NOTE TO USERS

This reproduction is the best copy available.

EFFECT OF LOCAL ANAESTHETIC BLOCK ON

NEUROGENIC TEMPOROMANDIBULAR

INFLAMMATION

Jason Kevin Wo ng

A thesis submitted in conformity with the requirements

for the Degree of Master of Science

Graduate Department of Dentistry

University of Toronto

O Copyright by Jason Kevin Wong 2001

National Library 1+1 of Canada Bibliothèque nationale du Cana&

Acquisiüons and Acquisitions et Bibliographie Services semices bibliographiques

The author has granted a non- exclusive licence aliowing the National Library of Canada to reproduce, loan, distribute or seil copies of this thesis in microfonn, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neitber the thesis nor substantial extracts fkom it may be printed or otherwise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire' prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

Local Anaesthet ic B lock Does Not Inhibit Mustard Oil Induced Temmromandibular

Edema Develovment . Jason Kevin Wong, Master of Science 200 1. Graduate Department

of Dentistry, University of Toronto.

Abstract

Temporomandibular joint (TMJ) disorders and rheumatoid arthritis are two

conditions in which neurogenic mechanisms are purported to play a critical role. The

purpose of this study was to uivestigate the neurogenic contribution underlying acute

TMJ inflammation by evaluating the effect of local anaesthetic blockade of afferent

innervation on the development of mustard oil (MO) induced edema in the rat TMJ area.

Adult male Sprague-Dawley rats were anaesthetized by intraperitoneal a-chloralose and

urethane. Saline injection into the right TMJ followed by MO ( 1 % to 60%) 6 minutes

later elicited dose-dependent edema development. Lidocaine (5%) or bupivacaine

(0.5%) followed by MO (1% or 40%) did not produce edema development different

from saline controls (p<0.05, Repeated measures ANOVA). Two conclusions may be

drawn. First, MO acts non-neuronally contrary to traditional perceptions or second, MO

selectively elicits edema through neurogenic mechanisms and therefore suggests a role

for neurogenic mec hanisrns in acute TMJ inflarnmat ion.

Acknowledgements

This work would not have been possible if not for the contributions of rny family,

fiiends, and professional mentors at this stage in my career but just as importantly in

years past which form the foundation on which 1 stand. The qualities 1 possess, 1 have

developed in the likeness of al1 these individuals.

My supervisors Dr. James Hu, Dr. Daniel Haas and Dr. Howard Tenenbaum 1

must thank for their time, efforts and valuable input fiom the outset to conclusion of this

thesis. Their guidance and attitudes have fostered my individual thought and abilities.

Professional growth and development at the graduate level I have them to thank.

Daily assistance in completing my experiments were provided by Ms. Susan

Carter and Mr. Ken Macleod. A technique and equipment intensive study such as this

would not be a possibility without these integral tearn members.

To Brian Cairns, Paolo Fiorentino, and Bonnie Cai who provided hours of

discussion regarding my project and other topics unmentionable, 1 thank. Their

professional expertise, aid, and fiiendships 1 value highly.

Financial support to bring this thesis to fniition was through NIH g a n t

Dec 1 1 995.

Last but not least 1 wish to thank my mother, father, my brother Jeff, and

Marianne, who gave me their understanding, support, and encouragement through al1 the

late nights and busy weekends when 1 know they missed me being with them. A final

thanks to Sacha, Andie, Stacey, Bruno, and Catherine who would peiiodically check on

me during wnting of this thesis to ensure 1 was exhibiting unassisted spontaneous

respirations.

iii

Table of Contents

. . ...................................................................................... Abstract -11

... ......................................................................... Acknowledgements .III

.................................................................................. List of Tables x

................................................................................ List of Figures xi

..* ............................................................................... Abbreviations xi11

1 Introduction and Review Of The Literature

1 . 1 Impetus and focus for the current investigation ................................. .1

1 .2 Temporomandi bular Disorders and Neurogenic Inflammation ................ .2

............................ 1.3 Rheumatoid Arthntis and Neurogenic Inflammation 8

1.4 Neurogenic Inflammation

1.4.1 Definition.. .................................................................... 1 1

1.4.2 HistoricalBackground ....................................................... I I

.......................................... 1.4.3 Concepts From Past to Present.. -14

1.4.4 The Sympathetic Nervous Systern and Neurogenic Inflammation

1.4.4.1 Sympathetic Intluences tncrease Severity of Chronic

............................................................ Arthritis.. 16

........ 1 -4.4.2 Sympathetic Contributions to Plasma Extravasation. .17

1.4.4.3 Sympathetically Mediated Increases in Plasma

Extravasation May Represent A Reparative

......................................................... Mechanism -20

.......... 1.4.5 Parasympathetic Influences on Neurogenic Inflammation.. .20

......... 1.4.6 The Central Nervous System and Neurogenic Inflammation.. 2 1

................................................ 1 .4.6.1 Local Spinal Circuits 2 1

1 A6.2 Central Descending Modulation ................................... 23

1.4.7 The Cellular Immune System and Neurogenic Inflammation ......... -25

1.5 Acute Versus Chronic lnflammation

1 .5 . 1 Acute and Chronic Inflammation: Traditional Methods

................................................................... Of Induction 27

1 S.2 Xcute Models of Inflammation And Study of

......................................................... Dorsai Root Reflexes 28

........................ 1 S.3 A Continuum of Inflammation: Acute to Chronic 30

1.6 Animal Models of Neurogenic Inflammation

........................................................................... 1.6.1 General 31

...................................... 1 6 2 Study of the Ternporomandibular Joint 34

1.7 Neurogenic Inflammation Involving The TMJ

..................................................................... 1.7.1 Substance P 35

.................................................... 1 . 7.2 Substance P and Arthritis -36

............................ 1.7.3 Substance P in the Ternporomandibular Joint -37

.......... 1.7.4 Direct Study of the Neural Influence on Joint Peptide Levels 38

.......................... 1.8 Electromyography In Pain and Inflammation Research 40

1.9 Mustard Oil As A Chemical Algogen in the Study of

.................................................... Neurogenic Inflammation 41

1.10 Innervation of the Temporomandibular Joint ............................. 43

1.1 1 Local Anaesthetics

................ 1.1 1.1 Physiology of Neuronal Depolarization and Conduction 46

1.1 1.2 Mechanism of Local Anaesthetic Activity ................................. 47

.................................. 1.1 1.3 Neuronal Excitation-Secretion Coupling 47

1.12 Statement of Rationale, Purpose, and Objectives

....................................................................... 1.12.1 Rationale 48

....................................................................... 1 . 1 2.2 Purpose -49

. . ........................................................... 1 . 12.3 Specific Objectives 49

1.12.4 Rationale for Use of Lidocaine, Bupivacaine,

............................................................... and Mustard Oil 51

2 Materials and Methods

2.1 Drug Itemization. and Preparation

................................................................. 2.1.1 Lidocaine HCl 53

.............................................................. 2.1.2 Bupivacaine HCl 53

.................................................................... 2.1.3 Mustard Oil 54

.............................................................. 2.1.4 Evan's Blue Dye 55

2.1.5 Urethane ....................................................................... 55

.................................................................. 2.1.6 a-Chioralose -56

................................. 2.1.7 Propnetary Heparin, Normal Saline, T-6 1 56

2.2 Experimental Models

2.2.1 General

................. 2.2.1.1 Set Up and Loading of Double Barrel Catheter 57

......................... 2.2.1.2 Confirmation of Final Catheter Position 57

2.2.2 Tissue Expansion Mode1 of Orofacial Inflammation

2.2.2.1 Setup .................................................................. 58

2.2.2.2 Dose Response Curves .............................................. 63

2.2.2.3 Conduction Blockade Using Local Anaesthetic .................. 63

2 -2.2.4 Examination of Contralateral Ternporomandibular

.................................................................... Joint 64

2.2.3 Electromyographic Jaw Reflex Mode1

2.2.3.1 SetUp ................................................................. 64

.................... 2.2.3.2 Confirming Complete Conduction Blockade 65

............... 2.2.3.3 Detmining Duration of Conduction Blockade -66

. . .................................................................... 2.3 Statistical Analysis 68

3 Results

................................................................ 3.1 Dose Response Cuwes 69

3.2 Effect of Local Anaesthetic Block On

....................................................... Mild and Severe Inflammation 72

............................................. 3.3 Confirmation of Conduction Blockade 72

.................................................. 3 -4 Duration of Conduction Blockade 77

........................... 3.5 Contralateral Temporomandibular Joint Examination 77

-

4 Discussion

4.1 Mustard Oil induces Acute Edema Development in

...................................................................... the Rat pTM Area 79

......................... 4.2 Effect of Local Anaesthetic Blockade On Inflammation 80

.............................................................. 4.3 Interpretation of Results 82

4.3.1 Does Mustard Oil Elicit Neurogenic Inflammation

Through Non-neurogenic Mechanisrns?. ................................. -83

4.3.2 Does Mustard Oil Initiate Direct Release of Mediators Of

Neurogenic Inflammation From Nociceptors?. ......................... .86

4.4 The Central Component to Neurogenic Inflammation In the TMJ

4.4.1 Are Dorsal Root Reflexes Fundamental to Acute Neurogenic

............................................................... Inflammation?. .88

............................ 4.4.2 Spinal Reflex Arcs In Acute Inflammation.. ..89

4.5 A Reverse Trend Toward Increased Plasma Extravasation With Local

Anaesthetic Blockade

.................................... 4.5.1 Inhibition Of Sympathetic Efferents.. .9 1

4.5.1.1 The Effect of Local Anaesthetic on

............................................. Sympathetic Neurons. -92

4.5.1.2 Summary: Does the Sympathetic Nervous System

................................ Play A Role In Producing Edema.. 93

4.5.2 Effect of Deafferentation on Activity of Pnmary Afferents and

Plasma Extravasation.. ...................................................... .94

.................. 4.5.2.1 The Gate Control Theory of Inflammation?. ..95

4.6 Other Studies Of Local Anaesthetics In Atternpts To Abolish

......................................................... Neurogenic Inflammation.. -96

.............................. 4.7 Vascular Effects of Lidocaine and Bupivacaine.. .98

4.8 Lidocaine and the Acute Local Cellular Immune Response

viii

.................................................... In The Tissue Expansion Mode1 100

........................................ 4.9 Strengths and Limitations of Methodology 103

....................................... 4.10 Future Directions For Investigation 105

4.1 1 Summary and Conclusions

........................................................................ 4.1 1 . 1 Summary 108

................................................... 4.1 1.2 Two Possible Conclusions 108

.............................................. 4.1 1.3 Clinical Implications of Results 110

.................................................................................... 5 References 1 1 1

List of Tables

Table

1

2

3

Page

Actual Mustard Oil Concentrations Utilized ....................................... 55

Experimental Groups .................................................................. 70

Mean Expansion Distance at time 0.20. 100. and 1 50 ........................... -70

List of Figures

Page Figure

1 A Dynamic Conceptual Model of the Etiology in

Temporomandibular Disorders ....................................................... 5

Common Pathways for Three Proposed Mechanisms .............................. 7

Cytokines and Other Factors Involved in the Pathogenesis of

.................................................................. Rheumatoid Arthritis 10

Evolution of Concepts of Antidromic Vasodilatation and

............................................................. Neurogenic Inflammation -15

Sympathetic Post-ganglionic Nerve Factors Elaborated Which

............................. Contribute to Plasma Extravasation and Joint Injury -18

Schematic: Set Up of Tissue Expansion Model of Neurogenic

........................................................................... Inflammation -59

Tissue Expansion Model of Neurogenic Inflammation: Timing of

Catheter Insertion, Injection of Local Anaesthetic or Saline and

............................................................................. Mustard Oil .62

EMG Jaw Reflex Model: Timing of Catheter Insertion, Baseline

............................................... Recording, and Mustard Oil Injection 67

9 Dose Response Cumes: increasing Mustard Oil Concentrations ................ 71

.................................... 10 Effect of 5% Lidocaine on Expansion Distance 73

............................... I l Effect of 0.5% Bupivacaine on Expansion Distance 74

........................ 12 Summary: Effect of 5% Lidocaine and 0.5% Bupivacaine 75

................................ 13 Evaluating Duration of Blockade By 5% Lidocaine 76

14 Evaluating Duration of Blockade By 0.5% Bupivacaine ........................... 78

1 5 Traditional Axon Retlex Theory ...................................................... 87

................................................... 16 The Trigeminal Dorsal Root Reflex 90

17 Expansion Distance Due to 0.9% Saline or 0.5% Bupivacaine .................. 101

........................ 18 Expansion Distance Due to 0.9% Saline or 5% Lidocaine 102

xii

Abbreviations

ANOVA

CGRP

CGRP-LI

CNS

CNQX

CSF

CVA

DRG

DRR

EB

EMG

GABA

GABAG

GABAB

IL- 1

i.p.

i.v.

LDI

LT

MO

NE

NI

analysis of variance

calcitonin gene related peptide

calcitonin gene-related peptide - like immunoreactivity

Centra[ Nervous System

6-cyano-7-nitroquinoxaline-2,3-dione

cerebrospinal fluid

cerebrovascular accident

dorsal root ganglion

dorsal root reflexes

Evan's Blue

electrom yographic

gamma-arnino-butync acid

gamma-amino-butyric acid receptor subtype A

gamma-amino-butyric acid receptor subtype B

interleukin - 1

intraperitoneal(1y)

intravenous(1y)

laser doppler perfusion imaging

leukotnene

mustard oil

norepinephrine (noradrenaline)

neurogenic inflammation

xiii

NKA

N U - L I

NO

non-NMDA

NPY

NPY-LI

NSAID

PAD

PE

PTM

RA

S.C.

SP

SP-LI

SPGN

TENS

TMD

TMJ

TNF-a

VIP

WGA-HRP

5-HT

6-OHDA

neurokinin A

neurokinin A - like immunoreactivity

nitric oxide

Non N-methyl-D-aspartate

neuropeptide Y

neuropeptide-Y like immunoreactivity

non-steroidal anti-in flanmatory drug

primary afferent depolarization

plasma extravasation

periarticular temporomandibular

rheumatoid arthritis

subcutaneous(l y)

substance P

substance P - like irnmunoreactivity

sympathetic post-ganglionic neuron

transcutaneous electrical nerve stimulation

temporomandibular disorders

temporomandibular joint

tumor necrosis factor - a

vasoactive intestinal polypeptide

wheat gem agglutinin-horseradish peroxidase

5-hydroxytryptarnine (serotonin)

6-hydroxydopamine

xiv

Chapter 1

Introduction and Review of the Literature

1.1 Impetus and focus for the current investigation

The suggestion that physical or functional abolition of nociceptive joint afferents

may prove to be a powerful means to arrest painful, destructive, inflammatory processes

is an exciting prospect. The therapeutic implications for rheumatoid arthritis (RA) and

degenerative temporomandibular joint (TMJ) disease are readil y apparent. Current

management approaches are unsatisfactory in tems of diagnostic criteria and long term

outcome subsequent to a poor understanding of relevant molecular and cellular

pathophysiology. The investigation into neural mechanisms underlying these

inflammatory diseases is still in its infancy.

The hypothesis that neurogenic inflammation plays a pivotal role in the

development of temporomandibular disorders (TMD) has been increasingly embraced in

recent years (Milam and Schmitz, 1995; McKay and Christensen, 1998). However,

evidence for pro-inflammatory neurogenic mechanisms in the TMJ has been

circumstantial at best. Cornparisons of TMJ aspirates from patients with intemal disk

derangement and control groups have revealed more intense expression of substance P-

like immunoreactivity (SP-LI, see section 1.8. l ) and pain ratings fkom affected patients

(Yoshida et al., 1999). The anatomical distribution of SP immunoreactive neurons

paraltels the trigeminal newe fibre distribution in the rat TMJ (Kido et al., 1993).

Carleson et al. (Carleson et al., 1996b) demonstrated SP injection into the TMJ raises

articular levels of other pro-inflarnrnatory peptides such as neuropeptide Y (NPY),

neurokinin A (NKA), and calcitonin gene related peptide (CGRP). Direct physiologic

examination of the TMJ to characterize the potential role of neurogenic mechanisms in

inducing inflammation appears to be the subsequent Iogical step for investigation. This

is the dnving impetus and focus of the current study.

"The one systern where a contribution of neurogenic inflammation

to a clinically relevant pathological process has been documented

is the migraine model, where there is a minimal non-neurogenic

component to meningeal and cerebrovascular inflammation.

In joints and skin it has proved much more difficult to unarnbiguously

demonstrate a major role for neurogenic inflammation."

(Woolf, 1995)

1995 Pain Forum Commentary

1.2 Temporomandibular Disorders and Neurogenic

Inflammation

Ternporomandibular disorders (TMD) are a vague1 y de fi ned and poorl y

understood heterogeneous collection of related conditions which present with common

symptoms that may include pain, joint clicking, and limitation of jaw movement

(Grosfeld et al., 1985; Locker and Slade, 1988; Rugh and Solberg, 1988; LeResche et

al., 199 1 ; Dworkin and LeResche, 1992; De Kanter et al., 1993; Dimitroulis et al., 1995;

NIH, 1996). An accurate estimate of the prevalence is difficult to make given the variety

of critena used to define a TMD (De Kanter et al., 1993; Deng et al., 1995). Joint pain

has been reported in 0.8- 12.9% of the general population with a higher prevalence in

women (Locker and Slade, 1988; Shiau and Chang, 199 1 ; De Kanter et al., 1993; Bibb

et al.. 1 995; Goulet et al., 1995; Hiltunen et al., 1995). Pain prevalence increases fiom

teen years to adulthood and then diminishes in geriatric populations (Greene, 1994;

Wanman, 1996). These statistics indicate that TMD as defined by joint pain is a

widespread phenornenon and is responsible for considerable pain, suffering, dysfunction

and reduction in quality of life for both young and elderly.

The use of symptomatology rather than objective clinical means of defining

TMD is ultimately a reflection of our limited appreciation of the pathophysiological

mechanisms underlying these conditions. Sternming fiom this ignorance are inadequate

subclassifications and consequent myriad arrays of surgical and non-surgical

management schernes and treatrnent protocols (Milarn and Schrnitz, 1995). Success in

treating disease requires first an ability to accurately and reliably diagnose the

underlying pathosis (Dimitroulis et al., 1995).

Our research efforts should focus on the integration of two related aspects of

disease management. First, a conceptual framework must be recognized that embraces

the multifactorial nature of TMD including physical, emotional and psychophysical

factors. Second, molecular biologic mechanisms that underlie physical contributions to

such disorders must be elucidated. Management modalities addressing the primax-y

biologic etiology of a M D while embracing secondary contributing factors will serve

patients and clinicians as a sound, and effective means to deal with TMD.

Parker (Parker, 1990) has proposed a dynamic conceptual model for approaching

the diverse etiological factors to TMD (Figure 1). The masticatory system is in dynamic

homeostatic balance between extreme conditions of 'orthofùnction' and 'pathofünction'.

Orthotiinction is a texm coined by Rugh (Rugh and Solberg, 1976) to describe a

physiologically Iünctional masticatory system including those simply adaptive in this

marner. Pathotùnction is dysfiinction involving injury of some sort: a TMD (Parker,

1990). Individuals are generally maintained in a state of orthohnction through

homeostatic mechanisms (Parker, 1990). The model further proposes that States of

orthofunction and pathofùnction have driving forces, which increase adaptability or

hyperfunction respectively. Various etiological factors either increase or decrease

adaptability or hyperfunction. The resulting sum of al1 influencing factors determines the

ultimate tendency toward orthoîùnction or TMD.

Physical trauma is held to be the single most important factor in reducing the

adaptability of the masticatory system and consequently a force predisposing toward

development of TMD (Parker. 1990). Trauma may take the form of excessive articular

loading through parafunction or may occur through surgical manipulation of the TMJ

(Parker, 1990; McKay and Christensen, 1998).

A recent hypothesis put forth by Milam and Schmitz (Milam and Schmitz, 1995)

regarding the molecular rnechanisms of TMD sirnilarly propose that mechanical trauma

may be the single most important initiating event in the process of joint degeneration.

Mechanical integrity of joint c m be envisioned to be the physical factor denominated in

Poor Health Structural Integrity

1 Female Gender

Male Gender Good Systemic

/kalthy Joint Structure 1

Low Stress s No Depression

Stable Occlusion No Sleep Disorders

Occlusion Depression

Life S tressors Sleep Disorders

Pathofunction

Figure 1 A Dynam ic Conceptual Mode1 of the Et iology in Tempommandibular Disorders Adapted fiom Parker, 1990

the aforementioiied mode1 (Parker, 1990) as imparting on the joint the property of

'adaptability'. Mechanical stresses rnay lead to TMJ tissue destruction and subsequent

reduced adaptability. A vicious cycle rnay ensue.

Currentl y, three h ypotheses have been proposed to ex plain how mechanical stress

rnay result in net tissue loss (Figure 2).

The first hypothesis subtends that mechanical stress causes direct darnage to

tissues. This rnay be through disruption of molecules and production of fiee radicals.

Free radicals rnay degrade important articular tissue components such as hyaluronic acid

via a propagated chain reaction (Milarn and Schrnitz, 1995). Free radicals rnay increase

arachidonic acid metabolism and consequent production of compounds such as

prostaglandins. Prostaglandins in tum rnay sensitize primary afferent neurons supplying

periarticular regions. Mechanical stress rnay also cause direct darnage at the cellular

level through disruption of cellular cytoskeletons (Haskin et a/., 1993).

The second mechanism proposed is production of a hypoxia-reperfusion injury

following mechanical stress. Articular loading rnay prûduce a transient localized hypoxia

and modification of certain enzyme species. Subsequent reperfusion rnay cause these

enzymes to catalyze reactions leading to fiee radical production and consequent tissue

damage as described previously.

The final mechanism that has been suggested to lead to tissue degradation

following mechanical insult to the TMJ is through a neurogenic inflammatory pathway.

1 Mechanical Stress

Direct Cellular Wury

Free Radical Hypoxia-Repemision

Arachidonic Acid Metabolkm

blatrix Degrading Enzymes

Figure 2 Common Pathways for Three Proposed Mechanisns of Degerierative TMJ Disease Adapted fmm Milan and Schmitz, 1995

1.3 Rheumatoid Arthritis and Neurogenic Inflammation

Rheumatoid arthritis (RA) is "a chronic multisystern inflammatory disease with

autoimmune features, and of unknown cause, associated with characteristic joint

deformities and increased mortality rate." (Odeh, 1997)

This definition of RA taken from a recent review highlights two key points. First,

the precise etiology of RA is presently unknown. Second, disease is only defined by

clinical signs and symptoms (Amett et of ., 1 988). The American Rheumatism

Association 1987 Criteria requires 4 of 7 defined signs or symptoms to be present in

order for the diagnosis of RA to be made (Amett, 1988). These include moming s t i f iess

in and around joints lasting at least 1 hour before maximal improvement, and symmetnc

swelling of joints (Amett, 1988). Additional important features of RA are its tendency to

involve more distal joints, and its increased severity the more distal the joint involved

(Mitchell and Fries, 1982b).

RA is a disease for which first and second line pharmacologic agents have

proven unsatisfactory in the long term due to limited effectiveness and signifiant side

effects (Iannuzzi et al., 1 983; Scott et al., 1 987; Pincus, 1 992). Complications include

non-steroidal anti-inflammatory drug (NSAID) gastropathy, osteoporosis, osteonecrosis,

fractures, cataracts, diabetes and other catabolic effects (Roubenoff et al., 1990; Hall et

al., 1993, Leigh and Fries. 1994). Outcornes usually consists of years of pain, suffering,

severe functional debilitation, work disability and premature death (tannuzzi et al., 1983;

Scott et al., 1987; Pincus, 19%).

RA is a condition recognized worldwide. The prevalence of RA is most

commonly reported to be between 0.3% to 1% in the adult population (Alarcon, 1995).

Peak onset is between the fourth and six decades of life with a predisposition for women

at a ratio of 3: 1 (Alarcon, 1995). RA is clearly recognized to shorten the life expectancy

of affected individuals (Pincus and Callahan, 1993; Pincus et al., 1994).

The diagnosis of TMJ involvernent by RA is made by signs and syrnptom as

outlined above in addition to signs and symptoms related to the joint itself including

clicking, crepitus, locking, and soreness (Holmlund et ai., 1 992; Celiker et al., 1 995).

Reported incidence of RA involvement in the TMJ varies fiom 5 to 86% (Syrjanen,

1985). A recent study of 20 patients reported a prevalence of 45% (Celiker, 1995).

Recent research into molecular mechanisms of RA implicate a number of key

inflammatory mediators expressed by immune cells (Figure 3). Key to this concept is the

identification of 'inflarnmatory stimuli' responsible for stimulating cells to elaborate

such factors. These 'inflarnmatory stimuli' may potentially take the form of a neurogenic

release of pro-inflammatory mediators.

Levine et al. (Levine, 1985; Levine et al., 1985a) make poignant remarks

regarding the potential role of neurogenic mechanisms in the pathogenesis of RA:

"The proposed pathogenic mechanisms for rheumatoid arthritis

in man - an abnomal immunologic response, a normal immunologic

response to an arthntogenic infectious agent, or a combination of these

two mechanisms - do not explain the important clinical features of these

diseases. . .We hypothesize that neural mechanisms are involved in the

pathophysiology of inflammation in rheumatoid arthritis."

1 ( Inflammatory Stimuli ) 1 1 Neurogenic Inflammation 1

LIF P GE, IL-1

PGE, Plasmin Plasmin

IL-6 IL-6 IL-1 IL-8 IL-8 IL-6 PGE, GM-CSF IL-8 MMPs LIF GM-CSF

MMPs L E IL-1 Mm's TNF- a =-a Others 1 CAM- 1

1 Others

='GE, IL- 1 Tm-@ TNF-a FGF MMPs

MMPs FcRm

Destruction /

Figure 3 Cytokines and Other f a d m Involved in the Pathogenesis o f Rheumatoid Arthritis Adapted fiom Ode4 1997

1.4 Neurogenic Inflammation

1.4.1 Definition

"Neurogenic inflammation is increased vascular permeability and

plasma extravasation elicited by antidromic stimulation of sensory nerve

fibres."

(Chahl, 1988)

1.4.2 Historie Background

Research and knowldge pertaining to neurogenic inflammation (NI) has its

ongins in the study of a related phenomenon known as antidromic vasodilatation.

Antidromic vasodilatation has been the subject of investigation for more than a century.

Stricker (Stricker, 1876) in 1876 noted that stimulating the cut ends of dorsal roots of

peripheral nerves induced an increase in the temperature of skin innervated by these

nerves secondary to vasodilatation. He concluded that these dorsal nerve tninks

contained efferent fibres, which contradicted the Bell-Magendie Law of separation

(Agnew et al., 1 965). Bayliss (Bayliss, 1 90 1 ) in 1 90 1 confimed these findings and

fûrther demonstrated that cutting these nerves between the ganglia and spinal cord had

no effect, but division distal to the ganglia caused degeneration of these nerve fibres and

abolished the vasodilatory response. Bayliss concluded the vasodilatation was in fact due

to the efferent activity of afferent nerves and coined the term 'antidrornic' to articulate

this concept. Bruce (Bruce, 19 13) in 19 13 demonstrated mustard oil (MO) induced

vasodilatation in the cat conjunctiva required intact nerve conduction but not necessarily

connection to the spinal cord. He suggested this phenornena of vasodilatation took place

completely within a single nerve fibre and its associated branches through a so-called

'mon reflex'. Langley (Langley, 192 1) in 192 1 suggested a physiological role for

antidrornic vasodilatation in the flare component of inflammatory reactions and therefore

not simply epiphenomena.

Lewis (Lewis, 1927) in 1927 described the 'triple response' of an inflammatory

reaction as having the components of redness, wheal, and flare. He rightly assumed that

the f lue response was distinct from the wheal or edema formation shown later to be due

to increased vascular permeability secondary to endothelial ce11 contraction (Majno et

al., 1969). Lewis suggested flare was mediated through an axon reflex, which delineated

the extent of vasodilatation according to the receptive field of that particular nerve. He

further suggested the role of a 'neurotransmitter' substance released fiom nerve

terminais to the site of action on the vasculature. This was to account for the delay of

vasodilatation observed fiom the time of nerve stimulation. Lewis suggested histamine

as this neurotransmitter but later concluded that other chemicals must be involved since

the reaction to histamine alone (including itch sensation) was not necessarily present

with vasodilatation.

Jancso and colleagues (Jancso et ai., 1967; Jancso and Jancso-Gabor, 1968) in

the 1960's and 1970's utilized capsaicin, an extract of hot peppers to induce a wheal and

flare response in the skin. Desensitization of entire animals was achieved with neonatal

parenteral capsaicin treatment indicating that capsaicin somehow acted selectively on

sensory nerve fibres (Jancso, 1967). Jancso (Jancso and Jancso-Gabor, 1968)

demonstrated that intact nerve conduction was necessary for induction of a flare

response since it was abolished by local anaesthesia. The wheal response though

requiring the presence of nerves did not depend on nerve conduction since local

anaesthetic block had no effect. Interestingly, capsaicin pretreatment of tissue was able

to abolish the ederna response to antidromic stimulation of the rat saphenous nerve

(Jancso, 1967). These studies provided the first strong direct evidence that sensory

neurons were responsible for producing the phenornena of antidromic vasodilatation and

associated vascular penneability.

Hinsey and Gasser (Hinsey and Gasser, 1930) noted antidromic vasodilatation

was elicited with voltages sufficient to produce a 'C' wave on a neurogram. Celander

and Folkow (Celander and Folkow, 1 953) were the first to provide direct evidence that

nociceptors were responsible for antidromic vasodilatation. Thermal stimulation only

into the noxious range elicited vasodilatation in the cat paw skin and this was abolished

with denervation. Chahl and Ladd (Chahl and Ladd, 1976) demonstrated that edema was

evoked with voltages sufficient to excite C-fibres in the rat saphenous nerve.

These studies and work by Jancso and Lewis al1 suggested that the mechanisms

and mediators of antidromic vasodilatation were similar if not identical to the ones

responsible for producing increased vascular permeability and plasma extravasation

(PE): that is, neurogenic inflammation.

1.43 Concepts From Past to present

The concept of neurogenic inflammation since its inception has embraced several

key aspects. Lewis (Lewis, 1927) coined the t e m 'nocifensor' systern to indicate his

belief in the existence of a separate neural network dedicated to host defense. n i e

recognition of the role of neurally derived mediators and involvement of mast cells was

similarly proposed (Lewis, 1927; Kieman, 197 1 ; Kiernan, 1972). Figure 4 depicts the

evolution of how these various components are thought to integrate to produce a

neurogenic inflarnmatory response. 4 (a) illustrates the axon reflex proposed by Lewis

(Lewis, 1927). The relegation of the role of mast cells fkom having a direct action on

afferent terminals (c) has changed to an action on vasculature as depicted in (d). It was

not until the 1970's that SP appeared to be the primary mediator of neurogenic

inflammation as illustrated in (e) (Lembeck et a/., 1977; Lembeck and Holzer, 1979).

Lembeck and Gamse (Lembeck and Gamse, 1982) proposed the action of an axon

cascade initiated by mediators which then propagated centrally as nociceptive

information and penpherally to release SP from neuronal terminals. The current day

concept (f ) perceives an initiating event to take place at the neuronal terminal resulting

in an eventual antidromic depolarization from the CNS and release of neurotransmi tters

such as SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) (Maggio

and Hunter, 1984; Gamse and Saria, 1985). SP and neurokinin A then act on mast cells

to release histamine (Jonzzo et al., 1983). CGRP in this context fbnctions to vasodilate

and facilitate the action of histamine, SP, and NKA (Gamse and Saria, 1985).

&on reflex £km Lewis, 1927

To CNS & N o acept ion

Mustard + 0 d I I

Histamine - -

~urratt Concept ]

I $ / To CNS From j

CNS ; 1 v ;

Mustard

CGRP SF'/-NKA

Figure 4 Evolution o f Concepts o f Antidromic Vasodilatation and Neumgenic Inflammation Adapted fim Chahl, 1988

Two other components of the neurogenic inflamrnatory response have received attention

in this decade. The role of the syrnpathetic nervous system and the role of central

influences are relatively unknown and the subject of current investigation.

1.4.4 The Sympathetic Newous System in Neurogenic Inflammation

1.4.4.1 Sympathetic Influences Increase Severity of Chronic Arthritis

The precise contribution of sympathetic efferents in arthritis has yet to be

elucidated. Levine et a/. (Levine et a/., 1986) dernonstrated that dorsal rhizotomy caused

an increase in arthritic severity in adjuvant induced rat knee arthritis. It was furiher

reveûled that prior sympathectomy abolished the exacerbation, suggesting a cntical role

of the sympathetic nervous system or large diameter afferents. In another study rat strains

with increased basal sympathetic tone (hypertensive rats) manifested more severe clinical

and radiographie evidence of arthritis (Levine, 1986).

Elucidation of specific receptors involved is further evidence for the role of

sympathetic post-ganglionic neurons (SPGN) in inflammatory disease. Epinephrine and

salbutamol (a specific Pz-agonist) increase severity of adjuvant induced arthritis

(Coderre, 1 990).

These data taken together strongly suggest a role of the syrnpathetic nervous

systern in exacerbating severity of chronic arthritis. The mechanism of sympathetic

influence on underlying inflammation has only recently becorne investigated using

models of acute inflammation and remains ambiguous.

1.4.4.2 Sympathetic Contributions to Plasma Extravasation

Lam and Ferrel (Lam and Ferrel, 1993) using laser doppler perfusion imaging

(LDI), demonstrated vasoconstriction in normal joints but vasodilatation in inflamed

joints following saphenous nerve stimulation. Supplernentation of acutely inflamed joints

with SP or CGRP resulted in a conversion of vasoconstriction to vasodilatation. These

experiments suggest an ontagonistic relationship between neuropeptides thought to be

released fiom small diameter unmyelinated afferents and vascular effects of the

sympathetic nervous system in modulating PE.

S tudies utilizing speci fic adrenergic receptor agonist and antagonists provide

good evidence for the sympathetic system in modulating PE (Figure 5). In a study by

Green et ai. (Green et al., 1993b), the effects of 6-OHDA and bradykinin (which both

release noradrenaline (NE) fiom SPGN terrninals) was to increase PE and not decrease it.

This finding is at odds with the observation in the sarne study that K' administration

(which also depolarizes and releases NE from SPGN terrninals) decreases PE. To

reconcile this apparent paradox, the authors point out that bradykinin and 6-OHDA while

releasing NE also release pro-inflarnmatory factors such as prostaglandin E2 (PGE?)

(Gonzales et al., 1989; Gonzales el ai., 1 99 1 ). Antidromic stimulation and K+

administration release inhibitors of PE such as NE and neuropeptide Y (NPY) but do not

release pro-inflammatory CO-transmitters such as PGE2. Pursuant to this theory, the

authors (Green et al., 1993a) proceeded to demonstrate in a mode1 of acute inflammation

induced by bradykinin that NE and NPY infiision diminish the PE response. NE was

1 LEGEND 1

O Mediators of p h a cxtmvasatioo

ln Release of medators

Ik inhibition of release

Joint 1 Joint

Figure 5 Sympathetic Post-gaaglionic Nerve Factors Elaboraîed Which Contriiute to PlasnaExtravasation and Joint Injury. Adapted h m Coderre et d. 1991

able to decrease baseline PE and perfusion with PGEz caused marked enhancement of PE

as theorized.

A multitude of other studies support the indication that catecholamines such as

NE reduce baseline PE and inhibit increases in PE (McKinney and Lish, 1966; Brown et

al., 1968; Green, 1972; Green, 1974; Svensjo et al., 1977; Joyner et al., 1979; Coderre et

al., 199 1 ) .

Komorowski et al. (Komorowski et al., 1 996) examineci the effect of

guanethedine sympathectomy on EB dye leakage in dental pulps of molars in response to

MO (a putative neurogenic algogen, see section 1.1 O). No difference was found between

in sympathectornized versus unsympathectomized rats in the degree of EB leakage. It

was concluded that SPGN have little role in MO induced PE. An expected decrease or

increase in PE to topical MO, and carrageenan injection was not seen after chernical

sympathectomy by Donnerer et al. (Domerer et al., 199 1 ) In both cases, MO induced

PE appear to be unaffected by sympathectorny.

Some authon suggest that this apparent lack of sympathetic influence is in reality

due to incomplete sympathectomy (Green, 1993b). This hypothesis is given support in a

study by Domerer et al. (Domerer et al., 199 1 ) who observed that guanethidine

treatment neonatally resulted in an 86% reduction in NE levels in skin and consequent

reduction in PE response to antidromic stimulation. In contrast when 6-OHDA was used

to induce sympathectomy, only a 66% reduction in NE levels were observed and

consequently no reduction in PE was seen in response to antidromic stimulation.

Two other studies finding signifiant roles of SPGN also confirmed the presence

of complete sympathectomy (Lam and Ferrell, 199 1 a; Green, 1993b).

1.4.43 Sympathetically Mediated Increases in Plasma Extravasation

May Represent A Reparative Mechanisrn

Increasing plasma extravasation can occur coincident with decreasing tissue

destruction. Clonidine (a specific a?-agonist) enhances PE while concomitantly reducing

severity of adjuvant induced arthritis (Coderre, 1990). The converse has also been

indicated experimentalIy: NE release fiom SPGN tenninals appears to inhibit PE (Green,

1993a) while epinephrine increases severity of adjuvant induced arthritis (Coderre et al.,

199 1). It has been suggested that PE serves a protective and reparative function by

diluting pro-inflarnrnatory peptides and enhancing their clearance (Coderre et al., 199 1).

The implication is that increased PE can CO-exist with decreased severity of joint injury

as evaluated radiographically. That is, PE is not to be equated with undesirable arthritic

changes.

1.4.5 Parasympathetic Influences on Neurogenk Inflammation

The role of parasympathetic influences in neurogenic inflammation are even less

well defined. Delepine and Aubineau (Delepine and Aubineau, 1997) found an increase

in PE in dura mater when electrically stimulating the sphenopalatine ganglion in rats.

This response was reduced with capsaicin pretreatment but completely abolished with

atropine infusion indicating activating parasympathetic fibres promotes NI.

1.4.6 The Central Newous System in Neurogenic Inflammation

Early concepts of NI incorporated the role of the central nervous system (CNS)

only to the extent of transmission or processing of nociceptive inputs (Chahl, 1988).

Current evidence suggests a role of the CNS in NI from two perspectives, either through

spinal reflex circuits and/or through descending modulatory influences from supraspinal

levels (Levine, 1 985).

1 A6. I Local Spinal Circuits

The bilateral nature and symrnetry of conditions such as RA suggest a central

influence on the disease process. More speci ficall y, this symrnetry suggests the existence

of local spinal circuits mediating responses to stimuli at the segmental level. Chahl and

Ladd (Chahl and Ladd, 1976) noted that antidromic stimulation of the saphenous nerve

in one limb produced an edema response in the contralateral limb. Levine et al. (Levine,

1985) afier three days of priming stimulus of saline in one rat paw applied a mild injury

and observed an increase in mechanical h yperalgesia and swelling in the contralateral

paw. The 'cross-swelling' response was elirninated in acute and chronically peripheral

denervated rats. Syrnpathectomized and capsaicin treated rats also failed to dernonstrate

contralateral swelling coined by the authors as "reflex neurogenic inflammation"

(Levine, 1985).

Houghton er al. (Houghton et al., 1998) in a mode1 of acute arthritis in the rat

knee induced by carrageenan and kaolin examined the effect of spinal administration of

both stereoisomers of 3-isobutylgaba (3-IBG). This compound originally designed as a

y-arninobutyricacid (GABA) analog acts at neither GABAA or GABAe receptors but is

thought to act rather at a subunit of voltage dependent calcium charnels (Gee et al.,

1996). Administration of either stereoisomer pior to inflammation induction reduced

maximal swelling demonstrating that intervention in the CNS cm reduce peripherally

evoked edema. Rees et al. (Rees et al., 1995) recorded activity in affierent fibres of the

media1 articular nerve in rat hindlimbs and demonstrated antidromic action potentials

after inflammation induction penpherally in the knee. Dorsal rhizotomy abolished

activity indicating action potentials were reflexively generated in the dorsal root. Sluka

et al. (Sluka et al., 1994b) in a mode1 of acute inflammation induced by carrageenan and

kaolin, demonstrated spinal cord transection had no effect on reducing inflammation in

the rat knee but dorsal rhizotomy did. These data indicate that local spinal circuits but

not descending influences were active in production and maintenance of articular

inflammation. Sympathectomy also had no effect.

Mapp et al.(Mapp et al., 1993) examined levels of SP and CGRP in the dorsal

hom and dorsal root ganglion (DRG) both in acute and chronic models. In the acute

phase, levels of CGRP and SP in the ipsilateral and contralateral dorsal hom and DRG

increased significantly. In the chronic study, no elevated levels of either SP or CGRP

were observed in the contralateral spinal cord or ganglion. Levels in the ipsilateral side

were significantly reduced.

This and other studies (Bileviciute et al., 1994) provide evidence of

neurochernical change in the CNS associated with the presence of an acute and chronic

inflarnmatory reaction. These changes, while highly suggestive of a role for supraspinal

elernents, are far from conclusive and leave many questions unanswered as to their exact

physiologic role and properties.

1.4.6.2 Central descending modulation

If segmental neural circuits at the spinal cord levef reflexively mediate

neurogenic inflammation then a subsequent inquiry would entai1 detexmining whether

descending controls could modiQ the peripheral inflammatory response. Morphine has

been dernonstrated to decrease firing of spinal cord neurons through brainstem

descending controls (Basbaum and Fields, 1984). In a chronic adjuvant mode1 of

experimental arthritis Levine et al. (Levine, 1986) found that intracerebroventricular

administration of morphine over 72 hours reduced the severity of arthritis compared to

controls. No control for the systemic activity of morphine was provided and therefore

local interactions of morphine on peripheral opioid receptors may have confounded

results.

One of the hallmark features of RA is its manifestation in joints bilaterally,

symmetricall y, and for more distal joints to exhibit more severe disease (Mitchell and

Fries, 1982a; Arnett, 1988). Perhaps one of the most fascinating phenornena to be

reported in the literature are cases of hemiplegia, in which RA develops unilaterally.

Hammoudeh et al. (Hammoudeh et al., 198 1) report a case in which a 53 year old

woman underwent craniotomy and left carotid ligation for brain aneurysms and was lefi

hemiplegic on her right side. Over the next six years she developed signs and symptoms

of RA on her lefi side exclusively. Hamilton (Hamilton, 1983) reports a case of a 64 year

old man who experienced a cerebrovascular accident (CVA) ten yean previously

resulting in right herniplegia. Two years following his CVA he began having bouts of

arthritic signs and symptoms - limited to his left and non-paretic side. A number of other

case reports in the literature descnbe similar instances of this apparent protective effect

of hemiplegia in RA (Thompson, 1962; Glick, 1967; Ueno et al., 1983; Zonin et al.,

1996).

Valayos and Cohen (Velayos and Cohen, 1970) describe a case in which a CVA

resulted in hemiplegia, in a patient with established RA. Signs and symptoms of arthritis

continued to worsen on the unaffected side and regressed on the paretic side.

A minority of reports have involved non-rheumatoid joint inflammation in the

presence of herniplegia. Mitchell and Capell (Mitchell and Capell, 1982) report two

cases of right-sided hemiplegia following CVA's. Development of acute episodes of

idiopathic sterile monoarthritis of joints ensud 7 to 10 days later in both cases with

signs and symptoms limited to the non-paretic side. The patients exhibited no

radiographie abnomali ties, never exhibi ted leukocytosis, and were negative for

rheumatoid factor. Goldberg et al. (Goldberg et al., 1980) describe a woman having right

armed paralysis secondary to poliomyelitis at age 3. At age 67 she began to experience

pain, swelling, and reduced functional capacity limited to left handed joints.

Radiographic evidence of osteoarthritis was evident in the functional hand but not the

paralyzed hand. Heberden and Bouchard nodes were present on the functional but not

the paralyzed hand. In 1995 Etherington and Spector (Etherington and Spector, 1995)

report a similar case of unilateral osteoarthritis in a hemiplegic patient.

It has been suggested that disuse of paretic joints spares hem of traumatic

loading and strain thereby exerting a protective effect. These reports cannot however be

explained on this basis since many have been ambulant for decades yet still exhibit

unilateral RA (iÏnompson, 1962).

These case reports taken together fùrther suggest that articular are heavily

dependent on intact descending central influences for initiation as well as maintenance.

1.4.7 The Cellular Immune System and Neurogenic Inflammation

Inflammation is a process initiated not through a single predictable pathway but

through multiple means of generation. The neurogenic release of mediators is only a

component of an integrated non-specific immune system involved in the process of

defense and repair of tissue after physical, chemical, or microbiological insult.

Neurogenic influences on the inflammatory response likely act in concert with other

inflammatory factors, most notably non-neuronal immune response systerns. This

immune response in general ternis may be described as innate non-specific or acquired

specific (Mayers and Johnson, 1998). In the former, mediators may be derived fiom

nerve terminals, immune cells, membrane fatty acids, vascular and tissue derived

precursors. Scott et al. (Scott et al., 1994) review these important classes of mediators

such as cytokines, fatty acid derivatives, completnent systems derived compounds,

histamine and serotonin. nie precise role of each of these mediators and their

relationship with the neurogenic inflammatory response is unknowr~, and the subject of

current debate and study.

Cytokines are glycoproteins secreted by virtually al1 nucleated ce11 types which

are involved in modulating activity of other host defense cells and processes such as

inflammation. Interleukin- l (IL- 1 ) is a primary cytokine released in the early stages

following acute injury and is chemotactic for lymphocytes, neutrophils, and

mononuclear phagocytic cells (Martin and Resch, 1988; Mayers and Johnson, 1 998).

Production ancilor release of IL- 1 may be stimulated by noxious stimuli, immune

complexes, and important1 y, neuropeptides (Oppenheim et al., 1986; Kimball et ai.,

1988; Scott et ai., 1994). IL- 1 increases phospholipase A?, arachidonic acid,

leukotriene, and eicosanoid levels such as prostaglandins, thromboxanes, and

prostacyclins: potent mediators of the inflammatory reaction. Notably, prostaglandins

Dz. El, Fz (PGD2, PGE?, PGF-) vasodilate and potentiate edema development.

Leukotriene B4 (LTB4) is a potent chemotactic agent promoting aggregation of

neutrophils while LTC4, LTD4, LTE4, and LTF4 cause a variety of actions including

increasing vascular permeability in post-capillary venules (Pace-Asciak, 1989).

Production of macrophage derived tumour necrosis factor-a (TNF-a) is stimulated

(Martin and Resch, 1988).

The complement system is compnsed of 14 plasma protein components that

interact to produce a number of pro-inflammatory effects. C3a and C5a components may

induce histamine secretion by mast cells and basophils. C4 and C2 cleavage result in

vascular permeability. Certain complement components are chemotactic for immune

cells (Scott et al., 1994).

The kinin system represents a set of peptides cleaved fiom a-globulin kininogen

precursors by kallikrein and non-specific proteases such as trypsin (Kadar, 1989).

Kallikrein itself is derived prekallikrein cleaved by Hageman factor, which is activated

as part of the blood-clotting cascade. Kinins cm aIso be produced from immune cells

such as mast and basophils directly as a result of cellular protease release in acute

inflammatory reactions (Dray, 1997). Exarnples of important kinins are bradykinin in

plasma and kallidin in tissue. Kinins are potent pain producing agents, vasodilators, and

cause increased vascular permeability and consequent edema formation (Kadar, 1989).

Bradykinin levels increased in acute inflammation (for example neurogenic

inflammation) ihemselves act to release neurotransmitten fiom sensory afferent nerve

terminais (Green er al., 1993~).

Histamine and serotonin (5-hydoxytryptamine or 5-HT) are two important

biogenic amines found in mast cells and platelets (Scott et al., 1994; Dray, 1997).

Release of histamine results in interactions with HI and Hz receptors to cause

vasodilatation, vascular permeability and PE (Lam and Ferrel, 1990). The role of mast

cells in neurogenic inflammation is unclear. There is evidence that SP released fiom

neural terminals causes increased vascular permeability and PE through the

degranulation of mast cells (Lam and Ferrel, 1990). Levine et al. (Levine et al., 1990)

hypothesizes that innervation of joints exerts a trophic effect on mast ce11 density which

suggests a critical role played by these cells in mediating neurogenic inflammation.

1.5 Acute Versus Chronic Inflammation

1.5.1 Acute and Chronic Inflammation: Traditional Methods of Induction

Acute algogenic models of inflammation have traditionally utilized carrageenan

and kaolin (Sluka et al., 1994b), MO, xylene, and capsaicin (Jancso, 1967).

Development of inflammation afier application of these compounds is immediate and

continues to develop for 3-4 hours (Sluka et al., 1995; Fiorentino et al., 1999;). In

contrast, chronic arthri tic models have traditional1 y utilized injection of Freund's

adjuvant in which inflammation develops slowly over 3-2 1 days (Sluka, 1995).

Adjuvant induced arthritis is suggested to be due to lymphatic activation by

mycobacterial proteins (Freund, 195 1 ). Carrageenan induced inflammation is generally

thought to be mediated by non-neurogenic cellular mechanisms (Fearn et al., 1965;

Willis, 1969), though others have reported evidence to the contrary (Lam and Ferrell,

199 1 a).

1.5.2 Acute Models of Infiammation And Study of Dorsal Root

Reflexes

The key interactions between neural and cellular components in the early acute

stages of inflammation cannot be expected to apply rigidly to later chronic stages.

Nonetheless, the hope is to gain insight into more chronic processes. However, one

primary reason for using an acute mode1 is its inherent simplicity and ability to lend

itseif to exarnination of fiindamental mechanisms of inflammation.

For example, acute models lend thernselves to the study of 'dorsal root reflexes'

(see section 4.4.1 ) (Sluka, 1995). With this in mind, the prospect that an acute

neurogenic inflammatory process rnay conceivably be initiated and maintained via

central and peripheral neurologic mechanisms is an exciting prospect. An initial step in

our survey for novel therapeutic approaches to TMD is the demonstration that

neurogenic inflammatory mechanisms play a significant role. The hope is that despite

the use of an artificial animal mode1 of acute inflammation, we may still gain insight into

those mechanisms that render neurogenic inflammation a potentially significant self-

sustaining phenomena.

"Acute inflammation in the absence of some sustained peripheral disease

is usually self limiting. A patient with acute staphylococcal septic

arthritis, if treated with the appropriate antibiotics, has a rapid resolution

of the inflamed joint. The sarne is tme for acute gout, and most non-

immune related inflammations. This leaves the intriguing question, can

the nervous system itself initiate peripheral inflammation, and if so, does

this occur via dorsal root reflexes? The jury is out."

(Woolf, 1995)

The majority view until now had been that dorsal root reflex is an artifact of

abnormal experimental situations. They have been demonstrated in laboratory animals

under very particular conditions such as the temperature of the animal, the anaesthetic

used, and the highly synchronized inputs fiom many afferents (Schmidt, 197 1 ; Linsey,

1995). There have always been doubts as to the significance dorsal root reflexes would

have in normal fùnction (Linsey, 1995).

1.53 A Continuum of Inflammation, Acute to Chronic

The inflammatory process initiated by the injection of MO in the TMJ of the rat

is an acute process. The issue of how and to what extent the study of acute inflammation

relates to chronic inflarnmatory processes for the most part is unknown and a matter of

current debate. In the context of inflammatory TMJ disease, that point at which an

'acute' phenornena becomes a 'chronic' becomes a philosophic question. ft is far more

likely that physical and functional manifestations of these arbitrary definitions exist on a

continuum and even CO-exist in transition. Certainly the physical elements within which

inflammation is induced and persist are present in the acute and chronic stages. For

example, the neuroanatomic relationships behveen primary and secondary afferent

neurons, sympathetic neurons, vasculature, synovial elements etc. are defining the

physical limits of edema, blood supply and so on. Arthritis-induced dorsal root reflexes

(see section 4.5.1) appear to be mediated by the sarne non-NMDA and GABAA receptors

in the spinal cord dorsal horn in both acute and chronic models (Sluka, 1995) This does

not mean that these elements remain static throughout the development and maintenance

of inflammation. They are more likely plastic and dynarnic, continually changing in

physical nature and function. For example, it is conceivable that in acute stages of

synovial inflammation, fiee nerve endings may be found (as they likely do in non-

pathologie states) in the superficial layers of synovium (Kido et al., 1995). Physically

poised to exert a secretory function, the afferents are promoted to secrete mediators such

as SP and CGRP into the articula space in response to an adequate stimulus. Mast ce11

degranulation in response to SP occurs (Barnes et al., 1986). Vascular endothelial

function is altered and vascular pemeability ensues with immune ceIl up-regulation. As

the inflarnmatory process progresses, the synoviurn may become hypertrophic and

'outgrow' the free afferent suppl y in some areas or the nerves may become atrophic and

microscopically begin to give the appearance of a non-imervated superficial synovium

(Mapp et al., 1990). Along with the synovium begiming to taking on a villous

hyperplastic appearance (Holmlund et al., 1992), pro-inflammatory sympathetic efferent

influences may now become proportionally more pronounced (Green, 1993a; Green,

1993b). Mast ce11 density decreases and consequently their role in the inflammatory

process senesces (Levine, 1990). As inflammation persists, macroscopic tissue changes

become apparent in the forrn of fibrosis, adhesions, and thickening of the capsule

(Holmlund et al., 1992).

Accompanying cellular and microscopic changes are associated clinical

fünctional manifestations. In acute stages, pain and physiologic reflex limitation of

mandibular movement may occur (Yu et al., 1995). As the inflammatory process evolves

and persists, physical limitation of movement may manifest or worsen.

1.6 Animal Models of Neurogenic Inflammation

1.6.1 General

A number of models have been utilized to investigate the physiologic

mechanisms and mediators of neurogenic inflammation as well as its specific

contribution to inflammatory diseases such as RA and TMD. In each case a proxy

measure of inflammation is chosen and change in this measure due to various agonists

and antagonists is evaluated.

Quantification of extravascular leakage of proteins labelled with dye in tissues

subject to an acute inflammatory reaction has been a traditional means of assessing PE.

The original method by Ramsdell (Ramsdell, 1928) utilizing Trypan Blue has been

modified and improved unto its most recent form using Evan's Blue (EB). Dye

extraction protocol by Harada et al. (Harada et al., 197 1) and spectrophotometric dye

measurement b y Saria and Lundberg (Saria and Lundberg, 1 983) comprises the method

most widely used and relatively unchanged. Use of a radiolabeled compound to quanti@

PE rather than spectrophotometry has also been implemented (Newbold and Brain,

1995)

The principle of using protein-binding dyes to quanti% PE is simple. Dyes such

as EB bind to plasma proteins such as albumin. As a consequence of an inflarnmatory

response, vascular permeability occurs allowing the extravascular outflow of these

plasma proteins into the sumounding tissue. Reduction in vascular content of dye is due

predominantly to this transvascular flux in the first 4 to 5 hours (Green et ai., 1988).

ï h e effect of various putative mediators and compounds on FE may be

quantified by first injecting them into tissues. If a joint is the tissue in question, the

contralateral one is typicall y utilized as a control for the trauma induced secondari1 y by

mechanical distention of fluid and penetration of the catheter used to introduce

compounds. EB is injected intravenously (i.v.) and the animal sacrificed at a

predetermined time. Tissue is then dissected out, and analyzed for EB content. The knee

has been studied extensively using this method (Lam and Ferrell, 199 1 a). Neurogenic

inflammation involving the TMJ (Haas et al., 1992; Yu et al., 1996;), skin (Louis et al.,

1989; Dux et al., 1996), tracheal mucosa (Lundberg and Saria, 1983), esophagus,

bladder, ureter, conjunctiva, and duodenum has also been examined in this way (Saria et

al., 1983).

A modification of this method has been utilized in studying the knee joint in

particular (Green. 1993a). Skin overlying the knee is excised to expose the joint capsule.

EB dye is injected intravenously. A catheter is introduced into the joint space and

perfusion initiated with a syringe pump while a second catheter is introduced into the

joint space to allow the outflow of perfusate. Compounds to be tested are added to the

perfusate, the resulting fluid is collected at predefined intervals, and analyzed

spectrophotometricall y for EB content. This method has the advantage of allowing

quantification of PE over continuous time course rather than being limited to single

points in time when sacnficing anirnals in techniques requiring tissue recovery.

A recent modification developed by Jonsson et al. (Jonsson et al., 1998)

examined P E induced by burn injury to the rat abdomen. Digital image colour analysis

was used to provide a continuous in vivo quantification over time of inflammatory PE.

Physical measurernent of swollen tissue has been another alternative associated

with varying degrees of accuracy and precision. In the rat, the knee joint lends itself to

direct circumferencial measurement with a device such as a measuring tape (Rees, 1995;

Houghton et al., 1998; Lawand et al., 1999). Measurement of mouse ear thickness has

been atternpted directly with fine micrometer calipers (Inoue et al., 1995). Volumetric

displacement of fluid by the extrernity in question has also been utilized to quantifi

changes in tissue volume (Lippe et al., 1993a).

1.6.2 Study of the TMJ

Study of neurogenic inflammation involving the TMJ area in an animal model

presents a number of difficulties. The joint does not lend itself to circumferential

measurement with measuring tape. Measuring ederna with calipers is difficuit if not

impossible. Volumetric displacernent of fluid is cornplicated by the fact that the joint is

approximated to the aiway which must not be compromised. Perfusion of the joint and

extraction of perfusate with a second catheter is extremely difficult within the TMJ for

several reasons. The joint capsule is deep to muscle and fascia whereas the knee joint

capsule is relatively accessible. To consistently place both catheters in the first attempt

into the sarne joint space (there are two compartments in the TMJ) would be difficult at

best. The sheer size of the knee joint relative to the TMJ allows placement of two

catheters. Attempts to pass two catheters through the TMJ may obliterate the capsule and

afferent supply.

For the aforernentioned reasons, study of ederna involving the TMJ area in the rat

has traditionally been perfomed with the fluorometric measurement of EB (Haas et al.,

1992; Yu, 1995) afier Saria and Lundberg (Saria and Lundberg, 1983). In 1999

Fiorentino et al. (Fiorentino, 1999) devised a simple yet effective means of

demonstrating edema development in periarticular temporomandibular (pTM) tissues of

the rat TMJ. The so-called 'tissue expansion' model was compared to traditional EB

methodology and was found to be simple, efficient, and equally effective in measuring

PE. Most noteworthy was that this model offered for the first time a means to study

neurogenic inflammation development in the pTM over a continuous time course

(Fiorentino, 1999).

1.7 Neurogenic Inflammation Involving the TMJ

1.7.1 Substance P

One prirnary line of evidence for the role of neurogenic inflammatory

mechanisms in the TMJ is the presence of mediator substances in nerves innervating the

articula tissues and in perfusate of inflarned joints. The prototypical neurotransmitter in

neurogenic inflammation is substance P (SP). Other putative mediators are calcitonin

gene related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neurokinin A

(NKA), and neuropeptide Y (NPY).

In 193 1 SP was isolated from equine brain and intestine by von Euler and

Gaddum (von Euler and Gaddum, 193 1). SP was found to cause hyperemia in tissues

when injected mimicking antidromic vasodilatation. It was hypothesized by Hellauer and

Umrath (Hellauer and Umrath, 1947) and tùrthered by Lembeck (Lembeck, 1953) that

SP was a neurotransrnitter released by sensory afferent nexves and the mediator of

antidromic vasodilatation. Andrews and Holton (Andrews and Holton, 1958)

demonstrated that afier section, levels of SP fell in a degenerating distal portion of

afferent neurons but rose in the proximal stump suggesting that SP was synthesized in

the ce11 body but transported to the nerve endings.

The determination of the peptide sequence and synthesis of SP in 197 1 was

followed soon afler by development of antibodies and radioimmunoassays (Powell et al.,

1973). A reliable means of characterizing the anatomical distribution of SP had been

bom. SP-like irnmunoreactivity (SP-LI) was found in primary afferent nerves of the

intestine (Nilsson et al., 19759, and ceIl bodies of dorsal root and spinal cord (Hokfelt et

al., 1975a; Hokfelt et al., 1975b). In 1975 it was demonstrated that SP is in fact

transported to the central terminais from ce11 bodies (Takahashi and Otsuka, 1975).

By 1980 SP appeared to be the primary neurotransmitter responsible for

mediating neurogenic inflammation (Chahl, 1988). Its potency, pharmacology,

disposition and activity were found to most closely mimic the effects of antidromic

stimulation of nerves (Chahl, 1988).

PE induced by SP is thought to involve specific interactions with vascular

receptors with the C-terminus of the peptide and related tachykinins (Foreman et al.,

1983).

1.7.2 Substance P and Arthritic Joints

Synovial fluid aspirates fiom knees, have been found to contain elevated

amounts of CGRP-LI, SP-LI, and VIP-LI in patients with RA as compared to

osteoarthritis (Hernanz et al., 1993). In contrast, a study by Marshall et al. (Marshall et

al., 1990) showed no difference in synovial fluid concentrations of SP in patients with

RA or osteoarthritis.

1.73 Substance P in the Temporomandibular Joint

Alstergren et al. (Alstergren et al., 1995) undertook radioimrnunoassay of

putative mediators of NI. Plasma and TMJ fluid aspirates were taken fkom 41 patients

with TMJ arthritis and analyzed for content of SP-LI, CGRP-LI, NPY-LI, and NKA-LI.

Al1 four peptides were present in al1 patient samples which is suggestive of an

association with a neurogenic inflarnmatory response but makes no argument for a

causal relationship. To address this ambiguity, Carleson et al. (Carleson et al., 1996a)

analyzed levels by radioimmunoassay in plasma, cerebrospinal fluid (CSF) and TMJ

perfusate before and after injection of adjuvant into the rat TMJ. Both monoarthritic

(unilateral TMJ injection of adjuvant only) and polyarthritic (adjuvant injected into the

tail base) models were studied. Injection of adjuvant into the right TMJ of rats induced a

significant increase in CGRP-LI, NPY-LI, NK A-LI, and SP-LI in both TMJ's at both 1

and 12 hours. A centrally mediated reflex was suggested by the authors to account for

the bilateral appearance of increased peptide levels. It is conceivable that a systemic

effect of adjuvant injected into the joint may account for this though plasma levels were

not significantly elevated.

These studies demonstrate that adjuvant induced monoarthritis in the TMJ is

associated with increased levels of peptides known to mediate inf ammation. A number

of confounding factors still penist however. The activity of adjuvant is not specific for

nociceptive neurons (known to be a primary source of SP (Nilsson et al., 1975)). It has

been suggested that peptidergic neurons are activated by mycobacterial proteins in

lymphoid structures and that polyarthntis is due to lymphatic activation (Freund, 195 1).

Furthemore, non-neuronal sources of peptides from immune cells is also a possibility

(Jakab et al., 1993).

Despite the suggestions drawn fiom al1 the above studies, the evidence for NI in

inflarnrnatory disease of the TMJ rernains equivocal. Study is required of the influence

of functional neural presence and absence in development of inflammation to make firm

arguments for NI in the TMJ.

1.7.4 Direct Study of the Neural Influence On Joint Peptide levels

Carleson et al. (Carleson et al., 1997) exarnined peptide levels in TMJ and

trigeminal ganglia of rats with adjuvant induced arthr;.tis afier 29 days. These peptides

such as SP (Lembeck and Holzer, 1979) and CGRP (Cambridge and Brain, 1992) have

been shown to have pro-inflarnmatory effects. Effects of pretreatment with subcutaneous

capsaicin and unilateral trigeminal surgical denervation on levels were studied.

Levels of SP-LI, and CGRP-LI were increased compared to controls in both

trigeminal ganglion and TMJ pemisate 29 days following adjuvant injection. This is

expected since the purpose of adjuvant was to stimulate an inflarnmatory reaction and

presurnably levels of inflammatory mediators. Surgical ligation of the third division of

the trigeminal nerve inhibited the increase in joint SP-LI, and CGRP-LI in response to

adjuvant injection. This result suggests that afferent neme viability is vital to be able to

increase intra-articular levels of pro-inflammatory mediators such as SP and CGRP.

Capsaicin pre-treatment prevented increase in levels of CGRP in response to adjuvant.

This similari y suggests afferent fibre viabili ty is necessary to increase intra-articular

CGRP in response to adjuvant.

Certain results from this study tempered conclusions regarding the neural

influence on inflammation. Rats receiving ligation of the mandibular nerves exhibited a

decrease of 50% in SP-LI but not CGRP-LI levels (both are thought to be CO-localized in

C-fibres (Alstergren, 1995). Othen have similarly demonstrated a reduction in CGRP

but not SP-immunoreactive fibres afier capsaicin treatrnent ( S m k i et al., 1997).

Capsaicin pre-treatrnent failed to inhibit the increase (80 to 90% increase) in joint SP-LI

in response to adjuvant. Finally, surgical ligation failed to reduce levels of CGRP-LI

though capsaicin treatrnent did.

One tùndarnental problem in measuring peptide levels (SP and CGRP) is the

speculation of the level at which point they induce clinically significant effects. To this

end, al1 dmgs have a subthreshold level at which their presence fails to produce a given

response. Increases in peptide levels during inflammation is more indicative of a causal

relationship but even changes in peptide levels is not absolutely indicative. It has been

demonstrated that CGRP levels in the TMJ following capsaicin injection fluctuate in

cyclic fashion from control levels to elevated levels throughout an inflammatory

reaction (Spears et al., 1998). This may indicate peptides such as SP and CGRP may in

some circumstances act as markers of inflammation.

Confounding the issue further are results fkom as a shidy by Mapp et al. (Mapp,

1990) who have described reduced numben of CGRP and SP immunoreactive nerves

imervating superficial synovium in patients with RA versus patients without RA.

1.8 Electrornyography In Pain and Inflammation Researcb

The orofacial musculature lacks muscle spindles and golgi tendon organs (Sessle,

1997). A compensatory mechanism to modifi afferent activity based upon efferent input

is the excitatory (and inhibitory) reflex connections made by a and y-motoneurons to

TMJ, penodontal ligament and other afferents (Sessle, 1997).

Stimulation of TMJ afferents in animals with algesic chemicais such as

potassium chloride, histamine, 7% sodium chloride, and MO, has been dernonstrated to

evoke reflex electromyographic (EMG) activity in jaw muscles such as the anterior

digastric, masseter, and middle ternporalis (Broton et al., 1988; Broton and Sessle, 1988;

Yu, 1995; Yu, 1996; Bakke et al., 1998).

Baseline activity may be quantified and compared to activity evoked by algesic

chemical stimulation of primary afferent neurons under a variety of conditions. For

example, these may be specific receptor antagonists such as the opiate receptor

antagonist naloxone (Bakke et al., 1 998), or non-NMDA antagonist MK-80 1 (Yu, 1996).

Complete conduction blockade of pnmary afferents imervating the TMJ should

theoreticall y abolish the typical MO evoked reflex jaw muscle activity. This was indeed

demonstrated by Yu et al. in 1995 (YU, 1995). Injection of 2% lidocaine 5 minutes prior

to injection of 20% MO into the TMJ of the rat completely negated the reflex EMG

activity in digastric and masseter muscles (Yu, 1995).

Based upon these experiments, the EMG mode1 provides a means by which to

evaluate the degree of conduction block of primary afferent fibres supplying the rat

TMJ.

1.9 Mustard Oil As A Chemical Algogen In The Study Of Neurogenic Inflammation

Mustard oil (allylisothiocyanate) has been used in the study of neurogenic

inflammation for decades. However, the precise molecular and cellular actions have yet

to be elucidated. No specific 'MO receptor' has been characterized. No endogenous

ligand with activity similar to MO has been identifiai. tIistologica1 examination of

tissues 30 minutes afier application of MO to mouse skin revealed edema in dermal and

subdermal layers but no tissue darnage and no blood ce11 leakage into extravascular

tissues (houe et al., 1997). Polymorphonuclear leukocyte infiltration is induced by MO

injection (Haas, 1992).

Antidromic stimulation produces increased vascular permeability and PE similar

to topical MO application, but does not sensitize nociceptors (Reeh et al., 1986). In

contrast, MO application to rat paw skin decreases mechanical and thermal thresholds as

well as increases spontaneous activity in C-fibres (Reeh, 1986). This suggests that the

mechanism of M O effect on afferents is similar with respect to production of PE but not

identical to antidromic stimulation of afferent C-fibres in other respects.

There is evidence to suggest that the MO induced vasodilatory response to be

nitric oxide (NO) dependent while not exudative component (Lippe et al., 1993b).

Mustard oil appears to cause vasodilatation and increased vascular pemeability

through a neurogenic mechanism. Jancso et al. (Jancso, 1967; Jancso et al., 1977)

demonstrated that systernic neonatal treatrnent of rats with capsaicin resulted in

degeneration of almost al1 pnmary afferent nociceptors. A consequent reduction in the

edema formation in the adult rat paw to topical MO was demonstrated (Jancso, 1977).

An extensive investigation into the mechanism of MO induced inflammation was

undertaken by houe et al. (Inoue, 1997) The effect of MO on wild type and mast

deficient mice was evaluated. No difference between mast ce11 deficient mice and

controls was obsewed as quantified by EB method or by direct measurement of edema.

This suggests mast cells and histamine release are not important factors in producing PE

by MO. Ederna was quantified in the mouse ear by direct measurement of tissue

thickness in response to 5% MO. Capsaicin pretreatment of ears (which has been shown

to reduce SP content of C-fibre nociceptors) reduced the edema response elicited by

MO. In contrast to this finding, pretreatment with cyclooxygenase inhibitors, S-

lipooxygenase inhibitors, antihistamines, 5-HT antagonists, NO inhibitors, ruthenium

red (functional inhibitor of the capsaicin receptor), or CGRP inhibitors, al1 failed to

modify the edema response to MO.

Lippe et al. (Lippe et al., 1993b) examined the response to topical MO using

measures of inflammation including skin temperature change (thermography), EB

spectroscopy, and paw volume change (edema). Neonatal pretreatment with capsaicin

resulted in abolition of the temperature increase, edema formation, and EB content in

adult rats. This indicates that MO requires functional capsaicin sensitive neurons to

produce vasodilatation, and increased vascular permeability. Similar to houe et al.

(Inoue, 1997) no statistically significant reduction of edema formation by NO inhibitors

was observed.

These studies taken together indicate that MO evoked vasodilatation and

increased vascular pemieability are dependent upon the existence of fùnctional C-fibre

afferents and that these effects are secondary to release of mediators fiom these neural

terminals. Additionally, indications are that direct endothelial injury, NO,

prostaglandins, leukotrienes, and histamine, do not necessarily play a role in these

responses. MO appears to act solely through a neurogenic mechanism. It is therefore an

invaluable tool for use in investigating the role of neurogenic inflammation in the

pathogenesis of inflammatory conditions involving tissues such as the TMJ.

1.10 Innervation of the Ternporomandibular Joint

In humans and the Macaque monkey, the TMJ is imervated by branches of the

third division of the trigeminal nerve. The auriculotemporal nerve is quantitatively most

important, imervating the posterior and lateral aspects of the TMJ capsule. The anterior

capsule is supplied by the masseteric nerve and occasionally the deep temporal. The

media1 aspect is innervated by a combination of auriculotemporal and masseteric

(Schmid, 1969).

Evidence for a role of neurogenic inflammation in the etiology of conditions such

as RA and especially TMD has been further strengthened by the specific identification of

nociceptors and localization of these neurons in and around cntical vascular and

synovial layers. Their appearance in this manner is pivota1 in suggesting a role in

inflammatory processes of the TMJ.

The general innervation of the TMJ has been described by light microscopy in

mouse and monkey models by Frommer and Monroe (Frommer and Monroe, 1966) and

Keller and Moffet (Keller and Moffett, 1968) respectively. Both demonstrated that

neurons actually terminateci in the synovial lining layer of the TMJ. Attempts to

specifically identiw these as sensory neurons was undertaken by several authors

(Ichikawa et al., 1989; Kido, 1993; Johansson et al., 1986). Each of these studies

demonstrated CGRP and SP like immunoreactivity in and around the synovial lining of

the TMJ of rat and monkeys alike. However, Heym et al. (Heym et al., 1993)

demonstrated that this SP and CGRP immunoreactivity is not specific for primary

afferent neurons since they may also be found in sympathetic fibres. This presented a

confounding factor since autonomic efferents had been identified in the TMJ of the rat in

addition to these sensory fibres (Widenfalk and Wiberg, 1990).

Kido et al. (Kido, 1995) resolved this ambiguity in 1995 using wheat germ

agglutinin-horseradish peroxidase (WGA-HRP) labelling. WGA-HRP is a compound

that once introduced into a neuron ce11 body, will be transported in an anterograde

fashion toward the neural terminal. This allows visuaiization by light microscopy of the

fine anatomical distribution of fibres. The authors were able to conclusively dernonstrate

the innervation of the TMJ of the rat with sensory afferents and detail the fine structure

as well as distribution of those fibres. Specifically, nerve bundles were observed entering

the joint from antenor, postenor, and lateral aspects but few medially. Frontal sections

demonstrated sensory afferents in the peripheral portions of the disk especially in the

lateral of the TMJ. Fine nerve fibres were observed in the synovial and subsynovial layer

of the membrane lining the joint compartments and overlying the cartilage of the

mandibular condyle. No fibres were observed in the thin central region of the disk. A

few fibres were observed in the penosteum of the condyle and temporal bone. Whole

mount preparations demonstrated the density of the neural network to be greatest in the

anterior aspect of the TMJ. Electron rnicroecopy demonstrated axons in close

approximation to post capillary venules, in superficial synovial lining at a minimum

distance from synovial cells of 75 nm. Kido (Kido, 1993) suggests that the significance

of the close anatomical location of afferent terminais to the synovial lining surface

points their ideal role to monitor the intrarticular environment and their proximity to

blood vessels indicate their potential neurogenic role (Kido, 1995).

Most recently, Casatti et al. in 1999 (Casatti et al., 1999) utilized a retrograde

axonal tracing study in order to characterize sensory and autonornic perikaryal profiles.

A tracer (EB) was deposited in the superior joint cornpartment of rats sacrificed and

analyzed 20 days later. The total proportion of autonornic labelled perikarya (56%) was

greater than that of sensory penkarya (44%). Of the sensory perikarya, 88% were tound

in the posterolateral aspect of the trigeminal ganglion and 12% found in C2-CS dorsal

root ganglia (DRG). In the autonornic ganglia, 66% were found in the supenor cervical,

19% in the stellate, and 15% in the otic. No profiles were found in the trigeminai

rnesencephalic nucleus. No large diameter perikarya were observed in this study

indicating the relative absence of a and P afferent fibres (The characteristic morphology

of neurons and associated conduction velocities has been established by Harper and

Lawson (Harper and Lawson, 1985)). This study dernonstrates that the innervation of

the rat TMJ is attributable equally to sensory afferents and autonornic efferents.

Autonornic innervation is predominantly sympathetic but 15% are fiom parasympathetic

fibres.

Retrograde immunocytochemistry has characterized this autonomic innervation

as originating fiom the sphenopalatine and otic ganglia (parasympathetic) and stellate

ganglion (sympathetic) (Uddman et al., 1998).

1.1 1 Local Anaesthetics

Physiology of Neuronal Depolarization and Conduction

The membrane potential exists in a neuron due to the selective penneability o f

the membrane to sodium ions and the existing concentration gradients across the neural

membrane for sodium, potassium, and chloride ion. Propagation of an action potential

requires first an initial stimulus to disturb the resting membrane potential of a neuron.

This disturbance takes the f o m of a depolarization in the membrane. This depolarization

is manifested chemically as an increase in penneability to sodium ions due to an opening

of sodium channels. If this depolarization is of sufficient magnitude (to bnng the

membrane to firing threshold), a larger depolarization occurs. ï h i s larger ciepolarization

is manifested as a massive influx of sodium. This massive influx changes the local

membrane potential and consequently sets up a current in the adjacent membrane at

resting potential. This current then opens sodium channels and similarly sets up a cument

in the next segment of membrane. This continuous creation of sequential currents along

the membrane is the propagation of a so-calleci 'action potential' (Malamed, 1990b).

1.11.2 Mechanism of local Anaesthetic Activity

The action of local anaesthetics is most widely held to be at the neural membrane

surface, at the site of specific receptors located on sodium channels. At least four

receptor sites may be bound in order to alter nerve conduction. In each case, the ability

of the sodium channe1 to allow the influx of sodium ions is inhibited. This is manifested

as the inability to produce a depolarization. Without depolarization of the membrane, no

action potential can be initiated and propagated. Tertiary amine local anaesthetics such

as Iidocaine bind at a site within the sodium channei to block membrane depolarization.

A small proportion (10%) of the blockade of sodium channels is thought to corne fiom a

nonspecific interaction with membrane lipids by the base form of tertiary-amine local

anaesthetic dmgs (Malarned, 1990b).

1,11.3 Neuronal Excitation-Secretion Coupling

Release of chernical mediators fiom the terminals of neurons are dependent upon

activation of voltage gated calcium channels and subsequent influx of calcium ions.

(Bicknell, 1988; McBumey and Kehl, 1988; Gonzalez Burgos et al., 1995; Losavio and

Muchnik, 1997) Opening or activation of calcium channels is accomplished through

depofarization arising fiom a propagated action potential (Branchaw et al., 1998). High

voltage-activated channels are mainly responsible for excitation-secretion coupling

(Jones, 1998). The brief (< 1 ms) delay between action potential arriva1 at a nerve

terminal and secretion initiation and termination suggests the cellular machinery

involved in neurochemical secretion is intimately associated with the calcium channel

(Jones, 1998). Secretion from neural terminais then does not occur without an initiating

depolarization of neural membranes by an action potential. Lt follows then that

neurogenic inflammation does not occur without depolarization of afferent nerves and

therefore may be blocked by a local anaesthetic.

1.12 Statement of Rationale, Purpose, Objectives

1.12.1 Ra tionale

TMJ involvernent in RA is well established (Levine et al., 1985a; Syjanen,

1985; Tegelberg, 1987; Ettala-Ylitalo et al., 1987). In these patients, the clinical signs

and syrnptoms exhibited involving the TMJ such as pain, clicking, crepitus, reduced

mandibular movement secondary to their arthritis are identical to criteria diagnostic of

so-called 'TMD'. McKay and Christensen (McKay and Christensen, 1998) summarize

the impact and current thoughts regarding the single somatic disease entity designated as

'TMD' :

"It is estimated that the insidious and progressive TMJ disease

entity of synovitis/osteoarthritis/intemal derangement/osteoarthrosis

exists and evolves throughout the adult life of 25%-3S% of the

population. TMJ synovitis/osteoarthritis is a vicious prolonged

neurogenic inflammation that affects the superficial synovial tissues and,

indirectly, the synovial fluid, . . 9 9

In light of the aforementioned body of evidence regarding TMD and RA

involvement of the TMJ this stud y h ypothesizes that neurogenic mechanisms play a

cntical role in the induction and maintenance of inflammation involving the TMJ.

Statement of Purpose

The purpose of this study was to investigate the neurogenic contribution

underlying acute TMJ inflammation by evaluating the effect of local anaesthetic

blockade of afferent innervation on the development of MO induced edema in the rat

TMJ area.

Specific Objectives

1. Confirm that a neurogenic component exists in acute TMJ inflammation.

Tissue expansion after MO injection into the pTM will be observed if a neurogenic

mechanisms play any role whatsoever in TMJ inflammation.

2. Evaluate the extent to which neurogenic inflammation may contribute to TMJ

inflammation.

This may be investigated by establishing a dose-response curve for varying

concentrations of MO.

3. To evahrate the relative neirrogenic contribution in mild and severe TMJ inflammation

indirced by MO.

This may be accomplished by evaluating the effect of local anaesthetic induced

conduction blockade on the response evoked by the appropriate MO concentrations

determined in (1).

4. Confirm that the local anaesthetic indirced condtrction blockade is complete-

This may be accomplished by observing whether the typical jaw reflex response to MO

injection is abolished at the time of MO injection.

5. Establish the duration of cornpiete local anaesthetic indirced condtiction blockade.

This may be accomplished by observing at various times when the jaw reflex response to

MO injection is abolished and retums following injection of 5% lidocaine and 0.5%

bupivacaine.

1.12.4 Rationale for Utilization of Lidocaine, Bupivacaine

and Mustard Oil

Lidocaine was chosen because it has been commonly used in previous studies

relating to the investigation of local anaesthetic effects on neurogenic inflammation.

(Yu, 1995; Dux, 1996; Midroni et al., 1996). A 5% lidocaine concentration was chosen

because it is a clinically relevant value. A 2% solution was not used since it was decided

to err on the side of a higher concentration than the minimal necessary to provide

conduction blockade. The minimal concentration required to produce complete

conduction blockade given the volumes utilized in this study were estimated based upon

in viiro and in vivo studies of the activity of lidocaine on rat sciatic nerves (Popitz-

Bergez et al., 1995; Jaffe and Rowe, 1996).

Bupivacaine was chosen because of its reported extended duration of effect as

compared to lidocaine (Malarned, 1990a). A 0.5% concentration was chosen since it is a

clinically utilized value.

This study is an exploration into the mechanism of the vasodilatory and increased

vascular permeability response induced by the small-fibre excitant and infiammatory

imtant MO. This and the study by Fiorentino et al. (Fiorentino, 1999) are based upon

expenments involving the nociceptive fibre activation of jaw muscle reflexes in response

to MO application to the TMJ (Yu, 1995; Yu, 1996; Bakke et al., 1998). Though

intimately related, NI is not identical to the dimension ofinflammatory pain. This study

hopes to provide insights into the mechanisms of neurogenic inflammation, placing

relevant findings in context with the associated dimension of inflammatory pain

explored with EMG studies. It follows then that the chemical algogen, doses, and

method of injection chosen for the neurogenic inflammatory investigation should ideally

approximate if not duplicate the apparatus utilized in the study of nociceptive fibre

activated jaw reflexes. MO, utilized in the EMG studies is considered a selective

activator of nociceptors and is ideally suited for studies relating to neurogenic

inflammation. The concentrations and volumes are therefore based upon those used in

the aforementioned studies.

Chapter 2

Materials and Methods

2.1 Drug Itemization and Preparation

Al1 drugs masses were measured with a Mettler PM2000 electronic scale. An error of + .O0 1 mg was assumed.

2.1.1 Lidocaine HCI

An aliquot for injection was prepared prior to each experiment. Lidocaine

hydrochlonde powder (2-(Diethylamino)-N-(S,6-dimethylphenyl)acetamide

hydrochloride, Sigma Chernical Co., St. Louis, MO, USA) 50 mg was measured out and

placed in a 1 ml vial. One millilitre of sterile normal saline for injection (0.9% sodium

chloride injection USP, Baxter, Toronto, Ontario) was then added. The mixture was then

agitated by hand for 2 minutes. This method provided lidocaine HC1 at a concentration

of 5%.

Bupivacaine HCI

An aliquot for injection was prepared pnor to each experiment. Bupivacaine

hydrochlonde powder ( 1 -buty l -n - (2 ,6 -d imethy lpheny1) -2 -p ipendide , Sigma

Chernical Co., St. Louis, MO, USA) 50 mg was measured out and placed in a 1 ml vial.

One millilitre of sterile normal saline for injection (0.9% sodium chloride injection USP,

Baxter, Toronto, Ontario) was then added. The mixture was then agitated by hand for 2

minutes. This mixture was then added to 9 ml of additional saline and again agitated by

hand. This method provided bupivacaine HCI at a concentration of 0.5%.

2.1.3 Mustard Oii

Mustard oil (allylisothiocyanate, Aldrich. Milwaukee, Wisconsin, USA) of varying

concentrations were prepared by diluting a volume of stock MO with the appropriate

volume of minera1 oil. The stock chernical was provided at a concentration of 95%. The

dilutions and subsequent designation of concentrations assumed a stock concentration of

100%. This was to facilitate preparation of solutions and labelling. Actual concentrations

were slightly less than the label by which they are referred to and are provided in Table 1

along with relative composition of minera1 and MO respective1 y. Mixtures were agitated

by use of a Maxi Mix Plus@ (Bernstead/Thermolyne, Dubuque, Iowa) mixer prior to

use.

Table 1. Reference labels and corresponding MO concentrations

1 Label (Referreà to in ~igÜres and Text) 1 Actual MO Concentration I

2.1.4 Evan's Blue Dye

Evan's Blue powder (Sigma Chemical Co., St. Louis, MO, USA) 100 mg was

measured and placed into an Erlenmeyer flask. 10 ml of distilled water was added to the

powder and stirred with a magnetic stimng pellet for 4 minutes. This provided a solution

of Evan's Blue dye at a concentration of 10 mg/ml.

2.1.5 Urethane

Urethane powder (Ethyl Carbarnate, Sigma Chemical Co., St. Louis, MO, USA)

20 g was measured and placed into an Erlenmeyer flask. 100 ml of distilled water was

added and the mixture stirred with a magnetic stimng pellet for 1 minute. The solution

was heated slowiy whilst being stirred until 30°C. 4 g of sodium borate was then added

and the mixture was heated to 45°C and stirred until al1 urethane powder had dissolved.

The solution was allowed to cool to room temperature. This method provided urethane

solution at a concentration of 200 mg/ml.

2.1.6 a-chloralose

1 g of a-chloralose powder (Fisher Scientific, Fairlawn, NJ, USA) was measured

out and placed in an Erienmeyer flask. 100 ml of distilled water was added and the

mixture stirred with a magnetic stirrer until al1 crystals of the chernical had been

dissolved. This method provided a-chloralose at a concentration of 10 mg/ml.

2.1.7 Proprietary Heparin, Normal Saline, and T-61

Hepaiean@ (Heparin Sodium U.S.P, Organon Teknika, Toronto. Ontario,

Canada) 1000 U/ml was utilized to aid in draining blood of the rat during saline

perfusion in order to visualize EB marking of PE.

Normal stenle saline for injection (0.9% sodium chloride solution, Baxter

Corporation, Toronto, Ontario)

T-6 1 8 (Hoechst, Regina, Saskatchewan, Canada) was utilized in euthanasia.

2.2 Experimental Models

2.2.1 General

2.2.1.1 Set Up and Loading of Double Barre1 Catheter

In early work with the tissue expansion and EMG models it was shown that

aqueous solutions would reflux back up the catheter and/or catheter tract when

attempting to inject them into tissues. To address this problem, both local anaesthetics

were drawn into the catheter in appropriate volumes with minera1 oil introduced prior.

The surface tension and viscous nature of minera1 oil ensured that the complete volume

of aqueous solution was expelled from the catheter tip and no quantity refluxed back into

the tubing. Its non-polar nature ensured that both phases were kept sequestered and

contamination was not a problern. This technique was employed in both models to allow

introduction of aqueous solutions into the TMJ.

2.2.1.2 Confirmation of catheter position

in both tissue expansion and EMG models, EB dye ( 1 0 mglml, 20 mg/kg) was

injected i.v. through the femoral vein cannula prior to euthanasia. Rats were then

euthanized and immediately perfUsed transcardially with 300 ml of 0.9% saline. Skin

and superficial muscle (masseter and temporalis) were dissected and retracted to expose

the periarticular tissues. Obvious bluing of the disk and capsule was taken to be

confirmation of correct placement of the catheter. Eight animals with bluing not

involving the joint capsule and disk were excluded from analysis.

2.2.2 Tissue Expansion Model of Orofacial Inflammation

2.2.2-1 Set Up (Figure 6)

Male Sprague-Dawley rats (Charles River, St-Constant, Quebec, Canada)

weighing 275-450 g were used in this study. Rats were housed in pairs under constant

humidity, temperature of 20°C, and light and dark cycles of twelve hours duration. Rats

were permitteci access to food and water ad libidzrm and allowed one week to become

acclimated to their surroundings before experimental procedures.

Rats were weighed immediately prior to anaesthesia. Anaesthesia was obtained

by intraperitoneal injection of a-chloralose (50 mgkg) and urethane (1 000 mg/kg)

solution. Animals maintained respirations spontaneously.

The hair on skin overlying the right TMJ area was shom with an electnc razor.

This area extended fiom the outer canthus of the right eye to just anterior to the right ear

antero-posteriorly, and above the zygomatic arch to the midpoint of the mandibular

rarnus supero-inferiorly.

Temperature was monitored rectally with an electronic probe. A heating pad was

manually activated and deactivated to keep core temperature maintained at 37.2 f 0.S0C.

To monitor heart rate, two 4 cm pieces of 30 gauge wires were tunneled subcutaneously

over the left and right chest wall with the use of a 2 1 gauge needle. Leads were attached

to the wires and the signal arnplified 1000 times before feeding into a real time Tektronic

TDS2 10 oscilloscope. Heart rates were maintained at 400 beats per minute.

Figure 6 Schematic: Set Up Of Tissue Expansion Mode1 of Neurogenic

Inflammation. Adapted f?om Fiorentino et al., 1999

The trachea was surgically exposed, separated from the esophagus, and an

incision made just below the third tracheal cartilage. A tracheal cannula was inserted

into the proximal airway and sutured in place with 2-0 silk. The surgical wound was

closed with 3-0 silk sutures. This tracheal cannula facilitated maintenance of a patent

airway during the experiment.

The lefi femoral vein was surgically exposed, separated from the femoral artery

and ligated distally. A 22-gauge blunted needle was inserted into the proximal segment

of the femoral vein and sutured in place with 2-0 silk suture. The intravenous line was

flushed with 0.5 cc of 0.9% saline. The surgical wound was closed with 3-0 silk suture.

The rats were then mounted on a sterotaxic appliance (David Kopf Instruments,

Tunjunga, CA, USA) with ear bars placed in the lefi and right extemal auditory meatus

(EAM), and central incisors engaged into an incisor bar. Mineral oiI was applied to each

eye to prevent dessication. A subpenosteal incision was made to expose the cranial

vault. Two pilot holes were placed in the left and nght parietal bones anterior to the

parieto-occipital suture and lateral to the inter-parietal sutures. Two 8 mm stainless steel

screws were placed into the pilot holes. A rigid mounting a m itself fixed to the

stereotaxic frame was approximated to the exposed screws and ligated with a piece of

30-gauge stainless steel wire. The three screws and wire were coated with kt" dental

acrylic and allowed to set.

Ear bars were removed and a polyether silicone mold was approximated to the

lefi side of the head and neck. Dental plaster was mixed, placed in the mold and allowed

to set while engaging the lefi side of the head and neck.

A double barre1 cannula constructed fiom two 27 gauge dental needles was

connected to PESO polyethylene tubing and attached to two 25 pl glass Hamilton

syringes (Hamilton Co., Reno, Nevada USA). The right zygomatic arch was palpated

and the posterior infero-lateral aspect of the zygomatic arch located. The catheter was

inserted at this point to a depth of 2 mm at which bony contact was obtained and then

advanceci anteriorly 0.5mm. The tubing and catheter position were fixed to the fiame

with masking tape. A paper support track was fixed lateral to the right TMJ area. A 50

mm tungsten microelectrode (FHC, Bowdoinham, ME, USA) was cut proximally and

bent 90" 3mm fiom the tip. The proximal end was attached to the skin overlying the

right TMJ area with a drop of cyanoacrylate adhesive and the distal end allowed to lie

passively on the support track. This served as a physical marker of lateral tissue

expansion. A second neural electrode was fixed to a micromanipulator and the tip lined

up the marker with the aid of a 1.6 X power dissecting microscope (Seiler Instruments,

St.Louis, Missouri). Following swelling of the periarticular tissues, the marker fixed to

the rat tissue becarne displaced laterally. Realigning the two electrode tips using the

micromanipulator allowed assessment of tissue expansion laterally.

An initial reading on the micromanipulator was taken and recorded along with

core body temperature, and heart rate. A baseline of 15 minutes was taken, recording the

value registered on the micromanipulator every 5 minutes to time O. These were denoted

time - 1 5, - 10, and O respectively. Injection 1 O pl of a compound at timeo was denoted as

'pre-load'. Injection of various concentrations of MO was perfonned at times.

Micromanipulator readings were taken every two minutes fiom timeo to tirne60 and then

every 5 minutes to timeiso. A timeiine schematic sumrnarizing these details is depicted

below in Figure 7.

Saline h h s tard or local 0 il End

T ime (minutes)

Figure 7 Tissue Expension Model of Nturogcnic Inflammation. Timing o f Caîheter insertion, injection of Local Anaesthetic or Saline and Mustard oil.

2.2.2.2 Dose Response Cumes

Introduction of a catheter into the TMJ produces a mild inflarnmatory response

with associateci edema and lateral tissue expansion. In addition, injection of even small

amounts of fluid causes volumetric distention of the joint. A control for trauma and

volumetic distension of the nght pTM was obtained by injecting 10 pl sterile normal

saline at time O followed by an injection of 20 pl mineral oil was six minutes later.

In order to induce a mild inflammatory reaction, an injection of 20 pl of 1 % MO

was utilized following a 10 pl saline injection (so called 'pre-Ioad'). MO concentration

was increased to 2%, 20%, 40% and 60% in subsequent series of experiments in order to

increase the magnitude of induced inflammation.

2.2.2.3 Conduction Blockade C'sing Local Anaesthetic

To determine the neurogenic contribution in mild and severe TMJ inflammation,

complete local conduction blockade was initially induced by pre-administration of 1 O pl

of 5% lidocaine into the right TMJ pnor to induction of the appropnate inflammatory

response.

To evaluate the effect of a prolonged complete neuronal conduction blockade on

inflammation, a solution of 0.5% bupivacaine was utilized in place of 5% lidocaine.

2-2-24 Contralateral Temporomandibular Joint Examination

The contralateral joint was also grossly exarnined for evidence of EB dye leakage after

dissection as outlined above.

2.23 Electromyographic Jaw Reflex Mode!

2-2.3.1 Set Up

Male Sprague-Dawley rats (Charles River, St-Constant, Quebec, Canada)

weighing 275-450 g were utilized in these series of experiments. Animals were similarly

housed, fed, and acclimated as descnbed for the tissue expansion model.

Induction of anaesthesia was pedormed with 4% halothane through a Fluotec III

vapourizer with the balance of the fiesh gas composed of oxygen. Anaesthesia was

maintained with a mixture of 152.0% halothane and a mixture of 66% N 2 0 and 33%

0 3 .

A tracheal cannula and femoral cannula were introduced appropriately and

sutured in place as previously described. The anterior digastric muscles were exposed

dunng placement of the tracheal cannula and an EMG electrode was placed in each

muscle bilaterally. The skull was fixed to a stereotaxic frame with ligature wire, screws,

and et@ dental acrylic. EMG electrodes were placed bilaterall y in each masseter muscle.

A dûuble barre1 cannula constructed fiom 27-gauge dental needles was placed

into the right TMJ as previously described. To this needle set was connect two 25 pl

Hamilton syringes by PESO polyethylene tubing.

Bilateral EMG activities were recorded in both anterior digastric and masseter

muscles continuously prior to, throughout and after catheter placement.

Increased reflex jaw muscle activity at the time of catheter insertion was taken as

confirmation that the catheter had entered the joint properly. In addition, the left or right

side was evaluated for strength o f signal. Pilot work had dernonstrated no EMG activity

if the catheter failed to pass through the joint capsule.

2.2.3.2 Confirm Complete Conduction Blockade

To confinn complete local anaesthetic conduction blockade at the time of MO

injection, 20 pl 40% MO was injected at 1 O minutes following injection of 10 pl 5%

lidocaine and the resultant EMG activity relative to baseline was evaluated. Presence of

increased EMG activity in anterior digastric andor masseter muscle above baseline

following injection was taken to be indication of failure of blockade. Relative magnitude

of EMG activity was not considered. Conversely, absence of increased EMG activity in

antenor digastric and/or masseter jaw muscle following injection of MO was taken to be

indication of complete blockade.

EMG activity was amplified (gain x500, bandwidth 30-1 000 Hz), displayed on

an oscilloscope and processeâ on-line with a data acquisition and processing system

sarnpling at 2000 Hz ( 140 1 plus@, and spike2" both by CED, Cambridge, UK). Al1 EMG

activities were nonnalized relative to baseline values and areas under the curve

calculated.

2 .2.3.3 Deter rnining the Duration of Conduction Blockade

In order to assess the duration of anaesthetic blockade induced by both lidocaine

and bupivacaine, the EMG mode1 was again utilized. To evaluate the duration of action

of lidocaine, injections of 20 pl of 40% MO were performed at 20,30, and 60 minutes

following injection of 10 pl 5% lidocaine. EMG activity assessed following each MO

injection. A timeline schematic summarizes this series of experiments (Figure 8)

To evaluate the duration of action of bupivacaine, injections of 20 pl of 40% MO

were performed at 60, and 30 minutes following injection of 10 pl 5% bupivacaine.

EMG activity assessed following each MO injection. EMG activity was recorded,

processed and interpreted as described in Objective 4.

5 CI- Begin Local h;lustard Recording .4mesthatic 0 il

T irne (minutes)

Basehne EMG Activlty

Figure 8 EMG Reflex Model. Timing of Catheter Insert ion, Badine Recording, and Mustard O il Injection to Confinn Conduction Blockade and Duration of Blockde for 5% Lidocaine and 0.5% bupiva~aine.

1 l

I I I 1

I

-1 O -5 - ! -- O 10 L 30 30

2.3 Statistical Analysis

Statistical analysis was performed with the aid of SPSS@ Software (SPSS Inc., Chicago,

IL, USA). Detemination of significant differences amongst mean expansion distances

from tirne-, 5 (t 5 ) to tirne, (t so)were accomplished with a general linear mode1

repeated measures ANOVA with Bonferroni correction. A p-value less than 0.05 was

used to determine statistical significance.

Chapter 3

Results

3.1 Dose Response Cuwes

Experimental groups are outlined in Table 2. Before the injection of saline at

t i m ~ (k), trauma fiom catheter insertion produced an equivalent mean baseline

expansion in al1 groups (ANOVA, p ~ 0 . 0 5 ; Table 3). Injection of MO in al1 groups at ts

produced an equivalent increase in tissue expansion until tzo (ANOVA, p <0.05).

Beginning at tn, saline/mineral oil controls (Group 1) exhibited a decrease in expansion

distance. This likely indicated the maximal effect of volumetric distention afler which

expansion demonstrated in other groups was due to neurogenic inflammation.

The entire time course for expansion across Groups 1 through 6 is plotted in

Figure 9. Tissue expansion after 150 minutes was not statisticaily significantly different

between minera1 oil, 1 %, and 2% MO (Groups 1,2, and 3) (ANOVA, p < 0.05). Minera1

oil (Group 1) differed significantly from 20%, 40% and 60% MO (Groups 4,5, and 6)

(ANOVA, p < 0.05). Expansion reached a plateau for 1 % MO (Group 2) approximately

at tito 130. Expansion continued throughout the 150 minute time course for Groups 3,4,

5, and 6.

Table 2 Experimental Croups (n=8 for each)

1 Croup 1 Descriptor 10 ul Saline + 20 ul Minerai Oil 1

- --

1 O pl Saline +20 pl 1 % Mustard Oil 10 pl Saline + 20 pl 2% Mustard Oil 10 pl Saline + 20 pl 20% Mustard Oil 10 pl Saline + 20 pl 40% Mustard Oil 10 ul Saline + 20 1.1160% Mustard Oil 1 O pl 5% Lidocaine + 20 pl 1 % Mustard Oil 10 ~ 1 5 % Lidocaine + 20 ~ 1 4 0 % Mustard Oil 10 pl 0.5% Bupivacaine + 20 pl 40% Mustard Oil

Table 3 Mean Expansion Distance (Mean) f Standard Error (SE) at time 0,20,100, and 150 (h, tz* tlO0, tlSO)

Figure 9 Dose Response Curves: Expansion Distance Versus Increasing Mustard Oil Concentrations

T h e (minutes)

+ Group 1 10 pl S a h e + 20 pl Mineral Oil Group 2 10 pl Saline + 20 pl 1 % Mustard oil

t Group 3 10 pl Saline + 20 pl 2% Mustard oil Group4 I O pl Saline + 20 pl 20% Mustard oil Group 5 10 pl Saline + 20 pl 40% Mustard oil

* Group 6 1 O pl Saline + 20 ~ 1 6 0 % Mustard oil

I Eight Rats Per Group

3.2 Effect of Local Anaesthetic Block On Mild and Severe

Inflammation

Baseline expansion and expansion to t2o after saline or lidocaine injection did not

differ significantly between groups as mentioned in 3.1. At tiso tissue expansion for

lidocaine pre-treated rats (Groups 7 and 8) did not differ significantl y from saline

controls for either 1 % or 40% MO (Figure 10, Groups 2 and 5) (ANOVA, p < 0.05).

Similarly, bupivacaine pre-treated rats (Group 9) did not differ signi ficantl y fiom saline

controls (Group 5) (Figure 1 1) (ANOVA, p < 0.05). Mean expansion distances and

standard m o r s are given in Table 3.

Tissue expansion for iidocaine and bupivacaine pre-treated rats (Groups 8 and 9)

did not differ significantly from each other at timeiso (ANOVA, p < 0.05). These same

groups continued to exhibit tissue expansion throughout the 150 minute time course. The

lidocaine pre-treated 1% MO group reached a plateau at approximately t100,150.

A summary plot is depicted in Figure 12.

3.3 Confirmation of Conduction Blockade

No significant increases in EMG activity were observed during baseline

recording between O and 10 minutes (koo) in either masseter or digastric. No significant

increases in EMG activity were evoked with local lidocaine injection at km. No increase

in EMG activity in either digastric or masseter muscles is evoked with 40% MO

injection I O minutes following lidocaine (at t (Figure 1 3 j. This confinns that

Figure 10 Effect of 5% Lidocaine On Expansion Distance

Time (minutes)

Group 2 10 pl Saline + 20 pl 1 % Mustard oil * Group 7 1 O pl 5% Lidocaine + 20 pl 1 % Mustard oil + Group 5 1 O pl Saline + 20 pl 40% Mustard oil v Group 8 IO pl 5% Lidocaine + 20 pl 40% Mustard oil

Eight Rats Per Group

Figure 11 Effect Of 0.5% Bupivacaioe On Expansion Distance

20 40 60 80 100 120 140 160

Time (minutes)

Group 5 IO pl Saline + 20 pl 40% Mustard Gil -0- Group 9 10 pl 0.5% Bupivacaine + 20 pl 40% Mustard Oil

Eight Rats Per Group

Figure 12 Expansion Distance: Effect Of 5% Lidocaine And 0.5% Bupivacaine

Time (minutes)

+ Group 5 1 O pl Saline + 20 pl 40% Mustard Oil -c+ Group 8 10 pl 5% Lidocaine + 20 pl 40% Mustard Oil + Group 9 10 pl 0.5% Bupivacaine + 20 pl 40?4 Mustard Oil

I Eight Rats Per Group

Evalunting duration of blockade by 5% Lidocaine:

Figure 13 (a) Right Digastric Muscle

+ lnjection o f MO 30 minutes following lidocaine (2400s) * lnjection o f MO I O minutes following lidocainc (1200s) 6 lnjection o f MO 20 minutes following lidocaine (1 800s)

O 600 1200 1800 2400 3000

Time (seconds)

Figure 13 (b) Right Masseter Muscle

-(I- lnjection o f MO 10 minutes following lidocaine ( 1200s) & Injection o f MO 20 minutes following lidocaine (1800s)

n -0.004

O 1 1

O 600 1200 1800 2400 3000

a Time (seconds)

neuronal conduction has been blocked at the time of MO injection (at b) in the dose

response experiments.

3.4 Duration of Conduction Blockade

EMG activity following MO injections at t1200 and longer, are shown in Figure

13. A sharp increase in activity is evident at 30 minutes post-lidocaine ( t Z 4 ~ )

demonstrating that the blockade is incomplete at this point in time. Complete conduction

blockade by lidocaine is at least 20 minutes in duration but no longer than 30 minutes

after administration.

EMG activity evoked by MO injections following bupivacaine are shown in

Figure 14. No activity is evident after injection of 40% MO at 30 minutes (t?400) post-

bupivacaine. A sharp increase in activity is evident after injection of 40% MO at 60

minutes (t4200) post-bupivacaine. Complete conduction blockade by bupivacaine is at

least 30 minutes in duration but not longer than 60 minutes afler administration.

3.5 Contralateral Temporomandibular Joint

Examination

No EB dye leakage was observed involving the left temporomandibular articular

or periarticular tissues.

Evaluating Duration Of Blockade By 0.5% Bupivacaine

Figure 14 (a) Right Digastric Muscle

* Injection of MO 30 minutes following bupivacaine (2400s)

+ Injection of MO 60 minutes following bupivacaine (4200s)

Time (seconds)

Figure 14 (b) Right Masseter Muscle

Time (seconds)

Chapter 4

Discussion

4.1 Mustard Oil induces acute edema development in the Rat

pTM area

This study demonstrates that MO induces acute edema development in the rat

pTM. in light of cument evidence which indicates MO acts purely neurogenically (see

section 1.9), this finding suggests that neurogenic mechanisms play a role in acute

inflammation of the rat pTM. It has been demonstrated in other studies that not al1

tissues exhibit a neurogenic PE response (Szolcsanyi, 1988). For exarnple, the vas

deferens, testes, prostate, plantar and abdominal muscles of the rat do not exhibit

extravascular accumulation of EB dye with antidromic stimulation of lumbar dorsal

roots (Szolcsanyi, 1988).

Neurogenic mechanisms have the potential to play a major role in acute TMJ

inflammation. Tissue expansion after 60% MO injection continued through 150 minutes,

having reached 1 193 f 226 pm at this tirne. This is approximately 50% the medio-lateral

width of a normal joint (Kido, 1995). Tissue expansion reached 1365 + 2 12 Fm at this

time with 0.5% Bupivacaine followed by 40% MO demonstrating that the maximal

potential for tissue expansion has not been defined.

Neither major nor minor degrees of neurogenic inflammation differed with local

anaesthetic conduction blockade. This suggests that the neurogenic contribution to MO

induced edema is consistent at both hi& and low concentrations.

4.2 Effect of Local Anaesthetic Block on Inflammation

Complete local anaesthetic blockade provided by lidocaine for 10 minutes or

bupivacaine for 30 minutes failed to inhibit the course of edema. Furthemore, though

complete conduction block was not achieved afier these, blockade was in no way fully

dissipated (Figure 13). Though a quantitative evaluation of EMG activity was not

undertaken, the relative magnitude and duration of responses to MO appeared to increase

with time afier local anaesthetic administration, returning to normal levels (Yu, 1996)

afier more than one hour. Despite this, no difference was observed between controls and

rats given local anaesthetic pre-treatment.

It was hypothesized that the relatively high concentration of MO (40% v/v)

utiiized to evoke a maximal degree of inflammation could be acting through

predominantly non-neurogenic means. A direct action on immune cells to release

inflarmnatory mediators andor direct action on vasculature to increase permeability is a

possibility. Lynn (Lynn and Shakhanbeh, 1988) utilized a 7.5% as well as a 25% MO

concentration in attempts to elicit NI on rabbit skin desensitized with capsaicin. It was

observed that PE as assessed by EB dye method was reduced compared to controls upon

application of 7.5% but not 25% MO. This phenomenon was similarly proposed to be

due to a non-neurogenic mechanism of highly concentrated MO by the authors.

In order to address this possibility in this study, a low concentration of MO ( 1 %)

was ernployed to evoke a perceivable edema response and local anaesthetic block again

applied. No difference between control animals receiving saline pretreatrnent and

experimental animals receiving 5% lidocaine pretreatment was observed. The hypothesis

that MO acts neurogenically at low but not hi& concentrations is not borne out by

observations in this study.

It is conceivable that the brief duration of activity of lidocaine was responsible

for the inability to dernonstrate a difference between anaesthetic and control groups.

EMG studies dernonstrated that complete block existed between 10 and 20 minutes after

lidocaine administration. Whether complete block was necessary, and whether 10

minutes duration was adequate to inhibit NI to a perceivable degree is a matter of debate.

To address the possibility that both factors were relevant it was assurned that complete

block was necessary and that 10 minutes was of insufficient duration to demonstrate a

difference in expansion distances. To this end, plain bupivacaine was ernployed in lieu

of lidocaine. EMG studies confirmed complete local nerve blockade of at least 30

minutes (3 tirnes that of lidocaine). No difference between bupivacaine pretreated rats

and saline pretreated rats was demonstrated. The hypothesis that duration of complete

local anaesthetic blockade by lidocaine was too brief to demonstrate a difference is not

consistent with observations from these additional investigations.

A proposa1 was made by Midroni (Midroni, 1996) to account for the apparent

lack of effect of lidocaine. It was suggested that the volume of imtant being greater than

anaesthetic may have resulted in diffusion to a greater extent as compared to local

anesthetic. This could have resulted in activation of afferent nociceptive fibres and

consequent neurogenic inflammation in tissues beyond that blocked by the anaesthetic.

However, the consideration that Molmineral oil compound is relatively viscous and

non-polar while lidocaine is in aqueous solution (thereby potentially allowing more rapid

and greater dif is ion through periarticular tissues) argues against this proposition. The

use of local anaesthetic of slightly higher concentration (5% lidocaine) in this study

would also argue against this factor. To account for this potential confounding factor,

Midroni (Midroni, 1996; see Section 1.6.2) utilized a volume of anaesthetic twice that o f

MO but again found no difference. The most compelling evidence is the abolition of

jaw muscle EMG reflex by lidocaine demonstrating absolute quiescence of nociceptive

afierents.

4.3 Interpretation of Results

The results of this study dernonstrate that MO (acting purely through neurogenic

means) continued to induce development of edema in the rat TMJ despite complete local

and sympathetic axonal conduction blockade. This paradoxical result may be interpreted

in two ways: MO acts non-neurogenically to produce edema or MO releases mediators

of NI by direct action on nociceptive terminais and does not require axonal

depolarization.

4.3.1 Does Mustard Oil Eücit Inflammation Through

Non-Neurogenic Mecbanisms?

These results may imply that MO acts predominantly through non-neurogenic

means contrary to popular belief. This is at odds to indications of selective neurogenic

activity of MO in the rat paw skin (Reeh, 1986; McMahon et al., 1989; Lippe, 1993a;

Lippe, 1993b;) and mouse ear skin (Inoue, 1997). There is no body of evidence to

support the suggestion that MO acts non-neurogenically to produce PE.

Nonetheless, to entertain this possibility is to suggest that the trigeminal system

is somehow unique with respect to the ability of afferent nerves to produce PE. This is

contrary to the observations of Jancso (Jancso, 1967; Jancso, 1977) and Szolcsanyi

(Szolcsanyi, 1988). Jancso utilized both topical MO painted on the rat muzzle and

antidromic stimulation of the trigeminal ganglion to elicit PE. It is mentioned that

stimulation of the ophthalmic and maxillary branches of the tngeminal nerve elicited an

inflammatory reaction. No mention of this effect in the mandibular branch is made. It is

speculation whether failure to mention action in the mandibuiar branch implied a

negative reaction. Later it is stated though that soon after i-v. injection of EB dye, "the

skin, conjunctiva, and mucous membranes supplied by the second and third branches of

the trigeminal nerve turned blue after a few minutes" (Jancso, 1967). Whether the

authors mistakenly meant to refer to the first and second branches of the trigeminal in

this latter statement is unknown. Regardless, it seems unusual to make specific reference

at any time to two and not al1 three branches of the trigeminal nerve.

Intuitively, there seems no reason to suspect that a neurogenic reaction mediated

by two branches of the same cranial nerve would somehow be non-neurogenic in the

third. Innervation of the TMJ however is predominantly h m the third division of the

tigeminal (Schmid, 1969). Interestingly, it has been found that receptive properties of

afferents are dependent on the properties of the tissues in which they are imbedded. For

exarnple, the threshold of nociceptive afferents innervating the comea are comparable to

those of low-threshold mechanoreceptors in skin (Belmonte and Giraldez, 198 1 ;

Tanelian and Beuerman, 1984).

Afferent nociceptive fibres (e.g. C-fibres) i~e rva t i ng one tissue are not

necessarily physically or functionally identical to those innervating others. It appears

that specific peptide complements are characteristic for subpopulations of afferents

(Hokfelt et al., 1975b; Leah et al., 1985a; Leah et al., 198513). For exarnple, one study

(McMahon et al., 1984) have demonstrated that antidromic stimulation of C-fibres fiom

muscle produces PE at a tiaction of the magnitude produced when stimulating those

innervating skin. Immunohistochernical analysis of these two groups of fibres show a

greatly reduced presence of charactenstic C-fibre markers (SP and fïuoride resistant acid

phosphatase) in t'le afferents supplying muscle as compared to skin.

McMahon (McMahon and Gibson, 1987; McMahon, 1989) demonstrated that

ability to produce NI by afferents was dependent upon the particular tissues innervated.

Cutaneous nerves were cross-anastarnosecl with afferent nerves to muscle as well as self-

anastarnosed with their distal stumps in a rat model. PE was elicited by topical MO

application to skin and also by capsaicin administration i.v. Self-anastamosed nerves

retained ability to produce PE after MO or capsaicin. Nerves that imervated skin

(originally able to exhibit PE) that were now imervating muscle lost the ability to

produce PE. Nerves that did not produce PE were now able to when innervating skin.

Assessrnent of neuronal levels of SP and CGRP (thought to mediate NI) showed that

ability to produce PE was correlated with these peptide levels. The authors concluded

that the particular tissue which afferents innervated exerted a trophic effect on peptide

production in these nerves. That is, the ability to generate neurogenic inflammation was

tissue specific.

This hypothesis is given consideration within the trigeminal system. Perhaps the

afferent fibres i~e rva t i ng the pTM are physiologicall y unable to produce NI. This

suggestion is given support by Uddman et al. (Uddman et al., 1998) who demonstrated

that sensory innervation in the rat TMJ consisted of only a few SP containing fibres. The

major neuropeptides were CGRP, VIP and NPY. Note that SP and CGRP alone has been

demonstrated to cause plasma extravasation (Louis et al., 1989; Cambridge and Brain,

1992).

Changes in peptide levels may be brought about by alterations in environment or

physical alteration of the nerve/tissue interaction (McMahon and Moore, 1988).

Separation fiom innervated targets or inhibiting axonal transport causes depletion of

peptides usually but sometimes increases in certain products (Barber el al., 1979;

Fitzgerald et al., 1984). Nerve growth factor when applied to sensory afferents results in

increases in protein content including SP (Goedert et al., 198 1 ). In inflammatory joint

disease, it is conceivable that products of inflammation may have a direct or indirect

trophic effect on pnmary afferents to increase content of pro-inflammatory mediators

such as SP.

4.3.2 Does Mustard Oïl Initiate Direct Release of Mediators

Of Neurogenic Inflammation From Nociceptive Terminals?

An alternate explanation for the paradoxical results observed is that NI elicited

by MO is through direct release of mediators from nociceptive teminals independent of

axonal conduction (See section 4.13).

To account for this suggestion, others have made a distinction between the axon

membrane potential and terminal membrane potential or "receptor potential"

(Szolcsanyi, 1988). Receptor potentials were provided as a graded response as opposed

to an 'al1 or none' that axonal action potential represented. This graded response was

described as resistant to local anaesthetics. These suggestions are contrary to the

majonty of literature conceming excitation-secretion coupling outlined in Section

1.1 1.3.

Assuming that generation of NI independent of axonal depolarization occurs, an

important implication is brought forth. n i e so-called 'axon reflex' in al1 of its entities

(Figure 15) thought to mediate flare reactions, is not necessary to produce NI contrary to

traditional dogrna. Due to local anaesthetic block, no action potentials were conducted

orthrodromically, so antidromic conduction of action potentials down bifurcations of the

same nerve could not have occurred. For similar reasons, the role of axo-axonal

conduction depicted in Figure 15 (c) (yet to be proven to occur) plays little role in

producing NI.

(a) Unidirectionai reflex (b) Bi-directional reflex

LEGEND

Receptor ending

Neumeffector collateral ending

4 Action potential

Normal vessel --

8 '

. : Dilated and pemeable vessel

ôil --. . .

- - . -

&on-axonal coupling and bi-directional mflex

Figure 15 Traditional Axon Reflex 'Iheory: Lewis, 1927 la) receptor activation of neuroeffector collateral. (b) and (c) bi-directional axon mflex via dual mode sençory-efferent nerve endings. Adapted h m SzolcsBnyi, 1988.

4.4 The Central Component to Neurogenic Inflammation

In the TMJ

4.4.1 Are Dorsal Root Reflexes Fundamental to Acute Neurogenic

Inflammation?

The results of this study impact on the current concept conceming 'dorsal root

reflexes' referred to briefly in Section 1.5.2. DRR are a pathological response to

hyperexciteability in dorsal horn neuronal circuits resulting in antidromic depolarization

of central terrninals of primary afferents and consequently causing release of pro-

inflarnmatory mediators fiom penpheral endings (Sluka, 1995).

Sluka et al. (Sluka, 1995) propose that this central mechanism is a fundamental

element in the development of neurogenic inflammation. This hypothesis has been based

upon studies of acute inflammation wherein it was observed that introduction of non-

NMDA and GABAA receptor antagonists (CNQX and bicuculline) into the spinal cord

dorsal hom produced a 50% reduction in joint circumference and temperature. The

process begins with sensitization of primary afferent nociceptors secondary to some

form of mechanical, chemical, or heat stimuli. As a result of the increased inputs to the

spinal cord dorsal hom, interneuronal circuits become sensitized. These central circuits

project to more rostral centers for pain perception and also to central terminals of

primary afferents. These circuits are hypothesized to cause primary afferent

depolarkation (PAD) sending antidromic action potentials to the periphery to release

pro-infiammatory mediators of NI. Theoretically, a positive feedback mechanism ensues

wherein the heightened peripheral inflammatory response M e r activates pnmary

afferents (Sluka, 1995).

Years ago, the trigeminal quivalent 'higerninal dorsal root reflex" (Figure 1 6)

was introduced (Calvin et al., 1977). This theory proposed that afferent inputs arising

from the TMJ excited neurons in the spinal tract of the trigeminal nerve and resulted in

depolarization of other joint afferents causing release of pro-inflammatory substances.

The present study provides contradictory evidence to the fundamental role of DRR in

generating NI in the trigeminal systern.

4.4.2 Spinal Reflex Arcs in Acute Inflammation

A signifiant role of spinal reflex arcs in this mode1 of acute inflammation is

unlikely. No dye was visible in the contralateral TMJ disk or capsule nor surrounding

periarticular area in saline control animals. Spectroscopic analysis of dye content of

both joints was not undertaken since the differences in dye content as perceived b y the

naked eye were obvious.

Levine et al. (Levine et al., 1985b) specifically exarnined the phenomena of

spatially rcmote generation of inflammation. Rats utilized in this study had to be

'pnmed' with acute injury stimulus in one paw for three consecutive days before the

phenomena of bilateral swelling following unilateral injury was manifested. Our results

support the proposition that spinal reflexes do not play a role in eliciting inflammation in

the spatial1 y remote tissues in the acute phase of injury.

# Trigeminal

1 Ganglion (

Ventral

Legend

Cell Body

Neuron Terminal

'-a Action potential direction

SICC Inflammation

Spinal Tract of V (Subnucleus

Caudalis)

Figure 16 The Trigemmal Dorsal Root Reflex.

This finding is contrasted with observations fiom an adjuvant mode1 of

inflammation. Carleson et al. (Carleson, 1996) found a bilateral increase in

immunoreactive peptides SP and CGRP in lefi and right TMJ afier adjuvant challenge in

only the right joint. Note that a systernic action of adjuvant was not mled out. This is of

particular relevance in light of the fact that polyarthritis is traditionally induced by

systernic injection of adjuvant.

4.5 A Reverse Trend Toward Increased Plasma

Extravasation With Local Anaesthetic Block

4.5.1 Inhibition of Sympathetic Efferents

The effect of lidocaine administration produced a non-statistically signifiant

difference in tissue expansion with both 1% and 40% MO (Figure 10). Nonetheless, a

trend is present toward a paradoxical increase in ederna as a consequence of local

anaesthetic administration. This trend is most apparent in the 40% MO curve with 5%

lidocaine pre-load. This apparent 'pro-inflammatory' effect of local anaesthesia is again

produced in the bupivacaine trials (Figure 1 1 ). A number of possibilities may be

entertained to account for such a response.

Sympathetic nervc terminals contain neuropeptide Y (NPY). They pervade the

synovial membranes, joint capsules, and articular disk in the rat TMJ (Uddman et al.,

1998). NPY is produced along with NE in sympathetic terminals and have a

vasoconstrictive effect (Lundberg et al., 1982). Increased NPY levels have been

documented in rat TMJ synovial fluid aspirates following inflammation induced acutely

by SP injection (Carleson, 1996b) and chronically by adjuvant injection (Carleson,

1996a). Increased levels have been found in arthritic human TMJ aspirates as compared

to non-rheumatic TMJ's (Alstergren, 1995). Indications are that NPY is CO-localizeà and

released with SP and CGRP and perform a regulatory role in the inflarnmatory process

( Alstergren, 1 995).

Presumably due in part to its vasoconstrictive effects, NPY has been observed to

reduce the SP and CGRP induced PE response (Gray and Morley, 1986). Conceivably,

if the primary action of sympathetic pst-ganglionic nerve (SPGN) terminals is to

diminuate the PE response, blockade of such fibres should result in increased PE.

Administration of adrenergic blockers by Ferrell et al. (Ferrell and Russell, 1986) prior

to antidromic C-fibre stimulation in the posterior articular nerve of the cat knee joint

resulted in an enhanced PE.

The Effect of Local Anaesthetic on Sympathetic Neurons

Release of mediators fiom primary afferent terminals was deemed to be resistant

to effects of local anaesthetic blockade. No direct measure of the degree of axonal

conduction block was assessed in Sf GN in this study. We speculate that since

sympathetic neurons teminate in the articular synovial lining as fine fiee endings, the

completeness of blockade can be assumed since primary afferents demonstrated block

with concentrations of lidocaine and bupivacaine utilized.

1s release of catecholarnines induced by MO in SPGN similarly independent of

conduction blockade? This study suggests that in fact syrnpathetic neurons differ in this

respect. Green et ai. (Green, 1993b) evaluated the effect of local anaesthetic on

bradykinin and 6-hydroxydopamine (6-OHDA) on PE response in the rat knee. 6-OHDA

acts specifically on syrnpathetic neurons to release stores of norepinephrine (NE)

(Tranzer, 1 97 1 ). The effects of both bradykinin and 6-OHDA on PE were attenuated

reversibly by 4% lidocaine infusion. Other studies have confinned the inhibitory effect

of lidocaine on NE secretion fiom SPGNs (Nakata et al., 1990; Du et al., 1993; Hogan et

al., 1994). In contrast, this as well as other studies (see section on effect of lidocaine) do

not appear to be able to inhibit neurosecretion fiom primary sensory afferents.

4.5.1 -2 Summary: Does the Sympathetic Newous System Play A

Role in Producing Edenia?

The focus of our study was not to examine sympathetic influences on acute PE

induced by MO. However, we may infer that blockade of SPGN terminals occurred with

local anaesthetic administration. Though we did not observe a statistically significant

difference between saline controls and rats receiving lidocaine pre-load, there appears to

be an unexpected trend toward increased inflammation. We speculate that this

paradoxical observation may be atîributed to inhibition of NE release fiom SPGN

terminals. There exists ample evidence that lidocaine can inhibit this NE release, that NE

acts to diminuate PE, and that reduction of NE release (by sympathectomy or adrenergic

antagonists) increases PE.

Despite this reasoning, evidence exists to the contrary. Sluka et al. (Sluka,

1994b) utilized chemical sympathectomy and dorsal rhizotomy to remove sympathetic

efferent effects in a model of acute inflammation (as quantifieci by tissue ederna) induced

by carrageenan and kaolin. No significant difference was found with chemical (a-

adrenergic blockade) or surgical sympathectomy indicating that an intact sympathetic

nervous system is not necessary for induction of acute arthritis.

Obviously, the issue of whether the sympathetic nervous systern participates in

neurogenic inflammation has yet to be reconciled. Sluka (Sluka, 1995) proposes that one

key distinguishing feature between so-called acute and chronic inflammatory models is

the relative role of the sympathetic nervous systern in producing inflammation. It may be

that pro-inflammatory sympathetic drive is responsible for perpetuation of inflammation,

in the absence of which results in self-limitation of the process (Sluka, 1995). That is, in

acute inflammation the sympathetic role is insignificant while in chronic stages, highly

signi ficant.

4.5.2 Effect of Deafferentation on Activity of Primary Afferents

and Plasma Extravasation

A more rapid onset and severe arthritis afier deafferentation by rhizotomy has

been observed in the adjuvant induced arthritic rat model (Levine, 1986). It was

suggested this results fiom enhanced activity of unmyelinated pnmary afferents afier

rhizotomy in increasing adjuvant induced arthritis. The present study implemented the

use of local anaesthetic which induced a fùnctional (and reversible) deafferentation. The

acute NI response induced under tùnctional deafferentation was no different than

without. It appears unli kel y that enhanced activity of unm yelinated afferents if present is

enough to influence acute PE responses.

4.5.2.1 The Gate Control Theory of Inflammation?

Local anaesthetic injection prior to MO induced a statistically insignificant trend

towards increased inflammation. This trend may possibl y be attributed to removal of

tonic inhibitions of afferent neuron excitability. If tonic inhibition from supraspinal

levels were normally present, lidocaine may have released nociceptors fiom these

influences and resulted in increased NI.

Altematively, this trend may be attribut4 to the role of large diarneter sensory

afferents through a modulatory effect on nociceptive afferent activity. Inhibition of large

diameter afferent inputs which may normally have reduced antidromic activation of

nociceptive afferents would theoretically have increased PE (Sluka, 1998). This is

suggestive of a 'gate control of inflammation' after the gate control theory of pain

proposed by Melzack and Wall (Melzack and Wall, 1965).

4.6 Other Studies Of Local Anaesthetic In

Attempts To Abolish Neurogenic Inflammation

Studies on the effect of local anaesthetic on PE have been performed with

conflicting results. Antidromic action potentials may conceivably arise not only from

descending central sources and spinal reflex Ioops, but also by antidromic conduction

from adjacent terminal nerve branches. This so-called 'mon reflex' as coined by

Langely and Anderson (Langley and Anderson, 1894) should theoretically be abolished

if axonal conduction is blocked locally. This blockade is appropriately accomplished

through the administration of local anaesthetic.

Dux et ai. (Dux, 1996) exarnined the neurogenic inflammatory response to MO

in rat skin afler pre-administration locally with lidocaine. A 5% MO / paraffin solution

was painted on abdominal skin. 0.1 %- 1 % lidocaine in 100 pl aliquots was injected

subcutaneously 15 minutes prior to MO injection. Rats were sacnficed 20 minutes afier

irritant application and PE measured with the EB method. An inhibition of PE by

lidocaine was demonstrated in a dose dependent fashion. Only concentrations of 0.5% to

1 % were statistically significant in inhibition but the concentrations of O. 1 % and 0.25%

Iidocaine showed an appropriate trend. The current study utilized concentrations 10

times (5% lidocaine) the minimum required to produce a significant effect reported by

Dux et al. (1 996). The current study examined the inflammatory reaction within the

acute phase (O to 2.5 hours) similar to Dux et al. (35 minutes). In contrast to the

aforementioned results, this study did not observe any inhibition or even trend toward

inhibition. The suggestion that the observed difference in results is due to the different

tissues examined (skin as opposed to TMJ) is a matter of debate (See section 4.3.1).

Dux et al. (1996) fùrther examined the effect of lidocaine on PE evoked upon

histamine injection and compound 48/80 injection (a histamine liberator). Lidocaine

similarly inhibited in a dose dependent fashion the PE response to both of these

compounds. The authors concluded that the site of action must be at the level of the

vascular endothelial ce1 1 since histamine acts direct1 y on postcapillary venules (Maj no et

al., 196 1). M i l e there is evidence that local anaesthetics modi& endothelial b c t i o n to

result in inhibition of vasodilatation (Johns, 1 989), there is Iittle if any evidence to show

a direct inhibitory effect on vascular penneability. Certainly Our results do not support

the claim that lidocaine acts directly on vascular endothelium to inhibit PE.

Jancso (Jancso and Jancso-Gabor, 1968) examined the effects of local anaesthesia on PE

in the rat trigeminal system (conjunctiva and skin). Pain reaction to capsaicin instilled in

the eye was abolished by local anaesthetic but EB dye leakage was no different from

controls. Procaine 1% injected S.C. prior to 5% MO topically applied to the dorsal aspect

of the rat paw failed to reduce the PE reaction seen with controls. This is in contrast to

findings previously mentioned by Dux et al.

Two further human experiments were performed by Jancso involving patients

who had sustained sensory nerve injury owing to accidents previously and were now

rendered insensitive unilaterall y on the skin of one m. In the first experiment capsaicin

and 10% MO were applied to the skin of both arms for 10 minutes. On the insensitive

skin there was a minimal inflammatory reaction. On the contralateral sensitive skin,

edema and intensive hypezmia was observed. In the second expenment, 1 % lidocaine

was injected subcutaneously into the volar forearm in normal volunteers pnor to

capsaicin or MO apptication to the same area. Contralateral arms were used as controls.

Though flare was abolished by lidocaine, edema and redness where the irritant had been

applied persisted. The identical reaction with the addition of flare was evident on the

contralateral a m .

The results of this study confirm the findings that local anaesthetic block does

not eliminate the ederna reaction to MO. It is interesting that no edema response was

seen in the human subjects with sensory nerve damage. Jancso suggests that while

axonal conduction is not necessary for induction of PE, release of mediators may still

occur at nerve teminals in response to algogenic substances such as MO. In the case of

nerve injury, these afferents having been darnaged, have lost the ability to produce

mediators of NI, and consequently, have none to release to cause PE upon stimulation by

MO. In our experiments, afferent nerves supplying the rat TMJ are and have been

functionally and physically intact. MO still elicited an edema response no different from

controls despite axonal conduction blockade. Release of mediators stored in afferent

teminals in response to MO injection appears to have occurred despite axon conduction

blockade.

4.7 Vascular Effects of Lidocaine and Bupivacaine

A possible masking effect of PE may be present since lidocaine has inherent

vasodilatory properties on the vessels in the TMJ. Studies have demonstrated that

lidocaine has a biphasic dose dependent effect. In the respiratory tract, high

concentrations are vasodilatory, and at low concentrations lidocaine is vasoconstrictive

(Pateromichelakis, 199 1 ). Concentration of 1 % and higher have been dernonstrated to

cause vasodilatation in rat cremaster muscle (Johns et a/., 1985). Guinard showed

increases in blood flow after clinically usehl concentrations of 0.05% to 2% lidocaine

and 0.025% to 0.075% bupivacaine in human skin (Guinard et al., 1992). Mepivacaine

however, did not induce vasodilatation greater than saline. Notably, needle stick or

saline injection alone increased blood flow to similar or greater extents respectively.

Midroni et al. demonstrated that lidocaine injected in the rat TMJ alone did not

produce any PE different from saline controls 30 minutes afier injection (Midroni,

1996). The vasodilating effect of local anaesthetic îs unlikely to have had a significant

impact on the results of this study. The current study utilized a six minute delay between

injection of local anaesthetic (b) and injection of MO (b). Within this time period, no

difference between tissue expansion between bupivacaine or lidocaine and saline

controls was seen (Figures 17 and 18; p < 0.05, ANOVA). in fact the average tissue

expansion of 25 pm for the six minute period ending at due to local anaesthetic

a fiom needle injection was no more than the five minute period ending at to due to traum

insertion.

Lam and Femell (Lam and Ferrell, 1 99 1 b) observed that CGRP had little if any

effect on facilitating PE in the rat knee in response to SP administration (despite causing

concomitant vasodilatation). This suggests that the extent of increased vascular

permeability predominantly influences PE and not the extent of vasodilatation.

4.8 Lidocaine and the Acute Local Cellular Immune

Response in the Tissue Expansion Mode1

Lidocaine has been shown to inhibit neutrophil aggregation in the lung exposed

to hyperoxia (Takao et al., 1996) and also has an inhibitory effect on release of oxygen

radical species fiom neutrophils (Peck et al., 1985; Stelzner et al., 1987; Sasagawa,

199 1). Lidocaine consequently causes a decrease in vascular permeability (Takao, 1996)

and reduces leukocyte adherence (Schmidt et al., 1997) by interfering with production of

oxygen radical species necessary for these activities (Suzuki et al., 199 1). Other local

anaesthetics similarly inhibit leukocyte adhesion (Martinsson et al., 199%) and release

of pro-inflammatory products (Martinsson et al., 1997a).

ure 17 pansion Distance Due to Saline or Bupivacaine Injection

Time (minutes)

+ Group 1 * Group 9

10 pl Saiine + 20 pl Mineral Oil 10 pl 0.5% Bupivacaine + 20 pl 40% Mustard Oil

Eight Rats Per Group

Figure 18 Expansion Distance Due To Saline Or Lidocaine Injection

2 4 6

Time (minutes)

+ Group 1 10 pl Saline + 20 pl Minera1 Oil -O- Group 8 10 pl 5% Lidocaine + 20 pI 40% Mustard Oil

Eight Rats Per Group

Lidocaine injection failed to decrease FE induced by mustard oil. This suggests

that inhibitory effects on neutrophils were not significant or conceivably, the relative

contribution of neutrophils on PE induced by MO is insignificant.

4.9 Strengths and Limitations of Methodology

These results of this study are similar to findings of Midroni (Midroni, 1996)

who, using the EB method, failed to demonstrate a difference 30 minutes after MO

administration to the rat TMJ. Thirty minutes was chosen as a time to sacrifice and

analyze TMJ sarnples based upon a study by Haas et al. (Haas, 1992) This latter study

had demonstrated a maximal degree of inflammation at this time as evident by EB dye

leakage. The EB method cames with it an inherent limitation since it requires the

sacrifice of animals at a single time point in order to retrieve inflammatory tissue for

spectrophotometric analysis. In contrast, the tissue expansion model used in this study

bears with it the distinct advantage of being able to follow the development of an

inflammatory process over a continuous time course. Consequently, a very brief duration

of inhibition of PE conceivably unresolvable using the EB method would more likely be

observed with the tissue expansion model. For example, an inhibition of extravasation

for 5 minutes fkom timeo to times may be apparent with the tissue expansion model but

rnay not be with EB method at time30.

The tissue expansion model is more efficient than the EB method for three

additional reasons. First, a single animal provides data over many time points rather than

requiring many animals each for a single point in time. Second, Fiorentino et ai. (1 999)

dernonstrated that ederna development as quantifid by the tissue expansion mode1

parallels the extravasation as measured by EB. The need to obtain, store, and analyze

tissue spectrophotometricall y is eliminated and therefore expedites data collection.

Finally, EB dye must be allowed approximately 10 minutes to distribute throughout the

intravascular compartment. Analysis is limited then to the 10 minutes afier injection of

dye and abrogates the ability to reliably observe inflammation development in these first

minutes following induction. This is likely the critical time period during which

compounds such as a lidocaine are likely to exhibit an effect, if at all.

The tissue expansion method has a few limitations. The apparatus requires a

plaster block to enable stabilization of the skull and is applied to the contralateral side to

the injection site. This abrogates use of this side as a control. Compensation by use of

additional animals to provide appropriate controls is necessary.

The rat TMJ itself is quite small and introduction of a catheter through the

capsule likely damages nociceptive afferents. The extent to which this influences results

is only speculation but it appears not to have a major effect. Electromyographic studies

have demonstrated that enough afferents are lefi functionally intact to provoke jaw

reflexes despite the physical trauma from catheterization (Yu, 1996). In contrast,

artifactual darnage to afferents in experiments utilizing topical application of MO to skin

is not a major consideration.

Inherent to animal studies is the question of interspecies extrapolation,

specifically the similarities between rats and hurnans with respect to biochemical

mechanisms of neurogenic inflammation. Recent evidence exists that tempers Our

tendency to immediately apply findings in animals to humans. Petersen et al. (1 997)

using microdialysis techniques, measured in vivo the levels of SP and histamine

following capsaicin application to the human volar forearm. Unlike rat skin, SP nor

histamine was unable to be recovered afier intradermal or topical capsaicin application

to human skin. SP injection did however release histamine and result in a similar

inflammatory reaction. The authors suggest that SP is not the mediator of NI in the

human skin. Shmelz et al. (1 997) using a similar technique confirms these observations.

4.10 Future Direction For Investigation

Release of mediators fiom afferent nerve teminals despite conduction blockade

suggests that axonal conduction and terminal receptor potentials are not necessarily

sequentially activated in response to MO. Depletion of mediators within afferent

neuronal teminals should therefore eliminate the PE response to MO. This assumes that

MO indeed acts purely neurogenically. Even in the likely case that MO has both

neurogenic and non-neurogenic potential to cause PE, the proportion of neurogenic

influence will be evident.

Depletion of mediators ('desensitization') may be selectively accomplished with

the use of capsaicin (Jancsb, 1967). Capsaicin application in adult rats has been

demonstrated to selectiveiy deplete primary afferent nociceptive neurons of mediators

such as SP by causing an initial violent release fiom teminals (Jancso et al., 1980). A

future study could utilize capsaicin to desensitize nociceptors within the TMJ and then

response to MO could be examined. Surgical denervation of the trigeminal nerve by

cauterization is an alternative rneans to cause degeneration of afferent supply to the TMJ

(Jancso, 1967). The relative neurogenic and non-neurogenic components of MO activity

could then be assessed. The lack of mediators in nociceptive terminals would be

expected to result in a significantly diminished edema response evoked by MO if our

hypothesis in this study are correct.

The relative role of inhibition or facilitation by sympathetic efferents on acute

inflammation induced by MO in the TMJ would be a pnmary focus of future study in

light of conflicting reports in the literature. Ideally, a variety of methods of obtaining

syrnpathectomy and confirming the extent of success by various means would be of

value. Reserpine treatment for 3 days prior to experimentation (Lam and Ferrell, 1 99 1 a),

neonatal guanethidine treatment (Domerer et al., 199 l), and chronic 6-OHDA

treatments are possible means by which this may be accomplished.

Considering the evidence that a large sympathetic component appears to be

present in chronic adjuvant arthritis (Levine, 1986; Codeme, 1990) the present

experiments could be repeated with and without sympathectomy as outlined above, but

with the animal induced into a state of adjuvant induced polyarthritis. The outcome of an

acute inflammatory challenge in a chronically inflamed joint could be investigated in

this way, and the relative proportional increase in the role of SPGN assessed and

compared to current results.

It appears that the cellular and biochernical mechanisms of NI play

proportionately different roles in different tissues and to different stimuli. Histamine

appears to be a major mediator of NI in rat skin (Lembeck and Holzer, 1979) but not in

the respiratory tract (Lundberg and Saria, 1983). Characterization of the role of

histamine and mast cells in the neurogenic inflammatory response in the TMJ is of

interest. Application of HI antagonists pnor to MO application would reveal whether the

mechanism of NI in the TMJ was heavily dependent on the release of histamine. A

viable alternative is to utilize rats who have been genetically manipulated to be mast ceIl

deficient. This method has been used in mice by houe et al. (houe, 1997).

Investigation into central influences on established acute inflarnmatory processes

is of interest. Sluka et al. (Sluka et al., 1994a) were able to reduce swelling afier

induction of acute inflammation by deposition of the non-NMDA antagonist CNQX into

the spinal cord dorsal hom. The parallel experiment within the trigeminal system would

be deposition of CNQX into brainstern trigeminal subnucleus caudalis of the trigeminal

spinal tract. In the current study we have dernonstrated that connections to spinal

neurons are not absolutely necessary for induction of edema. It would be of value to

examine whether they may potentialiy play a significant role in ampliS.ing or

maintaining neurogenic inflammation.

Alternative means by which to elicit PE is another future consideration. It

appears each chemical algogen canies with it a particular capacity to act neurogenicall y.

Carrageenan, kaolin, and bradykinin are examples. What best approximates the

neurogenic inflammatory stimulatory effect seen in patients with RA or TMD is a matter

of conjecture, to be clarified in the future. Capsaicin acts through a specific 'VR1' or

(vamilioid) receptor which has been recently cloned (Szallasi and Blumberg, 1999).

The action of local anaesthetic on inhibiting capsaicin as opposed to MO would provide

valuable information on the phannacology of two of the most useful tools in

neuroscience research today.

4.1 1 Summary and Conclusions

4.11.1 Surnmary

Tissue expansion (edema) increases dose dependently with increasing MO

concentration. Significant edema development is evident with concentration of MO as

low as 1%.

Local anaesthetics lidocaine and bupivacaine when injected into the TMJ area of

the r2t alone do not induce tissue expansion in this mode1 despite evidence that they are

inherentl y vasodilating.

MO induced edema development at low (1%) and high (40%) concentrations in

the rat TMJ area is not inhibited by pre-administration of local anaesthetics lidocaine or

bupivacaine. Edema development is independent of axonal conduction in primary

afferent nociceptors since tissue expansion occurred immediately and continued for

more than 2 hours despite confirmation that neuronal blockade was complete for at least

30 minutes (bupivacaine).

4.1 1.2 Two Possible Conclusions

Two possible conclusions may be drawn from the observations summarized in

section 4.1 1.1. First, if MO tnily acts through predominantly neurogenic mechanisms,

this study suggests that trigeminal dorsal root reflexes are not necessary to induce MO

induced ederna since afferent inputs into the spinal tract of the trigeminal nucleus were

blocked by local anaesthetic. In the same sense, traditional 'axon reflexes' are not

necessary to induce or maintain edema. Traditional concepts of NI which regard

antidromic conduction of action potentials as fundamental elements should be appended

in light of observations gleaned from this study. Neurogenic inflammation should be

redefined as increased vascular permeability and plasma extravasation secondary to

antidromic stimulation of afferent nerves or direct stimulation of afferent terminals.

A second conclusion may be made if MO is actually acting purely non-

neurogenically through direct damage to vasculature resulting in vascular permeability.

This is contrary to the existing body of evidence gained fiom previous studies which

point to the specificity of action of MO on C-fibre afferents. There is evidence however

that the tissues which are innervated by afferents exert a modulatory effect on neuronal

peptide content. (McMahon and Gibson, 1987). It is conceivable that afferents

innervating the TMJ of the rat do not nonnally produce a significant amount of peptides

mediating neurogenic inflammation. That is, the neurogenic contribution to TMJ

inflammation is smali. Local anaesthetic blockade then would not be expected to change

the edema response to MO acting non-neurogenically.

A statistically insignificant yet unexpected reverse trend toward increased

inflammation was observed with both lidocaine and bupivacaine preload as compared to

saline preload. It may be entertained that this is due to blockade of sympathetic tone

which nonnally exerts an inhibitory efiect on PE through release of mediators such as

neurokinin Y and NE. There is ample evidence in the literature îhat direct and indirect

adrenergic receptor stimulation results in decreased PE in acute inflammation. Similarly,

we speculate that local anaesthesia may block tonic inhibition of primary afferent

depolarization from supraspinal levels or large afferent inhibitory inputs and therefore

result in increased PE.

4.1 1.3 Clinical Implications Of Results

I f MO acts neurogenically, these results suggest that acute inflammation

involving the TMJ region in the context of arthritis or TMD camies with it the potential

of being neurogenic. Release of mediators appears to occur despite axonal block

indicating that anti-inflammatory therapy should either physically induce deafferentation

of the TMJ, or directly antagonize pro-inflarnmatory mediators phar~nacologically.

Altematively, MO acts non-neurogenically, which suggests that fûrther research

into the differential effect on skin versus the TMJ is of prime importance.

C hapter 5

References

Agnew LRC, et al., editors. Dorland's Ilustrated Medical Dictionary. Philadelphia and

London: W.B Saunders Company, 1 965.

Alarcon GS. Epidemiology of rheumatoid arthritis. Rheum Dis Clin North Am 1995; 2 1 :

589-604.

Alstergren P, Appelgren A, Appelgren B, et al. Co-variation of neuropeptide Y,

calcitonin gene-related peptide, substance P and neurokinin A in joint fluid from patients

with temporomandibular joint arthritis. Archs Oral Bi01 1995; 40: 127- 135.

Andrews TM, Holton P. The substance P and adenosine triphosphate (ATP) contents of

sensory nerve on degeneration. J Physiol 19%; 143: 45-46.

National Institutes of Health Technology Assessment Conference. Management of

temporomandibular disorders. J Am Dent Assoc 1996; 1 27: 1 595-606.

Amett FC, Edworthy SM, Bloch DA, McShane DJ, Fnes JF, Cooper NS, et al. The

American Rheumatism Association 1987 revised criteria for the classification of

rheumatoid arthritis. Arthritis Rheum 1988; 3 1 : 3 15-24.

Bakke M, Hu JW, Sessle BJ. Morphine application to peripheral tissues modulates

nociceptive jaw reflex. Neuroreport 1998; 9: 33 15-9.

Barber RP, Vaughn JEt Slemmon JR, Salvaterra PM, Roberts E, Leman SE. The origin,

distribution and synaptic relationships of substance P axons in rat spinal cord. J Comp

Neurol 1979; 184: 33 1-5 1.

Barnes PJ, Brown MJ, Dollery CT, Fuller RW, Heavey DJ, ind PW. Histamine is

released from skin by substance P but does not act as the final vasodilator in the axon

reflex. Br J Phannacol 1986; 88: 74 1-5.

Basbaum AI, Fields HL. Endogenous pain control systerns: brainstem spinal pathways

and endorphin circuitry. Annu Rev Neurosci 1984; 7: 309-38.

Bayliss WM. On the origin fiom the spinal cord of vasodilator fibres of the hind limb,

and on the nature of these fibres. J Physiol 190 1 ; 26: 1 73-209.

Belmonte C, Giraldez F. Responses of cat comeal sensory receptors to mechanical

substances of sensory afferent units in the cat's comea. J Physiol 1 98 1 ; 43 7: 3 5 5-368.

Bibb CA, Atchison KA, Pullinger GP, Bittar GT. Jaw fùnction status in an elderiy

community sample. Community Dent Oral Epidemiol 1995; 23: 303-8.

Bicknell RJ. Optimizing release from peptide hormone secretory nerve terminals. J Exp

Biol 1988; 139: 5 1-65.

Bileviciute 1, Lundeberg T, Ekblom A, Theodorsson E. Substance P-, neurokinin A-,

calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity (-LI) in rat

knee joint synovial fluid dunng acute monoarthritis is not correlated with concentrations

of neuropeptide-LI in cerebrospinal fluid and plasma. Neuroscience Letters 1994; 167:

145- 148.

Branchaw JL, Hsu SF, Jackson MB. Membrane excitability and secretion fiom

peptidergic nerve terminais. Ce11 Mol Neurobiol 1998; 18: 45-63.

Broton JG, Hu JW, Sessle BJ. EfTects of ternporomandibular joint stimulation on

nociceptive and non-nociceptive neurons of the cat's trigeminal subnucleus caudalis

(medullary dorsal hom). J Neurophysiol 1988; 59: 1575-89.

Broton JG, Sessle BJ. Reflex excitation of masticatory muscles induced by algesic

chernicals applied to the ternporomandibular joint of the cat. Arch Oral Bi01 1988; 33:

74 1-7.

Brown JH, Mackey HK, Riggilo DA, Schwartz NL. Studies on the acute inflammatory

response. II. Influence of antihistarninics and catecholarnines on formaldehyde-induced

edema. J Pharmacol Exp Ther 1968; 160: 243-8.

Bruce AN. Vas-dilator axon-reflexes. QJ Exp Physiol 19 13; 6: 339-354.

CaIvin WH, Loeser JD, Howe JF. A neurophysiological theory for the pain mechanism

of tic douloureux. Pain 1977; 3: 147-54.

Cambridge H, Brain SD. Calcitonin gene-related peptide increases blood flow and

potentiates plasma protein extravasation in the rat knee joint. Br J Pharmacol 1992; 106:

746-50.

Carleson J, Alstergren P, Appelgren A, et al. Effects of adjuvant on neuropeptide-like

immunoreactivity in experimentally induced temporomandibular arthritis in rats. Archs

Oral Bi01 1996a; 4 1 : 705-7 12.

Carleson J, Alstergren P, Appelgren A, Appelgren B, Kopp S, Theodorsson E, et al. A

mode1 for experimental induction of acute temporomandibular joint inflammation in

rats: eRects of substance P(SP) on neuropeptide-like immunoreactivity. Life Sci 1996b;

59: 1 193-20 1.

Carleson J, Kogner P, Theodorsson E, et al. Effects of capsaicin in temporomandibular

joint arthritis in rats. Arch Oral Bi01 1997; 42: 869-876.

Casatti CA, Frigo L, Bauer JA. Origin of sensory and autonomic innervation of the rat

temporomandibular joint: a retrograde axonal tracing study with the fluorescent dye fast

blue. J Dent Res 1999; 78: 776-83.

Celander O, Folkow B. The nature and the distribution of afferent fibres provided with

the axon reflex arrangement. Acta Physiol Scand 1953; 29: 359-370.

Celiker R, Gokce-Kutsal Y, Eryilmaz M. Temporomandibular joint involvement in

rheumatoid arthntis. Relationship with disease activity. Scand J Rheumatol 1995; 24:

22-5.

Chahl LA. Antidromic vasodilatation and neurogenic inflammation. Pharmac Ther 1988;

37: 277-300.

Chahl LA. Ladd RJ. Local oedema and general excitation of cutaneous sensory receptors

produced by electrical stimulation of the saphenous nerve in the rat. Pain 1976; 2: 25-34.

Coderre. Epinephrine exacerbates arthritis by an action at presynaptic beta-2

adrenoceptors. Neuroscience 1990; 34: 52 1-523.

Coderre TJ, Chan AK, Helms C, Basbaurn AI, Levine JD. Increasing sympathetic nerve

terminal dependent plasma extravasation correlates with decreased arthritic joint injury

in rats. Neuroscience 199 1 ; 40: 185- 189.

De Kanter RJAM, Truin GJ, et al. Prevalence in the Dutch aduit population and a meta-

analysis of signs and syrnptoms of temporomandibular disorder. J Dent Res 1993; 72:

1509-1 8.

Delepine L, Aubineau P. Plasma protein extravasation induced in the rat dura mater by

stimulation of the parasympathetic sphenopalatine ganglion. Exp Neurol 1997; 147: 389-

400.

Deng Y, Fu M, Hagg U. Prevalence of temporomandibular joint dysfunction (TMJD) in

Chinese children and adolescents. A cross -sectional epiderniological study. Europ J

Orth0 1995; 17: 305-9.

Dimitroulis G, Dolwick FM, Gremillion H. Temporomandibular disorders. 1. Clinical

evaluation. Australian Dental Journal 1995; 40: 30 1-5.

Donnerer J, Amann R, Lembeck F. Neurogenic and non-neurogenic inflammation in the

rat paw following chernical sympathectomy. Neuroscience 199 1 ; 45: 76 1 -5.

Dray A. Pharmacology of peripheral afferent tmninals. In: Yaksh T, editor. Anesthesia:

Biologic Foundations. Philidelphia: Lippincott-Raven, 1 997: 543-556.

Du XJ, Riemersrna RA, Fox KA, Dart AM. Propranolol and lidocaine inhibit neural

norepinephrine release in hearts with increased extracellular potassium and ischemia.

Circulation 1 993; 88: 1 885-92.

Dux M, Jancso G, S a m H, Pierau FK. Inhibition of the neurogenic inflamrnatory

response by lidocaine in rat skjn. Inflarnm Res 1996; 45: 10- 13.

Dworkin SF, LeResche L. Research diagnostic criteria for temporomandibular disorders:

Review, cnteria, examinations and specifications, critique. J Craniornand Dis 1992; 6:

301.

Etherington J, Spector TD. Assymetrical nodular osteoarthritis in a patient with a

hemiparesis. Ann Rheum Dis 1995; 54: 936-7.

Ettala-Ylitalo UM, Syrjanen S, Halonen P. Functional disturbances of the masticatory

system related to temporomandibular joint involvernent by rheumatoid arthritis. J Oral

Rehabil 1987; 14: 41 5-27.

Euler USv, Gaddum JH. An unidentified depressor substance in certain tissue extracts. J

Physiol 193 1 ; 72: 74-87.

Feam HJ, Karady S, West GB. The role of the newous system in local inflammatory

responses. J Pharm Pharmac 1965; 17: 76 1-765.

Ferre11 WR, Russell NJ. Extravasation in the knee induced by antidromic stimulation of

articular C fibre affèrents of the anaesthetized cat. J Physiol (Lond) 1986; 379: 407- 16.

Fiorentino PM, Cairns BE, Hu JW. Development of inflammation after application of

MO or giutamate to the rat temporomandibular joint. Arch Oral Biol 1999; 44: 27-32.

Fitzgerald M, Woolf CJ, Gibson SJ, Mallabum PS. Alterations in the structure, function,

and chemistry of C fibers following local application of vinblastine to the sciatic nerve

of the rat. J Neurosci 1984; 4: 430-4 1.

Foreman JC, Jordan CC, Oehme P, Renner H. Structure-activity relationships for sonie

substance P-related peptides that cause wheal and flare reactions in human skin. J

Physiol (Lond) 1983; 335: 449-65.

Freund J. The effect of paraffin oil and mycobacteria on antibody formation and

sensitization. Am J Clin Pathol 195 1 ; 2 1 : 645-656.

Frommer J, Monroe CW. The morphology and distribution of nenre fibers and endings

associated with the mandibular joint of the mouse. J Dent Res 1966; 45: 1762-6.

Gamse R, Saria A. Potentiation of tachykinin induced plasma protein extravasation by

calcitonin gene related peptide. Eur J Pharmac 1985; 1 14: 6 1-66.

Gee NS, Brown JP, et al. The novel anticonvulsant drug, Gabapentin (Neurontin), binds

to the alpha-2-delta subunit of a calcium channel. J Bi01 Chem 1996; 27 1 : 5768-5776.

Glick EN. Assyrnetrical rheumatoid arthritis after poliomyelitis. Bnt Med J 1967; 3: 26-

29.

Goedert M, Stoeckel K, Otten U. Biological importance of the retrograde axonal

transport of nerve growth factor in sensory neurons. Proc Natl Acad Sci U S A 198 1 ; 78:

5895-8.

Goldberg RP, Zulrnan JI, Genant HK. Unilateral primary osteoarthritis of the hand in

monoplegia. Radiology 1980; 1 3 5: 65-66.

Gonzales R, Goldyne ME, Taiwo YO, Levine JD. Production of hyperalgesic

prostaglandins by sympathetic postganglionic neurons. J Neurochem 1989; 53: 1595-8.

Gonzales R, Sherboume CD, Goldyne ME, Levine JD. NE-induced prostaglandin

production by sympathetic postganglionic neurons is mediated by alpha 2-adrenergic

receptors. J Neurochem 199 1 ; 57: 1 145-50.

Gonzalez Burgos GR, Biali FI, Cherksey BD, Sugimori M, Llinas RIZ, Uchitel OD.

Different calcium channels mediate transmitter release evoked by transient or sustained

depolarization at mammalian sympathetic ganglia. Neuroscience 1995; 64: 1 17-23,

Goulet JP, Lavigne GJ, Lund JP. Jaw pain prevalence among French-speaking

Canadians in Quebec and related syrnptoms of temporomandibular disorders. J Dent Res

1995; 74: 1 738- 1744.

Gray TS, Morley JE. Neuropeptide Y: anatornical distribution and possible function in

mammalian nervous system. Life Sci 1986; 38: 389-40 1.

Green KL. The anti-inflamrnatory effect of catecholamines in the peritoneal cavity and

hind paw of the mouse. Br J Phannacol 1972; 45: 322-32.

Green KL. Role of endogenous catecholamines in the anti-inflammatory activity of

alpha-adrenoceptor blocking agents. Br .J Pharmacol 1974; 5 1 : 45-53.

Green PG, Luo J, Heller P, Levine JD. Modulation of bradykinin-induced plasma

extravasation in the rat knee joint b y sympathetic CO-transmitters. Neuroscience 1 993a;

52: 45 1458.

Green PG, Luo J, Heller PH, Levine JD. Further substantiation of a significant role for

the sympathetic nervous system in inflammation. Neuroscience 1993b; 55: 1037- 1043.

Green PG, Luo J, Heller PH, Levine JD. Neurogenic and non-neurogenic mechanisms of

plasma extravasation in the rat. Neuroscience 1993c; 52: 735-743.

Green TP, Johnson DE, Marchessault RP, Gatto CW. Transvascular flux and tissue

accrual of Evans blue: effects of endotoxin and histamine. J Lab Clin Med 1988; 1 1 1 :

173-83.

Greene CS. Ternporomandibular disorders in the geriatric population. J Prosthet Dent

1994; 72: 507-9.

Grosfeld O, Jackowska M, Czarnecka B. Results of epiderniological examinations of the

temporomandibular joint in adolescents and young adults. Journal of Oral Rehabilitation

1985; 12: 95-105.

Guinard JP, Carpenter RL, Morell RC. Effect of local anesthetic concentration on

capillary blood flow in human skin. Reg Anesth 1992; 17: 3 1 7-2 1.

Haas DA, Nakanishi O, MacMillan RE, Jordan RC, Hu JW. Development of an

orofacial mode1 of acute inflammation in the rat. Arch Oral Bi01 1992; 37: 4 17-22.

Hall GM, Spector TD, Griffin Al, Jawad AS, Hall ML, Doyle DV. The effect of

rheumatoid arthritis and steroid therapy on bone density in postmenopausal women.

Arthritis Rheum 1993; 36: 15 10-6.

Hamilton S. Unilateral rheumatoid arthritis in hemiplegia. J Can Assoc Radio1 1983; 34:

49-50.

Hammoudeh M, Khan MA, Kushner 1. Unilateral rheumatoid arthritis. Arthritis Rheum

1981; 24: 1218.

Harada M, Takeuchi M, Fukao T, Katagiri K. A simple method for the quantitative

extraction of dye extravasatecl into the skin. J P h m Pharmacol 197 1 ; 23: 2 18-9.

Harper AA, Lawson SN. Conduction velocity is related to morphological cell type in rat

dorsal root ganglion neurones. J Physiol (Lond) 1 985; 3 59: 3 1 -46.

Haskin CL, Athanasiou KA, Klebe R, et al. A heat-shock-like response with cytoskefetal

disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells. Biochem

Ce11 Bi01 1993; 7 1 : 36 1.

Hellauer HF, Umrah K. The trammitter substances of sensory nerves. J Physiol 1947;

106: 20-2 1 P.

Hemanz A, De Miguel E, Romera N, Perez-Ayala C, Gijon J, Amalich F. Calcitonin

gene-related peptide II, substance P and vasoactive intestinal peptide in plasma and

synovial fluid fiom patients with inflammatory joint disease. Br J Rheumatol 1993; 32:

31-5.

Heym C, Liu N, Gleich A, Oberst P, Kummer W. Immunohistochemical evidence for

different pathways immunoreactive to substance P and calcitonin gene-related peptide

(CGRP) in the guinea- pig stellate ganglion. Ce11 Tissue Res 1993; 272: 563-74.

Hiltunen K, Schmidt K, Nevalainen J, Narhi T, Ainamo A. Prevalence of signs of

temporomandibular disorders among elderly inhabitants of Helsinki, Finland. Acta

Odontol Scand 1995; 53: 20-3.

Hinsey JC, Gasser HS. The component of the dorsal root mediating vasodilatation and

the Shemngton contracture. Am J Physiol 1930: 679-689.

Hogan QH, Stadnicka A, Stekiel TA, Bosnjak ZJ, Kampine JP. Mechanism of

mesenteric venodilatation afier epidural lidocaine in rabbits. Anesthesiology 1994; 8 1 :

939-45.

Hokfelt T, Kellerth J - 0 , Nilsson G, Pemow B. Experimental imrnunohistochemica~

studies on the localization and distribution of substance P in cat primary sensory

neurons. Brain Res 1975a; 100: 235-252.

Hokfelt T, Kellerth J-0, Nilsson G, Pemow B. Substance P: Localization in the central

nervous systern and in some primary sensory neurons. Science 1975b; 190: 889-890.

Holmlund A, Gynther G, Reinholt FP. Rheumatoid arthritis and disk derangement of the

temporomandibular joint: A comparative arthroscopie study. Oral Surg Med Path 1992;

73: 273-7.

Houghton AK, Lu Y, Westlund KN. S-(+)-3-Isobutylgaba and its stereoisomer reduces

the amount of inflammation and hyperalgesia in an acute arthritis mode1 in the rat. J

Pham Exp Therap 1998; 285: 533-538.

Iannuzzi L, Dawson N, Zein N, Kushner 1. Does drug therapy slow radiographie

deterioration in rheumatoid arthritis? New England Journal of Medicine 1983; 309:

1023-8.

Ichikawa H, Wakisaka S, Matsuo S, Akai M. Peptidergic innervation of the

temporomandibular disk in the rat. Expenentia 1989; 45: 303-4.

Inoue H, Asaka T, Nagata N, Koshihara Y. Mechanism of mustard oil-induced skin

inflammation in mice. Eur J Pharmacol 1997; 333: 23 1-40.

Inoue H, Nagata N, Koshihara Y. Participation of serotonin in capsaicin-induced mouse

ear edema. Jpn J Pharmacol 1995; 69: 61 -8.

Jaffe RA, Rowe MA. Differential nerve block. Direct measurements on individual

myelinated and unmyelinated dorsal root axons. Anesthesiology 1996; 84: 1455-64.

Jakab G, Webster HD, Salamon 1, Mezey E. Neural and non-neural origin of calcitonin

gene-related peptide (CGRP) in the gastric mucosa. Neuropeptides 1993; 24: 1 17-22.

Jancso G, Kiraly E, Jancso-Gabor A. Pharmacologically induced selective degeneration

of chernosensitive primary sensory neurones. Nature 1977; 270: 74 1-3.

Jancso G, Kiraly E, Jancso-Gabor A. Direct evidence for an axonal site of action of

capsaicin. Naunyn Schmiedebergs Arch Phannacol 1980; 3 13 : 9 1-4.

Jancso N, Jancso-Gabor A, Szolcsanyi J. Direct evidence for neurogenic inflammation

and its prevention by denervation and by pretreatment with capsaicin. Br J Pharmacol

1967; 31: 138-51.

Jancso N, Jascso-Gabor A. The role of sensory nerve endings in neurogenic

inflammation induced in human skin and in the eye and paw of the rat. Br J Pharmac

1968; 32: 32-4 1.

Johansson AS, Isacsson G, Isberg A, Granhoim AC. Distribution of substance P-like

immunoreactive nerve fibers in temporomandibular joint soft tissues of monkey. Scand J

Dent Res 1986; 94: 225-32.

Johns RA. Local anesthetics inhibit endothelium-dependent vasodilation.

Anesthesiology 1989; 70: 805- 1 1.

Johns RA, DiFazio CA, Longnecker DE. Lidocaine constricts or dilates rat arterioles in a

dose-dependent manner. Anesthesiology 1985; 62: 14 1 -4.

Jones SW. Overview of voltage-dependent calcium channels. J Bioenerg Biomembr

1998; 30: 299-3 12.

Jonsson A, Mattsson U, Cassuto J, Heyden G. Quantification of burn induced

extravasation of Evans blue albumin based on digital image analysis. Comput Bi01 Med

1998; 28: 1 53-67.

Jorizzo JL, Coutts AA, Eady RAJ, Greaves MW. Vascular responses of human skin to

injection of substance P and mechanism of action. Eur J Pham 1983; 87: 67-76.

Joyner WL, Svensjo E, Arfors KE. Simultaneous measurements of macromolecular

leakage and arteriolar blood flow as altered by PGE 1 and beta 2-receptor stimulant in the

hamster cheek pouch. Microvasc Res 1979; 18: 30 1 - 1 O.

Kadar D. The Autacoids. In: Kalant H and Roschlau WHE, editors. Principles of medical

phmacology. Toronto: B.C. Decker, 1989: 3 1 1-3 16.

Keller JH, Moffett BC, Jr. Nerve endings in the temporomandibular joint of the Rhesus

Macaque. Anat Rec 1968; 160: 587-94.

Kido MA, Kiyoshima T, Ibuki T, Shimizu S, Kondo T, Terada Y, et al. A topographical

and ultrastructural study of sensory trigeminal nerve endings in the rat

temporomandibular joint as demonstrated by anterograde transport of wheat genn

agglutinin-horseradish peroxidase (WGA-HRP). J Dent Res 1995; 74: 1 353-9.

Kido MA, Kiyoshima T, Kondo T, Ayasaka N, Moroi R, Terada Y, et al. Distribution of

substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the

rat temporomandibular joint. J Dent Res 1993; 72: 592-8.

Kieman JA. Degranulation of mast cells following antidromic stimulation of cutaneous

nerves. J Anat 197 1 ; 1 1 1 : 349-350.

Kieman JA. The involvement of mast cells in vasodilatation due to axon reflexes in

injured skin. Q J Exp Physiol 1972; 57: 3 1 1-3 17.

Kimball ES, Perisco FJ, Vaught JL. Substance p, neurokinin A and neurokinin B induce

generation of IL- 1 activity in P388D 1 cells. J lmrnun 1988; 14 1 : 3564-3569.

Komorowski RC, Torneck CD, Hu JW. Neurogenic inflammation and tooth pulp

innervation pattern in sympathectomized rats. J Endod 1996; 22: 4 14-7.

Lam FY, Ferrel W. Mediators of substance P induced inflammation in the rat knee

join. Agents and Actions 1990; 3 1 : 298-307.

Lam FY, Ferrel WR. Acute inflammation in the rat knee joint attenuates sympathetic

vasoconstriction but enhances neuropeptide-mediated vasodilatation assessed be laser

doppler perfùsion imaging. Neuroscience 1993; 52: 443-449.

Lam FY, Ferrell WR. Neurogenic component of different models of acute inflammation

in the rat knee joint. Ann Rheum Dis 199 1 a; 50: 747-5 1.

Lam FY, Ferrell WR. Specific neurokinin receptors mediate plasma extravasation in the

rat knee joint. Br J Pharmacol 199 1 b; 103: 1263-7.

Langley .IN. The autonomie nervous systern. Cambridge: Cambridge University Press,

192 1.

Langley .IN, Anderson HK. On reflex action fiom sympathetic ganglia. J Physiol 1894;

16: 410-440.

Lawand NB, Lu Y, Westlund KN. Nicotinic cholinergic receptors: potential targets for

inflammatory pain relief. Pain 1999; 80: 29 1 -9.

Leah JD, Carneron AA, Kelly WL, Snow PJ. Coexistence of peptide irnmunoreactivity

in sensory neurons of the cat. Neuroscience 1985a; 16: 683-90.

Leah JD, Carneron AA, Snow P.J. Neuropeptides in physiologically identified

marnmalian sensory neurones. Neurosci Lett 1 985b; 56: 257-63.

Leigh JP, Fries JF. Correlations between education and arthritis in the 197 1 - 1975

NHANES 1. Soc Sci Med 1994; 38: 575-83.

Lembeck F. Zur Frage der zentralen Ubertragung afferenter Impulse. III. Mitteilung. Das

Vorkommen und die Bedeutung der Spubstanz P in den dorsalen Wurzeln des

Ruckenmarks. Naunyn-Shmiedeberg's Arch Exp Pathol Pharmak 1 953; 2 19: 197-2 1 3.

Lembeck F, Gamse R. Substance P in penpheral sensory processes. In: Porter R and

O'Connor M, editors. Substance P in the Nervous System, Ciba Foundation symposium

9 1. London: Pitman, 1982: 35-54.

Lembeck F, Gamse R, Juan H. Substance P. In: Von Euler US and Pernow B, editors.

Substance P and sensory nerve endings. New York: Raven Press, 1977: 169- 1 8 1.

Lembeck F, Holzer P. Substance P as neurogenic mediator of antidromic vasodilation

and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 1979;

310: 175-83.

LeResche L, Dworkin SF, Somrners EE, Tnielove EL. An epiderniologic evaluation of

two diagnostic classification schemes for ternporomandibular disorders. J Prosthet Dent

1991; 65: 13 1-7.

Levine JD. The contribution of neurogenic inflammation in experimental arthritis.

Journal of Immunology 1985; 135: 843-7.

Levine JD, Coderre TJ, Covinsky K, Basbaum AI. Neural influences on synovial mast

ce11 density in rat. J Neurosci Res 1990; 26: 30 1-7.

Levine JD, Collier DH, Basbaum AI, Moskowitz MA, Helms CA. Hypothesis: the

nervous system may contribute to the pathophysiology of rheumatoid arthritis. J

Rheumatol 1985a; 12: 406-1 1.

Levine JD, Dardick SJ, Basbaum AI, Scipio E. Reflex neurogenic inflammation. 1.

Contribution of the penpheral nervous system to spatially remote inflammatory

responses that follow injury. J Neurosci 1985b; 5: 1380-6.

Levine JD, Dardick SJ, Roizen MF, Helms C, Basbaum AI. Contribution of sensory

afferents and sympathetic efferents to joint injury in experimental arthritis. J Neurosci

1986; 6: 3423-9.

Lewis T. The blood vessels of the human skin and their responses. London: Shaw and

Sons, 1927.

Linsey SJW. The Development of Peripheral Inflammation. Pain Forum 1995; 4: 153-

154.

Lippe IT, Stabentheiner A, Holzer P. Participation of nitric oxide in the mustard oi1-

induced neurogenic inflammation of the rat paw skin. Eur J Pharmacol 1993a; 232: 1 13-

20.

Lippe IT, Stabentheiner A, Holzer P. Role of nitric oxide in the vasodilator but not

exudative component of mustard oil-induced inflammation in rat skin. Agents Actions

1993b; 38 Spec No: C S 2 4

Locker D, Slade G. Prevalence of symptoms associated with temporomandibular

disorders in a Canadian population. Cornmunity Dent Oral Epidemiol 1988; 16: 3 10-3.

Losavio A, Muchnik S. Spontaneous acetylcholine release in mammalian neuromuscular

junctions. Am J Physiol 1997; 273: C 1835-4 1.

Louis SM, Jarnieson A, Russell NJW, Dockray GJ. The role of substance P and

calcitonin gene related peptide in neurogenic plasma extravasation and vasodilatation in

the rat. Neuroscience 1989; 32: 58 1-586.

Lundberg JM, Saria A. Capsaicin-induced desensitization of airway mucosa to cigarette

smoke, mechanical, and chemical irritants. Nature 1983; 307: 25 1-253.

Lundberg JM, Terenius L, Hokfelt T, Martling CR, Taternoto Ky Mutt V, et al.

Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and

effects of NPY on sympathetic function. Acta Physiol Scand 1982; 1 16: 477-80.

Lynn B, Shakhanbeh J. Substance P content of the skin, neurogenic inflammation and

numbers of C-fibres following capsaicin application to a cutaneous nerve in the rabbit.

Neuroscience 1988; 24: 769-75.

Maggio JE, Hunter JC. Regional distribution of kassinin-like immunoreactivity in rat

central and peripheral tissues and the effect of capsaicin. Brain Res 1984; 307: 370-373.

Majno Gy Palade GE, Shoefl GI. Studies on inflammation: II. The site of action of

histamine and serotonin along the vascular tree: A topographical study. J Biophys

Biochem Cytol 196 1 ; 1 1 : 607-625.

Majno G, Shea SM, Leventhal M. Endothelial contraction induced by histamine-type

mediators. An elecîron microscopie study. J Ce11 Bio 1969: 647-670.

Malamed SF. Clinical Action of Specific Agents. in: Malamed SF, editor. Handbook of

Local Anesthesia. St. Louis: Mosby-Year Book, Inc., 1990a: 46-76.

Malamed SF. NeurophysioIogy. In: Malamed SF, editor. Handbook of Local

Anaesthesia. St. Louis: Mosby-Year Book, Inc., 1990b.

Mapp PI, Kidd BL, Gibson SJ, Terry JM, Revel1 PA, Ibrahim NB, et al. Substance P-,

calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y-

immunoreactive fibres are present in normal synovium but depleted in patients with

rheumatoid arthritis. Ncuroscience 1 990; 37: 1 43-53.

Mapp PI, Terenghi G, Walsh DA. Monoarthntis in the rat knee induces bilateral and

time dependent changes in substance P and calcitonin gene-related peptide

immunoreactivity in the spinal cord. Neuroscience 1 993; 57: 1 09 1 - 1 096.

Marshall KW, Chiu B, Inman RD. Substance P and arthritis: analysis of plasma and

synovial fluid levels. Arthritis Rheum 1990; 33: 87-90.

Martin M, Resch K. Interleukin 1 : more than a mediator between leukocytes. TIPS 1988;

9: 171-177.

Martinsson T, Haegerstrand A, Dalsgaard CJ. Effects of ropivacaine on eicosanoid

release from human granulocytes and endothelial cells in vitro. Inflamm Res 1997a; 46:

398-403.

Martinsson TT Oda T, Femvik E, Roempke K, Dalsgaard CJ, Svensjo E. Ropivacaine

inhibits leukocyte rolling, adhesion and CD 1 1 b/CD 1 8 expression. J P h m a c o l Exp Ther

1997b; 283: 59-65.

Mayers 1, Johnson D. The nonspecific inflammatory response to injury. C m J Anaesth

1998; 45: 871-9.

McBumey RN, Kehl SJ. Electrophysiology of neurosecretory cells fiorn the pituitary

intermediate lobe. J Exp Bi01 1988; 139: 3 17-28.

McKay DC, Christensen LV. Whiplash injuries of the temporomandibular joint in motor

vehicle accidents: speculations and facts. J Oral Rehabil 1998; 25: 73 1-46.

McKinney GR, Lish PM. Interaction of beta adrenergic blockade and certain

vasodilators in dextran-induced rat paw edema. Proc Soc EXD Si01 Med 1966; 12 1 : 494-

6.

McMahon SB, Gibson S. Peptide expression is altered when afferent nerves reinnervate

inappropnate tissue. Neurosci Lett 1987; 73: 9- 15.

McMahon SB, Lewin GR, Anand P, Ghatei MA, Bloom SR. Quantitative analysis o f

peptide levels and neurogenic extravasation following regeneration of afferents to

appropriate and inappropriate targets. Neuroscience 1989; 33: 67-73.

McMahon SB, Moore CE. Plasticity of primary afferent acid phosphatase expression

following rerouting of afferents fiom muscle to skin in the adult rat. J Comp Neurol

1988; 274: 1-8.

McMahon SB, Sykova E, Wall PD, Woolf CJ, Gibson SJ. Neurogenic extravasation and

substance P levels are low in muscle as compared to skin the rat hindlimb. Neurosci Lett

1984; 52: 235-40.

Melzack R, Wall PD. Pain mechanisms: a new theory. Science 1965; 150: 97 1-9.

Meyer R, Cambell JN, Raja SN. Peripheral neural mechanisms of nociception. The book

of pain.

Midroni R. The effect of acetylsalicylic acid and acetaminophen on edema,

adrenocorticotropin, and beta-endorphin during orofacial inflammation. Department of

Anaestnesia. Toronto: University of Toronto, 1996: 165.

Milam SB, Schmitz JP. Molecular biology of ternporomandibular joint disorders:

Proposed mechanisms of disease. J Oral Maxillofac Surg 1995; 53: 1448- 1454.

Mitchell DM, Fnes JF. An analysis of the American Rheumatism Association criteria for

rheumatoid arthritis. Arthritis Rheum 1982a; 25: 48 1.

Mitchell DM, Fnes JF. An analysis of the Arnerican Rheumatism Association criteria for

rheumatoid arthritis. Arthritis Rheum 1982b; 25: 48 1-7.

Mitchell WS, Cape11 HA. Unexplained acute arthntis in herniplegia. British Medical

Journal 1982; 284: 86.

Nakata T, Berard W, Kogosov E, Alexander N. Microdialysis in the posterior

hypothalamus: sodium chlonde affects norepinephrine release, mean arterial pressure,

heart rate and behavior in awake rats. Brain Res Bull 1990; 25: 593-8.

Newbold P, Brain SD. An investigation into the mechanism of capsaicin-induced

oedema in rabbit skin. Br J Pharmacol 1995; 1 14: 570-7.

Nilsson G, Larsson L-1, et al. Localization of substance P-like imrnunoreactivity in the

mouse gut. Histochemistry 1975; 43: 97-99.

Odeh M. Short analytical review: New insights into the pathogenesis and treatment of

rheumatoid arthntis. Clin Immun01 and Irnmunopath 1997; 83 : 103- 1 16.

Oppenheim JJ, Kovaks EJ, et al. There is more than one IL- 1. immun Today 1986; 7:

45-46.

Pace-Asciak CR. The Eicosanoids. In: Kalant H and Roschlau WHE, editors. Principles

of medical pharmacology. Toronto: B.C. Decker Inc., 1989: 3 10-3 16.

Parker M. A dynamic mode1 of etiology in temporomandibular disorders. JADA 1990;

120: 283-290.

Pateromichelakis S. Effects of mepivacaine, prilocaine and lidocaine on the induced

constriction of the maxillofacial vasculature of the rat. Acta Anaesthesiol Scand 199 1 ;

35: 60-4.

Peck SL, Johnston RB, Jr., Horwitz LD. Reduced neutrophil superoxide anion release

after prolonged infusions of lidocaine. J Pharmacol Exp Ther 1985; 235: 41 8-22.

Petersen LJ, Winge K, Brodin E, Skov PS. No reiease of histamine and substance P in

capsaicin-induced neurogenic inflammation in intact human skin in vivo: a microdialysis

study. Clin Exp Allergy 1997; 27: 957-65.

Pincus T. The paradox of effective therapies but poor long-tem outcomes in rheumatoid

arthritis. Serninars in Arthritis & Rheumatism 1992; 2 1 : 2- 15.

Pincus T, Brooks RH, Callahan LF. Prediction of long-term mortality in patients with

rheumatoid arthritis according to simple questionnaire and joint count measures. Annals

of Intemal Medicine 1994; 120: 26-34.

Pincus T, Callahan LF. The 'side effects' of rheumatoid arthritis: joint destruction,

disability and early mortality. British Journal of Rheumatology 1993; 32: 28-37.

Popitz-Bergez FA, Leeson S, Strichartz GR, Thalhammer JG. Relation between

functional deficit and intraneural local anesthetic during peripheral nerve block. A study

in the rat sciatic nerve. Anesthesiology 1995; 83: 583-92.

Powell D, Leeman S, et al. Radioimmunoassay for substance P. Nature New Bi01 1973;

24 1 : 252-254.

Ramsdell SG. The use of trypan blue to demonstrate the immediate skin reaction in

rabbits and guinea pigs. J Immun01 1 928; 1 5: 305.

Reeh PW, Kocher L, Jung S. Does neurogenic inflammation alter the sensitivity of

unmyelinated nociceptors in the rat? Brain Res 1986; 384: 42-50.

Rees H, Sluka K, Westlund K, Willis WD. The role of glutamate and GABA receptors in

the generation of dorsal root reflexes by acute arthritis in the anaesthetized rat. J Physiol

1995; 484: 437-445.

Roubenoff R, Roubenoff RA, Ward LM, Stevens MB. Catabolic effects of hi&-dose

corticosteroids persist despite therapeutic benefit in rheumatoid arthritis. Am J Clin Nutr

1990; 52: 1 1 13-7.

Rugh JD, Solberg W. Psychological implications in temporomandibular pain and

dysfunction. Oral Sciences Rev 1976; 7: 3-30.

Rugh JD, Solberg W. Oral health status in the United States: Temporomandibular

Disorders. J Dent Edu 1988; 49: 398-404.

Saria A, Lundberg JM. Evans blue fluorescence: quantitative and morphological

evaluation of vascular perrneability in animal tissues. J Neurosci Methods 1983; 8: 4 1-9.

Saria A, Lundberg JM, Skofitsch G, Lernbeck F. Vascular protein leakage in various

tissues induced by substance P, capsaicin, bradykinin, serotonin, histamine, and by

antigen challenge. Naunyn-Schmiedberg's Arch Phannacol 1983; 324: 2 12-2 18.

Sasagawa S. hhibitory effects of local anesthetics on migration, extracellular release of

lysosomal enzyme, and superoxide anion production in human polymorphonuclear

leukocytes. Immunophamacol Imrnunotoxicol 1 99 1 ; 1 3 : 607-22.

Schmelz M, Luz O, Averbeck B, Bickel A. Plasma extravasation and neuropeptide

release in human skin as measured by intradermal microdialysis. Neurosci Lett 1997;

230: 1 17-20.

Schrnid F. On the nerve distribution of the temporomandibular joint capsule. Oral Surg

Oral Med Orat Pathol 1969; 28: 63-5.

Schmidt RF. Presynaptic inhibition in the vertebrate central nervous system. Ergeb

Physiol 197 1 ; 63: 20- 10 1.

Schmidt W, Schmidt H, Bauer H, Gebhard MM, Martin E. Influence of lidocaine on

endotoxin-induced leukocyte-endothelia1 ce11 adhesion and macromolecular leakage in

vivo. Anesthesiology 1997; 87: 6 1 7-24.

Scott DL, Syrnmons DP, Coulton BL, Popert AJ. Long-tem outcome of treating

rheumatoid arthritis: results afier 20 years. Lancet 1987; 1 : 1 108- 1 1 .

Scott DT, Lam F, Ferre11 WR. Acute joint inflammation - mechanisms and mediators.

Gen Pharmac 1994; 25: 1285-1 296.

Sessle BJ. Neural Basis of Oral and Facial Function. Encyclopedia of Human Biology.

Vol 6: Academic Press, 1997: 135- 146.

Shiau Y, Chang C. An epidemiological study of temporomandibular disorders in

university students of Taiwan. Community Dent Oral Epidemol 199 1 ; 20: 43-7.

Sluka KA, Bailey K, et al. Treatrnent with either hi& or low fiequent TENS reduces the

secondary hyperalgesia observed afier injection of kaolin and carrageenan into the knee

joint. Pain 1998; 77: 97-1 02.

Sluka KA, Jordan HH, Westlund KN. Reduction in joint swelling and hyperalgesia

following post-treatment with a non-NMDA glutamate receptor antagonist. Pain 1994a;

59: 95- 100.

Sluka KA, Lawand NB, Westlund KN. Joint inflammation is reduced by dorsal

rhizotomy and not by sympathectomy or spinal cord transection. Ann Rheum Dis 1994b;

53: 309-314.

Sluka KA. Willis WD, Westlund KN. The role of dorsal root reflexes in neurogenic

inflammation. Pain Forum 1995; 4: 14 1 - 149.

Spears R, Hutchins B, Hinton RJ. Capsaicin application to the temporomandibular joint

alters calcitonin gene-related peptide levels in the trigeminal ganglion of the rat. J

Orofac Pain 1998; 12: 108-15.

Stelmer TJ, Welsh CH, Berger E, McCullough RG, Moms K, Repine JE, et al.

Antiarrhythmic agents diminish thiowea-induced pulmonary vascular protein leak in

rats. J Appl Physiol 1987; 63: 1877-83.

Slricker S. Untersuchung uber die Gafasserwurzeln des Ischiadicus. Sitz Kaiser1 Akad

Wiss (Wein) 1 876; 3: 1 73.

Suzuki M, Asako H, Kubes P, Jemings S, Grisharn MB, Granger DN. Neutrophil-

derived oxidants promote leukocyte adherence in postcapillary venules. Microvasc Res

199 1 ; 42: 125-38.

Suzuki T, Kagoshima M, Shibata M, Inaba N, Onodera S, Yamaura T, et al. Effects of

several denervation procedures on distribution of calcitonin gene-related peptide and

substance P immunoreactive in rat stomach. Dig Dis Sci 1997; 42: 1242-54.

Svensjo E, Persson CG, Rutili G. Inhibition of bradykinin induced macromolecular

leakage fiom post- capillary venules by a beta2-adrenoreceptor stimulant, terbutaline.

Acta Physiol Scand 1977; 10 1 : 504-6.

Syrjanen SM. The ternporomandibular joint in rheumatoid arthritis. Acta Radio1 [Diagn]

(Stockh) 1985; 26: 235-43.

Szallasi A, Blumberg PM. Vanilloid (Capsaicin) receptors and mechanisms. Phannacol

Rev 1999; 5 1 : 159-2 12.

Szolcsanyi J. Antidromic vasodilatation and neurogenic inflammation. Agents Actions

1988; 23: 4-1 1.

Takahashi T, Otsuka M. Regional distribution of substance P in the spinal cord and

nerve roots of the cat and the effect of dorsal root section. Brain Res 1975; 87: 1 - 1 1 .

Takao Y, Mikawa K, Nishina K, Maekawa N, Obara H. Lidocaine attenuates hyperoxic

lung injury in rabbits. Acta Anaesthesiol Scand 1996; 40: 3 18-25.

Tanelian DL, Beuerman RW. Responses of rabbit corneal nociceptors to mechanical and

thermal nociception. Expenmental Neurology 1984; 84: 165- 178.

Tegelberg A. Temporomandibular joint involvement in rheumatoid arthritis. A clinical

study. Swed Dent J Suppl 1987; 49: 1 - 133.

Thompson M. Unilateral rheumatoid arthntis following hemiplegia. Ann Rheum Dis

1962; 2 1 : 370-377.

Tranzer JP. Fine structural aspects of the effect of 6-hydroxydopamine on peripheral

adrenergic nerves. in: Malmfors T and Thoenen H, editors. 6-Hydroxydopamine and

Catecholarnine Neurons. Amsterdam: North-Hoiland Publishing Co., 197 1 : 1 5-3 1.

Uddman R, Grunditz T, Kato J. Sundler F. Distribution and ongin of nerve fibers in the

rat temporomandibular joint capsule. Anat Embryof (Berl) 1998; 197: 273-82.

Ueno Y, Kenzo S, Imura K. Protective effect of neural lesion on rheumatoid arthritis.

Arthritis Rheum 1983; 26: 1 18.

Velayos EE, Cohen BS. n i e effect of stroke on well-established rheumatoid arthntis.

Md State Med J 1970; 19: 111-115.

Wanman. Longitudinal course of symptoms of craniomandibular disorders in men and

women. Acta Odontol Scand 1996; 54: 337-342.

Widenfalk B, Wiberg M. Origin of sympathetic and sensory innervation of the

temporomandibular joint. A retrograde axonal tracing study in the rat. Neurosci Lett

1990; 109: 30-5.

Willis AL. Release of histamine, kinin, and prostaglandins during carrageenan-induced

inflammation in the rat. In: Montegazza P and Horton EW, editors. Prostaglandins,

Peptides, and Amines. New York: Academic Press, 1969: 3 1-38.

Woolf CJ. Outside in or inside out: How and in what direction does C-fibre activity

influence pain and inflammation? Pain Forum 1995; 4: 150- 152.

Yoshida H, Fujita S, Nishida M, Iizuka T. The expression of substance P in human

temporomandibular joint sarnples: an immunohistochemical study [In Process Citation].

J Oral Rehabil 1999; 26: 338-44.

Yu XM, Sessle BJ, Vemon Fi, Hu JW. Effects of inflammatory imtant application to the

rat temporomandibular joint on jaw and neck muscle activity. Pain 1995; 60: 143-9.

Yu X-M, Sessle BJ, Haas DA, Izzo A, Vemon H, Hu JW. Involvement of NMDA

receptor mechanisms in jaw electromyographic activity and plasma extravasation

induced by inflamrnatory irritant application to temporomandibular joint region of rats.

Pain 1996; 68: 169-1 78.

Zorzin L, Sorgi ML, Palombi G. Unilateral Heberden's nodes in a case of Erb-Dechenne

paralysis. Ann Rheum Dis 1996; 55: 857-8.