Segmental, Axonal, and Demyelinative Lesions in the Trigeminal System Produce Neuropathic Pain

Segmental, Axonal, and Demyelinative Lesions in the Trigeminal System Produce

Neuropathic Pain

William R. Bauer, M.D.

Medical College of Ohio

2005

DEDICATION

To my wife Kate, my family, and patients, may they all suffer less pain.

ii TABLE OF CONTENTS

INTRODUCTION…………………………………………………………………...……1

LITERATURE REVIEW………………………………………………………...... 5

CHAPTER 1: Loose Infraorbital Nerve Ligatures Do Not Produce

Neuropathic Pain……………………………………………………………….….19

CHAPTER 2: Behavioral and Immunocytochemical Evidence of Trigeminal

Neuropathic Pain Produced by a Partial Infraorbital Nerve Lesion...... ……....…43

CHAPTER 3: Behavioral Evidence of Glycerol Induced Trigeminal Neuropathic

Pain in Sprague-Dawley Rats……………………………………………………..74

DISCUSSION/SUMMARY……………………………………………………………110

REFERENCES…………………………………………………………………………119

ABSTRACT…………………………………………………………………………….154

iii INTRODUCTION

One would expect that damage to a sensory nerve would produce sensory loss with the degree of loss roughly proportional to the amount of damage. This is the usual result.

Sometimes, when peripheral somatosensory nerves are damaged by disease or trauma, a small percentage of cases develop positive symptoms. These symptoms are almost always various kinds of pain rather than numbness or tingling. The pain is said to be neuropathic because it is believed to be due to a dysfunctional nervous system.

Neuropathic pain (NP) is commonly observed in the location of the primary sensory neuron as opposed to a more peripheral location. Neuropathic pain is considered to be a serious clinical problem that remains poorly understood and poorly treated by many physicians. The International Association for the Study of Pain (IASP) defines NP as

“pain initiated or caused by a primary lesion or dysfunction in the nervous system”

(Merskey and Bogduk, 1994). The IASP defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.” The etiologic classification of NP covers a variety of disease states including phantom limb, spinal cord injury, late-stage cancer, painful diabetic neuropathy, post-herpetic neuralgia, HIV, vinca-alkaloids, sciatica, multiple sclerosis, atypical facial pains, and trigeminal neuralgia (TN).

Neuropathic pain frequently becomes chronic in nature. Chronic pain (CP) is among the most disabling and costly afflictions in North America, Europe, and Australia. The IASP

1 provides a widely used definition of CP as “pain without apparent biologic value that persisted beyond the normal tissue healing time” (usually taken to be 3 mos.). The prevalence rate of CP was recently reviewed by the IASP (2003) in 13 studies and ranged from 10.1% to 55.2%. There is clinically significant CP found in 65% of persons diagnosed with multiple sclerosis (MS). These painful conditions include TN, painful optic neuritis and Lehrmitte’s syndrome (Kerns et al., 2002).

Trigeminal neuralgia is a well-recognized complication of MS and is thought to be due to plaques of demyelination in the trigeminal root entry zone, or less commonly, the brainstem (Jensen et al., 1982; Moulin et al., 1988; Soustiel et al., 1996; Moulin, 1998).

The examination by several laboratory groups of trigeminal rhizotomy specimens from patients with MS and intractable TN in the absence of a trigeminal compressive lesion reveal that demyelination has been found to extend along the proximal part of the trigeminal nerve root, in some cases right up to the junction with the peripheral nervous system (Love et al., 2001; Lazar and Kirkpatrick, 1979; Hilton et al., 1994). Moreover,

many of these rhizotomy specimens reportedly exhibited inflammatory disease activity,

as evidenced by the presence of lipid-laden macrophages. Another finding in most of these specimens is the closely juxtaposed bundles of thinly myelinated axons (Love et al.,

2001). As there is good experimental evidence that ectopic impulses can arise from demyelinated axons (Rasminsky, 1978; Smith and McDonald, 1980, 1982), it is possible

that the pathophysiology of TN is due to hyperactivity or abnormal discharges arising

from the trigeminal ganglion (Moller, 1991; Burchiel, 1993; Pagni, 1993; Rappaport and

Devor, 1994; Moulin, 1998). It is also possible that the abnormal generation of sensory

2 impulses from fibers conveying light touch to fiber pathways involved in the perception of pain, could cause the pathophysiologic effects of TN. It is more likely a combination of these effects contribute to the symptomatology of any given patient with TN. While it is important to diagnose the spectrum of disease conditions that can result in NP, disease diagnosis by itself is seldom helpful in selecting the optimal pain therapy. An important facet of the pain diagnosis is the proper identification of the neurobiological mechanisms responsible for neuropathic pain. The use of a mechanism-based approach to this condition offers the possibility of greater diagnostic sensitivity and a more rational basis for therapy.

Trigeminal neuralgia probably involves peripheral sensitization, alteration of ion channel expression, ectopic and spontaneous discharge, ephaptic conduction and sprouting of sympathetic axons into the gasserian ganglion. Central sensitization, cortical reorganization, spinal reorganization, and changes in inhibitory pathways are also likely to be involved in NP syndromes. These mechanisms reveal the complex nature of the response to any peripheral nerve injury. Current knowledge of peripheral, spinal, and brain events following disease or trauma suggest that many mechanistic aspects are involved in NP syndromes. The study of the mechanisms central to NP, especially that seen in patients with TN, is fundamental to the development of new therapies.

The overall goals of this dissertation project were to characterize three specific models of pain in the trigeminal system of the rat and evaluate the lesion site pathology associated with each model for evidence of changes that may contribute to the nature and

3 persistence of NP. Experimental methods used in these studies included anatomical pathology and behavioral measures in the male adult rat. The pain models used in these studies were chronic loose circumferential ligatures of the trigeminal infraorbital nerve

(ION), tight partial trigeminal ION ligature, and glycerol injection into the trigeminal ganglion.

4 LITERATURE REVIEW

One of the most common and debilitating clinical sensory disorders that can result from injury to primary afferent neurons is pain from mechanical contact with the skin (Bonica,

1990). Normally non-noxious stimuli such as brushing against clothing, or a puff of air might now elicit pain (tactile allodynia), however stimuli with sharp features, such as a stiff bristle, or the rough surface of sandpaper, will elicit considerable pain that outlasts the stimulus (mechanical hyperalgesia). In addition to chronic, spontaneous NP, the mechanical dysesthesia of allodynia and hyperesthesia are most troublesome because of our daily need to interact with objects in our environment. Given that these are the most common types of pain seen in clinical practice, elucidation of their mediators in the somatic or the trigeminal distribution of sensory nerve fibers, and their cellular sources and mechanisms of action is not only of scientific interest but also of significant clinical relevance.

Knowledge of the neuroanatomy of the somatosensory system is necessary for a clear understanding of the pathophysiology and mechanisms of NP. The anatomical pathways which transmit vision or hearing to the central nervous system normally involve four main components. These components include transduction of physical into electrical energy (receptor), the integration of the electrical signal (somal trigger zone), the conducting axon, and the signal output (synapse). Sensory signals are received by the receptor, which is a specialized structure that aids in the recognition and initiation of signal transmission. The receptor or generator potential is the transforming event that

5 changes the various stimuli (e.g. pressure, touch, temperature) into electrical and chemical signals for transmission. Signal integration occurs at the trigger zone whereby an action potential can be generated and sent down the axon. The action potential is transmitted centrally via the axon to the pre-synaptic terminal where it signals chemical transmitters to be released from vesicles into the synapse and then diffuse to the post- synaptic site on the central post-synaptic terminal. Binding of the neurotransmitter at the post-synaptic receptor permits additional signals to be generated and sent to thalamus and sensory cortex.

The ION of the trigeminal ganglion, like other sensory nerves, is composed of axons of different diameters and degrees of myelination. These axons convey modality specific signals, such as nociception, thermoreception, mechanoreception, and proprioception at different velocities to distinct second-order terminations in the brainstem nuclei.

The main types of primary sensory afferents include the Aα-, Aβ-, Aδ-, and C-type fibers. Table I provides a summary of fiber diameter, conduction velocity, presence of myelin and stimuli for activating these fibers (Martin, 1985; Fields, 1987). Note that diameter and conduction velocity will vary from species to species. These examples are from initial work done in the cat (Gasser, 1960; Burgess and Perl, 1973).

The variation in axon diameter and myelination affects the conduction properties of the neuron types. These variable factors can alter the speed at which different signals are transmitted and allow one to determine neuron fiber types based on the calculation of conduction velocity and evaluation of electrophysiological properties.

6 Table I Classification of Nerve Fibers

Sensory Largest Fastest Fibers Fiber Conduction General Comments Diameter Velocity (m/s) A-α 22 120 Myelinated - Muscle spindles A-α 22 120 Myelinated - Tendon organs, touch and pressure receptors A-β 13 70 Myelinated - Touch and pressure receptors, pacinian corpuscles (vibratory sensors) A-δ 5 15 Lightly Myelinated; Touch, pressure, pain, and temperature C 1 2 Unmyelinated; Pain and temperature

Nociceptors are receptors that are activated by damaging or potentially damaging stimuli

(Martin, 1985; Woolf, 1987). There are three classes of nociceptive fibers that contain

these nociceptors (1) the Aδ mechanosensitive nociceptor, (2) the mechanothermal

nociceptor, and (3) the C-type polymodal nociceptor (C-PMN or C-fiber) (Fields, 1987).

Aδ-nociceptors are primarily responsive to mechanical stimuli while the C-fiber

nociceptors are activated by a cascade of stimuli, including mechanical, chemical, and

thermal (Burgess and Perl, 1973; Torebjork, 1974; Fitzgerald and Lynn, 1977;

Georgopoulos, 1977; LaMotte et al., 1983). The first, fast, sharp pains are mediated by

the Aδ fibers, while the second, slow, burning and aching pains are mediated by C-fibers.

These classes of fibers have the ability to become sensitized (Woolf, 1987). Thus, Aδ

fibers can become responsive to lower threshold stimuli and have an increased response

to the stimuli following repeated noxious input (Fitzgerald and Lynn, 1977).

7 Additionally, antidromic stimulation allows the sensory axon to act as an efferent effector by releasing substances in the periphery (Otsuka and Yoshioka, 1993; Szolcsanyi, 1988).

This process is also known as neurogenic inflammation (Jancso, 1968).

In CP states, there is a conditon whereby other primary afferent fibers can contribute to the pain experience. In certain pain states like NP, Aβ fibers can begin to elicit painful responses when activated by a stimulus such as a light brushing stroke across its receptive field (Woolf, 1992). Clinically, this response is known as tactile allodynia. The development of tactile allodynia or tactile hyperesthesia may be initiated following an injury and tissue damage and involve numerous factors, including neural inputs, release of neuropeptides, activation of multiple receptors centrally, and activation of nuclear genes (Carstens, 1996).

While this thesis focuses on the peripheral aspects of nociception, it is important to understand that changes occurring peripherally are not without consequences centrally.

Within the dorsal horn of the spinal cord and subnuclei in the trigeminal system are multiple inputs which have excitatory effects on the overall signal process. These signals represent either fast or slow conduction of information being relayed. The excitatory post-synaptic potential is driven by actions of fast transmitters that include L-glutamate and L-aspartate, which produce a fast depolarization of the second order neuron that can last on the order of milliseconds (Woolf, 1987). The binding of these transmitters have differential effects depending on the post-synaptic receptor that they bind (e.g. kainate versus N-methyl-D-aspartate [NMDA]-receptors) (Watkins and Evans, 1981). Activation

8 of NMDA receptors centrally is intricately involved with hyperalgesia, hyperesthesia, and hyperexcitability of dorsal horn neurons (Coderre and Melzack, 1992). Slow transmitters would include Substance P (SP), which acts to produce long lasting effects on the magnitude of seconds to minutes (Woolf, 1987). When a brief high frequency stimulus is applied to primary afferents, the central neurons can respond by having sub-threshold, SP mediated prolonged depolarizations that can affect the response to other primary afferent inputs (Urban and Randic, 1984). Hyperalgesia following injury and inflammation can occur due to sensitization of the peripheral receptors and of the dorsal horn neurons

(Traub, 1996).

Volumes of information regarding the normal anatomy and components of disease and trauma-induced nerve injury have been collected on the somatosensory system below the foramen magnum. In sharp contrast, there is a relative paucity of information regarding the normal anatomy and pathology of the trigeminal system in the rodent or in the human.

Known features of the trigeminal system include a topographic map that reflects the distribution and density of receptors on the surface of the head. The relationship between peripheral and central structure was noted by Woolsey and Van der Loos (1970), to be correlated with the distribution of multi-cellular units in layer four of mouse somatosensory cortex with the distribution of mystacial vibrissae on the snout. Tactile sensation is subserved by a diverse group of mechanoreceptors distributed throughout the body surface in a nonrandom fashion (see Table I). In small rodents, a particularly important collection of receptors are located on the snout (Woolsey et al., 1975). These are associated with mystacial vibrissae. Each mystacial vibrissae follicle is completely

9 innervated at two levels of the hair shaft, and contains a rich complement of

mechanoreceptor types, including Merkel discs, Golgi-Manzoni, Lanceolate, and free

nerve endings (Andres, 1966; Renehan and Munger, 1986). The rat ION contains

approximately 33,000 nerve fibers (Jacquin et al., 1984).

Sensory Ganglia

The pseudounipolar somata of neurons in mammalian sensory ganglia do not have

synaptic contacts, and traditionally have been thought to play only supportive roles in the

maintenance of their axons that transmit sensory information from the periphery to

central terminations (Lieberman, 1976; Hall, 1992). This view is changing now with

evidence pointing to the dorsal root ganglia (DRG) and trigeminal ganglia (TRG) as

possible sites for modulating electrical signals in normal and pathological transmission

(Kirk, 1974; Lu et al., 1993; McLachlan et al., 1993; Devor et al., 1994). Without synaptic contacts, the somata of sensory neurons respond to many neurotransmitter and neuromodulators (Lieberman, 1976). Intraganglionic modulation of sensory signals may be chemically mediated by diffusible substances being released from cell bodies and affecting neighboring cell bodies via non-synaptic, cell-to-cell cross-excitation (Amir and

Devor, 1996; Shinder and Devor, 1994). During cross-excitation, sustained activity in one cell can lead to the discharge of neighboring cells (Devor and Wall, 1990). This interaction may be related to specific neurotransmitter actions within the ganglia (Amir and Devor, 1996; Devor and Wall, 1990).

10 Abnormal electrogenesis can occur after norepinephrine (NE) application either to the

DRG itself or to the injured site. This does not occur with normal, uninjured nerve

(LaMotte et al., 1996). The development of NE sensitivity of injured axons in these models also resembles human neuropathic conditions such as reflex sympathetic dystrophy (Burchiel, 1993; Sato and Perl, 1991; Xie et al., 1995; Perl 1999).

The generation of action potentials and development of NE sensitivity are not due to the release of extrinsic factors from the nerve terminals or from sympathetic efferent fibers

(LaMotte et al., 1996). This ectopic discharge may be due to the upregulation and expression of sodium channels leading to the increased ion conduction and firing of the neurons (Burchiel, 1984). Ectopic discharge can lead to sensory fiber cross-excitation, where abnormal activity from the injured nerve fiber can activate neighboring neurons

(Devor and Wall, 1990).

The identification of sensory ganglia as a modulatory site for transmission of sensory signals under normal physiological conditions now provides a new area of investigation into the development and maintenance of CP states. For example, peripheral nerve injury and chemical noxious stimuli have been shown to affect mRNA levels and synthesis of various neuropeptides, including galanin (GAL) levels in sensory ganglia (Zang and

Lundeborg, 1997, 2000; Rhoades et al., 1997). Electrophysiological evidence suggest that one possible contributing factor for allodynia and hyperalgesia commonly seen in peripheral nerve injuries may be due to increased amounts of GAL produced and released

11 from neurons in the sensory ganglia concomitant with an intraganglionic enhancement of

nocieceptive signal transmission.

Galanin is a 29 amino acid peptide which has important central nervous system actions

that may have a role in the therapy of Alzheimer’s disease, depression, eating disorders,

and NP. The actions are mediated via G-protein-coupled receptors and ion channels

usually producing inhibition of secretion of transmitters or hormones in the nervous and

endocrine system respectively. Galanin has been shown to have increased

immunoreactivity in the dorsal horn, gracile nuclei, and in sensory neurons following the

chronic constrictive injury (CCI) compared to complete sciatic transection. This suggests a facilitatory role in thermal and mechanical hypersensitivity or allodynia 9Ramer et al.,

1998). Galanin also is involved also in the development and regeneration of sensory neurons as exemplified by the reduced rate of peripheral regeneration after injury in a reduction of the number of neurons expressing SP in knockout mice (Holmes et al.,

2000). The expression of GAL in sensory neurons was more limited after partial injury

than after complete nerve injury where endogenous GAL plays an inhibitory role Xu

(2000). The increased GAL expression indicates injury to the infraorbital nerve (ION).

Galanin may have a role in the modulation of nociception after peripheral nerve injury

(Wiesenfeld-Hallin et al., 2004). This group has reported on GAL receptor subtypes with inhibitory and excitatory effects, and a third receptor with trophic actions on the DRG.

The role of GAL and modulation in ION transection and the trigeminal system has been studied extensively by Rhoades et al.(1997). The inhibitory, excitatory and trophic effects

12 of GAL could alter channel expression, ectopic discharge, ephaptic conduction, axonal

sprouting and brainstem reorganization resulting in suppression of the NP.

Animal Models of Neuropathic Pain

The recent development of animal models that mimic human NP conditions has greatly

advanced the study of NP. Most animal models of NP in humans produce mechanical dysethesia by axotomizing peripheral, spinal or dorsal root segments of primary sensory neurons (Bennett and Xie, 1988; Kim and Chung, 1992; Seltzer and Dubner, 1990; Wall,

1974). After axotomy, a subpopulation of injured neurons, including those with myelinated and those with unmyelinated axons, become hyperexcitable and exhibit abnormal, ectopic discharges (Devor, 1994). The peripheral axon terminals in nerve-end neuromas or the cell bodies in the DRG of these injured neurons may exhibit abnormal excitation to mechanical, thermal or to endogenous chemicals such as adrenergic agonists or inflammatory mediators. Mechanisms proposed to cause the enhanced excitability include the increased or decreased expression of neuronal neuropeptides (Noguchi, 1995;

Neumann, 1996; Jimenez-Andrade, 2004; Zhang, 1998), various cytokines and growth factors, for example from immune cells or glia (Lu and Richardson, 1993; Murphy et al.,

1995; Myers, 1995; Wagner and Myers, 1996; Watkins, 2002), altered expression or kinetics of sodium or potassium ion channels (Rizzo et al., 1995; Waxman et al., 1994;

Devor et al., 1994; Everill et al., 1998), second messengers and chemical, mechanical and thermal receptors (Devor, 1994; Birder, 1999). Spontaneous NP may be caused in part

13 by the abnormal spontaneous activity in injured primary sensory neurons that retain central connectivity with nociceptive spinal neurons, or that acquire a novel connectivity via central sprouting (Devor, 1994; Woolf, 1992). Primary sensory neurons that normally release SP, and those that acquire that capacity via an injury-induced phenotypic switch, may sensitize the responses in spinal neurons to input from low-threshold and nociceptive primary sensory neurons, thereby maintaining a neuropathic state of tactile allodynia and mechanical hyperalgesia (Noguchi, 1995; Neumann, 1996).

Peripheral injury to trigeminal nerve subdivisions, not unlike damage to sensory neurons in the lumbar DRG, will produce abnormal discharges (Bongenhielm, 1996, 1998;

Chudler, 1997; Tal, 1992) and alterations in behavior (Chudler, 1997; Berridge, 1986;

Imamura, 1997; Vos, 1994). Behavior changes include paresthesia and dysesthesia

(Imamura, 1997; Vos, 1994,1998).

The first attempt to produce an animal model of TN was reported by King et al. (1956).

They injected alumina gel into the caudal portion of the trigeminal brainstem complex of cats and showed over-reaction to tactile stimulation of the face. Experimental injury to the trigeminal nerve (e.g., transection, nerve crush, tooth pulp removal) in animals often causes anatomical, neurochemical, and electrophysiological changes in peripheral and central neurons (Fromm and Sessle, 1991). A previous study of trigeminal root injury by

Burchiel (1980) showed electrophysiological evidence of abnormal impulse generation in focally demyelinated trigeminal roots using implanted chromic sutures. This study lacks a detailed behavioral analysis of animals before and after experimental trigeminal injury.

14 Behavioral evidence of trigeminal NP following CCI to rat ION was first reported by Vos and Maciewicz, (1991,1994). This model reported behavioral changes they believe are typical of human trigeminal neuralgia with increased facial grooming and decreased response to mechanical stimulation in the injured nerve territory.

The majority of animal models have been based on peripheral nerve injury either complete or partial, but some recent models have tried to mimic individual disease states.

The streptozotocin model of peripheral diabetic neuropathy was reported by Malcangio and Tomlinson (1998). In this model a single injection of streptozotocin induces diabetes and neuropathic behavior, which was used extensively in the testing of gabapentin. A model of acute herpes zoster has been reported by Fleetwood–Walker et al.(1999), in which painful behavior follows the infection with varicella–zoster virus injected in rats.

Another example described by Idanpaan-Heikkila and Guilbaud (1999), used a CCI of the ION as a model for TN complete with pharmacological treatment. This group found that baclofen, but not carbamazepine, morphine or tricyclic antidepressants attenuated the

CCI-induced allodynia.

The suture material, along with placement and degree of constriction of the suture material play a significant role in the production of the lesion. In a study by Maves et al.

(1993) four ligatures of silk (4-0), plain gut (4-0), or chromic gut (3-0) were placed loosely around the sciatic nerve of male Sprague Dawley rats. Chromic-gut, but not plain gut or silk ligatures produced abnormal behavioral responses in the heel and paw of the affected limb.

15 The development of animal TN models also includes injections of glycerol. The use of

glycerol treatment was discovered during the development of a stereotaxic technique for

gamma irradiation of the gasserian ganglion to treat patients with TN (Leksell, 1971;

Hokanson and Leksell, 1979). It was used as a vehicle to deposit tantalum dust as a marker in the trigeminal cistern. Glycerol was chosen for its high viscosity and use with phenol for gasserian injections (Jefferson, 1963). Hokanson noted that the glycerol injection alone abolished pain completely in several patients for long periods, along with markedly well-preserved facial sensation. He developed a routine method of treating patients with trigeminal neuralgia with glycerol (Hokanson, 1981), but this procedure has been replaced by micro-vascular decompression and other methods of disruption of the ganglia.

Lunsford et al. (1985) first correlated the effects of glycerol injection into the trigeminal cistern of cats. Glycerol increased the average latencies and reduced the average amplitudes of the trigeminal brainstem evoked potentials. Histopathologic examination disclosed focal demyelination, axonal swelling, endoneurial fibrosis, and neuronal loss.

They concluded that pain abolition might be related to the effect of glycerol on the heavily myelinated large diameter fibers. The central portion of the trigeminal sensory root and the trigeminal tract are composed primarily of larger myelinated fibers, whereas the periphery of the root and a portion of the trigeminal tract just below the surface contained a higher percentage of smaller myelinated and unmyelinated axons (Rhoades et al., 1996).

16 Electron microscopic evidence of demyelination of axons with axonal swelling and proliferation of Schwann cells with accumulation of neurofilaments was reported by

Burchiel (1980). His study of 12 cats and two monkeys showed chronic focal injury to the trigeminal root with the implantation of chromic suture in the nerve near its entry into the brainstem. It was speculated with physiologic studies that the regions of focal injury with demyelination showed spike generation potentials as a mechanism of the neuropathic process.

Early human ultra-structural studies concentrated on the morphology of the trigeminal ganglia and nerve and described a range of abnormalities of the myelin sheaths, including proliferative degenerative changes and myelin disintegration (Kerr and Miller, 1966;

Beaver, 1967). Most of the changes were felt to be artifactual because similar findings were present in some of the control cases. In the first detailed description of ultrastructural abnormalities in the nerve root near the vascular compression, they found focal loss of myelin, close apposition of demyelinated axons, a few residual oligodendrocytes and no inflammatory cells (Hilton et al., 1994). Human rhizotomy specimens showed foci of apposition of demyelinated axons with a paucity of glial and inflammatory cells (Love et al. 1998). There were focal infiltrates of lipid-laden macrophages suggesting there had been recent demyelination. These authors also noted a few specimens from patients with visible vascular compression of the nerve at surgery with ultra-structurally normal studies which may constitute a sampling error rather than heterogeneity in the pathology and pathogenesis of the disorder.

17 The most common cause of TN in the human is focal compression of the trigeminal root

close to its point of entry into the pons by an aberrant loop of artery or vein. This was

first recognized as a cause of TN by Jannetta (1967) and it is now thought to account for

80–90% of the cases. Other rare causes include infiltration of the nerve root, gasserian ganglion or nerve by a tumor or amyloid, and small infarcts or angiomas in the pons or medulla. When all else has been excluded, there is a small proportion of patients in whom the etiology is undetermined (see Love and Coakham, 2001 for a review).

An important group of papers reported the consequences of transection and crush injury of the ION with electron microscopy identifying and characterizing the afferent innervation of the mystacial vibrissae, the response to ION transection and crush injury, and a third paper showing abnormal sensory reinnervation of rat guard hairs following the transection and crush injury (Renehan and Munger, (1986a,b); and Munger and

Renehan (1989). These lesions showed wallerian degeneration (WD) with reduction of the percentage of Merkel cells innervated. They found examples of dramatically unusual innervation patterns and abnormally reinnervated sensory receptors, which could explain the phenomena of faulty sensory localization as a common result of cutaneous nerve lesions in humans. Following ION transection the reinnervated sensory nerve terminals associated with guard hairs were markedly abnormal but following nerve crush, there was quantitatively less reinnervation of sensory terminals. These findings may in part explain the abnormal cutaneous sensory perceptions such as dysesthesia or paresthesia noted in human subjects following damage to nerves with subsequent sensory reinnervation of the skin.

CHAPTER 1

Loose Infraorbital Nerve Ligatures Do Not Produce Neuropathic Pain

Neuropathic pain (NP) is a common and serious problem that remains poorly understood

and not well treated by many physicians. Neuropathic pain frequently becomes chronic

pain (CP) and is among the most disabling and costly afflictions in North America,

Europe and Australia (IASP, 2003). The model of a chronic constrictive injury (CCI) of the infraorbital nerve (ION) proposed by Vos et al. (1994) reported behavioral alterations indicative of sensory disturbances within the territory of the injured nerve with mechanical allodynia and behavior signs of recurrent spontaneous aversive sensations.

This was deemed a reasonable approach to study the pathophysiology of trigeminal neuralgia (TN) and atypical facial pain. The CCI was applied to the ION of 30 male

Sprague-Dawley rats. They were behaviorally tested before and after the lesion with von

Frey monofilaments and pin stimuli to determine if the ligature lesions produced NP.

This lesion did not result in statistically significant behavioral effects. The immunocytochemistry for galanin (GAL) showed increased expression in the nucleus caudalis on the lesioned side. We confirmed a lesion but did not confirm behavioral evidence of trigeminal NP following CCI to the ION as reported by Vos et al. (1994).

INTRODUCTION

Injury to peripheral somatosensory nerves can sometimes result in NP. The International

Association for the study of Pain (IASP) defines NP as “pain initiated or caused by a primary lesion or dysfunction in the nervous system” (Merskey and Bogduk, 1994).

Neuropathic pain is associated with allodynia (non-painful stimulus produces intense and prolonged sensations with an explosive, radiating character) (Kugelberg and Lindblom,

1959) and hyperesthesia (an increased sensitivity to a stimulus) (Merskey and Bogduk,

1994). These somatosensory disorders are seen in the cutaneous region innervated by the damaged nerve.

Neuropathic pain research has concentrated on a group of behavioral animal models

(Wall et al., 1979; Bennett and Xie, 1988; Attal et al., 1990; Seltzer et al., 1990; Kim and

Chung, 1992). These models used a variety of experimental lesions including a transected tightly ligated nerve (neuroma model) (Wall et al., 1979), CCI of a nerve trunk or part of it (Bennett and Xie, 1988; Attal et al., 1990; Seltzer et al., 1990; Kim and Chung, 1992;

Vos et al., 1994).

Previous reports on the behavioral effects of trigeminal nerve injury (Burchiel, 1980), as well as studies of the behavioral effects of chemical irritation of the trigeminal nucleus caudalis (King et al., 1956; King, 1970; Black, 1974; Kryzhanovsky et al., 1974) both in

20 cats and rats, have described recurrent attacks of ipsilateral face grooming along with allodynia to light mechanical stimulation of the injured side of the face. This facial sensory testing is not permitted by humans with trigeminal neuralgia (TN) because of their allodynia.

The model of a CCI of the ION proposed by Vos and Maciewicz (1991), and Vos et al.

(1994) reported behavioral alterations indicative of sensory disturbances within the territory of the injured nerve with mechanical allodynia and behavior signs of recurrent spontaneous aversive sensations. This was deemed a reasonable approach for further study for the understanding of the pathophysiology of TN and atypical facial pain.

It is speculated that the CCI lesion to the ION may change receptive field properties, ION conduction and central activity. This might provide a basis for the pain behavior in the lesioned animals. Renehan et al. (1989) with physiological recordings after ION transection in the adult rat showed increased numbers of nociceptive, guard hair, and high velocity rapidly adapting units. They also reported abnormal sensory reinnervation of rat guard hairs following the transection and crush injury Renehan and Munger, (1986a,b); and Munger and Renehan (1989). These lesions showed changes of wallerian degeneration (WD) and the study found examples of dramatically unusual innervation patterns and abnormally reinnervated sensory receptors, which could explain the phenomena of faulty sensory localization as a common result of cutaneous nerve lesions in humans.

The aim of this study was to confirm that chronic ligature lesions will produce NP.

Materials and Methods

Ethical standards. The animals were treated and cared for according to the ethical standards and guidelines for investigations of experimental pain in animals outlined by the Committee for Research and Ethical Issues of the International Association for the

Study of Pain (IASP) (1983). The animals were maintained in a vivarium accredited by the American Association of Laboratory Animal Care at the Medical College of Ohio.

The animals were housed separately from other rats in the vivarium in a partial reversed dark/light cycle for testing purposes. Careful attention was given to the maintenance of similar care, attention, and handling to each and all of the animals.

Animals. Thirty male Sprague-Dawley rats (250–300 g) were used and housed under partially reversed 12-hour dark/light cycled with lights on at 1700 hours. They were housed in plastic cages in a colony room with food and water available at all times. The rats were randomly assigned to a CCI to the ION ligation, right or left side, or unilateral sham procedures on the right and left for the control condition. Each animal served as its own control using the non-operated side.

Surgery. Rats were anesthetized with ketamine (30 mg/kg, I.P.) and xylazine (15 mg/ kg,

I.P.) and placed in a standard stereotaxic head holder. All surgery was performed under direct visual control with the assistance of a Zeiss operating microscope. The ION in the orbit was exposed using a surgical procedure similar to that described by (Gregg, 1973;

22 Jacquin and Zeigler 1983; Vos et al., 1991 and 1994). The skull and nasal bone was

exposed with a midline scalp incision. The fascia at the edge of the orbit was dissected

free to give access to the ION with gentle deflection of the orbital contents laterally to

allow visualization of the ION. The ION was dissected free at the rostral portion of the

orbital cavity, just caudal to the infraorbital foramen. Two chromic gut (5-0) ligatures

were loosely tied around the circumference of the ION avoiding damage to the vasa

nervorum (Bennett and Xie, 1988). In rats of the sham control group, the ION was

exposed on one side using the same procedure except the exposed nerve was not ligated.

The scalp incision was closed and the animal observed carefully postoperatively and returned to the vivarium dark/light cycle room.

Behavioral testing. We modified the behavioral testing system reported by Vos and

Maciewicz (1991 and 1994). The animals were tested six times before and after surgery.

Testing was conducted from 0700 to 1700 hours. Each test session consisted of observation of free behavior and assessment of the behavioral response to the von Frey monofilaments and the pin.

We made some changes to the testing system reported by Vos and Maciewicz (1991 and

23 important to define the animal’s baseline response to these stimuli. We also took special care to differentiate between a “startle response” as a reflex movement of fright from an aversive movement in response to the mechanical or pin stimulus. In addition to the ordinal scale for behavior response we also used a category of either pain or no pain in response to the stimulus.

Mechanical stimulation. Stimuli. A graded series of three von Frey monofilamnts

(pressure Aesthesiometer, Stoelting Company, Chicago, IL) and a pin were used for mechanical stimulation. The stimuli were 2 gm, 6 gm, 9 gm, (converted to log units, 4.31,

4.74, and 4.93 units respectively), and a pin. A 24 gauge injection needle bent at a 30 degree angle and mounted on a wooden stick was used to apply pinprick stimulations.

The stimulus consisted of two consecutive placements of the stimulus monofilament or the pinprick under one second. The 2 gm light weight stimulus was selected to find allodynia. The more heavily weighted stimuli of 6 gm and 9 gm were chosen to elicit hyperesthesia, allodynia or pain compared to the pin stimulus which was chosen as the pain standard.

Stimulated areas. The stimulus was applied first within the ION territory, near the center of the vibrissa pad on the hairy skin surrounding the mystacial vibrissa, and a second outside the ION territory in the territory of the auriculotemporal nerve on the hairy skin under the ear. These areas were stimulated on both sides of the face, ipsilateral and contralateral to the side where surgery was performed.

24 Testing procedures. For mechanical stimulation the rats were placed in a transparent

plastic cage as described above and each session was recorded on video tape with the

recorder approximately 50 cm in front of the cage. The video tapes of behavior and

scoring at the time of each session were later independently reviewed. Stimulations were

delivered by reaching into the cage in a dark room with light provided by a 60 W

incandescent red bulb suspended one meter above the center of the test area. Reaching movements were performed slowly to avoid a startle response. The animal was adapted to the cage for ten minutes during which time the experimenter would reach into the cage approximately every 30 seconds touching the wall of the cage with a plastic rod similar to the von Frey instrument. Testing was accomplished when the rat was in a sniffing/no locomotion state with four paws on the ground, neither moving nor freezing. A new stimulus was applied only when the rat resumed this position or at least 30 seconds after the preceding stimulation. During one session the complete set of von Frey hair intensities was applied in a randomized order in an ascending/descending series. All four areas were explored with one level of stimulation before using the next intensity.

Response scoring. Responses were examined by the tester at the experiment and by independent evaluators by playing videotaped reactions in slow motion or in real time at a later date. We used a modified descriptive response category after Vos et al., 1994. (See

Table I)

Table I: Descriptive response category

Observed response elements

Response category Detection Withdrawal Escape/attack Face grooming Score

No response 1 0 0 0 1

Nonaversive response 1 1 0 0 2

Mild aversive response 1 1 1 0 3

Strong aversive response 1 1 1 1 4

Prolonged aversive behavior 1 1 2 1 5

(modified from Vos et al., 1994)

The categories were based on descriptions in prior reported behavioral studies in rats

(Blanchard and Blanchard, 1977; Marshall and Teitelbaum, 1974; Pinel and Treit, 1978;

26 escape/attack, rat avoids further contact with the stimulus either passively or moving its body away from the stimulus to assume a crouching position against the cage wall, sometimes with the head buried under the body or actively attacking the stimulus with biting or grabbing movements, sometimes accompanied with vocalizations; and

(4) asymmetric face grooming, in which the rat displays an uninterrupted series of at least three face wash strokes directed to the stimulated facial area (Vos & Maciewicz,1991 and

1994). We have modified the categories to show a score of 1 as no response, 2 as nonaversive, and 3 as mild aversive, 4 as strong aversive, and 5 as prolonged aversive behavior. The score of 5 includes an extra 1 point for vocalization scored under escape/attack plus 1 each for detection, withdrawal, escape/attack and face grooming. In addition, we have added a category of pain with the response. Ordinal values were assigned to each of the different categories for the purposes of statistical analysis and also for the pain or no pain category. The observed response scoring will also be categorized for each stimulus prior to and after the surgery comparing the lesion and the normal, non- lesioned side.

Immunocytochemistry for light microscopy. There is evidence for calcitonin gene- related peptide (CGRP) in the primary afferent fiber system as a messenger in the neuro- circuitry of the dorsal horn. Lazarov (2002) describes CGRP in the small to medium cells of the trigeminal ganglion and also in nucleus caudalis, nucleus oralis, and nucleus interpolaris (Sugimoto et al., 1997). Substance P (SP), the tachykinin, is also a messenger candidate in the primary afferent fibers and located in the small to medium cells of the trigeminal ganglion and together with CGRP is seen in the nucleus caudalis, oralis, and

27 interpolaris (see Weihe et al., 1991 for a review). C-fos is a proto-oncogene and is categorized as a cellular immediate early gene. The (Vos and Strassman, 1995) paper reported on expression in the medullary dorsal horn of the rat after the CCI to the ION.

Galanin (GAL) will be used in the experiments to monitor injury to the ION.

Neuropeptide Y (NPY) is widely distributed throughout the peripheral and central nervous system and modulates neurotransmitter release in a highly selective manner and is an inhibitor of SP and is affected with peripheral nerve injury and inflammation.

Processing of the brainstem for immunocytochemistry followed the procedures described by (Rhoades et al., 1990a). After fixation, the tissue was post-fixed for 12 - 36 hours.

Sections (50 micrometers) were cut through the brainstem and were incubated in primary antibody for CGRP (vendor: Chemicon) 1:1000 dilution in phosphate buffer) for 14 – 20 hours at room temperature, rinsed with phosphate buffer, incubated for one hour in goat- anti-rabbit IgG diluted 1:200, rinsed again, and incubated in AB complex (Vectastain) diluted 1:100. Following several rinses, sections were reacted with 0.03% 3, 3´ - diaminobenzedine (DAB) and 0.015% hydrogen peroxide in 0.1 M phosphate buffer.

After several rinses in phosphate buffer, sections were plated on gelatin-coated slides, air- dried, dehydrated in graded ethanol, cleared with Xylene, and cover slipped with

Permount. Similarly, tissue was processed for SP (vendor: INCstar 1:1000; C-fos

(vendor: Cambridge) 1:5000; GAL (vendor: Peninsula 1:3000; and NPY (vendor:

Peninsula 1:1000) were incubated and processed.

Statistical analysis. To assess potential behavioral differences from baseline between the operated, sham, and normal groups, we used nonparametric methods due to the ordinal

28 scaling of the behavioral measures. The data for the responses were collapsed across the

different von Frey monofilaments. The average response score to mechanical stimulation

was calculated for the lesion and the non lesion sides and sham animals for each week for

the animals with double CCI to the ION. The Friedman ANOVA was used to determine

the statistical significance of the response between the two sides.

Data analysis. Light microscopy was used to look at the immunocytochemistry of the brainstem.

RESULTS

The Mean Pain Score (MPS) results for the 2 gm, 6 gm, 9 gm, and pin stimuli are shown below in Figures 1 and 2. Only the pin stimulus showed a consistent pain behavioral response when comparing the MPS for the two weeks prior and the 4 weeks after the double constriction surgery. Surgery is marked on the timeline at the yellow arrow between the second and third week. The 2 gm stimulus for allodynia, the 6 and 9 gm stimulus for hyperesthesia showed no significant change in MPS in these animals for four weeks after the lesion compared to the presurgical baseline MPS two weeks prior to the

CCI.

Figure 1. Mean Pain Score for 2 gm and 6 gm stimuli.

2 Gram Stimulus: Double Constriction Lesion

Lesioned (N=30) 2.5 Non-Lesioned (N=30)

2 e r

o 1.5 c S 1 ean M 0.5

0 123456 Surgery

Week

6 Gram Stimulus: Double Constriction Lesion

Lesioned (N=30) 2.5 Non-Lesioned (N=30)

2 e r

o 1.5 c S 1 ean M 0.5

123Surgery 456

Week

The Mean Pain Score (MPS) following application of a 2 gm stimulus (upper panel) or 6 gm stimulus (lower panel) to face of control (blue) and lesioned (red) animals. There was no significant change in MPS in response to the 2 or 6 gm stimuli for 2 wks prior to

31 surgery (arrow) and 4 wks after surgery. Note the responses did not score over 2 while significant pain should score 3 or above for significant pain behavior, scored on the y- axis.

Figure 2. Mean Pain Score for 9 gm and pin stimuli.

9 Gram Stimulus: Double Constriction Lesion

Lesioned (N=30) 2.5 Non-Lesioned (N=30)

2 e r

o 1.5 c S 1 ean M 0.5

123Surgery 456

Week

Pin Stimulus: Double Constriction Lesion

Lesioned (N=30) 4 Non-Lesioned (N=30) 3.5 3 e r

o 2.5 c 2 S n

a 1.5

Me 1 0.5 0 123456 Surgery

Week

The Mean Pain Score (MPS) following application of a 9 gm stimulus (upper panel) or pin stimulus (lower panel) to face of control (blue) and lesioned (red) animals. Only the

32 pin stimulus showed significant pain responses (mean score >3) on the y-axis, but this was found both before and after surgery. Note that the 9 gm stimulus did not show significant pain behavior (mean score < 3) on the y-axis compared to the pin.

The above histograms show no significant change in MPS to the 2, 6, or 9 gm stimuli which reflects failure of the CCI to the ION lesion.

Immunocytochemistry for light microscopy. There was no change in expression for

CGRP, SP, c-Fos, or NPY. Galanin was definitely upregulated in the trigeminal spinal tract and nucleus caudalis. This is evidence that the double constriction lesion clearly damaged the ION producing this effect. Below is shown a right and left brainstem slice showing this uptake for a right and left ION lesion.

33 Figure 3. Galanin upregulation in nucleus caudalis.

Photomicrographs of coronal sections through nucleus caudalis of two double CCI to ION. A and B show a rostral (A) and caudal (B) sections through the brainstem of an animal that received a right double CCI. C and D show similar sections through the brainstem of an animal that received double CCI constriction of the left ION. Note the upregulation of galanin is seen on the lesioned side (right in A and B and left in C and D) compared to the normal side. Scale bars equal 1.0 mm.

34 DISCUSSION

The results of this study showed the CCI to the ION did not show significant change of

the MPS in response to the 2 gm, 6 gm, or the 9 gm stimuli. Only the pin stimulus

showed a consistent pain behavioral response when comparing the MPS for the two

weeks prior and the 4 weeks after the double constriction surgery.

It is possible this result was due to different placement of the ligatures around the ION.

We did use the same surgical approach as that of Vos et al., (1991 and 1994) and we did

confirm visually the presence of blood flow through the epineural vessels after placement

of the two sutures.

The increased GAL expression indicates injury to the ION. Galanin may have a role in

the modulation of nociception after peripheral nerve injury (Wiesenfeld-Hallin et al.,

2004). This group has reported on GAL receptor subtypes with inhibitory and excitatory

effects, and a third receptor with trophic actions on the dorsal root ganglion. The role of

GAL and modulation in ION transection and the trigeminal system has been studied

extensively by (Rhoades et al., 1997). It is possible that these effects of GAL may have altered the behavioral response in our animals and the animals of the (Chudler and

Anderson, 2002) experiment with CCI to the ION. The inhibitory, excitatory, and trophic effects of Gal could alter channel expression, ectopic discharge, ephaptic conduction, axonal sprouting and brainstem reorganization resulting in suppression of the NP expected as a result of the CCI lesion.

35 The (Chudler and Anderson, 2002) study of ten adult Sprague-Dawley rats had two 5-0

chromic sutures placed on the ION as reported in Vos and Maciewicz (1991 and 1994).

The rats with ION damage were unresponsive to mechanical stimulation of the facial area

for up to 56 days after surgery. Increased facial grooming was observed only in rats with

chronic ION constriction ten days after surgery. Free-ranging behavior was similar to that

of sham-injury animals. In contrast, increases in the number of spontaneously active

trigeminal ganglion neurons were observed in those rats with ION injuries at both 3 and

56 days. This data suggested that the CCI of the ION resulted in prolonged loss of low- threshold input from the periphery leading only to transient behavioral changes, despite the presence of spontaneous activity in trigeminal sensory neurons. They reported gross pathologic changes in the injured ION at three days with some translucency at 56 days, but no microscopic pathology was reported.

Galanin upregulation in our CCI to the ION injury and the finding of prolonged loss of low-threshold input from the periphery (Chudler and Anderson, 2002) study suggest mainly peripheral mechanisms of pain behavioral suppression in this model. Central sensitization and brainstem reorganization are also likely to follow peripheral ION, ganglion, and root injury.

After review of this experiment, it was decided to do a tight ligature partial ION injury to produce a reliable animal model of trigeminal neuropathic pain.

36 CONCLUSIONS

This study showed the double CCI lesion to the ION to be unreliable as a pain model.

Our study suggests GAL may have inhibitory, excitatory, and trophic effects that could alter channel expression, ectopic discharge, ephaptic conduction, axonal sprouting and brainstem reorganization resulting in suppression of the NP expected as a result of the

CCI lesion.

Further study of the lesion with light and electron microscopy along with electrophysiology and immunocytochemistry should give some insight into this unreliability. Central inhibitory mechanisms as well as the peripheral mechanisms discussed are likely to ultimately explain the failure of this lesion.

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42 CHAPTER 2

Behavioral and Immunocytochemical Evidence of Trigeminal Neuropathic Pain

Produced by a Partial Infraorbital Nerve Lesion

Chronic pain (CP) is among the most disabling and costly afflictions in North America,

Europe, and Australia (IASP, 2003). Trigeminal neuralgia (TN) and most other types of neuropathic pain (NP) remain an enigma. A reproducible animal pain model is necessary to study peripheral and central mechanisms for new medical and surgical pain intervention. Forty-four male Sprague-Dawley rats received a partial infraorbital nerve

(ION) ligature with behavioral testing prior to and after the surgical manipulation. The psychometric mechanical testing was done with pin and von Frey monofilaments with blinded testing, sham, and normal animal controls. At weekly intervals after surgery, the animals were sacrificed and studied with light and electron microscopy. The ION, trigeminal ganglia, and brainstem were evaluated for structural and inflammatory changes with Galanin (GAL). Microscopy showed segmental, axonal, demyelinative lesions and wallerian degeneration (WD). The GAL showed significant expression in the brainstem with changes on the surgically manipulated side. These immunopathologic findings were accompanied by a significant increase in aversive behavior, suggesting that inflammation may prolong chronic pain. This model should provide a basis for further study with neuroimaging techniques combined with pharmacologic and surgical manipulations in animals and humans.

43 INTRODUCTION

Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”. Neuropathic pain (NP) is defined as “pain initiated or caused by a primary lesion or dysfunction in the nervous system” by the (International Association for the Study of Pain (IASP, 1994).

Pain patients present to the clinic with a variety of symptoms and signs. They often report

NP as having lancinating or continuous burning characteristics. There are associated abnormal sensory signs, such as allodynia (pain as a result of a stimulus which does not normally provoke pain), or hyperesthesia (an increased sensitivity to a stimulus). Most

NP involves the cranial and spinal nerves with significant predilection to the proximal portion of the afferent fibers near entry to the spinal cord or brainstem. Common examples of these pains are TN, post-herpetic neuralgia, atypical and other facial pains as well as cervical, thoracic, lumbar, sacral and coccygeal root injuries and diseases.

Two types of painful neuropathy are present in the distribution of the trigeminal nerve.

The first and best known is tic douloureux characterized by paroxysms of spontaneous shock-like sensations that can be evoked by cutaneous stimulation. The second is idiopathic trigeminal neuropathy that is similar to the post-traumatic painful neuropathies that occur when other nerves are injured.

The first attempt to produce an animal model of TN was reported by (King et al., 1956).

They injected alumina gel into the caudal portion of the trigeminal brainstem complex of

44 cats and showed an over-reaction to tactile stimulation of the face. Experimental injury to

the trigeminal nerve (e.g. transection, nerve crush, and tooth pulp removal) in animals

results in anatomical, neurochemical, and electrophysiological changes in peripheral and central neurons (Fromm and Sessle, 1991). The study of trigeminal root injury by

(Burchiel, 1980) showed electrophysiologic evidence of abnormal impulse generation in focally demyelinated trigeminal roots using implanted chromic sutures. These models lack a detailed behavioral analysis of the animals before and after experimental trigeminal injury.

Behavioral evidence of trigeminal neuropathic pain following the chronic constriction injury (CCI) to the rat infraorbital nerve (ION) was reported by (Vos and Maciewicz,

1991 and 1994). This model showed behavioral changes atypical of human TN such as

“increased facial grooming” and “decreased” response to mechanical stimulation of the injured nerve territory. It was 15 days after surgery before allodynia and aversive behavior appeared. Patients with TN will not permit testing of the affected trigeminal division because of severe allodynia.

The trigeminal pain models have focused on the mechanical aspects of the lesions, some physiology, and one model (Vos et al., 1994) extensively on behavior. Review of these models does not show any attempt to look at the role of inflammation in the injured nerve. However, a recent study applied complete Freund’s adjuvant (CFA) to the orbital portion of the ION (Benoliel et al., 2002). They found behavioral changes and

45 inflammatory neuropathic changes in the CFA group and interestingly, also in the saline control group.

Both the central and the peripheral nervous system are susceptible to demyelinating disorders, including, multiple sclerosis (MS), Charcot-Marie-Tooth, and Guillain-Barre syndromes. These disorders often show segmental demyelination with slowing of nerve impulse transmission with impaired motor function and abnormal sensory phenomena.

This includes allodynia, hyperesthesia, and spontaneous pain with an increased occurrence of TN in the MS patients. Both the glycerol and chromic suture lesion is followed by WD (WD) (Waller, 1850; Ramon y Cajal, 1928). Wallerian degeneration is accompanied by an inflammatory response of the nervous system to myelin and axonal injury, primarily attributable to the production of cytokines, the mediator molecules of inflammation. The role of pro-inflammatory cytokines in the development and maintenance of persistent pain states has been extensively analyzed and characterized in the laboratory of S.M. Sweitzer (Winklestein et al., 2001).

To determine if segmental axonal demyelinative lesions produce NP, we placed a single

5-0 chromic suture as a partial ION ligation in the orbit. A trans-orbital approach and suture placement on the ION was performed in 44 adult Sprague-Dawley rats.

Observational and mechanical behavioral testing was accomplished prior to and subsequent to this manipulation. Light and electron microscopy was performed to define the pathologic process and immunocytochemistry was performed to look at injury and inflammatory changes.

46 MATERIALS AND METHODS

Ethical Concerns. Animals in this experiment were treated and cared for according to the ethical standards and guidelines for investigations of experimental pain in animals prescribed by the Committee for Research and Ethical Issues of the IASP (1983). The procedures for handling and testing described in this study were reviewed and approved by the Division of Laboratory Animal Medicine at the Medical College of Ohio.

Animals. Forty-four male Sprague-Dawley rats (250 – 300 gm) were used and housed under partially reversed 12-hour dark/light cycled with lights on at 1700 hours. They were housed in plastic cages in a colony room with food and water available at all times.

The rats were randomly assigned to a partial ION ligation, right or left side as well as unilateral sham procedures on the right and left for the control condition. Each animal served as its own control using the non-operated side.

Surgery. Rats were anesthetized with ketamine (30 mg/kg, I.P.) and xylazine (15 mg/ kg,

I.P.) and placed in a standard stereotaxic head holder. All surgery was performed under direct visual control with the assistance of a Zeiss operating microscope. The skull and nasal bone was exposed with a midline scalp incision. The fascia at the edge of the orbit was dissected free to give access to the ION with gentle deflection of the orbital contents laterally to allow visualization of the ION. The ION in the orbit was exposed using a surgical procedure similar to that described by (Gregg, 1973; Jacquin and Zeigler 1983;

Vos et al., 1991 and 1994). The ION was dissected free at the rostral portion of the orbital cavity, just caudal to the infraorbital foramen. A single chromic gut (5-0) ligature was

47 tightly tied around a portion of the ION carefully avoiding damage to any other part of the nerve. In rats of the sham control group, the ION was exposed on one side using the same procedure except the exposed nerve was not ligated. The scalp incision was closed and the animal observed carefully postoperatively and returned to the vivarium dark/light cycle room.

The single partial ligation was chosen because of the failure of two loose constricting 5-0 chromic sutures done in a prior experiment to produce a behavioral effect. It was further chosen because of the reported success of partial ligature of the sciatic nerve (Seltzer et al., 1990) and tight ligation of the L5 spinal nerve alone (Kim and Chung, 1992).

Behavioral testing. We modified the behavioral testing system reported by Vos and

Maciewicz (1991 and 1994). The animals were tested six times before and after surgery.

Testing was conducted from 0700 to 1700 hours. Each test session consisted of observation of free behavior and assessment of the behavioral response to the von Frey monofilaments and the pin.

We made some changes to the testing system reported by Vos and Maciewicz (1991 and

48 care to differentiate between a “startle response” as a reflex movement of fright from an aversive movement in response to the mechanical or pin stimulus. In addition to the ordinal scale for behavior response we also used a category of either pain or no pain in response to the stimulus.

Mechanical stimulation. Stimuli. A graded series of three von Frey monofilamnts

(pressure Aesthesiometer, Stoelting Company, Chicago, IL) and a pin were used for mechanical stimulation. The stimuli were 2 gm, 6 gm, 9 gm, (converted to log units, 4.31,

4.74, and 4.93 units respectively), and a pin. A 24 gauge injection needle bent at a 30 degree angle and mounted on a wooden stick was used to apply pinprick stimulations.

Testing procedures. For mechanical stimulation the rats were placed in a transparent plastic cage as described above and each session was recorded on video tape with the

49 recorder approximately 50 cm in front of the cage. The video tapes of behavior and

scoring at the time of each session were later independently reviewed. Stimulations were

delivered by reaching into the cage in a dark room with light provided by a 60 W

incandescent red bulb suspended one meter above the center of the test area. Reaching movements were performed slowly to avoid a startle response. The animal was adapted to the cage for ten minutes during which time the experimenter would reach into the cage approximately every 30 seconds touching the wall of the cage with a plastic rod similar to the von Frey instrument. Testing was accomplished when the rat was in a sniffing/no locomotion state with four paws on the ground, neither moving nor freezing. A new stimulus was applied only when the rat resumed this position or at least 30 seconds after the preceeding stimulation. During one session the complete set of von Frey hair intensities was applied in a randomized order in an ascending/descending series. All four areas were explored with one level of stimulation before using the next intensity.

Table I).

50 Table I: Descriptive response category

Observed response elements

Response category Detection Withdrawal Escape/attack Face grooming Score

No response 1 0 0 0 1

Nonaversive response 1 1 0 0 2

Mild aversive response 1 1 1 0 3

Strong aversive response 1 1 1 1 4

Prolonged aversive behavior 1 1 2 1 5

(modified from Vos et al., 1994)

The categories were based on descriptions in prior reported behavioral studies in rats

(Blanchard and Blanchard, 1977; Marshall and Teitelbaum, 1974; Pinel and Treit, 1978;

Schallert and Whishaw, 1978). The response of the rat to mechanical stimulation may consist of (1) detection, the rat turns head toward the stimulating object and the object is explored (sniffing or licking); (2) withdrawal reaction, the rat turns his head slowly away or pulls it briskly backwards after the stimulus, with or without a single face wipe ipsilateral to the stimulated area. This is considered a mild aversive response; (3) escape/attack, rat avoids further contact with the stimulus either passively or moving its body away from the stimulus to assume a crouching position against the cage wall, sometimes with the head buried under the body or actively attacking the stimulus with

51 biting or grabbing movements, sometimes accompanied with vocalizations; and

(4) asymmetric face grooming, in which the rat displays an uninterrupted series of at least three face wash strokes directed to the stimulated facial area (Vos & Maciewicz,1991 and

Ordinal values were assigned to each of the different categories for the purposes of statistical analysis and also for the pain or no pain category. The observed response scoring will also be categorized for each stimulus prior to and after the surgery comparing the lesion and the normal, non-lesioned side.

Immunocytochemistry. Immunocytochemistry permits the anatomic localization of specific peptides through their immunoreactivity as detected by specific anti-sera. A number of reviews have dealt with the use of immunocytochemical techniques for the localization of cellular antigens by direct and indirect methods (Nairn et al., 1969;

Sternberger et al., 1970; and Sternberger, 1974).

Prior trigeminal models and manipulations have evaluated some pathologic anatomy of the nerve lesions and the behavior, but very little, until recently, has been written on the inflammatory effects. The Benoliel et al. (2002) study applied complete Freund’s adjuvant (CFA) to the orbital portion of the ION and found behavioral and inflammatory neuropathic changes in the CFA group and interestingly also in the saline group. The

52 lesioned nerves, ganglia, and brainstem were evaluated for inflammatory changes and also for the expression of some neuro-peptides at the injury site. Calcitonin gene-related peptide (CGRP) in the primary afferent fiber system is a messenger in the neuro-circuitry of the dorsal horn. Similarly, substance P (SP), the tachykinin, is also a messenger candidate in primary afferent fibers, the intrinsic neurons and the descending fibers (see

Weihe et al., 1991, for a review). Neuropeptide-Y (NPY) is an inhibitor of SP and like

GAL should show increased activity after peripheral nerve injury or inflammation.

Processing of the infraorbital nerve, ganglia, and brainstem for immunocytochemistry, followed the procedures described by (Rhoades et al., 1990a). After perfusion, the tissue is post-fixed for 12 - 36 hours. Sections (50 micrometers) were cut through the brainstem, ganglia and nerve and incubated in the primary antibody GAL (vendor: Peninsula,

1:3000) dilution in phosphate buffer for 14 – 20 hours at room temperature, rinsed with phosphate buffer, incubated for one hour in goat anti-rabbit IgG diluted 1:200, rinsed again, and incubated in AB complex (Vectastain) . Following several rinses, sections were reacted with 0.03% 3, 3´-diaminobenzidine (DAB) and 0.01 M phosphate buffer.

After several rinses in phosphate buffer, sections were plated on gelatin-coated slides, air- dried, dehydrated in graded ethanols, cleared with Xylene, and cover slipped with

Permount. Similarly, the tissue will be processed for CGRP (vendor: Chemicon) 1:1000; and SP (vendor: INCstar) 1:1000.

Statistical Analysis. Calculation of the different behavioral response scores were introduced as an overall measure of altered responsiveness to mechanical stimulation and

53 the different score of an area on a preoperative or postoperative day was calculated as a mean of the four differences (one for each stimulus intensity) between pre and postoperative response scores evoked by stimulation of that area. Chi square analysis was performed on the 44 animals and the results of these analyses are presented below.

Changes in response scores over fourteen test sessions (six preoperative and eight postoperative) were studied with the Friedman ANOVA and Kendall Coefficient of

Concordance.

54 RESULTS

The general behavioral activity changed following the partial ION ligation and showed a noticeable increase in freezing-like behavior associated with hyperactivity, burrowing, and at times asymmetric face grooming. We did not see the dramatic grooming behavioral changes reported by (Vos et al., 1994).

We did see pain behavioral changes reported as an increase in the mean pain score (MPS) in the testing of 38 out of 44 rats to mechanical stimulation in the territory of the ligated nerve. This was statistically significant compared to the preoperative baseline MPS and when compared to the MPS of sham and normal animals.

We also saw animals where the lesion side had increased behavioral responsiveness that was accompanied by an increase of pain behavior on the non-lesioned or “normal” side.

This phenomenon of “allochiria” (sensation referred to the opposite side) is observed in humans and also has been reported in the animal pain literature. We saw 25 animals with allochiria. There were 25 animals with allodynia. There were 21 animals with allodynia and allochiria. There were 25 animals with allodynia and hyperesthesia, which likened the model to the clinical conditions of trigeminal neuralgia and atypical facial pain. There were 35 animals with hyperesthesia and 6 animals with no effect.

The time course of observed changes in behavior and evoked responses following ION ligation continued for eight weeks. Mechanical allodynia was a strong aversive response to the 2 gm stimulus and the most common response seen in this group. The response of

55 hyperesthesia to the 9 gm stimulus was next most common and the least common behavioral change was observed was an increase in MPS following the 6 gm and the pin stimuli. These responses were seen within the first week of testing and persisted throughout the eight week period of observation and therefore showed reliability of this model.

Allodynia and hyperesthesia to mechanical and thermal stimulation are well known consequences of partial tight ligation and CCI to the sciatic nerve (See Bennett, 1993 for a review). Our partial ION lesion showed allodynia and hyperalgesia similar to these models.

Shown below are the MPS for the 2 gm, 6 gm (Figure 1), 9 gm, and the pin (Figure 2) stimuli collapsed over a six week period before and after the surgery. The surgery was done after two weeks of baseline MPS observations (shown at the yellow arrow on the x- axis of all figures). The 2 gm stimulus showed allodynia post-lesion. The 6 gm stimulus did not show hyperesthesia, but the 9 gm stimulus did show hyperesthesia. The pin showed a consistent pain behavioral response before and after surgery.

Figure 1. Mean Pain Scores for 2 gm and 6 gm stimuli.

2 Gram Stimulus Partial ION

4 Lesioned (N=30) Non-Lesioned (N=30) * 3.5 * * * 3 e r 2.5 o c 2 S n

a 1.5

Me 1 0.5 0

12Surgery 3456 * P<0.01 Week

6 Gram Stimulus Partial ION

4 Lesioned (N=30) Non-Lesioned (N=30) 3.5 3 e r

o 2.5 2 Sc n

a 1.5

Me 1 0.5 0

12Surgery 3456

Week

The Mean Pain Score (MPS) following application of a 2 gm stimulus (upper panel) or 6 gm stimulus (lower panel) to face of control (blue) and lesioned (red) animals. Note that the 2 gram stimulus (allodynia) shows significant change (P<0.01) for 4 wks post-surgery compared to the 2 wks prior to surgery (arrow). The 6 gram stimulus did not show a statistically significant difference post-surgery compared to the pre-surgical testing and the normal side.

Figure 2. Mean Pain Scores for the 9 gm and pin stimuli

9 Gram Stimulus Partial ION

4 Lesioned (N=30) Non-Lesioned (N=30) 3.5 * * * * 3 e r

o 2.5 c 2 S n

a 1.5

Me 1 0.5 0

12Surgery 3456 * P<0.001 Week

Pin Stimulus Partial ION

3.9 Lesioned (N=30) Non-Lesioned (N=30) 3.8

3.7 e r o 3.6 Sc

n 3.5 a 3.4 Me 3.3

3.2

12Surgery 3456

Week

The Mean Pain Score (MPS) following application of a 9 gm stimulus (upper panel) or pin stimulus (lower panel) to face of control (blue) and lesioned (red) animals. Note that the 9 gm stimulus shows significant hyperesthesia (increased response) post-lesion compared to the 2 wks pre-surgical testing. The pin shows a consistent pain behavioral response before and after the surgery.

The light microscopy shows demyelination and axonal degeneration accompanied by WD

with macrophages and mast cells as part of an inflammatory process (See Figure 3).

Figure 3. Light micrograph of lesioned infraorbital nerve

High power micrograph (60X) stained for toluidine blue through the peripheral division of the ION of an animal that received a partial ligation. This nerve shows typical signs of inflammation and axonal damage indicated by the presence of macrophages (light green arrow), mast cells (white arrow), and degenerating axon at (red arrow). Scale bar = 20µm.

The lesion associated with the partial ION injury was reviewed with EM in six of the rats in the postoperative period. This study shows a significant loss of large myelinated (Aα

59 and Aβ) axons, while small myelinated (Aδ) and unmyelinated c-fibers were less affected

in these lesions. The lesions show characteristics of WD. There were shrunken axons and fragmentation occurring near the nodes of Ranvier. The unmyelinated fibers are typically more resistant to injury but in this lesion show varicosities, elongation and disintegration.

The myelin sheaths retracted from the axons at the nodes of Ranvier and showed

fragmentation. The loss of the large fibers and ephapsis is consistent with the hypothesis

that hyperalgesia and allodynia is found with this lesion (See Figure 4).

60 Figure 4. Electron micrograph of lesioned infraorbital.

Electron micrograph (4000X) shows a degenerating axon (yellow arrow) within the ligated ION fascicle. Note that the axon is surrounded by a foam cells (dotted lines) with lipid droplets.

61 The immunocytochemical markers did not show abnormal expression for TNF-α, IL-1β,

CGRP, SP, and NPY. Galanin was upregulated bilaterally in these animals. This GAL finding was confirmed with an additional study in five animals.

Five additional Sprague-Dawley rats (250-300 gm) were separately studied for GAL.

They were housed under the same partially reversed 12-hour dark/light cycle as described above. Behavioral testing was the same. Facial receptive fields were identified with a tungsten recording electrode prior to the lesion. The electrode was used to identify the receptive fields of the portion of nerve ligated for post-lesion testing. Between seven and fourteen days after surgery, the animals were sacrificed and studied for GAL in the ION, ganglia and brainstem. Two partial right ION, two left, a sham and a normal animal were tested over a four week period and sacrificed. The ION and ganglia from these animals showed increased GAL expression bilaterally with notable increase of ~5% in the ganglion on the lesion side. There was no significant GAL staining evident at 48 hours in the normal animal. This was consistent with the findings of (Wiesenfeld-Hallin and Xu,

1998; Xu, 2000) that the expression of GAL following peripheral nerve injury or inflammation is upregulated in primary afferents and spinal cord (See Figure 5).

62 Figure 5. Expression of galanin in trigeminal ganglia of partial ION injury.

Left Panel: Low power micrograph showing galanin immunoreactivity observed in the left and right V ganglion of a sham-operated (A and A’) and unoperated (B and B’) animal. Right Panel: Low power micrographs showing galanin immunoreactivity observed in several sections cut dorsal to ventral through the left (C –G) and right (C’- G’) V ganglion of left partial ION constriction injury. Note the upregulation of galanin immunoreactivity in the ganglion on the left (lesioned side) (C-G) compared to (C’-G’) in the normal ganglion. Panels G and G’ are a high power views of panels F and F’. Scale bars = 100µm for all.

63 DISCUSSION

The partial ION lesion produced significant NP with aversive behavioral changes on the

affected side and allochiria seen on the normal side of 25 animals. The 2 gm stimulus

showed allodynia and allochiria. The 9 gm, but not the 6 gm stimulus showed

hyperesthesia. The 6 gm stimulus just missed statistical significance (p=0.06). This may

represent an unusual response of this study population or an unusual peripheral or central

response associated with the tight partial ION lesion.

The partial single chromic ION ligation produced a segmental, axonal, and demyelinative

lesion as illustrated with light and EM. The light microscopy shows demyelination and

axonal degeneration accompanied by WD with macrophages and mast cells as part of an

inflammatory process and inflammatory changes. The inflammatory changes probably

contribute to altered ion channel expression, nociceptor sensitization and allochiria. The

EM demonstrated ephapsis is likely to produce abnormal sensory conduction, ectopic

discharges and altered ion channels causing the painful responses. The dysmyelination

and axonal injury could explain sprouting into the ganglion and brainstem reorganization that has been reported in the literature. The summation of these abnormalities could explain the persistence of pain.

It has been hypothesized that spontaneous activity in primary afferent fibers after injury to the sciatic nerve may alter the responsiveness of neurons in the central nervous system and explain these behavioral phenomena (Kajander and Bennett, 1992). The number of spontaneously active nerve fibers following experimental ION neuromas was less than

64 that found to result from sciatic neuromas (Tal and Devor, 1992). These authors found

that the spontaneous activity in the trigeminal neuromas was less sensitive to mechanical

stimulation than sciatic neuromas (Tal and Devor, 1992). These findings suggest that the

mechanisms underlying the generation of spontaneous activity following spinal and

trigeminal nerve injury may be different.

The increased GAL expression indicates injury to the ION. Galanin may have a role in

the modulation of nociception after peripheral nerve injury (Wiesenfeld-Hallin et al.,

2004). This group has reported on GAL receptor subtypes with inhibitory and excitatory

effects, and a third receptor with trophic actions on the dorsal root ganglion. The role of

GAL and modulation in ION transection and the trigeminal system has been studied

extensively by (Rhoades et al., 1997). It is possible that these effects of GAL may have altered the behavioral response in our animals and the animals of the (Chudler and

Anderson, 2002) experiment with CCI to the ION. The inhibitory, excitatory and trophic effects of Gal could alter channel expression, ectopic discharge, ephaptic conduction, axonal sprouting and brainstem reorganization resulting in suppression of the NP expected as a result of the CCI lesion.

The immunochemistry in this experiment with GAL was consistent with a segmental, axonal lesion and inflammation. Further information on the specific abnormalities and the timing in the acute process, as well as the multiplicity of pro-inflammatory cytokines will provide a basis for a “mixture” of anti-inflammatory and anti-nociceptive agents and future study. While this runs counter to the “magic bullet” approach to pain therapeutics,

65 it is probable that this concept is not valid in the approach to pain, neoplasm, neurodegenerative disease, and for that matter most neurologic diseases.

The pathology in our ION lesion reported here and in the sciatic nerve is similar to that seen in human cases of painful peripheral neuropathy (Dyck et al., 1976). Nerve biopsy is being replaced by skin biopsy to assess small fiber involvement allowing quantification of c-fibers and Aδ nerve fibers through the measure of the density of intra-epidermal nerve fibers (IENF). The loss IENF has been demonstrated in a variety of neuropathies, including small fiber sensory neuropathies (McCarthy et al., 1995; Holland et al., 1997 and 1998). Skin biopsy has recently been utilized to demonstrate mechano-receptors and their myelinated afferents (Nolano et al., 2003). Skin biopsy is planned for future experiments. We have been unable to find any other study that employed a partial orbital

ION lesion with a 5-0 chromic suture showing light or electron microscopy for comparison at this time.

The further definition of the nature of the mechanical aspects of the lesion, including ephapsis and its effect on neighboring normal axons, and the inflammatory aspects of the lesions should provide a rational basis for multiple medications at different times. The multi-faceted aspects of the pain behavior inclusive of the allochiria, which has been found in the animal models, clearly dispels the theory that people who perceive pain in expanded or unusual receptive fields are psychiatrically disturbed in their response to psychometric testing.

66 CONCLUSIONS

This study shows that a partial ION injury with a single 5-0 chromic suture produces statistically significant aversive pain behavioral changes in the distribution of the ION.

The partial ION injury produces a lesion that is a segmental, axonal, and demyelinative.

The light and electron microscopy showed signs of axonal damage and WD. The immunochemistry was positive for GAL expression which was consistent with the pain behavior and also demonstrates a possible explanation for allochiria.

Further study of the lesion with markers and immunocytochemistry is indicated to predict new methods of treatment of the NP with focus on structural and molecular mechanisms.

The partial orbital ION single 5-0 chromic tight ligature provides a reliable model for the study of pain behavior in the trigeminal system with functional magnetic resonance, PET scanning, pharmacologic, and surgical manipulation in animals and provides a basis for investigation in humans.

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73 CHAPTER 3

Behavioral Evidence of Glycerol Induced Trigeminal Neuropathic Pain in Sprague-

Dawley Rats

Neuropathic pain (NP) includes trigeminal neuralgia (TN) which is thought to be caused by partial injury from vascular compression near the root entry zone. To determine whether proximal demyelinating lesions are likely to cause trigeminal NP, 12 of 22 adult male Sprague-Dawley animals were randomly assigned to six different groups of two each to study the effects of 25% glycerol, 50% glycerol and 0.9% saline injected into the trigeminal root and ganglion. The animals were tested prior to and after surgery with von

Frey monofilaments and pin to test behavioral responses. This dosing study showed only the 50% glycerol injection into the root effective in producing pain behavior. Based on these results 10 animals were used to study 50% glycerol root injections. Using the

Kruskal-Wallis ANOVA, only the 50% glycerol injection in the trigeminal root caused statistically significant differences in response to behavioral testing (P =.0117, .0208,

.0278, .0445) over a four week period. Ultrastructural evaluation showed loss of large myelinated axons, ephapsis, degeneration and segmental axonal-demyelination compared to the saline injection. Immunocytochemistry showed no abnormal expression. This study provides behavioral evidence of trigeminal neuropathic pain due to a glycerol induced partial root injury. It provides a reproducible animal model for the study of pain in the trigeminal system.

74 INTRODUCTION

Neuropathic pain is a common and serious clinical problem that remains poorly understood and not well treated by many physicians. Neuropathic pain frequently becomes chronic pain (CP) and is among the most disabling and costly afflictions in

North America, Europe, and Australia. The prevalence rates range from 10.1 to 55.2%

IASP (2003).

There is clinically significant CP found in 65% of persons diagnosed with multiple sclerosis (MS). These conditions include TN, painful optic neuritis and Lhrmitte’s syndrome (Kerns et al., 2002). Trigeminal neuralgia is a well recognized complication of

MS and typically the plaque of demyelination is central in the root entry zone.

Neuropathic pain research has concentrated on a group of non-trigeminal behavioral animal models (Wall et al., 1979; Bennett and Xie, 1988; Attal et al., 1990; Seltzer et al.,

1990; Kim and Chung, 1992). These models used a variety of experimental lesions including a transected tightly ligated nerve (neuroma model) (Wall et al., 1979), chronic constriction injury (CCI) of a nerve trunk or part of it (Bennett and Xie, 1988; Attal et al.,

1990; Seltzer et al., 1990; Kim and Chung, 1992; Vos et al.).

The behavioral changes following the CCI to the sciatic nerve have been most similar to human NP. The CCI rats showed behavioral signs of (1) allodynia defined as reduced vocalization thresholds for mechanical stimulation of the foot and the reluctance to place the foot down on a normal or innocuously cold surface, (2) hyperesthesia , defined as

75 reduced withdrawal latency, prolonged flexion of the hind paw and occasional jerking/flailing or excessive licking of the affected paw in response to noxious thermal, mechanical, or chemical stimulation of the plantar surface of the foot. The animals in most of these studies showed behavioral abnormalities in the first week following ligation and peaked at about day 14 after the lesion.

Previous reports on the behavioral effects of trigeminal root injury (Burchiel, 1980), as well as studies of the behavioral effects to chemical irritation of the trigeminal nucleus caudalis (King et al., 1956; King, 1970; Black, 1974; Kryzhanovsky et al., 1974) both in cats and rats, have described recurrent attacks of ipsilateral face grooming along with allodynia to light mechanical stimulation of the injured side of the face. This facial sensory testing is not permitted by humans with TN because of allodynia.

Lesions of primary afferent fibers associated with pain can result from a variety of traumatic injuries or diseases. Trigeminal neuralgia is thought to be caused by partial injury to the trigeminal root at the root entry zone due to vascular compression or demyelination (for a review see Love and Coakham, 2001 or Jannetta, 1980).

To determine whether trigeminal root or ganglion lesions may be more powerful in producing trigeminal neuropathic pain, we did two experiments involving the trigeminal root and ganglion. The first experiment was a dosing experiment to compare the effect of glycerol 25%, glycerol 50%, and 0.9% saline on the trigeminal root and ganglion. A middle fossa craniotomy was used to approach the trigeminal ganglion and root for injection of either glycerol 25% or 50% or 0.9% saline. The second experiment consisted

76 of rats randomized into a group injected with 50% glycerol and a control group with

0.9% saline into the root and behaviorally tested.

Glycerol is a trivalent alcohol normally present in human tissue and plasma as an integral part of fats and triglycerides. It readily penetrates cell membranes and recently a glycerol conducting channel has been described (Stroud et al., 2000). Glycerol was used for injection of the trigeminal root because of its unusual use in the treatment of TN by injection into the trigeminal cistern. Glycerol treatment was discovered during the development of a stereotaxic technique for gamma irradiation of the gasserian ganglion to treat patients with TN (Leksell, 1971; Hokanson and Leksell, 1979). The glycerol was used as a vehicle to deposit tantalum dust as a marker in the trigeminal cistern. Hokanson noted that the glycerol injection alone abolished pain completely in several patients for long periods along with markedly well-preserved facial sensation. On that basis he developed a routine method of treating patients with TN with glycerol (Hokanson, 1981).

This procedure has been replaced by micro-vascular decompression and other methods of disruption of the ganglia.

In neurophysiologic experiments on the dorsal rootlets of cats, the thin unmyelinated fibers were more vulnerable to hypertonic solutions then larger ones. However, with increasing hypertonicity, A-delta fibers are also permanently blocked (King, 1972).

Rengachary (1983) studied the perineural and intraneural application of pure glycerol on rat sciatic nerve. Following intraneural injection, the histological changes were severe with extensive myelin swelling and axonal injury. The nerve fibers soaked in pure

77 glycerol were similar to the bathing of nerve fibers in the trigeminal cistern and showed less extensive damage. Major changes occurred in the larger myelinated fibers where the myelin sheets swelled causing axonal compression. It is likely that injection of the glycerol into the trigeminal ganglion is quickly diluted by the cerebral spinal fluid and is eventually washed out of the cistern and is therefore not comparable to intraneural injection.

The purpose of this study was to determine whether trigeminal root or ganglion lesions may be more powerful in producing trigeminal neuropathic pain and to simulate a lesion that might be seen in MS.

The results of the experiment with injection of glycerol into their trigeminal root was presented to Congress of Neurological Surgeons at the annual meeting in 1997 and this paper by Gouda, Bauer, Chiaia, and Brown was given the Tasker Award for the best presentation at that meeting. The abstract “Behavioral Evidence of Glycerol Induced

Trigeminal Neuropathic Pain in Sprague-Dawley Rats” was presented at the 1997 New

Orleans Congress of Neurosurgeons meeting.

78 MATERIALS AND METHODS

Ethical standards. The animals were treated and cared for according to the ethical

standards and guidelines for investigations of experimental pain in animals outlined by

the Committee for Research and Ethical Issues of the International Association for the

Study of Pain (IASP) (1983). The animals were maintained in a vivarium accredited by

the American Association of Laboratory Animal Care at the Medical College of Ohio.

The animals were housed separately from other rats in the vivarium in a partial reversed

dark/light cycle for testing purposes. Careful attention was given to the maintenance of

similar care, attention, and handling to each and all of the animals.

Animals. Zivic-Miller rats (250-300 grams) were used and housed under partially reversed twelve hour dark/light cycle with lights on 1700 hours. All animals were tested at least six sessions prior to experimental manipulations. The glycerol root and ganglion study consisted of 22 adult male Sprague-Dawley rats. There were twelve animals randomly assigned to six different groups; (1) two rats had the trigeminal ganglion injected with saline, (2) two rats were injected with 25% glycerol in the trigeminal ganglion, (3) two rats were injected with 50% glycerol in the trigeminal ganglion, (4) two rats had saline injected in the trigeminal root, (5) in two rats the trigeminal root was injected with 25% glycerol, and (6) two rats had the root injected with 50% glycerol.

Each group used the non-operative side as a control. A coding system was used to blind the investigator and others scoring the behavior of the animals to the surgical procedure

performed on individual animals.

79 Surgery. The animals were quietly and carefully transported to the surgical room prior to surgery. Anesthesia with xylazine (15mg/kg I.P.) and ketamine (30 mg/kg I.P.) was given intraperitoneally and the experimental lesions were performed in the surgical room. The trigeminal root was approached via a middle fossa craniotomy. The skull was drilled at the lambdoid suture and the opening widened to expose the transverse sinus on the corresponding side. The dura was opened just anterior to the sinus and the occipital lobe was gently retracted from the anterior surface of the petrous temporal bone exposing the trigeminal ganglia. For the trigeminal root, the dissection was continued medially to expose the tentorium where the root was identified beneath the tentorium and injected transtentorially. In each case a volume of 0.05 ml of either glycerol with a vital dye or saline was used for injection. Closure of the skin was done either with staples or 3-0 catgut sutures. Animals were given post-surgical evaluation with provisions made for observed pain and when stable were returned to the vivarium.

Behavioral testing. We modified the behavioral testing system reported by Vos and

Maciewicz (1991 and 1994). The animals were tested six times before surgery and for the trigeminal root experiment they were tested for five weeks after the surgery. Testing was conducted from 0700 to 1700 hours and the animals were transported from the dark room to the test room in a covered cage. The animal was placed in a transparent plastic cage and a video camera was placed approximately one meter in front of the cage and positioned so that the image of the animal’s body covered at least one-fourth of the recorded view and the recordings were analyzed by observers blinded to the surgical procedure. The rats were tested in the same quiet darkened room with a 60 watt

80 incandescent red bulb suspended one meter above the center of the test area, leaving the rest of the room dark. Each test session consisted of observation of free behavior and assessment of the behavioral response to the von Frey monofilaments and the pin.

We made some changes to the testing system reported by Vos and Maciewicz (1991 and

1994). They looked at the animals one day prior to the surgical event and we looked at the animals at least six times prior to surgery. We also looked at large numbers of normal animals to determine if there were animals who responded differently to the mechanical von Frey and the pin testing. We did notice that some animals responded with a more vigorous response to the mechanical and pin testing than others and it was determined important to define the animal’s baseline response to these stimuli prior to the independent variable. We did not feel that one day was adequate time to define the pre- surgical behavioral responses of the animals. We also took special care to differentiate between a “startle response” as a reflex movement of fright from an “aversive” movement in response to the mechanical or pin stimulus. In addition to the ordinal scale for behavior response we also used a pain category of either pain or no pain in response to the stimulus.

Mechanical stimulation. Stimuli. A graded series of three von Frey monofilamnts

(pressure Aesthesiometer, Stoelting Company, Chicago, IL) and a pin were used for mechanical stimulation. The stimuli were 2 gm, 6 gm, 9 gm, (converted to log units, 4.31,

4.74, and 4.93 units, respectively), and a pin A 24 gauge injection needle bent at a 30 degree angle and mounted on a wooden stick was used to apply pinprick stimulations.

81 The stimulus consisted of two consecutive placements of the stimulus monofilament or

the pinprick under one second. The 2 gm light weight stimulus was selected to find

allodynia. The more heavily weighted stimuli of 6 gm and 9 gm were chosen to elicit

hyperesthesia, allodynia or pain compared to the pin stimulus which was chosen as the

pain standard.

Stimulated areas. Stimuli were applied first within the ION territory, near the center of

the vibrissa pad on the hairy skin surrounding the mystacial vibrissa, and a second outside the ION territory in the territory of the auriculotemporal nerve on the hairy skin under the ear. These areas were stimulated on both sides of the face, ipsilateral and contralateral to the side where surgery was performed.

Testing procedures. For mechanical stimulation the rats were placed in a transparent plastic cage as described above and each session was recorded on video tape with the recorder approximately 50 cm in front of the cage .The tapes were independently reviewed for final scoring of observation recorded at the time of testing. Stimulations were delivered by reaching into the cage in a dark room with light provided by a 60 W incandescent red bulb suspended one meter above the center of the test area. Reaching movements were performed slowly to avoid a startle response. The animal was adapted to the cage for ten minutes. During this time the experimenter would reach into the cage approximately every 30 seconds to touch the wall of the cage with a plastic rod similar to the von Frey instrument. Testing was accomplished when the rat was in a sniffing/no locomotion state with four paws on the ground, neither moving nor freezing. A new

82 stimulus was applied only when the rat resumed this position or at least 30 seconds after the preceeding stimulation. During one session the complete set of von Frey hair intensities was applied in a randomized order in an ascending/descending series. All four areas were explored with one level of stimulation before using the next intensity.

Response scoring. Responses were video recorded and scored in real time . This was accomplished for the four different areas of the face inside or outside the trigeminal system (V1, V2, V3). The pinprick was always applied after completion of the von Frey hair series. The responses were categorized (See Table 1).

Table I: Descriptive response category

Observed response elements

Response category Detection withdrawal Escape/attack Face grooming score

No response 1 0 0 0 1

Nonaversive response 1 1 0 0 2

Mild aversive response 1 1 1 0 3

Strong aversive response 1 1 1 1 4

Prolonged aversive behavior 1 1 2 1 5

(modified from Vos et al., 1994)

83 The categories were based on descriptions in previously reported behavioral studies in

rats (Blanchard and Blanchard, 1977; Marshall and Teitelbaum, 1974; Pinel and Treit,

1978; Schallert and Whishaw, 1978). The response of the rat to mechanical stimulation

may consist of (1) detection, the rat turns head toward the stimulating object and the

object is explored (sniffing or licking); (2) withdrawal reaction, the rat turns his head

slowly away or pulls it briskly backwards after the stimulus, with or without a single face wipe ipsilateral to the stimulated area. This is considered a mild aversive response; (3) escape/attack, rat avoids further contact with the stimulus either passively or moving its body away from the stimulus to assume a crouching position against the cage wall, sometimes with the head buried under the body or actively attacking the stimulus with biting or grabbing movements, sometimes accompanied with vocalizations; and

(4) asymmetric face grooming, in which the rat displays an uninterrupted series of at least three face wash strokes directed to the stimulated facial area (Vos & Maciewicz,1991 and

1994). We have modified the categories to show a score of 1 as no response, 2 as nonaversive, and 3 as mild aversive, 4 as strong aversive, and 5 as prolonged aversive behavior. The score of 5 includes an extra 1 point for vocalization scored under escpape/attack plus 1 each for detection, withdrawal, escape/attack and face grooming. In addition, we have added a category of pain with the response. Ordinal values were assigned to each of the different categories for the purposes of statistical analysis and also for the pain or no pain category. The mean pain score (MPS) will also be categorized for each stimulus prior to and after the surgery comparing the lesion and the normal, non- lesioned side.

84 Microscopic anatomy. The animals from the glycerol experiment were studied for light and electron microscopy. The animal was deeply anesthetized with ether and perfused through the heart with a saline washout solution (0.9% containing 0.4% xylocaine and

0.4% heparin) followed by cold (4º C) 0.1 M Phosphate-buffered fixative (3% glutaraldehyde, 3% paraformaldehyde, and 0.1% Picric acid at pH 7.4). Following fixation (overnight) the brains are removed and the brainstem, ION, ganglion, and trigeminal root were dissected. The nerve, ganglion, and trigeminal root sections are rinsed, buffered and osmicated (1.5% Ferricyanide in 0.1 M Cacodylate buffer, pH 7.4) for 90 minutes (Langford and Coggeshall, 1980), rinsed in buffered and en block stained

(0.5% uranyl acetate in malate buffer, pH 6.0) for 75 minutes. After another buffer rinse, the tissue was dehydrated in ascending concentrations of ethanol, cleared in propylene oxide, and wafer-embedded in an Epon/Araldite mixture. Thick sections (1 micrometer) of the nerve, ganglion, root or brainstem are stained with toluidine blue for localization and orientation. Thin sections (80–100 nanometers) of the nerve, ganglion, or root were cut and mounted on 100 parallel bar grids and viewed using a Phillips CM 10 electron microscope. Toluidine blue sections are used to focus on the nerve, ganglion, or root distal to the lesion, at the lesion, and proximal to the lesion. Light and electron microscopic sections of the abnormal side will be compared to the normal side in normal similar sections.

85 of the trigeminal ganglion and also in nucleus caudalis, nucleus oralis, and nucleus

interpolaris (Sugimoto et al., 1997). Substance P (SP), the tachykinin, is also a messenger

candidate in the primary afferent fibers and located in the small to medium cells of the

trigeminal ganglion and together with CGRP is seen in the nucleus caudalis, oralis, and

interpolaris (see Weihe et al., 1991 for a review). C-fos is a proto-oncogene and is

categorized as a cellular immediate early gene. The (Vos and Strassman, 1995) paper

reported on C-fos expression in the medullary dorsal horn of the rat after the CCI to the

ION. Galanin (GAL) will be used in the experiments to monitor injury to the trigeminal

root. Neuropeptide Y (NPY) is widely distributed throughout the peripheral and central nervous system and modulates neurotransmitter release in a highly selective manner and is an inhibitor of SP and is affected with peripheral nerve injury and inflammation.

Processing of the brainstem for immunocytochemistry followed the procedures described by (Rhoades et al., 1990a). After fixation, the tissue was post-fixed for 12 - 36 hours.

Sections (50 micrometers) were cut through the brainstem and were incubated in primary antibody for CGRP (vendor: Chemicon) 1:1000 dilution in phosphate buffer for 14 – 20 hours at room temperature, rinsed with phosphate buffer, incubated for one hour in goat- anti-rabbit IgG diluted 1:200, rinsed again, and incubated in AB complex (Vectastain)

diluted 1:100. Following several rinses, sections were reacted with 0.03% 3, 3´ -

diaminobenzedine (DAB) and 0.015% hydrogen peroxide in 0.1 M phosphate buffer.

After several rinses in phosphate buffer, sections were plated on gelatin-coated slides, air-

dried, dehydrated in graded ethanol, cleared with xylene, and cover slipped with

Permount. Similarly, tissue was processed for SP (vendor: INCstar 1:1000; (vendor:

86 Cambridge) 1:5000; GAL (vendor: Peninsula 1:3000; and NPY (vendor: Peninsula

1:1000) were incubated and processed.

Statistical analysis. To assess potential behavioral differences from baseline between the operated, sham, and normal groups, we used nonparametric methods due to the ordinal scaling of the behavioral measures. The data for the MPS were collapsed across the different von Frey monofilaments. The average response score to mechanical stimulation was calculated for the injected and the non injected sides for each week for the animals tested in each of the six groups in the first part of the root study and also the two groups in the second part of the study. The Kruskal – Wallis ANOVA was used to determine the statistical significance of the response between the two sides.

Data analysis. Light microscopy will be used to look initially at the pathology of the infraorbital root, ganglion, or brainstem, as well as the immunochemistry. The electron microscopy was used to look at the small fiber damage and WD.

87 RESULTS

Non-provoked behavior and general behavioral activity. We did see a change in

behavior in the glycerol root injected animals when looking at face grooming,

exploratory behavior, freezing like behavior, and body grooming when comparing post- lesion to baseline behavior. There were increased MPS values compared to both presurgical evaluations and control animals

Evoked responses to mechanical facial stimulation. Change in responses following glycerol injection of trigeminal root. The 50% glycerol injected trigeminal root animals showed a significant MPS on the surgical side subsequent to the procedure and when compared to the pre-surgical testing and compared to the control saline injected trigeminal root animals. There was no significant correlation between the intensity of the stimulus and the behavioral response and the data was therefore collapsed across the different Von Frey hair intensities to give the proper degrees of freedom for the statistical testing. The aversive behavioral response was seen with either a strong or mild stimulus which is similar to allodynia and hyperesthesia and the painful responses seen in the testing of human trigeminal neuropathic pain where a non-noxious stimulus is perceived as pain. The MPS to mechanical stimulation were calculated for the injected and non- injected sides prior to the injection and in the post-surgical period. The difference in response to mechanical stimulation between the injected and non-injected sides was taken as the dependent variable. Using the Kruskal-Wallis ANOVA, the glycerol group was significantly different from the saline group over the postoperative testing period (first

88 week little p=0.0117, second week p=0.0208, the third week p=0.0278, the fourth week p=0.0445). The MPS for the 2 gm, 6 gm, 9 gm and the pin stimuli are shown below for each stimulus collapsed over the six week period.

89 Figure 1. Mean Pain Score for 2 gram and 6 gram stimuli, following ganglion injection.

Dosing: 2 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Ganglion Injection 25% Glycerol (N=2) 3 0.9% Saline (N=2) 2.5 50% Glycerol (N=2) e

r 2 o c 1.5 S

ean 1 M 0.5

0 123456 Surgery Week

Dosing: 6 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Ganglion Injection 25% Glycerol (N=2) 3 0.9% Saline (N=2) 2.5 50% Glycerol (N=2) e

r 2 o c 1.5 S

ean 1 M 0.5

0 123456 Surgery Week

The Mean Pain Score (MPS) following application of a 2 gm stimulus (upper panel) or 6 gm stimulus (lower panel) to face of 25% glycerol injection into ganglion (red), 0.9% saline control into ganglion (dark blue), and 50% glycerol into ganglion (light blue) animals. There was no significant change in MPS in response to either the 2 or 6 gm stimuli for 2 wks prior to surgery (arrow) and 4 wks after surgery. Note the responses did not score over 2 while significant pain should score 3 or above for significant pain behavior, scored on the y-axis.

90 Figure 2. Mean Pain Score for 6 gram and pin stimuli, following ganglion injection.

Dosing: 9 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Ganglion Injection 25% Glycerol (N=2) 3 0.9% Saline (N=2) 2.5 50% Glycerol (N=2) e

r 2 o c 1.5 S

ean 1 M 0.5

0 123456 Surgery Week

Dosing: Pin Stimulus: 25%, 50% glycerol, 0.9% saline Ganglion Injection 25% Glycerol (N=2) 4 0.9% Saline (N=2) 3.5 50% Glycerol (N=2) 3 e r

o 2.5 c 2 S 1.5 ean

M 1 0.5 0 123456 Surgery Week

The Mean Pain Score (MPS) following application of a 9 gm stimulus (upper panel) or pin stimulus (lower panel) to face of 25% glycerol injection into ganglion (red), 0.9% saline control (dark blue), and 50% glycerol into ganglion (light blue) animals. There was no significant change in MPS in response to the 2 or 6 gm stimuli for 2 wks prior to surgery (arrow) and 4 wks after surgery. Note the responses to the 9 gm stimulus did not score over 2 while significant pain should score 3 or above for significant pain behavior, scored on the y-axis. Note that only the pin stimulus elicited pain responses (mean score >3), but this was found both before and after surgery for all three groups.

91 Figure 3. Mean Pain Score for 2 gram and 6 gram stimuli, following trigeminal root injection.

Dosing: 2 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Root Injection 25% Glycerol (N=2) 4.5 0.9% Saline (N=2) * * * 4 50% Glycerol (N=2) 3.5 * e

r 3 o

c 2.5

S 2

ean 1.5 M 1 0.5 0 123456 Surgery Week * P<0.02

Dosing: 6 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Root Injection 25% Glycerol (N=2) 4.5 0.9% Saline (N=2) * * 4 50% Glycerol (N=2) * 3.5 * e

r 3 o

c 2.5

S 2

ean 1.5 M 1 0.5 0 123456 Surgery * P<0.02 Week

The Mean Pain Score (MPS) following application of a 2 gm stimulus (upper panel) or 6 gm stimulus (lower panel) to face of 25% glycerol injection into root (red), 0.9% saline control injection into root (dark blue), and 50% glycerol into root (light blue) animals. There was no significant change in MPS to the 2 or 6 gm stimuli for 2 wks prior to surgery (arrow) and 4 wks after surgery. Note that there is a statistically significant increase in MPS (p=0.02) in animals that received a 50% glycerol injection into the root (light blue) when tested with the 2 gm or the 6 gm stimulus.

92 Figure 4. Mean Pain Score for 6 gram and 6 Pin stimuli, following trigeminal root injection.

Dosing: 9 Gram Stimulus: 25%, 50% glycerol, 0.9% saline Root Injection 25% Glycerol (N=2) 4.5 0.9% Saline (N=2) * 4 * 50% Glycerol (N=2) * * 3.5 * e

r 3 o

c 2.5

S 2

ean 1.5 M 1 0.5 0 123456 Surgery * P<0.02 Week

The Mean Pain Score (MPS) following application of a 9 gm stimulus (upper panel) or pin stimulus (lower panel) to face of 25% glycerol injection into root (red),0.9% saline control injection into root (dark blue), and 50% glycerol into root (light blue) animals. Note that there is a statistically significant increase in MPS (p=0.02) for only the 50% glycerol into root (light blue) injection in response to application of the 9 gm stimulus. The response to application of the pin stimulus was unchanged from that observed before and after the root injection.

93 Figure 5. Mean Pain Score for 2 gram and 6 gram stimuli, following glycerol injection in the trigeminal root.

2 Gram Stimulus Glycerol Root

Glycerol (N=5) 3.5 Saline (N=5) * 3 * *

2.5 * e r o

c 2 S 1.5

ean 1 M 0.5

12345Surgery 6 * P<0.05 Week

6 Gram Stimulus Glycerol Root

Glycerol (N=5) 3.5 Saline (N=5) 3 * * 2.5 * * e r o

c 2 S 1.5

ean 1 M 0.5

12345Surgery 6 * P<0.05 Week

The Mean Pain Score (MPS) following application of a 2 gm stimulus (upper panel) or 6 gm stimulus (lower panel) to face of 50% glycerol into root (red) and 0.9% saline control injection into root (dark blue) animals. Note that there is a statistically significant (p=0.05) increase MPS to the 2 gm and the 6 gm stimuli after 50% glycerol injection into the root but not in the 0.9% saline group after surgery (arrow).

94 Figure 6. Mean pain response scores for 9 gram and pin stimuli, following glycerol injection in the trigeminal root.

9 Gram Stimulus Glycerol Root

Glycerol (N=5) 3.5 Saline (N=5) * * * 3 * 2.5 e r o

c 2 S 1.5

ean 1 M 0.5

123456Surgery * P<0.05 Week

Pin Stimulus Glycerol Root

Glycerol (N=5) 3.5 Saline (N=5) 3.4

3.3 e r o

c 3.2 S 3.1

ean 3 M 2.9

2.8

12345Surgery 6

Week

The Mean Pain Score (MPS) following application of a 9 gm stimulus (upper panel) or pin stimulus (lower panel) to face of 50% glycerol into root (red) and 0.9% saline control injection into root (dark blue) animals. Note that there is a statistically significant (p=0.05) increase MPS following application of the 9 gm stimulus beginning 1 week after 50% glycerol injection into the root relative to saline-injected control animals. The response to application of the pin stimulus was unchanged from that observed before and after the root injection.

Histology. Sections of the trigeminal root, including the injected segment and a margin of normal root were cut serially and examined under the microscope after staining with toluidine blue. The injection track was identified and in the case of the saline injection, there was separation of axons found without any significant injury to the nerve fibers or myelin adjacent to the injection (See Figure 7).

96 Figure 7. Effects of saline injection in the trigeminal root upon the morphology of trigeminal root fibers.

Saline injection sites (arrows) showing fiber separation and minimal damage that appears restricted to the injection site. Scale bar 25µm for both panels.

In contrast, the glycerol injected roots showed extensive axon and myelin disintegration affecting mainly the large myelinated fibers adjacent to the injection (See Figure 8).

97 Figure 8. Light micrographs of normal and glycerol injected tissue.

A. Normal trigeminal root (20X), B. Glycerol injection site in trigeminal root (4X), C&D. Higher magnifications (10X and 20X, respectively ) of injection site shown in B illustrating axonal degeneration and demyelination proximal to injection site. Scale bars 25µm.

At the electron microscopic level, ephapsis of C-fibers and axonal injury can be observed

(See Figure 9).

98 Figure 9. Electron micrographs of normal and glycerol injected tissue.

A. Non-treated trigeminal root (at 6610X) showing normal structure of C-fibers and myelinated Aδ-fibers, B. Glycerol-treated trigeminal root at same magnification showing ephapsis and axonal damage (arrows), C-E. Higher magnification micrographs, showing the sites of ephapsis and axonal damage (arrows). Scale bars = 2.0 µm.

Immunocytochemistry for light microscopy. Immunocytochemistry showed no increased expression for CGRP, SP, NPY or Galanin for the glycerol injected root animals.

99 DISCUSSION

We noted in the glycerol root injection animals that the MPS to the 2 gm, 6 gm and 9 gm

stimuli were strong aversive responses. This was consistent with allodynia and

hyperpathia compared to the pin and pre-surgical testing. The allodynia responses were similar to those reported by stroking the face of cats (King et al., 1956; King, 1970;

Black, 1974) or of rats (Kryzhanovsky et al., 1974) with an epileptiform-producing lesion placed in the trigeminal nucleus caudalis. Similar hyper-responsive behavior was seen in cats and monkeys following the insertion of chromic gut sutures near the trigeminal root

(Burchiel, 1980). This study also reported demyelination and abnormal impulse generation electro-physiologically in the region of focal demyelination (Burchiel, 1980).

The prolonged aversive responses seen with the glycerol root injections with light mechanical stimulation were similar to the allodynia and mechanical allodynia that is seen in patients with NP. Clinicians observed long ago in TN patients, that light touch of apparently normal skin could provoke an intense pain, which outlasted the stimulus. It was accompanied by the same affective and vegetative reactions as intense pain from trauma or visceral disease. The first quantitative data on allodynic pain was reported in

TN patients studied with amplitude controlled vibration of the trigger zone (Kugelberg and Lindblom, 1959). These abnormal pain sensations that are elicited by mechanical allodynia with von Frey testing are seen in patients with TN or “tic douloureux” has been further documented (Fromm and Sessle, 1991).

100 The mechanism of pain production in the glycerol root model probably relates to the

neuropathology observed in the root and associated with central changes in response to

that root lesion. The light and electron microscopic studies in the trigeminal glycerol

injected root showed significant damage to the myelinated Aβ, Aδ, and unmyelinated C-

fibers. There was relative preservation of the C-fibers and there was ephapsis seen. It is

probable that the glycerol induced pathology described and wallerian degeneration (WD)

are accompanied by electro-physiological and immunochemical changes. The abnormal

impulse generation in focally demyelinated trigeminal roots reported by Burchiel (1980)

documented pathology and showed abnormal electro-physiology at one week, three

weeks, and six weeks after surgery on twelve cats and two monkeys. As there is good

experimental evidence that ectopic impulses can arise from demyelinated axons

(Rasminsky, 1978; Smith and McDonald, 1980, 1982), it is possible that the pathophysiology of TN is due to hyperactivity or abnormal discharges arising from the trigeminal ganglion. (Moller,1991; Burchiel, 1993; Pagni, 1993; Rappaport and Devor,

1994; Moulin, 1998). In fact, it is quite possible that the abnormal generation of sensory impulses from fibers transmitting light touch to fiber pathways is involved in the perception of pain in TN.

Similar pathology is reported in the majority of cases of human TN where depression or contact with the trigeminal nerve or root by small vessels near the brainstem is the cause of the demyelination. Significant relief of the NP occurs when microvascular decompression takes place or if there is balloon compression of the ganglion or glycerol injection of the ganglion. It is reported in the animals that the pain behavior will go into

101 remission over weeks or months and similarly in humans the TN will go into remission spontaneously or with treatment with carbamazepine or other anti-epileptiform drugs.

The ectopic afferent activity from the root appears to be associated with a loss of inhibitory interneurons (Sugimoto, 1987; Sugimoto et al., 1989), with a decrease in afferent or segmental inhibition (Horch and Lisney, 1981; Sessle, 1987).

The glycerol root animals showed a partial, segmental, axonal, demyelinative lesion with

WD capable of producing abnormal, ectopic, unpredictable impulses. This pathology could explain the episodic, paroxysmal, severe painful nature of trigeminal neuralgia.

This should explain the response of the lesion to surgical removal of the root compressive process and the response to agents that suppress abnormal electrophysiology at the lesion site.

102 CONCLUSIONS

This study provides further evidence that a segmental, axonal, demyelinative lesion on the trigeminal root produces observable pain behavior indicative of NP similar to trigeminal neuropathic pain seen in humans.

The study showed allodynia and hyperpathic aversive behavior over a four week period after the trigeminal glycerol root injection associated with a segmental, axonal injury, demyelination with ephapsis and WD.

It is a reliable model and it should be possible to examine immuno-pathophysiologic changes in the trigeminal root, brainstem nuclei and central projections.

It should be possible to study the effect of medication and surgery in this behavioral model. Further studies are planned to look at this rodent model with PET scanning and f-

MRI and also image patients with trigeminal neuropathic pain with and without MS in parallel, translational studies.

103

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109 DISCUSSION/SUMMARY

The results of the three experiments presented in this dissertation demonstrate the following:

(1) A proximal glycerol root lesion produced a significant behavioral effect compared to the distal CCI to the ION lesions.

(2) A segmental, axonal, demyelinative lesion produced this significant behavioral effect.

(3) Allodynia and hyperpathic aversive pain behavior were associated with ephapsis and wallerian degeneration (WD) within the lesion pathology.

(4) The root lesion is a reliable model for study of immuno-pathophysiologic changes in the trigeminal root, brainstem and central connections.

(5) The double suture CCI to the ION did not produce statistically significant behavioral effects. The upregulation of galanin (GAL) in nucleus caudalis demonstrated the effect of the lesion.

(6) A partial ION injury with a single tight 5-0 chromic suture produced statistically significant pain behavioral responses in the ION sensory distribution.

(7) The partial ION injury produces a segmental, axonal, demyelinative lesion.

110 (8) The light and electron microscopy showed inflammation with positive

immunochemical markers consistent with pain behavior and demonstrates a possible mechanism for allochiria.

(9) The up-regulation of GAL reported could contribute to the persistence of chronic pain, central sensitization and the pain “pattern”.

(10) The partial ION ligature model provides a reliable model for the further study of pain behavior in the trigeminal and general somatosensory system.

(11) Further study with immunocytochemistry is necessary to predict new methods of medical and surgical treatment of NP focusing on peripheral and central mechanisms.

(12) Neuropathic pain is a local, national and worldwide “pandemic” problem.

The results of the double constriction orbital CCI to the ION did not show significant pain behavioral change in response to the 2 gm, 6 gm, or the 9 gm mechanical stimuli. It is possible that we did not get a behavioral response with the orbital CCI to the ION due to a different placement of the ligatures around the ION. We did use the same surgical approach as that of Vos et al., (1991 and 1994) and we did confirm visually the presence of blood flow through the epineural vessels after placement of the two sutures. The increased GAL expression indicates structural injury to the ION. Galanin may have a role in the modulation of nociception after peripheral nerve injury (Wiesenfeld-Hallin et al.,

2004). This group has reported on GAL receptor subtypes with (1) inhibitory and (2)

111 excitatory effects, and (3) a receptor with trophic actions on the dorsal root ganglion. The role of GAL and modulation in ION transection and the trigeminal system has been studied extensively by (Rhoades et al., 1997). It is possible that these effects of GAL may have altered the behavioral response in our animals and the animals of the (Chudler and

Further study of the lesion with light and electron microscopy along with electrophysiology and immunocytochemistry should give some insight into the unreliability of the double CCI to the ION. The (Chudler and Anderson, 2002) paper demonstrated one peripheral mechanism for the lesion which was loss of low threshold input from the periphery. Central inhibitory as well as the peripheral mechanisms discussed are likely to ultimately explain the failure of this lesion.

The glycerol root lesion produced a segmental, axonal, demyelinative trigeminal root lesion showing aversive pain behavior in the rat consistent with the behavior of human trigeminal neuropathic pain. The lesioned rats showed intense aversive pain behavior and frequently would attack the stimulus and in one instance “bit the von Frey filament into pieces”. A patient with TN will frequently push away any effort to test sensation over the affected trigeminal division and refuse to groom, clean or otherwise contact this area.

112 This was common with the root lesion but not with the partial ION lesion which is consistent with the nature of the proximal pathology.

The glycerol root lesion showed allodynia, hyperpathic, aversive pain behavior over a four week period associated with a segmental, axonal, demyelinating injury, WD and ephapsis. The ephapsis associated with abnormal impulse generation would provide a peripheral mechanism for the paroxysmal nature of the pain (Burchiel, 1980). The vascular mechanical, and demyelinative (multiple sclerosis) encroachment on the trigeminal root has been associated with this described pathology (Jannetta, 1980; Love and Coakam, 2001; Love et al., 2001) and similar pathology has been reported in human cases. The pathology with ephapsis and abnormal electrophysiology could explain the demonstrated pharmacologic effect on the neuralgia with anti-epileleptic and other inhibitory, channel altering agents. The severe paroxysmal pain of the neuralgia will only respond to “narcotizing” doses of the strongest narcotics which is consistent with ephapsis “driven” pain and central sensitization involving limbic and other cortical pain- associated areas.

The literature shows about 3,300 cases of microvascular decompression (MVD) with variable success of complete pain relief of pain ranging from 53% in a small series of 36 patients, 94% result in 178 patents, and 76% in the largest series of 1185 patients with a mean follow up period of 5-8.5 yrs. The small series of 10 multiple sclerosis patients, showed 50% complete pain relief (Broggi et al., 2000; Ashkenazi and Levin, 2004;

Resnick et al., 1996). A large series of about 10,500 patients with radiofrequency

113 rhizotomy (6,205), glycerol rhizotomy (1217), balloon compression (759), MVD (1417), and partial trigeminal rhizotomy (250) was reported by (Taha and Tew, 1996). The MVD had the lowest rate of technical success, radiofrequency rhizotomy and MVD had the highest rate of pain relief and lowest rate of recurrence. Balloon compression had the highest rate of trigeminal motor dysfunction and MVD had the lowest rate of corneal anesthesia or keratitis. MVD had the lowest rate of facial numbness or dysesthesia.

Glycerol rhizotomy had the highest rate of pain recurrence. The MVD appears to be the best procedure for the root pathology as it removes the vascular impingement producing the segmental, axonal dysmyelinative lesion. The glycerol produces a more destructive lesion as does the balloon compression which would account for the reported motor injury and high pain recurrence rate. This is consistent with our 50% glycerol root injection which caused significant light and electron microscopic abnormal findings. The radiofrequency lesion is reputed to cause selective destruction of the small unmyelinated fibers associated with a level of temperature applied to a peripheral nerve (Letcher and

Goldring, 1968). This would be reflected in reduced ephapsis and abnormal C-fiber input into the brainstem nuclei and centrally. The increased failure rate in multiple sclerosis can be explained by the summation of multiple dysmyelinative lesions in the root entry zone, trigeminal tract, and central white matter projections in addition to the vascular impingement increasing the lesion quantitatively and qualitatively. There is an effect on the blood brain or root peripheral nerve barrier as well. This might also explain the observation in MS that physical trauma to the head and neck is associated with increased symptoms or attacks.

114 The glycerol root lesion model is reliable as documented with behavioral statistics, light

and electron microscopy. It will provide a basis for further examination of

immunopathologic changes in the trigeminal receptive fields, ganglia, root, brainstem

nuclei and central projections.

The partial ION injury with a single tight 5-0 chromic ligature produces statistically

significant aversive behavioral changes in the distribution of the ION. This injury also

produced a segmental, axonal, demyelinative lesion with WD. The psychometric

mechanical von Frey stimuli and pin showed statistically significant pain behavioral

responses except for the 6 gram stimulus. This was different from the root lesion in which

all stimuli had significant allodynic and hyperesthetic responses over time. It seems

reasonable to explain this based on the pathology which is greatest at the root and

involves more abnormal receptive fields than with the partial ION. Thus, the stronger

stimulus of 9 grams is more likely to provoke hyperesthesia as it is closest to the pin in

nociception. It also could represent an unexplainable population characteristic or

biological phenomenon. However, this diversity of behavioral response is also seen in

human NP, particularly more distal pain as seen with carpal tunnel and diabetic NP.

There is less structural and inflammatory pathology seen in these distal lesions. It is further significant that severe distal NP is seen in diabetic and traumatic neuropathy where there is associated proximal root or root entry zone pathology. This produces summation of pathologic lesions as seen in multiple sclerosis and TN. This is also seen in the “double crush syndrome” with cervical root, brachial plexus and distal carpal, ulnar tunnel lesions. It should be kept in mind that all of the behavioral testing in the root and

115 partial ION was done blinded to the tester as to where the abnormal receptive fields were

located. Subsequent studies that were done with GAL had definition and mapping of the

receptive fields at the time of surgery with a tungsten electrode in the portion of the nerve

receiving the tight chromic ligature. This permits focus on more accurate mapping of

receptive field pain behavioral responses for further protocols.

There was increased GAL expression on the lesion and the normal “allochiric” side

suggesting that inflammation, excitation or inhibition might drive this pain rather than an

unexplained structural process. This behavior is seen with expanded receptive fields with

the structural root and partial ION lesions providing a pathophysiologic explanation of

the expanded receptive fields seen with allochiria and associated with clinical pain that is

often mistakenly attributed to psychiatric disorders (anxiety, conversion disorders). The

persistence of the pain behavior over an eight week period, twice as long as the more

powerful root lesion suggests something other than the mechanical effects of the lesion. It

is likely that the inflammatory immunopathology explains the persistence of the pain

behavior seen in these animals and other studies (Benoliel, et al., 2002). Further study of inflammatory markers and immunocytochemistry is indicated to predict new methods of treatment of NP with focus on structural and molecular mechanisms.

This project began based upon the epidemiologic observation that my practice area in

Northwest Ohio had a large amount of NP. This epidemiologic observation has been confirmed locally (Buenaventura et al., 20003), nationally (Pain in America, 1999; IASP,

2003), and worldwide (IASP, 2003). Neuropathic pain is therefore a worldwide

116 “pandemic problem” now and in the foreseeable future. The pervasive magnitude of this pain problem is multifactorial. Poor physical conditioning, faulty work ergonomics, an ageing work population, substandard physician pain management, rationing of pain health care, and a failed workers compensation system are the most significant factors.

Physician education, industrial ergonomic reform, health insurance reform, health legislative change, and repair of the “broken” worker compensation system are desperately needed to treat the “unmet needs” of pain patients.

It could be stated that these observations are “anecdotal reports”. The clinical anecdote however has been the cornerstone of medical education since antiquity. Further, it is important to remember the words of Schoenberg (1978) in his text on analytic, experimental, and theoretic epidemiology. His caveat states that “statistical significance does not equal biological significance and epidemiologic studies are most useful in providing etiologic clues, but cannot be used to prove or refute causality.”

Lastly, a plan is in place for the study of NP and in particular the unusual amount and nature of NP in multiple sclerosis patients. Our designated center for the treatment of multiple sclerosis and NP will provide PET scanning, functional magnetic resonance, and rigorous clinical data to design further experiments in the animal models in collaboration with other laboratories to develop a mixture of therapeutic agents to address the peripheral and central pain patterns. Clinical experience has already shown that multiple routes, i.e., oral, intramuscular, intravenous, epidural, and intrathecal, are necessary either alone or collectively in the relief of NP. It is planned to approach these problems with

117 parallel animal and patient studies such as facial pain, cervical and lumbar root pain for which there are models, and also in patients with and without multiple sclerosis with appropriate controls and Institutional Review Board review.

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153 ABSTRACT

Chronic pain (CP) is among the most disabling and costly afflictions in North America,

Europe, and Australia (IASP, 2003). Chronic pain afflicts approximately 2.3 million or

20% of the population of the State of Ohio (Buenaventura et al. 2003) and includes trigeminal neuralgia (TN) and other neuropathic pain (NP) states that are enigmatic. To determine whether proximal more than distal demyelinating lesions produce NP, we studied the effect of glycerol injected into the trigeminal root and identical chronic constrictive injury (CCI) to the infraorbital nerve (ION) in the orbit. Psychometric testing was performed with von Frey monofilaments and a pin prior to and after surgery to determine any change in pain behavioral responses from baseline presurgical behavioral testing. The 50% glycerol injection into trigeminal root caused statistically significant facial pain behavioral responses to tactile stimulation. Ultrastructural study revealed loss of large myelinated axons, ephapsis, degeneration and segmental, axonal, demyelination.

The glycerol root injection is a reproducible animal model for the study of pain in the trigeminal system. The double CCI to orbital ION showed no statistically significant effect on pain behavior. In a third study, forty-four male Sprague-Dawley rats received a partial ION ligature with pstchometric behavioral testing before and after surgical manipulation with sham and normal controls. At weekly intervals after surgery the animals were studied with light and electron microscopy. The ION, trigeminal ganglia, and brainstem were evaluated for structural and inflammatory changes with Galanin

(GAL). Microscopy showed segmental, axonal, demyelinative lesions and degeneration.

The immunocytochemistry with GAL showed significant expression in the affected ION,

154 ganglia and brainstem with similar changes on the normal side. The GAL upregulation was accompanied by a significant increase in aversive pain behavior. These two models will be studied with neuroimaging techniques, pharmacologic and surgical manipulations in animals and patients.

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