose-response curves. A synergistic interaction is indicated when the experimental A50 for the i.th./i.c.v. combination is significantly less than the calculated theoretical additive value for the combination. N = 6 to 10 rats per group. 93 Summery 1 In sham-operated rats, antinociceptive sjrnergy occurred between supraspinal/spinal morphine (approximately 30-fold increase) consistent with previous reports (Miyamoto et al., 1991; Yeung et al., 1980). In contrast, in Chung rats spinal/supraspinal morphine interaction against tactile allodynia or thermal nociception was additive. The loss of synergy may be responsible, in part, for the reported inability of morphine to provide adequate pain relief in neuropathic pain clinically. As the synergy was restored in Chung rats by dynorphind-i?) antiserum or by MK-801, which had no effects alone or in sham-operated rats, the loss of synergy may be due to the actions of elevated levels of spinal dynorphin acting directly or indirectly via NMDA receptors to produce a spinal sensitization. Sham-operated rats do not demonstrate tactile allodynia and i.th. morphine does affect the response to innocuous stimulation, preventing evaluation of "antiallodynic" morphine activity in non-injured rats. Supraspinal/spinal morphine antinociceptive synergy was suggested by the failure of either i.th. or i.c.v. naloxone to completely block systemic morphine antinociception (Yeung et al., 1980). Administration of a 1:1 fixed ratio of supraspinal/spinal morphine increased antinociceptive potency by about 30-fold compared to i.c.v. morphine alone (Yueng et al., 1980). Isobolographic analysis with multiple dose ratios resulted in the conclusion that synergy occurred at all spinal/supraspinal combinations of morphine which might be achieved after systemic injection. This observation has been extended to mice (Roering et al., 1988), and includes selective ^-agonists (Roering et al., 1989). Evidence that synergistic 94 antincx:iceptive activity of morphine is the resuh of site-site interaction was confirmed when synergy was determined based on spinal and supraspinal morphine content, rather than administered doses (Miyamoto et al., 1991). It is believed that the morphine synergy may be responsible for the clinical effectiveness of systemic morphine, which is greater than predicted based on its in vitro profile. The loss of activity of spinal morphine in Chung rats was shown here by the failure to block tactile allodynia in spite of dose-related antiallodynic actions seen with systemic or i.e.v. administration (Bian et al., 1995; Lee et al., 1995). Injury to lumbar afferents in Chung rats produced an unexpected loss of i.th. morphine activity against thermal nociception applied to the tail, which is ex-territory region of injured nerves of Chung rats. As afferent fibers from the paw and the tail respectively terminate in lumbar and sacral spinal cord without significant overlaps (Swett et al., 1991), multisegmental changes of neurotransmitters such as dynorphin levels in the spinal cord may occur in Chung model rats. Together with the finding that supraspinal morphine antinociception is not significantly altered by nerve injury, our results suggest that decreased spinal morphine antinociception leads to the loss of spinal/supraspinal synergistic antinociception after peripheral nerve injury. Further, increased dynorphin levels across multiple spinal segments may underlie the loss of morphine activity (Ibrahim et al., 1998). This hypothesis is supported by the present data indicating a significant elevation in the ipsilateral dorsal lumbar quadrant and sacral levels of dynorphin after injury to lumbar spinal nerves. Consistent with this hypothesis, a binding site for dynorphin on the NMDA receptor has been identified which appears. 95 however, to modulate the NMDA site negatively (Lai et al., 1998). Studies have shown dynorphin-mediated inhibition of NMDA current occurring at micromolar concentrations, suggesting that this inhibitory effect may not be physiologically relevant (Chen et al., 1995). However, such concentrations might be achieved at the synaptic junction (Chen et al., 1998). Some studies in vitro have demonstrated excitatory effects of dynorphin A(2-i7) which is unmasked by low glycine concentrations in cells of the periaqueductal gray (PAG) (Zhang et al., 1997). While dynorphin/NMDA interactions in vitro have generally suggested an inhibitory modulation (Chen et al., 1995; Lai et al., 1997), in vivo data support functional agonistic actions at the spinal cord. Whether the effects observed are due to a direct or indirect interaction of dynorphin or its fragments at the NMDA receptor is not known. However, considerable in vivo evidence exists to indicate that spinal dynorphin may act as a "functional" NMDA agonist. Both i.th. dynorphin A(i.i3) and dynorphin fragments that do not interact with opioid receptors produce hindlimb paralysis and depletion of neuronal cell bodies of intemeurons and sensory and motor neurons in the lumbosacral spinal cord (Faden et al., 1984; Przewlocki et al., 1983; Stewart et al., 1986; Long et al., 1988). Protection against dynorphin-induced hindlimb paralysis, loss of tail-flick reflex, or loss of neuronal cell bodies by either NMDA antagonists that modulate the NMDA-glycine receptor complex has been demonstrated (Bakshi et al., 1990; Bakshi et al., 1995; Long et al., 1994; Long et al., 1995). Such observations are consistent with an NMDA mediated action of dynorphin or its fragments. Further evidence that spinal dynorphin in conditions of nerve injury acts functionally as an NMDA agonist is provided by the 96 observations that both MK-801 and antiserum to dynorphin Ad-i?) when given i.th. restores the antinociceptive and antiallodynic action of spinal morphine (Faden et ai., 1990; Nichols et al., 1997;). Furthermore, both MK-801 and antiserum to dynorphin A{i.i7) given spinally reverse thermal hyperalgesia in Chung rats (Wegert et al., 1997). Evidence has shown that pre-emptive antagonism of dynorphin with a dynorphin antibody or MK-801 did not block the tactile allodynia associated with nerve injury, but did alter the expected neurochemical changes associated with sciatic nerve injury (Wagner et al., 1996). These observations are consistent with the observed lack of antiallodynic actions of i.th. antiserum to dynorphin when given alone. Here, we demonstrate that both i.th. antiserum to dynorphin A(i.i7) and MK-801 restore the i.th. antiallodynic and antinociceptive activity of morphine in Chung model rats, and more importantly, that both treatments also restore the morphine spinal/supraspinal synergy. It should be emphasized that the i.th. doses of antiserum to dynorphin A(i.j7) or of MK- 801 used in this experiment did not produce antinociceptive or antiallodynic effects when given alone. The significance of the loss of spinal/supraspinal synergy of morphine in Chung rats from this experiment may help explain its observed decreased activity against neuropathic pain in the clinic (Amer et al., 1988). Convergent evidence indicates that spinal dynorphin is associated with neuropathic pain states and the loss of activity of morphine. A. role in chronic pain is indicated by observations that spinal dynorphin is elevated after hindpaw inflammation (Riley et al., 1996). In situ hybridization revealed increased prodynorphin mRNA in laminae I, II, V and VI after peripheral inflammation (Ruda et al., 1996). Dubner and 97 Ruda (1992) demonstrated elevated spinal dynorphin mRNA after nerve injury. Immunocytochemistry revealed dynorphin in both lamina I projection and intemeurons, suggesting both local spinal and supraspinal functions of dynorphin (Lima et al., 1993). We have reported elevated dynorphin levels in the ipsilateral. dorsal quadrant of the spinal cord from the rostral lumbar to caudal sacral regions in Chung model rats (Ibrahim et al., 1998), fmdings corroborated here. Likewise, St nerve injury produced a significant increase in the lumbar dynorphin expression, associated with tactile allodynia and thermal hyperalgesia of the paw, again supporting the concept of multisegmental dynorphin changes following nerve injury (Ibrahim et al., 1998). It was shown that the i.th. injection of dynorphin A hyperalgesia in rats (Vanderah et al., 1996). These neuropathic symptoms were completely blocked by pretreatment with MK-801, but not by that with naloxone, indicating a non-opioid, NMDA-mediated action of dynorphin to elicit neuropathic pain symptoms (Vanderah et al., 1996). In conclusion. Study 1 demonstrates that the Chung model of peripheral neuropathy produces a complete loss of activity of i.th. morphine against tactile allodynia and a significant loss of efficacy against an acute thermal nociceptive stimulus. Critically, this loss of spinal activity correlates with a loss of spinal/supraspinal synergy of morphine. The spinal injection of antiserum to dynorphin A(i.i7) produces virtually identical effects to spinal MK-801. Neither substance alone blocks tactile allodynia, but both elicit a reversal of the loss of activity of spinal 98 morphine and reestablish the spinal/supraspinal synergy of morphine. Based on these and earlier observations discussed above, we suggest that peripheral nerve injury results in elevated levels of dynorphin in the spinal cord. The pathological role of elevated levels of spinal dynorphin in neuropathic pain states is most likely due to interactions with the NMDA receptor complex to cause central sensitization. This effect in turn leads to a loss of spinal morphine activity resulting in a loss of the spinal/supraspinal synergy of morphine. It is this interaction that is suspected to provide for the remarkable clinical analgesic effect of morphine, and its loss explains the relative lack of utility of morphine clinically against neuropathic pain symptoms. 99 Study 2 Tactile allodynia, but not thermal hyperalgesia, of the hindpaw is blocked by spinal transection in Chung model rats Experimental nerve injury of the Chung model produces consistent signs of allodynia and thermal hyperalgesia, which provides an animal model for some aspects of clinical neuropathic pain symptoms. Peripheral nerve injury is associated with a number of neuroanatomical changes, including the apparent formation of novel sjTiapses between myelinated large diameter, low-threshold primary afferent neurons and dorsal hom units normally transmitting nociceptive input, and development of sensitization of dorsal hom neurons (Woolf et al., 1992, 1994; Davis et al., 1987; Mao et al., 1992). Spinal levels of endogenous peptides such as dynorphin, enkephalins, and cholecystokinin (CCK) are elevated in neuropathic pain states (Stanfa et al., 1994; Xu et al., 1993). The chronic constriction model (CCI) has demonstrated a reduced anti- hyperalgesic effect of i.th. morphine and the Chung model has led to a virtually complete absence of antiallodynic activity of i.th. morphine (Mao et al., 1995; Bian et al., 1995; Chaplan et al., 1994; Lee et al., 1995). Additionally, the Chung model produced a loss of potency and efficacy of i.th. morphine to suppress an acute nociceptive stimulus applied to the tail (Ossipov et al., 1995). While spinal changes resultant from peripheral nerve injury are well investigated, there is a paucity of research into the supraspinal processes that may be involved in the development of allodynia and hyperalgesia. A strong suggestion of 100 supraspinal influence on hyperesthetic states, especially tactile allodynia, is found in observations that supramaximal doses of i.th. morphine are inactive against tactile allodynia, yet it is relatively active when administered either systemically or supraspinally (Bian et al., 1995; Lee et al., 1995). It is known that spinal nociceptive input involves ascending transmission of nociceptive information and is under modulatory control from supraspinal sites (Gebhart, 1982; Mayer et al., 1976; Rady et al., 1998). Spinopetal modulation of nociceptive responses arises from several sites in the brain. Electrical stimulation of, or microinjection of opioids into, various supraspinal loci, including the periaqueductal gray (PAG), the lateral reticular nucleus (LRN), and the medullary raphe nuclei (MRN) all produce an antinociceptive effect to noxious heat stimuli (Mayer et al., 1976; Carstens et al., 1995; Sandkuhler et al., 1984). Reversible spinal block produced a significant reduction in firing threshold of dorsal horn neurons to thermal stimulation of the tail, indicating a tonic descending inhibitory control (Necker et al., 1978). Finally, spinal transection facilitates the nociceptive tail flick reflex in rats (Advokat et al., 1987; Ghorpade et al., 1994). It is therefore reasonable to hypothesize that neuropathic pain symptoms due to peripheral nerve injury involve supraspinal, as well as segmental, neural circuits and that descending modulatory pathways will differentially affect neuropathic pain symptoms. In order to test this hypothesis, the involvement of ascending and descending pathways will be assessed by determining the relative importance of segmental spinal circuitry and of supraspinal loops in modulating nerve-injury related 101 tactile allodynia and thermal hyperalgesia using spinal cord transection. The possibility of tonic descending modulatory control of nerve-injury related responses would be explored by comparing baseline thermal responses of rats with Chung or sham- operation as well as baseline thermal responses of Chung rats with intact or transected spinal cords. Experimental Procedure A spinal transection surgery was performed in the thorathic region of the spinal cord in Chung and sham-operated rats twenty-four hours before testing. Mechanical allodynia of ipsilateral paw in Chung rats was determined by measuring paw withdrawal threshold in response to probing with von Frey filaments. Paw withdrawal latencies in warm water baths that maintained at six different temperature settings of 46, 47, 48, 49, 50 and 52°C were determined for each group of rats. A cut-off time for this paw withdrawal test was set to 10 seconds to avoid tissue injury. Mean withdrawal latencies were compared among groups and between ipsilateral and contralateral paws by analysis of variance followed by post-hoc analysis of least significant difference for multiple comparisons. Withdrawal latencies significantly less than those of intact, sham-operated groups were interpreted as hyperalgesia. Tail flick thresholds to mechanical stimulation were determined by probing with von Frey filaments. Mean tail flick thresholds were compared among intact and 102 transected groups. Tail flick thresholds significantly less than those of intact, sham- operated group were interpreted as hyperreflexia. Results Tactile allodynia and/or mechanical hyperreflexia: The cut-off value of paw withdrawal thresholds to probing with von Frey filaments was 15 grams. Mean response thresholds of sham-operated rats with either intact or transected spinal cords to probing with von Frey filaments were 15 ± 0 g. Chung rats with intact spinal cords exhibited mean ipsilateral paw withdrawal thresholds of 1.59 ± 0.36 g. Chung rats with spinal cord transections did not demonstrate any tactile allodynia. Mean response thresholds were 15 ± 0 g The lack of allodynic response in ipsilateral hindpaw in Chung rats was not due to the paralysis of hindpaw since a toe pinch produced a vigorous flexor reflex response in all spinally transected rats. The vigorous hyperreflexia of the tail that accompanies spinal cord transection precluded any accurate determination of tail flick latencies. Neither Chung nor sham-operated rats with intact spinal cords showed responses to probing the tail with von Frey filaments up to the cut-off value of 15 grams force. Spinal cord transections were accompanied by classic facilitation of flexor responses, with very robust reflexive responses of the tail and significantly different tail flick thresholds of 1.65 ± 0.1 g and 2.88 ± 0.37 g in Chung and sham-operated rats respectively (Fig. 2-1). 103 Thermal hyperalgesia Both Chung and sham-operated rats demonstrated an inverse relationship between water bath temperature and paw withdrawal latency. Ipsilateral paw withdrawal latencies of sham-operated rats with intact spinal cords ranged from 9.8 ± 0.26 sec at 46°C to 1.1 ±0.15 sec at 52°C, whereas those with transected spinal cords ranged from 10 ± 0 sec to 2.3 ± 0.52 sec (Fig. 2-2). The ipsilateral paw withdrawal latencies for sham-operated rats were significantly longer after spinal cord transection at all bath temperatures except 46°C (Fig. 2-2). Ipsilateral paw withdrawal latencies of Chung rats with intact spinal cords ranged from 9.8 ± 0.27 sec at 46°C to 1.2 ± 0.14 sec at 52°C, whereas those with transected spinal cords ranged from 10 ± 0 sec to 2.3 ± 0.52 sec (Fig. 2-3). The withdrawal latencies were significantly longer after spinal cord transection at ail bath temperatures except 46°C and 52°C (Fig. 2-4), but there was no significant difference between Chung and sham-operated rats. The withdrawal latencies of the contralateral paws were not different from ipsilateral ones between sham-operated and Chung rats either before or after spinal cord transection (Fig. 2-5 and Fig. 2-6). 104 Chung Sham-operated n = 6 **p<0.01 Intact Transected Figure 2-1. Allodynia of ipsilateral paw in Chung rats is abolished by spinal transection. 105 I I Sham-operated Chung n = 6 **p<0.01 Intact Transected Figure 2-2. Spinal transection produces a tail hyperreflexia to mechanical stimuli in Chung and sham-operated rats. The hyperreflexia in Chung rats is significantly higher than in sham-operated ones. Intact Transected n = 6 '=p<0.01 47 48 49 50 51 Water Bath Temperature (°C) Figure 2-3. Spinal transection increases ipsilateral paw withdrawal latencies of sham- operated rats to thermal stimuli in warm water at all temperature settings except 46°C. Intact Transected n= 6 '=p<0.01 Water Bath Temperature (°C) Figure 2-4. Spinal transection increases ipsilateral paw withdrawal latencies of Chung rats to thermal stimuli in warm water at all temperature settings except 46°C and 52°C. 108 Sham left Sham right g '^10- Chung left J r Chung right 1^ S e 5- I 0- —I— —I— —I— —I— —I— —I 46 47 48 49 50 52 Water Bath Temperature (°C) Figure 2-5. There is no significant difference between ipsilateral and contralateral paw withdrawal latencies to thermal stimuli in warm water at all temperature settings in Chung and sham-operated rats before spinal transections. 109 Sham left Sham right Chung left I '3^10- Chung right -> r n = 6 IR- I Ol. 0- —I— —I— —I 46 47 48 49 50 52 Water Bath Temperature (°C) Figure 2-6. There is no significant difference between ipsilateral and contralateral paw withdrawal latencies to thermal stimuli in warm water at all temperature settings in Chung and sham-operated rats after spinal transection. no Summary 2 The data presented here strongly suggest the importance of supraspinal processing in interpretation of normally non-noxious tactile stimuli as nociceptive. This suggestion is based on the observation that transection of the spinal cord completely abolishes tactile allodynic responses. If spinal processes alone were responsible for the enhanced tactile response after nerve injury, then transection would be expected to further enhance, rather than eliminate this response because of the loss of known tonic descending inhibitory controls (Advokat et al., 1987; Ghorpade et al., 1994; Villannueva et al., 1986). The fact that the nociceptive toe-pinch still produces a vigorous flexor reflex indicates that the lack of response to mild tactile stimuli is not due to paralysis of the hindlimb, and that spinal reflexes are intact. Tactile allodynia after peripheral tissue injury is preserved in decerebrate rats suggesting that at least subcortical structures are involved in processing some manifestations of chronic pain (Woolfetal., 1984). It is paradoxical that spinal cord transection inhibits paw-withdrawal reflexes, but facilitates the tail-flick reflex. In a different study, the application of mustard oil to a hindlimb produced tactile allodynia that also was abolished by spinalization or by the application of lidocaine to the MRN or LRN of intact rats (Mansikka et al., 1997). Spinalization also facilitated the tail flick reflex to radiant heat but raised hindpaw thresholds to mechanical stimuli in inflamed and non-inflamed hindpaws (Mansikka et al., 1997). Our present results extend these observations to include elevated thresholds to noxious heating of the hindpaw after spinalization, and agree with the conclusion that Ill there are differentia] descending tonic controls governing the tail and the hindpaw (Mansikka et al.. 1997). Since there are descending facilitatory and descending inhibitory pathways (Urban et al., 1996; Zhuo et al., 1992), it is possible that each of these opposing systems have a different degree of effect on either the tail or hindpaw. Also interesting is the fact that the tail demonstrates hyper-responsiveness after spinal transection in both Chung and sham-operated rats. This hyperreflexia is well documented and is generally believed to be the result of a loss of a tonic descending modulation on spinal reflexes originating from supraspinal sites. Reversible cold block of the spinal cord produces an increase in firing rate of dorsal horn neurons in response to noxious heating of the tail (Necker et al., 1978). Although the hindpaws also demonstrate robust reflexes to a nociceptive pinch, responses to nociceptive thermal stimuli or to normally innocuous tactile stimuli are lost after spinal transection. These observations suggest the possibility that different mechanisms govern responses of the tail and of the hindpaws (Necker et al., 1978). In this regard, evidence exists that different descending systems involving different neurotransmitters modulate the paws and tail (Fang et al., 1996). Another interesting observation is that thermally-evoked paw withdrawal latencies of Chung rats do not differ from those of sham-operated control rats. These results contrast with an earlier report that Chung model rats have a significantly lowered ipsilateral hindpaw response latency to radiant heat stimuli (Wegert et al., 1997). The explanation is that there is a fundamental difference between the two studies, however, in that the present study utilized water baths at constant temperatures (baseline latencies 112 from 2 to 10 sec) whereas the earlier study utilized a radiant heat source (baseline latencies of 16 sec). The radiant heat assay previously reported to demonstrate thermal hyperalgesia of the paw produced longer response latencies which correspond to an average rate of rise of l.l°C/sec (Wegert et al., 1997). Studies indicated that a slow (i.e. 0.9°C/sec) rate of rise is associated with selective activation of C, rather than AP—fibers (Yeumans et al., 1996). In contrast, a fast rate of heating (i.e. 6.5°C/sec) is associated with activation of myelinated nociceptors (Yeumans et al., 1996; Zacharious et al., 1997), a finding which may correlate well with the short response latencies found in the hot-water bath procedure of this experiment. An estimation of rate of rise can be obtained based on some assumptions and with the available data. For example, the average paw withdrawal latency at 48°C water bath is about 5 sec, and the ambient (and presumably the skin) temperature is 20°C. If the paw withdrawal occurs when the skin temperature reaches 48°C, then an approximate rate of rise of 5.6°C/sec is estimated; and higher bath temperatures are associated with faster rates. The present observations support the possibility of differential thermal activation of A5- and c-fibers with regard to nociceptive processing in neuropathic pain states. In conclusion, our data in Study 2 suggest that tactile allodynia resulting from peripheral nerve injury involves activation of supraspinal processes. It is uncertain as yet whether the changes in nociceptive processing of the tactile input are associated with alterations in ascending information, at the level of processing in the midbrain, or involve alterations in descending modulatory tone, both inhibitory and facilitatory. The present study extends the suggestion that reflexes of the hindpaws involve different 113 control mechanisms than the tail since spinalization facilitates the tail flick reflex but inhibits hindpaw reflexes. Control of hindpaw responses appears to involve both spinal and supraspinal processing of the nociceptive input. Finally, thermal activation of peripheral nociceptors in the state of peripheral nerve injury apparently produces differing results, depending on the type of thermal noxious stimulus applied. 114 Study 3 Lack of involvement of capsaicin-sensitive primary afferents in tactile allodynia in Chung model rats Non-noxious tactile stimulation is thought to be nomially transmitted chiefly through low-threshold, large diameter, myelinated Aji-fibers, while noxious thermal stimuli are transmitted to the spinal cord through high-threshold, thin, unmyelinated c- fibers and thinly myelinated A5-fibers (Yeomans et al., 1996). The large diameter, low threshold AP-fibers normally terminate in lamina III of the dorsal horn. Evidence has shown that after peripheral nerve injury the central terminals of AP-fibers sprout into the superficial laminae (i.e., lamina I and II) and may form novel pathophysiological synapses with transmission neurons (Lekan et al., 1997; Woolf et al., 1995). These second order transmission neurons, which normally code for nociceptive input, now receive non-noxious input from the low threshold fibers and in this way, innocuous tactile input might be interpreted as nociceptive. Evidence has shown that capsaicin-sensitive c-fibers are involved in the development of thermal hyperalgesia in the chronic constriction injury model (CCI) of neuropathic pain (Meller et al., 1992), while mechanical hyperesthesias were mediated by A-fibers in the Seltzer model (Shir and Seltzer, 1990). Kinnman et al., reported an attenuation of allodynic responses in neonatally capsaicin treated rats with L5 spinal nerve transection (1995). Their results contradicted the hypothesis that mechanical allodynia is mediated by large AP fibers in neuropathic pain states. !15 Spontaneously consistent discharges from sprouts and ectopic foci of injured peripheral nerves have been proposed to be the major cause of central sensitization (Laird, 1992). The consistent discharges from ectopic foci of injured peripheral nerves can cause excess release of excitatory amino acids such as glutamate in the central terminals of nociceptive neurons (c-fibers). Evidence has demonstrated glutamate release from c-fibers in the dorsal horn of the spinal cord and subsequent activation of NMDA receptors mediate central sensitization which may be responsible for allodynia and thermal hyperalgesia (Yamamoto et al., 1993). There is a correlation between ectopic discharge and behavioral signs of neuropathic pain (Han et al., 1995). Binding studies with [^H]-resiniferatoxin (RTX) and the identification of a competitive antagonist (capsazepine) provide evidence for a specific vanilloid receptor for capsaicin (Szallasi and Blumberg, 1996). The vanilloid receptor agonist capsaicin and its ultrapotent analog RTX have been used as selective tools to study the action of nociceptive sensory neurons. A [^H]-RTX binding autoradiographic study has revealed the specific distributions of vanUIoid receptors. Vanilloid receptor binding sites were detected by [^H]-RTX in somatic (trigeminal and dorsal root of the spinal cord) sensory ganglia (DRGs), peripheral nerves (sciatic nerve), dorsal horn of the spinal cord, as well as in nuclei of the CNS receiving sensory input (i.e. graciles nucleus) (Szallasi et al., 1995). Expression cloning strategies have led to the identification and expression of a cation channel mainly for Ca^, designated as the vanilloid receptor or VR1 (Caterina et al., 1997). Co-operativity shown in binding studies with normal tissue lead to speculation that more than one subtype of vanilloid receptor may exist. However, since 116 co-operactivity is also observed with the cloned VRl, the previously proposed subtypes are not due to multi-type genes; the possibility of functional subtypes composed of different configurations of VRl can not be rule out (Biro et al., 1997). Recently, a second vanilloid receptor, structurally related to VRl, but which does not respond to capsaicin or acids and with its own gene sequence, was reported, indicating the discovery of a molecular vanilloid receptor subtype, called (vanilloid-receptor-like protein 1) VRL-1 (Caterina et al., 1999). Earlier data have shown that VRl was expressed almost exclusively in afferent c-fibers involved with thermal nociceptive transmission and neurogenic inflammation (Szallasi and Blumberg, 1996). Recent evidence of immunocytochemical analysis of VRl and VRl mRNA distribution by using VRl antibody and is situ hybridization indicates that VRl is located in a neurochemically heterogeneous population of small-to-medium-sized diameter primary afferent fibers (Tominaga et al., 1998; Helliwell et al., 1998). Vanilloids such as capsaicin and RTX produce an initial activation of primary sensory neurons at low doses, which gives rise to the pungency and irritation. The short initial activation of the neurons is then followed by a long-lasting desensitization; this may be due to Ca^ accumulation. At high doses, irreversible long-lasting desensitization ultimately impairs the neuron's function and produces neuronal death due to neurotoxicity (Szallasi and Blumberg, 1996). RTX differs from capsaicin in that its initial nociceptive action is about equipotent to capsaicin but its potency to produce desensitization is several thousand-fold greater than capsaicin (Szallasi et al., 1989). Further, desensitization caused by RTX is of much longer duration than that of 117 capsaicin. Szallasi and colleagues (1989) reported that RTX was more efficacious than capsaicin, producing a greater degree of desensitization that was more rapid in onset and significantly more persistent against chemically induced nociception. The absence of sensitivity to chemical challenge after initial capsaicin or RTX exposure is attributed to a long-lasting desensitization of small diameter, unmyelinated nociceptive afferent c- fibers (Szallasi et al., 1989). Likewise, RTX also produced a long-lasting reduction in c-fiber mediated nociception in a model of visceral pain (Craft et al., 1995). In a comprehensive electrophysiological and behavioral study, the evidence has shown that RTX produces a long lasting irreversible (> 4 weeks) thermal hypoalgesia but does not alter nociceptive mechanical thresholds in normal rats. Wind-up induced by conditioning stimuli of c-fibers is attenuated by RTX and reflects RTX-induced desensitization of c-fibers (Xu et al., 1997). These studies support the use of RTX as a means of selectively depleting c-fiber primary afferents. The long-lasting desensitization and the depletion of c-fibers to further noxious stimuli make RTX the agent of choice for use in the study of the involvement of c-fibers in neuropathic pain states. For these reasons, RTX was selected in this study as a means to selectively desensitizing primary afferent c-fibers in order to explore the relative contributions of c- fibers to central sensitization and behavioral signs of neuropathic pain in Chung model rats (Kim and Chung et al., 1992). Study 3 will determine the involvement of capsaicin-sensitive c-fibers in behavioral responses to thermal, as well as mechanical, stimuli in the Chung model of neuropathic pain. 118 Experimental Procedure RTX administration (0.1 mg/kg i.p. or 0.3 mg/kg s.c) was performed under 2.5% halothane. After RTX administration rats were kept in 1.5 % halothane for half an hour. Tactile allodynia was determined by measuring the paw withdrawal threshold in response to probing with von Frey filaments. Thermal hyperalgesia was assessed in the Hargreaves' hot box (Hargeaves et al., 1988). Rats were allowed to acclimate in plexiglass enclosures on a clear glass plate maintained at 30°C. The heat intensity of the radiant heat source (i.e., high intensity projector lamp) was adjusted to distinguish thermal nociception and thermal hyperalgesia. The testing heat source was activated with a timer and focused onto the plantar surface of the affected paw of Chung or sham-operated rats. The paw withdrawal from the glass surface triggers a photocell that halts both lamp and timer. A maximal cut-off of 40 sec was used to prevent tissue damage. Thermal nociceptive testing was performed in both Chung and sham-operated rats by the warm water tail flick test. The hot plate test was performed on a thermostatically controlled metal plate maintained at 52°C. The latency to licking of hindpaw or jumping was taken as the thermal nociceptive endpoint at which time rats were removed from the plate. Data Analysis: In all tests, baseline data were obtained 1 hour before RTX injection (0.1 mg/kg i.p. or 0.3 mg/kg s.c.). Rats were tested at a 5-day interval for 40 days after RTX administration. Within each of the treatment groups, post-injection values were compared to the baseline values by analysis of variance (ANOVA), 119 followed by post-hoc analysis of "least significant difference for multiple comparisons". Comparisons between two means were performed by paired Student's t-test. A probability level of 0.05 indicated significance. Results The mean paw withdrawal thresholds of rats to probing with von Frey filaments of less than 5 g force would be considered as tactile allodynia. Sham-operated rats did not show allodynic responses to probing with von Frey filaments up to the maximal cut off of 15 g force either before or 4 days' after surgery throughout the entire 40-day observations, while Chung rats showed tactile allodynia in the ipsilateral paw 4 days after surgery. The ipsilateral paw withdrawal thresholds of Chung rats to probing with von Frey filaments varied from 1.5 ± 0.3 g to 4.6 ± 2.5 g from day 5 to day 40 after surgery. These values were significantly less than those of non RTX-treated sham-operated rats (15 ±0 g), indicating the presence of tactile allodynia in non RTX-treated Chung rats during the entire 40-day observation period (Fig.3-1). The ipsilateral withdrawal thresholds to probing with von Frey filaments in Chung rats remained in the range of tactile allodynia (less than 5 g) during the 40-day observation period after s.c. or i.p. RTX (Fig. 3-5 and Fig.3-9). RTX treatment (s.c. or i.p.) did not change ipsilateral paw withdrawal thresholds to probing with von Frey filaments in Chung rats significantly at each time point within the 40-day observation period compared to Chung rats without RTX treatment, indicating consistent tactile 120 allodynia. Allodynic responses does not differ significantly between RTX-treated and non RTX-treated Chung rats in 40-day observation period (Fig. 3-15). The ipsilateral paw withdrawal thresholds to von Frey filaments decreased significantly in rats after Chung surgery but not sham surgery with pre-RTX treatment and the lower paw withdrawal threshold in Chung rats remained up to 15 days of the observation period (Fig.3-I3). The baselines of ipsilateral paw withdrawal latencies of Chung and sham- operated rats to radiant heat were not significantly different. However, after surgery, Chung rats showed significantly lower paw withdrawal latencies at a 5 day interval compared with same day sham-operated rats up to 40 days indicating a long-lasting thermal hyperalgesia in the ipsilateral paw of Chung rats (Fig.3-2). Chung and sham-operated rats did not show a significant difference in tail and paw withdrawal latencies in the 52°C tail-flick test and hot-plate test respectively, before and up to 40 days after surgery (Fig. 3-3 and 3-4). The ipsilateral paw withdrawal thresholds to radiant heat in Chung and sham- operated rats were significantly increased after s.c. or i.p. RTX injection and remained significantly elevated throughout 40-day observation period (Fig. 3-6. and Fig. 3-10). The tail flick and paw withdrawal latencies in the 52° C tail-flick test and hot plate test in Chung and sham-operated rats were increased significantly after s.c. or i.p. RTX injection and remained at higher threshold levels up to 40-day of the observation period (Fig. 3-7, Fig. 3-8, Fig. 3-11 and Fig. 3-12). 121 The 52° C tail flick latencies were significantly increased after RTX injection and remained at higher threshold levels up to day 40 of the observation period in Chung and sham-operated rats. There was no significant difference between the 52° C tail flick latencies of Chung and sham-operated rats in the entire observation period (Fig. 3-14). 122 15- 2o 12- J= Chung Sham n = 6 Base 5 10 15 20 25 30 35 40 Time (Days After Surgery) Figure 3-1. Allodynia time course in Chung rats compared to sham-operated rats. The ipsilateral paw withdrawal threshold in Chung rats is less than 5 grams to probing with calibrated von Frey filaments, indicative of tactile allodynia. The same side paw withdrawal threshold in sham-operated rats is at the cut-off value of 15 grams, indicative of no allodynia. The allodynia in Chung rats lasts in the entire 40-day observation period. 123 40-1 30- ou 20- Chung p < 0.01 10- Sham n = 6 Base 5 10 15 20 25 30 35 40 Time (Days After Surgery) Figure 3-2. Thermal hyperalgesia time course in Chung rats. The ipsilateral paw withdrawal latency in Chung rats is shorter than that in sham-operated rats, indicative of thermal hyperalgesia. A cut-off time of 40 seconds is used to avoid tissue injury. The hyperalgesia in Chung rats lasts the entire 40-day observation period. 124 5n w 3- Chung Sham n = 6 Base 5 10 15 20 25 30 35 40 Time (Days After Surgery) Figure 3-3. Thermal ncx:iceptive response time course in Chung and sham-operated rats in the 52°C warm water tail-flick test. There is no significant difference between tail flick latencies of Chung and sham-operated rats during the entire 40-day observation period. A cut-off time of 10 seconds is used to avoid tissue injury. 125 40-1 30- SS 20- 10- Chung Sham n = 6 Base 5 10 15 20 25 30 35 40 Time (Days After Sugery) Figure 3-4. Thermal nociceptive response time course in Chung and sham-operated rats in the 52°C hot plate test. There is no significant difference between paw withdrawal latencies of Chung and sham-of)erated rats during the entire 40-day observation period. A cut-off time of 40 seconds is used to avoid tissue injury. 126 15- oooooo Sham 12- Chung 9- n = 6 T3 6- 3- Base 5 10 15 20 25 30 35 40 Time (Days After RTX ) Figure 3-5. A single dose of RTX (0.1 mg/kg i.p.) does not reverse allodynia in Chung rats during the entire 40-day observation period. 127 40-1 30H —o—Sham —•—Chung n = 6 ••p<0.01 loH a. Base 5 10 15 20 25 30 35 40 Time (Days After RTX) Figure 3-6. A single dose of RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the radiant heat paw withdrawal test in Chung and sham-operated rats over the entire 40-day observation period. There is thermal hyperalgesia in Chung rats compared to sham-operated ones before RTX administration (Base). 128 10- u v:u >v cU wa 5H u Sham a —1— —I— -I— —r- Base 10 15 20 25 30 35 40 Time (Days After RTX) Figure 3-7. A single dose of RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the 52°C warm water tail flick test in Chung and sham-operated rats over the entire 40-day observation period. 129 40-1 •§•< 30- Sham Chung n = 6 10- cu Base 5 10 15 20 25 30 35 40 Time (Days After RTX) Figure 3-8. A single dose of RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the 52°C hot plate test in Chung and sham-operated rats over the entire 40-day observation period. 130 15-|oo 12- Sham 9- Chung n = 6 6- 3- 4 0 T 1 1 1 1 1 1 1 r- Base 5 10 15 20 25 30 35 40 Time (Days after RTX) Figure 3-9. A single dose of RTX (0.3 mg/kg s.c.) does not reverse allodynia in Chung rats during the entire 40-day observation period. 131 40-1 30- —o— Sham • t/5U 20- —•—Chung n = 6 10- **p<0.01 Base 5 10 15 20 25 30 35 40 Time (Days after RTX) Figure 3-10. A single dose of RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effect in the radiant heat paw withdrawal test in Chung and sham-operated rats in the entire 40-day observation period. There is thermal hyperalgesia in Chung rats compared to sham-operated ones before RTX administration (Base). 132 Sham Chung n = 6 -r- —r— Base 5 10 15 20 25 30 35 40 Time (Days after RTX ) Figure 3-11. A single dose of RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effects in the 52°C warm water tail flick test in Chung and sham-operated rats for the entire 40-day observation period. 133 40- u cu « J 30- ;s^ uCJ 20- Sham 10- a.a 0- T" —r- —1— —t— —I— —I— Base 5 10 15 20 25 30 35 40 Time (Days after RTX) Figure 3-12. A single dose of RTX (0.3 mg/kg s.c.) produces a thermal antinociceptive effect in the 52°C hot plate test in Chung and sham-operated rats for the entire 40-day observation period. 134 Chung/Sham surgery 9- Chung Sham n = 8 Base I 5 10 15 Time (Days after RTX) Figure 3-13. A single dose of RTX (0.1 mg/kg i.p.) does not prevent the development of allodynia in Chung rats. Chung/Sham surgery Chung —o—Sham n= 8 I 5 10 Time (Days After RTX) Figure 3-14. A single dose of RTX (0.1 mg/kg i.p.) produces a thermal antinociceptive effect in the 52°C warm water tail flick lest in Chung and sham rats. 136 Non RTX-treated RTX (0.1 mg/kg i.p.) RTX (0.3 mg/kg s.c.) n = 6 1 1 1 1 1 1 1 1- Base 5 10 15 20 25 30 35 40 Time (Days After RTX) Figure 3-15. Allodynic response does not differ significantly between RTX-treated and non RTX-treated Chung rats in 40-day observation period. 137 Summary 3 The results demonstrated a thermal hyperalgesia of the ipsilateral paw in Chung rats to noxious radiant heat, but not in the 52° C hot plate test or tail flick test. The tail nick and hot plate tests of 52® C both provide fairly rapid responses to nociceptive thermal stimuli. Because both tests provide a noxious thermal stimulation with consistent high intensity, it is generally difficult to use these tests to differentiate a thermal hyperalgesia from a normal nociceptive response. In addition, the hot plate test provides additional difficulty in that Chung rats are able to shift their body weights from the injured paw to the contralateral one. In contrast, the radiant heat paw flick test presents the advantage of permitting control over the noxious heat intensity and rate of rise allowing for fine-tuning the paw withdrawal latency to be able to differentiate a thermal nociceptive response from a thermal hyperalgesic response. The paw flick test employed in this study corresponds to a slow rate of rise, preferentially activating thermal nociceptive c-fibers (Yeomans et al., 1996; Zacharious et al., 1997) and replicates the conditions which are consistently used in all thermal hyperalgesic tests in this dissertation. The desensitization of c-fibers to noxious thermal stimuli by systemic RTX was confirmed in the present study by the repeated observations of a significant loss of sensitivity to thermal nociceptive stimuli in all three thermal nociceptive tests in both Chung and sham-operated groups. The duration of desensitization to thermal nociception was maintained throughout the entire observation period and was seen following a single systemic RTX injection, either i.p. or s.c. The desensitization time- 138 course of systemic RTX correlates well with the long lasting desensitization induced by RTX in a number of nociceptive endpoints (Craft et al., 1995; Xu et al., 1997). VRl is a heat-activated nociceptor through which RTX may act to induce inward Ca^ flow. The similar pharmacology of VRl with regard to capsaicin and heat has led to the conclusion that VR1 acts as a physiological transducer for heat-activated nociceptors (Caterina et al., 1997). The experimental results here show that systemic treatment with a single dose of RTX significantly increases response latencies to noxious thermal stimuli in different settings, supportive of the idea of desensitization of c-fibers expressing VR 1 by RTX. Systemic injection of RTX has also been shown to reverse cold hyperalgesia, but not allodynia, in rats with injury to central, rather than peripheral nerves, again suggesting the involvement of c-fibers in cold, but not mechanical, hypersensitivity (Hao et al., 1996). A significant finding of this study is that in spite of the significant loss of responsiveness to noxious thermal stimuli after systemic RTX in both Chung and sham-operated rats, the tactile allodynia present in Chung rats was sustained throughout the entire observation period. Since RTX selectively desensitizes those neurons expressing VRl, and since this receptor has been identified as a physiologic thermal nociceptive transducer (Caterina et al. 1997), then tactile allodynia may well be expected to be mediated by other (i.e.; non-vanilloid receptor expressing) fibers in Chung neuropathic pain model rats. RTX pretreatment decreases nociceptive responses to noxious stimuli significantly in both Chung and sham-operated rats, but does not prevent the occurrence of allodynia in Chung rats. These results further support the 139 hypothesis that tactile allodynia and thermal hyperalgesia are mediated through different peripheral neuronal pathways. There are several f)ossible mechanistic interpretations that may be invoked to support this reasoning. It is strongly suggested, based on the observations, that tactile allodynia is mediated through the activation of capsaicin-insensitive myelinated large diameter neurons or AP-afferent fibers, since these afferent nerves are unlikely to conduct nociceptive thermal stimuli (Light et al., 1979; Sugiura et al., 1986; Maxwell et al., 1987) and are insensitive to desensitization by RTX (Szolcsanyi et al., 1990). This interpretation corresponds well with neuroanatomical studies. In the normal state, these fibers terminate in the deeper laminae (HI-V) of the dorsal hom and code for non-noxious stimuU, and they are not present in lamina U ( Suguira et al. 1968; Maxwell et al. 1987). After peripheral nerve injury to the L5/L6 nerve roots, Ap-fibers form axonal sprouts arising from intact adjacent nerves and from the DRG of the injured nerves, and form abnormal synaptic connections with second order neurons of lamina n (Woolf et al., 1992; 1995; Lekan et al., 1996; Doubell et al., 1997). This region normally receives almost exclusively nociceptive input from unmyelinated c-fibers and is associated with nociceptive transmission (Light et al., 1979; Sugiura et al., 1986). This neuroplastic change has been interpreted as representing an underlying mechanism in the development of neuropathic pain. Further evidence that AP-fiber sprouting might be associated with chronic pain states was suggested by Mannion and colleagues (1996), who showed infiltration of lamina II of AP-fibers after c-fiber injury induced by the application of capsaicin to the 140 sciatic nerve. Lekan and colleagues (1996) demonstrated similar sprouting of Ap-fibers into lamina II in Chung model rats. Although these studies provide strong evidence for a physiopathologic rearrangement of synaptic connections leading to neuropathic pain states, detailed time course analyses, especially in the early period after nerve injury, are not available and therefore it is not certain that the invasion of lamina II by AjJ-fibers correlates with the development of tactile allodynia. The possibility that the onset of tactile allodynia occurs too early to be the result of sprouting into the superficial laminae is open to speculation, since no strict correlation between neuroanatomical changes and the development of tactile allodynia have been reported. For exzmiple, although Lekan and colleagues showed significant infiltration of lamina Q by these fibers, this study was performed at a single time point, 11 days after L5/L6 ligation, whereas signs of tactile allodynia are typically evident within 2-3 days after ligation (Chaplan et al., 1994). Mechanistic differences between thermal hyperalgesia and tactile allodynia can also be demonstrated pharmacologically. Spinally administered morphine, for example, does not alter tactile responses to probing with von Frey filaments in Chung rats, even at doses considered to be supramaximal against other endpoints (i.e. lOO^ig) (Bian et al., 1995; Lee et al., 1995). A complete lack of effect of morphine is not indicated, since the supraspinal or systemic injection of morphine does produce a dose-dependent antiallodynic effect. On the other hand, spinal morphine is effective against noxious thermal stimuli, though at somewhat reduced potency, and produces effective antihyperalgesic actions (Wegert et al., 1997). Likewise, earlier studies also reported on 141 the efficacy of systemic administration of morphine in CCI rats, and implicated the possibility of a peripheral effect of morphine against signs of neuropathic pain (Lee et al.. 1995; Kayser et al., 1995). Based on these findings, it is suggested that thermal hyperalgesia is most likely mediated through slow-conducting, unmyelinated c-fibers, sensitive to RTX. These primary sensory neurons also have opioid receptors on their nerve terminals and this represents an important spinal site of action of opioids (Lombard et al., 1989; Yaksh et al., 1988). Activation of the opioid receptors residing on the nerve terminals of c-fibers will result in decreased transmitter release and thus attenuate nociceptive input and transmission to supraspinal sites (Yaksh et al., 1995, 1998). Evidence shows that AP-neurons do not possess opioid receptors on their nerve terminals (Taddese et al., 1995). There also exists a differential distribution of opioid receptors and mRNA for opioid receptors in the DRG cells based on the sizes of their cell bodies. Opioid receptor distributions are predominant in the superficial layers of the dorsal horn, corresponding with terminals of c-fibers (Mansour et al., 1994, 1995). Furthermore, activation of ^.-opioid receptors predictably inhibited Ca^ channels of small-diameter nociceptors and not of large-diameter cells indicating that ^.-opioid receptor activation selectively inhibits the activity of c-fibers (Taddesse et al., 1995). Therefore, morphine and other opioids may attenuate thermal nociception and thus thermal hyperalgesia through actions on opioid receptors on both the primary nerve terminals of c-fibers and on the cell bodies of second order neurons of the dorsal horn. In contrast to thermal stimuli, it appears that tactile allodynia is mediated through the non-opioid receptor containing A fibers. Therefore, the only population of 142 spinal opioid receptors that might be considered to be relevant to attenuation of transmission of allodynia is that existing post-synaptic to primary afferent neurons. Based on opioid binding studies after dorsal rhizotomy, these receptors may represent only 30% to 40% of the total spinal (i- and 6-opioid receptor population (Besse et al., 1990). Consequently, the ability of spinally injected opioids to block allodynia would be similar to the situation where the receptor reserve has been greatly decreased by a pharmacological manipulation, such as treatment with an irreversible antagonist (e.g., (3-funaltrexamine for opioid p. receptors) or the development of tolerance. Such a manipulation would be expected to displace the agonist dose-effect curve to the right, and for an agonist of limited efficacy such as morphine, produce a decrease in the maximal response, a pattern typical of partial, irreversible blockade of a fraction of the relevant receptor population. Such a pattern is seen for spinal morphine (Yaksh et al., 1995; Bian et al., 1995). On the other hand, a high efficacy opioid p. agonist such as DAMGO has been shown to elicit a significant antiallodynic effect though with somewhat reduced potency, as would be expected with decreased receptor reserve (Nichols et al., 1995). A clinical correlate is that morphine is generally considered to provide inadequate pain relief in different neuropathic pain states (Amer and Meyerson, 1988; Rowbotham et al., 1991). However, the highly efficacious p. opioid fentanyl given i.th. provided complete pain relief against established post-amputation stump pain (Jacobson et al., 1990). Finally, lesion studies have also indicated differences between anatomical circuitries involved in processing tactile allodynia and thermal hyperalgesia (Pertovaara 143 et al., 1996). It was found that microinjection of lidocaine into the periaqueductal gray (PAG) or rostroventral medulla (RVM) blocked tactile allodynia in Chung rats without changing thermal nociceptive tail-flick reflexes. Transection of the spinal cord abolished tactile allodynia of the affected hindpaw caused by mustard oil-induced neurogenic inflammation (Mansikka et al., 1997), but facilitated the tail-flick reflex. Most recently, it was found that complete transection of the spinal cord at T8 caused a complete ablation of tactile allodynia in Chung model rats (Bian et al., 1998). It was also found that thermal nociceptive responses of the injured hindpaw of spinally transected Chung rats were slightly elevated compared to that of ligated, non-spinalized rats, while the thermal nociceptive reflexes of the tail were exaggerated. It is unlikely that the loss of allodynia is an artifact of spinalization since spinal nocifensive responses to heat or pinch were present in the hindpaw and the tail. It was concluded that manifestation of tactile allodynia requires the activation of a spinal-supraspinal circuitry, possibly activating descending facilitatory and inhibitory systems arising from medullary sites, while thermal responses are principally a spinal reflex, with thermal hyperalgesia being driven by central sensitization of localized spinal circuitries, although it may also be under some degree of descending control (Bian et al., 1998). Similarly, transection of the spinal cord abolished tactile allodynia of the tail in rats with sectioned sacral (S1 or S2) spinal nerves but left spinal nociceptive reflexes intact (Na et al., 1997). An important differential pharmacology also exists between thermal hyperalgesia and tactile allodynia in addition to the differences in sensitivity to spinal 144 opioids. It was shown that systemic injection of the competitive NMDA antagonist dextrorphan reverses thermal hyperalgesia in CCI model rats (Tal and Bennett, 1994). In distinct contrast, dextrorphan injection produces no effect on tactile allodynia. It was also shown that i.th. injection of the non-competitive NMDA antagonist MK-801 normalized the thermal nociceptive baseline in CCI model rats, but not i.th. morphine dose-response curve for allodynia. There was a significant shift to the right of the morphine dose-response curve for CCI rats with or without MK-801 when compared to sham-operated rats (Mao et al., 1995). Similarly, i.th. injection of MK-801 did not change responses to tactile stimuli in Chung rats (Nichols et al., 1995), but did normalize the baseline of the affected hindpaw to radiant heat stimuli (Wegert et al., 1997). Again, the dose-response curve for i.th. morphine against thermal nociception was not changed in Chung rats (Wegert et al., 1997). This effect contrasts with the finding that i.th. pretreatment with MK-801 unmasked the anti-allodynic action of morphine, but did reverse thermal hyperalgesia in the affected paw in Chung rats (Nichols et al., 1997). Furthermore, pretreatment with i.th. dynorphin antiserum also unmasked the antiallodynic activity of i.th. morphine (Nichols et al., 1997). As mentioned previously in the main introduction, Dynorphin is an endogenous opioid peptide, increased after peripheral nerve injury in the spinal cord, and acts at NMDA receptors to cause central sensitization. It is believed that signs of neuropathic pain may be alleviated at least in part by blockade of NMDA receptors, which would modify the development of central sensitization driven by spontaneous activity of injured primary afferent neurons (Tal et al., 1994; Woolf et al., 1994). This interpretation is supported 145 partially by the observation that L5/L6 spinal nerve ligation also reduces the antinociceptive activity of i.th. morphine in the tail-flick test (Ossipov et al., 1995), and that the potency and efficacy of morphine is restored by i.th. dynorphin antiserum (Nichols et al., 1997), i.th. MK-801 or application of bupivacaine at the site of injury (Ossipov et al., 1995). In seeming contradiction to the results presented here, a clinical investigation reported that a high (5% to 10%) dose of capsaicin provided relief against neuropathic pain in the foot or chest wall arising from a variety of causes (Robbins et al., 1998). However, interpretation of the data is complicated in that the type of pain (i.e. hyperalgesia vs. allodynia vs. spontaneous pain) was not reported. Additionally, a local anesthetic was applied prior to capsaicin, and this maneuver alone may have influenced the outcome. For example, it was shown that secondary c-fiber mediated hyperalgesia may be blocked by anesthesia with pentobarbital, but not isoflurane (Cieland et al., 1994). Also, peripheral nerves neighboring an injured nerve have shown increased spontaneous and evoked activity, and this excitation was blocked by application of lidocaine to the injured nerve (Sotgiu et al. 1997). Finally, lidocaine may not have been an agent of choice here since capsaicin acts initially by opening cation channels, whereas lidocaine exerts its local anesthetic activity by blocking Na"*" channels, thus opposing the action of capsaicin. The results presented here strongly suggest that different mechanisms may underlie the development of different signs of neuropathic pain behavior. No single model is adequate to investigate the possible mechanisms that may be exploited in order 146 to develop treatment modalities for complex pain symptoms. It is clear that different signs of neuropathic pain may be due to different and unique pathophysiological mechanisms. The present study, in concert with recent observations, suggests that treatment of hyperalgesia or other c-fiber related manifestations might be amenable to resolution with vanilloids, such as RTX, but one must be cautious in extrapolating such conclusions to the entire array of neuropathic pain states, especially to mechanical stimuli which appear to be resistant to manipulations resulting in changes in c-fiber activity. Capsaicin-sensitive afferent fibers are believed to induce a state of central sensitivity in the dorsal horn of the spinal cord after peripheral nerve injury, which is responsible for neuropathic pain symptoms such as hyperalgesia and allodynia (Mendell, 1966; Adriaensen et al., 1983; Steen, et al., 1992; Woolf, 1994). Recent evidence has shown that selective elimination of c-fibers by neonatal capsaicin treatment results in the disappearance of allodynia induced by i.th. PGE2 but not by PGFia indicating two pathways for induction of allodynia at the spinal level (Minami et al., 1999). The evidence has also shown that there is increased PN3 expression in the medium-sized neurons in the DRG after Chung surgery. PN3 antisense knock-down of the expression of PN3 protein in the DRG of Chung model rats abolishes allodynia and thermal hyperalgesia (Lai et al., 1999). Here our data that pretreatment with systemic RTX abolishes nociceptive responses to noxious thermal stimulation, but not the appearance of allodynia in Chung model rats, indicate that there is little, if any involvement of c-fibers in the development of allodynia in Chung model neuropathy. 147 In conclusion, Ap-fibers could be responsible for the development of neuropathic pain symptoms by causing central sensitization or by changing the receptive modalities. In addition, c-fibers are not solely responsible for causing central sensitization, which is responsible for neuropathic symptoms such as allodynia and thermal hyperalgesia after peripheral nerve injury. 148 Study 4 Antisense oligodeoxynucleotides against a TTX-resistant sodium channel, PN3, prevent and reverse chronic, inflammatory and neuropathic pain In the rat Peripheral nerve injury results in a number of significant neuroanatomical and neurophysiological changes to the injured nerve. An important consequence of nerve injury is the development of spontaneous neuronal activity. The injured nerve fires spontaneously, giving rise to spontaneous pain. Also, normally quiescent high-threshold nociceptors alter their electrophysiological activity so that constantly active nociceptors lead to spontaneous pain and hyperalgesia. It is thought that this constant afferent drive leads to the development of central sensitization in the spinal cord, which drives the development of the manifestations of neuropathic pain. Early studies in rats and rabbits have shown that chronic nerve injury produces spontaneous firing of sensory nerves (Wall et al., 1974; Kirk et al., 1974). The primary sites of this abnormal, ectopic firing were determined to be the site of injury itself and the dorsal root ganglion of the injured nerve (Devor, 1994, Kajander et al., 1992). The ectopic site is also believed to be associated with the generation of discharges which can be initiated by noxious or by normally non-noxious stimuli (Devor, 1994, Fields et al., 1997). Ectopic firing contributes to neuropathic pain directly, by eliciting abnormal sensation (parensthesias from increased firing of large fibers) and dysesthesias and pain (from increased firing of small fibers), as well as indirectly by the resulting sensitization of neurons in the spinal cord (Fields et al.. 149 1997). Damaged peripheral nerves may also form peripheral collateral sprouts and ectopic foci, resulting in spontaneous discharges that are perceived as nociception (Kajander et al., 1992) as well as sprouting in central connections. Thus, damaged large fiber terminals sprout into the superficial lamina of the spinal dorsal horn to form pathophysiologic synapses that may be the basis whereby normally innocuous inputs now drive nociceptive transmission cells (Woolf et al., 1992; Woolf et al., 1994). Sodium channels located in peripheral sensory neurons are considered to play an important role in the development of spontaneous and/or evoked hyperexcitability of the peripheral nerve, a primary feature of many chronic pain symptoms in particular neuropathic pain symptoms (Devor, 1994; Woolf, 1994). The increased, persistent afferent drive then triggers a process of central neuronal sensitization and, consequently, the parensthesias and pain associated with either peripheral nerve or prolonged tissue injury (Woolf, 1983; Cook et al., 1987; Woolf et al., 1994). A prominent molecular basis for the abnormal, repetitive firing of injured primary afferents in many neuropathic conditions is an accumulation and increased membrane density of sodium channels at focal sites of injury (Devor et al., 1993; England et al., 1996). The resultant membrane remodeling contributes to a lower threshold for action potential generation at these sites and, consequently, precipitates ectopic impulse generation in a chronically injured nerve (Wall et al., 1974; Matzner et al., 1994). It was recently demonstrated that the degree of ectopic firing in Chung rat correlates well with behavioral signs of neuropathic pain (Han et al., 1995). Rats were subjected to L5 and L6 spinal nerve root ligation and assessed for neuropathic pain 150 behavior and for ectopic discharges by electrophysiological recordings from the L5 dorsal root. It was found that the number of units showing ectopic discharge and behavior indicative of neuropathic pain both reached a peak at one week, and were reduced considerably at 10 weeks (Han et al., 1995). The generation of spontaneous, ectopic discharges after nerve injury strongly implicates voltage-gated sodium channels in neuropathic pain. These channels are located in the plasma membrane and permit entry of sodium ions into the cell causing depolarization and generation of the action potential (Catterall, 1992; Kallen et al., 1993). There is an increase in the concentration of sodium channels at the site of injury and in the dorsal root ganglia of injured nerves. The importance of sodium channel activity in clinical neuropathic pain is underlined by the key observation that essentially all of the drug categories that are clinically useful for neuropathic pain exhibit a significant degree of sodium channel blocking activity (Devor, 1994). Categories of drugs that are clinically important against neuropathic pain include the anti-seizure drugs, local anesthetics, tricyclic antidepressants and orally active antiarrhythmic agents. For example, carbamazepine and sodium valproate are useful in the treatment of trigeminal neuralgia and show promise against lancinating types of neuropathic pain (Fields et al., 1997). Phenytoin has shown effectiveness in diabetic neuropathy. The orally active antiarrythmic agent mexiletine has reduced parensthesias, dysthesia and pain in double blind studies of patients with diabetic neuropathies (Stracke et al., 1992; Dejgard et al., 1988). The use of tricyclic antidepressants has also met with some degree of clinical success. For example, amitryptyline has relieved pain 151 resulting from diabetic neuropathy and postherpetic neuralgia (Max et al., 1987; 1988). The antiallodynic efficacy of desipramine has been attributed to both its sympathetic activity and also its sodium channel blocking properties (Jett et al., 1997). Importantly, a common theme of all these compounds is their demonstrated ability to block TTX- sensitive and TTX-resistant sodium channels non-selectively (Tanelian et al., 1991; Jett et al., 1997; Delpon et al., 1993). A crucial problem with these medications is that relief of pain usually occurs at doses that are very close to the production of toxic effects. Additionally, adequate pain relief is obtained only by combinations of several medications at the same time, which also results in increased, and often barely tolerable, side effects and decreased quality of life for patients. It is clear that the non-selective blockade of different classes of sodium channels by these agents produces severe side effects, and that pain relief might be accomplished with minimal side effects if the specific sodium channel types driving the firing of the injured nerve could be selectively blocked. One clinically useful compound that does not readily fit the profile of sodium blocking agents is gabapentin, an anti-seizure medication structurally related to GABA, but without activity at the GABAA and GABAB receptors (Singh et al., 1996). Although the mechanism of gabapentin to relieve neuropathic pain is not known, it has been suggested to involve a spinal site of action and has been hypothesized to involve an indirect potentiation of GABAergic modulation. It was recently shown that gabapentin produces a does-dependent inhibition of evoked c-fiber activity in normal rats (Stanfa et al., 1997). Gabapentin was also shown to inhibit calcium currents in neurons. 152 presumably by binding to the (*25 subunit of calcium channels (Stefani et al., 1998; Felix, 1999). Recent clinical studies have indicated that gabapentin is useful against reflex sympathetic dystrophy (Mellick et al., 1995) and some forms of neuropathic pain (Rosner et al., 1996). It appears that gabapentin may present a means of specifically inhibiting hyperexcited, but not normally active, nociceptive c-fibers. A significant amount of evidence exists to indicate local anesthetics may abolish neuropathic pain for short to occasionally extended time periods, sometimes after a single application. It is especially important to note that systemically injected local anesthetics abolish neuropathic pain for short periods of time at concentrations that do not block normal nerve conduction (Devor et al., 1992). Clinically, it has been demonstrated that application of lidocaine to a primary lesion resulted in reversal of allodynia extending from the injury (Gracely et al., 1992). Local anesthetic block has also relieved postherpetic neuralgia without blocking normal sensation (Rowbotham et al., 1991). It has been suggested that spontaneous, ectopic activity arising from the site of injury drives central sensitization which causes normally innocuous A^-input to be translated as nociceptive (Fields et al., 1997). Similar observations have been made experimentally, where a peripheral nerve injury produced thermal hyperalgesia and tactile allodynia in the rat and these signs of neuropathic pain were abolished by the application of non-paralytic doses of lidocaine at the site of injury. These observations clearly implicate sodium channel activity in the manifestation of neuropathic pain. Further, it has been shown that there is an abnormal increase in sodium channels in the 153 neuronal membrane at the site of injury. This increased amount of sodium channel results in a lowered threshold for the generation of action potentials at these sites, thus forming foci for ectopic impulse generation (Devor, 1994). In fact, Devor and his colleagues have calculated that the increase in sodium channel density and consequent increased sodium conductance could theoretically account for the hyperexcitability of injured nerves (Matzner and Devor, 1992). These observations provide some explanations for the particular efficacy of local anesthetics against neuropathic pain at doses that do not block conduction in normal nerves. A number of voltage-gated sodium channel subtypes have been identified based on biophysical properties and sensitivity to TTX. Membrane excitability in different tissues is predominantly controlled by the tissue-dependent expression of distinct genes encoding individual voltage-gated sodium channel subtypes that have been distinguished on the basis of primary structure but which can also be differentiated by their biological kinetics and sensitivity to low, nanomolar concentrations of TTX. These sodium channels include rat brain types I, IIA and HI, skeletal muscle type I (Catterall, 1992; Kallen et al., 1993), and the recently described novel channels, sodium channel protein 6 (SCP6; Schaller et al., 1995), the closely homologous peripheral nerve 4 (PN4; Dietrich, et al., 1998) and peripheral nerve 1 channels (PNl; Sangameswaran et al., 1997) of which the latter appears to be the rodent orthlog of the human neuroendocrine channel (Toledo-Ara et al., 1997). However, most persistent, slowly inactivating sodium currents have also been described, especially in heart and sensory ganglia. The cardiac channel HI (Rogart et al., 1989) exhibits low, micromolar 154 (1-5 pM) sensitivity to TTX. More recently, a novel TTX-resistant (TTX-r) sodium channel has been identified and cloned from rat dorsal root ganglia. This TTX-r sodium channel is expressed primarily in small-diameter unmyelinated peripheral nerves, and is not found in other peripheral or central neurons or non-neuronal tissues (Akopian et al., 1996). This TTX-r sodium channel is identified as either peripheral nerve 3 (PN3; Sangameswaran et al., 1996) or sensory nerve specific (SNS; Akopian et al., 1996). Targeting individual voltage-gated sodium channel subtypes that may be either specific to sensory nociceptive neurons or selectively regulated (Waxman et al., 1994) in response to peripheral nerve injury may serve as a unique and novel approach to the treatment of neuropathic pain. In general, the neurons of dorsal root ganglia express two main types of sodium currents: a rapidly inactivating, TTX-sensitive sodium current (TTX-s Isa) and a slowly inactivating TTX-resistant sodium current (TTX-r iNa) (Roy et al., 1992; Caffrey et al., 1992; Elliott et al., 1993; Ogata et al., 1993). The relative proportions of these sodium currents in individual DRG neurons determine the wide range of firing behaviors and, consequently, fiinctional properties of the various classes of DRG neurons. TTX-sensitive currents are the predominant iNa in all types of DRG cells at all stages of development and represent the main iNa associated with the large diameter, fast conducting, myelinated AP-fibers (Roy et al., 1992; Rizzo et al., 1994). Whole cell patch clamp studies have indicated that large neurons of the DRG may have TTX-sensitive (TTX-s), kinetically fast Isa and a combination of TTX-s fast and slow currents, indicating a heterogeneity of channels responsible for these currents 155 (Rizzo et al., 1994). In contrast, the TTX-r Inq normally has a distribution restricted to the small diameter, unmyelinated, capsaicin-sensitive nociceptors (Roy and Narahashi, 1992; Elliott and Elliott, 1993; Arbuckle, 1995). The slow inactivation and rapid repriming properties of TTX-r Inu appear particularly well suited to sustained firing at the depolarized potential characteristic of an injured peripheral nerve. Several sodium channel proteins have been identified from DRG tissue with inactivation kinetics and sensitivity to TTX suggesting mediation of the TTX-s Inq- These channels include brain type I, HA, HI, SCP6, PN4 and PNl. In contrast, only PN3 appears to be a suitable channel for mediation of the TTX-r iNa- To summarize, it appears that the novel sodium channel PN3, which was cloned from a rat DRG cDNA library, is expressed predominantly in small sensory neurons and may contribute to the TTX-r Ins that is believed to be associated with central sensitization in chronic neuropathic pain states (Akopian et al., 1999). A recent study employed electrophysiological, in situ hybridization, and immunohistochemical methods to monitor changes in the TTX-r sodium current and the distribution of PN3 in normal and peripheral nerve injured rats. It was found that there were no significant changes in the TTX-r and TTX-s sodium current densities of small DRG neurons and no changes in the expression of PN3 mRNA in the DRG up to 14 days in the CCI model (Sangameswaran et al., 1996). However, the intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral 156 axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain (Novakovic et al., 1998). PN3, as a neuropathic pain target, may be useful in the selective block of chronic neuropathic pain states. Further, a PN3 inhibitor may produce pain relief in these states without many of the limiting CNS and other side effects associated with current therapies. In order to test this hypothesis and in the absence of selective inhibitors, we will use two 23-mer antisense (AS 1 and 2) and mismatch (MM I and 2) oligodeoxynucleotides (ODNs) to specific sequences on the 5' end of the coding region of PN3 and test their effects on behavioral manifestations, i.e. tactile allodynia and thermal hyperalgesia, in different pain model rats. The expression of PN3 will be seen and compared in the Chung nerve injury model, carrageenan, and CPA inflammatory pain models, and diabetic neuropathic pain model. These models offer advantages for studying different aspects of neuropathic pain. Chung model rats produce spontaneous pain, thermal hyperalgesia, and reliable mechanical allodynia, corresponding well with clinical neuropathic pain states caused by peripheral nerve injury (Kim and Chung, 1992). It is also believed that the Chung model produces ectopic discharges from the injured spinal nerves and associated DRGs that trigger central sensitization. Afferent input arrives from the low threshold AP-fibers of the undamaged LA branch of the spinal nerve root and the behavioral response of the animal to single stimulation with a von Frey filament is consistent with allodynia (Fields et al., 1997). The carrageenan 157 inflammatory pain model has been used as an acute inflammatory pain model (Hargreaves et al., 1988). Generally, 3 or 4 hours after intra-paw carrageenan injection, the inflamed paw shows a dramatic thermal hyperalgesia (Tsuruoka et al., 1996; Hargreaves et al., 1988). Advantages of this model are that it is capable of identifying analgesic effects of non-steroid anti-inflammatory drugs (NSAIDS) and morphine and it is shorter and less painful compared to other inflammatory pain models such as formalin and CFA inflammatory models. Also importantly, the carrageenan inflammatory pain model can be used as a control to investigate gene changes in the DRG after short-term peripheral inflammation. The CFA model is a relatively long- lasting inflammatory pain model. Four days after challenging with intra-paw CFA, there is an obvious thermal hyperalgesia in the inflamed paw caused by a delayed type hypersensitivity immunoreaction (Ma et al., 1996). This model can be used to investigate gene changes for chronic inflanmaatory pain states. The streptozotocin induced diabetic neuropathic pain model has been widely used in pain pharmacology research. This model mimics the common neuropathic pain symptoms of diabetes in the clinic. It is an experimental animal model with a long-term deteriotive painful diabetic neuropathy. It is reported that three to four days after one large dose of streptozotocin intraperitoneal injection, animals start to have diabetic symptoms and four to six weeks after the injection, animals start to exhibit thermal and mechanical hyperalgesia. The hyperalgesic symptoms, which appeared in diabetic model animals last for almost the rest of their lives (Rittenhouse et al., 1996). 158 Experimental Procedure Chung and other inflammatory model rat preparations Chung nerve ligation injury rats were prepared five days before experiment procedures. Acute and chronic inflammations were induced by the intraplantar administration of 150 (il of 2% carrageenan (CAR) and 150 (il Complete Freund's Adjuvant (CFA), respectively. Peak changes in paw withdrawal latencies (thermal hyperalgesia) were obtained after either 3hrs for CAR or 4 days for CFA. Antisense and mismatch oiigodeoxynucleotide sequences Chronically indwelling intrathecal (i.th.) catheters were implanted for administration of the ODNs. AS I (5'-TCC-TCT-GTG-CTT-GGT-TCT-GGC-CT-3 ). AS2 (5'-TCC-ACG-GAC-GCA-AAG-GGG-AGC-T-3'), MM 1 (5-TCC-TTC-GTG-CTG-TGT-TCG-TGC-CT-3) MM2 (5'-TCC-AGC-GAC-GAC-AAG-GGA-GGC-T-3') phosphodiester ODNs were reconstituted in nuclease free water and i.th. injections made in a volume of 5 (il followed by a 9 fil distilled water flush. Rats received twice daily (b.i.d.), i.th. injections (45 ^g/5 fil) of either AS or MM ODNs to the PN3 for either 3 days prior to and for 5 days after, Chung surgery (preventative) or from days 5-10 post-surgery (ameliorative). The degree of allodynia and hyperalgesia was assessed on the day of surgery and then either on days 1, 4, 5 and 6 (preventative) or on days 5, 6, 7, 8 and 10 (ameliorative) post-surgery. In the CAR and CFA inflammatory pain models, AS and MM ODNs were administered for two days (45 ng, i.th., b.i.d.) prior to and continuing 159 in the presence of either an acute, injection of CAR (three hours) or during four days of CFA administration. Tactile allodynia and thermal hyperalgesia were assessed after three hours (CAR) and on day five (CFA), respectively. Immunohistocheniistry procedure Rats were anesthetized with 10% chloral hydrate (300 mg/ kg) and subsequently perfused through the aorta with sodium phosphate-buffered saline (PBS, 200 ml, pH 7.5, 4°C), followed by 10% formalin (500 ml, pH 7.8, 4°C). DRGs (L4. L5 and L6) were removed, 10 |am thick sections cut, mounted on slides and incubated with site- directed, polyclonal antibodies raised in rabbits against peptide sequences unique to either PN3, PNl or NaCh6/SCN8A/PN4. Immunolocalization of each antiserum utilized the ABC method with the staining pattern visualized by the DAB substrate reaction for light microscopy (Some DRG tissues were sent to Dr Patrick Mantyh's lab at the University of Minnesota, Department of Molecular biology and Dr. John Hunter s group at Roche Bioscience, Department of Analgesia for immunocytochemistry. Results .\ntisense (ASl), not mismatch (MMl), ODN to PN3 prevents the development of allodynia and thermal hyperalgesia in Chung rats The Chung nerve ligation injury, but not sham-surgery, produced a state of tactile allodynia and thermal hyperalgesia within four days of surgery (Chaplan et al., 1994). MMl ODN (45|ig, i.th., b.i.d.) treatment, beginning three days prior to Chung or sham surgery and continuing for 6 days post-surgery, neither altered the development 160 of allodynia in Chung rats (range 1.9 - 2.5 g, 4-6 days post-surgery) nor had any significant effect (P>0.05) in the sham-operated rats (Fig. 4-1; range 11.6 - 12.9 g). In contrast, AS 1 ODN (45|ig, i.th., b.i.d.) treatment completely prevented the development of tactile allodynia in Chung rats with paw withdrawal thresholds (range 10.9 - 12.1 g) not significantly different (P>0.05) from those of the sham-operated group (range 11.6 - 13.6 g) throughout the post-surgery period (Fig.4-1). A similar profile was observed with respect to the prevention of thermal hyperalgesia (Fig. 4-2). Cessation of ASl ODN treatment at day six after Chung surgery resulted in the development of tactile allodynia and thermal hyperalgesia within four days (Fig. 4-1 and Fig. 4-2), demonstrating reversibility of the ODN effect. ASl, not mismatch MMl, ODN to PN3, reverses tactile allodynia and thermal hyperalgesia in Chung rats. The effect of ODN post-treatment was also measured on established tactile allodynia and thermal hyperalgesia (Fig. 4-3 and 4-4). MMl ODN treatment had no significant effect (P>0.05) on the level of tactile allodynia observed in Chung rats and paw withdrawal thresholds remained in the allodynic range (2.1 - 3.3 g) throughout the course of the study (Fig. 4-1 and Fig. 4-3). In contrast, ASl ODN administration caused a significant elevation in paw withdrawal thresholds of the Chung group (range 11.2 - 13.1 g) within two days of treatment and for four further days during continued exposure to the ODN, indicating a reversal of tactile allodynia (Fig. 4-1 and Fig. 4-3). A similar profile was observed with respect to reversal of thermal hyperalgesia (Fig. 4-2 and Fig. 4-4). Pre-ASl ODN treatment levels of tactile allodynia (Fig. 4-1 and Fig. 4-3) 161 and thermal hyperalgesia (Fig. 4-2 and Fig. 4-4) were re-established within two days of cessation of treatment, demonstrating the reversibility of the ASl ODN effect. The equivalent time course for the onset and reversibility of the ASl ODN effect was consistent with the reported twenty-six hour half-life for the rate of sodium channel turnover and biosynthesis (Waechter et al., 1983). Additionally, it should be noted that the ASl ODN treatment reversed the thresholds to non-noxious tactile and noxious thermal stimuli to, but not above, the control (i.e., MMl) range, suggesting that inhibition of PN3 protein expression does not change normal non-noxious or noxious sensory thresholds. ASl or MMl ODN to PN3 does not affect nociception in acute pain models As a comparison to neuropathic pain states caused by a peripheral nerve ligation injury, the effect of PN3 antisense or mismatch ODN to acute nociceptive stimulation has been investigated in the most commonly used nociceptive tests. Two days pre- treatment with intrathecal ASl or MMl (45 jig, i.th., b.i.d.) before testing does not affect acute nociception. PN3 ASl or MMl ODN does not affect CAR caused allodynia and thermal hyperalgesia as well as nociceptive responses to thermal and mechanical stimuli in the tail and paws in both CAR (3 hrs) and CFA (4 days) inflammatory model rats (Fig. 4-5, Fig. 4-6, Fig. 4-7, Fig. 4-8, Fig. 4-9, Fig. 4-10 and Fig. 4-11). 162 AS2, not MM2 ODN to PN3, prevents and reverses tactile allodynia and thermal hyperalgesia in Chung rats A second antisense sequence (AS2 ODN) unique to PN3 was also investigated and produced a very similar profile when administered under identical procedures. AS2, but not MM2, ODN (45|ag, i.th., b.i.d.), administered using either the preventative or ameliorative paradigms for the treatment of the Chung injury, produced a significant reduction in the signs of both tactile allodynia and thermal hyperalgesia in a manner similar to that observed with the AS 1 ODN and again, the effects of the AS2 ODN were completely reversible (Fig. 4-11 and Fig. 4-12). At no time during treatment with either antisense or mismatch ODN sequences were any overt behavioral, particularly CNS, side effects observed; animals gained weight and behaved normally. ASl ODN, not MMl ODN to PN3 inhibits chronically, but not acutely developed neuropathic pain in inflanunatory pain models PN3 has also been implicated in the hyperesthesias that result from tissue injury. We therefore also investigated the effect of the ASl and MMl ODNs on tactile allodynia and thermal hyperalgesia induced acutely by CAR or chronically by CFA. Pretreatment with either MMl or ASl ODN for two days (45 jig, i.th., b.i.d.) prior to CAR had no significant effect on the development of either tactile allodynia or thermal hyperalgesia of the hindpaw, as determined three hours after the injection of CAR (Fig. 4-5 and Fig. 4-6). Paw withdrawal thresholds to tactile stimuli for the MM 1 and ASl groups were 3.7 ± 0.9 g (n=6) and 2.4 ± 0.5 g (n=6), respectively, values which did not differ significantly (P < 0.05) between each other, but were significantly 163 reduced from the pre-carrageenan baseline value of 15 g for each group, indicating the presence of tactile allodynia. Paw withdrawal latencies to thermal stimuli for the MM 1 and ASl groups were 16.8 ±0.8 sec (n=6) and 15.7 ± 1.3 sec (n=6), respectively, values which did not differ significantly (P > 0.05) between each other but were significantly reduced from the pre-carrageenan baseline of 22.2 ± 0.8 sec (n=6) and 22.3 ± 0.5 sec (n=6), respectively, indicating the presence of thermal hyperalgesia. In the CFA treated groups, rats received twice-daily injections (45 ng, i.th.) of either MMl or ASl ODN for two days prior to CFA with administration continuing in the presence of CFA for four further days. On the fourth day after commencing CFA, the animals that received MMl demonstrated clear tactile allodynia and thermal hyperalgesia while, in contrast, those that received ASl ODN showed no signs of having developed either tactile allodynia (Fig. 4-13) or thermal hyperalgesia (Fig. 4-14). Finally, neither MMl nor ASl ODN affected acute, high threshold nociceptive responses in CFA and CAR rats. In groups scheduled to receive either MMl or ASl ODN, tail-flick latencies from the 52°C water bath were 3.1 ± 0.2 sec and 3.1 ± 0.1 sec, respectively. After two days of receiving either MMl or ASl ODN (45ng, i.th., b.i.d.), the tail-flick latencies were 2.8 ±0.1 sec and 3.7 ± 0.3 sec, respectively. These values were not significantly different (p > 0.05) from the pre-ODN values. ASl, not MMl ODN to PN3, reverses tactile allodynia and thermal hyperalgesia in diabetic neuropathic pain model rats Rats which received one injection of streptozotocin (50 mg/kg, i.p.) developed stable neuropathic pain states characterized by consistent allodynia and thermal 164 hyperalgesia in the paw (Fig.4-15 and Fig 4-16). The diabetes mellitus states which developed in streptozotocin injected rats were confirmed by polydipsia, polyphagia, polyuria, gradually body weight loss and chronic hyperglycemia (Fig. 4-17). The development of neuropathic pain states is confirmed by measuring allodynia and thermal hyperalgesia in the left paw (Fig. 4-15 and Fig. 4-16). PN3 AS I, ODN (45 ^g i.th.), not MM I (45 ng i.th.), reverses tactile allodynia and thermal hyperalgesia in diabetic rats (range 11.2 - 13.1 g) within four days of continuos FN3 ASl treatment. Tactile allodynia and thermal hyperalgesia were re-established within two days of cessation of treatment in ASl ODN treated diabetic group, demonstrating the reversibility of the ASl ODN effect (Fig. 4-18 and Fig. 4-19). Effects of AS or MM ODN to PN3 on immunohistochemical studies In normal rats, immunohistochemical analysis of the distribution of FN3 in LA DRG neurons shows predominant iimnunolabeling of the small diameter cells (78% of all small diameter cells <700nm", n=l04; 33% of all medium-large diameter cells >700^1 m", n=l39. A similar profile is observed in either L5 or L6 DRG. In comparison, 7 days after Chung surgery, a substantial reduction in PN3 irmnunostaining was observed in both small (18% of total, n=l 15) and medium to large (10% of total, n=112) diameter cells of the L5 and L6 DRG on the side ipsilateral to the injury, compromising an accurate assessment of the ODN effect on channel protein at this level. For this reason we chose to analyze the DRG taken from the intact L4 spinal root. Iimnunohistochemical analysis of the ipsilateral or contralateral (relative to the side of nerve injury) L4 DRG tissue following administration of saline showed that 55% 165 of all small diameter (n=54) and 77% of all medium to large diameter (n=76) cells labeled with PN3 antibody. A similar immunolabeling pattern was observed after forty- eight hours of treatment with MMl ODN (post-surgery day 7), with 43% of all small diameter (n=157) and 81% of all medium to large diameter cells (n=196) labeled with PN3 antibody. AS I ODN, however, produced a substantial "knock-down" of PN3 protein in both small (8% of total, n=271) and medium to large (18% of total, n=I99) diameter cells of the ipsilateral and contralateral L4 DRG (Table 4. and Fig. 4-21). In order to determine the specificity of the AS I ODN effect on PN3 protein expression, immunohistochemical analysis was also performed on two prominent TTX-s sodium channels present in the DRG: PNl and NaCh6 (Toledo et al., 1997; Sangameswaran et al., 1997; Schaller et al., 1995). In contrast to PN3, neither ASl nor MMl ODN treatment affected the level of expression of either PN1 or NaCh6 protein (Fig. 4-22). Meanwhile, dynorphin content was measured to determine if PN3 protein knockdown by AS ODN in Chung model rats could prevent increase in spinal dynorphin levels. There are elevated levels of dynorphin in the ipsilateral dorsal quadrant of the lumbar spinal cord in Chung model rats 10 days after Chung surgery. ASl, but not MMl, to PN3 (45|ig, twice daily, i.th. for 5 days) reverses increased dynorphin levels in the lumbar spinal cord back to normal in Chung model rats (Fig 4- 23). In diabetic rats there was a dramatically increased PN3 protein staining in the LA, L5 and L6 DRGs in both sides compared to the normal age-matched control group (Fig. 4-24, only shows L4 DRGs). PN3 AS I ODN treatment knocked down the PN3 166 protein in DRGs in diabetic rats and age-matched control rats, but not MMl (Fig. 4-24 and Fig. 4-25). Cessation of PN3 AS I treatment in diabetic rats for four days caused PN3 expression in L4, L5 and L6 to return to previous levels (Fig. 4-25). 167 Start ODN Stop ODN Start ODN Stop ODN injections injections injections injections B Chung AS1 Sham ASl -Q- Chung MM1 ShamMMl Chung or Sham surgery 18 20 22 Time (Days) Figure 4-1. Pretreatment with ASl (45 jig, twice daily, i.th.), but not MMl, ODN to PN3 prevents the development of tactile allodynia and continuous ASl, but not MMl, ODN reverses the existing tactile allodynia in Chung model rats. The antiallodynic effect of AS 1 is reversible. 168 Start ODN StopOCW Start ODN Stop ODN injections injections injections injectiors 26r Chung ASl -•-ShamASl O -OQu^MMl o -O- Sham MMl os 'S -J "3 -a S > Qungor Sham surgery 12- i 10- ' ' 8 10 12 14 16 18 20 22 Tune (Days) Figure 4-2. Pretreatment with ASl (45 ^.g, twice daily, i.th.), but not MMl, ODN to PN3 prevents the development of thermal hyperalgesia and continuous ASl ODN reverses the existing thermal hyperalgesia in Chung rats. Neither ASl nor MMl ODN changes the threshold to thermal stimuli. The anti-thermal hyperalgesic effect of AS I is reversible. 169 on 15 ^ 12 Chung ASl •3 9 -•-Chung MMl Start ODN Last ODN -A-Sham MMl IL_ injections injections •5 6 -Ch Chung Saline -O-Sham Saline C3 a. 0 0 3 4 5 8 Time (Days) Figure 4-3. ASl, but not MMl, ODN to PN3 (45 |ig, twice daily, i.th.) reverses tactile allodynia in Chung rats. Tactile allodynia reappears in Chung rats after cessation of ASl ODN. 170 ou 22 :A. >»U c Figure 4-4. ASl, but not MMl, ODN to PN3 (45 Hg, twice daily, i.th.) reverses thermal hyperalgesia in Chung rats. Thermal hyperalgesia reappears in Chung rats after cessation of AS 1 ODN. 171 ASl MMl Base Carraseenan Figure 4-5. ASl and MMl ODNs to PN3 (45 [ig, twice daily, i.th.) do not prevent allodynia of the affected paw in carrageenan inflammatory pain model rats. 172 ^•ASl [ZZIMMl Base Carraseenan Figure 4-6. ASl and MMl ODNs to PN3 (45 \Lg, twice daily, i.th.) do not prevent thermal hyperalgesia of the affected paw in carrageenan inflammatory pain model rats. 173 ASl MMl Figure 4-7. ASl and MMl ODNs to PN3 (45 ng, twice daily, i.th.) do not change the 52°C tail flick latency in carrageenan inflammatory pain model rats. 174 ^•ASl (ZZDMMl Figure 4-8. ASl and MM I ODNs to PN3 (45 p.g, twice daily, i.th.) do not prevent mechanical hyperalgesia of the affected paw in carrageenan inflammatory pain model rats. 175 Figure 4-9. ASl and MMl ODNs to PN3 (45 |ig, twice daily, i.th.) do not change the 52°C tail flick latency in CPA inflammatory pain model rats. 176 2 20-1 o SI. o•J-. Base Carraseenan Figure 4-10. ASl and MMl ODNs to PN3 (45 |ig, twice daily, i.th.) do not prevent mechanical hyperalgesia in the affected paw in CPA inflammatory pain model rats. 177 12 —ChungAS2 -•-Sham AS2 9 —Q— Chung MM2 •a Start ODNs —O- Sham MM2 6 —Chung Saline —Sham Saline cu 3 0 0 2 3 4 Time (Days) Figure 4-11. AS2, but not MM2, ODN to PN3 (45 ^ig, twice daily, i.th.) reverses tactile allodynia in Chung rats. The antiallodynic profile of AS2 is similar to that of ASl ODN to PN3. 178 CZIDASl ^•MMl Base CFA Figure 4-12. AS2, but not MM2, ODN to PN3 (45 ^g, twice daily, i.th.) reverses thermal hyperalgesia in Chung rats. The anti-thermal hyperalgesia profile of AS2 is similar to that of AS 1 ODN to PN3. 179 ASl MMl Base CFA Figure 4-13. ASl, but not MMl, ODN to PN3 (45 ng, twice daily, i.th.) prevents tactile allodynia in CFA inflammatory pain model rats (p < 0.01). 180 T3 30-1 o ASl MMl Base CFA Figure 4-14. ASl, but not MMl, ODN to PN3 (45 (ig, twice daily, i.th.) prevents thermal hyperalgesia in CFA inflammatory pain model rats (p < 0.01). 181 —Diabetic Rats Normal Rats Time (Weeks) Figure 4-15. A single dose of streptozotocin (50 mg/kg, i.p.) causes tactile allodynia in the paw with time in streptozotocin-induced diabetic model rats. 182 —Diabetic Rats -Q— Normal Rats Time (Weeks After Streptozotocin) Figure 4-16. A. single dose of streptozotocin (50 mg/kg, i.p.) causes thermal hyperalgesia in the paw with time in streptozotocin-induced diabetic model rats. 183 500-1 'oo 400" Diabetic Rats 300- Normal Rats ^200- CQ 100- O-" 1 1 1 1 1— 0 2 4 6 8 Time (Weeks After Streptozotocin) Figure 4-17. A single dose of streptozotocin (50 mg/kg, i.p.) causes a significant body weight loss with time in streptozotocin-induced diabetic model rats. 184 15r O- -0—o—o o—o—o- BO 12 2o •y.u g .Start injections Diabetic ASl H Stop injections EMabetic MMl r3 Normal Saline •a w -ts- Diabetic Saline s 31 OL_u J I I L. J L. _L 3 4 5 6 7 8 9 Time (Days) Figure 4-18. ASl, but not MMl, ODN to PN3 (45 ng, twice daily, i.th.) reverses tactile allodjmia in diabetic rats. Aliodynia reappears in diabetic rats after cessation of AS I ODN. 185 30»- Diabetic ASl Diabetic MMl 3: 20- NchitoI Saline Di^tic Saline Start injections Stopuqections 3 4 5 6 7 1ime(DE^) Figure 4-19. ASl, but not MMl, ODN to PN3 (45 Jig, twice daily, i.th.) reverses thermal hyperalgesia in the paw in streptozotocin-induced diabetic rats. Thermal hyperalgesia reappears in streptozotocin-induced diabetic rats after cessation of ASl ODN. 186 Figure 4-20. ASI, but not MMI, ODN to PN3 (45 J.Lg, i.th., twice daily for 5 days) knocks down the PN3 protein in the ipsilateral L4 DRG of Chung model rats. 187 Figure 4-21. ASl, but not MMl, ODN to PN3 (45 J..Lg, i.th., twice daily for 5 days) knocks down the PN3 protein in the Ls DRG of Chung tnodel rats. 188 Figure 4-22. ASl, but not MMl, ODN to PN3 (45 J.Lg, i.th., twice daily for S days) does not alter PNl or PN4 labeling in the L4 DRG of normal rats. 189 -Q 1200 Figure 4-23. Dynorphin content increases in the ipsilateral dorsal quadrant of the lumbar region of the spinal cord in Chung model rats (10 days after Chung surgery). ASl, but not MMl, ODN to PN3 (45(ig, twice daily i.th. for 5 days) reverses increased dynorphin levels back to normal in Chung model rats (p < 0.01). (Shown with permission of Dr. T. Philip Malan Jr. and Mr. Mohab Ibrahim at the University of Arizona, Department of Anesthesiology). 190 Figure 4-24. The PN3 staining increases in the L4 DRG in streptozotocin-induced diabetic model rats compared to that in age-matched normal rats. ASl to PN3 ODN (45 ~g, twice daily for 5 days, i.th.) knocks down the PN3 protein in the L4 DRG of streptozotocin-induced diabetic and age-matched normal rats. 191 Saline 5 ~~ x 2 for 5 days, i.th. MMl 45 ~Lg X 2 for 5 days, i.th. Cessation of ASl for 5 days Figure 4-25. ASl, but not MMl, ODN to PN3 (45 Jlg, i.th., twice daily for 5 days) knocks down the PN3 protein in the L4, L5 and L6 DRGs in streptozotocin-induced diabetic model rats. The knock-down effect is reversible. 192 % Small diameter neurons, % Large diameter neurons, which are PN3 positive (n) which are PN3 positive (n) Normal L4/L5/L6 78% (104) 33% (139) DRG Chung lA DRG 55% (54) 77% (76) With Saline Chung L5/L6 18% (115) 10% (112) With Saline Chung L4 DRG 8% (271) 18% (199) With ASl to PN3 Chung L4 DRG 43% (157) 81% (196) With MMl toPN3 Table 4. PN3 staining increases in large diameter neurons in the ipsilateral LA DRG and decreases in large and small diameter neurons in Chung model rats 7 days after Chung surgery. I.th. ASl, but not MMl, ODN to PN3 (45ng, twice daily for 5 days) knocks down PN3 protein in L4 DRGs significantly (p < 0.01). 193 Summary 4 The present study has demonstrated that spinal administration of specific AS ODNs to the TTX-r sodium channel, PN3, produces a selective block of channel protein expression and prevents the development of tactile allodynia and thermal hyperalgesia, principle manifestations of neuropathic pain in Chung model rats. The AS ODNs also reversed these symptoms once they had become established after the Chung injury. The lack of effect of the corresponding MM ODNs, together with the lack of effect on baseline (i.e., uninjured) responses to non-noxious or noxious stimuli, suggests that this was not a non-specific artifact due to repeated injections of an oligodeoxynucleotide. Intrathecal administration of ODNs have previously been demonstrated to alter the expression of proteins in the spinal cord, the DRG and peripheral nerve terminals (Bilsky et al., 1996; Khasar et al., 1996). Further, the ASl, but not MMl, ODN affected the expression level of the PN3 channel protein in the DRG and neither ODN had an effect on the TTX-s sodium channels PNland NaCh6/SCN8A/PN4 (Toledo et al., 1997; Sangemeswaran et al., 1997; Schaller et al., 1995; Burgess et al., 1995; Dietrich et al., 1998). The present data suggest that the behavioral consequences of Chung peripheral nerve injury, chronic inflammation and diabetic painful neuropathy require de novo PN3 protein synthesis. This view is bome out by noting that inhibition of PN3 protein expression by AS ODN in the DRG does not affect thresholds to normal noxious and non-noxious sensory inputs, and fails to affect the hyperalgesia seen following carrageenan, an acute inflammation with a time-course which would not likely be 194 associated with new channel protein synthesis (Waechter et al., 1983). However, prevention of protein synthesis by pretreatment with AS ODN prior to chronic injuries, such as Chung or CFA, results in a complete inhibition of the development of the behavioral consequences of the injury. Similarly, the increased number of large diameter cells expressing PN3 protein in the Chung model may relate to the observed allodynia which is rapidly reversed (within 2 days) following AS ODN administration. The evidence also provides additional support for the involvement of uninjured primary afferents in adjacent segments of the sciatic nerve in mediating certain types of neuropathic pain behaviors (Yoon et al., 1996). In this respect, the effect of Chung injury on the immunolabeling pattern of PN3, at the level of the DRG, is more complex than that described previously for either the chronic, sciatic nerve constriction injury (CCI) or transection (neuroma) models (Novakovic et al., 1998). Thus, in the CCI and neuroma models, much of the labeling of PN3 disappears from all sizes of DRG cells in L4-L6, indicative of an injury to the main trunk of the sciatic, with a subsequent translocation of the predominantly intracellular pool of channel protein to the site of injury. It has been argued that the increased density of PN3 channel protein, as well as that of other sodium channels at the site of injury is a major contributor to the hyperesthesias observed in these models (Devor et al., 1993; England et al., 1996). In this regard, the loss of protein from the L5 and L6 DRG cells might be reflecting a similar re-distribution process of PN3 protein at this level following Chung peripheral nerve ligation injury. On the other hand, the PN3 protein level of in the L4 DRG was preserved or even increased. However, this most likely reflects the intact nature of the 195 sensory input at the level of L4 and therefore the differential availability of factors, e.g. NGF, released from peripheral tissues and retrogradely transported to the ganglion where they have been suggested to control mRNA expression of PN3 as well as other sodium channels (Black et al., 1997; D'Arcanggelo et al., 1993; Zur et al., 1995). It remains to be determined the extent to which the immunolabeling pattern observed in the L4 ganglion translates into axonal accumulation of the channel protein. Collectively, therefore, the data I Study 4 suggest that the primary symptoms of neuropathic pain may be significantly attenuated by interfering with the expression and, consequently, the function of PN3. The ASl ODN mediated prevention of thermal hyperalgesia induced by CFA and diabetic neuropathy, but not by carrageenan, treatment indicates that the clinical potential for a selective inhibitor of PN3 may extend to pain resulting from chronic tissue, as well as nerve, injury. Such an effect is also consistent with recent evidence indicating that hyperalgesic substances associated with tissue injury can alter the function of PN3 (England et al., 1996; Gold et al., 1996). The lack of an effect of the AS 1 ODN in the tail flick and paw-flick, and paw- pressure tests, together with the lack of effect of the AS 1 ODN on the consequences of an acute inflammation (i.e., carrageenan) where the time-course of the response makes new expression of the PN3 protein unlikely, suggests that this channel does not play a role in normal nociceptive function. Clinical evidence has shown that the most effective therapy for neuropathic pain caused by diabetes mellitus is sodium channel antagonists such as antidepressants (Wright, 1994). Therefore, it is reasonable to hypothesize that chronic diabetic 196 neuropathy could be caused by accumulations or phenotype switches of certain type of sodium channels in DRGs. The results from study 4 that there is an increased expression of PN3 in DRGs and that PN3 AS specifically and reversibly knocks down the PN3 expression in DRGs and reverses neuropathic pain in streptozotocin diabetic model rats provide solid evidence that PN3 is at least responsible for neuropathic pain states in diabetic neuropathy. Phenotype switches of PN3 in the diabetic neuropathy is an interested research topic in future studies. The study 4 has shown that after peripheral nerve injury there is dramatically increased expressions of PN3 in large diameter neurons in the DRG. Consequently, the data in Study 4 strongly suggest that selective inhibition of PN3 will not result in changes in normal, nociceptive function. The collective profile of the AS ODNs against PN3 therefore clearly implicates this channel in the pathophysiology of pain following nerve and tissue injury. The lack of any overt, particularly CNS, adverse events with either AS ODN was consistent with the previously described discrete localization of the channel to sensory nerve fibers with an absence of staining in CNS and cardiac tissue (Novakovic et al., 1998). It also reaffirms the potential that a selective inhibitor of PN3 may be clinically analgesic, providing not only an improved therapeutic window over existing therapies, but offering relief from neuropathic pain symptoms of various disease states, which are mostly resistant to current clinical therapies. 197 CONCLUSIONS Central sensitization, the hyperexcitability of spinal processing that often accompanies peripheral nerve injury is a major component of many neuropathic pain states (Kerr et al., 1999). Our electrophysiological data (unpublished) from flexor reflex experiment have shown an increased wind-up effect to high-intensity electric stimuli in the spinal cord of spinal transected Chung model rats compared to sham or normal rats, suggesting central sensitization. Findings from study 1 provide fiirther support to the hypothesis that peripheral nerve injury results in increased dynorphin levels, which in turn contributes to central sensitization by acting at NMDA receptors in the spinal cord. The clinical evidence has shown that morphine causes neuropathic pain symptoms in patients (Heger et al., 1999; Weber, 1998). Research evidence has also shown that high doses of morphine i.th. produce algesia and hyperalgesia as opposed to the analgesia found at lower doses and that this hyperalgesia is not naloxone reversible (Woolf, 1981). Either acute large doses of morphine that produce neuropathic pain symptoms or chronic exposure to morphine that produces morphine tolerance results in increased levels of dynorphin in the spinal cord. Furthermore, the morphine analgesic dose-response shifts to the right in either neuropathic pain model animals or morphine tolerant animals (Wang et al., 1999; Georges et al., 1999; Wan et al., 1998; Nichols et al., 1997). Study 2 provides further evidence to support the hypothesis that increased dynorphin levels in the spinal cord may be a common pathological factor in neuropathic 198 pain states caused by peripheral nerve injury as well as morphine tolerant states. The increased levels of dynorphin in the spinal cord are not only responsible for central sensitization to cause neuropathic pain due to peripheral nerve injury or large doses of morphine, but also for the decreased analgesic potency of morphine in these states (Mayer etal., 1999). Besides central sensitization, study 2 provides evidence that neuropathic symptoms due to peripheral nerve injury involve different spinal or supraspinal processes and different central mechanisms for allodynia and thermal hyperalgesia. Study 2 supports the hypothesis that the neuronal mechanisms in the spinal cord underlying two different manifestations of neuropathic pain such as tactile allodynia and thermal hyperalgesia are different, which is consistent with another report in the literature (Jett et al., 1997). Our previous data have shown that i.th. high doses of MK- 801 only block thermal hyperalgesia, not allodynia in Chung model rats, suggesting the involvement of different afferent fibers for different neuropathic pain manifestations and differential impacts of central sensitization on these manifestations, such as allodynia and thermal hyperalgesia (Wegert et al., 1997). A single large dose of systemic RTX irreversibly depletes vanilloid receptor 1 (VRl) containing c-fibers by neurotoxicity (Goso et al., 1993; Farkas et al., 1996). Study 3 reveals the lack of afferent c-fiber involvement in allodynia in the Chung neuropathy. Plenty of evidence has shown that VRl containing c-fibers are composed of small neurons in the DRG, which transfer noxious heat stimulation signals to the central nervous system. Some thermal nociceptive sensory neurons are not VRl 199 positive (Snider et al., 1998). Since there are reciprocal inhibitory interactions between A(3 fibers and c-fibers, the lack of VRl positive c-fibers due to RTX treatment may augment AP fiber-mediated allodynic response to some extent (Fields et al., 1997). The cloning of a TTX-r sodium channel PN3 (SNS) exclusively expressed in the DRG primary sensory neurons indicated a breakthrough for the further understanding of peripheral mechanisms of neuropathic pain (Sangameswaran et al., 1996: Akopian et al., 1996). Study 4 used antisense to PN3 to eliminate neuropathic pain symptoms in Chung model and diabetic model rats, which indicates the role of PN3 in manifestations of neuropathic pain due to nerve injury and diabetic neuropathy. Therefore, selective blockade of the PN3 sodium channel may represent a new therapy for neuropathic pain with less or without common side effects from non-selective sodium channel blockers. Results from Study 4 suggest that the PN3 sodium channel protein does not play a physiological role in maintaining normal nociceptive functions, but becomes important in mediating spontaneous firing after [jeripheral nerve injury and in diabetic neuropathy, and that further understanding mechanisms of increased expression of PN3 or of phenotype switch would be valuable. Study 4 provides a successfiil example of in vivo antisense therapy for neuropathic pain and indicates antisense strategy as a powerful tool to validate therapeutic targets in pharmacological research and as a possible therapeutic agent. Above all, the main research work in this dissertation has provided evidence to elucidate mechanisms for neuropathic pain states from the periphery to the spinal cord. 200 REFERENCES Aanonsen, L.M. and Wilcox, G.L., Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and sigma agonists. J. Pharmacol. Exp. 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