Sex Differences in Allodynia:

A Complex Interaction Between 17beta-Oestradiol and the Innate Immune System

Lauren Louise Nicotra

Discipline of Pharmacology School of Medical Sciences University of Adelaide

A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Adelaide, South Australia

January 2015

Lauren Nicotra Sex Differences in Allodynia 1

TABLE OF CONTENTS ABSTRACT 4 ACKNOWLEDGEMENTS 7 LIST OF FIGURES 10 LIST OF TABLES 12 ABBREVIATIONS 13 CHAPTER 1. LITERATURE REVIEW AND AIMS 16 1.1 PAIN VS. NOCICEPTION 16 1.2 THE SEX ISSUE: THE FEMALE PREVALENCE OF CHRONIC PAIN 18 1.2.1 SEX DIFFERENCES IN CHRONIC PAIN CONDITIONS 20 1.2.1.2 NEUROPATHIC PAIN 20 1.2.1.3 BACK PAIN 21 1.2.1.4 FIBROMYALGIA 22 1.2.1.5 HEADACHE AND MIGRAINE 23 1.2.2 SEX DIFFERENCES IN ANIMAL MODELS OF HYPERNOCICEPTION 24 1.2.2.1 ANIMAL MODELS OF PERSISTENT HYPERNOCICEPTION 25 1.2.3 CLINICAL SEX DIFFERENCES IN RESPONSES TO EXPERIMENTAL PAIN 26 1.2.4 SUMMARY 29 1.3 MECHANISMS UNDERLYING SEX DIFFERENCES IN PAIN 29 1.3.1 COGNITIVE-AFFECTIVE FACTORS 30 1.3.2 SEX ROLES 30 1.3.3 HORMONAL INFLUENCES 32 1.3.3.1 PHYSIOLOGY OF GONADAL STEROID HORMONES 33 1.3.3.1.2 OESTROGENS 34 1.3.3.1.2.1 OESTROGEN RECEPTORS 37 1.3.4 THE ROLE OF GONADAL SEX STEROID HORMONES IN CHRONIC PAIN: OESTROGEN AND EXACERBATED FEMALE PAIN 39 1.3.4.1 SEX DIFFERENCES IN THE PREVALENCE OF CHRONIC PAIN CONDITIONS: PREPUBESCENT VS. ADULT SUBJECTS 39 1.3.4.2 SEVERITY OF PAIN SYMPTOMS ACROSS THE MENSTRUAL CYCLE 40 1.3.4.3 EXOGENOUS E2 ADMINISTRATION 43 1.3.4.4 PAIN ACROSS THE OESTRUS CYCLE 45 1.3.4.5 GONADAL HORMONE MANIPULATION 47 1.3.4.6 SUMMARY 48 1.4 MODULATION OF PAIN BY OESTROGENS 49 1.4.1 SUMMARY 53 1.5 THE INNATE IMMUNE SYSTEM IN CHRONIC PAIN 54 1.5.1 THE ROLE OF GLIA IN CHRONIC PAIN 54 1.5.1.2 GLIAL CELL REACTIVITY 58 1.5.1.3 NEURON-TO-GLIA SIGNALS IN NEUROPATHIC PAIN 59 1.5.1.4 REACTIVE GLIA PRODUCE PATHOLOGICAL PAIN 61 1.5.2 TOLL LIKE RECEPTORS IN CHRONIC PAIN 63 1.5.3 SUMMARY 79 1.6 OESTROGEN AND THE INNATE IMMUNE SYSTEM: A ROLE IN THE FEMALE PREVALENCE OF CHRONIC PAIN 79 1.7 PHD SUMMARY AND AIMS 82 1.8 PHD SYNOPSIS 85

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CHAPTER 2. THE EXAMINATION OF SEX AND THE ROLE OF THE FEMALE OESTRUS CYCLE IN A CLINICALLY TRANSLATABLE PRECLINICAL CHRONIC PAIN MODEL 87 2.1 SEX DIFFERENCES IN MECHANICAL ALLODYNIA: HOW CAN IT BE PRECLINICALLY QUANTIFIED AND ANALYSED? 91 CHAPTER 3. THE CRITICAL ASSOCIATION BETWEEN E2 AND THE NEUROIMMUNE SYSTEM IN CHRONIC PAIN 107 3.1 ESTROGEN-PRIMED MICROGLIA ARE CENTRAL TO SEX DIFFERENCES IN ALLODYNIA 112 CHAPTER 4. THE NEUROIMMUNE SWITCH: THE CREATION OF DIVERGENT PAIN PROCESSING IN THE ABSENCE OF TOLL LIKE RECEPTORS 167 4.1 THE NEUROIMMUNE SWITCH: THE CREATION OF DIVERGENT PAIN PROCESSING IN THE ABSENCE OF TLRS 171 CHAPTER 5. SUMMARY AND DISCUSSION OF PHD FINDINGS 217 BIBLIOGRAPHY 228

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ABSTRACT

Chronic pain is a debilitating and costly disease that is well known to preferentially affect women, particularly in their child bearing years. In spite of the obvious differences in pathophysiology between the sexes, the underlying mechanisms causing this sex difference in chronic pain have not yet been determined. Contributing to this gap in knowledge is the unrelenting use of preclinical animal pain models along with pain behavioural assessment techniques, which fail to best replicate the clinical pain experience. This translatability issue, along with the continued preferential use of male subjects in preclinical chronic pain investigations has meant that researchers have not yet uncovered what contributes to this sex difference in pain sensitivity, resulting in half the population (females) being inadequately treated for their pain.

Among the numerous mechanisms that have been proposed to underlie this sex difference in chronic pain are both the gonadal hormones, particularly 17β-oestradiol, along with the innate immune system. Although seperately known to play a role in chronic pain, prior to this PhD project, the relationship between these two systems had not be investigated in this debilitating condition. Recent evidence however has revealed not only the presence of oestrogen receptors on the key neuroimmune cells in pain responsive regions of the CNS, but also the exaggerated production of pain producing pro-inflammatory mediators in response to their activation.

Given therefore that an association between these two pain-producing systems does exist, this

PhD project investigated the relationship between 17β-oestradiol and the innate immue system in exaggerated female chronic pain. Furthermore, based on the significant and reported translatability issues relating to preclinical pain studies in light of the currently

Lauren Nicotra Sex Differences in Allodynia 4

available chronic pain experimental models and assessment techniques, this study further examined this relatinship for the first time using novel translatable preclinical methodologies.

Through a series of key in vitro and in vivo studies our findings not only present and validate a novel preclinical chronic pain model and behavioural assessment technique that better produces and examines a preclincal neuropathy, but our findings also substantiate previous work demonstrating a significant role of 17β-oestradiol in chronic pain. Importantly this PhD project reveals for the first time not only the capability of the oestrogens to directly bind to key innate immune signalling receptors involved in chronic pain, but that 17β-oestradiol priming of CNS innate immune cells via such direct binding is in part responsible for the exaggerated female pain phenotype.

Overall, the findings of this body of work highlight a fundamental difference in chronic pain pathophysiology between the sexes and further emphasize the importance of treating chronic pain in women during their reproductive years differently from prepubesent female, postmenopausal and male chronic pain sufferers.

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DECLARATION

I certify that this work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work will, in the future, be used in a submission for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint-award of this degree.

I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

……………………………………… Date:

Lauren Nicotra

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ACKNOWLEDGEMENTS

I would like to sincerely thank and express my heartfelt appreciation for the tireless efforts, hours and constant support my two supervisors, Professor Mark Hutchinson and Professor

Paul Rolan have given me throughout my time and journey as a postgraduate PhD student in the Pharmacology department. Not only did your enthusiasm for the project make every individual experimental day enjoyable, but your patience and constant reassurement provided me with every faith throughout my research journey, in turn resulting in the discovery of an exciting story that this thesis has become. As well as your time, the most important gift you have both given me is the ability to trust in myself and to know that although the journey may not be mapped, there is an exciting one to be discovered.

The research presented in this thesis would not be possible without the financial support of the Australian Postgraduate Award, along with the Faculty of Health Sciences Travel Awards that allowed me to present this exciting story on an international stage. Most importantly, I would also like to thank Professor Paul Rolan for his financial support, which made this research journey possible.

I would especially like to thank the following people for their contributions: Dr. Peter Grace for his endless hours in teaching me the Chronic Constriction Injury model and von Frey testing; Dr. Heilie Kwok for not only her support as a friend and fellow colleague throughout my research journey, but for the hours she provided me with in vivo experiments; Dr.

Jonathan Tuke for miraculously teaching me the statistical program that is R; Dr. Zahied

Johan and Dr. Danielle Glynn for teaching me the ovariectomy and oestrogen pellet insertion procedure; Gordon Crabb for all of his technical support; Vicky Staikopoulos for her

Lauren Nicotra Sex Differences in Allodynia 7

countless hours of help with immunohistochemistry, western blots, animal surgeries and essentially every kind of experiment performed in Mark Hutchinson’s lab- without you Vicky how would I have survived?!; Dr. Jacinta Johnson for her help throughout the western blot difficulties in the Pharmacology Department; my beautiful and selfless friend Dr. Nicole

Sumracki for not only being my constant support and rock throughout my Honours and PhD years but for the hours she has given me in listening to my experimental troubles and proof reading, as well as proof listening to my husky voice for all those seminars; Dr. Benjamin

Harvey for being the best desk neighbour one could ask for as well as for his time helping me with day to day PhD struggles and statistical analyses; Jake Gordon for not only being a wonderful and supportive friend and colleague but essentially for being one of the normal ones in that nerd office and Jacob Thomas for just generally putting up with my constant abuse and torture for the last four or so years. Nicole, Ben, Heilie, Jacob, Jacinta, James and

Jake, thank you all for making my time in the department so much fun. The Pharmacology postgraduate room became my home away from home because of you guys. I couldn’t love and appreciate you more!

To my friends outside of the PhD walls, thank you for your support and understanding over the past four years. Your encouragement that I could do this was not unnoticed and it will not be forgotten.

I would sincerely like to thank Dr. Deborah Rathjen and Dr. Sue O’Connor for giving me the most incredible opportunity of completing this PhD thesis whilst undertaking my new role in

Strategic Alliances at Bionomics. Without this opportunity, I would not be in the fortunate position I am in today. Thank you both further for your support and encouragement

Lauren Nicotra Sex Differences in Allodynia 8

throughout the past twelve months. Your patience and reassurement made completion not only possible but also enjoyable.

I am extremely thankful to my family for their financial and emotional support throughout my educational journey. Mum, you are and always have been my rock and my greatest advocate.

Dad, I couldn’t thank you more for teaching me the value of persistence, hard work, humility and love. Without understanding the value of these merits I wouldn’t be the person I am today and I would not have had the courage and drive to undertake and complete this PhD. To

James, I could not thank you more for being the best, most supportive and loving best friend and brother I could ask for. Thank you for accepting me for the person that I am and for loving me unconditionally.

Last, but not least, I would like to thank my fiancé Matt. For all of the hours that would equate to weeks, months and years of listening to me talk, complain and cry about chronic pain, oestrogen and microglia, I cannot thank you more. Thank you for putting up with the tears, the anger, the frustration and the mood swings. Thank you for faking the enthusiasm and the excitement whenever I would talk about the exciting new concept of oestrogenic microglial cell priming, something which you had completely no understanding of (and probably still don’t!) Thank you for all the proof reading, the proof listening and the help with statistical coding which again, you really had no concept of or desire to learn! Most of all thank you for being the most loving, supportive and selfless person I have ever encountered.

Without your constant encouragement and patience this would not have been made possible. I love you and I look forward to seeing where our journey takes us after all the Honours, PhD and Physicians Training years!

Lauren Nicotra Sex Differences in Allodynia 9

LIST OF FIGURES

Figure 1. Ascending modulation of pain...... 17

Figure 2. Average Annual Increase in the Number of Publications Investigating Sex

Differences in Chronic Pain Post 1980...... 19

Figure 3. A Review of the Global Population-Based Data for Migraine Prevalence...... 23

Figure 4. Hypothalamic-Pituitary-Gonadal Axis Production of Gonadotrophins and the

Regulation of Steroidogenesis...... 34

Figure 5. E2 Synthesis in the Ovary...... 35

Figure 6. Neuronal and Astrocytic E2 Synthesis in the Brain...... 36

Figure 7. Plasma Levels of Hormones Throughout a Woman’s Menstrual Cycle...... 41

Figure 8. Plasma Steroidal Hormone Concentrations Throughout the Rodent Oestrus Cycle

Compared to the Human Menstrual Cycle...... 46

Figure 9. The Tripartite Synapse...... 58

Figure 10. Glial Cell Activation in Chronic Pain...... 63

Figure 1.1 Toll-Like Receptor Activation in Astrocytes ...... 70

Figure 1.2 Toll-Like Receptor Activation in Dorsal Root Ganglion and Trigeminal Ganglion

Neurons ...... 71

Figure 2.1 Inability of von Frey Method 1 to Replicate Healthy Human Pain Thresholds ..... 95

Figure 2.2 Von Frey Test 3 Demonstrates the Ability to Investigate Chronic Pain Mechanisms

Without Inflicting Maximal Pain ...... 99

Figure 2.3 von Frey Test 3: A Behavioural Technique Refining the Investigation of the Role of Sex and Rodent Oestrus Cycle in Mechanical Hypersensitivity ...... 100

Figure 2.4 Exacerbated Female Pain is Influenced by the Rodent Oestrus Cycle ...... 101

Figure 3.1a. Sex differences in chronic pain: Greater mechanical allodynia in females following nerve injury...... 129

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Figure 3.1b. Exacerbated female mechanical allodynia is associated with 17β-estradiol. ... 130

Figure 3.2. Exacerbated female rodent mechanical allodynia is mediated by 17β-estradiol primed microglial cells...... 134

Figure 3.3. Female microglia are inherently primed to subsequent pain signals...... 137

Figure 3.3c. Inherent Estrogenic microglial-priming is dependent upon MyD88, TLR2/4 signaling...... 138

Figure 3.4. Exposure of HEK-TLR2&4 cells to estrogens in vitro activates NFκB signaling, likely via actions at TLR2&4...... 141

Figure 3.5. 17β-Estradiol mediated NFκB activation in HEK-TLR4 cells is inhibited by TLR4 antagonists...... 143

Figure 3.6. Interaction Between 17β-estradiol and the Innate Immune System...... 145

Figure 4.1. Neuroimmune Independent Exaggerated Female Mechanical Allodynia...... 185

Figure 4.2. Neuroimmune independent pain is subject to gonadal hormone modulation...... 186

Figure 4.3. Balb/c Female mice are more sensitive to neuroimmune modulation...... 189

Figure 4.4. In the Genetic Absence of TLRs and their Adaptor Protein, Mechanical Allodynia is Pro-inflammatory Independent...... 190

Figure 4.5. In the Genetic Absence of TLRs and their Adaptor Protein, Mechanical Allodynia is Pro-inflammatory Independent...... 191

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LIST OF TABLES

Table 1. Toll-Like Receptor Activation in Astrocytes, Microglia and Dorsal Root Ganglion

(DRG) Neurons ...... 71

Table 2.1 Traditional Statistical Analysis at Independent von Frey Filaments for von Frey

Test 2 ...... 95

Table 2.2 Traditional Statistical Analysis at the Independent 2 g von Frey Filament ...... 96

Table 2.3 Traditional Statistical Analysis at the Independent 12 g von Frey Filament for vn

Frey Test 2 Revealed the Absence of an Oestrus Cycle Effect Prior to Nerve Injury (Pre-

Surgery) ...... 96

Table 2.4 Responses Generated from von Frey Test 2 were Further Used to Examine the

Estimate of the Slope of the Linear Relationship Between the Response and von Frey

Filament ...... 97

Table 2.5 Examining the Estimate of the Slope of the Linear Relationship Between the

Response and von Frey Filament for von Frey Test 2 ...... 98

Table 2.6 von Frey Test 3 Examined the Relationship Between the Response and the Force of the von Frey Filament, Across a Range of Filaments...... 98

Table 2.7 von Frey Test 3 Examined the Relationship Between the Response and the Force of the von Frey Filament, Across a Range of Filaments...... 99

Table 2.8. The Ability of von Frey Test 3 to Estimate the Difference in Allodynia for Diverse

Covariates Across Individual von Frey Filaments...... 102

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ABBREVIATIONS

ABBREVIATION DEFINITION 5-HT 5-Hydroxytrptamine, serotonin AIC Akaike’s Information Criterion AMPA Alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole-Propionic Acid AP1 Activating Protein 1 ATP Adenosine Triphosphate BDNF Brain-Derived Neurotrophic Factor CCI Chronic Constriction Injury CCL2 Chemokine Ligand 2 CD11b Cluster of Differentiation Molecule 11B CFA Complete Freund’s adjuvant CNS Central nervous system COX Cyclooxygenase CX3CL1 Fractalkine CX3CR1 CX3CL1 Receptor DAMPs Damage-Associated Molecular Patterns Di Dioestrus DRG Dorsal Root Ganglion E Oestrogen E1 Estrone E2 17β-Oestradiol E3 Estriol ER Oestrogen Receptor ERK Extracellular Signal-Regulated Kinase ERα Oestrogen Receptor Alpha ERβ Oestrogen Receptor Beta FSH Follicle-Stimulating Hormone g Grams Force GABA Gamma Aminobutyric Acid GFAP Glial Fibrillary Acidic Protein GLAST Glutamate–Aspartate Transporter GLT1 Glutamate Transporter 1 GnRH Gonadotropin Releasing Hormone GPR G Protein-Coupled Receptor HEK-293 Human Embryonic Kidney-293 HMGB1 High Mobility Group Box-1 HRT Hormone replacement therapy

HSPs Heat Shock Proteins

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i.t. Intrathecal IBS Irritable Bowel Syndrome IFN-β Interferon-Beta IL Interleukin IL-1ra IL-1 Receptor Antagonist i.p. Intraperitoneal JNK c-jun N-Terminal Kinase LAL Limulus Amebocyte Lysate LH Luteinizing Hormone LPS Lipopolysaccharide LTA Lipoteichoic Acid MAL MyD88 Adaptor Like MAPK Mitogen-Activated Protein Kinase MCP-1 Monocyte Chemotactic Protein-1 MD-2 Myeloid Differentiation Factor 2 Met Metoestrus MHC Major Histocompatibility Complex MIP-2 Macrophage Inflammatory Protein 2 MMPs M atrix Metalloproteinases MPQ McGill Pain Questionnaire MyD88 Myeloid Differentiation Primary Response Gene 88 N Sciatic Nerve NF-κB Nuclear Factor-κB NLRs NOD-Like Receptors NMDA N-methyl-D-aspartate NOD Nucleotide Binding Oligomerization Domain 1 NR Nuclear Receptor OC Oral Contraceptive Pill Oest Oestrus OV Ovulation OVX Ovariectomy P Progesterone PAG Periaqueductal Gray PAMPs Pathogen-Associated Molecular Patterns PBS Phosphate-Buffered Saline

PGE2 Prostaglandin E2 PGN Peptidoglycan PNS Peripheral Nervous System PO Postoperative POPNO Pain of Predominantly Neuropathic Origin Lauren Nicotra Sex Differences in Allodynia 14

Pro Pro-oestrus PSNL Partial Sciatic Nerve Ligation ROS Reactive Oxygen Species S Subcutaneous Suture SARM Sterile ∝ and Armadillo Motifs siRNA Small Interfering RNA T Testosterone TIR Toll Interleukin-1 Receptor TIR Toll/IL1 Receptor TIRAP TIR Domain Containing Adaptor Protein TLR Toll-Like Receptor TMD Temporomandibular TNF-α Tumor Necrosis Factor-α TRAM TRIF Related Adaptor Molecule TRIF Toll-Receptor-Associated Activator of Interferon TRPV1 Transient Receptor Potential Cation Channel Subfamily V Member 1

Lauren Nicotra Sex Differences in Allodynia 15

CHAPTER 1. LITERATURE REVIEW AND AIMS

1.1 Pain vs. Nociception

Although often incorrectly interchanged, nociception and pain are two distinct processes. A nociceptive stimulus results in a nocifensive response, whereby both the peripheral (PNS) and central (CNS) nervous systems process noxious information following nociceptor activation to the spinal cord, brainstem, thalamus, and subcortical structures1. Through the activation of ion channels, nociceptors are responsible for transducing energy from noxious signals into action potentials, which are conducted along the fibre’s axon to laminae I, IV and V in spinal cord dorsal horn neurons2. From here, the central terminals of nociceptors synapse onto nociceptive interneurons and second order neurons, carrying information regarding the intensity and duration of the message through the release of neurotransmitters and neuropeptides 3.

In contrast, pain is the result of supra-brain center processing1 (Figure 1). Axons of noxious information-carrying interneurons and second order neurons ultimately join ascending fibres4, where their projection to, and synapse with, cortical regions of the brain, results in the perception of pain5. Importantly, as well as integrating nociceptive information in the CNS generating the perception of pain, higher brain regions are also involved in the modulation of pain transmission, contributing to the magnitude of pain experienced. Supraspinally organised descending pathways adapt ascending nociceptive pathways in the spinal cord dorsal horn, consequently altering the point at which nociceptive information is integrated before being transmitted to higher brain centers in the CNS6 (For an overview of the basic physiology of pain please see Marchand and Colleagues7. Lauren Nicotra Sex Differences in Allodynia 16

Although generally perceived as bad, pain serves as an alert danger signal, a vital protective and adaptive warning of actual or potential tissue damage8. Unlike this necessary protective

CNS function of pain whose physiological underpinnings are well entrenched and understood, pain that serves no physiological function is defined as chronic pain and is still the subject of great exploration, with its complete pathophysiology still to be determined8, 9.

(a) (b) (c)

Figure 1. Ascending modulation of pain.

The main pathways include the spinothalamic tract (a), the spinoreticular tract (b) and the spinohypothalamic pathways. For a detailed neuroanatomical review, please see Almeida and

Tufik5. Figure acquired with permission5.

Lauren Nicotra Sex Differences in Allodynia 17

Nociception involves peripheral and central nervous system processing of peripheral information following nociceptor activation to the spinal cord, brainstem, thalamus, and subcortical structures. Through the transduction of energy from noxious signals into action potentials, nociceptors transmit this information to the spinal cord dorsal horn. Information that is ultimately sent to the cerebral cortex is however, perceived as pain. Figure used with permission from the National Research Council1.

1.2 The Sex Issue: The Female Prevalence of Chronic Pain

Contrasting the vital protective role of acute pain, chronic pain is maladaptive and dysfunctional, having no identifiable noxious stimulus or damage to the nervous system10.

Although most often hallmarked by both hyperalgesia; an exaggerated pain response to noxious stimuli, and allodynia; pain in response to normally innocuous stimuli, brought about by central sensitisation11, the underlying mechanisms contributing to the induction and maintenance of this pathological pain are far more complicated than those involved in acute pain (discussed Section 1.3), and are also less understood1.

One unresolved problem plaguing millions across the globe is the excess female burden of chronic pain. Epidemiological studies have revealed that although chronic pain is a extremely common affecting millions around the world, there is a higher prevalence in women12-15. In

2007, chronic pain was found to affect 20 % more Australian females than males, with 1.4 million male and 1.7 million female sufferers16. Prevalence rates have also been found much higher in females living in the United States, with a 2010 epidemiological report also evidencing a female predominance (females, 34.3%; males, 26.7%)17.

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Estimated to affect approximately 1.5 billion people worldwide, the global burden of this condition is far-reaching and expensive. Assessed at cost of 34.4 billion Australian dollars in

200716 and at 635 billion in the USA18, the economic impact of this female prevalent chronic disease condition is not to be dismissed. Consequently, although research investigating sex differences in pain has increased substantially over the years (Figure 2), the mechanisms underlying this debilitating and high-priced condition must be determined in order to find new and improved therapies to aid chronic pain sufferers and in turn, the global economy.

Figure 2. Average Annual Increase in the Number of Publications Investigating Sex

Differences in Chronic Pain Post 1980.

Percentages are calculated following literature searches using PubMed. For the year 2008, the

first 6 months of information was gathered and doubled in order to acquire an annualized estimate. Figure acquired from Hurley and colleagues with permission13.

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1.2.1 Sex Differences in Chronic Pain Conditions

The clinical research literature demonstrates that women report more severe pain, more recurrent pain and more enduring pain compared to men19. Importantly, this sex difference is not dependent upon female specific disorders or conditions, with a female prevalence among the majority of chronic pain conditions, including in the absence of female particular pathologies19. Epidemiological research indicates that women form a greater representation of neuropathic pain, back pain, temporomandibular disorder pain, headache, migraine and non- migranious headache, fibromyalgia and abdominal pain sufferers13, 20. In addition to the above-mentioned chronic pain disorders, a sex prevalence has been identified across many other various pain disorders (for a comprehensive list please see Berkley and colleagues21). A summary of the more common and disabling chronic pain conditions is provided below.

1.2.1.2 Neuropathic Pain

There exist few studies that have examined the prevalence of chronic pain with neuropathic qualities in general populations, mostly due to the absence of appropriate tools required to identify pain of neuropathic origin22. Despite this, those studies which have examined the prevalence of neuropathic pain, have reported chronic pain with neuropathic origin to be more prevalent in women13.

In a study by Torrance and colleagues23 utilizing a self-completed survey enabling the identification of pain that is dominated by neuropathic mechanisms (pain of predominantly neuropathic origin; POPNO), individuals with chronic POPNO were found more likely to be women taken from a sample of 2957 residents of the United Kingdom. Comparable findings were also demonstrated in a large sample of the French population22. In 2008 Bouhassira and

Lauren Nicotra Sex Differences in Allodynia 20

colleagues22 further revealed that chronic pain with neuropathic characteristics was female predominant, with 60.5 % of respondents representing women. Moreover, more recently, a telephonic survey determining the prevalence of chronic pain with neuropathic characteristics in the Moroccan general population further revealed similar findings, with neuropathic pain prevalence higher in the female sex24. Accordingly, although few studies have investigated the incidence of neuropathic pain, current epidemiologic literature establishes that women have a greater probability for developing neuropathic pain than men.

1.2.1.3 Back Pain

In developed countries, musculoskeletal conditions cause a great amount of disability and contribute a substantial financial burden to healthcare and social resources25. In Australia, there have been few reliable population studies conducted which have investigated back pain prevalence. In 2004, a study by Walker and colleagues26 however, investigated the prevalence and severity of lower back pain in the Australian adult population. This study demonstrated a female predominance of lower back pain, back pain persisting for 1 month, six months and also one year.

A number of European studies have also investigated the prevalence of back pain. In 2004, a

Swedish population study revealed that women reported back pain to a greater extent than males27. Moreover, in 2006 a German investigation identified a greater number of female back pain sufferers, with a female prevalence for back pain persisting for 7 days28. Similar findings have further been demonstrated in Spanish populations25 and throughout the United

Kingdom29 as well as in a non-Caucasian population in China30.

Lauren Nicotra Sex Differences in Allodynia 21

Despite these findings, investigators have also revealed a male prevalence in addition to the lack of a sex difference in back pain. One Nigerian study for example failed to find a sex difference in chronic back pain between men and women31. Furthermore, another study determined that the prevalence of low back pain was much higher among males (44.7%) compared to females (35.6%)32, although this finding was attributed to occupational factors.

Male subjects in this study were Nigerian farmers, opposed to the female participants who were not exposed or involved in laborious employment. Therefore, on balance, the literature clearly suggests that there is higher prevalence of back pain in women.

1.2.1.4 Fibromyalgia

Fibromyalgia, a chronic pain disorder of unknown cause characterised by widespread body pain, is typically diagnosed in women at a 3 to 6 fold rate higher than in men33. A number of studies have revealed not only significantly lower female pressure pain thresholds in fibromyalgia tender-point areas compared to males34, but also higher prevalence rates across all age groups in women compared to men when examining widespread body pain, across several geographic locations and hence cultural factors35-39. Further supporting this sex difference, self-report investigations have likewise revealed increased fibromyalgia prevalence rates among females25, 40, 41 along with direct clinical evidence, with the London

Fibromyalgia Study finding a female prevalence among Canadian adults42.

Despite the majority of studies indicating a higher prevalence of fibromyalgia and lower pressure pain tolerance in women, sex differences have not always been consistently found. In both 2002 and 2007, two studies did not identify a sex difference between males and females examining chronic widespread pain43, 44, although it must be noted that both studies did not

Lauren Nicotra Sex Differences in Allodynia 22

identify the race of their subjects. Notwithstanding these few investigational anomalies, the majority of studies have revealed a sex difference in the prevalence of fibromyalgia, with women being found to be at a much greater risk than men41, 42.

1.2.1.5 Headache and Migraine

Headache and migraine have been described as two of the most common and debilitating pain conditions experienced by both men and women13. Although common in both sexes, epidemiological research indicates that women have higher prevalence rates for both headache and migraine and experience enhanced headache severity, in addition to increased lifetime prevalence of headache compared to men45. In 2007, all of the existing evidence for the global prevalence of headache disorders was reviewed as part of the ‘Lifting The Burden:

The Global Campaign to Reduce the Burden of Headache Worldwide’ Initiative46. This meta analysis revealed a higher prevalence of migraine in women across the globe (Figure 3), with the same identified for headache.

Figure 3. A Review of the Global Population-Based Data for Migraine Prevalence.

Lauren Nicotra Sex Differences in Allodynia 23

Headache prevalence was found lowest in Africa and highest for Australia (not depicted).

Migraine was determined most prevalent in Europe and least prevalent in Africa (illustrated).

For both migraine and headache, a female predominance was established across the globe.

Figure acquired from Stovner and colleagues46 with permission.

1.2.2 Sex Differences in Animal Models of Hypernociception

Because using human subjects is fraught with challenges and are in their nature subjective, animal models have been widely used to examine sex differences in nociceptive sensitivity47.

Although animals cannot self report, their behaviours in response to noxious stimuli can be objectively examined47. The most commonly recorded behaviours are reflexes or instinctive responses to the stimuli such as licking of the afflicted area. Despite such behaviours reported as lacking clinical translatability, their use has significantly contributed to our current understanding of chronic pain pathophysiology48.

Through the use of animal hypernociception models it has become well accepted that female rodents display lower pain thresholds and tolerance for the majority of chronic pain modalities, in turn replicating the clinical pain scenario (detailed above, Section 1.2.1).

Methodological variations as well as the utilisation of diverse rodent populations19 has resulted in some variability. However, the general consensus is that the exaggerated female pain found using animal persistent pain models replicates the female prevalence of chronic pain as well the elevated pain sensitivity across the majority of chronic pain conditions.

Investigations examining sex differences across a diverse range of persistent animal pain models is presented below.

Lauren Nicotra Sex Differences in Allodynia 24

1.2.2.1 Animal Models of Persistent Hypernociception

Experimental pain models are generally subdivided into acute and persistent, with persistent models often inclusive of neuropathic injuries49. Most commonly, acute tests utilise a more severe thermal, mechanical or chemical insult, often measuring nociceptive threshold. In contrast, persistent models inflict long lasting chemical agents or injuries, examining ensuing behavior, or hyperalgesia and/or allodynia.

Sex differences in pain sensitivity have been thoroughly investigated in several persistent injury models, including neuropathic injuries. Unfortunately however, although described as the most clinically relevant, persistent pain models have presented the most inconsistent findings50, 51. Using the L5 nerve transection model for example, DeLeo and colleagues50 revealed variable results with greater female mechanical allodynia compared with males dependent on rodent strain. This strain effect has also been replicated by others using the rodent model of lumbar radiculopathy50 . Further contributing to this variability are the studies which have failed to reveal a sex effect52, 53, with others failing to demonstrate a difference in the amplitude of pain between the sexes, revealing a more persistent pain in females compared with males50, 54.

Amongst these discrepancies however, several investigations using numerous disease models have revealed a robust sex difference. Pain models which have established exacerbated female pain sensitivity include the formalin paw model, an inflammatory pain model where the second phase of ‘pain’ examines central sensitisation55, 56, where rats were assessed for licking and flexing duration along with paw-jerk frequency and females were demonstrated to exert a greater number of behavioural signs of hypersensitivity; capsaicin-induced allodynia57, where capsaicin-induced hyperalgesia was found greater in females than males; the complete

Lauren Nicotra Sex Differences in Allodynia 25

Freund’s adjuvant (CFA) model58, another inflammatory pain model, whereby female rats exhibited significantly elevated thermal hyperalgesia compared to their male counterparts; the temporomandibular joint (TMJ) disorder chronic pain model59, where females demonstrated enhanced rubbing of the orofacial region and the hind paw; the infraorbital nerve injury model53, a model of chronic europathic pain, where female rats developed greater and longer- lasting facial hypersensitivity compared with males; the standard neuropathic pain L5 spinal nerve transection model50, where DeLeo and colleagues revealed greater mechanical allodynia in female Sprague Dawley rats; hot water tail immersion60, where tail- flick/withdrawal from hot water test revealed sex differences between male and female

Sprague-Dawley rats and Swiss-derived mice; as well as the chronic constriction injury model

(CCI)61, where gonadally intact females revealed robust thermal hyperalgesic symptoms.

Evidently therefore, these preclinical findings replicated the clinical chronic pain scenario of a female predominance of pain.

1.2.3 Clinical Sex Differences in Responses to Experimental Pain

Although the clinical evidence presented above (Section 1.2.1) convincingly demonstrates that females are at a much greater risk of developing chronic pain and experience greater pain in relation to these chronic pain conditions compared to males, the findings in relation to experimental pain in healthy men and women are far less consistent. A large range of stimulus modalities have been used to investigate sex differences in response to experimental pain, including, but not limited to, pressure pain29, 62-74, electrical pain75-79, heat and cold pain70, 80-90 and has been examined using a range of outcome measures, with threshold and tolerance the most common measure.

Lauren Nicotra Sex Differences in Allodynia 26

In 2012, Racine and colleagues presented a 10-year review of the sex effect on experimental pain91. Their conclusions were that throughout this 10-year period, studies have been unsuccessful in producing a clear pattern of sex differences in human pain sensitivity. Prior to this review, Fillingim and colleagues assessed this sex effect in 200913 and preceding this meta analysis, Riley and colleagues review the experimental pain literature to determine a role of sex20. Both these earlier publications revealed a large degree of inconsistency, corresponding with Racine’s findings.

Although those investigations examining electrical, thermal heat, cold pain and mechanical stimuli modalities produced great variability, the general pattern of results revealed greater pain sensitivity in females. Interestingly, the magnitude of those sex differences was not universal, with some modalities such as ischemic pain producing small effect sizes and electrical pain resulting in larger differences.

Of specific note are those studies that have examined mechanically evoked pain. Although studies examining sex differences in mechanical pain thresholds have yielded variable findings, investigations using phasic noxious stimuli (as compared to sustained stimulation) consistently demonstrate equivalent healthy male and female thresholds in humans92, 93.

Considering that a great number of preclinical rodent chronic pain investigations utilise mechanical stimuli to investigate pain sensitivity (measuring pain thresholds, tolerance, hyperalgesia and allodynia), the distinction between equivalent pain sensitivities in healthy human individuals, compared with exaggerated pain sensitivity in the chronic setting using this pain induction modality must be noted. With the lack of translational success in regards to pain partly attributed to the continued use of suboptimal preclinical pain methodologies47, it is not only important to highlight this distinction between baseline (healthy) pain scores and

Lauren Nicotra Sex Differences in Allodynia 27

those found in chronic pain patients, but also to emphasize the need for preclinical pain studies to replicate this distinction between healthy mechanical pain thresholds and such thresholds in the chronic pain population94.

Preclinical animal chronic pain models must also best mimic the human scenario in order to elucidate the mechanisms underlying the female prevalence of chronic pain so to aid in the discovery of potential therapeutics to treat this female burdened disease94. The recent development of a graded neuropathy model has been shown to not only detect a heterogenous pain response, but replicate the heterogeneous magnitude of triggers of chronic pain95.

Moreover, this novel preclinical model also deliberately activates both neuronal and peripheral immune signals, thereby better mimicking the immune system response in clinical chronic pain populations96. Although only previously utilized to examine male rodent mechanical pain sensitivity, this novel preclinical pain method may provide an improved means to more appropriately preclinically investigate the mechanisms underlying sex differences in pain sensitivity. Moreover, with this preclinical chronic pain model capable of investigating pain sensitivity across a range of nociceptive intensities, this model has refined the traditional preclinical CCI animal neuropathic pain model, thereby also satisfying the

‘refinement’ category of the 3 R’s. With the 3 R’s well embedded throughout the legislature governing the use of animals in science, continued use of the graded neuropathy pain model will no doubt aid in the future use of rodents in preclinical pain investigations, thereby potentially unveiling therapeutics to treat this female burdened disease94

Lauren Nicotra Sex Differences in Allodynia 28

1.2.4 Summary

Together, the epidemiological evidence revealing a greater female representation within the chronic pain community as well as exacerbated pain sensitivity in both humans and rodents across the majority of chronic pain conditions and preclinical persistent injury models respectively, reveal a fundamental difference in pain processing between the sexes. Although this female prevalence and exaggerated sensitivity does not lie true for all existing chronic pain conditions and across all experiential investigations, the vast majority of studies emphasize a significant sex difference in pain.

Chronic pain places a large financial burden on the global economy, with a substantial amount of sufferers living with chronic pain across the globe16. In spite of this, the precise mechanisms contributing to this sex difference remain unknown however, somewhat attributable to this are the inappropriate preclinical methodologies that continue to be used by pain researchers47, 94. Consequently, in order to discern what makes females more susceptible to the development of chronic pain and in turn produce novel therapeutics in order to adequately treat the disproportionate female chronic pain population97, the pathophysiology underlying this sex difference must continue to be explored and most importantly, use clinically translatable preclinical methodologies47, 98.

1.3 Mechanisms Underlying Sex Differences in Pain

Although sex differences in pain clearly exist, the pathophysiology underlying this difference remains far from clear. Several biological and psychological mechanisms have been both proposed and investigated and together, have been found to contribute to the underlying disparity in pain sensitivity and prevalence. Many of the best-characterised contributing factors are presented below. Lauren Nicotra Sex Differences in Allodynia 29

1.3.1 Cognitive-Affective Factors

Pain coping strategies and emotional stressors may place another layer of complexity and also contribute to the clinical sex differences identified in pain sensitivity. A subject’s coping strategies have been found to modulate pain threshold outcomes. The maladaptive coping strategy catastrophising for example, has been found to translate to increased sensitivity and lower tolerance to experimental pain across a range of chronic pain patients99-102. Importantly, higher rates of catastrophising have been identified among women103, which may contribute to this sex difference.

Clinical psychological disorders such as depression and anxiety are two commonly identified pain comorbidities104, of which both have also been associated with enhanced pain sensitivity105-108. As well as their concurrent incidence, several antidepressants, although not specifically developed or intended for the treatment of chronic pain, are mainstay therapies for chronic pain sufferers109, 110. Although outside of the scope of this thesis, the relationship between disorders such as depression and pain have been well investigated and are reviewed elsewhere104, 111, 112. Importantly however, both anxiety and depression are far more prevalent among the female sex113, suggestive of a complex association which may be partially accountable for clinically identified nociceptive sex differences15.

1.3.2 Sex Roles

Although used interchangeably, the terms ‘sex’ and ‘gender’ are not synonymous. Often used to describe ‘sex’, gender is a social role, where one or others identify somebody as male or female based on their characteristics such as the way that they dress or behave114.

Sociocultural beliefs regarding one’s gender have been shown to affect pain responses in the

Lauren Nicotra Sex Differences in Allodynia 30

clinic, with women who identify themselves as ‘feminine’ found more likely to encourage pain behaviours, and ‘masculine’ men, found to discourage pain expression, with pain expression found to be more generally acceptable among women67 . In a study by Pool and colleagues for example, males who identified themselves as masculine were found to tolerate a greater amount of pain compared with males who did not define themselves as masculine

115. Similarly, Otto and colleagues revealed a correlation between elevated masculinity scores and higher pain thresholds in males67.

Gender-related motivation has also been identified to influence pain expression between the sexes, with expectations regarding one’s performance based on their sex found to influence pain outcomes. This was specifically identified by Fowler and colleagues116, who revealed that men primed with a feminine gender role reported increased cold pain, as indicated by the

McGill Pain Questionnaire (MPQ) pain rating. Additionally, culture-related stereotypes have also been found to modulate pain expression, with specific cultures reporting pain sensitivities dependent upon gender117.

The sex of the experimenter has also been shown to influence clinical responses to painful stimuli. Although the results presented are not entirely convincing, investigations have revealed that male subjects report less pain when in the presence of a female experimenter118,

119. Interestingly, the female pain response has been found both dependent on experimenter gender, with lower pain thresholds in the presence of an attractive male or female examiner, as well completely independent of experimenter gender or desirability118. Although the findings presented have been inconstant, the role of the examiner in clinical pain studies may be possible. Interestingly, in a recent study by the Mogil laboratory120, pain researchers extended these clinical findings and revealed that exposure to male experimenters produced

Lauren Nicotra Sex Differences in Allodynia 31

pain inhibition in mice. Mogil demonstrated that this did not occur in the presence of a female experimenter and found that the physiological stress response resulted in stress- induced analgesia120.

1.3.3 Hormonal Influences

The above-mentioned influences and potential sources of divergence leading to sex differences in pain sensitivity are potential contributing explanations for differences in pain response specific to the clinic. Methodological variations have also been found to influence the pain outcome in non-human experimental studies. These causative factors however, do not completely explain the large degree of divergence in pain responses observed between the sexes in both clinical and experimental studies13. Perhaps one of the most obvious distinctions between males and females that has been thoroughly investigated as a main contributor to sex differences in pain sensitivity are the gonadal sex steroid hormones121.

The sex steroid hormones play an important role in several CNS and peripheral functions.

Although once previously defined by their role as reproductive hormones, variability in ligand availability, the discovery of previously unrecognised gonadal hormone receptors, as well as the identification of novel targets has altered what is currently known of gonadal hormone physiology and function122. While the progestins and the androgens are known to influence several physiological processes and have also been identified to play a role in CNS, particularly neuronal, processing and inflammation, the oestrogens are now well recognized to play a key regulatory role in pain (please see Section 1.4). Several lines of clinical and experimental evidence support and suggest a role for gonadal hormones, particularly oestrogen, in the sex differences observed in chronic pain. Prior to examining this large body

Lauren Nicotra Sex Differences in Allodynia 32

of evidence, the physiology of the oestrogens, along with their receptor proteins will be presented. Important to note however is not only the role of gonadal hormones in pain and chronic pain, but that sex differences in chronic pain are also driven by genetic/sex dependent mechanisms. A discussion of these genetic influences is outside of the scope of this thesis but has been discussed by several others123-125.

1.3.3.1 Physiology of Gonadal Steroid Hormones

Classically, the sex steroid hormones include oestrogens, progestins and androgens, with the ovaries and the testis primarily responsible for the production of these hormones. The testis produce androgens: testosterone and dihydrotesterone, whereas the ovaries are responsible for the production of both oestrogens (e.g. oestradiol, oestrone and oestriol) and progestins (e.g. progesterone)126. As well as the reproductive sex organs, the adrenal cortex is also responsible for androgen production127, the testes are capable of producing oestrogens128 and the ovaries are a source of testosterone129. Accordingly, both oestrogens and androgens are found in each sex130.

The major sex steroid hormones, oestradiol, progesterone and testosterone are secreted in response to the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone

(FSH), which occurs at puberty131. Unlike in males, the female reproductive system is quiescent from birth until puberty, occurring approximately at 12 years of age when gonadotropin-releasing hormone (GnRH) is released from the hypothalamus, in turn stimulating the anterior pituitary to release LH and FSH into the bloodstream132. These gonadotrophins act on receptors on the gonads to stimulate oogenesis, spermatogenesis and

Lauren Nicotra Sex Differences in Allodynia 33

steroidogenesis. Once produced, these steroids negatively feedback to the hypothalamus and pituitary133 (Figure 4).

Figure 4. Hypothalamic-Pituitary-Gonadal Axis Production of Gonadotrophins and the

Regulation of Steroidogenesis.

GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E, oestrogen; P, progesterone; T, testosterone. Figure acquired without the need for permission from Wierman’s sex steroid review122

1.3.3.1.2 Oestrogens

Naturally occurring oestrogens (oestradiol, oestrone and oestriol) are steroids derived from cholesterol134. Although all three oestrogens play a physiological role in the CNS and periphery, 17beta-oestradiol (17β-oestradiol or E2) is the predominant physiological oestrogen in mammals and the principle oestrogen produced from the biosynthesis pathway134.

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E2 is primarily produced by the ovary (approximately 90%)135. In addition to ovarian synthesized E2 however, this principal oestrogen is also produced at extragonadal sites that act locally at the site of synthesis135.

E2 synthesis in the ovary begins at puberty, with the production of pregnenolone, derived from cholesterol, which with the aid of several enzymes is catalyzed to progesterone by the thecal and granulosa cells of the ovary134. Following further enzymatic catalysis, progesterone is transformed into the androgen testosterone, with the cytochrome P450 enzyme aromatase ultimately responsible for the final catalysis step in the E2 biosynthesis pathway134 (Figure 5).

From the time of puberty when hypothalamic GnRH activity increases for the first time in females, until menopause, a phase of ovarian failure, this period is termed the prime ovarian years.

Figure 5. E2 Synthesis in the Ovary.

Ovarian thecal and granulosa cells are involved in oestradiol (E2) production, with the aromatase enzyme responsible in the final phase of synthesis. This figure was acquired from

Cui and colleagues134 with permission. Lauren Nicotra Sex Differences in Allodynia 35

The cytochrome P450 enzyme aromatase is widely expressed, with its presence along with several other E2 synthesis requiring enzymes also in the brain. Much like E2 production in the ovary, brain derived E2 is produced from cholesterol134 (Figure 6). Unlike microglia and astrocytes, both neurons and astrocytes have been found capable of producing oestrogens, with neurons the major location of E2 production in the brain. Upon their release, ovarian and brain derived E2 travel via the bloodstream targeting oestrogen responsive reproductive and non-reproductive organs134.

Figure 6. Neuronal and Astrocytic E2 Synthesis in the Brain.

Both neurons and astrocytes possess all of the required enzymes for E2 synthesis including the aromatase enzyme. Unlike these cells however, both microglia and oligodendrocytes are not capable of E2 synthesis from cholesterol. This figure was acquired from Cui and colleagues134 with permission.

With E2 primarily produced by the ovary135 however, the surgical technique in animals whereby the ovaries are excised, termed ovariectomy (OVX), rapidly ablates cycling hormone Lauren Nicotra Sex Differences in Allodynia 36

production and female rodents lose most of their ability to produce E2, along with progesterone136, 137. In effect, this technique induces females to enter a “surgical menopause”, in comparison with “natural menopause,” which is a natural effect of the ageing process in women. Interestingly, although oestrogenic influences have still been documented in models of OVX due to elevated peripheral tissue production of oestrogen from androgen precursors138, 139, researchers have shown that OVX in young rodents (3-6 month old mice) does not alter brain levels of E2140. Consequently, although several peripheral sites and the brain are well known capable of synthesising E2, with aromatase expressed widely throughout the periphery and CNS, the surgical OVX technique is widely utilised in the preclinical literature and has been proven an effective surgical tool to investigate the many roles of this gonadal hormone.

1.3.3.1.2.1 Oestrogen Receptors

The predominant functions of E2 are mediated by the nuclear oestrogen receptors (ER’s) ERα and ERβ141. Unlike the intracellular signalling mediated by these nuclear receptors that occurs over hours or days, oestrogens have also recently been identified to signal via membrane receptors such as GPR30, whose signalling transduction occurs far more rapidly142. ER’s are involved in ER-dependent signalling pathways, which regulate transcription of oestrogen responsive genes, ion channels and second messenger systems as well as cell proliferation134.

(a) ERα and ERβ

First characterised in 1973, the ERα was found to be the receptor for oestrogen identified in rat uterus/vaginal extracts143. Identification of ERβ followed closely, characterized in 1986144.

Lauren Nicotra Sex Differences in Allodynia 37

The ER’s, α and β belong to the nuclear receptor (NR) family of transcription factors142. Both receptors share similar structure homology, containing six functional domains necessary for ligand and DNA binding, as well as protein-protein interactions and nuclear translocation145.

The classical ER’s are located throughout the CNS as well as the periphery, with distinct functional tissue-specific distribution locations134. Since sex hormones, particularly oestrogens, not only contribute to sexual development and behaviour, but also modulate many

CNS functional roles including, but not limited to, neuronal development and growth146-152, synaptic plasticity and neuronal regeneration153, it is not surprising that the classical ER’s are widely located throughout the CNS (for a review please see Cui and colleagues134).

(b) GPR30

Although steroid receptors are generally known to be ligand-activated transcription factors, steroids are now also recognised to mediate G protein-coupled and growth factor associated signalling. In the year 2000, the G protein-coupled receptor, GPR30 was identified as an oestrogen receptor, capable of rapid signalling transduction independent of the steroid

ER’s154. Filardo and colleagues specifically found that this GPCR was involved in oestrogen- mediated signalling in cells devoid of nuclear ERs154. Since that time, further evidence has revealed a role of GPR30 in oestrogen cellular responses141. Moreover, similar to the classical

ER’s, GPR30 is expressed in several CNS and peripheral locations155-157.

Together, upon their activation, the ER’s are subsequently involved in oestrogen-mediated signalling processes, with the majority of such responses resulting in transcriptional activity

(For a review of oestrogen mediated signalling please see Cui and colleagues134) Lauren Nicotra Sex Differences in Allodynia 38

1.3.4 The Role of Gonadal Sex Steroid Hormones in Chronic Pain: Oestrogen and Exacerbated Female Pain

A substantial body of experimental and clinical evidence supports a major role for gonadal hormones, particularly E2, in the sex differences observed in chronic pain. Examining incidence rates of chronic pain across the lifecycle of a woman; the incidence of chronic pain syndromes and pain sensitivities in relation to the female menstrual cycle; and the effect of exogenous hormone therapy on pain outcomes, are a few of the ways in which gonadal sex steroid hormones have been shown to influence chronic pain in the clinical setting. The use of rodents has also provided a convenient and valuable mode to investigate the role of gonadal hormones. The ability to examine hypersensitivity across the rodent oestrus cycle, experientially manipulate the circulating levels of E2 in rodents via surgical removal of the ovaries (OVX) and the reintroduction of oestrogens at desirable concentrations makes rodents an exceptionally useful tool in preclinical pain investigations. Numerous studies utilising these techniques and procedures have undoubtedly revealed a role for E2 in chronic pain. A summary of these clinical and experimental findings is presented below.

1.3.4.1 Sex differences in the Prevalence of Chronic Pain Conditions: Prepubescent vs. Adult subjects

As previously reviewed and discussed (Sections 1.2.1-1.2.1.5), women are overrepresented amongst the majority of chronic pain conditions, particularly amongst headache/migraine and temporomandibular disorder (TMD) sufferers. Interestingly however, in a number of studies that have investigated migraine prevalence amongst prepubescent boys and girls, the distinct female predominance in adults has not been replicated in children. Across several migraine investigations, a female prevalence was found to increase with age, failing to establish a sex difference prior to the age of 12, and a spike in female migraine incidence at the age of

Lauren Nicotra Sex Differences in Allodynia 39

puberty158-160. Comparable to these studies for migraine, the sex differences found in adults for TMD prevalence are also not so apparent in children, with rates of pain in the temporomandibular region similar for girls and boys across a range of investigations161-163.

Moreover, just as for several of the aforementioned migraine studies, the prevalence of TMD has also been found to increase for females after the age of puberty164. Prevalence of back pain and stomach pain has also been shown to increase dramatically with pubertal development in girls165.

Evidently, these investigations support an association between the gonadal hormones and the prevalence of several chronic pain conditions in the female sex. Although prepubescent pain scores have not been examined across several other chronic pain conditions in which a greater proportion of females are affected, the abovementioned findings for migraine and TMD in particular, support several other lines of evidence implicating gonadal hormones in chronic pain.

1.3.4.2 Severity of Pain Symptoms across the Menstrual Cycle

The menstrual cycle is a progression of ovarian and uterine changes enabling sexual reproduction in women166. Throughout a normal menstrual cycle, hormone levels fluctuate, preparing the uterus for a possible pregnancy166 (Figure 7). Classically divided into three distinct phases, the menstrual cycle is defined by the events occurring in both the ovary and the uterus. The ovarian cycle is segregated into the follicular phase, characterised by increasing levels of E2 in response to the release of estrogens and androgens from the thecal and granulosa cells of the ovary following FSH and LH release from the anterior pituitary; ovulation, where a surge in LH results in ovum release and is characterised by declining

Lauren Nicotra Sex Differences in Allodynia 40

levels of E2 and slowly increasing levels of progesterone; and the luteal phase, which occurs just after ovulation, is the period in which the remnants of the ruptured follicle following ovulation is transformed into the progesterone producing corpus luteum167. In the event that pregnancy does not occur, lysis of the corpus luteum results in a decrease in progesterone and

E2 levels. Together these phases describe the role of the sex hormones that are fundamental for the changes throughout the women’s reproductive system allowing for pregnancy. In contrast, the uterine cycle describes the changes throughout a female’s uterus in preparation for the implantation of a fertilized egg. This cycle is classified into menstruation, proliferation and the secretory phase, which correspond with the follicular phase, ovulation and the luteal phase respectively166.

Figure 7. Plasma Levels of Hormones Throughout a Woman’s Menstrual Cycle.

Described by the events occurring in both the ovaries and the uterus, the menstrual cycle is divided into three distinct phases; the follicular phase, ovulation and the luteal phase, in the

Lauren Nicotra Sex Differences in Allodynia 41

ovary; and menstruation, the proliferative phase and the luteal phase, in the uterus. Figure acquired from Brisken and colleagues168 with permission.

The intensity of pain symptoms in healthy females and the incidence of pain associated with particular chronic pain conditions has been shown to vary across the menstrual cycle. In a meta-analysis conducted in 1999, Riley and colleagues169 reviewed sixteen published studies that investigated experimentally induced pain across the menstrual cycle in healthy female subjects. Riley investigated pain thresholds for pressure pain170-175, cold pain176, 177, thermal heat pain178-180, ischemic pain175, 180, 181, and electrically stimulated pain177, 182, 183, where it was revealed that for most methodologies (excluding electrical stimulation), pain thresholds were largely found to increase during the follicular phase (characterised by rising levels of E2 and low levels of progesterone) of the menstrual cycle.

Since that time, the effect of menstrual cycle has been further investigated. However, more recent findings have generated great inconsistency. Pain sensitivity following electrical stimulation for example, has revealed lower pain tolerance throughout the luteal phase, in comparison with the follicular phase of the menstrual cycle184, with others failing to find an affect of cycle for electric pain induction185, 186. This variability has been found amongst several other pain induction methods, with some methodologies also failing to reveal an effect of cycle208, 209, or demonstrate greater pain sensitivity at limes of low E2 and higher progesterone187 (please refer to Fillingim and colleagues for a relatively recent review13).

In addition to tolerance and threshold, the impact of menstrual period on the prevalence of particular chronic pain conditions has also revealed an association between gonadal hormones and chronic pain. Most intriguing are those studies that have examined headache/migraine

Lauren Nicotra Sex Differences in Allodynia 42

prevalence; revealing an association between declining E2 levels and the incidence of migraine attack. In a study by Keenan and Lindamer188, occurrence of non-migraine headache was found to occur most commonly throughout the peri-menstrual phase (two days prior to the menses, characterized by declining levels of E2). Others have replicated this finding189, 190.

Interestingly however, researchers have revealed that the association between E2 and headache/migraine is not in fact dependent upon the relative levels of E2 but rather, the drop in this hormone. Following an investigation of E2 supplementation, Sommerville demonstrated that blocking E2 withdrawal was effective in preventing headache191, 192.

Supporting this theory are the investigations that have examined headache/migraine prevalence during pregnancy193, 194. With pregnancy characterized by a dramatic increase in plasma E2 levels, the findings of less migraine or a total remission of migraine attack throughout pregnancy, as well as a spike in migraine incidence postpartum195 not only support

Somerville’s ideology regarding the fall in E2 level and migraine, but also the association between gonadal hormones and chronic pain. Further supporting this notion are those studies reporting a relationship between the incidence of other chronic pain conditions such as

TMD196 and fibromyalgia197-199 and specific phases of the menstrual cycle.

1.3.4.3 Exogenous E2 Administration

Although considerable attention has been paid to the effect of endogenous hormones and clinical pain (such as those investigating the effects of the female menstrual cycle), the effects of exogenous hormones, particularly E2 has revealed a strong association between this gonadal sex steroid hormone and pain. Both the administration and the withdrawal of the combined oral contraceptive pill (OC’s) (containing both E2 and progestin) and hormone

Lauren Nicotra Sex Differences in Allodynia 43

replacement therapy (HRT) have been shown to affect the incidence of pain associated with several chronic pain conditions, as well as pain severity.

The prime ovarian years occur prior to the age of 30. As previously described (Section

1.3.3.1), throughout these years, the hormones of the anterior pituitary, FSH and LH, are responsible for the release of E2, the maturation of follicles containing ova, and the liberation of such ova along with the resultant production of progesterone. The period at which there are fewer functioning follicles and ova, along with a corresponding decrease in E2 and progesterone level is defined as the perimenopausal period200. During this perimenopausal period, there is a reduction of menstrual flow, whereas menopause is the period defined as the time at which there is complete and permanent cessation of menstruation for one year200.

Despite some variability, epidemiological evidence suggests that pain severity and the onset of painful symptoms for both fibromyalgia and migraine peak during the reproductive years and drastically reduce after menopause201, 202 160, 203, 204. Interestingly, throughout this stage of life characterised by low incidence and reduced severity of painful attack for TMD and migraine, the use of HRT, a hormone therapy comprised of E2 or conjugated oestrogens with progesterone, used to supplement the lack of naturally occurring hormones205, has been found to not only increase incidence of attack for these chronic pain conditions206-208, but increase the severity of symptoms related to these conditions as well as many others including back pain209, 210 and orofacial pain211. In addition to HRT, combined OC’s have also been adversely linked to migraine206 as well as associated with an increase risk of pain accompanying

TMD212 and pain associated with carpal tunnel syndrome213.

Lauren Nicotra Sex Differences in Allodynia 44

Interestingly and contradictory to the above findings, the withdrawal of HRT in postmenopausal women has also been found to elevate pain and precipitate migraine214, 215.

Despite this opposing finding in migraine, it must be noted that this result does in fact support

Sommerville’s suggestion of an association between the drop in E2 plasma concentration and pain that is specifically associated with headache191, 192.

Studies that have examined exogenous hormone therapy in transsexual individuals not only extend the above findings but also reveal that this association between the gonadal hormones and chronic pain highlighted above is not female specific. In a study by Aloisi and colleagues216, a correlation was demonstrated between hormone treatment and pain, with approximately one third of the male-to-female subjects (47 subjects) developing chronic pain concurrently with combined oestrogen and anti-androgen treatment. With the need for exogenous E2 administration and therefore supraphysiological concentrations of E2 required to elevate pain sensitivity in the male sex, this study highlights that sex differences in chronic pain may be attributable to high circulating levels of E2, rather than just the presence of E2 itself.

1.3.4.4 Pain Across the Oestrus Cycle

In addition to the abundant clinical evidence supporting a correlation between gonadal hormones and chronic pain, are the experimental rodent studies that reveal fluctuating pain sensitivity across the oestrus cycle. The oestrus cycle is the rodent equivalent of the menstrual cycle, commonly divided into four stages, which are the sequence of recurring physiological changes occuring in response to fluctuating hormone levels. These four phases of variable gonadal hormone levels include: met-oestrus, where plasma progesterone levels are elevated

Lauren Nicotra Sex Differences in Allodynia 45

and E2 remains low; di-oestrus, characterised by increasing levels of E2 and decreasing concentrations of progesterone; pro-oestrus, where both E2 and progesterone plasma concentrations peak; and oestrus, characterised by declining levels of both these steroidal gonadal hormones121 (Figure 8).

Figure 8. Plasma Steroidal Hormone Concentrations Throughout the Rodent Oestrus Cycle

Compared to the Human Menstrual Cycle.

LH, luteinizing hormone; FSH, follicle stimulating hormone; OV, ovulation. Figure acquired with approval from the Fillingim and Ness review of hormonal influences on pain and analgesic responses121.

Studies that have examined pain sensitivity differences across the female oestrus cycle, using a diverse range of nociceptive assays, examining a variety of response measures, have revealed an association between the gonadal hormones and pain. Although a concise meta analysis published in the year 2000121; reported exaggerated pain sensitivity during oestrus217, characterised declining levels of E2, as well as throughout metoestrus218, defined by low concentrations of this gonadal hormone; the great majority of investigations, across a wide

Lauren Nicotra Sex Differences in Allodynia 46

range of pain assays including acute, inflammatory and neuropathic assays, have demonstrated elevated pain sensitivity during the pro-oestrus stage, corresponding with peak plasma concentrations of E2219-224. Elevated pain sensitivity throughout pro-oestrus has been replicated by others since this review94, 225-227.

While the majority of studies reveal elevated sensitivity throughout periods of higher plasma

E2 levels, the inconsistencies between findings has been attributed to methodological variables such as the stimulus modality investigated121. Moreover, variability in cycle phase nomenclature has also been described as attributable to menstrual cycle differences13. With each preclinical study defining oestrus cycle periods somewhat differently, the determination of which phase is most prone to elevated sensitivity is somewhat difficult and open to inaccuracy.

1.3.4.5 Gonadal Hormone Manipulation

By eliminating the gonadal hormones via gonadectomy, researchers have identified a role for the gonadal hormones in pain. Specifically, the surgical OVX technique (described above

Section 1.3.3.1.2) that dramatically reduces the circulating levels of E2, has been shown to decrease pain threshold in acute pain assays228, 229. Results following OVX however have not been entirely straightforward or consistent in several persistent and chronic pain models, with reports of increased, decreased and no effect or change in response to inflammatory and chronic pain insults. Following carrageenan administration (a persistent inflammatory pain model) for example, OVX rodents displayed reduced thermal hyperplasia in one particular investigation57 however, using another inflammatory pain model, formalin injection, OVX increased the amount and duration of licking of the affected paw56. Along with these

Lauren Nicotra Sex Differences in Allodynia 47

investigations, several other studies have also failed to determine a difference between intact and OVX females58, 230, 231,232. The above examples however, are only a sample of the myriad of investigations that have examined a role of these hormones using OVX (for additional information please refer to the reviews which have discussed these findings49, 121, 126).

Furthermore, although the literature has produced some inconsistent findings and the OVX technique cannot specifically segregate the role of any one particular gonadal hormone, it is most evident that the gonadal hormones generally, are influencing acute and chronic pain processing.

Supplementing OVX females with E2, using several differing administration techniques

(injection or surgical pellet insertion for example), has allowed researchers to isolate the role of specific hormones in pain modulation. E2 administration in OVX (and intact) female rodents has been shown to increase mechanical allodynia, thermal hyperalgesia, decrease tailflick latency, reduce paw withdrawal threshold, increase muscle reflex activity, as well as elevate or potentiate pain symptoms associated with chronic pain conditions such as TMJ219,

224, 233-235. Supporting these findings, several studies have suggested that elevated plasma/local

E2 levels correspond to increased pain sensitivity in the rodent236-241. In direct contrast however, others have found E2 to reduce pain related behaviours219, 229, 242-247 across acute and chronic pain assays237, 263-269.

1.3.4.6 Summary

Examining pain between the sexes in humans is fraught with complications since pain outcome is affected by coping, expectation, environment and mood, along with many other factors. Consequently, sex differences reported in pain sensitivity have been attributed to many of these influences13, 125. With gonadal hormones, particularly oestrogens, recognised to Lauren Nicotra Sex Differences in Allodynia 48

not only play a role in reproduction and sexual diversification, but several other homeostatic processes; and the brain recognised to be a major target of oestrogen action, it is not surprising that these hormones have also been found to contribute to the differences in acute and chronic pain observed between the sexes248. Although abundantly clear that the gonadal hormones, particularly E2 have a modulatory role in pain and chronic pain, both the clinical and animal findings are highly equivocal and the effects of E2 can not be easily delineated as antinociceptive or pronociceptive130. The diverse and tissue-specific actions of E2 in the PNS and CNS, where E2 has been shown to modulate several physiological and neurochemical systems, makes for understanding and isolating the specific roles of estrogen in pain modulation exceptionally difficult130. A brief introduction to these oestrogenic actions and their role in pain are presented below. Importantly however, with the recent appreciation of the neuroimmune interface in chronic pain, currently identified mechanisms may only partly explain an extremely complicated story.

1.4 Modulation of Pain by Oestrogens

Although research regarding the relationship between sex and pain has proliferated in the past thirty years and is far from understood, the neuroanatomy and neurochemistry of pain processing itself is not yet entirely defined. As outlined previously, pain transmission involves both ascending and descending PNS and CNS integrating processes, and the gonadal hormones have been shown to interact both peripherally and centrally121. The pain modulatory effects of gonadal hormones however cannot be simplified to a single mechanism or outcome130, 248. A complete review of the literature in regards to hormonal and oestrogenic modulation of pain is beyond the scope of this thesis, however the mechanisms presented below are to highlight not only the complexity of this interaction but that the majority of

Lauren Nicotra Sex Differences in Allodynia 49

better defined and investigated mechanisms center around the modulation of classical nociceptive processing and analgesic responses, with gonadal hormone and neuroimmune interactions being largely overlooked as having a role in chronic pain.

As discussed in Section 1.3.3.1.2.1, the ER’s have a wide distribution throughout the periphery and the CNS, specifically found in pain responsive and processing regions. When evaluating oestrogenic pain modulation in the periphery, it is important to highlight that receptors for this gonadal hormone are specifically located on primary afferent nerve fibers249 and are therefore able to influence nociception and ultimately nociception from its very origin121. E2 has in fact been shown to affect nociception at the level of the primary afferent nerve, altering the receptive field of mechano-receptive neurons for example 250, 251 as well as sensitise primary afferents making them more sensitive to nociceptive stimuli252.

As well as their effects at primary afferents, E2 is known to modulate the release of several neurotransmitters and neuromodulators important in nociception and pain transmission such as GABA, glutamate, substance P, dopamine and serotonin 207, 253 208. The dopaminergic system specifically has been shown to play an important role in neurotransmission, modulating the pain outcome. Although the mechanisms are outside the scope of this thesis, oestrogens play an important role in regulating dopaminergic activity and functionality within the CNS254-257, and this system has been found linked to pain associated with fibromyalgia and chronic widespread pain258. Moreover, although having a complex mechanism of action in pain, serotonin (5-hydroxtryptamine (5-HT)) has been shown to be both pronociceptive and antinociceptive13 as well as linked to several sex-related painful conditions such as migraine and irritable bowel syndrome (IBS)259-262. Oestrogen specifically modulates 5HT function in both the periphery and the CNS256, 257 and therefore this additional modulatory role of E2 may

Lauren Nicotra Sex Differences in Allodynia 50

contribute or partly explain the female predominance of particular chronic pain conditions that are associated with 5-HT.

One of the most crucial pain modulatory systems, the endogenous opioid system, has also been recognised subject to gonadal hormone control. Interestingly, men and women display divergences in the distribution of opioid receptors in CNS pain regions13. Opioid receptor binding has also been found to differ between the sexes, with greater cortical and subcortical mu-opioid receptor binding in females compared to males for example263. E2 specifically has been shown to modulate peripheral, spinal and supraspinal mu-opioid receptor activity248. In women, low E2 states, such as menopause, have been associated with reduced mu-opioid receptor biding activity, and both high and low E2 states have been found to modulate pain outcome264. In rodents, the modulation of oestrogens, via OVX and E2 supplementation, has also been found to modulate opioid nociception, with the effects dependent on several factors including timing of oestradiol administration amongst others265.

In addition to the aforementioned peripheral and central oestrogenic actions, this gonadal hormone is also known to affect disease pathophysiology, having tissue-specific actions in multiple systems of the body, ultimately affecting disease-related pain. For example, the oestrogens regulate bone deposition and cartilage degradation, and have accordingly been shown to influence pain associated with osteoarthritis266, 267, a heterogeneous group of conditions related to defective articular cartilage function and modifications of joint bone268.

Oestrogenic modulation of the cardiovascular system has also been found contributable to migraine pathophysiology. As previously detailed (Section 1.2.1.5), migraine is a female predominant disease and is under strict gonadal hormone control. One mechanism by which oestrogen has been found to modulate migraine is via ER-mediated increases in nitric oxide in

Lauren Nicotra Sex Differences in Allodynia 51

vascular endothelial cells261, 262. In addition to these peripheral oestrogenic effects, the effects of this hormone on the endogenous opioid system, discussed previously, have further been shown to contribute to migraine specifically261, 262. While the mechanisms by with the oestrogens alter vasodilation and vascular tone, and endogenous opioids are outside the scope of this thesis, these effects are to be noted and highlight that the role of oestrogens in pain are far reaching, tissue and disease specific.

Although a relationship between the gonadal hormones, sex and chronic pain is well established, with the majority of evidence provided in Sections 1.3.4.1 to 1.3.4.5, the mechanisms underlying the precise oestrogenic involvement in chronic pain is not as well understood or investigated as it is in classical pain signalling. Of the few potential mechanisms, the influence of oestrogen on the cyclooxygenase (COX) enzyme and corticosterone levels49, as well as particular neuromodulator expression and the N-methyl-D- aspartate (NMDA) receptor, have been better recognized and proposed attributable to sex differences in chronic pain. Of particular note, are the oestrogenic effects on NMDA receptor function. Of the mechanisms which underlie the initiation and maintenance processes contributing to chronic pain, are the changes in spinal cord dorsal horn nociceptive neurons, resulting in a change in NMDA receptor function and ultimately amplification of the nociceptive message signaled to higher brain centers269. This ultimate sensitisation of spinal cord neurons and ongoing pathological synaptic activity is referred to as central sensitization and is one of the leading causes of chronic pain8. The NMDA receptor plays a principal role in central sensitization. Continuous activation of spinal cord neurons, leads to NMDA phosphorylation, increased NMDA receptor trafficking to the synaptic membrane, and the removal of magnesium that is normally present in the NMDA channel269. Subsequent to these events, is the production of nitric oxide and prostaglandins, successively responsible for

Lauren Nicotra Sex Differences in Allodynia 52

increasing spinal cord dorsal horn neuron excitability and an exaggerated release of neurotransmitters, amplifying the response to the original noxious stimulus270. Interestingly,

E2 has been found to elevate NMDA receptor currents however the ability to do so is significantly more substantial in females compared to males271. This oestrogenic enhancement of NMDA receptor excitability has been proposed to produce grater central sensitization in the female sex and ultimately lead to sex differences in chronic pain92, 93, 272, 273.

1.4.1 Summary

Oestrogenic effects on nociception, pain and chronic pain signalling result from actions at several peripheral and CNS locations and systems. Of the few mentioned in Section 1.4, it is evident that the role of oestrogen is complex, contributed to by the widespread distribution of

ER’s in pain relevant areas, as well as disease specific mechanisms and the dose- and timing- dependency of oestrogenic pain modulation. The aforementioned mechanisms focus on the modulation of classical nociceptive processing and analgesic responses. To note however, a relationship between oestrogen and the immune system generally has also been found to play a role in pain related conditions such as rheumatoid arthritis and osteoarthritis274. Moreover, the oestrogens have also been identified to contribute to inflammatory pain specifically, with systemically and centrally administered E2 found to modulate nociceptive responses242, 275-277.

One way in which this gonadal hormone has been linked to inflammatory driven pain is via its neuromodulatory role in substance P release. Oestrogen treatment has been shown to modulate the expression of substance P, which is not only a neuropeptide, but also a strong inflammatory stimulus278-280. A complete review of the effects of oestrogen on systemic inflammation and the role of this association in pain is outside the scope of this thesis and is discussed elsewhere274. Important to highlight however is the unknown relationship between gonadal hormones, particularly E2 and the innate immune system in chronic pain. The role of Lauren Nicotra Sex Differences in Allodynia 53

this system in chronic pain is summarized below and its association with E2 is discussed in regards to its potential role in the sex differences observed in chronic pain.

1.5 The Innate Immune System in Chronic Pain

In the acute setting, pain and inflammation have been long associated. It was not until the

1970’s however that immunity and persistent pain were found connected, with chronic pain patients experiencing additional symptoms to classical hyperalgesia, which resembled the sickness response281. Soon following was the identification of elevated peripheral interleukin

1β (IL-1β) that was capable of inducing classical hyperalgesia as well as symptoms associated with the immune derived sickness response282, 283. A central action of IL-1β in addition to other cytokines was identified subsequently284, 285, and an appreciation for cytokine signalling in the CNS developed, with residing CNS glial cells found capable of releasing many of the cytokines involved in the sickness response286.

Elevated glial cell markers following peripheral nerve injury and the ability of intrathecally administered glial cell attenuators to reduce pain behaviours first implicated these non- neuronal cells in chronic pain287, 288. From this time, the role of glia in chronic pain has been substantially extended and reciprocal signaling between neurons and these immunocompetent cells has been identified as a key contributor to both the initiation and maintenance mechanisms underlying chronic pain.

1.5.1 The Role of Glia in Chronic Pain

Spinal cord glial activation is now well appreciated to not only play a role in neuropathic pain, but several other pain syndromes including, but not limited to diabetic neuropathy289,

Lauren Nicotra Sex Differences in Allodynia 54

spinal cord inflammation290 and chemotherapy-induced neuropathy291, 292.

Glia (microglia, astrocytes and oligodendrocytes) are resident non neuronal cells of the nervous system that are now not only recognized for providing structural integrity but also for their bidirectional communication with neurons that has been shown to regulate neurotransmission in the CNS, known as the neuroimmune interface281. Although oligodendrocytes, which are best known for myelinating axons, are now acknowledged to modulate neuronal activity, both astrocytes and microglia are best known for modifying nociception8. Hence the role of astrocytes and microglia will be the focus of this thesis when describing the role of glia in pathological pain.

Astrocytes are the predominant glial cell type in the CNS. Among their many roles, including creation of the blood-CNS barrier, maintenance of homeostasis in the extracellular environment and repair of neuronal systems293, 294, astrocytic encapsulation of neurons provides structural neuronal support and further allows them to modify synaptic transmission and thereby neuronal communication295. This now recognized bidirectional communication between astrocytes and neurons has been conceptualized as the tripartite synapse. Through this encapsulation, the exchange of information allows for the control of synaptic transmission and plasticity (Figure 9).

Astrocytes express various receptors for several neurotransmitters including NMDA receptors, glutamate and substance P receptors8, 296. Astrocytic reactivity is often correlated with increased expression of glial fibrillary acidic protein (GFAP) and involves stimulation of the extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathways Lauren Nicotra Sex Differences in Allodynia 55

(amongst others) leading to the activation of the transcription factor nuclear factor-κB (NF-

κB), resulting in the release of several pro-inflammatory mediators including, but not limited

8, 281 to IL-1β, IL-6, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) (Figure 11) .

Astrocytes are now well known to contribute to the maintenance phase of neuropathic conditions297, 298.

Microglia are considered the macrophages of the CNS but perform a myriad of further functions. In the healthy CNS, microglial cells continuously sample their environment, surveying the extracellular space, thereby maintaining homeostasis299. In response to damage in the CNS however, microglia transition from this maintenance role to a reactive phenotype by a process known as microgliosis. This conversion to a more active state involves an increase in cell number, morphology, phenotype, motility, increased expression of signaling proteins and the release of pro-inflammatory mediators281, characterized by increased expression of cluster of differentiation molecule 11B (CD11b) and other cell surface antigens such as major histocompatibility complex (MHC) classes I and II, CD11b, cellular adhesion molecules, CD4, CD45 and allograft inflammatory factor 1300-305. During pathological conditions, similar pathways are activated in microglia as astrocytes, however many of the cytokines released and therefore the enzymes responsible for their activation are distinct for microglia. Diverse matrix metalloproteinases (MMPs) that facilitate pro-inflammatory IL-1β activity, for example, are distinct between the two types of glial cells. MMP2 induces pro- IL-

1β cleavage and is responsible for maintenance of astrocyte activation throughout the later stages of neuropathic pain however, MMP9 induced pro- IL-1β cleavage is specific for microglia, resulting in the activation of p38 mitogen-activated protein kinase (p38 MAPK) in microglial cells throughout the early stages of neuropathic pain (Figure 10)306. Distinctly, pro-

Lauren Nicotra Sex Differences in Allodynia 56

inflammatory mediators released from microglial cells activate other nearby glial cells as well as neighboring neurons, potentiating the pro-inflammatory response known to play a role in pathological pain8.

Similar to astrocytes, microglia contribute to plastic changes within the CNS following injury and their recognition as contributors to synaptic transmission has led some to now define this concept as the tetrapartite synapse8. Despite their shared involvement in the changes which occur post injury, resulting in central sensitization and chronic pain, the distinct roles of microglia and astrocyte activation in pain facilitation are now well known. Minocycline for example, which has been shown to inhibit microglial activation307-311 rather than astrogliosis, prevents, rather than reverses, both hyperalgesia and allodynia310-314. Divergent timing in the expression of microglial and astrocytic markers has further distinguished microglial and astrocyte roles. Opposed to CD11b expression, which has been identified in the induction phase of neuropathy, elevated GFAP expression has been found from postoperative days 4 to

28315. Evidently, these studies have recognized that microglia participate in the induction of neuropathic pain whereas astrogliosis is important for the maintenance of pathological pain310.

Lauren Nicotra Sex Differences in Allodynia 57

Figure 9. The Tripartite Synapse.

Encapsulation of neurons by astrocytes allows for direct communication, contributing to the synaptic plasticity involved in the development and maintenance of chronic pain. As well as astrocytes, microglia form part of the tripartite synapse, contributing to plastic changes within the CNS following injury. Their involvement has led some to now define this concept as the tetrapartite synapse. Figure acquired with permission from glial communication review by

Smith, K316.

1.5.1.2 Glial Cell reactivity

Following tissue injury and/or damage, several nociception facilitating factors are released which are able to activate glial cells, including, but not limited to, nitric oxide (NO) and reactive oxygen species (ROS)8. Following peripheral nerve injury specifically, several neuromodulators and neurotransmitters are capable of activating microglia, including MMP9, neuregulin 1, chemokines, adenosine triphosphate (ATP) and endogenous danger signals

(damage-associated molecular patterns (DAMPs) (Figure 10)306, 317. The transition to an

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activated state, results in microglial release of several pro-inflammatory mediators, which are in turn further responsible for activating nearby astrocytes, microglia and neurons8.

1.5.1.3 Neuron-to-glia signals in Neuropathic pain

Many of the neuromodulatory mediators that neurons release in chronic pain states are capable of triggering glia to enter reactive nociception facilitatory states. Upon activation, glial responses to these neurotransmitters and neuromodulatory mediators are augmented, contributing to pathological pain281.

(a) ATP

Neuronally derived ATP is a crucial mediator involved in microgliosis and the etiology of

318 neuropathic pain. Neuronal ATP is known to active P2X4 receptors on microglia , in turn inverting the polarity of currents generated by the inhibitory neurotransmitter gamma aminobutyric acid (GABA)319, as well as activating p38 mAPK, which is involved in microglial intracellular signaling, leading to the production of pro-inflammatory cytokines

320 (Figure 10) . Microglial cells are also known to express the P2X7 receptor. Although known to play a role in inflammatory pain, this ATP receptor has been shown to contribute to both chronic inflammatory and neuropathic pain hypersensitivity321. Both the genetic and pharmacological blockade of these ATP receptors have implicated this neuronally derived mediator in pathological pain321, 322. Moreover, peripheral nerve injury induced allodynia has been further shown to result in a transient increase in spinal microglial expression of P2X4 receptors323.

(b) Chemokines

Following noxious stimulation, neuronal chemokine release is also recognized to play a role

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in glial cell reactivity. The chemokine fractalkine (CX3CL1) for example can be released following strong neuronal activation such as under pathological conditions and is known to be a neuron to glia signal324. Fractalkine is released from neurons by MMP9 and MMP2. Both genetic and pharmacological blockade of the CX3CL1 receptor (CX3CR1) has been shown to attenuate peripheral nerve injury induced microgliosis and the resulting nociceptive hypersensitivity. Astrocytic fractalkine and CX3CR1 expression has also been shown upregulated throughout chronic pain states, suggestive of a role for this chemokine in ongoing pathological pain325, 326. Furthermore, chemokine (C-C motif) ligand 2 (CCL2), known as

MCP-1, released from the central terminals of injured neurons, is further involved in microglial activation following nerve injury (Figure 10)327, 328. Not only is microglial CCR2 upregulated following peripheral nerve injury329, 330, its upregulation precedes microglial activation and administration induces microglial activation, which is abolished in CCR2 KO mice328.

(c) Danger Signals

Glia are CNS immunocompetent cells, becoming reactive upon pathogen invasion8. In order to discriminate between self and non-self pathogen-associated structures and endogenous danger signals, glial cells express a wide range of pattern recognition receptors, including toll- like receptors (TLRs)331. As well as becoming activated in response to pathogen invasion,

TLRs also recognize many endogenous signals that are produced throughout chronic pain states98. The activation of TLRs and their contribution to chronic pain is discussed in detail below (Section 1.5.2) (Figure 10).

Lauren Nicotra Sex Differences in Allodynia 60

(d) Other Mechanisms of Reactive Gliosis

Astrocytic expression of ionotropic non-NMDA and NMDA receptors, in addition to metabotropic glutamate receptors8, 296 provides for the production and release of pain producing pro-inflammatory cytokines following neuronal glutamate release through the activation of intracellular pathways including ERK (also known as MAPK1) and JNK (also known as MAPK8) signalling pathways8.

332 Both microglia and astrocytes are further activated by neuronally and glially derived PGE2

(Figure 10). PGE2 exerts its effects via several GPCRs; EP1, EP2, EP3, EP4. Microglia

332 however express EP1 receptors, whilst both glial cell types express EP2 .

Along with the above neuromodulators and neurotransmitters, glia are also activated by the pro-inflammatory cytokines which they release upon activation themselves. Mechanisms underlying glial activation and their contribution to neuropathic pain has been thoroughly reviewed by Milligan and Watkins8 as well as the recent Grace and colleagues neuroimmune interface publication281.

1.5.1.4 Reactive Glia Produce Pathological Pain

Activated glial cells contribute to both the induction and maintenance of chronic pain through several diverse mechanisms. Through the direct activation of cytokine receptors expressed on neurons333, released pro-inflammatory cytokines from activated glia enhance neuronal transmission of nociception8. Several lines of evidence have supported this concept. The injection of pro-inflammatory cytokines such as TNFα and IL-1β have been shown to enhance nociception as well as neuronal excitability. Conversely, particular cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra) and soluble anti-TNF receptor antibodies

Lauren Nicotra Sex Differences in Allodynia 61

prevents as well as reverses both hyperalgesia and allodynia across most animal models of inflammation and nerve injury334-337.

Glial derived pro-inflammatory cytokines are further known to contribute to pathological pain by altering synaptic transmission in dorsal horn neurons. Astrocytic TNFα and IL-1β have been shown to increase neuronal excitability and strength by directly increasing neuronal

AMPA and NMDA receptor number as well as their conductivity338-341. IL-1β specifically, has been found to induce the phosphorylation of NMDA receptors thereby increasing the inward calcium conductance of such channels, resulting in an exaggerated release of pro- inflammatory mediators known to increase the excitability of spinal cord dorsal horn neurons342, 343.

Astrocyte activation further contributes to central immune signalling and the induction and maintenance of chronic pain through the modification of glutamate signalling. Nerve injury induced downregulation of the excitatory amino acid transporters glutamate transporter 1

(GLT1) and the glutamate–aspartate trans- porter (GLAST), results in an elevation of extracellular glutamate and spontaneous nociceptive hypersensitivity344-346. Furthermore, by facilitating disinhibition, glially derived pro-inflammatory cytokines have been further found to invert the polarity of currents generated by inhibitory GABA receptors and decreasing the frequency and amplitude of currents activated by glycine319.

Lauren Nicotra Sex Differences in Allodynia 62

Figure 10. Glial Cell Activation in Chronic Pain.

The tetrapartite synapse where neurons, astrocytes and microglia are in close proximity allowing for bidirectional communication, contributes to pathological pain processing.

Several neuronally released mediators are capable of activating nearby astrocytes and microglia. Upon their activation, several released pro-inflammatory mediators contribute to the maintenance phase of chronic pain. In turn, glially derived substances have now been identified as ligands for neurons as well as other glial cells. This exchange of pro- inflammatory information between these neuronal and non-neuronal cells contributes to central sensitization and the plastic changes which result in chronic pain. This figure has been acquired from the Milligan and Watkins review8 with permission.

1.5.2 Toll Like Receptors in Chronic Pain

Critical to the interface between neurons and immunocompetent cells, termed central immune signalling are toll-like receptors (TLRs). TLR central immune signalling is now well established in both the initiation and maintenance of chronic pain as well as several other

Lauren Nicotra Sex Differences in Allodynia 63

CNS pathologies98. Evidence implicating TLRs and their associated signalling constituents in reactive gliosis and their contribution to chronic pain states has been recently reviewed as part of this PhD project and presented here in its peer reviewed and published form.

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Lauren Nicotra Sex Differences in Allodynia 65

Nicotra, L., Loram, L.C., Watkins, L.R. & Hutchinson, M.R. (2012). Toll-like receptors in chronic pain. Experimental Neurology, v. 234 (2), pp. 316-329

NOTE:

This publication is included on pages 66 - 78 in the print

copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1016/j.expneurol.2011.09.038

1.5.3 Summary

Chronic Pain processing is a plastic phenomenon, receiving both neuronal and immune input.

Immnocompetent cells, such as glia are no longer viewed as passive bystanders, but rather neuro- and nociceptive modulators, with bidirectional signalling between neurons and immnocompetent cells now well recognised to contribute to pathological pain. The recognition of the immune system and the discoveries implicating TLRs specifically have contributed to our understanding of the pathology chronic pain. Interestingly, although a relationship between gonadal hormones, particularly the oestrogens, sex and pain, including chronic pain undoubtedly exists, and the oestrogens are well known to modulate pain signalling and inflammation, the relationship between these sex steroid hormones and the innate immune system in chronic pain has not yet been examined. As briefly referred to in my

TLR review paper (located above, Section 1.5.2), several lines of evidence reveal a strong association between E2 and these pattern recognition receptors now well characterised to mediate the central immune signalling events contributing to chronic pain. Deviations in TLR signalling between the sexes under the hormonal control of E2, may partially elucidate the differences in pain sensitivity between the sexes. Supporting evidence identifying this connection between E2 and the innate immune system is presented below.

1.6 Oestrogen and the Innate Immune system: A Role in the Female Prevalence of Chronic Pain

The susceptibility to infectious diseases is biased towards the female sex, with females generally producing greater protective humoral and cell-mediated immune responses347. This more robust response to antigenic challenges has been specifically identified following infection with the herpes simplex viruses348 and influenza349. Interestingly however, sex differences in the response to microbial challenges have also been identified, with sexual

Lauren Nicotra Sex Differences in Allodynia 79

dimorphism in the inflammatory response in the event of TLR4 mediated sepsis, with a 70% mortality rate identified in males, compared with only 26% in infected females350. Important to note are the studies which have extended this finding and specifically revealed enhanced

TLR4 mediated responses generally in females, both clinically and in rodents347, 351-354.

These differences in immune response have been long thought attributable to the gonadal sex steroids. Although androgens are known to suppress immune reactions to bacterial endotoxin challenges355, the oestrogens provide for elevated resistance to bacterial infections and have also been shown to augment and elevate immune responses to such an extent as to increase mortality rates following particular immune challenges356, 357. Further evidencing an association between oestrogens and TLR4 are those studies that have identified elevated E2 levels in sepsis patients and that the severity of the sepsis infection is reliant upon circulating

E2 plasma levels350, 358-360.

As previously discussed in Section 1.3.3.1.2.1, the ER’s are widely located throughout the periphery and CNS, with all cells within the CNS expressing both the classical ER’s, ERα and

ERβ 361, 362. Imperative to emphasize here are the studies that have revealed microglial and astrocytic expression of these steroid receptors, as well as the novel ER, GPR30363-367 368, 369.

ERα astrocyte immunoreactivity was first identified in the guinea pig preoptic area and median eminence in 1992370, with expression of this receptor also identified in cultures of rat astrocytes371, 372. It wasn’t until years later however, that E2 was found to mediate microglial release of pro-inflammatory mediators, as well as stimulate MAPK activation373-378, as well as the expression of microglial neuro-inflammatory genes379, 380. Interestingly, these studies investigating the acute effects of oestrogen revealed anti-inflammatory effects in the brain as well as microglia in vitro.

Lauren Nicotra Sex Differences in Allodynia 80

More recent in vivo studies however, have identified opposing findings. Chronic in vivo E2 administration was first found to enhance the transcription of pro-inflammatory mediators in response to LPS, specifically via the microglial ERα381. These results were later extended upon, further revealing exacerbated release of pro-inflammatory cytokines and NO in microglia following chronic E2 administration to OVX female mice in response to LPS activation382. Others have further extended these findings, with chronic in vivo E2 treatment found to interact with TLR4 signalling, potentiating NFκB activation and amplifying pro- inflammatory cytokine gene expression and release354, 383.

Together, these findings reveal an association between TLR4 signalling and the gonadal steroid hormone explicitly identified to play a role in pain. Given that TLRs, particularly

TLR4 is well characterised as a key contributor in the initiation and maintenance of chronic pain310, 384-395, that E2 is acknowledged to modulate nociception, pain and chronic pain pathophysiology, and that recent studies have recognized an interaction between 17β-estradiol and TLR signaling; perhaps this gonadal hormone-innate immune interaction may contribute to the sex differences seen in pain sensitivity. Although it is currently unknown whether the oestrogens bind to TLRs specifically, this interaction between E2 and microglial TLRs may possibly occur directly at the receptor level.

With the current pain management strategies focused on suppressing neuronal activity and broadly lacking clinical efficacy396, the neuroimmune system is a potential and promising clinical target for future chronic pain therapeutics. Since aberrant microglial TLR signalling exists between the sexes, due to the influence of E2, and microglial TLR activation and signalling is so important in chronic pain, the neuroimmune mechanisms underlying chronic

Lauren Nicotra Sex Differences in Allodynia 81

pain may be divergent between the sexes, which may provide evidence of a need to treat female chronic pain patients differently to males, potentially using glial targeted therapies.

1.7 PhD Summary and Aims

The mechanisms that underlie sex differences in chronic pain whereby females are unfortunately overrepresented in the chronic pain population are still to be determined.

Although the oestrogens have long been known to influence acute pain processing and inflammatory pain, their involvement in chronic pain is far from understood. Contributing to the inability thus far to completely understand the pathophysiology underlying sex differences in chronic pain, as well as the translational issues in regards to chronic pain therapeutics, is the continued use of preclinical methodologies which do not best replicate the clinical pain scenario. Consequently, future preclinical investigations examining the underlying contributions to exaggerated female chronic pain must utilise behavioural testing techniques and animal models that translate well to the clinical setting. In addition to the methodological issues, the failure to investigate or acknowledge the strong association between the oestrogens and the innate immune system and the potential role for this relationship in chronic pain may have resulted in researchers overlooking a fundamental contributor to the female chronic pain problem.

Accordingly, the aims of this PhD project were:

1. To investigate sex differences in chronic pain for the first time using preclinical

methodologies not only capable of replicating the heterogeneity in pain severity

Lauren Nicotra Sex Differences in Allodynia 82

observed clinically but also able to refine the preclinical pain investigation,

thereby adhering to the 3 R’s.

(a) Examine sex differences in chronic pain using the novel graded model of graded

neuropathy

(b) Investigate the role of the female rodent oestrus cycle in this graded neuropathy

model

(c) Develop and test a novel quantitative mechanical allodynia assessment technique

2. To examine the contribution of gonadal hormones, specifically E2 in the novel

graded neuropathy model

(a) Examine sex differences in chronic pain following the removal of ovarian hormones

in the female sex

(b) Investigate rodent mechanical allodynia following the re-introduction of E2 in OVX

females

(c) Investigate the ability to phenocopy the exaggerated female pain phenotype in male

rodents via the supplementation of E2

3. To investigate the association between E2 and the innate immune system in the

novel graded neuropathy model

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(a) Examine the role of female spinal microglial cells in neuropathy induced mechanical

allodynia via the intrathecal transfer of microglia taken from the spinal cords of nerve-

injured female, ovariectomised, and E2 supplemented female mice

(b) Examine whether female microglial cells are inherently pre-disposed to over-respond

to nerve injury via the intrathecal transfer of microglial cells taken from the spinal

cords of non-nerve injured female, ovariectomised, and E2 supplemented female mice

(c) Extend the previous preclinical studies and genetically and pharmacologically

investigate TLR’s and their MyD88 dependent signalling in the graded nerve injury

model

(d) Investigate a potential interaction between innate immune signalling elements TLR’s

2 and 4 and their adaptor protein Myd88, and female rodent microglia in the graded

nerve injury model

(e) Examination of the ability of E2 to bind directly to TLRs in vitro

(f) Examine neuroimmune modulation in exacerbated female mechanical allodynia in the

graded neuropathy model

The overall aim of this body of work is to best investigate the role of sex and the interaction between the gonadal hormones, specifically E2 and the innate immune system in chronic pain using refined and clinically translatable preclinical methodologies. Based on a strong body of literature revealing an association between the oestrogens and pain, the discoveries in the past

20 years detailing a pivotal role of the innate immune system in chronic pain, along with the recent identification of an interaction between these gonadal hormones and this system; this work aims to extend these findings and uncover a role for this gonadal hormone/neuroimmune relationship in the overrepresentation of females in the chronic pain

Lauren Nicotra Sex Differences in Allodynia 84

population and thereby potentially unveil a new avenue for treating female chronic pain patients.

1.8 PhD Synopsis

In Chapter 1 the problem of sex in the chronic pain population was introduced along with a review of the literature establishing the strong relationship between sex, gonadal hormones and chronic pain. Examination of the newly recognised association between the oestrogens and the innate immune system, two systems independently known to contribute to chronic pain pathology, identified the need to investigate this association in chronic pain. Based on the existing literature the aims to be examined in this thesis are then outlined.

Subsequent to this chapter, Chapter 2 details the investigation of sex differences and the role of female gonadal hormones in the novel preclinical graded nerve injury model. The results of this study are presented in a publication which further discusses the importance of investigating future chronic pain investigations using the novel methodologies developed and employed in order to not only uncover the mechanisms underlying sex differences in chronic pain, but to ensure the future use of rodents in preclinical pain investigations.

Extending the findings in Chapter 2, Chapter 3 presents a series of in vivo and in vitro experiments that were designed and executed in order to examine the aims presented in

Section 1.7. This chapter investigates a role not only for E2 in the graded nerve injury model, but the interaction between this gonadal hormone and the innate immune system in the exaggerated female pain phenotype established in Chapter 2.

Lauren Nicotra Sex Differences in Allodynia 85

The final experimental chapter, Chapter 4 extends the pharmacological investigations presented in chapter 3 and genetically investigates the role of the innate immune system in the graded nerve injury model and the function of this system in nerve injury induced exacerbated female mechanical allodynia.

Lastly, the final chapter summaries the findings of this large body of work, relates these discoveries to the current understanding of chronic pain pathophysiology and details the significance of this contribution to the female chronic pain population.

As is the etiquette of presenting a thesis of this style, short linking statements are provided between each published chapter to aid in the flow of the communication of the scientific journey.

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CHAPTER 2. THE EXAMINATION OF SEX AND THE ROLE OF THE

FEMALE OESTRUS CYCLE IN A CLINICALLY TRANSLATABLE

PRECLINICAL CHRONIC PAIN MODEL

It is now well recognised that sex differences exist in chronic pain pathologies (outlined extensively in Chapter 1). The precise mechanisms underlying the female prevalence of chronic pain however still remain unknown. Although the sex steroid hormones have been found contributable to the modulation of pain and chronic pain processing, their potential role in the pathophysiology of exacerbated female chronic pain is still to be determined and more than half the chronic pain population (females) remain inadequately treated.

Contributing to this problem is the continued use of preclinical animal pain models and behavioural techniques that do not best reflect the clinical pain scenario. Many of the existing animal chronic pain models are binomial in their approach (pain vs. control), potentially overlooking subtle changes in the pain response that may unveil mechanisms and potential analgesic targets. Furthermore, with the stringent statutory policies in place restricting the use of animals in pain research there is a definite need to develop and utilise preclinical animal pain models that better reflect the heterogeneity of clinical pain which also reduce animal suffering.

For these reasons this study sought to appropriately investigate sex differences in chronic pain for the first time using the novel graded neuropathy model. This model has been previously demonstrated to better replicate the heterogeneous severity of pain in the chronic pain population. In a further attempt to refine preclinical chronic pain testing, this study also tested

Lauren Nicotra Sex Differences in Allodynia 87

an adapted quantitative allodynia assessment technique, with the aim to successfully investigate the role of sex without having to inflict maximal pain.

This study is the first to examine a role of sex and oestrus cycle in a refined heterogeneous pain model. On comparison with other common von Frey techniques, the adapted von Frey test was the sole method to conclusively demonstrate a sex difference across a heterogeneous pain population and further reveal a role of sex and oestrus cycle in less allodynic animals.

Consequently, this adapted von Frey method combined with the use of a graded model of neuropathy, not only allowed for sensitive determination of subtle influences on female chronic pain, but satisfies the ‘Refinement’ category of the 3 R’s thereby helping to ensure the continued use of rodents in chronic pain research.

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Lauren Nicotra Sex Differences in Allodynia 90

2.1 Sex Differences In Mechanical Allodynia: How Can It Be Preclinically

Quantified And Analysed?

METHODS ARTICLE published: 13 February 2014 BEHAVIORAL NEUROSCIENCE doi: 10.3389/fnbeh.2014.00040 Sex differences in mechanical allodynia: how can it be preclinically quantified and analyzed?

Lauren Nicotra 1*,JonathanTuke2,PeterM.Grace3,PaulE.Rolan1 and Mark R. Hutchinson 4

1 Department of Pharmacology, Neuroimmunopharmacology, The University of Adelaide, Adelaide, SA, Australia 2 Faculty of Engineering, School of Mathematical Sciences, Computer Science and Mathematics, The University of Adelaide, Adelaide, SA, Australia 3 Department of Psychology and Neuroscience, The University of Colorado, Boulder, CO, USA 4 Department of Physiology, Neuroimmunopharmacology, The University of Adelaide, Adelaide, SA, Australia

Edited by: Translating promising preclinical drug discoveries to successful clinical trials remains a Larry J. Young, Emory University significant hurdle in pain research. Although animal models have significantly contributed School of Medicine, USA to understanding chronic pain pathophysiology, the majority of research has focused on Reviewed by: male rodents using testing procedures that produce sex difference data that do not align Michael M. Morgan, Washington State University Vancouver, USA well with comparable clinical experiences. Additionally, the use of animal pain models Dayna L. Averitt, US Army Institute presents ongoing ethical challenges demanding continuing refinement of preclinical of Surgical Research, USA methods. To this end, this study sought to test a quantitative allodynia assessment *Correspondence: technique and associated statistical analysis in a modified graded nerve injury pain model Lauren Nicotra, Department of with the aim to further examine sex differences in allodynia. Graded allodynia was Pharmacology, Neuroimmunopharmacology, The established in male and female Sprague Dawley rats by altering the number of sutures University of Adelaide, Rome Road, placed around the sciatic nerve and quantified by the von Frey test. Linear mixed effects Adelaide, 5000 SA, Australia modeling regressed response on each fixed effect (sex, oestrus cycle, pain treatment). On e-mail: lauren.nicotra@ comparison with other common von Frey assessment techniques, utilizing lower threshold adelaide.edu.au filaments than those ordinarily tested, at 1 s intervals, appropriately and successfully investigated female mechanical allodynia, revealing significant sex and oestrus cycle difference across the graded allodynia that other common behavioral methods were unable to detect. Utilizing this different von Frey approach and graded allodynia model, asinglesutureinflictinglessallodyniawassufficienttodemonstrateexaggeratedfemale mechanical allodynia throughout the phases of dioestrus and pro-oestrus. Refining the von Frey testing method, statistical analysis technique and the use of a graded model of chronic pain, allowed for examination of the influences on female mechanical nociception that other von Frey methods cannot provide.

Keywords: neuropathic pain, von Frey, sex, oestrus cycle

INTRODUCTION stimuli; and allodynia, pain in response to normally innocu- It is now well recognized that sex differences exist in chronic pain ous stimuli (Woolf, 1995). Recent preclinical investigations have pathologies (Ruau et al., 2012). Clinical and experimental studies provided for a greater understanding of the initiation and main- have demonstrated that women are overrepresented in numerous tenance processes underlying neuropathic pain. However, due chronic pain conditions compared to males (Riley et al., 1998; to the continued use of suboptimal preclinical methodologies, Hurley and Adams, 2008; Fillingim et al., 2009). Animal studies substantial mechanistic questions remain, with the reasons for have replicated the clinical pain experience, with female rodents the female prevalence of this chronic pain condition not yet characteristically exhibiting lower thresholds to painful stimuli fully understood. Contributing to the translational issues associ- and exaggerated pain responses following nerve injury compared ated with preclinical pain studies, many investigations examining to males (Lacroix-Fralish et al., 2006a). Evidently, chronic pain is chronic pain mechanisms have done so utilizing animal models, predominantly a female problem. such as chronic constriction injury (CCI), partial sciatic nerve lig- Neuropathic pain is one chronic pain condition with a pre- ation (PSNL) and L5 spinal nerve ligation that do not in fact best dominant number of female sufferers (Riley et al., 1998; Fillingim reflect the clinical heterogeneity of clinical pain (Wang and Wang, et al., 2009). Neuropathic pain is specifically is defined as damage 2003; Mogil, 2009; Grace et al., 2010; Berge, 2011). Consequently, or inflammation of the peripheral nervous system, characterized in order to draw improved information from preclinical stud- by both hyperalgesia, exaggerated pain in response to noxious ies and extrapolate those findings to the clinical situation it is imperative for future studies to develop and utilize novel animal Abbreviations: CCI, chronic constriction injury; CNS, central nervous system; pain models and behavioral testing techniques that more closely PAG, periaqueductal gray; PSNL, partial sciatic nerve ligation; N, sciatic nerve; S, subcutaneous; PO, postoperative; pro, pro-oestrus; oest, oestrus; met, metoestrus; resemble clinical chronic pain and to standardize such procedures di, dioestrus; AIC, Akaike’s Information Criterion. to enable suitable comparisons and improved interpretation of

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results thereby allowing appropriate future investigation into the This study aimed to investigate for the first time the role of sex role of sex in chronic pain pathologies (Bove, 2006). and oestrus cycle in a graded sciatic CCI pain model that produces In addition to the various translational issues associated with heterogeneous degrees of mechanical allodynia. Additionally, the utilizing animal chronic pain models, the use of animals in pain von Frey testing approach employed to assess mechanical allo- research poses numerous ethical dilemmas. Inflicting pain in dynia was also examined combined with trialing of different order to ultimately understand and prevent it is the ethical para- statistical analysis approaches. dox the medical and scientific community have largely accepted in order to validate intentional animal suffering (Zimmermann, METHODS 1986; Dubner, 1987; Tannenbaum, 1999). In the interest of pro- SUBJECTS tecting the welfare of animals used in scientific research the Pathogen-free adult male and female Sprague-Dawley rats deliberate infliction of pain in animals requires strong justifica- (300–350 g; University of Adelaide, Laboratory Animal Services, tion (Carbone, 2012). This stringency is transparent throughout Waite Campus, Urrbrae, Australia) were utilized in all experi- the various global statutory policies that govern the use of animals ments in this study. Rats were housed in temperature- (18–21◦C) in science. Embedded within this legislation are the guiding prin- and light-controlled (12h light/ dark cycle; lights on at 07:00h) ciples of the three R’s, which form a structure ensuring animals rooms where standard rodent food and water was available ad- are only used when absolutely necessary (Replacement), as few libitum. Preceding experimentation, rats were habituated to the animals are used as required to achieve the scientific and statisti- animal holding care facility for 1 week, followed by 1 week cal objectives of the investigation (Reduction) and the models and of extensive experimenter handling and acclimatization to the testing procedures reduce or preclude potential harm, pain and von Frey testing apparatus in order to reduce successive han- distress (Refinement) (Gad, 1990). Despite easily satisfying the dling stress. All procedures were approved by the Animal Ethics replacement parameter of the three R’s with strong evidence for a Committee of the University of Adelaide and were performed in reliance on and a necessity to use animals in pain research (Mogil accordance with the NHMRC Australian code of practice for the et al., 2010), scientists are faced with great difficulty in support- care and use of animals for scientific purposes and adhered to the ing the refinement category in their endeavor to obtain animal guidelines of the Committee for Research and Ethical Issues of ethics with chronic pain investigations which in their very nature the IASP. intentionally inflict persistent pain. Consequently, as the high economic and social stress of exaggerated female pain becomes GROUPS AND DESIGN more apparent, and the interest and need to research the etiol- This study utilized a novel graded sciatic nerve injury model of ogy and management of chronic pain becomes more demanding allodynia (Grace et al., 2010), a modified CCI model in the rat (Casey and Dubner, 1989), there is an evident need to develop (Bennett and Xie, 1988), where 0, 1, 3, or 4 chromic gut sutures animal pain models that not only better reflect the heterogeneity were placed around the sciatic nerve (N), to develop graded of clinical chronic pain, but that are also capable of reducing the behavioral allodynia (varying degrees of allodynia) as described extent of animal suffering. in detail previously (Grace et al., 2010). To ensure the systemic One of the most established chronic pain models is the CCI chromic gut challenge was equivalent between animals (Maves of the sciatic nerve model developed by Bennett and Xie (1988). et al., 1993), additional equivalent chromic gut lengths were Despite the wide application of this rodent pain method within placed subcutaneously (S) over the hip, enabling each treated ani- the preclinical chronic pain literature, one key limitation is the mal to be exposed to a total of four equivalent chromic gut pieces. binary all-or-none nature of the model. That is, the typical exper- Consequently, the treatment groups included N0S0, N0S4, N1S3, imental design consists of a sham control that displays minimal N3S1, and N4S0 animals, with eight males and eight females in alteration in mechanical allodynia, and the four chromic gut each treatment group. Male and female rodents were followed to suture CCI group that display statistically significant and often postoperative (PO) day 21 to determine whether the extent of maximal responses on a behavioral measure such as the von nerve injury (number of chromic gut pieces around the sciatic Frey test. Considering human chronic pain is derived from het- nerve) modified the degree of allodynia. erogeneous injuries, ranging from mild to severe, a model that deliberately generates only marked allodynia in itself is not a true PRE-SURGICAL AND TEST PROCEDURES representation of clinical pain (Grace et al., 2010) and is also Prior to CCI surgery and behavioral testing, a vaginal smear was unable to detect heterogeneity in the pain response. However, the taken from female rats using the common pipette smear tech- recent development of a modified CCI model has been shown to nique to determine oestrus cycle phase (Marcondes et al., 2002). successfully produce graded allodynia in male rodents, through Smears were taken from eight females per phase, with results gen- variation in the number of sutures tied around the sciatic nerve erated from a minimum of four consecutive oestrus cycles, with (Grace et al., 2010). This innovative chronic pain model devel- males “matched” according to assigned animal number and tested oped by Grace and colleagues not only better replicates the on the corresponding female test day. Vaginal smears were taken heterogeneous magnitude of triggers of chronic pain, but is able between 0800 and 1000 each testing and surgical day to minimize to detect a heterogenous pain response owing to differing degrees the incidence of transitional or “missed” stages (Sahar et al., 1997; of pathology, that may provide further investigations a new and Goldman et al., 2007). The four stages of the oestrous cycle pro- improved means to preclinically investigate sex differences in pain oestrus (pro), oestrus (oest), metoestrus (met), and dioestrus (di) sensitivity without having to inflict maximal allodynia. could be recognized by the presence, absence or proportion of

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epithelial, cornified, and leucocyte cells (Goldman et al., 2007). threshold) by fitting a Gaussian integral psychometric func- In an attempt to replicate the small amount of stress experienced tion using a maximum-likelihood fitting method (Harvey, 1997; by female rats during the smear procedure, male rats were given a Treutwein and Strasburger, 1999)asdescribedpreviouslyby parallel injection of 1 ml/kg isotonic saline intraperitoneally. Milligan et al. (2000, 2001).

CHRONIC CONSTRICTION INJURY SURGERY Colburn von Frey method (Test 2) The CCI model of chronic pain was performed at the mid-thigh Mechanical allodynia was also assessed utilizing a von Frey test level of the left hindleg as previously described (Grace et al., which employed phasic application of stimulus at 2 point esti- 2010). Rats were anaesthetized with isoflurane (3% in oxygen), mates along the rodent von Frey logarithmic force scale (the fur shaved over the left mid-thigh and the skin cleaned. The sci- Colburn method; Test 2) (Colburn et al., 1997). Following previ- atic nerve was aseptically exposed and isolated at mid-thigh level. ous pain investigations, all rodents were assessed for mechanical One, three, or four loose chromic gut suture ligatures (cuticular allodynia utilizing a 2 and 12 g von Frey filament (von Frey fil- 4-0 chromic gut, FS-2; Ethicon, Somerville, NJ, USA) were placed aments; Stoelting, Wood Dale, IL, USA), which were applied around the sciatic and once the superficial muscle overlying the within the sciatic innervation region of the left and right hind nerve was sutured, additional chromic gut was placed subcuta- paws. Rats were subjected to a series of three sets of ten stim- neously. Whilst rescue opioid analgesia was on hand to administer ulations per filament, with filaments applied at 1 s intervals. to animals following surgery if an adverse event occurred, no 10 min break was provided in between each set of stimulations such additional analgesia was provided following surgery, as to avoid sensitization (Tanga et al., 2004). Behavioral responses commonly employed opioids have recently been demonstrated were recorded as the average number of responses out of 30 for to exacerbate nerve injury-induced mechanical hypersensitivity either the 2 or 12 g stimulus. (Watkins et al., 2009). Animals were monitored postoperatively until fully ambulatory prior to return to their homecage and von Frey Test 3 checked daily for any sign of infection. No such cases occurred By combining aspects of von Frey Tests 1 and 2, von Frey Test in this study. 3 investigated mechanical allodynia using phasic stimulation of von Frey filaments across a range of thresholds, including lower ALLODYNIA BEHAVIOR ASSESSMENT threshold filaments than those usually examined. Briefly, rats Throughout the study, testing was performed blind with regard to were subjected to 10 stimulations with 6 calibrated von Frey fila- group assignment and oestrus cycle phase. Three diverse von Frey ments (2.83, 0.07; 3.61, 0.40; 4.08, 1; 4.31, 2; 4.74, 6; 5.07, 10 g), approaches were assessed in both male and female rats. All ani- chosen from the series of 10 utilized in von Frey Test 1. von Frey mals were examined using each von Frey technique, with each test filaments were applied for 1 s at 1 s intervals. Filaments were not separated by at least 2 h. Each von Frey test examined mechanical applied in ascending order of force, but rather random assign- allodynia utilizing von Frey filaments across a range of thresh- ment each test session. In order to avoid sensitization, 10 min olds in a sustained or phasic fashion. von Frey analyses examined break was given between each set of stimulations, with 10 stim- in this study included: the classical 8-S method (Milligan et al., ulations per filament. von Frey Test 3 investigated the response 2000)andColburnmethod(Colburn et al., 1997) (henceforward frequency at each von Frey filament and behavioral responses termed Tests 1 and 2 respectively) introduced in detail below, were recorded as the average number of responses out of 10 for in addition to a different von Frey approach, which combined each von Frey stimulus. aspects of Tests 1 and 2 using lower threshold stimuli, henceforth Results for all three von Frey tests that are provided fol- termed Test 3. For all von Frey techniques, testing was performed lowing nerve injury are at the timepoint where allodynia was within the sciatic innervation region of the hindpaws as previ- demonstrated stable (days 17–21). ously described in detail (Chacur et al., 2001; Milligan et al., 2001). Allodynia was characterized in all three behavioral tests STATISTICS as an intense paw withdrawal or licking of the stimulated hind For each of the von Frey tests, 1, 2, and 3, the relationship paw. Assessments were made before surgery (baseline) and on between the percent response and the variables sex, oestrus postoperative days 3, 7, 10, 14, 17, and 21. cycle phase, von Frey filament stimulus and surgery was assessed using linear modeling fitted using the statistical package R (R 8-S von Frey method (Test 1) Development Core Team, 2011)viathegraphicaluserinterface: This test was performed as described previously (Milligan et al., Rstudio (RStudio). 2000). Briefly, a logarithmic series of 10 calibrated Semmes- Initially a linear model was fitted that aimed to predict the per- Weinstein monofilaments (von Frey filaments; Stoelting, Wood cent response (number of positive responses out of a maximum Dale, IL, USA) were applied for 8 s randomly to the left and of 30 (von Frey Test 2) or 10 (von Frey Test 3) by using the pre- right hindpaws of all animals in order to characterize the thresh- dictors sex, oestrus cycle phase, von Frey filament stimulus and old stimulus intensity necessary to produce a paw withdrawal surgery. The goal was to estimate the average increase in percent response. Log stiffness of the filaments was determined by log10 response for each increase in von Frey filament stiffness. We also (milligrams 10) and ranged from manufacturer designated 2.83 wanted to estimate how this average increase is influenced by the × (0.07 g) to 5.18 (15.136 g) filaments. Behavioral responses were various levels of sex, oestous cycle phase and surgery. As well, a used to calculate absolute threshold (the 50% paw withdrawal modified linear modeling method was used, called mixed effect

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linear models, that had an extra term to account for the fact that Again the model used accounted for the repeated measures obtain we had repeated measures on the rats. This method allows us to from each rat. account for the variation seen in the percent response both within An example R input of such subsets to investigate certain and between the rats. Believing that there was a possibility for the predictors: influence of surgery and oestrous to depend on the rat’s sex, we Example 1. In order to investigate the effect of the female also added two terms to account for these interactions. The initial rodent oestrus cycle, male rodent data was excluded from the model (M1) was, therefore, analysis. Below if the example R input in order to investigate this predictor individually: response ~ sex + oestrus + surgery + von Frey stiffness + sex:oestrus + sex:surgery #Modeldataexcludingmales:specifically +(1|rat) investigating the effect of the oestrus cycle predictor: where response is the response rate expressed as a percentage (% response), sex is male or female, oestrus is pro-oestrus, M1<- lmer(response~(von Frey stiffness/ oestrus, metoestrus or dioestrus, surgery is N0S0, N0S4, N1S3, surgery/sex/(oestrus)+ (1|rat), data=subset N3S1, N4S0, von Frey stiffness is 1–6 and rat is the unique rat ID. (data, sex==’female’)) The model was fitted using the lmer() function from the lme4 summary(M1) package in R. The code is available on request from the authors. An example R input would appear as: where response is the response rate expressed as a percent- age (% response), sex is female, oestrus is pro-oestrus, oestrus, M1 <- lmer(response ~ sex + oestrus + metoestrus or dioestrus, surgery is N0S0, N0S4, N1S3, N3S1, surgery + von-Frey stiffness + sex:oestrus N4S0, von Frey stiffness is 1–6 and rat is the unique rat ID. +sex:surgery+(1|rat),data=data) von Frey Test 2 was also examined using two different summary(M1) approaches. For each predictor (sex, oestrous cycle phase, surgery and von Frey filament stimulus), the difference between the allo- To ensure the simplest model to predict the percent response, the dynia (percentage response for the 2 g von Frey filament) for each initial model (M1) was tested to assess if any for the predictors level of the predictor was estimated. This was also repeated for could be removed as there are not statistically significant predic- the 12 g von Frey filament. Alternatively, an analysis model for tors. The ability of the model to predict the percent response was covariance approach was utilized to assess the difference in inter- measured using the Akaike’s Information Criterion (AIC). The cept and slopes for linear regression lines fitted with allodynia AIC measures how well the model fits the observations with a (percentage response) as the response variable and von Frey fila- penalty term for the number of terms in the model. The penalty ment stiffness as the predictor, with difference regression lines for term is to try and ensure the most parsimonious model. Each pre- each level of the fixed effect. The different approaches are equiva- dictor was removed from the model and the AIC measured to see lent but give difference contexts to compare the levels of the fixed if this caused a change in the AIC. The model with the small- effects. est AIC was chosen. This process is repeated until the simplest model that predicts the percent response the best is obtained. This RESULTS process is automated by the stepAIC() function from the MASS EXPERIMENT 1: IMPORTANCE OF von Frey APPROACH: CHOICE OF von package (Venables and Ripley, 2002). This procedure indicated Frey TEST DETERMINES SEX DIFFERENCE AT BASELINE that none of the predictors could be removed without reducing Statistical differences for covariates generated utilizing von Frey the predictive ability of the model. Test 1 are expressed as the average difference in absolute thresh- There is controversy regarding P-values for mixed effects mod- old (the 50% paw withdrawal threshold). Investigating the role els (Bates, 2006)andthus,wereportobservedt-values rather of sex in the graded nerve injury model revealed the inability than P-values and use an observed t-value with absolute value of this von Frey test to distinguish between a female rodent paw of less than negative two or greater than two to indicate statis- withdrawal that was indicative of allodynia, or a paw withdrawal tical significance. Owing to the statistical power of the model arising from repeated filament stimulation. As a consequence, statistically significant but behaviorally small differences can be females tested utilizing von Frey Test 1 were found to respond identified. As such, we only report statistical differences that are at the lowest von Frey filament examined (0.04 g force) and dis- behaviorally relevant, representing for Test 2 a difference in per- played extremely low absolute thresholds prior to nerve injury, centage response of greater than 3.3, and 1.6% for von Frey responding significantly more than males prior to nerve injury test 3, as this represents a change in 1 response out of 10 on (t 14.0) (Figure 1). Accordingly, this von Frey technique failed = the tests. to differentiate between factual observation (positive paw with- To further elucidate differences in allodynia (percentage drawal) and the inference (pain) and as a consequence females response) and each of the predictors (sex, oestrous cycle phase, were inappropriately deemed in pain, which did not resemble the surgery and von Frey filament stimulus), subsets of the data factual scenario at baseline. In light of the inability to appro- were considered (e.g., males only) and models fitted that pre- priately determine a female paw withdrawal response, this sex dicted percent response for each of the predictors individually. difference was thereby rendered meaningless and further results

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obtained using this von Frey approach are subsequently excluded model. This study followed the traditional statistical approach from discussion. whereby responses generated were independently exam- ined at the 2 and 12 g von Frey filaments (DeLeo and EXPERIMENT 2: von Frey TEST 2 DISPLAYS NO SEX DIFFERENCES AT Rutkowski, 2000; Sweitzer et al., 2001; LaCroix-Fralish et al., BASELINE, REPLICATING HEALTHY HUMAN PAIN SEX DIFFERENCES 2005a,b, 2006b). Results generated at individual von Frey Mechanical allodynia was examined using von Frey Test 2 filaments are expressed as the average difference in percent for the first time in a graded allodynia preclinical animal response. Prior to nerve injury, baseline responses generated by von Frey Test 2 reflected healthy male and female nociceptive thresh- olds, displaying no significant difference between the sexes (Sarlani and Greenspan, 2002; Sarlani et al., 2004). Matched male and female rodents were found to respond equivalently at both the 2 and 12 g von Frey filaments prior to nerve injury (2 g, t 0.20; 12 g, t 0.43; Table 1), with the average dif- = = ference in percent response failing to reach behavioral signifi- cance (2 g: 0.21%; 12 g: 0.42%; Table 1). Prior to nerve injury, this testing method further revealed the absence of an oestrus cycle effect. At baseline, female responses were unaffected by the rodent oestrus cycle, with an average percent difference in response of only 0.40% between phases at both the 2g (t 2 ≥− but 1.7, Table 2)and12g(t 2but 0.27, Table 3)vonFrey ≤ ≥− ≤ filaments.

von Frey TEST 2 REVEALS GRADED CHRONIC PAIN IN THE FEMALE RAT Application of von Frey Test 2 and statistical analysis at the FIGURE 1 | Inability of von Frey method 1 to replicate healthy human independent 2 and 12 gram von Frey filaments demonstrated pain thresholds. Prior to nerve injury female mechanical allodynia was unable to be appropriately assessed utilizing the common 8-S method (von graded chronic pain for the first time in both the male and Frey Test 1). Results demonstrated the inability to distinguish female paw female rat. Increasing the number of sciatic sutures significantly withdrawals indicative of allodynia, or those arising from repeated filament increased the number of responses indicative of allodynia for stimulation. Consequently, female rats displayed exceptionally low absolute both sexes (2 g: t 4.4; 12 g: t 11.0, Table 1). Furthermore, thresholds compared to males across oestrus cycle phases prior to nerve ≤− ≤− chromic gut itself was demonstrated to cause allodynia, with injury (t 14), contrasting healthy human pain thresholds. Assessments = were made prior to CCI and behavioral responses were used to calculate N0S4 males and females having significantly greater allodynia absolute threshold (the 50% paw withdrawal threshold) by fitting a compared to N0S0 rats (t 2.6, Table 1). Consequently, exam- ≤− Gaussian integral psychometric function using a maximum-likelihood fitting ining responses at independent von Frey filaments replicated method. A t-value of < 2or>2 was determined statistically significant − previous findings of a graded chronic pain model in the male (p < 0.05). n 8peroestrusphase,persex.Di,dioestrus;Met, = metoestrus; Oest, oestrus; Pro, pro-oestrus. rodent and demonstrated an allodynia dose-response relationship for the first time in females.

Table 1 | Traditional statistical analysis at independent von Frey Filaments for von Frey Test 2 revealed equivalent male and female baseline (Pre-Surgery) responses.

2gMale 2gFemale 2gDifference 2gIsthesex 12gMale 12gFemale 12gDifference 12gIsthesex (% response) (% response) in percent difference (% response) (% response) in percent difference response (%) significant? response (%) significant? male:female (t )male:female male:female (t )male:female = = Pre-surgery 3.8 0.12 4.0 0.18 0.21 0.20 9.0 0.18 9.2 0.14 0.42 0.43 ± ± ± ± − − N0S0 4.6 0.23 4.8 0.19 0.21 0.20 9.2 0.12 10.0 0.17 0.42 0.43 ± ± ± ± − − N0S4 9.4 0.21 12.0 0.24 2.5 1. 6 11 .0 0.24 18.0 0.22 3.5 2.3 ± ± − − ± ± − − N1S3 14.0 0.31 17.0 0.30 2.9 2.7 26.0 0.33 32.0 0.32 6.5 6.3 ± ± − − ± ± − − N3S1 27.0 0.31 41.0 0.31 14.0 7. 3 38.0 0.22 60.0 0.24 22.0 11.0 ± ± − − ± ± − − N4S0 36.0 0.38 51.0 0.42 15.0 8.1 52.0 0.31 78.0 0.38 27.0 13.0 ± ± − − ± ± − − Once allodynia became stable (days 17–21) N4S0 and N3S1 females were significantly more allodynic than matched males however, a significant sex difference was not confirmed for N1S3 rodents, failing to demonstrate this sex effect using the 2 g von Frey filament. Results generated at individual von Frey filaments are expressed as the average difference in percent response, generated from the response rates (the percent response /30; Ta b l e s A 1 , A2). A t-value of < 2or>2 − was determined statistically significant (p < 0.05) when difference in percentage response were greater than 3.3%. n 8pertreatmentgroup,persex.N,number = of sciatic sutures, S, number of subcutaneous sutures; g, grams force.

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Table 2.1 Traditional Statistical Analysis at Independent von Frey Filaments for von Frey Test 2

Figure 2.1 Inability of von Frey Method 1 to Replicate Healthy Human Pain Thresholds

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Table 2 | Traditional statistical analysis at the independent 2g von Frey Filament for von Frey Test 2 revealed the absence of an oestrus cycle effect prior to nerve injury (Pre-Surgery).

PRO:OEST PRO:MET PRO:DI OEST:MET DI:OEST DI:MET

% t % t % t % t % t % t ======Pre-surgery 0.77 0.19 0.60 0.58 0.53 0.15 0.20 0.39 0.25 0.34 0.051 1.7 − − − − − − − − N0S0 0.30 1.91.11.70.80 0.80.80 1.7 0.51 0.83 0.30 1.5 − − − N0S4 2.11.02.01.7 1.11.1 0.11 1.33.20.86 3.10.67 − − − N1S3 5.1 6.7 5.8 6.3 0.51 0.60 0.27 0.42 7. 6 6 . 7 7. 3 6 . 3 − − N3S1 1.20.97 1.21.0 0.93 0.57 7.81.12.2 1.32.20.87 − − − − N4S0 0.70 1.63.11.7 6.0 0.073 2.70.64 2.70.74 2 .31.2 − − Once allodynia became stable (days 17–21) females (N1S3) in pro-oestrus and dioestrus displayed heightened mechanical sensitivity. Results generated at individual von Frey filaments are expressed as the average difference in percent response, generated from the response rates (the percent response /30; Ta b l e A 1 ). A t-value of < 2or>2 was determined statistically significant (p < 0.05) when difference in percentage response were greater than 3.3%. n 8pertreatmentgroup,per − = sex. N, number of sciatic sutures, S, number of subcutaneous sutures; g, grams force. %, the average difference in percent response; t , is there a significant = effect of oestrus cycle?

Table 3 | Traditional statistical analysis at the independent 12 g von Frey Filament for von Frey Test 2 revealed the absence of an oestrus cycle effect prior to nerve injury (Pre-Surgery).

PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

% t % t % t % t % t % t ======Pre-surgery 1.7 0.39 1.3 0.29 1.10.27 0.44 0.79 0.48 0.64 0.10 1.3 − − − − − − − − − − N0S0 0.90.42 0.44 1.20.48 0.40 0.54 0.71 0.77 0.32 1.01.3 − − N0S4 1.12.1 0.11 1.7 1.00.90 1.6 0.71 2.70.22 1.42.4 − − − − N1S3 2.42.2 6.3 3.7 0.25 1.2 0.18 0.24 2.61.92.03.1 − − N3S1 0.27 0.19 2.72.1 0.32 0.14 0.44 1.70.43 0.83 0.43 2.2 − − − N4S0 0.12 2.30.81 1.9 1.40.024 0.50 0.92 1.22.12.12.1 − An oestrus cycle effect could not be conclusively established following nerve injury with females responding significantly more throughout pro-oestrus, but only when compared to metoestrus and no other cycle phase. Results generated at individual von Frey filaments are expressed as the average difference in percent response, generated from the response rates (the percent response /30; Ta b l e A 2 ). A t-value of < 2or>2 was determined statistically significant (p < 0.05) when − difference in percentage response were greater than 3.3%. n 8pertreatmentgroup,persex.n 8peroestrusphase.N,numberofsciaticsutures;S,numberof = = subcutaneous sutures; g, grams force. Di, dioestrus; Met, metoestrus; Oest, oestrus; Pro, pro-oestrus. %, the average difference in percent response; t , is there = asignificanteffectofoestruscycle?

ASSESSMENT OF MECHANICAL HYPERSENSITIVITY AT INDIVIDUAL INDEPENDENT FILAMENT ANALYSIS FAILS TO DETERMINE AN von Frey FILAMENTS FAILS TO APPROPRIATELY INVESTIGATE THE OESTRUS CYCLE EFFECT ROLE OF SEX IN ALLODYNIA Traditional statistical analysis at independent von Frey filaments Sex differences in mechanical allodynia were subsequently exam- demonstrated the inability for von Frey Test 2 to conclusively ined in the graded chronic pain model utilizing von Frey Test 2. demonstrate an oestrus cycle effect on female mechanical allody- When allodynia became stable, females (N4S0) were significantly nia following nerve injury. When allodynia became stable, females more allodynic than matched males (2 g: 15.0%, t 8.1; 12 g: (N1S3) displayed heightened mechanical sensitivity throughout = − 27.0%, t 13.0, Table 1), replicating previous studies that have both dioestrus and pro-oestrus (2g: 5.1%, t 6.3, Table 2). = − ≥ ≥ utilized the traditional CCI four-suture approach. This study fur- These results however, were not replicated using the 12 g von Frey ther demonstrated for the first time a sex difference in moderately filament, where females responded significantly more throughout allodynic animals, with N3S1 females responding significantly pro-oestrus, but only when compared to metoestrus and no other more than matched N3S1 males (2 g: 14.0% t 7.3; 12 g: cycle phase (Pro:Met, 6.3%, t 3.7, Table 3). = − = 22.0%, t 11.0, Table 1). This sex difference, however, was not = − conclusively established in N1S3 rodents, failing to refine pre- INVESTIGATING THE ROLE OF SEX AND THE OESTRUS CYCLE USING clinical chronic pain testing. Although N1S3 females were found von Frey METHOD 2: EXAMINING DIFFERENCES ACROSS von Frey significantly more allodynic than matched males when tested uti- FILAMENTS lizing the 12 g von Frey filament (6.5%, t 6.3, Table 1), this Considering the inability of von Frey Test 2 to conclusively estab- = − sex difference was not replicated with the 2 g filament (2.9%, lish both sex and oestrus cycle differences using the traditional t 2.7, Table 1). statistical approach whereby mechanical allodynia is assessed at = −

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Table 2.2 Traditional Statistical Analysis at the Independent 2 g von Frey Filament

Table 2.3 Traditional Statistical Analysis at the Independent 12 g von Frey Filament for vn Frey Test 2 Revealed the Absence of an Oestrus Cycle Effect Prior to Nerve Injury (Pre- Surgery)

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independent von Frey filaments, this study examined an alterna- (male:female, 21.0%, t 6.9, Table 4)andN3S1rats = − tive statistical approach whereby the relationship between the two (male:female, 18.0%, t 7.5, Table 4). This sex difference = − point estimates was examined (slope). Considering the ability to however, was not demonstrated between N1S3 rodents (1.0%, accurately estimate a difference in slope utilizing data from upper t 1.7, Table 4) and female rodent mechanical allodynia was = − and lower limits of a scale, this study investigated the estimate of further demonstrated unaffected by the oestrus cycle for all pain the slope of the linear relationship between the response and von treatment groups ( 3.1% but 3.0%, t 1.8but 1.1; ≥− ≤ ≥− ≤ Frey filament. Results are expressed as the percent difference in Table 5). slope between the response and two von Frey filaments. Following an examination of a difference in slope, von Frey EXPERIMENT 3: von Frey TEST 3: A BEHAVIORAL TECHNIQUE TO Test 2 revealed baseline rodent allodynia scores reflected healthy INVESTIGATE THE ROLE OF SEX AND THE RODENT OESTRUS CYCLE IN human pain thresholds (Sarlani and Greenspan, 2002; Sarlani MECHANICAL HYPERSENSITIVITY et al., 2004), with males and females responding equivalently von Frey Test 3 examined the relationship between the response prior to nerve injury (male:female, 0.25%, t 0.71, Table 4). and the force of the von Frey filament. Results generated by von − = − Moreover, prior to nerve injury, the rodent oestrus cycle also Frey Test 3 are expressed as the percent difference in response per failed to influence female allodynia scores, with less than a 1% dif- increase in von Frey hair stiffness. ference in slope between phases, replicating the findings at both Utilizing von Frey Test 3, graded mechanical allodynia was the 2 and 12 g von Frey filaments (Section Experiment 2: von Frey established in both the male and female rat once allodynia became Test2DisplaysNoSexDifferences atBaseline,ReplicatingHealthy stable. Following nerve injury, increasing the number of sci- Human Pain Sex Differences) (t 1.1but 1.4, Table 5). atic sutures significantly increased the level of allodynia in both ≥− ≤ Agradedallodyniaresponsewasalsorevealedwhendetermin- rodent sexes ( 1.6%, t 4.9; Table 6, Figure 2). Of particular ≤− ≥ ing a slope difference between pain treatment groups following importance, a significant differentiation was established between nerve injury. Increasing the number of sciatic sutures was demon- N0S0 and N0S4 males and females specifically, where N0S4 strated to significantly increase the number of responses indica- rodents responded significantly more compared to N0S0 rats tive of allodynia for both sexes ( 11.0%, t 5.6, Table 4). ( 2.3%, t 2.4, Table 6), indicating chromic gut itself causes ≥ ≤− ≥ ≥ This robust mechanical allodynia was observed in both male and allodynia. female rats once allodynia became stable. Prior to CCI, male and female rats examined using this von Post nerve injury, von Frey Test 2 demonstrated females Frey testing method were found to respond equivalently, with an were considerably more allodynic than males in the graded average difference in response per increase in von Frey hair stiff- nerve injury model. Once allodynia became stable, a signif- ness of only 0.83% (Table 6, Figure 2), thereby replicating the icant difference in slope was demonstrated between N4S0 clinical pain scenario. Female rodent mechanical allodynia was also demonstrated unaffected by the rodent oestrus cycle prior to nerve injury, with an average difference of merely 1% between Table 4 | Responses generated from von Frey Test 2 were further used phases (t 1.5but 1.6, Table 7). ≥− ≤ to examine the estimate of the slope of the linear relationship In contrast to Test 2, von Frey Test 3 not only revealed a sex between the response and von Frey filament. difference in mechanical sensitivity following nerve injury but Male Female Difference in Is the sex further revealed the ability of the novel sciatic nerve injury model (% response) (% response) slope (%) difference to refine the traditional CCI technique (Figure 3). Once allodynia male:female significant? became stable, application of von Frey testing method 3 revealed (t ) exacerbated clinical female pain, with a significant difference in = male:female response per increase in von Frey hair stiffness between N1S3 and N3S1 males and females. For the first time, N1S3 females Pre-surgery 6.46.6 0.25 0.71 were found to respond 3.6 % more than matched males per von − − N0S0 7.17.2 0.10 0.12 Frey filament (t 3.4; Table 6, Figure 3A)andN3S1females − − = − N0S4 12.015.0 3.0 2.1 responding 3.4% more than matched males per von Frey fil- − − N1S3 20.0 21.0 1.0 1.7 ament (t 7.7; Table 6, Figure 3B). von Frey Test 3 further − − = − N3S1 33.0 50.0 18.0 7. 5 revealed a hormonal influence on female mechanical hypersen- − − N4S0 44.0 65.0 21.0 6.9 sitivity once allodynia became stable. Unlike Test 2, this von Frey − − testing method revealed exacerbated female mechanical allodynia This alternative statistical analysis revealed equivalent male and female throughout the phases of dioestrus and pro-oestrus in both N1S3 responses prior to nerve injury (Pre-Surgery) however a sex difference was and N3S1 rodents (N1S3: 1.7%, t 2.2; N3S1: 2.1%, t 3.3, confirmed once allodynia became stable (days 17–21) where N3S1 and N4S0 ≥ ≥ ≥ ≥ Table 7, Figure 4). females were found significantly ore allodynic than matched males. Results are expressed as the percent difference in slope between the response and two von Frey filaments, generated from response rates (the percent response /30 across DISCUSSION von Frey filaments; Ta b l e A 3 ). A t-value of < 2or> 2 was determined statisti- This study aimed to investigate for the first time the role of sex and − cally significant (p < 0.05) when difference in percentage response were greater oestrus cycle in a graded sciatic CCI pain model that produces than 3.3%. n 8pertreatmentgroup,persex.N,numberofsciaticsutures;S, heterogeneous degrees of mechanical allodynia. Additionally, = number of subcutaneous sutures; g, grams force. the von Frey testing approach employed to assess mechanical

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Table 2.4 Responses Generated from von Frey Test 2 were Further Used to Examine the Estimate of the Slope of the Linear Relationship Between the Response and von Frey Filament

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Table 5 | Examining the estimate of the slope of the linear relationship between the response and von Frey filament for von Frey Test 2 revealed the absence of an oestrus cycle effect across all surgery groups.

PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

% t % t % t % t % t % t ======Pre-Surgery 0.44 1.4 0.38 1.1 0.018 0.050 0.059 0.19 0.29 1.2 0.37 1.1 − − − − − − N0S0 0.42 0.29 0.56 0.51 1.3 1.1 0.11 0.10 1.7 1.1 1.8 0.86 − − − − − − − − − N0S4 2.41.10.10 1.21.1 0.19 1.0 0.86 0.11 1.80 0.91 0.97 − − − − − − − N1S3 1.80.78 1.9 1.21.10.36 0.21 0.029 2.9 0.65 3.1 0.73 − − − − − − − − − N3S1 1.9 0.22 2.5 0.43 1.8 0.31 1.0 0.11 0.025 0.0030 1.08 0.79 − − − − − − − − − − − N4S0 1.1 0.16 2.70.61 1.80.28 3.00.71 3.0 0.40 0.012 0.32 − − − − − Results are expressed as the percent difference in slope between the response and two von Frey filaments, generated from response rates (the percent response /30 across von Frey filaments; Ta b l e A 3 ). A t-value of < 2or>2 was determined statistically significant (p < 0.05) when difference in percentage response were greater − than 3.3%. n 8pertreatmentgroup,persex.n 8peroestrusphase.N,numberofsciaticsutures;S,numberofsubcutaneoussutures;g,gramsforce.Di, = = dioestrus; Met, metoestrus; Oest, oestrus; Pro, pro-oestrus. %, the average difference in percent response; t , is there a significant effect of oestrus cycle? =

with similar conclusions able to be drawn employing only one Table 6 | von Frey Test 3 examined the relationship between the suture. response and the force of the von Frey filament, across a range of In order to preclinically investigate the mechanisms underly- filaments. ing the female prevalence of chronic pain, studies need to employ Male (% Female (% Difference in Is the sex models and behavioral testing methods that best replicate the response) response) response per difference clinical scenario. To examine sex differences in chronic pain uti- increase in von significant? lizing the novel graded sciatic nerve injury model, this study Frey hair male:female investigated male and female mechanical nociception using three stiffness von Frey testing approaches. Despite numerous studies exploit- male:female ing the technique of von Frey to investigate rodent mechanical hypersensitivity, this study revealed the profound differences in Pre-surgery 3.9 0.11 4.7 0.10 0.83 1.1 ± ± − − conclusions that can be drawn from the use of different von Frey N0S0 4.0 0.23 4.9 0.28 0.64 1.8 ± ± − − procedures, as well as the importance of the statistical approach N0S4 6.3 0.24 8.1 0.30 0.80 1.6 ± ± − to investigate female rodent mechanical hypersensitivity. N1S3 6.4 0.29 10.1 0.30 3.6 3.4 ± ± − − Experiment 1 investigated the role of sex prior to nerve injury N3S1 8.6 0.32 12.0 0.30 3.4 7. 7 ± ± − − utilizing von Frey Test 1. This study demonstrated the inabil- N4S0 12.1 0.48 13.6 0.50 1.5 1.7 ± ± − − ity of this test to appropriately infer female pain from a positive Prior to nerve injury (Pre-Surgery), von Frey Test 3 determined equivalent male paw withdrawal given that females were erroneously found sig- and female responses. Once allodynia became stable (days 17–21) a sex differ- nificantly more allodynic than males. Since adapted by Milligan ence was established, with N1S3 and N3S1 females significantly more allodynic et al. (2000), von Frey Test 1 has successfully investigated both than matched males. Results generated by von Frey Test 3 are expressed as the the attenuation and the exacerbation of male mechanical hyper- percent difference in response per increase in von Frey hair stiffness, generated sensitivity (Milligan et al., 2003, 2004; Ledeboer et al., 2005; from the estimated increase in percent response per increase in von Frey stiff- Hutchinson et al., 2008, 2009, 2010; Grace et al., 2010; Loram ness; Ta b l e A 4 .At-valueof< 2or>2 was determined statistically significant et al., 2011). To our knowledge however, within the very few − (p < 0.05) when differences in percentage response were greater than 1.6%. investigations that have utilized this particular von Frey test to n 8pertreatmentgroup,persex.N,numberofsciaticsutures;S,numberof = examine sex differences in chronic pain, some have also reported subcutaneous sutures; g, grams force. non-sensically high female baseline mechanical thresholds (prior to any pharmacological, genetic or surgical intervention). Such allodynia was also examined combined with trialing of different was demonstrated in a study by Mogil and Colleagues investi- statistical analysis approaches. gating sex differences in mechanical allodynia in C57BL/6 mice This aim was successfully achieved utilizing a modified von utilizing a comparable von Frey technique (Mogil et al., 2006). Frey testing approach, accompanied by unique statistical analy- In our study, observed higher baseline female activity misinter- sis. Here we demonstrated that testing the number of responses preted by this particular application of von Frey as a pain response out of 10 tests across all 6 von Frey hairs in N1S3 sciatic injury may potentially elucidate lower female mechanical thresholds was sufficient to observe significant sex and oestrous phase compared with males. Interestingly, in a discussion of the issues effects. In contrast, the other two approaches were not able to associated with monofilament pain testing, Bove identifies that achieve this degree of behavioral sensitivity. Importantly, the the application of sustained filament stimuli may in fact be use of the graded constriction injury identified that the cur- examining different sensory modalities such as itch, rather than rent four suture model produces unnecessary maximal allodynia pain (Bove, 2006). Considering the already known translatability

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Table 2.5 Examining the Estimate of the Slope of the Linear Relationship Between the Response and von Frey Filament for von Frey Test 2

Table 2.6 von Frey Test 3 Examined the Relationship Between the Response and the Force of the von Frey Filament, Across a Range of Filaments. Lauren Nicotra Sex Differences in Allodynia 98

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FIGURE 2 | von Frey test 3 demonstrates the ability to investigate without having to inflict maximal pain (t 3.4) when investigating the = − chronic pain mechanisms without inflicting maximal pain. difference in percent response per increase in von Frey filament stiffness. Appropriately investigating the role of sex in chronic pain became von Frey Test 3 data is presented as a 3D surface plot of the response possible utilizing von Frey Test 3. Using this adapted behavioral test this rate out of 10, recorded as a percentage. 3D surface plots were study demonstrated the successful generation of a graded chronic pain generated using the R Studio lattice package. Results were analyzed model in both the male and female rat. Increasing the number of sciatic utilizing repeated measures linear mixed effects modeling. A t-value sutures was found to significantly increase the percentage response rate of < 2or>2 was determined statistically significant (p < 0.05). n 8 − = per von Frey filament in both males (A) and females (B) ( 1. 6 % , per treatment group, per sex. von Frey filaments: 6: 10, 5: 6, 4: 2, 3: ≤− t 4.9). Illustrating refinement of the traditional CCI procedure, females 0.6, 2: 0.4, 1: 0.07 g. N, number of sciatic sutures; S, number of ≥ (N1S3, N3S1) were demonstrated statistically more allodynic than males subcutaneous sutures; g, grams force.

Table 7 | von Frey Test 3 examined the relationship between the response and the force of the von Frey filament, across a range of filaments.

PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

% t % t % t % t % t % t ======Pre-Surgery 0.83 0.88 1.43 1.5 0.95 0.83 0.59 0.69 0.20 0.98 0.41.6 − − − − − − − N0S0 0.10 0.68 0.10 1.20.20 0.68 0.20 0.45 1.1 0.67 0.91.2 − − − − − − N0S4 1.1 1.1 0.60 0.63 1.0 0.34 0.50 0.60 0.80 0.94 0.30 0.40 − − − − − − − − − − N1S3 1.7 2.2 2.1 2.4 0.20 0.020 0.40 0.49 2.0 2.6 1.9 2.4 − N3S1 2.1 3.5 2.9 3.4 0.097 0.12 0.80 1.4 2.1 3.3 3.0 3.0 − − N4S0 1.01.31.00.39 1.00.74 0 .010 0.94 0.013 0.60 0.012 0.40 − − − Prior to nerve injury (Pre-Surgery), the rodent oestrus cycle did not influence female rodent responses. Following nerve injury, once allodynia became stable (days 17–21) von Frey Test 3 revealed an oestrus cycle effect, with N1S3 and N3S1 females significantly more allodynic throughout pro-oestrus and dioestrus.Results generated by von Frey Test 3 are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness; Ta b l e A 4 .At-valueof< 2or>2 was determined statistically significant (p < 0.05) when differences in − percentage response were greater than 1.6%. n 8peroestrusphase.N,numberofsciaticsutures;S,numberofsubcutaneoussutures;g,gramsforce.Di, = dioestrus; Met, metoestrus; Oest, oestrus; Pro, pro-oestrus. %, the average difference in percent response; t , is there a significant effect of oestrus cycle? =

issues associated with preclinical animal pain studies, the contin- oestrus cycle. Following the traditional statistical approach to ued use of behavioral paradigms that fail to appropriately infer this method whereby allodynia was examined at two indepen- pain casts further doubt on the validity of animal pain studies dent von Frey filaments, a large degree of variability between von (Mogil, 2009). As a direct result, this study questions the valid- Frey hairs led to the need for an alternative statistical analysis. ity of this method’s use in future pain investigations examining Although the majority of chronic pain investigations conven- female mechanical allodynia. tionally analyse mechanical allodynia using a logistic equation Although previously utilized by chronic pain investigations (using essential features of the stimulus response function such to examine rodent mechanical allodynia, this study employed as the maximal response, the response threshold and the 50% von Frey Test 2 for the first time in a graded allodynia preclin- of maximal response), data generated from von Frey Test 2 at ical animal model to investigate the role of sex and the rodent only two points along the von Frey logarithmic force scale did

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Table 2.7 von Frey Test 3 Examined the Relationship Between the Response and the Force of the von Frey Filament, Across a Range of Filaments.

Figure 2.2 Von Frey Test 3 Demonstrates the Ability to Investigate Chronic Pain Mechanisms Without Inflicting Maximal Pain

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FIGURE 3 | von Frey test 3: a behavioral technique refining the (A) and the same was found for N3S1 female rodents (B).Results investigation of the role of sex and rodent oestrus cycle in generated by von Frey Test 3 are expressed as the percent difference in mechanical hypersensitivity. Through to 21 days following nerve injury, response per increase in von Frey hair stiffness. Results were analyzed von Frey Test 3 was the sole behavioral testing method examined that utilizing repeated measures linear mixed effects modeling. A t-value identified a contributing role of sex and oestrus cycle in female of < 2or>2 was determined statistically significant (p < 0.05). n 8 − = mechanical allodynia examined in the graded nerve injury model. N1S3 per treatment group, per sex. N, number of sciatic sutures; S, number of females were found significantly more allodynic than matched males subcutaneous sutures; g, grams force.

not permit this conventional statistical analysis (owing to the lack cycle differences in less allodynic rodents (N1S3 subjects). In of data). Rather the estimates generated from upper and lower order to compare the findings between von Frey Tests 2 and 3, limits of this scale enabled the capacity to accurately estimate the and distinguish where von Frey Test 3 in fact identified sex and slope between response and von Frey filament. In doing so, this oestrus cycle differences that Test2 was unable to detect, this study unconventional statistical approach provided an improved means unconventionally examined the relationship (slope) between the to investigate the relationship between the sexes and oestrus response and the force of the von Frey filament for responses gen- cycle phases. To our knowledge this study is the first to exam- erated by von Frey Test 3. As previously mentioned, historically, ine a difference in slope in rodent mechanical allodynia. Using chronic pain investigations conventionally use a logistic approach this alternative statistical approach, significantly greater female (reporting maximal response, the response threshold and the 50% mechanical hypersensitivity was demonstrated in N3S1 recipi- of maximal response). Although von Frey Test 3 could have been ents post neuropathic injury, replicating previous findings in an statistically examined conventionally, and may be examined as array of animal chronic pain models (Coyle et al., 1995; DeLeo such in future investigations, this was not possible with von Frey and Rutkowski, 2000; LaCroix-Fralish et al., 2005a,b; Li et al., Test 2 (see above). Consequently, in order to make comparisons 2009). Despite bypassing the large degree of inconsistency asso- between the two von Frey methods, von Frey Test 3 investigated ciated with a point estimate approach, examining a difference in the relationship between the response and the force of the von slope failed to demonstrate well-founded sex differences in N1S3 Frey filament (the slope). rats as well as an oestrus cycle effect in all pain treatment groups The inability of von Frey Method 2 to refine preclinical chronic post neuropathic injury. pain testing, failing to identify sex and oestrus cycle differences in In the interest of continuing preclinical animal pain research less allodync animals, upon further analysis came as a result of the global aim of this study was to investigate male and female testing at the high end of the von Frey logarithmic force scale. mechanical nociception in a model capable of reducing ani- Parameterization of results generated by von Frey Test 3 revealed mal suffering compared to existing models. As demonstrated in the particular von Frey filaments at which sex and oestrus cycle Experiment 3, von Frey Test 3 was the only testing method to where statistical differences occurred, with numerous filaments reveal the ability of the novel sciatic nerve injury model to refine at the low end of the von Frey logarithmic force scale found to the traditional CCI technique, firmly establishing sex and oestrus contribute to the overall statistical effects of this von Frey test

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Figure 2.3 von Frey Test 3: A Behavioural Technique Refining the Investigation of the Role of Sex and Rodent Oestrus Cycle in Mechanical Hypersensitivity

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healthy male and female thresholds (Sarlani and Greenspan, 2002; Sarlani et al., 2004). Application of phasic rather than sustained noxious force utilsing von Frey Tests 2 and 3 duplicated such clinical findings, with both male and female rodents respond- ing equivalently prior to neuropathic injury. Although both von Frey tests revealed the ability to detect heterogenous chronic pain with the level of mechanical allodynia found proportional to the number of sciatic sutures, as previously mentioned the adapted von Frey Test 3 was the only method to demonstrate exacerbated female mechanical hypersensitivity in recipients with significantly reduced pain (N1S3 recipients) or demonstrate that the rodent oestrus cycle influenced female mechanical allodynia. Numerous avenues have been investigated to explain exagger- ated female pain. One theorized explanation of the differences in pain response between the sexes is the hormonal milieu. The sex steroids estrogens, androgens and progesterones are primarily produced by the gonads from cholesterol, where their receptors have a wide distribution in the body including throughout the central nervous system (CNS) (McEwen and Alves, 1999; Aloisi, 2003; Craft, 2007). Oestrogen receptors specifically have been located in trigeminal neurons, dorsal root ganglion cells, as well as in brain areas such as the hypothalamus and the periaqueduc- tal gray (PAG) (Shughrue et al., 1997; Yang et al., 1998; Kruijver et al., 2003; Merchenthaler et al., 2004; Bereiter et al., 2005; Loyd and Murphy, 2008)suggestingestrogeninfluencesonboth ascending and descending nociceptive pathways. This divergent localization of estrogen receptors enables a variety of functional roles within the CNS, which are not limited to the regulation of FIGURE 4 | Exacerbated female pain is influenced by the rodent reproductive behavior. Despite modulating pain-processing sys- oestrus cycle. von Frey Test 3 demonstrated female mechanical allodynia was heavily dependent on the rodent oestrus cycle. Post nerve injury, tems such as the endogenous opioids (enkephalins) and GABA moderate pain females (N3S1) were significantly more allodynic during pathways (Amandusson et al., 1999; McEwen and Alves, 1999; dioestrus and pro-oestrus t 7.7. The data presented is generated from = − Craft, 2007), the precise mechanisms underlying estrogen’s role in von Frey Test 3. Results generated by von Frey Test 3 are expressed as the pain remains unclear. Estrogens however, have also been demon- percent difference in response per increase in von Frey hair stiffness. strated anti-inflamamtory, particular in vitro and are not the only Results were analyzed utilizing repeated measures linear mixed effects modeling. A t-value of < 2or>2 was determined statistically significant sex hormones known to influence pain. Progesterone for exam- − (p < 0.05). n 8pertreatmentgroup.vonFreyfilaments:6:10,5:6,4:2,3: ple has also been demonstrated anti-inflamamtory in numerous = 1.0, 2: 0.4, 1: 0.07g. Di, dioestrus; met, metoestrus; oest, oestrus; pro, diseases and in animal spinal cord injury models (Garcia-Ovejero pro-oestrus. et al., 2005; Muller and Kerschbaum, 2006; Labombarda et al., 2011). Although great inconsistency remains in the literature, there appears to be a strong association between the steroid (Table 8). The ability to assess the effect of covariates on the allo- hormones and pain. dynia response for a range of von Frey filaments is not achievable Literature demonstrates prepubescent boys and girls display using von Frey Test 2, where differences can only be assessed at equal prevalence in the majority of chronic pain conditions, until the two point estimates or by inferring differences for von Frey puberty where a female prevalence is henceforward established filaments between these values on interpolation based on the until menopause (Ogura et al., 1985; Abu-Arefeh and Russell, assumption of linearity. Consequently, parameterization revealed 1994; Lipton et al., 2001; Sonmez et al., 2001; Bigal et al., 2007). the advantages of testing over a range of von Frey filaments, most Numerous studies have also demonstrated fluctuating hypersen- importantly lower threshold filaments when examining female sitivity throughout the female menstrual (LeResche et al., 2003; rodent mechanical sensitivity, emphasizing the effectiveness of Pamuk and Cakir, 2005; Crawford et al., 2009)androdentoestrus von Frey Test 3. cycles (Frye et al., 1992; Kayser et al., 1996; Giamberardino et al., It is hypothesized that poorly translatable preclinical method- 1997). Consequently, by assessing mechanical allodynia across a ologies are partly responsible for the incomplete understanding range of von Frey filaments using von Frey Test 3, this study’s of exacerbated female pain. Therefore, chronic pain investiga- finding of an oestrus cycle effect replicates many other preclini- tions need to utilize methods which best replicate the clinical pain cal investigations as well as the clinical situation and indicates a scenario. Although studies examining sex differences in mechan- hormonal sensitivity within the pain pathway. ical pain thresholds have yielded variable findings, investigations Until recently, neuronal mechanisms were thought to solely using phasic noxious stimuli consistently demonstrate equivalent contribute to pathological pain (Nicotra et al., 2012). Our

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Figure 2.4 Exacerbated Female Pain is Influenced by the Rodent Oestrus Cycle

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Table 8 | The ability of von Frey test 3 to estimate differences in allodynia for diverse covariates across individual von Frey filaments.

von Frey filament Statistical sex effect Statistical sex effect Statistical oestrus effect Statistical graded (grams force) (t < 2 or >2) N1S3 (t < 2 or >2) N3S1 (t < 2 or >2) N3S1 surgical effect − − − subjects subjects subjects (t < 2 or >2) −

Filament 1 (0.07) !!  Filament 2 (0.4) !! !  Filament 3 (1.0) !! ! ! Filament 4 (2.0) !! ! ! Filament 5 (6.0) !! ! ! Filament 6 (10.0) !! ! !

Parameterization of repeated measures linear mixed effects modeling exposed the specific von Frey filaments at which the adapted von Frey Test 3 demonstrated sex, oestrus cycle and surgical statistical differences. The modified von Frey technique demonstrated the successful generation of a graded chronic pain model by which sex differences were established for the first time in rodents with reduced pain, and the rodent oestrus cycle found to undoubtedly influence female pain (Section Experiment 3: von Frey Test 3: A Behavioral Technique to Investigate the Role of Sex and the Rodent Oestrus Cycle in Mechanical Hypersensitivity). The sole ability of this von Frey test to indisputably demonstrate such effects resulted from testing over a range of von Frey filaments. Parameterization revealed statistical differences at the low end of the von Frey logarithmic force scale, thereby demonstrating their contribution to the positive findings utilizing this method. The data presented is generated from von Frey Test 3. Results were analyzed utilizing repeated measures linear mixed effects modeling, where the repeated measures of the experimental design was also reparameterized using the contrast() function in the contrast R package (Kuhn, 2011) and the glht() function in the multcomp R package (Hothorn et al., 2008). This enabled the assessment of the difference in the percentage responses for different levels of each fixed effect, sex, oestrous, surgery, for different weights of von Frey filaments. A t-value of < 2or>2 was determined statistically significant (p < 0.05). To demonstrate the von Frey filaments − at which statistical differences occured, -indicate a positive finding, whereas -signify the failure to determine a significant effect. n 8pertreatmentgroup,per ! = sex. Smears were taken from eight females per phase, with results generated from a minimum of four consecutive oestrus cycles. von Frey filaments: 2.83:0.07, 3.61: 0.4, 4.08: 1, 4.31: 2, 4.74: 6, 5.07: 10.

understanding of chronic pain pathophysiology however, has binary in nature pain models, which are more likely to reveal since developed, with non-neuronal immune cells now also effective analgesics for the treatment of severe pain, the graded known to play role in both the initiation and maintenance of nerve injury model has the ability to detect heterogeneous pain, chronic pain (Haydon, 2001; Milligan and Watkins, 2009; Grace specifically moderate reductions in allodynia thereby unveiling et al., 2011). Recent evidence demonstrates not only an inter- potential analgesics for a range of chronic pain patients (Grace action between steroid hormones and innate immune receptors et al., 2010). Although utilizing several animals in order to gener- known to play a role in chronic pain (Calippe et al., 2010; Loram ate the diverse pain treatment groups in this study, it is essential et al., 2010), but exacerbated proinflammatory responses follow- to note that this investigation extends the findings by Grace and ing steroid hormone-immune cell priming (Soucy et al., 2005; Colleagues, demonstrating graded nerve injury in the female sex. Rettew et al., 2009; Calippe et al., 2010). Considering the impor- In order to establish graded chronic pain for the first time in the tant role innate immune signaling plays in chronic pain process- female rodent, it was essential for this study to consider numer- ing, this interaction between neuroimmune function and the sex ous pain treatment groups. The successful determination of sex steroids may partially explain sex differences in pain sensitivity and oestrus cycle effects without having to inflict maximal allo- and requires further investigation (Nicotra et al., 2012). dynia using von Frey Test 3, endorses the use of less allodynic The importance of utilizing preclinical techniques capable of animals in order to investigate chronic pain mechanisms in the reducing animal pain and stress has become increasingly appar- future. Consequently therefore, although having to compromise ent through the stringent statutory policies governing animal the “Reduction” component of the three 3 R’s in this particu- use in pain research (Gad, 1990). With chronic pain investiga- lar investigation, using von Frey Test 3 has allowed “Refinement” tions in their very nature intentionally inflicting persistent pain, in future investigations without disturbing the “Reduction” con- researchers face great difficulty in satisfying the Refinement cat- stituent. Consequently, in the hope of discovering novel analgesics egory of the three R’s unless ongoing refinement of experimental and to preserve the use of animals in pain studies, this study com- approaches continues. The ability of von Frey Test 3 to investigate mends the use of the graded model of graded sciatic nerve injury, both the role of sex and the influence of the rodent oestrus cycle specifically recommending the use of less allodynic animals in fur- utilizing the refined and novel graded model of neuropathy sug- ther preclinical pain research and the use of von Frey Test 3, which gests that the use of the traditional CCI four-suture approach cre- proved sensitive enough to identify contributing factors that other ates unnecessary suffering for the animal. In addition to reducing common von Frey tests could not reveal. animal suffering, this refined preclinical methodology may also lead to the discovery and development of new analgesics. Existing CONCLUSION pain models are limited in their binomial approach reducing To our knowledge, this study is the first to examine a role of sex the statistical power to investigate underlying pain mechanisms. and oestrus cycle in a refined heterogeneous allodynia model. In Unlike currently utilized two group, sham controlled, and hence comparison to two commonly utilized von Frey testing methods,

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Table 2.8. The Ability of von Frey Test 3 to Estimate the Difference in Allodynia for Diverse Covariates Across Individual von Frey Filaments.

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the adapted von Frey Test 3 whereby mechanical allodynia was DeLeo, J. A., and Rutkowski, M. D. (2000). Gender differences in rat neuro- assessed utilizing phasic application of lower threshold filaments, pathic pain sensitivity is dependent on strain. Neurosci. Lett. 282, 197–199. doi: alone demonstrated a role for sex and the rodent oestrus cycle in 10.1016/S0304-3940(00)00880-6 Dubner, R. (1987). Research on pain mechanisms in animals. J. Am. Vet. Med. Assoc. female mechanical allodynia, without having to inflict maximal 191, 1273–1276. pain. Considering the emphasis animal ethics committee’s place Fillingim, R. B., King, C. D., Ribeiro-Dasilva, M. C., Rahim-Williams, B., and on the three R’s and the difficulty pain researchers face in satisfy- Riley, J. L. 3rd. (2009). Sex, gender, and pain: a review of recent clinical and ing the “refinement” category to gain ethical approval, the ability experimental findings. J. Pain 10, 447–485. doi: 10.1016/j.jpain.2008.12.001 to investigate chronic pain mechanisms using this modified von Frye, C. A., Bock, B. C., and Kanarek, R. B. (1992). Hormonal milieu affects tailflick latency in female rats and may be attenuated by access to sucrose. Physiol. Behav. Frey approach in a model capable of inflicting less pain without 52, 699–706. doi: 10.1016/0031-9384(92)90400-V compromising overall statistical significance is invaluable. Gad, S. C. (1990). Recent developments in replacing, reducing, and refining animal use in toxicologic research and testing. Fundam. Appl. Toxicol. 15, 8–16. doi: ACKNOWLEDGMENTS 10.1016/0272-0590(90)90157-F Lauren Nicotra is the recipient of an Australian Postgraduate Garcia-Ovejero, D., Azcoitia, I., Doncarlos, L. L., Melcangi, R. C., and Garcia- Award. Mark Hutchinson is an Australian Research Council Segura, L. M. (2005). Glia-neuron crosstalk in the neuroprotective mecha- nisms of sex steroid hormones. Brain Res. Brain Res. 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Venables, W. N., and Ripley, B. D. (2002). Modern Applied Statistics with S. 4th Edn. Conflict of Interest Statement: The authors declare that the research was con- New York, NY: Springer. ducted in the absence of any commercial or financial relationships that could be Wang, L. X., and Wang, Z. J. (2003). Animal and cellular models of chronic pain. construed as a potential conflict of interest. Adv. Drug Deliv. Rev. 55, 949–965. doi: 10.1016/S0169-409X(03)00098-X Watkins, L. R., Hutchinson, M. R., Rice, K. C., and Maier, S. F. (2009). Received: 25 September 2013; accepted: 27 January 2014; published online: 13 The “toll” of opioid-induced glial activation: improving the clinical effi- February 2014. cacy of opioids by targeting glia. Trends Pharmacol. Sci. 30, 581–591. doi: Citation: Nicotra L, Tuke J, Grace PM, Rolan PE and Hutchinson MR (2014) Sex dif- 10.1016/j.tips.2009.08.002 ferences in mechanical allodynia: how can it be preclinically quantified and analyzed? Woolf, C. J. (1995). Somatic pain–pathogenesis and prevention. Br. J. Anaesth. 75, Front. Behav. Neurosci. 8:40. doi: 10.3389/fnbeh.2014.00040 169–176. doi: 10.1093/bja/75.2.169 This article was submitted to the journal Frontiers in Behavioral Neuroscience. Yang, Y., Ozawa, H., Lu, H., Yuri, K., Hayashi, S., Nihonyanagi, K., et al. (1998). Copyright © 2014 Nicotra, Tuke, Grace, Rolan and Hutchinson. This is an open- Immunocytochemical analysis of sex differences in calcitonin gene-related pep- access article distributed under the terms of the Creative Commons Attribution License tide in the rat dorsal root ganglion, with special reference to estrogen and its (CC BY). The use, distribution or reproduction in other forums is permitted, provided receptor. Brain Res. 791, 35–42. doi: 10.1016/S0006-8993(98)00021-3 the original author(s) or licensor are credited and that the original publication in this Zimmermann, M. (1986). Ethical considerations in relation to pain in animal journal is cited, in accordance with accepted academic practice. No use, distribution or experimentation. Acta Physiol. Scand. Suppl. 554, 221–233. reproduction is permitted which does not comply with these terms.

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APPENDIX Table A3 | Percent response values von Frey test 2. Table A1 | Response rates for von Frey test 2 generated by the 2g filament. PRO OEST MET DI PRO OEST MET DI Pre-surgery 6.9 0.22 7.3 0.18 7.3 0.22 7.0 0.16 ± ± ± ± Pre-surgery 4.2 0.30 5.0 0.22 4.9 0.24 4.7 0.22 N0S0 7.4 0.20 7.0 0.16 8.0 0.22 8.7 0.26 ± ± ± ± ± ± ± ± N0S0 5.2 0.24 4.9 0.18 4.1 0.43 4.4 0.44 N0S4 17.0 0.24 16.0 0.11 17.0 0.21 16.0 0.26 ± ± ± ± ± ± ± ± N0S4 8.6 0.44 6.5 0.42 6.6 0.56 9.7 0.41 N1S3 23.0 0.33 25.0 0.38 25.0 0.41 22.0 0.23 ± ± ± ± ± ± ± ± N1S3 20.0 0.52 15.0 0.46 14.3 0.55 17.0 0.51 N3S1 48.0 0.32 50.0 0.48 51.0 0.46 50.0 0.41 ± ± ± ± ± ± ± ± N3S1 42.0 0.44 41.0 0.41 41.0 0.38 43.0 0.51 N4S0 63.0 0.44 64.0 0.49 61.0 0.33 41.0 0.40 ± ± ± ± ± ± ± ± N4S0 54.0 0.33 53.0 0.44 51.0 0.56 48.0 0.53 Responses generated from von Frey Test 2 were used to examine the estimate ± ± ± ± Responses generated utilizing von Frey Test 2 were examined following the tradi- of the slope of the linear relationship between the response and von Frey fila- tional statistical approach whereby responses were independently examined at ment. The results presented throughout this study are expressed as the average the 2 and 12 g von Frey filaments. For each predictor (sex, oestrus cycle phase, difference in percent response, which are generated from the percent response surgery and von Frey filament stimulus), the difference between the allodynia values (the percent response /30 across von Frey filaments). (percentage response for the 2 g von Frey filament) for each level of the pre- dictor was estimated. This value obtained is referred to as the response rate, the percent response /30 (% response). The results presented throughout this Table A4 | Response rates for von Frey test 3. study are expressed as the average difference in percent response, which are PRO OEST MET DI generated from the response rates. Pre-surgery 4.1 0.20 4.9 0.20 5.5 0.80 5.1 0.21 ± ± ± ± N0S0 5.1 0.60 5.0 0.72 5.2 0.50 6.1 0.60 ± ± ± ± Table A2 | Response rates for von Frey test 2 generated by the 12g N0S4 5.0 0.44 6.1 0.60 5.6 0.30 5.3 0.50 ± ± ± ± filament. N1S3 9.8 0.52 8.1 0.52 7.7 0.38 10.0 0.60 ± ± ± ± PRO OEST MET DI N3S1 11.0 0.42 8.9 0.80 8.1 0.80 11.0 0.50 ± ± ± ± N4S0 12.0 0.58 11.0 0.74 11 .0 1.011.0 0.85 Pre-surgery 8.3 0.24 10.0 0.22 9.6 0.28 9.4 0.21 ± ± ± ± ± ± ± ± von Frey Test 3 examined the relationship between the response and the force N0S0 11.0 0.21 10.0 0.22 10.0 0.26 11.0 0.22 ± ± ± ± of the von Frey filament. Results generated by von Frey Test 3 are expressed as N0S4 22.0 0.38 21.0 0.28 22.0 0.31 22.0 0.26 ± ± ± ± the percent difference in response per increase in von Frey hair stiffness, which N1S3 34.0 0.32 32.0 0.24 26.0 0.38 34.0 0.33 ± ± ± ± are generated from the estimated increase in percent response per increase in N3S1 61.0 0.44 61.0 0.51 64.0 0.51 62.0 0.57 ± ± ± ± von Frey stiffness. N4S0 79.0 0.51 79.0 0.43 78.0 0.44 81.0 0.56 ± ± ± ± Responses generated utilizing von Frey Test 2 were examined following the tradi- tional statistical approach whereby responses were independently examined at the 2 and 12 g von Frey filaments. For each predictor (sex, oestrus cycle phase, surgery and von Frey filament stimulus), the difference between the allodynia (percentage response for the 2 g von Frey filament) for each level of the pre- dictor was estimated. This value obtained is referred to as the response rate, the percent response /30 (% response). The results presented throughout this study are expressed as the average difference in percent response, which are generated from the response rates.

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CHAPTER 3. THE CRITICAL ASSOCIATION BETWEEN E2 AND THE NEUROIMMUNE SYSTEM IN CHRONIC PAIN

The finger of blame for the female predominance of chronic pain has pointed to the gonadal sex steroid hormone 17β-estradiol (E2). Although Chapter 2 revealed a role for the ovarian hormones in the graded neuropathy model, with the female oestrus cycle found to modify female rodent mechanical allodynia pain scores, these findings do not specifically indicate E2.

The mechanisms underlying heightened female chronic pain remain elusive, with no scientific justification available to treat several female-predominant chronic pain conditions differently to that experienced by males, despite the hypothesized E2-dependent mechanistic differences presented in Chapter 1. Consequently, the remainder of this research journey aimed to discover the elusive mechanism underpinning the predominance of female chronic pain and to do so using the refined methodologies adapted and presented in Chapter 2. With the more recent developments in the neuroimmune field, the interaction between E2 and the neuroimmune interface was investigated in Chapter 3 and the origins of female chronic pain were found not in the classical neuronal explanations of chronic pain, but in the association between this gonadal hormone and innate immune signalling receptors.

As detailed in Chapter 1, microglia are seen as the initiators and propagators of chronic danger in the CNS and facilitate the sensitization of nociceptive pathways that lead to chronic pain. Thus, we hypothesized that female microglia are more reactive than male microglia and hence would contribute significantly more to sensitization of nociceptive pathways.

Importantly, we also believed that E2-priming of microglial innate pattern recognition TLR signaling systems was responsible for this microglial sensitization, due to the now known association between E2 and these innate immune receptors. Lauren Nicotra Sex Differences in Allodynia 107

Herein this chapter, we establish for the first time that intrathecal transfer of female microglia is sufficient to create exaggerated pain following nerve injury in males, and that this occurs in a TLR2/4-MyD88 dependent fashion. These conclusions can be drawn from a series of key in vivo studies, which demonstrate the connection between E2 and innate immune chronic pain contributions. To support these extensive behavioral studies we have employed a range of pharmacological, cellular and molecular techniques to further interrogate the 17β-estradiol priming of the innate immune signaling pathways.

This study establishes that female pain has a greater proportion of neuroinflammation during the reproductive/ estrogenic years, consequently advocating that chronic female pain may be treated more effectively using neuroimmune-targeted pharmacotherapies.

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3.1 Estrogen-Primed Microglia are Central to Sex Differences in Allodynia

The manuscript provided here is currently undergoing a second round of revision at Nature

Neuroscience.

Estrogen-Primed Microglia are Central to Sex Differences in Allodynia

Nicotra, LL1, Loram, LC2, Tuke, S.J.3, Kwok, YH1, Zhang, Y2, Rice, KC4, Staikopoulos,

V5, Rolan, PE1, Watkins, LR2, Hutchinson, MR5,6

1. Discipline of Pharmacology, School of Medical Sciences, University of Adelaide,

South Australia, 5005, Australia

2. Department of Psychology and Center for Neuroscience, University of Colorado at

Boulder, Boulder, Colorado, 80309, USA

3. School of Mathematical Sciences, The University of Adelaide, Adelaide, SA,

Australia

4. Chemical Biology Research Branch, National Institute on Drug Abuse and National

Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville,

Maryland 20892

5. Discipline of Physiology, School of Medical Sciences, The University of Adelaide,

South Australia, 5005, Australia

6. ARC Centre of Excellence for Nanoscale BioPhotonics, The University of Adelaide,

South Australia, 5005, Australia

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Chronic pain is a world wide epidemic problem, predominantly affecting females, with gonadal sex steroids implicated in the increased pain prevalence. The recent acceptance of innate immune involvement in chronic pain opens a new avenue to explore spinal immunology as a mediator of sex differences in chronic pain. Here we demonstrate for the first time that 17β-estradiol priming of spinal cord microglia contributes to exacerbated female mechanical allodynia in a TLR2/4- MyD88-dependent fashion. Moreover, “female- like” allodynia was phenocopied in males solely by intrathecal transfer of female microglia.

Excitingly, we also demonstrate that neuroimmune targeted pharmacotherapies have greater potency in female mice. These are groundbreaking results with wide reaching basic science and clinical implications and support that female chronic pain should be treated differently from that in males.

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Chronic pain is a worldwide epidemic, accounting for over six hundred billion USD each year397. Females are over-represented within the chronic pain population20. However, the underlying pathophysiology remains undetermined. Autoimmune diseases often present with a female prevalence, suggesting immune and sex hormone interactions. Here, we explore whether such interactions extend to chronic pain, where central immune cell involvement is an important contributor98.

Communication between neurons and non-neuronal immunocompetent cells, including glia, contributes to several neuropathologies398. Spinal glial pro-inflammatory phenotypes

(particularly microglia) are upregulated in numerous chronic pain models399, 400 with several neurotransmitters and neuromodulators responsible for activating neuron-to-glia signaling, resulting in pathological pain8.

Newly appreciated contributors to this signaling process are specialized pattern recognition receptors named toll-like receptors (TLRs)401. TLRs are distributed in several cell types throughout pain responsive CNS regions98. Upon microglial TLR activation, release of pro- inflammatory mediators contributes to heightened pain8. Preclinical pain studies demonstrate upregulated TLR expression and their requirement for the development and maintenance of chronic pain384-386, 388-391, 402. Gonadal sex steroid hormones, particularly 17β-estradiol are implicated in exaggerated pain in females126. One possible mechanism underlying sex differences in chronic pain is the mechanistic link between 17β-estradiol and TLR4383. TLR4 mediated inflammatory responses are exaggerated in females354, likely mediated by signaling downstream of microglial estrogen receptors366 interacting with TLR4 signaling383. By cooperation or complementation403 with TLR4 signaling, chronic, in vivo, estrogen potentiates

NFκB activation, amplifying pro-inflammatory cytokine gene expression and release347, 383.

Although demonstrated to interact with TLR4 signaling, whether estrogen binds to TLRs

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specifically is unknown. Given the importance of TLRs in chronic pain, and the interaction between 17β-estradiol and TLR signaling, it is hypothesized that exaggerated pain in females arises, in part, from the interaction between 17β-estradiol and TLRs, possibly directly at the receptor level. Accordingly, we investigated for the first time whether this sex difference in neuroinflammation may contribute to exaggerated pain in females.

Methods

Subjects

Behavioral studies:

Pathogen-free prepubescent male and female Balb/c mice (3 weeks of age) were obtained from The University of Adelaide Laboratory Animal Services (Adelaide, SA, Australia). Two null mutant mouse strains, TLR4-/- and Myeloid differentiation primary response gene 88

-/- (MyD88) back crossed onto Balb/c 10 times, were originally sourced from Professor Akira

(Osaka University, Osaka, Japan) via Dr. Paul Foster from University of Newcastle

(Newcastle, NSW, Australia). Mice were housed in temperature- (18 - 21 °C) and light- controlled (12 h light/ dark cycle; lights on at 07:00 h) rooms where standard rodent food and water was available ad libitum. Preceding experimentation, mice were habituated to the animal holding care facility for 1 week, followed by 1 week of extensive experimenter handling and acclimatization to the von Frey testing apparatus in order to reduce successive handling stress. Study experimentation for all mice began at 6 weeks of age. All procedures were approved by the Animal Ethics Committee of the University of Adelaide and were performed in accordance with the NHMRC Australian code of practice for the care and use of animals for scientific purposes.

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Pre-surgical and test procedures

Prior to chronic constriction injury (CCI) surgery and behavioral testing, a vaginal smear was taken from female mice using the common pipette smear technique to determine estrus cycle phase. Vaginal smears were taken between 0800 and 1000 each testing and surgical day to minimize the incidence of transitional or ‘missed’ stages. The four stages of the estrus cycle pro-estrus (pro), estrus (est), metestrus (met) and diestrus (di) could be recognized by the presence, absence or proportion of epithelial, cornified and leucocyte cells. Smears were taken from 8 females per phase, with results generated from a minimum of 4 consecutive estrus cycles. In order to attempt to replicate the small amount of stress experienced by female mice during the smear procedure, male mice were given a parallel injection of isotonic saline intraperitoneally.

Surgery

Ovariectomy (OVX) and 17β-estradiol treatment

To investigate the influence of steroid gonadal hormones in a model of graded nerve injury, female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy (OVX). The specific role of 17β-estradiol was determined by the simultaneous surgical insertion of slow release 17β-estradiol pellets (Estrogen) (0.18 mg/kg), and placebo pellets as a control (Estrogen). One week following OVX/hormone replacement, male and female mice underwent nerve injury (N3S1) and mechanical allodynia was examined once stable (at days 17-21 following nerve injury).

At 6 weeks of age, mice were anaesthetized with isoflurane (3% in oxygen), fur shaved bilaterally over the lumbar spine and the skin cleaned. A single midline dorsal incision (0.5

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cm) was made to locate the ovaries and ovarian duct, which were then pulled through the hole in the abdominal wall. The ovary and surrounding adipose tissue were removed and the ovarian duct reinserted into the abdominal cavity. Following OVX and prior to skin closure, mice were implanted with a 90-day release subcutaneous pellet containing 17β-estradiol (0.18 mg/pellet) or vehicle-placebo pellet containing a mixture of cholesterol, cellulose, alpha- lactose, calcium phosphate, calcium and magnesium stearate, and stearic acid (0.18 mg/pellet)

(Innovative Research of America, Sarasota, FL). Mice were allowed one week recovery before experimentation.

Chronic Constriction Injury (CCI)

The CCI model of chronic pain was performed at the mid-thigh level of the left hindleg as previously described95. Mice were anaesthetized with isoflurane (3% in oxygen), fur shaved over the left mid-thigh and the skin cleaned. The sciatic nerve was aseptically exposed and isolated at mid-thigh level. One, three or four loose chromic gut suture ligatures (cuticular 4-0 chromic gut, FS-2; Ethicon, Somerville, NJ, USA) were placed around the sciatic (N) and once the superficial muscle overlying the nerve was sutured, an additional length of chromic gut was placed subcutaneously to replicate the graded model of neuropathy previously published (S)95. Consequently, nerve injury treatment groups included N0S0, N0S4, N1S3,

N3S1 and N4S0 animals. Rescue analgesia (Buprenorphine) was available post surgical intervention in cases of adverse events, however was not required in these investigations.

Following all surgical procedures mice were monitored postoperatively until fully ambulatory prior to return to their homecage and checked daily for any sign of infection. No such cases occurred in this study.

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Drugs

(+)-naltrexone was obtained from Kenner C Rice, the National Institute on Drug Abuse

(Research Triangle Park, NC and Bethesda, MD, USA). Human IL-1ra (Kineret, Amgen Inc.,

Thousand Oaks, CA, USA) was purchased from the Queen Elizabeth Hospital Pharmacy

(Woodville, SA, Australia). Sterile endotoxin-free isotonic saline (0.9% sodium chloride) was the vehicle for both neuroimmune targeted therapies. Estrone (E1), Estradiol (17β-estradiol,

E2) and Estriol (E3) were purchased from Sigma (St. Louis, MO, USA). LPS-RS (a TLR4 antagonist naturally produced by R. sphaeroides) was purchased from Invivogen (San Diego,

CA, USA).

For animal behavioral studies (+)-naltrexone and IL-1ra were freshly diluted in endotoxin- free saline (0.9% sodium chloride) and injected intraperitoneally (i.p.). A dose response relationship was established with (+)-naltrexone administered at 30, 3 and 0.3 mg/kg, and IL-

1ra at 100, 10 and 1 mg/kg. Vehicles were administered in equal volumes to the drugs under test.

Endotoxin assay:

A limulus amebocyte lysate (LAL) assay (Bio Whittaker QCL-1000) was performed on all the drugs tested to ensure no detectable endotoxin contamination. The assay was performed according to manufacturer’s instructions.

In vitro studies of TLR2 and TLR4 signaling in HEK-TLR2/TLR4 cells

A human embryonic kidney-293 (HEK-293) cell line stably transfected to express human

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TLR4 was purchased from Invivogen (293-htlr4a-md2cd14; here referred to as HEK-TLR4,

San Diego, CA, USA). In addition, these cells stably express an optimized alkaline phosphatase reporter gene under the control of a promoter inducible by several transcription factors such as NF-κB and activator protein 1 (AP-1). A parallel HEK-TLR2 cell line was also purchased from Invivogen. HEK-TLR4 and HEK-TLR2 cells were grown at 37 °C, 5%

CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), HEK-TLR4 selection

(Invivogen), penicillin 10,000 U ⁄ mL (Invitrogen), streptomycin 10 mg/mL (Invitrogen), normocin (Invivogen), and 200 nm l-glutamine (Invitrogen). The cells were plated for 48 h in

96-well plates. E2 (0.01-100 µM), E1 (1-100 µM), E3 (1-100 µM), LPS-RS (10 ng/ml), (+)- naltrexone (100 µM) or vehicle were added individually and in combination, and incubated for 24 h. Secreted alkaline phosphatase (SEAP) in the supernatants was assayed using the

Phospha-Light System (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions.

Microglial cell preparation

Donor mice (Study 4: 11 weeks of age, 21 days post nerve injury; Study 5: 8 weeks of age, 1 week post OVX surgery) were anaesthetized with sodium pentobarbital and perfused transcardially with isotonic saline. The lumbar (L4–L6) segment of the spinal cord

(innervating the sciatic nerve) was carefully excised and homogenized in phosphate-buffered saline pH 7.4 (PBS). The lumbar spinal cord was dissociated using a loose fitting glass homogenizer and the MACS Neural Tissue Dissociation Kit (Miltenyi biotec, Bergisch,

Germany). The spinal cord cell suspension was centrifuged at 130xg for 10 min at room temperature, re-suspended in PBS at a maximum cell concentration of 107 total cells. To Lauren Nicotra Sex Differences in Allodynia 119

isolate microglial cells, the spinal cord cell suspension was incubated with MACS CD11b

(microglia) microbeads (Miltenyi biotec, Bergisch, Germany 130-093-634). Labeled cells were positively selected through a MACS MS column (Miltenyi Biotec, Bergisch Gladbach,

Germany 130-094-802) as directed by the manufacturer, with similar protocols obtaining a purity of 95%.

Intrathecal transfer of spinal cord microglial cells

Intrathecal (i.t.) injections were performed as previously described404. Briefly, under isoflurane anesthesia a 30-gauge sterile needle was inserted between the L5 and L6 vertebrae, evoking a tail flick. The injection, over 1 min, consisted 1 µl of dead-space air followed by microglial cells (6 x 103) prepared from the spinal cords of donor mice in 10 µl of PBS.

Injections were performed with a 25 µl glass Hamilton syringe with 30-gauge needle for delivery. Mice were monitored until fully ambulatory, prior return to their home cage. No abnormal motor behavior was observed after any injection. Each recipient animal received spinal microglial cells prepared entirely from the spinal cord of another donor mouse and microglial cells were not pooled from within treatment groups. Behavioral testing was conducted following adoptive transfer as described below.

Western Blot

At day 21 following microglial delivery and final behavioral assessment, mice were anaesthetized with sodium pentobarbital and perfused transcardially with isotonic saline. The lumbar (L4–L6) segment of the spinal cord (innervating the sciatic nerve) was carefully excised and homogenized in 1 % protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, Lauren Nicotra Sex Differences in Allodynia 120

USA P8340). Homogenates were centrifuged at 15,000 rpm at 4 °C. Supernatant protein concentrations were determined by a BCA assay (Pierce Biotechnology, Inc., Rockford, IL) as per the manufacturer’s directions and supernatants stored at -70 °C until required. Each well was loaded with 25 µl of spinal cord sample containing 20 µg of protein. Each gel contained a control sample from the same source (combination of all animals from all treatment groups) to account for inter-gel variability. Following transfer, the membrane was incubated with antibodies: Goat-anti-GFAP (Santa Cruz biotechnology, Texas, USA N-18) at

1:2000, Rabbit-anti-betaActin (Sigma Aldrich, St. Louis, MO, USA A2668) 1:5000 and Goat- anti-Intergrin alphaM (CD11b) (Santa Cruz biotechnology M-19) 1:2000 overnight at 4 °C.

Secondary antibodies were applied at room temperature for 1 hour: GFAP: Donkey anti Goat

IRdye 800 (Rockland Immunochemicals, PA 605-731-002) (1:10,000); Beta Actin: Donkey anti Rabbit Irdye 700 (Rockland Immunochemicals, PA 611-730-127) (1:10,000). For

CD11B, biotinylated Donkey anti Goat 1:500 was applied for 1hr at room temperature followed by Streptavidin Irdye 700 (Rockland Immunochemicals, PA 610-730-124)

(1:10,000). Membranes were scanned on the Licor Infrared Odyssey (Li-Cor Biosciences,

Cambridge, UK).

Behavior

Throughout the study, testing was performed blind with regard to group assignment and estrus cycle phase.

Briefly, mice were subjected to 10 stimulations with 6 calibrated von Frey filaments (0.008 g,

0.02 g, 0.04 g, 0.07 g, 0.16 g, 0.4 g), within the sciatic innervation region of the hindpaws as previously described in detail94. In order to avoid sensitization, 10 min break was given

Lauren Nicotra Sex Differences in Allodynia 121

between each set of stimulations, with ten stimulations per filament. Allodynia was characterized as an intense paw withdrawal or licking of the stimulated hind paw. Behavioral responses were recorded as the average number of responses out of 10 for each von Frey stimulus and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments94.

Behavioral Experimental Design

The role of sex and 17β-estradiol in exacerbated female mechanical hypersensitivity

Male and female mice were behaviorally assessed prior to CCI surgery (baseline) and from postoperative days 3 to 21. To produce graded chronic pain for the first time in Balb/c mice, male and female pain treatment groups included N0S0, N0S4, N1S3, N3S1 and N4S0. Up to

21 days post nerve injury, female mice were behaviorally examined at each phase of the estrus cycle (taken from a minimum of 4 consecutive cycles), with males “matched” according to animal number and tested on the corresponding female test day.

To control for gonadal hormones, females were ovariectomised (OVX), with males receiving a sham ovariectomy (OVX) and both sexes receiving slow release 17β-estradiol pellets

(Estrogen) or placebo treatment (Estrogen). Prior to OVX/hormone replacement surgery

(baseline), mechanical allodynia was assessed in male and female Balb/c mice. Following baseline assessments over 4 consecutive female estrus cycles, males and females underwent

OVX/hormone treatment followed by a one week resting period before nerve injury (N3S1).

Mechanical allodynia was assessed in N3S1 subjects from postoperative days 3-21.

The role of female microglia in CCI-induced mechanical hypersensitivity

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N3S1 nerve injured male and female Balb/c spinal microglial cells were intrathecally transferred between sexes, and hormone-treatment groups, to determine the role of female microglia in exaggerated female nociceptive hypersensitivity. Following baseline assessments

(prior to OVX/ hormone replacement) over 4 consecutive female estrus cycles, donors and recipients underwent OVX/hormone treatment followed by a one week resting period before nerve injury (N3S1). Mechanical allodynia was assessed in N3S1 subjects from postoperative days 3-21. At day 21 post N3S1 nerve injury, donor N3S1 Balb/c mice were sacrificed, and microglial cells were intrathecally transferred to corresponding N3S1 Balb/c recipients using the protocol outlined above. Recipient mechanical allodynia was assessed through to 21 days post intrathecal transfer.

Are female microglia inherently primed?

To determine whether the presence of 17β-estradiol primed microglial cells at the time of injury were capable of inducing exacerbated female hypersensitivity, naïve (non-nerve injured) female microglia taken from Balb/c, TLR4-/- and MyD88-/- donor mice were transferred to Balb/c recipients at the time of nerve injury. Prior to OVX surgery/hormone treatment (baseline), mechanical allodynia was assessed in male and female Balb/c recipients.

Following baseline assessments over 4 consecutive female estrus cycles, male and female recipients underwent OVX/ hormone treatment. Following a one week resting period, recipient mice underwent N3S1 nerve injury and simultaneous intrathecal delivery of donor naïve microglial cells. Mechanical allodynia was assessed up to 21 days post N3S1 nerve injury/microglial transfer.

The effect of sex and 17β-estradiol on neuroimmune targeted interventions

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To investigate the effect of sex and the presence of 17β-estradiol on neuroimmune-targeted therapies mechanical allodynia was assessed in male and female Balb/c mice prior to CCI surgery (baseline). Following OVX/hormone treatment and subsequent N3S1 nerve injury, mechanical allodynia was assessed from postoperative days 3-21. 21 days post nerve injury, mechanical allodynia was further assessed 1 h following i.p. injection of (+)-naltrexone (30 mg/kg, 3 mg/kg, 0.3 mg/kg) and IL-1ra (100 mg/kg, 10 mg/kg, 0.1 mg/kg). Each neuroimmune targeted therapy agent was assessed on separate testing days, with each dose examined once, bi-daily.

Statistics

The relationship between the percent response and the variables sex, estrus cycle phase, von

Frey filament stimulus and surgery was assessed using linear modeling fitted using the statistical package R via the graphical user interface: Rstudio405.

Initially a linear model was fitted that aimed to predict the percent response (number of positive responses out of a maximum of 10 by using the predictors sex, estrus cycle phase, von Frey filament stimulus and surgery. The goal was to estimate the average increase in percent response for each increase in von Frey filament stiffness. We also wanted to estimate how this average increase is influenced by the various levels of sex, estrus cycle phase and surgery. As well, a modified linear modeling method was used, called mixed effect linear models, that had an extra term to account for the fact that we had repeated measures on the rats. This method allows us to account for the variation seen in the percent response both within and between the rats. Believing that there was a possibility for the influence of surgery and estrous to depend on the rat’s sex, we also added two terms to account for these

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interactions. The initial model (M1) was, therefore,

response ~ sex + estrus + surgery + von Frey stiffness + sex:estrus + sex:surgery + (1|rat) where response is the response rate expressed as a percentage (% response), sex is male or female, estrus is pro-estrus, estrus, metestrus or diestrus, surgery is N0S0, N0S4, N1S3,

N3S1, N4S0, von Frey stiffness is 1-6 and rat is the unique rat ID.

The model was fitted using the lmer() function from the lme4 package in R. The code is available on request from the authors.

An example R input would appear as

M1 <- lmer(response ~ sex + estrus + surgery + von-Frey stiffness + sex:estrus + sex:surgery

+ (1|rat), data=data) summary(M1)

To ensure the simplest model to predict the percent response, the initial model (M1) was tested to assess if any for the predictors could be removed as there are not statistically significant predictors. The ability of the model to predict the percent response was measured using the Akaike’s Information Criterion (AIC). The AIC measures how well the model fits the observations with a penalty term for the number of terms in the model. The penalty term is to try and ensure the most parsimonious model. Each predictor was removed from the model and the AIC measured to see if this caused a change in the AIC. The model with the smallest AIC was chosen. This process is repeated until the simplest model that predicts the percent response the best is obtained. This process is automated by the stepAIC() function from the MASS package406. This procedure indicated that none of the predictors could be removed without reducing the predictive ability of the model.

Lauren Nicotra Sex Differences in Allodynia 125

There is controversy regarding P-values for mixed effects models407 and thus, we report observed t-values rather than P-values and use an observed t-value with absolute value of less than negative two or greater than two to indicate statistical significance. Owing to the statistical power of the model statistically significant but behaviorally small differences can be identified. As such, we only report statistical differences that are behaviorally relevant, representing 1.6%, as this represents a change in 1 response out of 10 on the tests.

To further elucidate differences in allodynia (percentage response) and each of the predictors

(sex, estrus cycle phase, surgery and von Frey filament stimulus), subsets of the data were considered (e.g. males only) and models fitted that predicted percent response for each of the predictors individually. Again the model used accounted for the repeated measures obtain from each rat.

An example R input of such subsets to investigate certain predictors:

Example 1. In order to investigate the effect of the female rodent estrus cycle, male rodent data was excluded from the analysis. Below if the example R input in order to investigate this predictor individually:

# Model data excluding males: specifically investigating the effect of the estrus cycle predictor:

M1<- lmer(response~(von Frey stiffness/surgery/sex/(estrus)+ (1|rat), data= subset(data, sex=='female'))

Lauren Nicotra Sex Differences in Allodynia 126

summary(M1) where response is the response rate expressed as a percentage (% response), sex is female, estrus is pro-estrus, estrus, metestrus or diestrus, surgery is N0S0, N0S4, N1S3, N3S1, N4S0, von Frey stiffness is 1-6 and rat is the unique rat ID.

Results

Results were analyzed using linear mixed effects modeling (utilizing the lmer() function) fitted using the statistical package R408. Findings are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response, Supplementary materials).

There is controversy about P-values for linear mixed effect models407. Consequently, the author of the lmer() function does not report them. Hence, we report the t-value with each fixed effect, with absolute value of less than negative two or greater than two indicating statistical significance. Only statistical differences representing a behaviorally relevant are presented. This threshold of behavioral relevance is considered to be a difference in percentage response of greater than 1.6%, as this equals a change in 1 response out of 10 on the tests.

Sex differences in chronic pain: Greater mechanical allodynia in females following nerve injury

A graded sciatic nerve injury model95 was utilized for the first time in mice. By varying the number of sciatic sutures (N), this model produces graded chronic pain. Additional chromic gut was placed subcutaneously (S) so all suture groups had a total of four segments, Lauren Nicotra Sex Differences in Allodynia 127

designated as NxSy where x and y are the number of nerve versus subcutaneous sutures, respectively95. Consequently, treatment groups included N0S0, N0S4, N1S3, N3S1 and N4S0 animals. Before nerve injury, there were no differences between male and female Balb/c nociceptive response scores (0.027% t= 0.02; Table 1) and female nociceptive response scores were unaffected by the estrus cycle (t≥-1.1 but ≤1.5; Table 2).

After nerve injury, increasing sensitivity to mechanical stimuli was positively correlated with increasing numbers of sciatic sutures (t≤-2.7; Table 1; Figure 3.1a). Once allodynia stabilized

(days 17-21 following nerve injury), female mice in all pain treatment groups (Figure 3.1a right) displayed significantly greater mechanical allodynia compared to males (Figure 3.1a left), responding 2.4% more than males per increase in von Frey filament stiffness (t≤-3.4;

Table 1). N1S3 and N3S1 females in both diestrus and pro-estrus further demonstrated exacerbated mechanical allodynia relative to estrus (Di:Est, ≥2.3%, t≥2.7; Pro:Est, ≥1.7%, t≥2.2) and metestrus (Di:Met, ≥1.9%, t≥2.2; Pro:Met, ≥2.1%, t≥2.4) (Table 2; Figure 3.1b).

(a)'

Male Female 80' 100# 100# # # 80# 80# # # 60# 60' 60# # # 40# 40# #

Response'(%)' # 20# 40' 20# # # 0# 0#

20'

0'

Lauren Nicotra Sex Differences in Allodynia 128

Figure 3.1a. Sex differences in chronic pain: Greater mechanical allodynia in females following nerve injury.

Male (left panel) and female (right panel) responses were found to closely resemble the clinical healthy pain experience, with equivalent nociceptive scores at baseline and a sex difference post nerve injury. By varying the number of chromic gut lengths of suture placed around the sciatic nerve, graded mechanical allodynia was established in both rodent sexes.

Pain treatment was designated as NxSy, where x and y are the number of chromic gut lengths of suture placed around the sciatic vs. subcutaneously, respectively. Consequently pain treatment groups included N0S0, N0S4, N1S3, N3S1 and N4S0 animals, Once allodynia became stable (days 17-21 following nerve injury), statistical analysis revealed female mice across all pain treatment groups displayed significantly greater mechanical allodynia compared to males, responding on average 2.4 % more than matched males per von Frey filament ((t ≤ - 3.4). Data is presented as a 3D surface plot using the R Studio lattice package.

Results were analyzed utilizing repeated measures linear mixed effects modeling. A t-value of

< -2 or > 2 was determined statistically significant (P < 0.05). n=8 per treatment group, per sex (Balb/c). von Frey filaments : 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g, 1: 0.008 g. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

Lauren Nicotra Sex Differences in Allodynia 129

(b)

20 20

(b) 15 15

10 10 Proestrus Estrus Metestrus Diestrus N1S3 Difference in Response per Increase in von Frey Stiffness (%) 5 N3S1 Difference in Response per Increase in5 von Frey Stiffness (%) Pre- 3 7 10 14 17 21 Pre- 3 7 10 14 17 21 Nerve Injury Nerve Injury Days Post Nerve Injury Days Post Nerve Injury

Figure 3.1b. Exacerbated female mechanical allodynia is associated with 17β-estradiol.

Following nerve injury, female mechanical allodynia was demonstrated heavily dependent on the rodent estrus cycle. Once allodynia became stable (days 17-21 following nerve injury),

N1S3 (left panel) and N3S1 (right panel) female mice were significantly more allodynic throughout both proestrus and diestrus compared to all other estrus cycle phases (Pro:Est, ≥

1.7 %, t ≥ 2.2; Pro:Met, ≥ 2.1 %, t ≥ 2.4; Di:Est, ≥ 2.3 %, t ≥ 2.7; Di:Met, ≥ 1.9 %, t ≥ 2.2).

Results were analyzed utilizing repeated measures linear mixed effects modeling and are expressed as the percent difference in slope, representing a difference in response per increase in von Frey filament stiffness. A t-value of < -2 or > 2 was determined statistically significant

(p < 0.05). n=8 Balb/c mice per estrus phase, with results generated from a minimum of 4 consecutive estrus cycles.. Di: diestrus, Met: metestrus, Est: estrus, Pro: proestrus). N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

17β-estradiol induces exacerbated mechanical allodynia

Female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy (OVX). Slow release 17β-estradiol pellets (Estrogen)

Lauren Nicotra Sex Differences in Allodynia 130

(0.18mg/kg) or placebo pellets (Estrogen) were simultaneously implanted. 17β-estradiol pellets generate plasma levels of 17β-estradiol ~125 pg/ml409 (greater than homeostatic 17β- estradiol 20-100 pg/ml)410. One week following OVX/pellet insertion, mice underwent nerve injury (N3S1).

Once allodynia stabilized, freely cycling nerve injured females (N3S1-OVX-Estrogen) responded 3.3% more per increase in von Frey filament stiffness compared to nerve injured ovariectomised female mice (N3S1-OVX-Estrogen) (t=-3.6). 17β-estradiol supplemented

N3S1 male (N3S1-OVX-Estrogen) and female mice (N3S1-OVX-Estrogen) also responded significantly more per increase in von Frey stiffness compared to vehicle (≥2.9%, t≥-9.1 and

≤8.7 (Table 3)).

Exacerbated female rodent mechanical allodynia is mediated by 17β-estradiol primed microglia

Given microglia express estrogen receptors374,411 and the microglial involvement in chronic pain, we investigated spinal microglia in 17β-estradiol-induced exacerbated pain. Spinal microglia were isolated 21 days after nerve injury from “donor” N3S1 mice (female donors at pro-estrus). Upon isolation, microglia were immediately intrathecally transferred to stably allodynic N3S1 recipients (day 17-21 post nerve injury) by lumbar puncture (description in online methods). Within animal (day 17-21 to day 38-42) and between microglial treatment group comparisons (at day 38-42) were conducted to determine the impact of microglial delivery and microglia phenotype on mechanical allodynia, respectively.

Lauren Nicotra Sex Differences in Allodynia 131

Donor mice were of 5 different in vivo states (phenotypes); A) male N3S1 OVX (Estrogen vs

Estrogen); B) female N3S1 OVX Estrogen; or C) female N3S1 OVX (Estrogen vs Estrogen).

Donor microglia were administered to 2 different recipient groups; male N3S1 OXV Estrogen or female N3S1 OVX Estrogen. Herein, post microglial transfer recipients are designated with a “Microglia” prefix.

A significant time and in vivo treatment state interaction was demonstrated post microglia delivery. Microglia isolated from freely cycling N3S1 female mice (N3S1-OVX-Estrogen) transferred the exaggerated female pain phenotype to male recipients (N3S1-OVX-Estrogen).

Intra-microglia phenotype comparisons (day 17-21 to day 38-42) revealed male recipients of freely cycling nerve injured female microglia (Microglia-N3S1-OVX-Estrogen) responded

2.3% more per increase in von Frey stiffness post female microglia delivery (t=-7.9). Inter- microglia phenotype comparisons (at day 38-42) determined that male recipients (Microglia-

N3S1-OVX-Estrogen) of these freely cycling nerve injured female microglial cells responded

2.1% more per increase in von Frey stiffness compared to intrathecal delivery of nerve injured ovariectomised female microglia (N3S1-OVX-Estrogen) (t=7.2) and delivery of nerve injured male microglia (N3S1-OVX-Estrogen) (t=7.1) (Table 4; Figure 3.2a).

Exacerbation of female pain following the transfer of female microglia (N3S1-OVX-

Estrogen) was gonadal steroid hormone dependent. Intra-microglia phenotype comparisons

(day 17-21 to day 38-42) revealed the intrathecal transfer of freely cycling nerve injured female microglia (N3S1-OVX-Estrogen) to ovariectomised N3S1 female recipients (N3S1-

OVX-Estrogen) restored exaggerated female mechanical hypersensitivity. Following microglia delivery, such female recipients (Microglia-N3S1-OVX-Estrogen) responded 2.8% more per increase in von Frey stiffness post-microglia delivery (t=-9.6; Table 4; Figure 3.2).

Lauren Nicotra Sex Differences in Allodynia 132

Inter microglial phenotype comparisons (at day 38-42) further demonstrated that these female recipients (Microglia-N3S1-OVX-Estrogen) responded significantly more per increase in von

Frey stiffness than female recipients (N3S1-OVX-Estrogen) of ovariectomised female microglia (N3S1-OVX-Estrogen) (5.2%, t=12.0) and nerve injured male microglia (N3S-

OVX-Estrogen) (4.9%, t=12.0) (Table 4; Figure 3.2b).

To determine whether this ability to transfer exaggerated mechanical allodynia was 17β- estradiol mediated, male (N3S1-OVX-Estrogen) and female (N3S1-OVX-Estrogen) recipients intrathecally received nerve injured spinal microglia taken from mice supplemented in vivo with 17β-estradiol. Intra-microglia phenotype comparisons (day 17-21 to day 38-42) demonstrated that subsequent to microglial delivery, both male (Microglia-N3S1-OVX-

Estrogen) and female (Micro-N3S1-OVX-Estrogen) recipients responded significantly more per increase in von Frey stiffness post microglia delivery (≥4.0 % t≥13.0; Table 4). At day 38-

42, male (Figure 3.2a) and female (Figure 3.2b) mice that received intrathecal spinal microglia supplemented in vivo with 17β-estradiol responded significantly more compared to the intrathecal delivery of all other microglial phenotypes (≥1.9 %, t≥7.6; Table 4).

Importantly, the transfer of nerve injured male (N3S1-OVX-Estrogen) and ovariectomised nerve injured female (N3S1-OVX-Estrogen) microglia to male (N3S1-OVX-Estrogen) (Table

4; Figure 3.2A) and female (N3S1-OVX-Estrogen) (Table 4; Figure 3.2B) recipients was unable to modify recipient rodent mechanical allodynia (day 17-21 to day 38-42) (t>0.20 but

<2). Consequently, simply transferring spinal microglia to recipient animals does not alter mechanical allodynia. Only in the presence of female microglia or 17β-estradiol primed microglia could recipient mechanical allodynia be modified.

Lauren Nicotra Sex Differences in Allodynia 133

Female OVX-V N3S1 + Female OVX-E N3S1 + Female N3S1 OVX-Estrogen Female OVX-V N3S1 + Female OVX-V N3S1 + Female N3S1 OVX-Estrogen Female OVX-V N3S1 + Female ShamOVX-V N3S1 + Female N3S1 OVX-Estrogen Female OVX-V N3S1 + Male ShamOVX-V N3S1 + Male N3S1 OVX-Estrogen Female OVX-V N3S1 + Male ShamOVX-E N3S1

+ Male N3S1 OVX-Estrogen (b) Recipient: Female N3S1-OVX-Estrogen (a) Recipient: Male N3S1-OVX-Estrogen Origin of Donor Microglial Cells: Origin of Donor Microglial Cells: + Female Female N3S1 N3S1 OVX-Estrogen OVX-Estrogen + Female Female N3S1 N3S1 OVX-Estrogen OVX-Estrogen + Male N3S1 N3S1 OVX OVX-Estrogen-Estrogen +Male Male N3S1 N3S1 OVX OVX-Estrogen-Estrogen

15 15 + FemaleFemale N3S1 N3S1 OVX OVX-Estrogen-Estrogen + FemaleFemale N3S1 N3S1 OVX OVX-Estrogen-Estrogen

+ Male N3S1 N3S1 OVX OVX-Estrogen-Estrogen + MaleMale N3S1 N3S1 OVX OVX-Estrogen-Estrogen +Female Female N3S1 N3S1 OVX- OVX-EstrogenEstrogen + Female N3S1 N3S1 OVX- OVX-EstrogenEstrogen

10 10

Difference in Response per Increase in von Frey Stiffness (%)5 Difference in Response per Increase in von Frey Stiffness (%)5 Pre-CCI N3S1 3 7 10 14 17 21 Pre-CCI N3S1 3 7 10 14 17 21 Days Post Microglial Delivery Days Post Microglial Delivery

Figure 3.2. Exacerbated female rodent mechanical allodynia is mediated by 17β- estradiol primed microglial cells.

Spinal microglia were intrathecally transferred from N3S1 “donor” male and female Balb/c mice 21 days following nerve injury to “recipient” N3S1 nerve injured Balb/c male (3.2a) and female (3.2b) mice. Male recipients were N3S1 sham ovariectomised (OVX) vehicle pelleted

(Estrogen) Balb/c mice (3.2a). Female recipients were N3S1 ovariectomised (OVX) vehicle pelleted (Estrogen) Balb/c mice (3.2b). Donor microglia were acquired from a number of

Balb/c male and female mice that received (in vivo) ovariectomy (OVX) or sham ovariectomy

(OVX) in addition to 17β-estradiol supplementation (Estrogen) or placebo (Estrogen). The addition of donor microglia is designated with a “+”. Microglial cells intrathecally transferred from freely cycling nerve-injured females (N3S1-OVX-Estrogen) exacerbated nerve injured male (N3S1-OVX-Estrogen) (t=- 7.9;Figure 3.2a) and ovariectomised female (N3S1-OVX-

Estrogen) (t=- 9.6;Figure 3.2b) mechanical allodynia. Microglial cells acquired from nerve injured 17β-estradiol treated (in vivo) females (OVX-Estrogen) further exacerbated male

(N3S1-OVX-Estrogen) (Figure 3.2a) and female (N3S1-OVX-Estrogen) (Figure 3.2b) recipient mechanical allodynia (t < -13.0). Recipient mechanical allodynia was recorded through to 21 days post delivery of microglial cells and reported once allodynia stabilized

(days 38-42 post N3S1 nerve injury). 6 x 103 microglial cells in 10µl PBS were intrathecally transferred to recipients by lumbar puncture. Results were analyzed utilizing repeated Lauren Nicotra Sex Differences in Allodynia 134

measures linear mixed effects modeling. A t-value of < -2 or > 2 was determined statistically significant (P<0.05). n=8 per treatment group. von Frey filaments: 6:0.40 g, 5:0.16 g, 4:0.07 g, 3:0.04 g, 2:0.02 g, 1:0.008 g. N:number of sciatic sutures, S:number of subcutaneous sutures.

Female microglia are inherently primed to subsequent pain signals

To determine if microglially-derived exaggerated pain was because female microglia were inherently pre-disposed to over-respond to nerve injury, naïve (not nerve injured) spinal microglia were intrathecally transferred to recipients at naïve the time of recipient N3S1 nerve injury (Microglia+N3S1) and mechanical allodynia recorded once allodynia stabilized (17 to

21 days after combined nerve injury and microglia injection). Pain scores from days 17-21 after nerve injury were compared between nerve-injured mice receiving no microglia (N3S1) and mice additionally receiving microglia at the time of nerve injury (Microglia+N3S1).

The mere presence of female microglia was sufficient to exacerbate recipient mechanical allodynia. Male recipients (OVX-Estrogen) of naïve female microglia (OVX-Estrogen) at the time of nerve injury (Microglia+N3S1 OVX-Estrogen) demonstrated elevated mechanical allodynia, responding 3.1% more per increase in von Frey filament stiffness than male nerve injured mice (N3S1 OVX-Estrogen) (t=-12.0) (Table 5; Figure 3.3a). Female microglia’s ability to elevate recipient mechanical allodynia was gonadal steroid hormone dependent.

Intrathecal delivery of microglia from naïve freely cycling females (OVX-Estrogen) at the time of nerve injury significantly enhanced mechanical allodynia in ovariectomised female recipients (N3S1 OVX-Estrogen), with such female microglia recipients (Microglia+N3S1

OVX-Estrogen) responding 3.2% more per increase in von Frey filament stiffness compared

Lauren Nicotra Sex Differences in Allodynia 135

to ovariectomised N3S1 females who did not receive microglia (N3S1 OVX-Estrogen) (t =-

13.0) (Table 5; Figure 3.3b). In contrast, intrathecal transfer of naïve ovariectomised female microglia (OVX-Estrogen) at the time of nerve injury failed to increase male

(Microglia+N3S1 OVX-Estrogen) (Figure 3.3a) and female (Microglia+N3S1 OVX-

Estrogen) (Figure 3.3b) recipient mechanical allodynia compared to control N3S1 male

(N3S1 OVX-Estrogen; 0.21 %, t=0.17; Table 5) and female (N3S1 OVX-Estrogen; 0.06%, t=0.12; Table 5) mice.

To determine if this freely cycling female microglia effect was mediated by 17β-estradiol, naïve microglia taken from female mice supplemented in vivo with 17β-estradiol (OVX-

Estrogen) were transferred to the spinal cord of male recipients (OVX-Estrogen) at the time of nerve injury. Male recipients (Microglia+N3S1 OVX-Estrogen) of these 17β-estradiol in vivo supplemented microglia responded 5.2% more per increase in von Frey filament stiffness than corresponding N3S1 male controls (N3S1 OVX-Estrogen) (t=24.0; Table 5, Figure 3.3a).

Analogous findings were demonstrated in ovariectomised female recipients (OVX-Estrogen).

Inter-treatment comparisons demonstrated that these ovariectomised female recipients

(Microglia+N3S1 OVX-Estrogen) of in vivo 17β-estradiol supplemented microglia responded

5.3% more per increase in von Frey filament stiffness than N3S1 ovariectomised female controls (N3S1 OVX-Estrogen) (t=23.0) (Table 5; Figure 3.3b).

Lauren Nicotra Sex Differences in Allodynia 136

Female OVX-V N3S1 Female OVX-V + N3S1 Female ShamSurg-V + N3S1

(b) Female OVX-E + N3S1 (a)

20 20

Male OVX-Estrogen + Female OVX-Estrogen Female OVX-Estrogen + Female OVX-Estrogen

Male OVX-Estrogen + Female OVX-Estrogen Female ShamSurg-V OVX-Estrogen + N3S1 + Female OVX-Estrogen 15 15

Male OVX-Estrogen + Female OVX-Estrogen Female OVX-Estrogen + Female OVX-Estrogen Male N3S1 OVX-Estrogen FemaleFemale OVX-V N3S1 N3S1 OVX-Estrogen 10 10

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 3 7 10 14 17 21

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 3 7 10 14 17 21 Baseline Days Post Surgical Intervention

Baseline Days Post Surgical Intervention

Figure 3.3. Female microglia are inherently primed to subsequent pain signals.

Naïve (non-nerve injured) ovariectomised vehicle-treated Balb/c female mice (OVX-

Estrogen) (Figure 3.3b) and naïve male Balb/c recipients (OVX-Estrogen) (Figure 3.3a) received female microglia taken from naïve freely cycling and/or 17β-estradiol supplemented

Balb/c donors at N3S1 nerve injury. The presence of freely cycling female microglia (OVX-

Estrogen) at nerve injury restored exacerbated female allodynia in ovariectomised female recipients (OVX-Estrogen) and exaggerated mechanical hypersensitivity in males (OVX-

Estrogen). Female mice were ovariectomised (OVX) and males received Sham ovariectomy as a control (OVX). 17β-estradiol (Estrogen) and vehicle (Estrogen) was administered in pellet form (0.18 mg). Responses between N3S1 mice and mice that had additionally received microglia (Micro+N3S1) were compared. Results were analysed utilising repeated measures linear mixed effects modelling. A t-value of < -2 or > 2 was determined statistically significant (P< 0.05). 6 x 103 microglial cells in 10 µl PBS were intrathecally transferred to recipients simultaneously with N3S1 nerve injury. Allodynia was recorded through to 21 days following nerve injury/microglial delivery and reported once stable (days 17-21). All recipients received N3S1 nerve injury. n=8 per treatment group. von Frey filaments: 6:0.40 g,

5:0.16 g, 4:0.07 g, 3:0.04 g, 2:0.02 g, 1:0.008 g. OVX:ovariectomy, Estrogen:17β-estradiol,

Estrogen:vehicle, N:number of sciatic sutures, S:number of subcutaneous sutures. CCI- injured mice (N3S1) that have not received microglial delivery are signified by - - - Lauren Nicotra Sex Differences in Allodynia 137

Estrogenic microglial-priming is dependent upon MyD88, TLR2/4 signaling

This study further determined whether the ability for 17β-estradiol primed spinal microglia to exacerbate recipient rodent mechanical allodynia was dependent on innate immune signaling.

Spinal microglia were taken from naïve (non-nerve injured) female TLR2/4 (Tlr2/4–/–) and

Myd88 (Myd88–/–) knockout (KO) mice and intrathecally transferred to females of their wildtype (WT) (Balb/c) strain at the time of recipients’ N3S1 nerve injury. Nociceptive responses between WT and KO microglia recipients were directly compared. Once allodynia stabilized (days 17-21 after combined N3S1 and microglia injection), the preceding ability for in vivo estrogen supplemented microglia to exacerbate recipient mechanical allodynia

** same time microglial transfer using myd88 and tlr2/4 –/– (t≥23.0) was abolished inFemale the Balb/c genetic N3S1 OVX-V absence + N3S1 of MyD88 and TLR2/4 signaling (Myd88 , Female Balb/c OVX-E + N3S1 t≥0.18 but ≤0.21; Tlr2/4–/– ,Female t≥0.12 MyD88 butOVX-V ≤+ N3S10.18; Table 6; Figure 3.3c). Female MyD88 OVX-E + N3S1 Female TLR2/4 OVX-V + N3S1 Female TLR2/4 OVX-E + N3S1 (C) Recipient: Female Balb/c OVX-Estrogen

25 Origin of Donor Microglial Cells:

20 FemaleFemale Balb/cBalb/c OVX-E OVX-Estrogen + N3S1

15 FemaleFemale Balb/c Balb/c N3S1 OVX- OVX-VEstrogen + N3S1 Female MyD88 OVX-Estrogen Female MyD88 OVX-E + N3S1 Female MyD88MyD88 OVX-VOVX-Estrogen + N3S1 10 FemaleFemale TLR2/4 TLR2/4+ OVX-OVX-EEstrogen + N3S1 FemaleFemale TLR2/4 TLR2/4+ OVX-EstrogenOVX-V + N3S1

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 3 7 10 14 17 21

Baseline Days Post Surgical Intervention

Figure 3.3c. Inherent Estrogenic microglial-priming is dependent upon MyD88, TLR2/4 signaling.

Lauren Nicotra Sex Differences in Allodynia 138

The previous ability for female and 17β-estradiol primed wildtype (Balb/c) microglia to exacerbate recipient allodynia (Figure 3.3a and 3.3b) was abolished in the genetic knockout of toll-like receptor 2 and 4 (TLR2/4) and MyD88 (Figure 3.3c). Female mice were ovariectomised (OVX) and males received Sham ovariectomy as a control (OVX). 17β- estradiol (Estrogen) and vehicle (Estrogen) was administered in pellet form (0.18 mg).

Responses between N3S1 mice and mice that had additionally received microglia

(Micro+N3S1) were compared. Results were analysed utilising repeated measures linear mixed effects modelling. A t-value of < -2 or > 2 was determined statistically significant (P<

0.05). 6 x 103 microglial cells in 10 µl PBS were intrathecally transferred to recipients simultaneously with N3S1 nerve injury. Allodynia was recorded through to 21 days following nerve injury/microglial delivery and reported once stable (days 17-21). All recipients received

N3S1 nerve injury. n=8 per treatment group. von Frey filaments: 6:0.40 g, 5:0.16 g, 4:0.07 g,

3:0.04 g, 2:0.02 g, 1:0.008 g. OVX:ovariectomy, Estrogen:17β-estradiol, Estrogen:vehicle,

N:number of sciatic sutures, S:number of subcutaneous sutures. CCI-injured mice (N3S1) that have not received microglial delivery are signified by - - -

In vitro exposure of HEK-TLR2 and HEK-TLR4 cells to estrogens activates NFκB signaling, potentially by cooperation or complementation with TLR2 and TLR4

To determine estrogen’s ability to induce TLR signaling alone or potentiate TLR-induced signaling, estrogens were added to human embryonic kidney (HEK) cell lines transfected to over-express TLRs. TLR4-overexpressing cells displayed a dose dependent activation of

TLR signaling by LPS (0.01-100ng/ml), with activation of NFκB (reflected by SEAP expression) that was potentiated by estradiol (17β-estradiol, Figure 3.4a). NFκB activation occurred by all three estrogens at 100 µM in the absence of LPS and by estrone at 10 µM Lauren Nicotra Sex Differences in Allodynia 139

(F=269.6, P<0.0001) compared to vehicle. Figure 3.4d-f demonstrates analogous findings in

TLR2-overexpressing cells, with a dose-dependent activation of NFκB (reflected by SEAP expression) by PAM3CSK4 (0.01-100 ng/ml, TLR2 agonist), which was potentiated by estradiol (17β-estradiol, Figure 3.4d), estriol (Figure 3.4e) and estrone (Figure 3.4f).

Significant NFκB activation occurred using all three estrogens at 100 µM in the absence of

PAM3CSK4 (F=41.77, P<0.0001) and 10µM Estriol (P<0.05) compared to vehicle in TLR2 over-expressing cells. Therefore, it is possible that 17β-estradiol can bind to TLR2 and 4 or potentiate the intracellular signaling mediating by TLR activation, tested below.

Lauren Nicotra Sex Differences in Allodynia 140

Vehicle (a)$ vehicle (d)$ 17β-Estradiol 1 uM Estradiol 1 uM 17β-Estradiol 10 uM 20000 Estradiol 10 uM 80000 17β-Estradiol 100 uM Estradiol 100 uM

1500 B signal)

B signal) 15000 B signal) 60000 κ 1000 κ κ SEAP 500

0 10000 expression (NF 40000 0 1 uM 10 uM 100 uM SEAP SEAP

5000 20000 expression (NF 0 expression (NF 0 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 LPS (ng/ml) PAM3CSK4 (ng/ml)

Vehicle Vehicle (b)$ Estriol 1 uM (e)$ Estriol 1 uM Estriol 10 uM Estriol 10 uM Estriol 100 uM 80000 Estriol 100 uM 20000 2000

1500 B signal) κ B signal)

B signal) 60000

κ 1000 15000 κ SEAP

500

0 40000 expression (NF

SEAP 10000 0 1 uM 10 uM 100 uM SEAP

5000 20000 expression (NF 0 expression (NF 0 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 LPS (ng/ml) PAM3CSK4 (ng/ml)

Vehicle Vehicle (c)$ Estrone 1 uM (f)$ Estrone 1 uM Estrone 10 uM Estrone 10 uM Estrone 100 uM 80000 Estrone 100 uM 20000

1500 B signal)

B signal) 60000 B signal)

κ 1000 κ 15000 κ SEAP 500 40000 0 expression (NF SEAP 10000 SEAP 0 1 uM 10 uM 100 uM

5000 20000 expression (NF 0 expression (NF 0 0 0.01 0.1 1 10 100 0 0.01 0.1 1 10 100 LPS (ng/ml) PAM3CSK4 (ng/ml)

Figure 3.4. Exposure of HEK-TLR2&4 cells to estrogens in vitro activates NFκB signaling, likely via actions at TLR2&4.

Estriol, 17β-Estradiol, and Estrone (1-100 µM) administered to HEK-TLR4 (a-c) in the presence and absence of LPS (0.01-100 ng/ml) and TLR2 (d-f) cells in the presence and absence of PAM3CSK4 (0.01-100 ng/ml) for 24 hours. In HEK-TLR4 and TLR2 cells all three estrogens induced SEAP expression alone at 100 µM (P < 0.0001). 17β-Estradiol

Lauren Nicotra Sex Differences in Allodynia 141

potentiated SEAP expression induced by both LPS (TLR4) at 100 µM (P < 0.05) and

PAM3CSK4 (TLR2) at both 100 µM and 10 µM (P < 0.01). Estriol potentiated SEAP expression induced by PAM3CSK4 (TLR2) at 100 µM (P < 0.05) and 10 µM (P < 0.05).

Estrone potentiated SEAP expression induced by PAM3CSK4 (TLR2) at 100 µM (P < 0.05).

Each experiment was repeated at least twice and similar results obtained. Results are presented from 1 experiment only. Data are expressed as mean ± SEM.

17β-Estradiol mediated NFκB activation in HEK-TLR4 cells is inhibited by TLR4 antagonists.

To identify whether SEAP expression induced by 100 µM of the estrogens through direct receptor activation, TLR4 HEK cells and 100µM 17β-estradiol (E2) were treated with two

TLR4 antagonists: the high affinity LPS-RS and the low affinity (+)-naltrexone390. 17β- estradiol induced comparable SEAP expression to that of 1 ng/ml LPS (Figure 3.5), which were both significantly greater than vehicle (F=21.91, P<0.0001). 17β-estradiol induced

SEAP expression was significantly reduced utilizing LPS-RS (post hoc, P<0.001) and 1-

100µM (+)-naltrexone (post hoc P<0.001, Figure 3.5). Neither antagonist had any effect on

SEAP expression in the absence of 17β-estradiol (P>0.05).

Lauren Nicotra Sex Differences in Allodynia 142

30000

***" ***"

20000 B signal)

κ 10000 (NF SEAP expression 0

Vehicle E2+vehicle LPS 1 ng/ml

E2+10 ng/ml LPS-RS E2+1 uM (+)-Naltrexone E2+10 uM (+)-Naltrexone Vehicle+10ng/ml LPS-RS E2+100 uM (+)-Naltrexone Vehicle+100 uM (+)-Naltrexone

Figure 3.5. 17β-Estradiol mediated NFκB activation in HEK-TLR4 cells is inhibited by

TLR4 antagonists.

17β-Estradiol (E2) (100 µM) induced comparable SEAP expression, a measure of NFκB activation, to that of 1 ng/ml LPS in HEK-TLR4 overexpressing cells. LPS-RS, a specific

TLR4 receptor antagonist and 1-100 µM (+)-Naltrexone, a putative TLR4 receptor antagonist significantly reduced SEAP expression induced by 17β-Estradiol (E2) (100 µM). Data are expressed as mean ± SEM (n=4 per group). Each experiment was repeated at least twice and similar results obtained. Results are expressed from one experiment only. All antagonists significantly attenuated E2-induced NFκB activation. *** P < 0.0001 against all other groups.

E2 (100 µM) was not significantly different to 1 ng/ml LPS.

Lauren Nicotra Sex Differences in Allodynia 143

Female mice are more sensitive to neuroimmune modulation

Extending these in vitro findings, established exacerbated female mechanical allodynia was subsequently reversed utilizing neuroimmune modulators. Intraperitoneal injection of the blood brain barrier permeable TLR4 receptor antagonist (+)-naltrexone (Table 7; Figure 3.6a) and interleukin-1 (IL-1) receptor antagonist (IL-1ra) (Table 8; Figure 3.6b) reversed established mechanical allodynia in a dose-dependent manner at day 21 post N3S1 nerve injury ((+)-naltrexone: males ≥ 2.9%, t≥7.3; females ≥2.4 %, t≥10.0; IL-1ra: males≥2.6, t≥7.0; females ≥2.7, t≥7.8). Interestingly, despite females being significantly more allodynic than males, both neuroimmune agents brought males and females to a common non-allodynic post- drug state.

Interaction between 17β-estradiol and the innate immune system

17β-estradiol induced exacerbated mechanical allodynia was further demonstrated neuroimmune dependent using (+)-naltrexone and IL-1ra. Both neuroimmune-targeted agents reduced mechanical allodynia across all OVX/gonadal steroid hormone treatment groups

(Tables 7 and 8). The allodynia of previously hypersensitive freely cycling females (OVX-

Estrogen) and 17β-estradiol pelleted mice was found to fall to an equivalent state as in ovariectomized females (OVX-Estrogen) and vehicle treated mice using (+)-naltrexone

(Figure 3.6c) and IL-1ra (Table 9; Figure 3.6d).

Lauren Nicotra Sex Differences in Allodynia 144

(a)$ (b)$ 20 20 Female&N3S1& Male&N3S1&

15 15

10 10

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 5 3 30 0.3 1 10 Vehicle mg/kg (+)-naltrexone 100 Vehicle mg/kg IL-1ra No Injection No Injection (c)$ (d)$

20 20 Female&OVX/Estrogen&N3S1& Female&OVX/Estrogen&N3S1& Male&OVX/Estrogen&N3S1& Male&OVX/Estrogen&N3S1&

15 15

10 10

5 Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 1 3 10 0.3 30 100 Vehicle Vehicle mg/kg (+)-naltrexone mg/kg IL-1ra No Injection No Injection

Figure 3.6. Interaction Between 17β-estradiol and the Innate Immune System.

(+)-naltrexone and IL-1ra reduced female nerve injury induced mechanical allodynia in a

17β-estradiol dependent manner, independent of sex and OVX surgery. (+)-naltrexone (3.6a) and IL-1ra (3.6b) reduced mechanical allodynia significantly more in freely cycling (OVX)

Lauren Nicotra Sex Differences in Allodynia 145

females compared to ovarictomized (OVX) females. A greater reduction in allodynia was demonstrated in 17β-estradiol (Estrogen) treated male and female mice utilising (+)- naltrexone (Figure 3.6c) and IL-1ra (Figure 3.6d) compared to vehicle (Estrogen). Allodynia was recorded 1 hour post intraperitoneal administration. (+)-naltrexone, 30 mg/kg, 3 mg/kg and 0.3 mg/kg; IL-1ra, 100 mg/kg, 10 mg/kg and 1 mg/kg. Results were analysed utilising repeated measures linear mixed effects modelling and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von

Frey filaments. von Frey filaments: 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g, 1:

0.008 g. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05). Moderate pain (N3S1) Balb/c male and female mice were used in all experiments. n=8 per treatment group, per sex. OVX:ovariectomy, Estrogen:17β-estradiol, Estrogen:vehicle, N:number of sciatic sutures, S:number of subcutaneous sutures.

Exacerbated female rodent mechanical allodynia is not accompanied by a change in astrocyte or microglial markers

Given the ability of female and 17β-estradiol primed microglia to exacerbate recipient rodent mechanical allodynia, L5 spinal cords of these recipient mice were processed for astrocyte

(GFAP) and microglia (CD11b/c) activation markers. Twenty-one days following microglial transfer/nerve injury, no significant difference in GFAP or CD11b expression was demonstrated between estrogen and vehicle treated microglia from the spinal cords of either

Balb/c (CD11b/c: t=0.71, GFAP: t=0.30), MyD88–/– (CD11b/c: t=0.55. GFAP: t=1.22) or

Tlr2/4–/– (CD11b/c: t=0.55, GFAP: t=1.22) recipients.

Lauren Nicotra Sex Differences in Allodynia 146

Discussion

This study established that spinal microglia exacerbated female mechanical allodynia through

17β-estradiol-priming, which was dependent on TLR2/4-MyD88 signaling. We utilized a novel model of graded nerve injury412 and unique statistical analysis94, applied here for the first time in mice. Importantly, this graded nerve injury model intentionally engages both neuronal and peripheral immune signals, resulting in preclinical immune responses that parallel those of clinical chronic pain populations413. We show that female mice were significantly more allodynic than males and this was exacerbated during raised 17β-estradiol estrus cycle phases. The importance of 17β-estradiol was confirmed as removal of the ovaries reversed exaggerated female allodynia and 17β-estradiol supplementation restored allodynia in females and elevated allodynia in males. Critically, we established that exaggerated allodynia could be adoptively transferred to males and OVX females via intrathecal administration of spinal microglia from nerve injured mice supplemented with 17β-estradiol.

Our studies are the first to demonstrate that the presence of naïve female microglia at the time of nerve injury is sufficient to exacerbate mechanical allodynia, revealing a contribution of

17β-estradiol-female-microglial-priming to the exacerbated female pain phenotype. 17β- estradiol primed microglia exacerbated recipient allodynia through TLR2 and/or TLR4 and their MyD88-dependent signaling. In the genetic absence of TLR2/4 MyD88, female and

17β-estradiol-primed microglia failed to exacerbate recipient mechanical allodynia, demonstrating an association between 17β-estradiol and microglial TLRs in nerve injury- induced exacerbated female allodynia.

In vitro, estrogens alone were capable of triggering NFκB activation in over-expressing TLR2 and TLR4 HEK cells. This interaction was TLR4 dependent, with NFκB activation inhibited

Lauren Nicotra Sex Differences in Allodynia 147

by the TLR4 antagonists (+)-naltrexone and LPS-RS, suggestive of 17β-estradiol binding directly to TLR4. In vitro reversal of estrogenic pro-inflammatory effects was further replicated in vivo. Neuroimmune modulation preferentially reduced female and 17β-estradiol- induced nociceptive hypersensitivity with male and female; and estrogen and vehicle nociceptive responses reduced to a common non-allodynic post-drug state.

These results extend previous investigations establishing TLR4 involvement in nerve injury- induced allodynia390. We demonstrated exacerbated female nociceptive sensitivity was linked to TLR2/4-MyD88-dependent 17β-estradiol-priming of spinal microglia.

Before nerve injury, male and female nociceptive responses were equivalent, reflecting the clinical pain scenario in healthy human populations. Replicating the disproportionate female representation within the chronic pain population13, exacerbated female mechanical allodynia was established following nerve injury. Supporting previous investigations414,218, a correlation was demonstrated between exaggerated female mechanical allodynia and the high estrogenic phases of pro-estrus and diestrus in N1S3 and N3S1 females.

Despite animal models improving our understanding of chronic pain pathophysiology, most studies employ preclinical methodologies that produce sex difference findings that are not clinically comparable94. The inability to determine an estrus cycle effect in maximally allodynic animals highlights issues with using the traditional two group, sham controlled, and hence binary in nature pain models which are more likely to only reveal profound deviations in the pain response. Conversely, the graded nerve injury model can detect heterogeneous pain and moderate reductions in allodynia, with sex and estrus cycle differences determined

Lauren Nicotra Sex Differences in Allodynia 148

here without requiring maximal allodynia94. Moreover, the use of the graded pain model provides greater translation to clinical chronic pain populations, owing to similar neuroimmune repsonses32. Consequently, to investigate the underlying chronic pain pathophysiology and potentially unveil analgesics for a range of chronic pain patients, we utilized moderately allodynic animals in the remaining investigations.

Replicating previous studies415, exacerbated female mechanical allodynia was ovarian steroid hormone-dependent in the graded nerve injury model. Despite inconsistency in the literature, gonadal steroid hormone, particularly 17β-estradiol, modulation of nociception is observed both clinically and experimentally130. The ovaries represent the major source of estrogens in females, 17β-estradiol being the most potent416. Estrogen is not only critically involved in reproduction, but modulates neuronal function, acting as a neurosteroid, potentiating inflammatory responses via NFκB activation in innate immune cells, including microglia417.

We investigated whether this association between 17β-estradiol and TLR4 was responsible for nerve injury induced exacerbated female pain, given that NFκB is downstream of TLRs, including TLR4418, TLR4 mediated pro-inflammation is greater in females,354 and chronic pain is maintained in part by an upregulation and increased signaling of TLR498.

The association between 17β-estradiol and TLR4 appeared to be microglia-dependent. Given that mechanical allodynia can be potentiated by intrathecal transfer of activated peripheral and innate immune cells412 and females and 17β-estradiol supplemented mice displayed exacerbated mechanical allodynia in the graded nerve injury model, we investigated the ability for their microglia to phenocopy exaggerated pain when transferred to less allodynic mice412. Intrathecal delivery of these microglia elevated recipient mechanical hypersensitivity.

Lauren Nicotra Sex Differences in Allodynia 149

As this was 17β-estradiol dependent, this established an interaction between this hormone and the innate immune cells of the CNS that is pivotal in the establishment of the exaggerated female pain phenotype.

Although never before investigated in the sexual dimorphism of chronic pain, glial sensitization or “priming” is now known to contribute to several neuroinflammatory diseases419. In several domains, glial-priming contributes to persistent neuroinflammation resulting in exaggerated expression of brain IL-1β420,421. Here, we extended glial-priming to female chronic pain, with naïve microglia, taken from freely cycling or 17β-estradiol supplemented mice exaggerating recipient nociceptive sensitivity. Replicating findings from

TLR4 priming and two-hit dosing regimen studies422, we demonstrated a secondary requirement of nerve injury in recipients for 17β-estradiol ’primed’ microglia to exacerbate recipient mechanical allodynia. Evidently, these studies have revealed a fundamental sex difference in the innate immune cells known to mediate chronic pain. Although others have previously shown that microglia are highly sexually dimorphic, with sex differences in the number, morphology and responses of microglia between males and females362, 423, 424 our results demonstrate for the first time that female microglia are inherently primed by 17β- estradiol, predisposing them to over-respond in nerve injury, in turn contributing to the exacerbated female pain phenotype.

The ability for 17β-estradiol primed microglia to exacerbate female mouse sensitivity was dependent on TLR2 and/or TLR4 and their MyD88-dependent signaling. Following TLR binding, activation of MyD88 signaling results in NFκB activation and pro-inflammatory cytokine production contributing to chronic pain425. Although microglial TLR activation is

Lauren Nicotra Sex Differences in Allodynia 150

implicated in pathological pain98, we are the first to uncover an interaction between 17β- estradiol and TLRs in exaggerated female nociception. In the genetic absence of TLR2/4 and their adaptor protein MyD88, female and 17β-estradiol primed microglia failed to exacerbate recipient mechanical allodynia. These findings demonstrated a role for 17β-estradiol spinal microglial-priming in exaggerated female pain but could not be explained using glial activation markers. Considering the strong behavioral evidence documented here for TLR2/4

MyD88 dependent 17β-estradiol microglial-priming, in addition to microglial activation in other preclinical pain models426, these findings emphasize the importance of not discounting a role for glial involvement when reactive surface expression markers are not obvious.

Although unable to support this study’s behavioral findings utilizing glial activation markers, these priming studies identified for the first time that nerve injury-induced exacerbated female nociceptive sensitivity lies in part to an association between 17β-estradiol and microglial

TLRs. Important to note however are the limitations of this study in regards to the contributors to the exaggerated female nociceptive sensitivity. Although female spinal microglia have been elucidated and investigated in these particular studies, the contribution of peripheral immune responses such as a possible role of mast cells427, or the role of supraspinal immune responses428 in the generation of exacerbated mechanical allodynia were not investigated. Although this limitation does not detract from or reduce the significant role of spinal female microglia in mechanical allodynia, these immune responses should be examined in future studies.

To elucidate the mechanisms underlying 17β-estradiol in exaggerated female nociception, we explored whether 17β-estradiol directly bound TLRs or exacerbated TLR signaling. Here we provide the first evidence of 17β-estradiol possibly directly activating TLR4 signaling and

NFκB activation.

Lauren Nicotra Sex Differences in Allodynia 151

In vivo studies replicated the in vitro findings, with neuroimmune inhibition of established mechanical allodynia in the graded nerve injury model. Following nerve injury, sexual dimorphism in the pharmacological attenuation of neuroimmune signaling was demonstrated using (+)-naltrexone and IL-1ra, replicating several in vivo pharmacological studies390,422. The preferential reduction of female nociceptive sensitivity in the graded nerve injury model, however, was 17β-estradiol dependent, thereby supporting not only previous findings of 17β- estradiol’s ability in vivo, to potentiate IL-1β mRNA responses in rodent microglia429, but also our in vitro findings demonstrating nerve injury-induced exacerbated female nociceptive sensitivity depended, in part, on 17β-estradiol interaction at TLR4.

Until recently, studies investigating the role of TLR4 in chronic pain utilized only males.

Publications from Mogil430 and Yaksh and colleagues,431 however, have demonstrated in the

LPS and SNL pain model, respectively, that female mechanical allodynia is TLR4 independent in CD-1 and C57BL6 mice. Several important distinctions can be made from our approach that provides further mechanistic insights into chronic pain processes. Firstly, we have established that combined neuronal (N3) and peripheral immune challenges (S1) are required to more closely mimic the immune system response in clinical chronic pain populations413. Interestingly, LPS (immune) and SNL (neuronal) challenges appear to dissect out a profound, unrecognized selectivity in the male neuroimmune system, causing responses divergent to females. However, our previous data suggest the clinical chronic pain scenario is not as delineated as this and therefore the choice of pain model is key413, 432. Nevertheless, these studies highlight the need for female specific glial research.

Lauren Nicotra Sex Differences in Allodynia 152

In summary, utilizing a novel graded model of nerve injury, we demonstrated for the first time that female mechanical allodynia had greater neuroimmune sensitivity than males, and nerve injury-induced exaggerated female mechanical allodynia was attributed in part to the association between 17β-estradiol and the innate immune system, particularly TLR4. By potentially directly activating TLR4, 17β-estradiol was demonstrated to prime female microglia, resulting in exaggerated female pain in the subsequent event of nerve injury. The translational significance of this work suggests that females experiencing chronic pain during their estrogenic years should be treated differently to prepubescent females, post-menopausal females, and males. There is precedent for this with the irritable bowel treatment, alosetron, only being effective and hence licensed in women433. Hence chronic female pain may be treated more effectively using glial-targeted pharmacotherapies.

Acknowledgments: Thank you to Prof Paul Foster, University of Newcastle, for provision of

TLR and MyD88 null mice, and to Dr. Danielle Glynn and Mohammad Zahied Johan for their help with ovariectomy and hormone replacement surgeries. Lauren Nicotra is the recipient of an Australian Postgraduate Award. This work was supported by an NIH grant (DA023132). A portion of this work was supported by the Intramural Research Programs of the National

Institute on Drug Abuse and the National Institute on Alcohol Abuse and Alcoholism.

Lauren Nicotra Sex Differences in Allodynia 153

Supplementary Material

Male Female Is the sex difference significant? Difference in Percent Response % Response % Response Male:Female Male:Female (t=)

Pre- CCI 6.8 ± 0.13 6.8 ± 0.57 0.021 0.027

N0S0 6.7 ± 0.18 6.6 ± 0.24 0.042 0.10

N0S4 8.3 ± 0.33 8.4 ± 0.28 0.37 - 0.12

N1S3 10.0 ± 0.31 12.0 ± 0.43 - 3.4 -1.9

N3S1 12.0 ± 0.36 16.0 ± 0.56 - 9.3 - 3.3

N4S0 14.0 ± 0.52 18.0 ± 0.57 - 4.2 - 2.0

Supplementary Table 1. Prior to nerve injury (Pre-CCI), male and female Balb/c mice responded equivalently. Once allodynia stabilized (days 17-21), female mice were significantly more allodynic than males across all CCI surgery groups. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per surgery group, per sex. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

Lauren Nicotra Sex Differences in Allodynia 154

PRO:EST PRO:MET PRO:DI EST:MET DI: EST DI:MET % t= % t= % t= % t= % t= % t=

Pre- - 0.33 - 1.1 - 0.14 - 0.49 0.46 1.5 - 0.21 - 0.48 - 0.83 0.46 - 0.69 1.2 CCI ± 0.29 ± 0.28 ± 0.31 ± 0.28 ± 0.28 ± 0.27

N0S0 0.20 0.48 - 0.092 - 1.0 0.010 0.56 - 0.29 - 0.61 0.20 0.56 - 0.091 - 0.47 ± 0.22 ± 0.31 ± 0.20 ± 0.28 ± 0.33 ± 0.31

N0S4 - 1.1 - 1.4 - 0.61 - 1.2 - 0.29 - 0.41 0.49 0.22 - 0.79 -1.0 - 0.28 - 0.49 ± 0.41 ± 0.38 ± 0.31 ± 0.31 ± 0.38 ± 0.33

N1S3 1.7 2.2 2.1 2.4 - 0.17 - 0.42 0.40 0.38 2.3 2.7 1.9 2.2 ± 0.42 ± 0.44 ± 0.38 ± 0.41 ± 0.51 ± 0.48

N3S1 4.7 3.7 3.5 3.3 0.092 1.0 2.1 1.35 4.2 3.5 2.9 2.8 ± 0.55 ± 0.24 ± 0.13 ± 0.17 ± 0.34 ± 0.12

N4S0 1.1 1.2 0.97 0.86 0.88 1.1 - 0.060 0.21 0.86 1.1 0.84 1.0 ± 0.44 ± 0.31 ± 0.34 ± 0.44 ± 0.41 ± 0.36

Supplementary Table 2. Prior to nerve injury (Pre-CCI), the rodent estrus cycle did not influence female rodent responses. Following nerve injury, once allodynia became stable

(days 17-21) N1S3 and N3S1 females were significantly more allodynic throughout pro- estrus and diestrus. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table A). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Di: diestrus, Met: metestrus, Est: estrus, Pro: pro-estrus.

%: the average difference in percent response, t=: is there a significant effect of estrus cycle?

Lauren Nicotra Sex Differences in Allodynia 155

Female Female Female Female Male OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen Vs. Vs. Vs. Vs. Vs. OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen

% t= % t= % t= % t= % t=

Pre-CCI 0.20 0.93 0.21 0.87 0.21 0.51 - 0.19 0.48 0.19 0.013 ± 0.21 ± 0.18 ± 0.16 ± 0.16 ± 0.20

N3S1 3.0 - 3.1 3.1 8.7 0.11 0.28 3.8 - 3.6 2.9 - 9.1 ± 0.24 ± 0.50 ± 0.44 ± 0.46 ± 0.24

Supplementary Table 3. Prior to nerve injury all surgical treatment groups responded equivalently. Once allodynia became stable (days 17-21 post nerve injury) however, freely cycling females that underwent sham OVX surgery (OVX) responded significantly more than ovariectomised (OVX) females. Furthermore, 17β-estradiol supplemented mice (Estrogen) were also found to respond significantly more compared to vehicle (Estrogen) irrespective of sex or OVX surgery. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table B. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy (OVX). The simultaneous surgical insertion of slow release 17β-estradiol pellets is designated as ‘Estrogen’ (0.18 mg) and vehicle (placebo) pellet designated as ‘Estrogen’ (0.18 mg) (detailed in online methods).

%: the average difference in percent response, t=: is there a significant effect between treatment groups? Lauren Nicotra Sex Differences in Allodynia 156

Recipient Donor Cells N3S1 % Response Micro-N3S1 % Difference in Percent Did the glial (Day 17-21 post N3S1 Response Response transfer nerve injury) (Day 38-42 post N3S1 between N3S1 and significantly nerve injury) Microglia-N3S1 alter the pain (%) response? (t=) ♂ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.38 12.0 ± 0.12 0.21 0.17 Estrogen Estrogen

♀ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.44 12.0 ± 0.32 - 0.090 - 0.11 Estrogen Estrogen

♂ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.38 14.0 ± 0.24 - 2.3 - 7.9 Estrogen Estrogen

♀ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.44 15.0 ± 0.33 -2.8 - 9.6 Estrogen Estrogen

♂ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.38 17.0 ± 0.35 - 4.5 - 13.0 Estrogen Estrogen

♀ N3S1 OVX- ♀ N3S1 OVX- 12.0 ± 0.44 16.0 ± 0.46 - 4.0 - 13.0 Estrogen Estrogen

♂ N3S1 OVX- ♂ N3S1 OVX- 12.0 ± 0.38 17.0 ± 0.51 - 4.4 - 13.0 Estrogen Estrogen

♀ N3S1 OVX- ♂ N3S1 OVX- 12.0 ± 0.44 17.0 ± 0.46 - 4.6 - 14.0 Estrogen Estrogen

♂ N3S1 OVX- ♂ N3S1 OVX- 12.0 ± 0.38 12.0 ± 0.21 0.10 0.12 Estrogen Estrogen

♀ N3S1 OVX- ♂ N3S1 OVX- 12.0 ± 0.44 12.0 ± 0.26 -0.090 0.10 Estrogen Estrogen

Supplementary Table 4. The ability to transfer the exaggerated female pain phenotype was demonstrated utilizing nerve injured (N3S1) spinal mouse microglial cells. Intrathecal delivery of microglial cells taken from nerve injured (N3S1) freely cycling females (OVX-

Estrogen) elevated male recipient (Microglia-N3S1-OVX-Estrogen) mechanical allodynia, in a 17β-estradiol dependent fashion. Spinal microglial cells were isolated using magnetic bead separation, from nerve injured (N3S1) mice. 6 x 103 microglial cells in 10 µl PBS were intrathecally transferred to recipients by lumbar puncture 21 days following nerve injury

(detailed description in online methods). Within animal (day 17 - 21 to day 38- 42) and between microglial treatment group comparisons (at day 38-42) were conducted to determine the impact of the microglial delivery and microglia phenotype on mechanical allodynia, respectively. Results are expressed as the percent difference in response per increase in von

Lauren Nicotra Sex Differences in Allodynia 157

Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (P< 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. OVX: Female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy (OVX). The simultaneous surgical insertion of slow release 17β-estradiol pellets is designated as ‘Estrogen’ (0.18 mg) and vehicle (placebo) pellet designated as ‘Estrogen’ (0.18 mg) (detailed in online methods). ♀: Female, ♂: Male.

%: the average difference in percent response per increase in von Frey filament stiffness following microglial transfer, t=: is there a significant effect of the microglial transfer?

Recipient Donor Cells N3S1 Microglia+N3S1 Difference in Percent Did the glial transfer (Microglia+N3S1) % % Response Response between significantly alter the pain Response N3S1 and response? Microglia+N3S1 ♂ OVX-Estrogen + ♀ OVX- 12.0 15.0 - 3.1 - 12.0 N3S1 Estrogen ± 0.32 ± 0.38

♀ OVX-Estrogen + ♀ OVX- 12.0 15.0 - 3.2 - 13.0 N3S1 Estrogen ± 0.42 ± 0.36

♂ OVX-Estrogen + ♀ OVX- 12.0 12.0 0.21 0.17 N3S1 Estrogen ± 0.37 ± 0.41

♀ OVX-Estrogen + ♀ OVX- 12.0 12.0 0.060 0.12 N3S1 Estrogen ± 0.40 ± 0.44

♂ OVX-Estrogen + ♀ OVX- 12.0 18.0 - 5.2 - 19.0 N3S1 Estrogen ± 0.40 ± 0.46

♀ OVX-Estrogen + ♀ OVX- 12.0 18.0 - 5.3 - 20.0 N3S1 Estrogen ± 0.41 ± 0.38

Supplementary Table 5. The concept of glial ‘priming’ was investigated via the intrathecal transfer of naïve (non-CCI) mouse microglial cells to N3S1 recipients at the time of N3S1 nerve injury (Microglia+N3S1) and mechanical allodynia recorded once allodynia became stable (days 17 to 21 post microglial transfer/nerve injury). The presence of naïve female

Lauren Nicotra Sex Differences in Allodynia 158

microglial cells at the time of N3S1 nerve injury was sufficient to exacerbate male recipient

(OVX-Estrogen) mechanical allodynia. This effect of female microglial cells was demonstrated 17β-estradiol dependent. 6 x 103 microglial cells in 10 µl PBS were intrathecally transferred to recipients by lumbar puncture. Responses between nerve-injured mice (N3S1) and mice that had additionally received microglial delivery at the time of N3S1 nerve injury (Microglia+N3S1) were directly compared. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant P< 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. OVX: Female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy

(OVX). The simultaneous surgical insertion of slow release 17β-estradiol pellets is designated as ‘Estrogen’ (0.18 mg) and vehicle (placebo) pellet designated as ‘Estrogen’ (0.18 mg)

(detailed in online methods). ♀: Female, ♂: Male. %: the average difference in percent response per increase in von Frey filament stiffness following microglial transfer, t=: is there a significant effect of the microglial transfer?

Lauren Nicotra Sex Differences in Allodynia 159

Recipient KO WT WT N3S1 KO WT Difference Did the Difference Was there a significant Donor Cells Donor % Microglia Microglia between KO glial transfer between difference between the Cells Response +N3S1 +N3S1 WT N3S1 significantly alter WT WT glial transfer and the % % % Response the pain response? Microglia KO glial transfer? Response Response and KO t= +N3S1 t= Microglia and KO +N3S1 % Microglia Response +N3S1

♀ ♀ Myd88–/– ♀ Balb/c 12.0 12.0 12.0 0.48 0.21 0.60 0.25 OVX- OVX- OVX- ± 0.32 ± 0.32 ± 0.44 Estrogen + Estrogen Estrogen N3S1

♀ ♀ Myd88–/– ♀ Balb/c 12.0 12.0 19.0 0.40 0.18 7.8 19.0 OVX- OVX- OVX- ± 0.32 ± 0.34 ± 0.38 Estrogen + Estrogen Estrogen N3S1

♀ ♀ Tlr2/4–/– ♀ Balb/c 12.0 12.0 12.0 0.40 0.18 0.51 0.21 OVX- OVX- OVX- ± 0.32 ± 0.41 ± 0.44 Estrogen + Estrogen Estrogen N3S1

♀ ♀ Tlr2/4–/– ♀ Balb/c 12.0 12.0 19.0 0.22 0.12 7.6 16.0 OVX- OVX- OVX- ± 0.32 ± 0.36 ± 0.38 Estrogen + Estrogen Estrogen N3S1

Supplementary Table 6. The ability for female 17β-estradiol ‘primed’ microglial cells to exacerbate recipient rodent mechanical allodynia was abolished in the genetic absence of

MyD88 and TLR2/4 signaling. 6 x 103 microglial cells in 10 µl PBS were intrathecally transferred to recipients by lumbar puncture. Responses between recipients of wildtype

(Balb/c) naïve microglial cells and recipients of Tlr2/4–/– and Myd88 Myd88–/– knockout (KO) mice were directly compared. Responses between nerve-injured mice (N3S1) and mice that had additionally received microglial delivery at the time of N3S1 nerve injury

(Microglia+N3S1) were directly compared. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. OVX: Female Balb/c mice were ovariectomized (OVX) and as a control, male and female mice underwent sham ovariectomy (OVX). The simultaneous surgical insertion of slow release 17β-estradiol pellets is designated as

Lauren Nicotra Sex Differences in Allodynia 160

‘Estrogen’ (0.18 mg) and vehicle (placebo) pellet designated as ‘Estrogen’ (0.18 mg) (detailed in online methods). ♀: Female, ♂: Male. %: the average difference in percent response per increase in von Frey filament stiffness following microglial transfer, t=: is there a significant effect of the microglial transfer?

Subject N3S1 Vehicle % Dose 1 (+)- Dose 2 (+)- Dose 3 (+)- V: Dose 1 V: Dose 2 V: Dose 3 % Response nal nal nal Response % Response % Response % Response % t= % t= % t= ♀ OVX- 16.0 16.0 10.0 12.0 15.0 5.9 16.0 4.1 10.0 1.1 1.2 Estrogen ± 0.26 ± 0.18 ± 0.33 ± 0.44 ± 0.31 N3S1

♀ OVX- 19.0 19.0 11.0 14.0 18.0 8.2 27.0 5.7 17.0 1.0 1.6 Estrogen ± 0.41 ± 0.28 ± 0.40 ± 0.41 ± 0.33 N3S1

♀ OVX- 12.0 12.0 8.4 9.7 12.0 3.7 14.0 2.4 12.0 0.60 1.1 Estrogen ± 0.32 ± 0.30 ± 0.39 ± 0.44 ± 0.31 N3S1

♀ OVX- 19.0 19.0 11.0 14.0 18.0 8.4 27.0 5.0 19.0 1.3 1.6 Estrogen ± 0.44 ± 0.38 ± 0.44 ± 0.38 ± 0.41 N3S1

♂ OVX- 12.0 12.0 8.3 9.0 12.0 3.8 7.3 2.9 7.6 0.04 0.076 Estrogen ± 0.33 ± 0.23 ± 0.30 ± 0.31 ± 0.38 N3S1

♂ OVX- 18.0 18.0 9.6 13.0 17.0 8.4 22 5.2 16 0.83 1.4 Estrogen ± 0.41 ± 0.24 ± 0.30 ± 0.41 ± 0.44 N3S1

Supplementary Table 7. 17β-estradiol induced exacerbated mechanical allodynia was demonstrated neuroimmune dependent using the specific TLR4 antagonist (+)-naltrexone.

Mechanical allodynia was recorded 1 hour post intraperitoneal administration. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness

(% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. Dose 1: 30 mg/kg, Dose 2: 3 mg/kg, Dose 3: 0.3 mg/kg. As a control, mice were also given an intraperitoneal injection of isotonic saline (Vehicle). Moderate pain (N3S1) male and female mice were used in all

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experiments. n=8 per treatment group, per sex (Balb/c). n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. OVX: Ovariectomy, OVX:

Sham ovariectomy, Estrogen: 17β-estradiol pellet (0.18 mg), Estrogen: vehicle pellet (0.18 mg), Vehicle: isotonic saline, V: vehicle, (+)-nal: (+)-naltrexone, ♀: Female, ♂: Male.

Subject N3S1 Vehicle Dose Dose 2 Dose 3 V: Dose 1 V: Dose 2 V: Dose 3

% % 1 IL-1ra IL-1ra IL-1ra % t= % t= % t=

Response Response % % %

Response Response Response

♀ OVX-Estrogen 16.0 14.6 10.0 12.0 16.0 6.0 13.0 4.3 10.0 0.41 1.3

N3S1 ± 0.26 ± 0.18 ± 0.29 ± 0.44 ± 0.31

♀ OVX-Estrogen 19.0 19.0 11.0 13.0 18.0 8.1 25.0 5.9 17.0 0.55 1.8

N3S1 ± 0.41 ± 0.28 ± 0.32 ± 0.41 ± 0.33

♀ OVX-Estrogen 12.0 12.0 8.0 9.4 11.0 4.0 18.0 2.7 7.8 0.26 0.84

N3S1 ± 0.32 ± 0.30 ± 0.31 ± 0.44 ± 0.31

♀ OVX-Estrogen 19.0 19.0 11.0 14.0 18.0 8.2 30.0 5.1 11.0 0.21 0.76

N3S1 ± 0.44 ± 0.38 ± 0.40 ± 0.38 ± 0.41

♂ OVX-Estrogen 12.0 12.0 8.3 10.0 12.0 3.7 7.1 2.6 7.0 0.02 0.082

N3S1 ± 0.33 ± 0.23 ± 0.33 ± 0.31 ± 0.38

♂ OVX-Estrogen 19.0 19.0 10.0 14.0 18.0 8.7 21 5.3 15 0.92 1.7

N3S1 ± 0.41 ± 0.24 ± 0.30 ± 0.41 ± 0.44

Supplementary Table 8. 17β-estradiol induced exacerbated mechanical allodynia was demonstrated neuroimmune dependent using the cytokine inhibitor IL-1ra. Mechanical allodynia was recorded 1 hour post intraperitoneal administration. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t- value of < -2 or > 2 was determined statistically significant (P< 0.05) when differences in percentage response were greater than 1.6%. n=8 per estrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. OVX: Ovariectomy, OVX: Sham Lauren Nicotra Sex Differences in Allodynia 162

ovariectomy, Estrogen: 17β-estradiol pellet (0.18 mg), Estrogen: vehicle pellet (0.18 mg).

Dose 1: 100 mg/kg, Dose 2:10 mg/kg, Dose 3: 0.1 mg/kg. As a control, mice were also given an intraperitoneal injection of isotonic saline (Vehicle). Moderate pain (N3S1) male and female mice were used in all experiments. n=8 per treatment group, per sex (Balb/c). N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force,Vehicle: isotonic saline, V: vehicle. ♀: Female, ♂: Male.

Female Female Female Female Male OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen Vs. Vs. Vs. Vs. Vs. OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen

30 2.2 ± 0.44 4.7 ± 0.42 2.3 ± 0.40 - 0.20 ± 0.37 4.6 ± 0.41 mg/ml (t= 5.4) (t= 5.6) (t= 6.0) (t= 0.39) (t= 9.4)

3 mg/ml 1.7 ± 0.33 2.6 ± 0.44 1.9 ± 0.40 0.70 ± 0.33 2.3 ± 0.38

(t= 4.3) (t= 6.1) (t= 3.8) (t= 1.7) (t= 4.0) altrexone n -

(+) 0.3 0.50 ± 0.28 0.70 ± 0.30 1.0 ± 0.4 - 0.10 ± 0.33 0.80 ± 0.23 (t= 1.4) (t= 0.051) (t= 1.3) mg/ml (t= 1.6) (t= 1.2)

100 2.0 ± 0.38 4.2 ± 0.44 2.2 ± 0.37 - 0.10 ± 0.37 4.9 ± 0.42 mg/ml (t= 3.4) (t= 5.4) (t= 5.1) (t= 0.79) (t= 8.9)

10 1.6 ± 0.30 2.4 ± 0.38 1.6 ± 0.41 0.81 ± 0.36 2.7 ± 0.44

1ra - mg/ml (t= 3.1) (t= 3.8) (t= 2.8) (t= 0.42) (t= 4.1) IL

1 mg/ml 0.15 ± 0.22 - 0.051 ± 0.28 0.14 ± 0.28 0.34 ± 0.21 0.91 ± 0.30

(t= 0.8) (t= 0.9) (t= 1.2) (t= 1.0) (t= 1.1)

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Supplementary Table 9. Neuroimmune-modulation of nerve injury induced mechanical allodynia was demonstrated 17β-estradiol dependent. Increasing does of (+)-naltrexone and

IL-1ra reduced established mechanical allodynia significantly more in freely cycling (OVX-

Estrogen) females and 17β-estradiol treated male and female mice compared to ovariectomised (OVX-Estrogen) females and vehicle treated mice respectively. Mechanical allodynia was recorded 1 hour post intraperitoneal administration. (+)-naltrexone, 30 mg/kg, 3 mg/kg and 0.3 mg/kg; IL-1ra: 100 mg/kg, 10 mg/kg, 0.1 mg/kg. Results were analyzed utilizing repeated measures linear mixed effects modeling and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments. von Frey filaments : 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g,

1: 0.008 g. A t-value of < -2 or > 2 was determined statistically significant (P < 0.05).

Moderate pain (N3S1) male and female mice were used in all experiments. n=8 per treatment group, per sex (Balb/c). OVX: Ovariectomy, OVX: Sham ovariectomy, Estrogen: 17β- estradiol pellet (0.18 mg), Estrogen: vehicle pellet (0.18 mg).

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PRO EST MET DI

Pre-CCI 6.9 7.2 7.0 6.4 ± 0.22 ± 0.21 ± 0.18 ± 0.24

N0S0 6.7 6.5 6.8 6.7 ± 0.61 ± 0.72 ± 0.53 ± 0.63

N0S4 8.0 9.1 8.6 8.3 ± 0.44 ± 0.60 ± 0.30 ± 0.50

N1S3 11.8 10.1 9.7 12.0 ± 0.52 ± 0.52 ± 0.38 ± 0.60

N3S1 16.0 13.9 13.1 16.0 ± 0.42 ± 0.80 ± 0.80 ± 0.50

N4S0 18.0 17.0 17.0 17.0 ± 0.58 ± 0.74 ± 1.0 ± 0.85

Supplementary Table A: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of estrus cycle in Balb/c mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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♀ ♀ ♀ ♀ ♂ ♂

OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen OVX-Estrogen

Pre-CCI 6.7 ± 0.22 6.5 ± 0.18 6.5 ± 0.24 6.7 ± 0.17 6.8 ± 0.18 6.7 ± 0.22

N3S1 16.0 ± 0.36 12.0 ± 0.32 19.0 ± 0.44 19.0 ± 0.38 12.0 ± 0.34 17.0 ± 0.46

Supplementary Table B: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of ovariectomy and 17β- estradiol supplementation in Balb/c mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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CHAPTER 4. THE NEUROIMMUNE SWITCH: THE CREATION OF DIVERGENT PAIN PROCESSING IN THE ABSENCE OF TOLL LIKE RECEPTORS

In order to better elucidate the complex relationship between E2, the neuroimmune system and chronic pain; this chapter extends the in vitro and in vivo pharmacological findings of

Chapter 3 and presents the findings of a genetic inquiry into the importance of key neuroimmune contributors in the exaggerated female chronic pain phenotype.

Although others have genetically investigated TLRs in chronic pain, the majority of these studies have only utilized males as well as purely neuronal or immune challenges. As detailed in Chapters 1 and 2, chronic pain preferentially affects the female sex and to our knowledge, the novel graded neuropathy model is the only preclinical chronic pain model to replicate the immune responses in chronic pain patients. With the lack of translational success in regards to chronic pain somewhat attributed to the continued use of suboptimal preclinical pain methodologies47, this study aimed to extend these previous efforts and genetically examine neuroimmune modulation of exaggerated female mechanical allodynia in the graded neuropathy model.

Opposing several lines of evidence that have revealed a role for TLRs and their signalling components in chronic pain, male and female neuroimmune null mutant mouse strains surprisingly developed mechanical allodynia in the graded neuropathy model. Female mice were found to display greater mechanical allodynia compared with their male counterparts and female mechanical allodynia was further found pro-inflammatory independent in the genetic absence of TLRs and their adaptor protein MyD88. This female pro-inflammatory independent pain was further revealed subject to gonadal hormone modulation.

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These findings not only reveal a complex interplay between the gonadal hormones and pain circuitry in chronic pain, but also demonstrate for the first time using the graded nerve injury model, the development of divergent pain circuitry in the genetic absence of key neuroimmune contributors. Accordingly, this chapter highlights an obvious evolutionary determination underlying the burden of chronic pain in the female sex. Although one can only speculate to what advantage this encumbrance may provide the female sex, these findings reveal the unrelenting nature of the exaggerated female chronic pain phenotype.

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Submitted to PLoS

Lauren Nicotra Sex Differences in Allodynia 169

Lauren Nicotra Sex Differences in Allodynia 170

4.1 The Neuroimmune Switch: The Creation Of Divergent Pain Processing In The Absence Of TLRs

The manuscript provided here has been submitted to PLoS ONE

The Neuroimmune Switch: The Creation Of Divergent Pain Processing In The

Absence Of TLRs

Nicotra, LL1, Tuke, S.J.2, Rice, KC3, Rolan, PE1, Hutchinson, MR4, 5

1. Discipline of Pharmacology, School of Medical Sciences, University of Adelaide,

South Australia, 5005, Australia

2. School of Mathematical Sciences, The University of Adelaide, Adelaide, SA,

Australia

3. Chemical Biology Research Branch, National Institute on Drug Abuse and National

Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville,

Maryland 20892

4. Discipline of Physiology, School of Medical Sciences, The University of Adelaide,

South Australia, 5005, Australia

5. ARC Centre of Excellence for Nanoscale BioPhotonics, The University of Adelaide,

South Australia, 5005, Australia

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The innate immune system is now well recognized to contribute to chronic pain pathophysiology. This study aimed to extend the previous pharmacological and genetic investigations revealing a TLR involvement in chronic pain, and genetically examine the contribution of these pattern recognition receptors in the exaggerated female pain phenotype using a preclinical animal model that better replicates not only the heterogeneous severity of pain in chronic pain patients but also the immune responses in the chronic pain population.

Surprisingly, a TLR2/4 and MyD88 independent pain was generated in both the male and female sex in contrast to recent publications. This pain was further found insensitive to IL-1ra and (+)-naltrexone, but subject to gonadal hormone modulation. The development of a compensatory divergent pain circuitry highlights the significance of the exaggerated female pain phenotype, with the persistence of exacerbated female mechanical allodynia in the absence of innate immune signaling elements.

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Neuropathic pain is a prevalent and complex chronic pain condition resulting from pathological changes in the central and peripheral nervous systems8. In addition to being poorly responsive to the currently available analgesics434, neuropathic pain preferentially affects the female sex13, 20, although the precise mechanisms underlying this sex difference in chronic pain remain unknown.

Until recently, our understanding of chronic pain was limited to neuronal mechanisms98.

Under pathological circumstances, when tissue injury or damage is prolonged, several chemical and neuronal changes occur, collectively referred to as central sensitization8. These chemical and neuronal changes at the site of injury, in the spinal cord dorsal horn and supra- spinal pain processing centres, enhance the excitability of spinal cord neurons, resulting in an exaggerated release of sensitizing pro-inflammatory mediators. Together, these result in normal pain processing being sensitized to an excitatory state, causing an amplification of the nociceptive signal, contributing to chronic pain8.

Exclusively considering the underlying neuronal mechanisms provides an incomplete understanding of chronic pain pathophysiology. The innate immune system is now also recognized to play a considerable role in chronic pain states, with communication between neurons and non-neuronal immunocompetent cells, such as glia, contributing to both the initiation and potentiation of several neuropathologies281. Critical to this innate neuroimmune involvement are toll-like receptors (TLRs)98. These specialized pattern recognition receptors are extensively distributed throughout pain responsive CNS regions, allowing them to form a critical component of pain signaling. Upon their activation, microglial TLRs contribute to chronic pain pathology through the dysregulation of homeostatic cellular functions and the release of numerous pro-inflammatory mediators, which together, increase the

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neuroexcitatory tone98 and within nociceptive pathways lead to hypernociception.

Several preclinical neuropathy studies have demonstrated a role of the innate immune system in chronic pain. Investigations by the DeLeo group were the first to identify a critical role of

TLR4 in the induction of neuropathic pain310. Since then, numerous other research groups have pharmacologically and genetically revealed a requirement for TLRs, especially TLR4 in the development and maintenance of chronic pain315, 384-386, 388-392, 394, 395, 402, 435.

Although others have demonstrated a role for TLRs in chronic pain pathology, the majority of these investigations have only done so in the male sex. Current research indicates that persistent pain presents with a skewed prevalence towards females12-15. While the underlying mechanisms remain unknown, a vast majority of studies indicate a role for gonadal hormones, particularly 17beta-oestradiol (17β-oestradiol)15, 49, 121, 123, 125, 126, 436. One possible mechanism underlying this sex difference in pain hypersensitivity is the strong association between 17β- oestradiol and TLR4. 17beta-oestradiol has been shown to interact with the TLR4 signaling pathway, amplifying pro-inflammatory responses implicated in persistent pain354, 381-383.

Interestingly, those that have genetically identified a role for the TLRs in chronic pain have utilised either nerve injury (L5 nerve transection, CCI) or immune challenges (LPS)315, 384-386,

388-392, 394, 395, 402, 435. A novel preclinical model of graded neuropathic pain95 however is capable not only of reproducing and detecting the heterogenous nature of chronic pain, but deliberately activating both neuronal and peripheral immune signals, thereby better replicating the immune responses of clinical chronic pain patients413. Although utilised to investigate sex differences in mechanical hypersensitivity94, this preclinical chronic pain Lauren Nicotra Sex Differences in Allodynia 174

model has never before genetically investigated neuroimmune modulation of nociceptive mechanical hypersensitivity.

Accordingly, this current study was undertaken to extend the previous preclinical pharmacological and genetic studies and determine the role of TLRs 2&4 and their myeloid differentiation primary response gene 88 (MyD88) dependent signaling in the female sex in a novel graded nerve injury model that provides greater translation to the chronic pain setting, owing to similar neuroimmune responses.

2. Methods

2.1. Subjects

Pathogen-free prepubescent male and female Balb/c mice (3 weeks of age) were obtained from The University of Adelaide Laboratory Animal Services (Adelaide, SA, Australia).

Three male and female null mutant mouse strains, TLR4-/-, TLR2/4-/- and MyD88-/- (3 weeks of age) back crossed onto Balb/c 10 times, were originally sourced from Professor Akira (Osaka

University, Osaka, Japan) via Dr. Paul Foster from University of Newcastle (Newcastle,

NSW, Australia). Mice were housed in temperature- (18 - 21 °C) and light- controlled (12 h light/ dark cycle; lights on at 07:00 h) rooms. Typical rodent food and water was available ad libitum. In order to reduce successive handling stress, mice were habituated to the animal holding care facility for 1 week, followed by 1 week of extensive experimenter handling and acclimatization to the von Frey testing apparatus prior to experimentation. Investigatory procedures for all mice began at 6 weeks of age. All procedures were approved by the Animal

Ethics Committee of the University of Adelaide and were performed in accordance with the

Lauren Nicotra Sex Differences in Allodynia 175

NHMRC Australian code of practice for the care and use of animals for scientific purposes and adhered to the guidelines of the Committee for Research and Ethical Issues of the IASP.

2.2. Pre-Surgical and Test Procedures

Preceding chronic constriction injury (CCI) surgery and behavioral testing, a vaginal smear was taken from female mice using the common pipette smear technique to determine oestrus cycle phase437. Smears were taken from 8 females per phase, with results generated from a minimum of 4 consecutive oestrus cycles, with males “matched” according to assigned animal number and tested on the corresponding female test day. Vaginal smears were taken between 0800 and 1000 each testing and surgical day to minimize the incidence of transitional or ‘missed’ stages438, 439. The four stages of the oestrus cycle pro-oestrus (pro), oestrus (est), metoestrus (met) and dioestrus (di) were recognized by the presence, absence or proportion of epithelial, cornified and leucocyte cells. In an attempt to replicate the small amount of stress experienced by female mice during the smear procedure, male mice were given a parallel injection of 1 ml/kg isotonic saline intraperitoneally.

2.3. Surgery

Chronic Constriction Injury (CCI) Surgery

The CCI model of chronic pain was performed at the mid-thigh level of the left hindleg as previously described95. Mice were anaesthetised with isoflurane (3 % in oxygen), fur shaved over the left mid-thigh and the skin cleaned. The sciatic nerve was aseptically exposed and isolated at mid-thigh level. One, three or four loose chromic gut suture ligatures (cuticular 4-0 chromic gut, FS-2; Ethicon, Somerville, NJ, USA) were placed around the sciatic and once

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the superficial muscle overlying the nerve was sutured, additional chromic gut was placed subcutaneously. Whilst rescue opioid analgesia was on hand to administer to animals following surgery if an adverse event occurred, no such additional analgesia was provided following surgery, as commonly employed opioids have recently been demonstrated to exacerbate nerve injury-induced mechanical hypersensitivity440. Animals were monitored postoperatively until fully ambulatory prior to return to their homecage and checked daily for any sign of infection. No such cases occurred in this study.

2.4 Drugs

(+)-naltrexone was obtained from Kenner C Rice, the National Institute on Drug Abuse

(Research Triangle Park, NC and Bethesda, MD, USA). Human IL-1ra (Kineret, Amgen Inc.,

Thousand Oaks, CA, USA) was purchased from the Queen Elizabeth Hospital Pharmacy

(Woodville, SA, Australia). Sterile endotoxin-free isotonic saline (0.9 % sodium chloride) was the vehicle for both neuroimmune targeted therapies

(+)-naltrexone and IL-1ra were freshly diluted in endotoxin-free saline (0.9 % sodium chloride) and injected intraperitoneally to both N1S3 and N3S1 male and female mice. A dose response relationship was established with (+)-naltrexone administered at 30, 3 and 0.3 mg/kg, and IL-1ra at 100, 10 and 1 mg/kg. Vehicles were administered in equal volumes to the drugs under investigation.

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2.5. Behavior

Throughout the study, testing was performed blind with regard to group assignment and oestrus cycle phase.

Briefly, mice were subjected to 10 stimulations with 6 calibrated von Frey filaments (0.008 g,

0.02 g, 0.04 g, 0.07 g, 0.16 g, 0.4 g), within the sciatic innervation region of the hindpaws as previously described in detail94. In order to avoid sensitization, 10 min break was given between each set of stimulations, with ten stimulations per filament. Allodynia was characterized as an intense paw withdrawal or licking of the stimulated hind paw. Behavioral responses were recorded as the average number of responses out of 10 for each von Frey stimulus and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments94.

Results presented following nerve injury are at the timepoint where allodynia was demonstrated to be stable (days 17 to 21).

2.6 Behavioral Experimental Design

Graded Mechanical Allodynia in the Genetic Absence of TLR2, TLR4 and their MyD88

Dependent Signaling

Male and female Balb/c, TLR4-/-, TLR2/4-/- and MyD88-/- mice were behaviorally assessed prior to CCI surgery (baseline) and from postoperative days 3 to 21. Through variation in the number of sciatic sutures, pain treatment groups included N0S0, N0S4, N1S3, N3S1 and

N4S0. Through to 21 days post nerve injury, female mice were behaviorally examined at each

Lauren Nicotra Sex Differences in Allodynia 178

phase of the oestrus cycle (taken from a minimum of 4 consecutive cycles), with males

“matched” according to animal number and tested on the corresponding female test day.

Greater Female Mechanical Allodynia is Independent of TLRs and their MyD88 Dependent

Signaling

21 days post nerve injury, mechanical allodynia was assessed 1 hour following intraperitoneal injection of the neuroimmune targeted therapies (+)-naltrexone (30 mg/kg, 3 mg/kg, 0.3 mg/kg) and IL-1ra (100 mg/kg, 10 mg/kg, 0.1 mg/kg). Each neuroimmune agent was assessed on separate testing days, with each dose examined once, bi-daily.

2.7 Statistics

The relationship between the percent response and the variables sex, strain, oestrus cycle phase, pharmacological treatment, dose, and surgery was assessed using linear modeling fitted using the statistical package R408 via the graphical user interface: Rstudio405.

Initially a linear model was fitted that aimed to predict the percent response (number of positive responses out of a maximum of 10 by using the predictors sex, strain, oestrus cycle phase, von Frey stiffness, pharmacological treatment, dose and surgery. The goal was to estimate the average increase in percent response for each increase in von Frey filament stiffness. We also wanted to estimate how this average increase is influenced by the various levels of sex, strain, estrus cycle phase, pharmacological treatment, dose and surgery. As well, a modified linear modeling method was used, called mixed effect linear models, that had an extra term to account for the fact that we had repeated measures on the mice. This method

Lauren Nicotra Sex Differences in Allodynia 179

allows us to account for the variation seen in the percent response both within and between the mice. Believing that there was a possibility for the influence of pharmacological treatment, surgery and oestrous to depend on the subject’s sex, we also added two terms to account for these interactions. The effect of pharmacological treatment may also depend on the female rodent’s oestrus cycle phase and accordingly, this term was added to the model.

The possibility for the influence of sex, oestrus phase, pharmacological treatment and surgery on the strain of the subject was also considered. The initial model (M1) was, therefore,

response ~ sex + oestrus + pharmacological treatment + surgery

+ von Frey stiffness + sex:oestrus + sex:pharmacological treatment + sex:surgery + oestrus:pharmacological treatment +

(1|id)

Where response is the response rate expressed as a percentage (% response); sex is male or female; strain is Balb/c, TLR4-/-, TLR2/4-/- or MyD88-/-; oestrus is pro-oestrus, oestrus, metoestrus or dioestrus; pharmacological treatment is (+)-naltrexone, IL-1ra or Vehicle; surgery is N0S0, N0S4, N1S3, N3S1, N4S0; dose is 30 mg/kg, 3 mg/kg or 0.3 mg/kg for (+)- naltrexone and 100 mg/kg, 10 mg/kg or 0.1 mg/kg for IL-1ra; and von Frey stiffness is 1-6 and id is the unique mouse ID.

The model was fitted using the lmer() function from the lme4 package in R. The code is available on request from the authors.

An example R input would appear as

M1 <- lmer(response ~ sex + oestrus + pharmacological treatment + surgery + von Frey stiffness + sex:strain +

Lauren Nicotra Sex Differences in Allodynia 180

sex:oestrus + sex:pharmacological treatment + sex:surgery + oestrus:pharmacological treatment + strain:oestrus + strain:pharmacological treatment + strain:surgery (1|id), data=data) summary(M1)

To ensure the simplest model to predict the percent response, the initial model (M1) was tested to assess if any for the predictors could be removed as there are not statistically significant predictors. The ability of the model to predict the percent response was measured using the Akaike’s Information Criterion (AIC). The AIC measures how well the model fits the observations with a penalty term for the number of terms in the model. The penalty term is to try and ensure the most parsimonious model. Each predictor was removed from the model and the AIC measured to see if this caused a change in the AIC. The model with the smallest AIC was chosen. This process is repeated until the simplest model that predicts the percent response the best is obtained. This process is automated by the stepAIC() function from the MASS package406. This procedure indicated that none of the predictors could be removed without reducing the predictive ability of the model.

There is controversy regarding P-values for mixed effects models407 and thus, we report observed t-values rather than P-values and use an observed t-value with absolute value of less than negative two or greater than two to indicate statistical significance. Owing to the statistical power of the model statistically significant but behaviorally small differences can be identified. As such, we only report statistical differences that are behaviorally relevant, representing 1.6%, as this represents a change in 1 response out of 10 on the tests.

Lauren Nicotra Sex Differences in Allodynia 181

To further elucidate differences in allodynia (percentage response) and each of the predictors

(sex, strain, oestrus cycle phase, pharmacological therapy, dose, surgery and von Frey filament stimulus), subsets of the data were considered (e.g. males only) and models fitted that predicted percent response for each of the predictors individually. Again the model used accounted for the repeated measures obtain from each rat.

An example R input of such subsets to investigate certain predictors:

Example 1. In order to investigate the effect of the female rodent oestrus cycle, male rodent data was excluded from the analysis. Below if the example R input in order to investigate this predictor individually:

# Model data excluding males: specifically investigating the effect of the oestrus cycle predictor:

M1<- lmer(response~(von Frey stiffness/strain/pharmacological treatment/dose/surgery/sex/(oestrus)+ (1|id), data= subset(data, sex=='female')) summary(M1)

Where response is the response rate expressed as a percentage (% response); sex is male or female; strain is Balb/c, TLR4-/-, TLR2/4-/- or MyD88-/-; oestrus is pro-oestrus, oestrus, metoestrus or dioestrus; pharmacological treatment is (+)-naltrexone, IL-1ra or Vehicle; surgery is N0S0, N0S4, N1S3, N3S1, N4S0; dose is 30 mg/kg, 3 mg/kg or 0.3 mg/kg for (+)- naltrexone and 100 mg/kg, 10 mg/kg or 0.1 mg/kg for IL-1ra; and von Frey stiffness is 1-6 and id is the unique mouse ID.

Lauren Nicotra Sex Differences in Allodynia 182

3. Results

Results in this study were analyzed using linear mixed effects modeling (utilizing the lmer() function) fitted using the statistical package R408. Findings are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness (% response,

Supplementary materials). Due to the controversy concerning P-values for linear mixed effect models407, the author of the lmer() function does not report them. Hence, in this paper, we report the observed t-value associated with each fixed effect, with an absolute value of less than negative two or greater than two to indicate statistical significance. Moreover, only statistical differences that represent a behaviorally relevant change in response rate are presented. This threshold of behavioral relevance is considered to be a difference in percentage response of greater than 1.6%, as this equals a change in 1 response out of 10 on the tests.

3.1 Graded Mechanical Allodynia in the Genetic Absence of TLR2, TLR4 and their

MyD88 Dependent Signaling

Prior to nerve injury (baseline), there were no significant differences between male and female nociceptive response scores across all mouse strains, with an average difference between slopes of 0.074 % (t ≥ 0.010 but ≤ 0.032; Tables 1-4; Figure 4.1). At baseline, female nociceptive responses were also found unaffected by gonadal steroid hormones independent of strain (t ≥ - 0.094 but ≤ 0.64; Tables 5-8), based on oestrus cycle recordings taken across four consecutive oestrus cycles.

Lauren Nicotra Sex Differences in Allodynia 183

Surprisingly, following nerve injury, graded allodynia was not only demonstrated in wild-type

Balb/c mice, but across all mouse strains in both males and females. Increasing the number of sciatic sutures significantly increased the number of responses per increase in von Frey filament stiffness indicative of allodynia across all mouse strains in both sexes (≥ 2.1%, t ≥

4.0; Tables 1-4; Figure 4.1). Mechanical allodynia was observed across all mouse strains, across all pain treatment groups 3 days following nerve injury and maintained until day 21 for both rodent sexes, with no significant difference across the strains (Males: > - 1.1 but < 0.011

%, t > -1.2 but < 0.76, Table 9; Females: > - 0.96 but < 0.010 %, t > - 0.92 but < 0.98, Table

10; Figure 4.1).

3.2 Sex Differences in the Graded Nerve Injury Model: Greater Female Mechanical

Allodynia is Independent of TLRs and their MyD88 Dependent Signaling in Genetic

Knockout Animals

Once allodynia became stable (days 17-21 following nerve injury), female mice across all strains in all pain treatment groups were significantly more allodynic than males. Balb/c females responded on average 2.9 % more than males per increase in von Frey stiffness (t ≤ -

4.1; Table 1; Figure 4.1) and N1S3 and N3S1 Balb/c females further demonstrated exacerbated mechanical allodynia throughout both pro-oestrus and dioestrus relative to oestrus (Di:Est, ≥ 4.0 %, t ≥ 3.0; Pro:Est, ≥ 3.9 %, t ≥ 3.0) and metoestrus (Di:Met, ≥ 3.5 %, t

≥ 3.1; Pro:Met, ≥ 3.3 %, t ≥ 2.9; Table 5; Figures 4.2A and 4.2B).

Remarkably, TLR4-/-, TLR2/4-/- and MyD88-/- females also responded significantly more than males, with TLR4-/- and TLR2/4-/- females on average responding 2.8 % more, and MyD88-/- females responding on average 2.6 % more than males per increase in von Frey stiffness (t ≤ -

Lauren Nicotra Sex Differences in Allodynia 184

3.6; Tables 2-4; Figure 4.1). Analogous to Balb/c females, an oestrus cycle effect was also found across these knockout strains following nerve injury. TLR4-/-, TLR2/4-/- and MyD88-/-

N1S3 and N3S1 females displayed exacerbated mechanical allodynia throughout both pro- oestrus and dioestrus compared with oestrus (Di:Est, ≥ 3.1 %, t ≥ 2.7; Pro:Est, ≥ 2.8 %, t ≥

2.7) and metoestrus (Di:Met, ≥ 2.1 %, t ≥ 2.0; Pro:Met, ≥ 2.6 %, t ≥ 2.4; Tables 5-8; Figures

4.2A and 4.2B).

20 Male Balb/c Female Balb/c Male TLR4-/- Female TLR4-/- Male TLR2/4-/- Female TLR2/4-/- 15 Male MyD88-/- Female MyD88-/-

10

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 Pre-CCI N0S0 N0S4 N1S3 N3S1 N4S0 Pain Treatment

Figure 4.1. Neuroimmune Independent Exaggerated Female Mechanical Allodynia.

Replicating previous findings and the clinical pain experience, male (represented in blue) and female (represented in pink) mice responded equivalently prior to nerve injury. Surprisingly, graded allodynia was found across all mouse strains, along with a sex difference following chronic constriction injury (CCI), with females responding more than males up to 21 days

Lauren Nicotra Sex Differences in Allodynia 185

following nerve injury. The development of graded mechanical allodynia was achieved by varying the number of chromic gut lengths of suture placed around the sciatic nerve. Pain treatments were designated as NxSy, where x and y are the number of chromic gut lengths of suture placed around the sciatic vs. subcutaneously, respectively. As a result, pain treatment groups included N0S0, N0S4, N1S3, N3S1 and N4S0 animals. Once allodynia became stable

(days 17-21 following nerve injury), female mice across all strains displayed significantly greater mechanical allodynia compared to males (t ≤ - 4.1). Results were analyzed utilizing repeated measures linear mixed effects modeling. A t-value of < -2 or > 2 was determined statistically significant (P < 0.05). n=8 per treatment group, per sex (Balb/c). von Frey filaments : 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g, 1: 0.008 g. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

(A)$ (B)$ 20 18

18

16

16

14

14

12 Balb/c Balb/c N3S1 12 -/- TLR4-/- TLR4 N3S1 -/- TLR2/4-/- TLR2/4 N3S1 MyD88-/- N3S1 MyD88-/- Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 10 10 pro oest met di pro oest met di Estrus Cycle Phase Estrus Cycle Phase

Figure 4.2. Neuroimmune independent pain is subject to gonadal hormone modulation.

Following nerve injury, female Balb/c mechanical allodynia was found affected by the rodent oestrus cycle. Once allodynia became stable (days 17-21 following nerve injury), N1S3

(Figure 4.2A) and N3S1 (Figure 4.2B) female Balb/c mice were significantly more allodynic Lauren Nicotra Sex Differences in Allodynia 186

throughout both pro-oestrus and dioestrus compared to all other oestrus cycle phases ((t >

2.9). Mechanical allodynia established in the knockout strains was also determined affected by the oestrus cycle (t > 2.0) Results were analyzed utilizing repeated measures linear mixed effects modeling and are expressed as the percent difference in slope, representing a difference in response per increase in von Frey filament stiffness. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05). n=8 Balb/c mice and null mutant mice (TLR4-/-

, TLR2/4-/- and MyD88-/-) per oestrus phase, with results generated from a minimum of 4 consecutive estrus cycles.. Di: dioestrus, Met: metoestrus, Est: oestrus, Pro: pro-oestrus). N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

3.3 Graded Mechanical Allodynia is Innate Immune Independent in the Genetic

Absence of TLR2 and 4 and Their Adaptor Protein MyD88

Once allodynia stabilized, male and female wild type Balb/c mechanical allodynia was reversed utilizing the interleukin-1 (IL-1) receptor antagonist (IL-1ra) (N1S3 ≥ 2.0 %, t ≥ 2.1;

N3S1 ≥ 2.3 %, t ≥ 2.3; Table 11; Figure 4.3A and 4.3B) and the blood brain barrier permeable

TLR4 receptor antagonist (+)-naltrexone (N1S3 ≥ 2.1 %, t ≥ 2.0; N3S1 ≥ 2.3 %, t ≥ 2.2; Table

12; Figures 4.3C and 4.3D). A dose-response relationship was determined in both male and female Balb/c mice however, despite females being significantly more allodynic than males prior to drug administration, both neuroimmune agents brought males and females to a common non-allodynic post-drug state.

Surprisingly, established male and female mechanical allodynia could not be reversed using the neuroimmune modulator IL-1ra in TLR4-/-, TLR2/4-/- and MyD88-/- mice (≥ 0.011 but ≤

0.26 %, t ≥ 0.014 but ≤ 0.33; Tale 11; N1S3: Figure 4.4A; N3S1: Figure 4.4B). As expected,

Lauren Nicotra Sex Differences in Allodynia 187

the TLR4 receptor antagonist (+)-naltrexone did not significantly alter male or exacerbated female mechanical allodynia in TLR4-/- and TLR2/4-/- mice (≥ 0.011 but ≤ 0.26 %, t ≥ 0.014 but ≤ 0.33; Table 12; N1S3: Figure 4.5A; N3S1: Figure 4.5B). Contrasting the findings in

Balb/c mice however, this neuromodulator also unpredictably failed to modify mechanical allodynia in MyD88-/- male and female subjects (≥ 0.097 but ≤ 0.21 %, t ≥ 0.10 but ≤ 0.26;

Table 12; N1S3: Figure 4.5A; N3S1: Figure 4.5B).

Lauren Nicotra Sex Differences in Allodynia 188

(A)$ (B)$ 15 20 Balb/c Male Balb/c Female

15

10

10

Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 10 0.1 100 10 0.1 100 Vehicle Vehicle No Injection mg/kg IL-1ra mg/kg IL-1ra No Injection (C)$ (D)$

20 15

15

10

10 Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 5 (%) Stiffness Frey von in Increase per Response in Difference 5 3 30 3 0.3 0.3 30

Vehicle Vehicle mg/kg (+)-naltrexone mg/kg (+)-naltrexone No Injection No Injection

Figure 4.3. Balb/c Female mice are more sensitive to neuroimmune modulation.

IL-1ra and (+)-naltrexone preferentially reduced Balb/c female nerve injury induced mechanical allodynia. IL-1ra (Figure 4.3A and 4.3B) and (+)-naltrexone (Figure 4.3C and

3D) reduced mechanical allodynia significantly more in freely cycling females compared to

Lauren Nicotra Sex Differences in Allodynia 189

males. Allodynia was recorded 1 hour post intraperitoneal administration. IL-1ra, 100 mg/kg,

10 mg/kg and 1 mg/kg; (+)-naltrexone, 30 mg/kg, 3 mg/kg and 0.3 mg/kg. Results were analysed utilising repeated measures linear mixed effects modelling and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments. von Frey filaments: 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2:

0.02 g, 1: 0.008 g. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05).

Moderate pain (N3S1) Balb/c male and female mice were used in all experiments. n=8 per treatment group, per sex. N:number of sciatic sutures, S:number of subcutaneous sutures.

(A) (B) -/- TLR4 Male -/- 18 TLR4-/- Female TLR4 Male -/- TLR4 Male TLR2/4-/- Male TLR4 Female TLR4 Female MYD88 Male TLR2/4-/- Female -/- TLR2/4 Male MYD88 Female -/- 14 MyD88 Male TLR2/4 Male -/- -/- 16 MyD88 Female TLR2/4 Female TLR2/4 Female MyD88-/- Male MyD88-/- Female

14

12

12 Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 10 (%) Stiffness Frey von in Increase per Response in Difference 10 10 100 10 0.10 0.10 100 Vehicle Vehicle mg/kg IL-1ra mg/kg IL-1ra No Injection No Injection

Figure 4.4. In the Genetic Absence of TLRs and their Adaptor Protein, Mechanical

Allodynia is Pro-inflammatory Independent.

Female (N1S3, Figure 4.4A; N3S1, Figure 4.4B) and male (N1S3, Figure 4.4A; N3S1, Figure

4.4B) mechanical allodynia could not be reversed using IL-1ra in TLR4-/-, TLR2/4-/- and

MyD88-/- mice (≥ 0.011 but ≤ 0.26 %, t ≥ 0.014 but ≤ 0.33). Allodynia was recorded 1 hour post intraperitoneal administration. IL-1ra, 100 mg/kg, 10 mg/kg. Results were analysed

Lauren Nicotra Sex Differences in Allodynia 190

utilising repeated measures linear mixed effects modelling and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments. von Frey filaments: 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g,

1: 0.008 g. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05). n=8 per treatment group, per strain , per sex. N:number of sciatic sutures, S:number of subcutaneous sutures.

TLR4-/- Male (A) (B) TLR4-/- Female -/- 18 TLR2/4 Male TLR2/4-/- Female TLR4 Male -/- 14 TLR4 Female MyD88 Male 16 TLR2/4 Male MyD88-/- Female TLR2/4 Female MYD88 Male MYD88 Female 14 12

12

10 10 Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference Difference in Response per Increase in von Frey Stiffness (%) Stiffness Frey von in Increase per Response in Difference 3 3 0.3 30 0.3 30

Vehicle Vehicle mg/kg (+)-Naltrexone mg/kg (+)-Naltrexone No Injection No Injection

Figure 4.5. In the Genetic Absence of TLRs and their Adaptor Protein, Mechanical

Allodynia is Pro-inflammatory Independent.

Female (N1S3, Figure 4.5A; N3S1, Figure 4.5B) and male (N1S3, Figure 4.5A; N3S1,

Figure 4.5B) mechanical allodynia could not be reversed using (+)-naltrexone in TLR4-/-,

TLR2/4-/- and MyD88-/- mice (≥ 0.011 but ≤ 0.26 %, t ≥ 0.014 but ≤ 0.33;. Allodynia was recorded 1 hour post intraperitoneal administration. (+)-naltrexone, 30 mg/kg, 3 mg/kg and

0.3 mg/kg. Results were analysed utilising repeated measures linear mixed effects modelling

Lauren Nicotra Sex Differences in Allodynia 191

and are expressed as the percent difference in slope, representing a difference in response per von Frey filament across a range of von Frey filaments. von Frey filaments: 6: 0.40 g, 5: 0.16 g, 4: 0.07 g, 3: 0.04 g, 2: 0.02 g, 1: 0.008 g. A t-value of < -2 or > 2 was determined statistically significant (p < 0.05). n=8 per treatment group, per strain , per sex. N:number of sciatic sutures, S:number of subcutaneous sutures.

4. Discussion

This study aimed to investigate the role of TLRs 2 and 4 and their MyD88 dependent signaling in exacerbated female mechanical hypersensitivity in a clinically translatable preclinical chronic pain model. Here, we demonstrated for the first time that if genetic deletions in innate immune signaling elements are present at the time of nerve injury, the type of pain generated is one that is not pro-inflammatory dependent, which however does remain subject to gonadal hormone modulation. Our findings extend previous in vivo pharmacological and genetic investigations that have demonstrated a role for the TLRs in male chronic pain pathology310,310, 379-381, 383-387, 389, 390, 397, 425. Here we reveal the generation of a model and strain specific, non-neuroimmune type of pain that was magnified in the female sex.

Translating successful preclinical findings to the clinic, resulting in promising analgesic candidates for chronic pain populations remains at the mercy of preclinical chronic pain models47. Although animal models such as chronic constriction injury (CCI) and L5 spinal nerve ligation have been well utilised within the chronic pain literature, such models do not best reflect the clinical heterogeneity of clinical pain47, 95, 441, 442. Considering that chronic pain Lauren Nicotra Sex Differences in Allodynia 192

is heterogeneous, utilising preclinical methodologies which produce marked allodynia are less clinically translatable and subsequently suboptimal when investigating the mechanisms underlying persistent pain95. The novel graded nerve injury model95 however, has been demonstrated by our laboratory to produce graded allodynia in both male and female rodents.

Owing to the ability to not only replicate but detect a heterogenous pain response, this novel nerve injury model has been demonstrated superior in the investigation of chronic pain mechanisms and has been further described as having the potential to unveil prospective analgesics for a range of chronic pain patients95. Consequently, although previous investigations have investigated the role of TLRs in pathological pain310,315, 384-386, 388-392, 394,

395, 402, 435, this study is the first to genetically do so using this clinically translatable chronic pain model.

Using this novel preclinical pain model, Balb/c and null mutant male and female mice responses were found to shadow the clinical scenario in the healthy human population96, 97, responding equivalently prior to nerve injury. Following nerve injury however, our results were unexpected. Considering the now large degree of preclinical pharmacological and genetic evidence validating a role for TLRs in the initiation of persistent pain, the development of mechanical allodynia across both sexes and across all mouse strains following nerve injury was a surprising determination. An extensive range of TLR4 antagonists have now been shown to prevent preclinical models of male neuropathic pain, including (-) and (+) naloxone and naltrexone, FP-1, mutant forms of LPS and small interfering RNA (siRNA)388-391. Several investigations using TLR loss-of-function mutant mice have also implicated TLRs in initiating male neuropathic pain behaviors across a range of preclinical chronic pain methodologies. Moreover, a range of preclinical animal nerve injury models have also established a role for these pattern recognition receptors in neuropathic pain. In 2005, Tanga first revealed using the L5 sciatic nerve ligation (SNL) Lauren Nicotra Sex Differences in Allodynia 193

model, a role for TLR4 in the initiation of neuropathic pain, which was supported years later by Bettoni and colleagues using the CCI model of nerve injury388. TLR involvement in preclinical pain was further extended to TLRs 2 and 3 following L5 nerve transection injury.

Utilizing loss-of-function mutant mice, TLR3 was demonstrated essential to the resulting tactile allodynia following peripheral nerve injury435 and in 2007, Kim identified a role for

TLR2 in the initiation of both mechanical and thermal pain hypersensitivity402. Consequently, our findings whereby TLR4-/-, TLR2/4-/- and MyD88-/- male and female response rates were indistinguishable from Balb/c male and female subjects respectively, are in direct contrast to an array of findings that have revealed a role for the TLRs in chronic pain.

With chronic pain known to predominantly affect the female sex, recent investigations by both the Mogil430 and Yaksh431 research labs have extended the abovementioned studies in an attempt to cast light and unravel the pathophysiology underlying the female burden of chronic pain. In direct contrast with our findings in the graded nerve injury model, whereby male and female TLR4-/- mice developed profound mechanical allodynia, Mogil and Yaksh determined female mechanical allodynia to be TLR4 independent in CD-1 and C57BL6 mice, using the

LPS and SNL pain models respectively. Extending these findings, Yaksh431 also investigated a role for the myeloid differentiation primary response gene 88 (MyD88) along with TLR2 in his laboratories’ recent publication. In direct opposition to our findings in the graded nerve injury model, Yaksh evidenced a partial reduction in tactile allodynia in MyD88 and TLR2 null mutant male mice compared to wild type controls. Opposingly, our findings revealed a preclinical neuropathic pain that was determined to be TLR2/4 and MyD88 independent in both male and female mice. Dissimilar to the purely neuronal and immune challenges, which have been responsible for uncovering a role for TLRs in the initiation of several preclinical

Lauren Nicotra Sex Differences in Allodynia 194

chronic pain methodologies, the graded nerve injury model deliberately activates both neuronal and peripheral immune signals to more closely mimic the immune system response in clinical chronic pain populations413. Accordingly, we surmise that the generation of this

TLR2/4 and MyD88 independent pain may in fact be model specific and warrants further investigation.

In an attempt to determine the pathophysiology underlying this newfound TLR2/4 and

MyD88 independent pain in the graded nerve injury model, a neuroimmune contribution was considered. With the detection of elevated TH1- and TH2-type cytokine expression in TLR4- deficient mice following infection443, activation of alternative immune signalling pathways was considered across our null mutant strains. From the time when an association between the sickness response and nociception was identified, IL-1B has been recognised to play a role in pain-related behaviours273, 274. Through the phosphorylation of NMDA receptors, leading to enhanced excitatory synaptic processing, as well as a reduction in GABA release from inhibitory descending central projections resulting in reduced inhibitory synaptic transmission, IL-1B has been found to influence neural pain processing circuitry and contribute to the bidirectional, modulatory signaling between immune cells and neurons that ultimately results in pathological pain340, 444. Confirming a central nervous system (CNS) mechanism of action of IL-1β, previous investigations have revealed the ability of IL-1RA to attenuate pain behaviors associated with several preclinical pain methodologies337, 444, 445.

Contradictory to these findings however, IL-1RA failed to attenuate mechanical hypersensitivity across our null mutant male and female mice, indicating the generation of an

IL-1 independent type of pain in the graded nerve injury model. With IL-1RA found to significantly attenuate both male and female Balb/c mechanical allodynia however, and the

Lauren Nicotra Sex Differences in Allodynia 195

TLR4 antagonist (+)-naltrexone also demonstrated unable to reverse the mechanical allodynia generated in TLR4-/-, TLR2/4-/- and MyD88-/- mice, our results demonstrate for the first time, the generation of a pro-inflammatory independent type of pain when genetic deletions in neuroimmune signaling components are present at the time of nerve injury, in the graded nerve injury model

Although our findings are in direct contrast to previous investigations that have utilised neuronal preclinical chronic pain models, the results do not rule out the possibility of the generation of a purely neuronal type of pain in our null mutant mice. Previous investigations, including Yaksh431 that have utilised neuronal injuries have in fact done so using C57BL/6 mice. As the infectious diseases literature suggests that Balb/c mice closely mimic human pro-inflammatory responses432, our study deliberately utilised this strain in order to improve clinical translability and in doing so, may have revealed a strain specific effect. In order to further examine the possibility of a purely neuronal pathology and thereby potentially identify the mechanisms underlying this nociceptive presentation however, pharmacotherapies capable of modulating neuronal activity must be examined in future investigations.

Extending our anomalous findings in the graded nerve injury model, the pro-inflammatory independent pain generated across our null mutant female mice following nerve injury, was found to be regulated by the female rodent oestrus cycle, replicating several previous investigations revealing a correlation between exaggerated female nociceptive responses and the high oestrogenic phases of pro-oestrus and dioestrus following nerve injury94, 169, 220-227.

Interestingly however, observed neuroimmune modulation of this mechanical allodynia was

Lauren Nicotra Sex Differences in Allodynia 196

in fact determined to display a female bias, with both (+)-naltrexone and IL-1ra found to preferentially reduce female Balb/c exacerbated nociceptive hypersensitivity, with male and female nociceptive responses reduced to a common non-allodynic post-drug state in wild-type mice. (+)-naltrexone and IL-1ra however failed to do the same across the null mutant mouse strains.

Although known for their role in reproduction, the sex steroids, particularly the oestrogens, have also been shown to regulate numerous homeostatic processes in addition to nociceptive processing130, 248. The role of oestrogen in the CNS however, is not easily designated as pro or antinociceptive. Oestrogens are however now known to modulate several neuronal pain mechanisms and pain processing systems such as the endogenous opioids (enkephalins) and

GABA pathways130, 446, 447. Despite such research developments, the precise pathophysiology underlying the role of gonadal hormones in nociceptive processing remains undetermined.

Recently however, an interaction between gonadal steroid hormones, particularly oestrogen and innate immune receptors known to play a role in chronic pain, such as TLR4, have been identified, resulting in exacerbated pro-inflammatory responses381, 382, 345, 374. Such findings have led to researchers hypothesizing a role for this interaction between neuroimmune function and the sex steroids in exacerbated female nociceptive sensitivity.

Both the neuroimmune dependent and pro-inflammatory independent types of pain generated in this study were subject to gonadal hormone modulation. Considering the now known interaction between the oestrogens and innate immune cells and their signaling receptors, it is possible that the exaggerated female nociceptive sensitivity found in our Balb/c mice following nerve injury was partly attributable to oestrogenic innate immune cell priming and warrants further investigation. Lauren Nicotra Sex Differences in Allodynia 197

5. Conclusion

To our knowledge, this is the first study to genetically investigate the role of the innate immune system in a refined heterogeneous chronic pain model. Here, we demonstrate using mice that more closely mimic the immune responses of clinical chronic pain populations, the possibility of developing divergent pain circuitry when innate immune signaling elements are not present at the time of nerve injury. Our results not only highlight a complex interplay between gonadal steroid hormones, neuronal pain processes and the innate immune system which requires further examination, but further emphasizes the complexities of interpreting and comparing findings across studies that have utilised diverse chronic pain injuries and rodent strains.

Lauren Nicotra Sex Differences in Allodynia 198

Supplementary Material

Male Female Is the sex difference Difference between slopes significant? Male:Female Male:Female (t=)

Pre-Surgery 6.5 ± 0.21 6.6 ± 0.22 0.032 0.11

N0S0 6.6 ± 0.24 6.7 ± 0.26 0.040 0.10

N0S4 8.6 ± 0.36 8.4 ± 0.32 0.41 0.21

N1S3 10.0 ± 0.42 13.0 ± 0.44 - 4.1 -2.5

N3S1 13.0 ± 0.40 16.0 ± 0.54 - 8.6 - 3.1

N4S0 15.0 ± 0.47 19.0 ± 0.51 - 9.2 -3.2

Supplementary Table 1. Prior to nerve injury (Pre-CCI), male and female Balb/c mice responded equivalently. Once allodynia stabilized (days 17-21), female mice were significantly more allodynic than males across all CCI surgery groups. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per surgery group, per sex. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

Lauren Nicotra Sex Differences in Allodynia 199

Male Female Is the sex difference Difference between TLR4 KO TLR4 KO significant? slopes Male:Female Male:Female (t=)

Pre-Surgery 6.7 ± 0.24 6.8 ± 0.21 0.020 -0.081

N0S0 6.6 ± 0.24 6.6 ± 0.26 0.032 0.091

N0S4 8.8 ± 0.36 8.6 ± 0.32 0.40 0.19

N1S3 10.0 ± 0.51 12.0 ± 0.48 - 3.6 -2.0

N3S1 13.0 ± 0.40 16.0 ± 0.54 - 8.3 - 3.0

N4S0 15.0 ± 0.47 18.0 ± 0.51 - 9.3 -3.3

Supplementary Table 2. Male and female TLR4-/- mice responded equivalently prior to nerve injury (Pre-CCI), Surprisingly, male and female TLR4-/- mice developed stable allodynia from day three following nerve injury. Once allodynia stabilized (days 17-21), female TLR4-/- mice were significantly more allodynic than males across all CCI surgery groups. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness

(% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per surgery group, per sex. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

Lauren Nicotra Sex Differences in Allodynia 200

Male Female Is the sex difference Difference between slopes TLR2/4-/- KO TLR2/4-/- KO significant? Male:Female Male:Female (t=)

Pre-Surgery 6.6 ± 0.22 6.6 ± 0.24 0.010 -0.012

N0S0 6.8 ± 0.24 6.7 ± 0.26 0.031 0.063

N0S4 9.1 ± 0.28 8.9 ± 0.30 0.42 0.18

N1S3 11.0 ± 0.47 13.0 ± 0.48 - 3.8 -2.2

N3S1 13.0 ± 0.47 16.0 ± 0.55 - 8.0 - 3.1

N4S0 16.0 ± 0.47 19.0 ± 0.51 - 8.4 -3.0

Supplementary Table 3. Male and female TLR2/4-/- mice responded equivalently prior to nerve injury (Pre-CCI), Surprisingly, male and female TLR4-/- mice developed stable allodynia from day three following nerve injury. Once allodynia stabilized (days 17-21), female TLR4-/- mice were significantly more allodynic than males across all CCI surgery groups. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von

Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p

< 0.05) when differences in percentage response were greater than 1.6%. n=8 per surgery group, per sex. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

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Male Female Is the sex difference Difference between slopes MyD88 KO MyD88 KO significant? Male:Female Male:Female (t=)

Pre-Surgery 6.5 ± 0.26 6.4 ± 0.22 0.022 -0.091

N0S0 6.6 ± 0.27 6.6 ± 0.28 0.014 0.021

N0S4 9.6 ± 0.37 9.4 ± 0.41 0.48 0.22

N1S3 10.0 ± 0.41 13.0 ± 0.44 - 4.8 - 3.0

N3S1 13.0 ± 0.47 15.0 ± 0.55 - 6.9 - 2.4

N4S0 16.0 ± 0.51 18.0 ± 0.56 - 7.1 - 2.4

Supplementary Table 4. Male and female MyD88-/- mice responded equivalently prior to nerve injury (Pre-CCI). Surprisingly, male and female MyD88-/- mice developed stable allodynia from day three following nerve injury. Once allodynia stabilized (days 17-21), female MyD88-/- mice were significantly more allodynic than males across all CCI surgery groups. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von

Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p

< 0.05) when differences in percentage response were greater than 1.6%. n=8 per surgery group, per sex. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force.

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PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

Pre-Surgery - 0.090 ± 0.22 - 0.090 ± 0.24 0.70 ± 0.22 - 0.21 ± 0.21 - 0.78 ± 0.24 - 0.72 ± 0.22

t= - 0.094 t= - 0.93 t= 0.64 t= - 0.41 t= - 0.69 t= -0.70

0.49 ± 0.29 - 0.088 ± 0.31 - 0.10 ± 0.30 - 0.59 ± 0.28 0.59 ± 0.28 - 0.031 ± 0.30 N0S0 t= 0.51 t= - 0.91 t= - 0.22 t= - 0.61 t= 0.60 t= - 0.44

- 0.089 ± 0.38 - 0.30 ± 0.38 0.20 ± 0.34 - 0.19 ± 0.38 - 0.30 ±0.34 - 0.50 ± 0.62 N0S4 t= - 0.091 t= 0.49 t= 0.40 t= - 0.39 t= - 0.49 t= - 0.62

3.9 ± 0.48 4.4 ± 0.39 - 0.17 ± 0.38 0.22 ± 0.40 4.0 ± 0.38 4.6 ± 0.34 N1S3 t= 3.0 t= 3.4 t= 0.26 t= 0.41 t= 3.0 t= 3.5

5.2 ±0.40 3.3 ± 0.42 0.65 ± 0.40 -1.3 ± 0.38 4.4 ± 0.40 3.5 ± 0.41 N3S1 t= 4.1 t= 2.9 t= 0.64 t= 1.4 t= 3.4 t=3.1 - 0.089 ± 0.51 0.29 ± 0.55 0.10 ± 0.48 0.32 ± 0.40 - 0.19 ± 0.55 0.13 ± 0.53 N4S0 t= 0.090 t= 0.40 t= 0.22 t= 0.49 t= 0.29 t= 0.24

Supplementary Table 5. Female Balb/c responses were found unaffected by the rodent oestrus cycle prior to nerve injury (Pre-CCI). Following nerve injury, once allodynia became stable (days 17-21) N1S3 and N3S1 Balb/c females were significantly more allodynic throughout pro-oestrus and dioestrus. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table A). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per oestrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Di: dioestrus, Met: metoestrus,

Oest: oestrus, Pro: pro-oestrus. %: the average difference in percent response, t=: is there a significant effect of oestrus cycle?

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PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

Pre-Surgery 0.089 ± 0.20 0.079 ± 0.21 0.28 ± 0.21 - 0.0090 ± 0.22 - 0.19 ± 0.21 - 0.21 ± 0.20

t= 0.091 t= 0.087 t= 0.44 t= - 0.047 t= - 0.39 t= - 0.38

0.29 ± 0.28 - 0.21 ± 0.22 - 0.28 ± 0.24 - 0.47 ± 0.20 0.60 ± 0.24 - 0.091 ± 0.22 N0S0 t= 0.31 t= - 0.31 t= - 0.39 t= - 0.57 t= 0.61 t= - 0.10

- 0.091 ± 0.32 - 0.10 ± 0.37 0.19 ± 0.32 - 0.098 ± 0.34 - 0.17 ±0.34 - 0.27 ± 0.30 N0S4 t= - 0.094 t= - 0.22 t= 0.25 t=- 0.092 t= - 0.27 t= - 0.30

3.1 ± 0.44 3.6 ± 0.48 -0.17 ± 0.38 - 1.0 ± 0.44 4.0 ± 0.38 4.6 ± 0.41 N1S3 t= 2.7 t= 3.4 t= 0.26 t= - 1.1 t= 3.0 t= 4.1

4.2 ±0.46 2.8 ± 0.44 0.45 ± 0.41 - 1.5 ± 0.44 3.2 ± 0.47 3.2 ± 0.44 N3S1 t= 4.2 t= 2.6 t= 0.54 t= 1.4 t= 2.9 t= 3.2

0.090 ± 0.50 0.34 ± 0.51 0.62 ± 0.47 0.39 ± 0.47 - 0.71 ± 0.51 - 0.33 ± 0.51 N4S0 t= 0.089 t= 0.41 t= 0.67 t= 0.51 t= - 0.72 t= 0.46

Supplementary Table 6. Female TLR4-/- responses were found unaffected by the rodent oestrus cycle prior to nerve injury (Pre-CCI). Following nerve injury, once allodynia became stable (days 17-21) N1S3 and N3S1 TLR4-/- females were significantly more allodynic throughout pro-oestrus and dioestrus. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table B). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per oestrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Di: dioestrus, Met: metoestrus,

Oest: oestrus, Pro: pro-oestrus. %: the average difference in percent response, t=: is there a significant effect of oestrus cycle?

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PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

Pre-Surgery - 0.035 ± 0.21 0.11 ± 0.20 - 0.15 ± 0.21 0.11 ± 0.20 0.04 ± 0.20 0.28 ± 0.21

t= 0.041 t= 0.22 t= - 0.23 t= 0.22 t= - 0.042 t= 0.33

- 0.54 ± 0.22 - 0.59 ± 0.21 - 0.22 ± 0.24 - -0.051 ± 0.24 0.070 ± 0.24 - 0.30 ± 0.24 N0S0 t= - 0.62 t= - 0.56 t= - 0.29 t= - 0.054 t= 0.077 t= - 0.35

- 0.33 ± 0.30 0.30 ± 0.30 0.079 ± 0.31 - 0.098 ± 0.34 - 0.22 ±0.30 0.19 ± 0.32 N0S4 t= - 0.40 t= 0.39 \t= 0.072 t=- 0.092 t= - 0.31 t= 0.26

3.1 ± 0.44 3.0 ± 0.42 - 0.17 ± 0.38 0.51 ± 0.47 3.9 ± 0.44 3.9 ± 0.48 N1S3 t= 2.7 t= 2.8 t= 0.26 t= 0.53 t= 3.6 t= 3.7

3.4 ± 0.41 2.6 ± 0.47 1.05 ± 0.50 - 1.1 ± 0.52 3.1 ± 0.50 2.1 ± 0.54 N3S1 t= 3.1 t= 2.4 t=1.0 t= - 1.0 t= 2.7 t= 2.0

- 0.52 ± 0.48 1.03 ± 0.50 0.0093 ± 0.48 - 0.34 ± 0.48 - 0.80 ± 0.48 - 1.2 ± 0.50 N4S0 t= 0.54 t= 0.98 t= 0.091 t= - 0.42 t= - 0.84 t= - 1.1

Supplementary Table 7. Female TLR2/4-/- responses were found unaffected by the rodent oestrus cycle prior to nerve injury (Pre-CCI). Following nerve injury, once allodynia became stable (days 17-21) N1S3 and N3S1 TLR2/4-/- females were significantly more allodynic throughout pro-oestrus and dioestrus. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table C). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per oestrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Di: dioestrus, Met: metoestrus,

Oest: oestrus, Pro: pro-oestrus. %: the average difference in percent response, t=: is there a significant effect of oestrus cycle?

Lauren Nicotra Sex Differences in Allodynia 205

PRO:OEST PRO:MET PRO:DI OEST:MET DI: OEST DI:MET

Pre-Surgery 0.19 ± 0.21 0.38 ± 0.21 0.21 ± 0.20 0.11 ± 0.20 0.14 ± 0.21 0.19 ± 0.22

t= 0.29 t= 0.36 t= 0.31 t= 0.22 t= 0.19 t= 0.21

- 0.53 ± 0.24 0.43 ± 0.28 - 0.41 ± 0.27 - -0.051 ± 0.24 - 0.060 ± 0.27 - 0.30 ± 0.24 N0S0 t= - 0.55 t= - 0.44 t= - 0.42 t= - 0.054 t=- 0.068 t= - 0.35

-0.063 ± 0.40 - 0.25 ± 0.38 - 0.60 ± 0.39 - 0.098 ± 0.34 0.43 ±0.36 0.39 ± 0.40 N0S4 t= -0.061 t=- 0.22 t=- 0.62 t=- 0.092 t= 0.49 t= 0.41 2.8 ± 0.40 2.7 ± 0.41 - 0.90 ± 0.42 0.51 ± 0.47 3.6 ± 0.42 3.5 ± 0.46 N1S3 t=2.7 t= 2.6 t= - 1.0 t= 0.53 t= 3.5 t= 3.3

3.6 ±0.52 3.6 ± 0.50 - 0.78 ± 0.53 - 1.1 ± 0.52 4.0 ± 0.54 4.2 ± 0.52 N3S1 t=3.3 t= 3.3 t=- 0.80 t= - 1.0 t= 3.7 t= 3.9

-0.57 ± 0.49 - 0.96 ± 0.50 0.14 ± 0.52 - 0.34 ± 0.48 -0.84 ± 0.50 - 1.1 ± 0.49 N4S0 t=-0.53 t= 0.97 t= 0.091 t= - 0.24 t= - 0.86 t= - 1.2

Supplementary Table 8. Female MyD88-/- responses were found unaffected by the rodent oestrus cycle prior to nerve injury (Pre-CCI). Following nerve injury, once allodynia became stable (days 17-21) N1S3 and N3S1 MyD88-/- females were significantly more allodynic throughout pro-0estrus and dioestrus. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table D). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per oestrus phase. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force. Di: dioestrus, Met: metoestrus,

Oest: oestrus, Pro: pro-oestrus. %: the average difference in percent response, t=: is there a significant effect of oestrus cycle?

Lauren Nicotra Sex Differences in Allodynia 206

Male Balb/c Male Balb/c Male Balb/c Male TLR4-/- Male TLR4-/- Male TLR2/4-/- VS VS Vs VS VS Vs Male TLR4-/- Male TLR2/4-/- Male MyD88-/- Male TLR2/4-/- Male MyD88-/- Male MyD88-/-

Pre-Surgery - 0.20 ± 0.22 0.11 ± 0.18 0.012 ± 0.11 0.11 ± 0.18 0.18 ± 0.22 0.079 ± 0.17

t= - 0.40 t= 0.041 t= 0.014 t=0.26 t=0.31 t=0.14

N0S0 0.011 ± 0.20 0.18 ± 0.27 0.021 ± 0.21 0.22 ± 0.28 - 0.010 ± 0.21 0.20 ± 0.24

t=0.012 t=0.31 t=0.10 t=0.40 t=- 0.14 t= 0.34

N0S4 -0.18 ± 0.21 0.47 ± 0.41 0.97 ± 0.44 0.17 ± 0.31 - 0.98 ± 0.32 0.49 ± 0.33

t= - 0.38 t=0.51 t=0.76 t= 0.31 t=- 0.70 t= 0.49

N1S3 0.11 ± 0.28 0.91 ± 0.41 0.20 ± 0.31 0.61 ± 0.41 0.43 ± 0.44 0.57 ± 0.41

t=0.038 t=0.67 t=0.42 t= 0.55 t=0.54 t=0.50

N3S1 0.21 ± 0.31 0.32 ± 0.38 -0.53 ± 0.44 0.090 ± 0.33 - 0.43 ± 0.38 0.76 ± 0.48

t= 0.44 t=0.44 t=- 0.67 t= 0.092 t= - 0.52 t= 0.61

N4S0 0.11 ± 0.27 -1.1 ± 0.55 - 0.80 ± 0.51 0.21 ± 0.34 - 0.45 ± 0.44 -0.44 ± 0.43

t= 0.032 t= - 1.2 t= - 0.81 t=0.40 t= - 0.56 t= - 0.42

Supplementary Table 9. Mechanical allodynia was observed across all male mouse strains, for all pain treatment groups from day 3 following nerve injury and maintained until day 21, with no significant difference across strain. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table A). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per sex, per strain and per treatment group.

N: number of sciatic sutures, S: number of subcutaneous sutures.

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Female Balb/c Female Balb/c Female Balb/c Female TLR4-/- Female TLR4-/- Female TLR2/4-/- VS VS Vs VS VS Vs Female TLR4- Female TLR2/4- Female MyD88-/- Female TLR2/4- MyD88 Female MyD88-/- /- /- /- Pre- - 0.18 ± 0.17 0.079 ± 0.12 0.19 ± 0.17 -0.037 ± 0.22 0.39 ± 0.20 0.39 ± 0.21 Surge ry t= - 0.32 t= 0.12 t= 0.34 t=- 0.021 t=0.41 t=0.44

N0S0 0.010 ± 0.18 0.011 ± 0.20 0.098 ± 0.28 0.070 ± 0.21 0.030 ± 0.22 0.067 ± 0.28

t=0.011 t=0.014 t=0.094 t=0.074 t=0.019 t= 0.034

N0S4 -0.17 ± 0.20 0.50 ± 0.38 - 0.51 ± 0.40 0.38 ± 0.37 - 0.80 ± 0.38 -0.45 ± 0.39

t= - 0.38 t=0.53 t=- 0.62 t= 0.41 t= - 0.92 t= - 0.52

N1S3 0.11 ± 0.38 - 0.10 ± 0.36 - 0.96 ± 0.38 0.98 ± 0.44 -0.75 ± 0.42 0.57 ± 0.41

t=0.032 t=- 0.041 t=- 0.74 t= 0.97 t=- 0.84 t=0.50

N3S1 0.11 ± 0.32 0.21 ± 0.41 0.67 ± 0.44 0.11 ± 0.48 0.69 ± 0.40 0.10 ± 0.44

t= 0.12 t=0.34 t=0.66 t= 0.12 t= 0.67 t= 0.10

N4S0 0.11 ± 0.32 0.012 ± 0.30 0.87 ± 0.46 -0.86 ± 0.51 0.20 ± 0.40 0.12 ± 0.48

t= 0.030 t= 0.014 t= t=- 0.82 t= 0.34 t= 0.23 0.92

Supplementary Table 10. Mechanical allodynia was observed across all female mouse strains, for all pain treatment groups from day 3 following nerve injury and maintained until day 21, with no significant difference across strain. Results are expressed as the percent difference in response per increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response; Supplementary Table A). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. n=8 per sex, per strain and per treatment group.

N: number of sciatic sutures, S: number of subcutaneous sutures.

Lauren Nicotra Sex Differences in Allodynia 208

Subject N3S1 Vehicle % Dose 1 Dose 2 Dose 3 IL- V: DOSE 1 V: Dose 2 V: Dose 3 % Response IL-1RA IL-1RA 1RA % t= % t= % t= Response % % % Response Response Response ♀ 13.0 13.0 8.1 10.0 13.0 5.1 3.8 3.0 2.6 0.87 1.0 Balb/c ± 0.44 ± 0.41 ± 0.48 ± 0.50 ± 0.48 N1S3 ♀ 16.0 16.0 9.8 13.0 16.0 6.3 5.4 3.3 3.6 1.0 1.4 Balb/c ± 0.54 ± 0.38 ± 0.48 ± 0.52 ± 0.46 N3S1 ♂ 10.0 10.0 6.8 8.2 10.0 3.2 2.8 2.0 2.1 0.87 1.0 Balb/c ± 0.42 ± 0.40 ± 0.46 ± 0.50 ± 0.44 N1S3 ♂ 13.0 13.0 9.7 11.0 13.0 3.3 3.6 2.3 2.3 0.50 0.69 Balb/c ± 0.40 ± 0.40 ± 0.46 ± 0.51 ± 0.50 N3S1 ♀ 12.0 12.0 12.0 12.0 12.0 0.12 0.16 0.0 0.04 0.08 0.10 TLR4-/- ± 0.48 ± 0.38 ± 0.38 ± 0.34 ± 0.33 24 6 6 N1S3 ♀ 16.0 16.0 16.0 16.0 16.0 0.24 0.29 0.1 0.23 0.03 0.05 TLR4-/- ± 0.54 ± 0.40 ± 0.34 ± 0.38 ± 0.40 7 0 2 N3S1 ♂ 10.0 10.0 10.0 10.0 10.0 0.31 0.36 0.1 0.16 0.04 0.06 TLR4-/- ± 0.51 ± 0.52 ± 0.40 ± 0.42 ± 0.40 2 4 1 N1S3 ♂ 13.0 13.0 13.0 13.0 13.0 0.11 0.17 0.2 0.26 0.01 0.02 TLR4-/- ± 0.40 ± 0.41 ± 0.41 ± 0.40 ± 0.36 0 7 7 N3S1 ♀ 13.0 13.0 13.0 13.0 13.0 0.20 0.26 0.2 0.26 0.08 0.11 TLR2/4 ± 0.48 ± 0.51 ± 0.38 ± 0.36 ± 0.38 1 7 -/- N1S3 ♀ 16.0 16.0 16.0 16.0 16.0 0.17 0.25 0.1 0.17 0.07 0.09 TLR2/4 ± 0.55 ± 0.40 ± 0.50 ± 0.48 ± 0.40 1 6 4 -/- N3S1 ♂ 11.0 ± 0.47 11.0 11.0 ± 0.48 11.0 11.0 0.24 0.29 0.0 0.08 0.02 0.04 TLR2/4 ± 0.46 ± 0.51 ± 0.41 67 2 4 6 -/- N1S3 ♂ 13.0 13.0 13.0 13.0 13.0 0.12 0.17 0.0 0.10 0.03 0.05 TLR2/4 ± 0.47 ± 0.50 ± 0.46 ± 0.44 ± 0.40 87 2 2 -/- N3S1 ♀ 13.0 13.0 13.0 13.0 ± 0.34 13.0 0.20 0.26 0.1 0.17 0.07 0.09 MyD88- ± 0.44 ± 0.46 ± 0.40 ± 0.38 2 6 3 /- N1S3 ♀ 15.0 15.0 15.0 15.0 ± 0.42 15.0 0.14 0.17 0.1 0.14 0.09 0.14 MyD88- ± 0.55 ± 0.42 ± 0.40 ± 0.36 0 8 /- N3S1 ♂ 10.0 10.0 10.0 10.0 ± 0.42 10.0 0.26 0.32 0.1 0.17 0.02 0.04 MyD88- ± 0.41 ± 0.42 ± 0.44 ± 0.41 1 4 7 /- N1S3 ♂ 13.0 13.0 13.0 13.0 ± 0.46 13.0 0.12 0.16 0.2 0.26 0.09 0.13 MyD88- ± 0.47 ± 0.48 ± 0.48 ± 0.40 2 6 /- N3S1

Supplementary Table 11. Male and female Balb/c mechanical allodynia was reversed utilizing the interleukin-1 (IL-1) receptor antagonist (IL-1ra). Established male and female TLR4-/-,

TLR2/4-/- and MyD88-/- mechanical allodynia however, could not be reversed using the neuroimmune modulator IL-1ra. Mechanical allodynia was recorded 1 hour post intraperitoneal administration. Results are expressed as the percent difference in response per

Lauren Nicotra Sex Differences in Allodynia 209

increase in von Frey hair stiffness, generated from the estimated increase in percent response per increase in von Frey stiffness (% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than

1.6%. Dose 1: 100 mg/kg, Dose 2:10 mg/kg, Dose 3: 0.1 mg/kg. As a control, mice were also given an intraperitoneal injection of isotonic saline (Vehicle). n=8 per treatment group, per sex, per strain. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force,Vehicle: isotonic saline, V: vehicle. ♀: Female, ♂: Male.

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Subject N3S1 Vehicle Dose 1 Dose 2 Dose 3 V: DOSE 1 V: Dose 2 V: Dose 3 % % (+)-nal (+)-nal (+)-nal % t= % t= % t= Response Response % % % Response Response Response ♀ Balb/c 13.0 13.0 7.8 10.0 13.0 5.3 4.0 3.2 3.5 1.0 1.4 N1S3 ± 0.44 ± 0.32 ± 0.44 ± 0.48 ± 0.48

♀ Balb/c 16.0 16.0 10.0 13.0 16.0 6.2 5.4 3.1 3.2 0.97 1.2 N3S1 ± 0.54 ± 0.33 ± 0.46 ± 0.50 ± 0.40

♂ Balb/c 10.0 10.0 7.1 8.1 10.0 3.0 3.1 2.1 2.0 0.61 0.97 N1S3 ± 0.42 ± 0.41 ± 0.40 ± 0.51 ± 0.38

♂ Balb/c 13.0 13.0 9.7 11.0 13.0 3.3 3.6 2.3 2.2 0.52 0.76 N3S1 ± 0.40 ± 0.36 ± 0.41 ± 0.46 ± 0.44

♀ TLR4-/- 12.0 12.0 12.0 12.0 12.0 0.09 0.09 0.01 0.01 0.04 0.07 N1S3 ± 0.48 ± 0.32 ± 0.38 ± 0.34 ± 0.33 8 7 1 4 2 4

♀ TLR4-/- 16.0 16.0 16.0 16.0 16.0 0.11 0.20 0.20 0.23 0.03 0.06 N3S1 ± 0.54 ± 0.24 ± 0.34 ± 0.38 ± 0.40 8 4

♂ TLR4-/- 10.0 10.0 10.0 10.0 10.0 0.17 0.26 0.21 0.25 0.02 0.08 N1S3 ± 0.51 ± 0.38 ± 0.40 ± 0.42 ± 0.40 6 6

♂ TLR4-/- 13.0 13.0 13.0 13.0 13.0 0.19 0.27 0.24 0.31 0.02 0.07 N3S1 ± 0.40 ± 0.40 ± 0.41 ± 0.40 ± 0.36 1 5

♀ TLR2/4- 13.0 13.0 13.0 13.0 13.0 0.24 0.31 0.26 0.33 0.11 0.21 /- N1S3 ± 0.48 ± 0.34 ± 0.38 ± 0.36 ± 0.38

♀ TLR2/4- 16.0 16.0 16.0 16.0 16.0 0.12 0.17 0.17 0.20 0.08 0.15 /- N3S1 ± 0.55 ± 0.41 ± 0.50 ± 0.48 ± 0.40 6

♂ TLR2/4- 11.0 11.0 11.0 ± 11.0 11.0 0.18 0.24 0.20 0.24 0.07 0.13 /- N1S3 ± 0.47 ± 0.44 0.48 ± 0.51 ± 0.41 6

♂ TLR2/4- 13.0 13.0 13.0 13.0 13.0 0.08 0.09 0.07 0.11 0.02 0.04 /- N3S1 ± 0.47 ± 0.31 ± 0.46 ± 0.44 ± 0.40 7 2 4 4 6

♀ MyD88- 13.0 13.0 13.0 13.0 13.0 0.14 0.18 0.10 0.16 0.06 0.86 /- N1S3 ± 0.44 ± 0.38 ± 0.40 ± 0.34 ± 0.38 8

♀ MyD88- 15.0 15.0 15.0 15.0 15.0 0.09 0.10 0.11 0.16 0.08 0.01 /- N3S1 ± 0.55 ± 0.37 ± 0.40 ± 0.42 ± 0.36 7 7 0

♂ MyD88- 10.0 10.0 10.0 10.0 10.0 0.21 0.26 0.14 0.18 0.06 0.09 /- N1S3 ± 0.41 ± 0.40 ± 0.44 ± 0.42 ± 0.41 5 7

♂ MyD88- 13.0 13.0 13.0 13.0 13.0 0.17 0.21 0.17 0.23 0.09 0.14 /- N3S1 ± 0.47 ± 0.41 ± 0.48 ± 0.46 ± 0.40 8

Supplementary Table 12. Male and female Balb/c mechanical allodynia was reversed utilizing the specific TLR4 antagonist (+)-naltrexone. (+)-naltrexone did not significantly alter male or exacerbated female mechanical allodynia in TLR4-/- ,TLR2/4-/- and MyD88-/-mice.

Mechanical allodynia was recorded 1 hour post intraperitoneal administration. Results are expressed as the percent difference in response per increase in von Frey hair stiffness,

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generated from the estimated increase in percent response per increase in von Frey stiffness

(% response). A t-value of < -2 or > 2 was determined statistically significant (p < 0.05) when differences in percentage response were greater than 1.6%. Dose 1: 30 mg/kg, Dose 2: 3 mg/kg, Dose 3: 0.3 mg/kg. As a control, mice were also given an intraperitoneal injection of isotonic saline (Vehicle). n=8 per treatment group, per sex, per strain. N: number of sciatic sutures, S: number of subcutaneous sutures, g: grams force, Vehicle: isotonic saline, V: vehicle, (+)-nal, (+)-naltrexone. ♀: Female, ♂: Male.

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PRO OEST MET DI

Pre-Surgery 6.7 6.8 6.8 6.0 ± 0.24 ± 0.20 ± 0.25 ± 0.21

N0S0 6.8 6.3 6.9 6.9 ± 0.31 ± 0.28 ± 0.30 ± 0.31

N0S4 8.4 8.5 8.7 8.2 ± 0.40 ± 0.38 ± 0.41 ± 0.32

N1S3 15.2 11.0 10.7 15.4 ± 0.51 ± 0.44 ± 0.38 ± 0.32

N3S1 18.2 12.7 14.1 17.6 ± 0.41 ± 0.40 ± 0.41 ± 0.51

N4S0 19.0 19.1 18.7 18.9 ± 0.58 ± 0.74 ± 1.0 ± 0.85

Supplementary Table A: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of oestrus cycle in Balb/c mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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PRO OEST MET DI

Pre-Surgery 6.9 6.8 6.8 6.6 ± 0.22 ± 0.24 ± 0.21 ± 0.24

N0S0 6.6 6.3 6.8 6.9 ± 0.30 ± 0.28 ± 0.21 ± 0.24

N0S4 8.6 8.6 8.7 8.4 ± 0.39 ± 0.38 ± 0.30 ± 0.32

N1S3 14.2 11.0 10.6 15.2 ± 0.47 ± 0.44 ± 0.51 ± 0.48

N3S1 16.4 12.1 13.7 16.9 ± 0.46 ± 0.50 ± 0.51 ± 0.47

N4S0 18.0 18.1 17.7 17.4 ± 0.52 ± 0.47 ± 0.53 ± 0.54

Supplementary Table B: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of oestrus cycle in TLR4-/- mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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PRO OEST MET DI

Pre-Surgery 6.5 6.6 6.4 6.7 ± 0.20 ± 0.26 ± 0.24 ± 0.22

N0S0 6.3 6.9 6.9 6.6 ± 0.27 ± 0.24 ± 0.21 ± 0.26

N0S4 8.9 9.2 8.6 8.8 ± 0.31 ± 0.30 ± 0.27 ± 0.33

N1S3 14.0 10.8 10.9 14.7 ± 0.42 ± 0.50 ± 0.47 ± 0.44

N3S1 17.4 13.7 14.8 16.9 ± 0.50 ± 0.52 ± 0.57 ± 0.56

N4S0 18.5 19.1 19.5 18.3 ± 0.50 ± 0.51 ± 0.55 ± 0.47

Supplementary Table C: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of oestrus cycle in TLR2/4-/- mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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PRO OEST MET DI

Pre-Surgery 6.6 6.4 6.2 6.4 ± 0.22 ± 0.21 ± 0.22 ± 0.24

N0S0 6.2 6.8 6.7 6.6 ± 0.28 ± 0.22 ± 0.29 ± 0.27

N0S4 9.1 9.2 9.4 9.7 ± 0.40 ± 0.42 ± 0.41 ± 0.39

N1S3 14.0 11.4 11.3 14.9 ± 0.40 ± 0.42 ± 0.46 ± 0.43

N3S1 16.4 12.7 12.8 17.2 ± 0.51 ± 0.56 ± 0.55 ± 0.53

N4S0 18.5 19.1 19.5 18.3 ± 0.50 ± 0.51 ± 0.55 ± 0.47

Supplementary Table D: The relationship between the response and the force of the von Frey filament was examined in all studies. When examining the effect of oestrus cycle in MyD88-/- mice results are expressed as the percent difference in response per increase in von Frey hair stiffness, which are generated from the estimated increase in percent response per increase in von Frey stiffness.

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CHAPTER 5. SUMMARY AND DISCUSSION OF PHD FINDINGS

Chronic pain is a disease process with wide reaching implications, inflicting huge health and economic encumbrances not only on the 1.5 billion global chronic sufferers, but also the worldwide economy. Although several diverse first, second and third-line therapies are available for chronic pain patients, these pharmacotherapies are symptom-modifying rather than disease-correcting and at present, there does not exist an analgesic which preferentially treats the female burden of chronic pain. While there is precedence for this sex- pharmacotherapy bias433, the incomplete understanding of the pathophysiology underlying the female prevalence of chronic pain, is fundamental to why females are still inadequately treated for their chronic pain and further, why researchers have not yet found a way to prevent this health adversity affecting the ‘fairer’ sex.

Contributing to the inability to underpin the mechanisms underlying sex differences in chronic pain is not only the continued preferential use of male rodents in preclinical investigations but also the preclinical methodologies that fail to mirror the clinical pain situation. Although animal models are responsible for our current understanding of pain and chronic pain processes, those utilised to examine the mechanisms underlying chronic pain are limited in their ability to mimic the range of clinical hypersensitivities. Furthermore, with the ethical dilemmas associated with pain research, and the difficulty of approving and conducting pain research in light of the three R’s that are strongly embedded within not only the Australian, but global legislation governing animal use in science, it has become increasingly apparent that preclinical pain models are in need of a makeover. This preclinical pain model revolution began in 2010, when Grace and colleagues developed a modified CCI

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model of neuropathic injury, which was demonstrated to replicate the heterogeneous range of clinical chronic pain, thereby addressing the translational and ethical concerns of previously used animal pain models. Consequently, in an attempt to uncover the pathophysiology underlying sex differences in chronic pain, the first aim of the work presented in this thesis was to investigate potential mechanisms for the first time using translatable and refined preclinical methodologies.

One way in which this work attempted to appropriately investigate sex differences in pain sensitivity and therefore better replicate the clinical pain experience, was to investigate mechanical allodynia not only using the novel graded neuropathy model for the first time in females, but to examine mechanical allodynia using a range of von Frey methods. Since their development during the 1890’s, von Frey hairs have been used as a nociception assay, with varying protocols producing diverse findings. By examining and contrasting pain responses generated using three diverse von Frey methodologies, our findings highlight the importance of using the correct behavioural assessment technique. Allodynia recordings generated using the 8-second method (von Frey Test 1) support published arguments that have highlighted the problems associated with examining nociception with sustained noxious stimuli. Moreover, comparisons between von Frey methods 2 and 3 highlighted the significance of examining mechanical allodynia across the von Frey logarithmic force scale, with only von Frey Test 3 revealing a sex difference in less allodynic animals, as well as an oestrus cycle effect following nerve injury. Consequently, our findings in Chapter 2 extend those first presented by Grace and colleagues, with von Frey test 3 the only testing method to reveal the ability of the novel graded neuropathy model to refine the traditional CCI technique in the female sex, and therefore preclinical pain testing generally.

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Continued use of pain models and behavioral assessment techniques that do not best reflect the clinical pain scenario will limit the progress of pain research, with the pathophysiology underlying the female prevalence of chronic pain remaining to be determined. The ability to detect modest reductions in pain using the graded nerve injury model combined with the novel von Frey approach presented in Chapter 2 however, allowed our studies to determine both an effect of sex and a gonadal hormone influence thereby addressing the first set of aims presented in Chapter 1.7. Although together the graded neuropathy model and von Frey Test

3 have the ability to refine preclinical investigations examining sex differences in chronic pain, it must be noted that our published studies presented in Chapter 2 sacrificed the

Reduction parameter of the three R’s in order to satisfy the Refinement component of these guiding principles. In spite of this, the findings of Chapter 2 encourage the use of less allodynic animals in preclinical pain research thereby allowing for the refinement of future preclinical pain studies. On the whole, when utilised together, the combination of the graded nerve injury model and von Frey approach 3 not only help to ensure the use of animals in future pain research in an era where inflicting pain in order to understand it is less tolerated, but this methodology, has the ability to shed new light on potential contributors to chronic pain that may have been previously overlooked and may also lead to the discovery of novel analgesics. As a direct result of our findings in Chapter 2, the remainder of the investigative work presented in this thesis utilised these novel methodologies in the hope of better understanding the underlying contributors fundamental to the skewed female prevalence of chronic pain.

Amongst the numerous mediators and systems that have been proposed to underlie sex differences in chronic pain, one of the most investigated are the gonadal hormones, with E2

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definitively known to influence nociception and pain processing both peripherally and centrally. In spite of this, the precise contribution of E2 in chronic pain remains far from understood. With poorly translatable preclinical methodologies partly to blame for an incomplete understanding of chronic pain pathophysiology, the second aim of this body of work became to more appropriately examine the role of E2 in the graded nerve injury model.

In light of the findings in Chapter 2, it was hypothesized that the chance of uncovering a gonadal hormone influence was far greater using preclinical methods that could determine modest to large deviations in the pain response. Following nerve injury, von Frey Test 3 revealed that E2 significantly influenced female rodent responses however, this effect was in fact only visible in both slight (N1S3) and moderately (N3S1) allodynic Balb/c female mice.

Non-maximally allodynic (N1S3 and N3S1) females were found to display greater mechanical allodynia throughout the high oestrogenic level phases of pro-oestrus and dioestrus, thereby providing further validation for the use of less allodynic animals in future pain research. Although revealing a correlation between elevated mechanical hypersensitivity and E2, it must be noted that several other gonadal hormones known to influence nociception and pain are in fact present throughout the oestrus cycle. As a consequence, the exaggerated female hypersensitivity documented throughout pro-oestrus and dioestrus could not be definitively attributed to E2. Accordingly, with the ovaries the major source of E2 output in females, the surgical procedure of ovariectomy was later utilised in Chapter 3 in an attempt to isolate the specific effects of this gonadal hormone. Reduced female response rates following

OVX and the return of hypersensitivity following the reintroduction of E2 via slow release pellets, found E2 alone responsible for the exaggerated female pain response in the graded nerve injury model. This effect was later confirmed to be an E2 mediated phenomenon rather than sex specific, with the surgical implantation of E2 pellets in our male Balb/c subjects resulting in female-like exacerbated mechanical allodynia following nerve injury. Together,

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the principal findings of Chapter 3 conclusively established a role for E2 in nerve-injury induced exaggerated female mechanical allodynia.

While our OVX and E2 replacement studies definitively isolated a specific effect of this female gonadal hormone in the graded nerve injury model, it must be noted that serum levels of E2 were not in fact recorded and confirmed for those animals having undergone these surgical procedures. Irrespective of this, both the OVX procedure and the silastic capsule method are experimental tools that have been well utilised throughout the preclinical literature to examine the role of gonadal hormones in several pathologies. While the ovaries are not the only source of oestrogens in females, they are, as previously stated, the principal generator of

E2 in females and their bilateral removal reliably and significantly reduces serum estradiol levels compared with those found in intact females. Likewise, the silastic pellets chosen to replace E2 in our ovariectomised females and sham-operated males, have been well documented to reliably produce stable serum concentration of E2448, with supraphysological doses of E2 also strategically chosen accross our own studies to ensure a visible effect of E2.

Despite these assurances, a

In an attempt to determine the mechanisms by which E2 intensified the female pain response in the graded nerve injury model, Chapters 3 and 4 investigated the newly identified interaction between this gonadal hormone and the innate immune system described in Chapter

1.6 and a possible role for this interface in chronic pain. Since identified as a contributor to the chronic pain process in the 1970’s, the immune system has steadily received greater attention and recognition as a key player in persistent pain states. It wasn’t until the early

1990’s however, that the resident non-neuronal cells of the nervous system, glia, were

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identified as anything other than the glue supporting neurons within the CNS. Proliferation in glial research over the last 20 or so years has resulted in not only the discovery but also the appreciation of bidirectional communication between neurons and these immuncompetent cells in the initiation and maintenance of pathological pain. In an attempt to examine a role for a gonadal hormone innate-immune interaction in exacerbated female chronic pain and thereby address the third aim of this thesis described in Chapter 1.7, Chapter 3 first examined the contribution of female spinal microglia in neuropathy-induced mechanical allodynia.

Throughout this chapter it was established that exaggerated female allodynia could be adoptively transferred via intrathecal administration of spinal microglia from nerve injured female mice. This ability to transfer exaggerated ‘pain’ was further determined E2-dependent, supporting several investigations which have shown a role for glial sensitization or “priming” in several neuroinflammatory diseases. Although inspired by previous investigations who have reported potentiated mechanical allodynia following intrathecal transfer of activated peripheral and innate immune cells412, the later findings of Chapter 3, whereby the mere presence of naïve, and therefore non ‘activated’ female microglia at the time of nerve injury were sufficient to exacerbate the pain response, are to our knowledge unprecedented.

Moreover, our findings whereby it was demonstrated possible to transfer the exaggerated female pain phenotype to the male sex using naïve female resident CNS cells are also unparalleled. We demonstrated for the first time that intrathecal transfer of naïve female microglial cells that had been exposed to E2 elevated recipient mechanical allodynia in the subsequent event of nerve injury. Importantly, the inability for naïve male microglia and more so, naïve female microglia transferred from ovariectomised subjects, to alter the recipient pain response confirmed that this was a female microglial phenomenon. Accordingly, the discoveries of Chapter 3 revealed that E2-female-microglial cell-priming was contributing to the exacerbated female pain phenotype in the graded nerve injury model.

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As detailed in our recently published review presented in Chapter 1.5.2, crucial to glia’s role in chronic pain are the pattern recognition receptors, TLRs, with TLR4 the most characterized in pathological pain. In light of recent evidence demonstrating a strong association between

E2 and TLR4 (detailed Chapter 1.6), and known role of both this gonadal hormone and this pattern recognition receptor in pain and chronic pain independently, it was hypothesized that perhaps the female microglial cell-priming revealed in Chapter 3 was occurring via E2 interactions with TLR4. In spite of previous studies identifying E2 potentiation of TLR transcriptional factors such as NFκB, resulting in the exaggeration of pro-inflammatory cytokine release347, 383, whether oestrogens bind to TLRs specifically is at present unknown.

From a series of key in vivo and in vitro studies throughout Chapter 3 however, our findings revealed a strong interface and potential binding between this gonadal hormone and pattern recognition receptor in the graded nerve injury model. E2-female-microglial cell-priming was revealed to occur in a TLR2/4-MyD88 dependent fashion. Moreover, oestrogens were found to initiate NFκB activation in over-expressing TLR2 and TLR4 HEK cells contingent upon

TLR4, suggestive of direct E2 TLR4 binding rather than mere potentiation or alteration of

TLR signalling. Together, these findings not only reveal for the first time, the ability for E2 to potentially directly activate TLR4, but that the nerve injury induced exaggerated female pain phenotype seen in the graded nerve injury model in fact resulted from this E2 TLR4 interaction. Although our unprecedented findings are suggestive of E2 binding to TLR4, it must be emphasised that binding studies are in fact required in order to confirm a direct interaction between this gonadal hormone and pattern recognition receptor. In spite of this, the in vivo and in vitro findings of Chapter 3 reveal a novel mechanism underlying preclinical sex differences in neuropathic pain, whereby E2 priming of female microglia via TLR4 is

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responsible for the resulting exacerbated mechanical pain sensitivity in the subsequent event of nerve injury.

Further substantiating the discoveries of Chapter 3, were the later pharmacological interventions of this chapter, which confirmed a role for the E2 TLR4 association in the exacerbated female pain phenotype. Using the blood brain barrier permeable TLR4 receptor antagonist (+)-naltrexone along with the cytokine inhibitor IL-1ra, neuroimmune modulation of mechanical allodynia was not only evidenced to preferentially reduce female nociceptive hypersensitivity, but was also shown to be E2 dependent. These in vivo findings, along with the earlier behavioural studies and in vitro discoveries of chapters 2 and 3 therefore, not only established that exaggerated Balb/c female pain in the graded nerve injury model resulted from interactions between this gonadal hormone and innate immune receptor, but that there is a clear distinction in pathophysiology between female chronic pain during the oestrogenic years compared with males, prepubescent and post-menopausal females.

In an attempt to further characterize this association between the female sex and the innate immune system in chronic pain, Chapter 4 extended the pharmacological investigations of

Chapters 2 and 3 and undertook a genetic approach to investigating a role for the TLRs and other key neuroimmune contributors in the female prevalence of chronic pain. Somewhat contradictory to the findings of Chapters 2 and 3, Chapter 4 revealed the development of a preclinical chronic pain that was not only pro-inflammatory, but also TLR2/4 and MyD88 independent. Surprisingly, this pro-inflammatory and neuroimmune independent pain was also revealed to remain subject to the female sex bias, with mechanical allodynia preferentially affecting the female sex irrespective of the presence of key neuroimmune

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contributors such as TLR4. While the findings of this final experimental chapter may appear to be in direct contrast to those presented in earlier chapters, it must be stressed that opposed to the Balb/c subjects utilised in Chapters 2 and 3, the subjects of Chapter 4 were null mutants and therefore had genetic deletions in innate immune signaling elements at the time of nerve injury. Moreover, although several other previous genetic investigations have shown a role for the TLRs and other key neuroimmune components in preclinical chronic pain, it must be noted that mechanical allodynia was generated accross our male and female neuroimmune null mutant subjects using a diverse chronic pain model and strain of mouse compared to those utilised in such previous investigations. Consequently, using both the graded nerve injury model and Balb/c mice, thereby attempting to investigate sex differences in chronic pain using translatable preclinical methodologies, Chapter 4 revealed the unexpected creation of a compensatory, potentially neuronal, pro-inflammatory independent type of pain. With chronic pain now known to result from activation of both the neuronal and innate immune systems however, the results of Chapter 4 should not cast doubt on the findings of earlier chapters, which reveal a strong association between the female sex, oestrogen and the innate immune system in a translatable chronic pain model. In spite of this, this novel and completely uncharacterized type of chronic pain does warrant further investigation, perhaps with the use of neuronal-targeted therapies such as minocycline. Moreover, although failing to validate or extend the findings of previous chapters, Chapter 4 did reveal an evolutionary pressure, whereby the exacerbated female pain phenotype is one that is both predetermined and persistent. Accordingly, the findings of Chapter 4 do highlight the significant and unrelenting problem of the female burden of chronic pain. Although at present the evolutionary advantage of this sex bias is unknown, the findings across all three experimental chapters highlight the necessity to treat the overwhelming female burden of chronic pain.

.

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In summary, this body of work significantly advances the knowledge of female chronic pain mechanisms. Here we establish for the first time using translatable preclinical methodologies in vivo and in vitro evidence of a direct interaction between the sex steroid E2 and the innate immune system in pain. Exaggerated female pain hypersensitivity was found to result from

E2 priming of spinal cord microglial cells in a TLR2/4 MyD88 dependent fashion.

Importantly, these studies undoubtedly establish that female pain has a greater proportion of neuroinflammation during the reproductive/ oestrogenic years, thereby advocating that chronic female pain may be treated more effectively using glial-targeted pharmacotherapies in the future. Consequently, these findings encourage further investigation into the efficacy of neuroimmune modulators in treating the exacerbated female pain phenotype, along with further characterization of the interface between the E2 steroid and the pattern recognition receptor TLR4. Binding investigations along with pharmacological intervention studies using oestrogen receptor antagonists such as tamoxifen for example, are required in order to determine a direct interface between this gonadal steroid hormone and this neuroimmune receptor. Moreover, in order to determine whether our findings in chronic pain are unique to female microglia, future examination of the impact of other cells resident in the CNS is also required. Important to highlight however, is the need to conduct these future investigations, along with any other future preclinical chronic pain studies, using the novel and translatable preclinical methodologies presented here for the first time. Utilisation of these novel methodologies will aid in not only appropriately investigating mechanical allodynia in the female rodent but will greatly assist in elucidating modest to large deviations in the pain response and thereby aid in the determination of the mechanisms underlying chronic pain.

Notwithstanding the need for these future investigations, this body of work clearly demonstrates how the cellular and molecular generators of chronic pain are fundamentally

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different between the sexes. Fundamentally, these novel findings reveal the potential for not only improving the treatment of female chronic pain patients, but also potentially preventing this female-biased clinical pain adversity.

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