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 i 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 immunocytochemistry, and for her kind advice throughout my study My sincere ii 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 iii Table of Contents Acknowledgements ii Summary ix List of Tables xi List of Figures xii List of Abbreviations xiv List of Publications xvii List of Conference Papers xvii Chapter Introduction 1.1 Chronic Pain 1.1.1 Epidemiology of Chronic Pain 1.1.2 Pathophysiology of Chronic Pain 1.1.2.1 Role of Glial Cells in Chronic Pain 1.1.2.1.1 T98G Cell Line as an in vitro Astrocytic Model 1.1.3 Treatment Strategies for Chronic Pain and their Challenges 1.1.4 Conditions related to Chronic Pain 11 1.1.4.1 Post-Herpetic Neuralgia (PHN) 11 1.1.4.2 Osteoarthritis 12 1.2 Neurotransmitters/Neuromodulators in Chronic Pain 1.2.1 Small Molecule Neurotransmitters 14 14 1.2.1.1 Amino Acids 14 1.2.1.2 Prostaglandins 16 1.2.2 Neuropeptides 16 1.2.2.1 Substance P 17 iv 1.2.2.2 Nociceptin/orphanin FQ and Nocistatin 1.2.3 Pro-inflammatory Cytokines 17 18 1.2.3.1 Tumour Necrosis Factor-α (TNF-α) 19 1.2.3.2 Interleukin-1β (IL-1β) 21 1.2.3.3 Interleukin-6 (IL-6) 22 1.3 Importance of Cerebrospinal Fluid (CSF) in Chronic Pain Research 1.3.1 Biological Markers in the CSF 1.4 Aim and Scope of Study Chapter 23 24 26 Release of Pro-inflammatory Cytokines in T98G Cells Upon Exposure to CSF of PHN Patients 2.1 Objectives of Chapter 29 2.2 Materials and Methods 31 2.2.1 Materials 31 2.2.2 CSF Samples 32 2.2.3 Cell Culture 33 2.2.4 Measurement of TNF-α, IL-1β and IL-6 Release 34 2.2.5 Measurement of Dexamethasone in CSF 35 2.2.6 Statistical Analyses 36 2.3 Results 37 2.3.1 TNF-α, IL-1β and IL-6 Releasing Activity in T98G Cells Upon Exposure to CSF of PHN Patients 37 2.3.2 Comparison of IL-6 Releasing Activity Between CSF of Different PHN Treatment Groups 39 2.3.3 Effect of in vitro Steroid on IL-6 Releasing Activity 2.4 Discussion 41 44 v Chapter Comparison of IL-6 Releasing Activity between CSF of Chronic Pain Patients and Pain-free Patients 3.1 Objectives of Chapter 49 3.2 Materials and Methods 50 3.2.1 Materials 50 3.2.2 CSF Samples 50 3.2.3 Culture of Primary Astrocytes 51 3.2.4 Immunocytochemistry 52 3.2.5 Measurement of IL-6 in T98G and Primary Astrocyte Cell Culture 53 3.2.6 Statistical Analyses 53 3.3 Results 54 3.3.1 Comparison between PHN Therapy Effective, Therapy Ineffective and Control Group 54 3.3.2 Comparison between Osteoarthritis and Control Group 56 3.3.3 IL-6 Release from Primary Astrocytes Upon Exposure to CSF of Chronic Pain Patients 57 3.4 Discussion Chapter 59 Purification of Protein-like Compounds from CSF of Chronic Pain Patients 4.1 Objectives of Chapter 64 4.2 Materials and Methods 65 4.2.1 Materials 65 4.2.2 CSF Samples 65 4.2.3 Preliminary Experiments on CSF 66 4.2.3.1 Separation of CSF by Molecular Weight vi 66 4.2.3.2 Treatment of CSF with Pronase 66 4.2.3.3 Measurement of IL-6 in T98G Cell Culture 67 4.2.4 Separation of CSF into Different Fractions 67 4.2.4.1 CSF Fractionation using HPLC 68 4.2.4.2 Measurement of IL-6 in T98G Cell Culture 70 4.2.5 IL-6 Release from T98G Cells When Exposed to Lignocaine and Albumin 70 4.2.5 Statistical Analyses 71 4.3 Results 72 4.3.1 CSF Fraction >10 kDa Molecular Weight Triggered IL-6 Release in T98G Cells 72 4.3.2 Pronase Attenuated IL-6 Release in T98G Cells 74 4.3.3 Separation of CSF by HPLC 75 4.3.3.1 HPLC on Pooled CSF 75 4.3.3.2 HPLC on Pooled Active Fractions 77 4.3.4 Comparison between Chromatograms Obtained from HPLC of Osteoarthritis CSF and Control CSF 4.3.5 Effects of Lignocaine and Albumin on IL-6 Release 4.4 Discussion Chapter 80 81 83 Signaling Mechanism of Chronic Pain in the T98G Cell System 5.1 Objectives of Chapter 89 5.2 Materials and Methods 90 5.2.1 Materials 90 5.2.2 CSF Samples 90 5.2.3 Cell Culture and NF-κB Inhibition 91 vii 5.2.4 Cell Fractionation and Cell Lysis 91 5.2.5 Western Blot 93 5.2.6 Statistical Analyses 93 5.3 Results 95 5.3.1 Inhibition of NF-κB led to Reduced IL-6 Release in T98G Cells 95 5.3.2 NF-κB Activation in T98G Cells Upon Exposure to CSF of Chronic Pain Patients 97 5.4 Discussion Chapter 99 Conclusion and Future Directions 6.1 Conclusion 104 6.2 Limitations of Study 106 6.3 Future Directions 107 References 109 viii 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 proinflammatory 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 ix 6.1 Conclusion Chronic pain is a major healthcare problem due to its negative impact on work performance and daily activities Effective therapeutics for chronic pain requires targeting the cellular mechanisms that individually or collectively produce pain, instead of targeting superficial symptoms Hence, studying the mechanisms underlying chronic pain and the search for potential targets for chronic pain treatment have become top priority in pain research in recent years The importance of biological markers in chronic pain research is apparent, be it in assessing the degree of chronic pain, in enabling the early diagnosis of painrelated diseases, or in predicting the outcome of pain treatment There are no reports on in vitro markers that may aid in chronic pain research at present In this pursuit in understanding the complex mechanisms of chronic pain, this study focused on establishing an in vitro biological marker that may aid in the management of chronic pain, and also on isolation and identification of substances involved in chronic pain The first objective of the study was to investigate pro-inflammatory cytokine release in a T98G cell culture system as a potential marker for chronic pain IL-6 release from T98G cells was found to be elevated upon exposure to CSF of chronic pain patients as compared to CSF of pain-free controls IL-6 release was also elevated upon exposure to CSF collected before steroid therapy as well as upon exposure to CSF of patients whose steroid therapy was ineffective IL-6 release upon exposure to CSF of patients whose therapy was effective was attenuated completely when in vitro steroid was added to the cell culture All 104 these results showed the effectiveness of IL-6 release in the T98G cell culture system upon addition of CSF as in vitro biological marker for chronic pain The second objective of the study was to fractionate and isolate painrelated substances in the CSF and to establish the signaling pathway involved in chronic pain CSF from chronic osteoarthritic pain patients was fractionated by a two-step HPLC procedure, using the previously established cell culture system to analyze the IL-6 releasing ability of each peak Three peaks that could trigger IL6 release in T98G cells were isolated and collected at the end of the procedures The HPLC chromatogram of chronic pain CSF was compared against that of painfree control CSF and the peaks that showed differences were also collected We have effectively isolated fractions of chronic osteoarthritic pain CSF that contain protein-like substances that could contribute to chronic pain In addition, in investigating the possible signaling mechanism that leads to the IL-6 release, it was found that exposure to chronic osteoarthritic pain CSF led to NF-κB activation in T98G cells and this activation acts upstream of IL-6 In summary, the present results of the study demonstrate another perspective in chronic pain research Utilizing CSF of chronic pain patients, IL-6 have been successfully established as an in vitro biological marker that can provide an objective evaluation of the extent of chronic pain, which is an advantage over subjective methods such as pain score measurements, and also to predict an individual’s response to i.t steroid therapy The isolation of fractions from chronic osteoarthritic pain CSF that can trigger IL-6 release paves a way for the identification and characterization of pain-related substances in the CSF 105 These collected fractions, after analysis by mass spectrometry, would provide highly useful and detailed information on the protein-like substances in the CSF that contribute to chronic pain Establishing that CSF of chronic pain patients trigger NF-κB activation in T98G cells upstream of IL-6 is the first step towards elucidating the mechanisms behind IL-6 release in this T98G cell system NF-κB could be yet another useful in vitro biological marker for the objective assessment of chronic pain 6.2 Limitations of Study The study involves in vitro work using T98G cells as an astrocytic model The disadvantage of in vitro studies is that it is difficult to extrapolate the results achieved from in vitro studies to the actual physiology of humans The results obtained using T98G cells could not be replicated using primary astrocytes As such, the IL-6 release from T98G cells acts uniquely as a read-out, providing an unbiased assessment of the extent of chronic pain in patients Another limitation of our study was that due to difficulties in obtaining more CSF samples, we were only able to focus on two conditions related to chronic pain: PHN and osteoarthritis In addition, due to the lack of more PHN CSF samples, isolation of pain-related protein-like substances using HPLC could only be carried out on osteoarthritis CSF samples Encompassing more chronic pain conditions in our study would widen the scope of our work Comparisons can be made between various pain conditions, thereby establishing a more complete 106 picture of the relationship between substances in the CSF and the perception of chronic pain due to different conditions 6.3 Future Directions The separation and purification of chronic pain patients’ CSF into different fractions is just the first step in the characterization of substances involved in chronic pain More in depth studies have to be performed on these purified fractions MALDI-TOF mass spectrometry would be the next step to determine the molecular weight of the peaks, and the peaks would be subsequently sequenced to identify and characterize the protein-like substances in the CSF that play a role in chronic pain Subsequently, these substances could be isolated and animal bioactivity studies carried out to determine the pain perception mechanisms of these substances Further work should also be carried out on the signaling mechanism of IL6 release Identification of NF-κB activation as upstream of IL-6 release is only the first step in unraveling the signaling mechanism of chronic pain in this T98G cell system Other substances known to be involved in IL-6 activation and chronic pain mechanisms should also be analyzed 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asparagine, tyrosine and valine were also demonstrated to be elevated in the CSF of chronic pain patients as compared to that of acute pain patients On the other hand, GABA... 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... Pain The International Association for the Study of Pain defines chronic pain as pain that persists for at least months Chronic pain is normally triggered by injury or disease, which damages the