INTERLEUKIN 6 RELEASE FROM t98g HUMAN GLIAL CELL LINE AS a PREDICTIVE MARKER FOR CHRONIC PAIN, AND THE CHARACTERIZATION OF SUBSTANCE(S) INVOLVED IN PAIN

1.2.2.2 Nociceptin/orphanin FQ and Nocistatin 171.3 Importance of Cerebrospinal Fluid CSF in Chronic Pain Research 23 Chapter 2 Release of Pro-inflammatory Cytokines in T98G Cells Upon..

INTERLEUKIN-6 RELEASE FROM T98G HUMAN GLIAL CELL LINE AS A PREDICTIVE MARKER FOR CHRONIC PAIN, AND THE CHARACTERIZATION OF SUBSTANCE(S) INVOLVED IN CHRONIC PAIN

TAY SUAN ANNABEL B.Sc (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF ANAESTHESIA NATIONAL UNIVERSITY OF SINGAPORE

2013

Acknowledgements

I would like to express my sincere gratitude to my supervisor A/P Low

Chian Ming, for his support, advice and constant encouragement throughout my

research career at the National University of Singapore Throughout the course of

my project, I have not only learned many laboratory techniques, but also learned many invaluable life skills that will benefit me for life I would also like to

express my gratitude to Prof Shinro Tachibana, for his guidance and support

throughout the course of my project Without his directions, this work would not

have come to fruition My heartfelt thanks are also due to A/P Liu Hern Choon

Eugene and Prof Lee Tat Leang, for supporting my work constantly, for their

helpful advice when things did not go well, and for helping me procure the valuable samples from National University Hospital for the project Thank you for your supervision, guidance and support throughout my study My sincere thanks

also goes out to Prof Toshiaki Minami for helping me procure the precious samples from Osaka Medical University Hospital for my research work

I would also like to express my heartfelt thanks to Mdm Li Chunmei for

her constant support, advice and encouragement Thank you for all your help with

my HPLC work and other technical support My sincere thanks go out to Ms

Wang Anni for her assistance in western blot and for brightening up my days as a

researcher Thank you both so much for all the fun and laughter you have brought

to my days in the laboratory I am also very grateful to Ms Jeyapriya Raja

Sundaram for her help and guidance in primary astrocyte culture and

thanks are also due to Mdm Karen Ho Ban Shian and Mrs Mariam Mathew for their administrative support and assistance

Lastly, I am grateful to my family and friends for their understanding,

endless support and encouragement throughout the course of my research work

Table of Contents

List of Abbreviations xiv List of Publications xvii List of Conference Papers xvii

Chapter 1 Introduction

1.1.3 Treatment Strategies for Chronic Pain and their Challenges 8

1.2.2.2 Nociceptin/orphanin FQ and Nocistatin 17

1.3 Importance of Cerebrospinal Fluid (CSF) in Chronic Pain Research 23

Chapter 2 Release of Pro-inflammatory Cytokines in T98G Cells Upon

Chapter 3 Comparison of IL-6 Releasing Activity between CSF of Chronic

Pain Patients and Pain-free Patients

3.3.1 Comparison between PHN Therapy Effective, Therapy Ineffective

3.3.3 IL-6 Release from Primary Astrocytes Upon Exposure to CSF of

Chapter 4 Purification of Protein-like Compounds from CSF of

Chronic Pain Patients

4.2.3.2 Treatment of CSF with Pronase 66

4.2.5 IL-6 Release from T98G Cells When Exposed to Lignocaine and

4.3.4 Comparison between Chromatograms Obtained from HPLC of

5.2.4 Cell Fractionation and Cell Lysis 91

Summary

Besides neurons, the central nervous system (CNS) consists of glial cells, which are mainly microglia and astrocytes Chronic pain is classically viewed as being mediated solely by neurons, but there is mounting evidence that glial cells also play a part Glial cells, responding to stimulation by neurotransmitters and peptides, are activated and release pain-enhancing substances like pro-inflammatory cytokines These cytokines have been shown to play a role in enhancing pain by their actions in the spinal cord

This research work focuses firstly on investigating pro-inflammatory cytokine release in a cell culture system as a potential marker for chronic pain Two conditions related to chronic pain were studied: post-herpetic neuralgia (PHN) and osteroarthritis Cerebrospinal fluid (CSF) from patients suffering from either condition was used to trigger astrocytic cell line T98G cultures, and subsequent pro-inflammatory cytokine release was measured by enzyme-linked immunosorbent assay (ELISA) IL-6 release in the chronic pain patient groups was found to be significantly higher compared to pain-free controls, as well as in the PHN patient group whose steroid treatment was ineffective compared to those whose treatment was effective CSF samples collected before steroid treatment

also triggered higher IL-6 release than after treatment samples These in vitro tests

provide an objective evaluation on the extent of chronic pain as well as the efficacy of steroid therapy

Secondly, we attempted to separate and isolate pain-related substances in

chronic pain patients’ CSF, making use of the in vitro T98G cell system

established earlier The CSF was separated by a two-step high performance liquid chromatography (HPLC) technique, and the fractions that triggered IL-6 release

in T98G cells were isolated We narrowed down to three peaks that could trigger IL-6 release and these peaks would be subjected to future mass spectrometry analysis to identify the protein-like substances in the CSF, which could be potential markers for chronic pain Lastly, we attempted to elucidate the IL-6

signaling pathway in this in vitro model of chronic pain NF-κB inhibition studies

and western blot analysis confirmed that NF-κB acts upstream of IL-6 in this T98G cell system

In conclusion, we have established an effective in vitro assay system to

quantify chronic pain utilizing CSF and have taken the first steps in isolating pain-related protein substances in the CSF of chronic pain patients In an attempt

to better understand the complex mechanisms, we hope to contribute to the management of unrelenting chronic pain

List of Tables

Table 1-1 Common pharmacological treatments for chronic pain 10

List of Figures

Figure 1-1 Schematic diagram of how chronic pain is transmitted 6 Figure 2-1 TNF-α, IL-1β and IL-6 release in T98G cells upon addition 37

of PHN CSF Figure 2-2 Comparisons in IL-6 release between before and after 39

treatment groups, and between therapy effective and ineffective groups

Figure 2-3 IL-6 release in T98G cells upon addition of PHN CSF and 41

methylprednisolone

Figure 3-1 Comparisons in IL-6 release between control, PHN therapy 54

effective and PHN therapy ineffective groups Figure 3-2 Comparison in IL-6 release between control and 55

osteoarthritis groups

Figure 3-3 Immunocytochemical analysis of primary astrocytes 56 Figure 3-4 IL-6 release in primary astrocytes upon addition of 57

PHN CSF Figure 4-1 IL-6 release in T98G cells upon addition of pooled 71

osteoarthritis CSF of < 10 kDa molecular weight CSF and pooled osteoarthritis CSF of > 10 kDa molecular weight Figure 4-2 IL-6 release in T98G cells upon addition of pooled 72

osteoarthritis CSF pre-treated with pronase Figure 4-3 HPLC of pooled osteoarthritis CSF and IL-6 releasing 73

activity of the peaks Figure 4-4 HPLC of pooled active fractions from previous HPLC 76

run, and IL-6 releasing activity of the peaks Figure 4-5 Chromatograms obtained from HPLC of (A) pooled 78

osteoarthritis CSF and (B) pooled pain-free control CSF Figure 4-6 IL-6 release in T98G cells upon addition of lignocaine 79 Figure 4-7 IL-6 release in T98G cells upon addition of albumin 80 Figure 5-1 Effect of NF-κB inhibitor Bay 11-7082 on IL-6 release 92

Figure 5-2 Effect of NF-κB inhibitor Bay 11-7082 on IL-6 release 93

in T98G cells upon addition of control CSF

Figure 5-4 Possible signaling pathway of IL-6 release in T98G cells 99

List of Abbreviations

EBSS Earle's Balanced Salt Solution

GFAP Glial Fibrillary Acidic Protein

MALDI-TOF Matrix-assisted Laser Desorption/ionization–time-of-flight

PMSF Phenylmethylsulfonyl Fluoride

SSNRIs Selective Serotonin and Norepinephrine Reuptake Inhibitors

List of Publications

A.S Tay, E.H Liu, T.L Lee, S Miyazaki, W Nishimura, T Minami, C.-M Low,

S Tachibana, Cerebrospinal fluid of postherpetic neuralgia patients induced interleukin-6 release in human glial cell-line T98G (Manuscript submitted)

List of Conference Papers

A.S Tay, C Li, A Wang, T.L Lee, S Tachibana, C.-M.Low, Cerebrospinal fluid from post-herpetic neuralgia Japanese patients triggers interleukin-6 release in glioblastoma cells 2nd Singapore-Duke Anaesthesia Update (Singapore, 2010) Poster presentation

A.S Tay, C Li, A Wang, E.H Liu, T.L Lee, S Chiang, S Fujiwara, W Nishimura, T Minami, C.-M Low, S Tachibana, Cerebrospinal fluid from post-herpetic neuralgia Japanese patients triggers interleukin-6 release in glioblastoma cells, J Neurochem 115 Supplement 1 (2010) 10th Biennial Meeting of the Asian-Pacific Society of Neurochemistry (Phuket, Thailand, 2010) Poster presentation

A.S Tay, E.H Liu, T.L Lee, S Miyazaki, W Nishimura, T Minami, C.-M Low,

S Tachibana, Interleukin-6 release from human glial cell line T98G as a predictive marker on the effectiveness of steroid therapy in reducing neuropathic pain of post-herpetic neuralgia patients Yong Loo Lin School of Medicine 2ndAnnual Graduate Scientific Congress (Singapore, 2012) Oral presentation

Chapter 1 Introduction

1.1 Chronic Pain

The International Association for the Study of Pain defines chronic pain as pain that persists for at least 3 months Chronic pain is normally triggered by injury or disease, which damages the nervous system in such a way that it is unable to restore its normal physiological functions to homeostatic levels It can persist for months or years, long after the original injury has healed (Gao and Ji, 2010) Chronic pain is heterogeneous, and multiple molecular and cellular mechanisms act in combination within the peripheral nervous system (PNS) and central nervous system (CNS) to produce the chronic pain (Scholz and Woolf, 2002)

1.1.1 Epidemiology of Chronic Pain

Chronic pain has become a major healthcare challenge as it interferes with normal daily life The World Health Organization (WHO) approximates that one

in five people worldwide experiences chronic pain In a large-scale survey involving 16 countries, 19% of respondents over 18 years old had suffered pain for more than 6 months 61% of these chronic pain sufferers were unable to work outside the home, and 19% had lost their jobs 40% of them had insufficient pain

management and only 2% were seeing a pain specialist (Breivik et al., 2006)

A study carried out in Singapore in 2009 showed that the prevalence of chronic pain was 8.7% Chronic pain afflicted women at a higher rate than men, and the prevalence increased sharply beyond 65 years of age Although a

seemingly lower prevalence of chronic pain was seen in this study, it remains a healthcare problem of high importance, due to a rapidly ageing population in many developed countries including Singapore, and is likely to consume an increasingly large amount of healthcare resources within the next few years (Yeo and Tay, 2009)

1.1.2 Pathophysiology of Chronic Pain

Chronic pain can be classified as either nociceptive or neuropathic Nociceptive pain is derived from mechanical, chemical or thermal irritation to the peripheral sensory nerves, and is typically well-localized Neuropathic pain, on the other hand, is pain initiated by damage to or a lesion of the nervous system, and is characterized by poor localization (Goucke, 2003)

Chronic pain results from the development of neural plasticity in the PNS and CNS It was traditionally believed that only neurons and their neural circuits were responsible for pain development and maintenance Hence current therapeutics for chronic pain focuses on neuronal targets, which include N-methyl-D-aspartic acid (NMDA) receptor antagonists and opioid analgesics Such therapies provide transient pain relief and do not resolve the underlying pathological processes that lead to chronic pain Hence, studies on non-neuronal cells, in particularly glial cells in chronic pain conditions, have increased tremendously in recent years

1.1.2.1 Role of Glial Cells in Chronic Pain

Recent studies and review articles have highlighted a communication cross-talk that exists between the immune system and the nervous system (Scholz and Woolf, 2007) Neurons and glial cells in the nervous system have close interactions with each other on a cellular and molecular level Injury to peripheral nerves triggers an inflammatory response in peripheral glia and immune cells The vital role of inflammation in the development of chronic pain is demonstrated in a study whereby injection of pro-inflammatory agents such as carrageenan or complete Freund’s adjuvant around the sciatic nerve induced mechanical

allodynia in animal models (Sorkin and Schafers, 2007) The peripheral inflammatory response then sets off a series of downstream reactions through the release of neurotransmitters or neuromodulators from primary afferents These substances in turn lead to activation of glial cells that are in close proximity to the

afferent neuron terminals (Vallejo et al., 2010)

Glial cells, forming 70% of the total cell population in the brain and spinal cord, have long been viewed as performing mainly neuronal housekeeping and support functions, providing insulation and protection to the neurons They do not have axons, and hence they have been perceived to have no role in nerve signal transmission However, this view has slowly been changing Pathological pain is classically viewed as being mediated solely by neurons, but there is mounting evidence that glial cells also play a part in exaggerated pain states created by

inflammation and neuropathy (Hashizume et al., 2000; Watkins and Maier, 2002)

Astrocytes, in particular, play an important role in pain processing They are the

most abundant cells in the CNS (constituting 40-50% of all glial cells) in terms of number and volume, forming networks with themselves via gap junctions and

making very close contacts with neuronal synapses and blood vessels (Halassa et

al., 2007) Astrocytes express receptors for numerous neurotransmitters,

neuroactive substances and amino acids, and hence provide support and nourishment for neurons as well as regulate the external chemical environment during synaptic transmission (Verkhratsky and Steinhauser, 2000; Hanson and Ronnback, 2004)

In normal physiological conditions, glial cells are in a quiescent state In most cases of early glial response to injury or disease conditions, activation of microglia occurs first Activated microglia display a change in surface markers and membrane proteins, and this triggers the production and release of pain-enhancing substances such as reactive oxygen species, excitatory amino acids, nitric oxide, prostaglandins and pro-inflammatory cytokines (Wieseler-Frank, Maier and Watkins, 2004) This subsequently leads to activation and proliferation

of astrocytes, correlating with the release of even more pain enhancing substances These substances modulate pain processing by influencing either presynaptic release of neurotransmitters and/or postsynaptic excitability, leading to

persistence in hypersensitivity and chronic pain (Guo et al., 2007) Figure 1-1

shows the schematic diagram of chronic pain transmission

Figure 1-1 Schematic diagram of how chronic pain is transmitted After

receiving a pain stimulus, peripheral neurons transmit signals to the primary afferent neuron, causing the release of neurotransmitters and neuromodulators that activate the receptors on microglia and astrocytes The glial cells are activated and release pro-inflammatory cytokines and other pain enhancing substances This affects the presynaptic release of neurotransmitters, postsynaptic excitability, as well as a self-propagating mechanism of enhanced production of pain enhancing

substances by the glial cells

Astrocytic reaction is more persistent than microglial reaction after nerve injury, lasting more than 150 days after nerve injury Activation of astrocytes results in prolongation of the pain state, and is accompanied by a reduction in

microglial activity over time (Tanga et al., 2004) Thus, microglia are involved in

the early development of chronic pain, while astrocytes function in sustaining the

pain (Vallejo et al., 2010) An interesting finding was that nerve injury induces an

increase in interleukin-18 (IL-18) and IL-18 receptors in activated microglia and astrocytes respectively, suggesting an interaction between microglia and

astrocytes in the physiology of chronic pain (Miyoshi et al., 2008)

However in some cases, astrocytes are activated without microglial activation, and are shown to be sufficient on their own to produce chronic pain

Davies et al (2008) showed that transplantation of astrocytes derived from

glial-restricted precursor cells triggered the onset of mechanical allodynia and thermal

hyperalgesia after spinal cord injury in rats Hald et al (2009) also demonstrated

that development of hypersensitivity and activation of astrocytes occurred without microglial activation in chronic pain mouse models

1.1.2.1.1 T98G Cell Line as an in vitro Astrocytic Model

The cell line used in this project is the T98G human glial cell line T98G cell line is a derivative of glioblastoma and is of astrocytic origin T98G cells have the transformed characteristics of immortality and anchorage independence However at the same time, they act like normal human cells in that they become

arrested in G1 phase under stationary phase conditions of high cell density or low serum concentration (Stein, 1979) They have also been shown to have higher proliferative activity and low susceptibility to the effects of external environmental factors

The cells express glial fibrillary acidic protein (GFAP), the specific marker of astrocytes, and also share other phenotypes seen in primary astrocytes such as CD68- and HLA-I- Due to its biological resemblance to primary astrocytes, it is a widely used cell line model for the study of astrocytes (de

Joannon et al., 2000; Gasque et al., 1996)

1.1.3 Treatment Strategies for Chronic Pain and their Challenges

Chronic pain management requires a multifaceted approach that addresses both the biological and psychosocial factors that contribute to and maintain the chronic pain Pharmacological treatment remains the most common therapy for many chronic pain conditions, despite the emergence of other approaches such as physical therapy and cognitive behavioural therapy (Ho and Siau, 2009)

Currently, there are no drugs that are able to control prolonged pain without harmful side effects Drugs that are most commonly used for the treatment of chronic pain are non-steroidal anti-inflammatory drugs (NSAIDs) including cyclooxygenase type-2 (COX-2) inhibitors, opioids, and antidepressants that include tricyclic antidepressants (TCAs) and selective serotonin and

norepinephrine reuptake inhibitors (SSNRIs) Table 1-1 details the adverse

effects of these drugs

Due to rapid advancements in disease prevention, diagnosis and interventions, the life span of human beings and the survival rates of patients with pain-inducing disorders have increased This brings about even higher demands for mechanism-based treatments for chronic pain that can improve the quality of life Chronic pain treatment should not be targeted solely at the general symptom, which is pain relief, but rather at the fundamental mechanisms that are responsible for the pain

Table 1-1 Common pharmacological treatments for chronic pain

Class of drugs Side effects Precautions

Myocardial infarction, stroke

Nausea/vomiting, drowsiness, urinary retention

Cardiac disease, glaucoma, suicide risk, seizures

Hepatic dysfunction, renal insufficiency, withdrawal syndrome with abrupt discontinuation

1.1.4 Conditions related to Chronic Pain

This study focuses on two conditions that are related to chronic pain: herpetic neuralgia, an example of neuropathic pain, and osteoarthritis, an example

post-of nociceptive pain Both post-of these illnesses give rise to chronic, intractable pain that can last for months or even years, and the treatment processes are often difficult and slow

1.1.4.1 Post-Herpetic Neuralgia (PHN)

Herpes zoster is a disease due to infection by the varicella zoster virus Patients with herpes zoster develop rashes confined to a specific dermatomic region of the skin and suffer from pain that typically resolves after four weeks However, approximately 20 percent of patients experience pain even after rash healing, and this chronic pain syndrome, which may last for years, is known as

post-herpetic neuralgia (PHN) (Wu et al., 2000) PHN is the most common

complication of herpes zoster in patients who are immunocompetent or have a

serious infection (Whitley et al., 1999) The most important risk factor for PHN is

age; it occurs 15 times more often in patients who are more than 50 years old

whereby nociceptors get activated by stimuli that would normally feel innocuous (peripheral sensitization) Prolonged nociceptor activation leads to enhanced response and abnormal state of excitability of dorsal horn neurons, enlarging the

neuron’s receptive field to external stimuli (central sensitization) (Opstelten et al.,

2004)

Patients with PHN usually report constant burning or stabbing pain, and may also suffer pain in response to non-noxious stimuli At present, there are many proposed therapies targeted at PHN pain control, including antiviral agents, TCAs and opioid agonists, but all of them have their own side effects and very

few treatments achieve high success rate (Dworkin et al., 2007) PHN is often

accompanied by physical, social and occupational disabilities, hence impacting the quality of life of patients suffering from it

1.1.4.2 Osteoarthritis

The most common form of arthritis, osteoarthritis is one of the main causes of chronic pain and long-term disability in populations over the age of 65 worldwide Osteoarthritis, sometimes known as degenerative arthritis, is a clinical syndrome involving the degradation of joints and eventual loss of cartilage and subchondral bone, leading to joint pain and other symptoms such as stiffness, tenderness and increase in intra-articular fluid (Conaghan, 2008) Knees, hips and hand joints are the most commonly affected sites, although any synovial joint can

develop osteoarthritis Osteoarthritis is twice as prevalent in women as compared

to men, and its incidence increases with age (Felson, 1997)

The pathophysiology of osteoarthritis is complex, although loss of articular cartilage plays a main role in the disease Physiologically, cell surface integrins modulate cell/extra-cellular matrix (ECM) interactions, and this is crucial for normal growth and homeostasis of articular cartilage During osteoarthritis, uncharacteristic integrin expression leads to abnormal cell/ECM signaling, altering the growth and differentiation of chondrocytes, which are cells found in the cartilage This is coupled with increased expression of pro-inflammatory cytokines such as interleukin-1 (IL-1) and enzymes involved in ECM breakdown such as metalloproteinases, which eventually lead to the degradation and loss of cartilage (Iannone and Lapadula, 2003; Goldring and Goldring, 2006)

The most common surgery that patients with advanced osteoarthritis of the knees undergo is total knee arthroplasty, or knee replacement, when other treatment interventions such as corticosteroids or NSAIDs have been exhausted The surgery involves removing the damaged cartilage, and replacing worn surfaces with artificial metal and plastic bearings (Leopold, 2009) However, not all patients are relieved of symptoms; 8 to 23 percent of patients complain of prolonged residual pain or stiffness in the knee even after knee replacement

surgery (Lingard et al., 2006)

1.2 Neurotransmitters/Neuromodulators in Chronic Pain

Many neurotransmitters and neuromodulators are involved in the transmission of chronic pain They are widely distributed throughout the CNS, co-existing in different regions, and are involved in complex interactions that eventually lead to the perception of chronic pain This section will focus on small molecule neurotransmitters, neuropeptides and pro-inflammatory cytokines, which have been well established to play important roles in chronic pain

1.2.1 Small Molecule Neurotransmitters

Many small molecule transmitters, which are chemicals that are released

by neurons for communication within the nervous system, are involved in chronic pain transmission They are synthesized locally within the axon terminal Besides the common small molecule neurotransmitters such as serotonin, dopamine and acetylcholine, there are many others that play an important role in modulating chronic pain Amino acids and prostaglandins are two such examples

1.2.1.1 Amino Acids

Glutamate is the main excitatory amino acid in the mammalian CNS Its

interaction with glutamate receptors mediates excitatory synaptic transmission in the CNS, hence playing an important role in both normal and pathophysiological nociception Chronic pain can be maintained by a state of sensitization in the CNS, which is partly mediated by glutamate binding to NMDA receptors Prolonged

activation of nociceptors, for example due to tissue damage or nerve injury, leads

to a continuous release of glutamate, causing a long-lasting membrane depolarization that releases the voltage-dependent magnesium block of NMDA receptors, therefore activating them (Chizh, 2002)

D-serine has also been identified as a neuromodulator and co-agonist for

NMDA receptors, and is involved in nociception, binding to the glycine site on the NR1 subunit of the NMDA receptor The presence of glutamate increases the

glycine site affinity Intrathecal (i.t.) administration of D-serine has been

demonstrated to be pro-nociceptive in the tail-flick assay, which is blocked by administration of a selective NMDA receptor antagonist that targets the glycine

co-site (Kolhekar et al., 1994)

Conversely, γ-amino-butyric acid (GABA) is a major inhibitory

neurotransmitter in the brain Neuropathic pain following peripheral nerve injury, which can be modeled using the chronic constriction model (CCI), is subject to

GABAergic control I.t injection of GABA-A receptor antagonist bicuculline,

when given within a few days of CCI, was demonstrated to cause a dependent increase in the magnitude of hyperalgesia (Yamamoto and Yaksh,

dose-1993) Furthermore, a single i.t injection of GABA has been shown to reverse the chronic neuropathic pain induced by nerve injury (Eaton et al., 1999)

1.2.1.2 Prostaglandins

Prostaglandins, a group of C20 fatty acids each containing a 5-carbon ring, are synthesized from arachidonic acid by the COX enzyme, the rate-limiting enzyme in the arachidonate cascade As mentioned in Section 1.1.3, COX inhibitors are commonly used as a pharmacological treatment strategy for chronic pain Prostaglandin E2 (PGE2) plays a major role in mediating inflammation and pain, and this was demonstrated when peripherally administered PGE2 produced hyperalgesia in mice Anti-PGE2 monoclonal antibodies inhibited the nociceptive

response of mice to phenylbenzoquinone (Mnich et al., 1995) The nociceptive

response was associated with the production of prostaglandins and was blocked

by inhibitors of prostanoid synthesis In addition, COX inhibitors as well as prostaglandin receptor blockers injected subcutaneously into the affected hind paw produced significant relief of mechanical and thermal hyperalgesia observed

in chronic neuropathic pain produced by partial sciatic nerve ligation in rats

(Syriatowicz et al., 1999) This provides further evidence that PGE2 is involved in peripheral mechanisms underlying hyperalgesia following nerve injury

1.2.2 Neuropeptides

Neuropeptides, another category of neurotransmitters, differ from molecule neurotransmitters in both size and in the way that they are synthesized Many studies involving CNS diseases and pain facilitation have focused on

small-neuropeptides, and these neuropeptides range from 3 to 36 amino acids in length and are synthesized in the cell body

1.2.2.1 Substance P

Substance P belongs to the tachykinin neuropeptide family and is a key element in chronic pain perception Substance P coexists with the excitatory amino acid glutamate in the primary afferents that respond to painful stimulation,

and also increases glutamate activity (De Felipe et al., 1998) Substance P acting

at its receptor neurokinin 1 (NK1) has been reported to evoke thermal hyperalgesia It was demonstrated that mice carrying a disruption of the preprotachykinin A gene, which encodes substance P, significantly reduced responses to moderate to intense nociceptive pain Pain behaviours caused by thermal, mechanical and chemical stimulation of somatic and visceral tissues

were all reduced in the mutant mice (Cao et al., 1998) The role that substance P

plays in nociception was also proven by the increase in response to noxious stimuli by administration of NK1 receptor agonists Hence, it has been proposed that substance P receptor NK1 antagonists could be developed as analgesic drugs (Saravana Kumar and Gandhimathi, 2012)

1.2.2.2 Nociceptin/orphanin FQ and Nocistatin

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are derived from the same precursor protein, prepronociceptin (ppN/OFQ), and they play important

roles in pain transmission and learning and memory processes in the CNS The two neuropeptides play antagonizing roles in pain transmission N/OFQ is an endogenous ligand for the orphan opioid-like receptor, and induces both hyperalgesia and allodynia when administered intrathecally in mice NST, on the other hand, blocks nociceptin-induced allodynia and hyperalgesia, and also

attenuates pain due to prostaglandins In addition, Joseph et al (2007) found

increased levels of ppN/OFQ, N/OFQ and NST in the brains of rats with partial sciatic nerve ligation as compared to naive rats Their spinal cords also showed a significant increase in ppN/OFQ level These findings demonstrate the important roles of these neuropeptides in the mechanisms that give rise to chronic neuropathic pain

1.2.3 Pro-inflammatory Cytokines

The role of pro-inflammatory cytokines, namely tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), in the initiation and facilitation of chronic pain has been a major field of interest in pain research Physiologically, these cytokines play a role in cell proliferation, differentiation, gene expression and the synthesis of matrix proteins essential for cell growth and tissue regeneration Most importantly, they are responsible for triggering the early immune response to infection by recruiting and activating immune cells at the site

of action (Wieseler-Frank, Maier and Watkins, 2005) The first evidences of the role that pro-inflammatory cytokines play in pain facilitation were derived from

studies using cytokine injections in rats Ferreira et al (1988) and Cunha et al

(1992) found out that intraplantar injection of IL-1β and TNF-α in rats caused inflammatory hyperalgesia in a prostaglandin-dependent process

In the spinal cord, each of these cytokines has been shown to be associated with the development of pain In response to peripheral nerve injury and spinal nerve injury, TNF, IL-1 and/or IL-6 mRNA expression and protein levels were

shown to be elevated in the spinal cord, enhancing pain response (Arruda et al.,

1998) The mRNA and protein expression levels of these cytokines were also elevated in the lumbar spinal cord of rats and mice in neuropathic pain models

like CCI (Lee et al., 2004) and sciatic cryoneurolysis (DeLeo, Colburn and Rickman, 1997) Milligan et al (2001) also found out that these cytokines,

released into lumbar cerebrospinal fluid (CSF) from activated glia, induced exaggerated pain states These cytokines have also been found to be elevated in the temporomandibular joint fluid of patients suffering from temporomandibular disorders as well as the circulating blood of patients with widespread chronic pain

(Gur et al., 2002; Bazzichi et al., 2007)

Each of the main pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) will be elaborated on in the following subsections

1.2.3.1 Tumour Necrosis Factor-α (TNF-α)

In the family of pro-inflammatory cytokines, TNF-α is widely considered the main prototype, due to its role in initiating a cascade of cytokine and growth factor activation TNF-α is synthesized and released by many different cell types,

including Schwann cells, in response to injury or inflammation A correlation in TNF-α expression level and the development of hyperalgesia or allodynia in chronic pain models have been demonstrated in many studies Endoneurial administration of TNF-α increases the extent of chronic pain, whereas inhibition

of TNF-α has the opposite effect (Wagner and Myers, 1996; George et al., 1999; Schafers et al., 2003)

A study by Sommer and colleagues in 2001 demonstrated that preemptive treatment with etanercept, a recombinant TNF receptor-Fc fusion protein that competitively inhibits TNF-α, was able to inhibit mechanical allodynia in neuropathic models This shows that TNF-α is particularly crucial in the initiation

of neuropathic pain Ren et al (2011) demonstrated that the upregulation of

TNF-α in adult Sprague-Dawley rats led not only to aggravated chronic neuropathic pain, but also working memory deficits following peripheral nerve injury It was speculated that TNF-α produced in the dorsal root ganglia (DRG) might diffuse into the CSF, triggering the production of more TNF-α in other brain regions including the hippocampus, thus leading to aggravated neuropathic pain

TNF-α may act by inducing the production of other cytokines and

activating the prostanoid pathways, or directly on nociceptors In vitro perfusion

of TNF-α to the DRG stimulates neuronal discharges in A- and C- fibers, which are significantly prolonged and higher after nerve injury, showing a marked sensitivity of injured afferent neurons to TNF-α Furthermore, administration of

TNF-α into rat DRGs induces allodynia in vivo, proving its direct action on nociceptors (Schafers et al., 2003)

1.2.3.2 Interleukin-1β (IL-1β)

IL-1β is a potent pro-inflammatory cytokine implicated in chronic neuropathic pain Its expression was shown to be elevated after nerve injury, and neutralizing antibodies against IL-1β receptors significantly reduced pain-related

behaviours in neuropathic mouse models (Sommer et al, 1999) Recombinant

human IL-1β injected in low amounts in the brain was found to induce hyperalgesia in rats, and this action was completely abolished by pre-treatment with either IL-1 receptor antagonist or sodium salicylate, a COX inhibitor This suggests that the IL-1β-induced hyperalgesic behaviour is receptor-mediated and

prostaglandin-dependent (Oka et al., 1993) In addition, IL-1β administered via

intracerebroventricular injection was demonstrated to enhance nociceptive neuronal responses of the dorsal horn of the trigeminal nucleus caudalis in rats, and this enhancement was inhibited by pre-treatment with IL-1 receptor antagonist, sodium salicylate, or α-melanocyte stimulating hormone, a potent anti-

cytokine peptide (Oka et al., 1994)

IL-1β acts via a complex signaling cascade that leads to the production of pronociceptive compounds from immune cells or Schwann cells, causing changes

in gene expression and neuronal excitability There is evidence that IL-1β also acts directly on nociceptive fibers or increase their responses to heat stimuli through an IL-1β receptor type I/tyrosine kinase/protein kinase C (PKC)-

dependent mechanism (Obreja et al., 2002)

1.2.3.3 Interleukin-6 (IL-6)

IL-6 is one of the major neuropoietic cytokines involved in various neuronal functions that are important in pain maintenance IL-6 is secreted by many cells, including neurons, immune cells and fibroblasts in the spinal cord and

DRG after nerve injury in the peripheral nerves (De Jongh et al., 2003) A large

amount of IL-6 is produced at the site of a surgical wound, and its systemic concentration correlates with the severity of surgery and with the extent of tissue injury (Holzheimer and Steinmetz, 2000)

I.t anti-IL-6 antibodies attenuated the hyperalgesic reaction in rat CCI

models, thus showing the involvement of IL-6 in pain facilitation (Wieseler-Frank,

Maier and Watkins, 2005) Oka et al (1995) have also shown that

intracerebroventricular injection of IL-6 stimulates thermal hyperalgesia in rats, and this IL-6-induced hyperalgesia is not dependent on the actions of IL-1 IL-6 has also been found to be significantly upregulated in serum and CSF of rats

following peripheral nerve injury (Zanjani et al., 2006) In addition, Ito et al

(1998) have reported that IL-6 receptor mRNA levels increased after sciatic nerve injury, accompanied by the increase in the expression of IL-6 signal transducing protein, gp130 Rats with persistent allodynia after this type of nerve injury also had more IL-6 expression in the DRG than rats with allodynia that had subsided

Clinically, IL-6 has been shown to be markedly upregulated in various pathologic situations associated with heightened pain and hyperalgesia For instance, elevated IL-6 levels have been detected in serum of chronic neuropathic