FACULTY OF MEDICINE
Infra-slow oscillations in chronic orofacial neuropathic pain and
the effects of palmitoylethanolamide
Zeynab Alshelh
A thesis submitted to the University of Sydney in fulfilment of the
requirement for the degree of Doctor of Philosophy
Laboratory of Neural Imaging
Department of Anatomy and Histology
June 2018 “There is something fascinating about science. One gets such wholesome
returns of conjecture out of such a trifling investment of fact.”
Mark Twain (1883) Table of Contents
Originality statement...... vi
Abstract ...... vii
Acknowledgements ...... x
Publications and awards ...... xi
Co-authors contributions ...... xiv
Chapter one: Introduction ...... 1
1. Chronic pain ...... 2
1.1. Chronic neuropathic pain ...... 4
1.2. Trigeminal neuropathic pain ...... 4
1.2.1. Background and presentation ...... 4
1.2.2. Incidence and prognosis ...... 5
1.2.3. Treatments ...... 6
1.3. Models of chronic neuropathic pain ...... 6
1.3.1. Central sensitization ...... 6
1.3.2. Pain matrix ...... 7
1.3.3. Thalamocortical dysrhythmia ...... 8
1.4. Nervous system ...... 9
1.4.1. Pain pathways ...... 9
1.5. Central nervous system ...... 10
1.5.1. Dorsal horn ...... 10
iii 1.5.1. Dorsal horn ...... 10
1.5.2. Thalamus ...... 14
1.5.3. Primary somatosensory cortex ...... 20
1.6. Astrocytes in neuropathic pain ...... 22
1.6.1. Anatomy and function of astrocytes ...... 22
1.6.2. Role of astrocytes in neuropathic pain ...... 24
1.7. Neuroimaging: Magnetic resonance imaging ...... 27
1.7.1. Functional magnetic resonance imaging ...... 27
1.7.2. T2 relaxometry ...... 28
1.8. Overarching aims of the thesis ...... 29
1.8.1. Specific aims of the thesis ...... 29
References ...... 30
Chapter two: Chronic neuropathic pain: It’s about the Rhythm ...... 39
2.1. Abstract ...... 40
2.2. Introduction ...... 40
2.3. Materials and Methods ...... 41
2.4. Results ...... 44
2.5. Discussion ...... 46
2.6. References ...... 48
Chapter three: Altered regional brain T2 relaxation times in individuals with chronic orofacial neuropathic pain ...... 51
3.1. Abstract ...... 52
iv 3.2. Introduction ...... 52
3.3. Methods ...... 53
3.4. Results ...... 53
3.5. Discussion ...... 56
3.6. Acknowledgements ...... 57
3.7. References ...... 58
Chapter four: Effects of the glial modulator palmitoylethanolamide on chronic pain intensity and
brain function ...... 59
4.1. Abstract ...... 61
4.2. Introduction ...... 62
4.3. Results ...... 63
4.4. Discussion ...... 68
4.5. Online Methods ...... 71
4.7. References ...... 74
Chapter five: Conclusions ...... 80
5.1. Main findings ...... 81
5.2. Implications ...... 83
5.3. Recommendations for future research ...... 92
5.4. References ...... 94
Appendix ...... 116
v Originality statement
The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution.
……………………………………………………..
Zeynab Alshelh
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
vi Abstract
For centuries, chronic pain was denied as being real by physicians, mainly because there was no
evidence of tissue damage. More recently, the lack of understanding of the neural mechanisms
underlying chronic pain, particular that arising from nervous system damage, has hindered treatment
development which has led to the over-prescription of opioids even though they are generally
ineffective and have serious side-effects including addiction. Chronic pain of neuropathic origin is exceptionally difficult to treat, with this inability largely due to our lack of understanding of the underlying mechanisms.
Whilst the brain circuitry responsible for the perception of acute painful stimuli have been mapped in both animal studies and studies using brain imaging in awake humans, the circuitry responsible for the initiation and maintenance of chronic pain remain unknown. Over the past few decades, many human brain imaging investigations have reported regional changes in brain anatomy and function in individuals with chronic pain. Surprisingly, these studies have shown that chronic neuropathic pain is not simply on-going acute pain, or at least chronic pain is not associated with constant activation of acute pain circuits. They have shown that neuropathic pain is associated with altered brain rhythms and in particular thalamocortical dysrhythmia. In addition, animal studies have shown that neuropathic pain is associated with altered non-neural function including microglial and astrocyte activation at the level of the primary afferent synapse. These results have led to theories that non- neuronal cells may be crucial for the initiation and maintenance of chronic pain, particularly chronic neuropathic pain.
It has been a long held view that astrocytes mainly play the role of neural support in the central nervous system, however, these cells are also capable of controlling neural function. In fact, astrocytes have access to every neural synapse and animal models of chronic neuropathic pain have shown that
vii targeting astrocytes can control pain intensity. As such, the focus of this thesis is to identify the role of astrocytes in modulating neural function in chronic neuropathic pain and to determine whether reducing astrocyte activity can reduce pain intensity.
The first investigation (Chapter two) describes an experimental procedure whereby on-going patterns of neural activity were assessed in patients with orofacial neuropathic pain using resting state functional magnetic resonance imaging. Chronic orofacial neuropathic pain was associated with an increase in on-going infra-slow oscillatory activity (ISO, <0.1Hz) in regions of the ascending pain pathway, i.e. primary afferent synapse, the somatosensory thalamus and the somatosensory cortex.
Interestingly, astrocytes display calcium wave oscillations at similar frequencies, and these calcium oscillations can lead to oscillatory release of neurotransmitters. Therefore, in this research series, it is speculated that the increase in ISOs in patients with chronic neuropathic pain are due to an increase in astrocyte activity causing the oscillatory release of neurotransmitters which modulate neural activity.
While the results from Chapter two strongly suggest that the changes in neural activity in patients with chronic neuropathic pain are a result of increases in astrocyte activity, chapter three of this thesis, attempts to measure an anatomical marker of astrocyte activation. T2 relaxation time is dependent upon the tissue make-up and an increase in astrocyte activity will have an effect on T2 relaxation time.
Indeed, post-mortem investigations have shown that an increase in astrocyte activity causes a decrease in T2 relaxation time. Using this indirect, non-invasive measure of astrocyte activation, there are decreases in T2 relaxation times, indicative of astrocyte activation in specific regions of the pain pathway in patients with chronic neuropathic pain, that is, in the primary afferent synapse and the primary somatosensory cortex.
viii Finally, if chronic neuropathic pain is caused, at least in part, by increases in astrocyte activity, then reducing astrocyte activation should reduce the intensity of pain. Chapter four describes an experimental procedure whereby patients with orofacial neuropathic pain were administered an astrocyte modulator, palmitoylethanolamide (PEA) and neural activity was compared before and after treatment. The results showed that PEA decreased pain intensity in over half of these patients (73%), and decreased ISO in regions of the primary afferent synapse and the somatosensory thalamus, that is, the same areas that showed increases in ISOs in these patients when compared to healthy controls in Chapter two. Chapter four indicates that decreases in ISOs due to PEA were correlated to decreases in pain intensity at the regions of primary afferent synapse. Importantly, a decrease in the circuitry strength between regions of the primary afferent synapse and the somatosensory thalamus is essential for the decrease in pain intensity.
Overall, this thesis provides an increased understanding of the role of astrocytes in chronic neuropathic pain. More importantly, it describes the effect of an astrocyte modulator in neural activity and pain intensity in patients with chronic neuropathic pain. Unlike other drugs, PEA has no side- effects and can be used to treat chronic neuropathic pain in the future.
ix Acknowledgements
I would like to acknowledge the many people along my journey in finalising this thesis. My supervisor, Professor Luke Henderson is my guiding principle. The support and encouragement you afforded me during the years of study taught me so much. Your direct and honest approach, though at times blunt, assisted in reaching my goals and become a better version of myself. I will forever cherish the wisdom and lessons learnt.
To Associate Professor, Kevin Keay, thank you for looking out for me throughout all the years. Your support has been incredible. I also thank the Neural Imaging Laboratory for making my time enjoyable even during demanding days. Special acknowledgement goes to Emily for her loyalty and patience, and for taking on my tasks in the lab. Thank you Emily for being a good friend and support and for appreciating all the Arabic food I brought in. Though Danny joined the team later, thank you for continuing on my project and for trying to solve the mystery that is me without any friends.
Lastly, I could not have completed my thesis without the eagle eye editing skills of Flavia. You taught me valuable lessons in writing and those skills are forever with me. You are an amazing role model.
To my best friend, Ifdal, thank you for your patience, support and simply putting up with me during the ups and downs over the years. Special thanks for caring for my birds during my hectic schedules.
And for forgiving me when I couldn’t spend time during the preparations of your wedding. I promise to make it up to you.
The enduring support in my journey is my wonderful and dedicated parents. My father is my biggest fan and is the rock in my life. Thank you for teaching me leadership skills, life lessons and to stand up for what I believe in. Without you, this thesis would not be possible. Finally, a huge thank you is reserved for my mother. She is the gentle and calming soul with patience and fortitude to deal with my many requests. Thank you for being my friend and being steadfast and unwavering during some of the most challenging times. You are a role model and guide. This thesis is dedicated to both my parents and I hope it brings pride and joy to you both.
x Publications and awards
Publications contained in this thesis
Z. Alshelh, F. Di Pietro, A. M. Youssef, J. M. Reeves, P. M. Macey, E.R. Vickers, C.C. Peck G.M. Murray
and L.A. Henderson. Chronic Neuropathic Pain: It’s about the Rhythm. The Journal of Neuroscience,
January 20, 2016, 36(3):1008 –1018. PMID: 26791228
Z. Alshelh, K.K. Marciszewski, R. Akhter, F. Di Pietro, E.P. Mills, E.R. Vickers, C.C. Peck, G.M. Murray
and L.A. Henderson. Disruption of default mode network dynamics in acute and chronic pain states.
NeuroImage: Clinical, 17 (2018) 222-231. PMCID: PMC5683191
Z. Alshelh, F. Di Pietro, E.P. Mills, E.R. Vickers, C.C. Peck, G.M. Murray and L.A. Henderson. Altered regional brain T2 relaxation times in individuals with chronic orofacial neuropathic pain. NeuroImage:
Clinical, 2018. Accepted “PMC Journal-In Process”
Other publications during candidature
E.P. Mills, F. Di Pietro, Z. Alshelh, C.C. Peck, G.M. Murray, E.R. Vickers and L.A. Henderson. Brainstem
pain controls circuitry connectivity in chronic neuropathic pain. The Journal of Neuroscience,
November 24, 2017, 1647-17. PMID: 29175957
F. Di Pietro, P. M. Macey, C. Rae, Z. Alshelh; V. Macefield, E.R. Vickers; L.A. Henderson. The relationship between thalamic GABA content and resting cortical rhythm in neuropathic. Human Brain Mapping,
May 2018, 39(5): 1945-1956. PMID: 29341331.
xi Conference proceedings during candidature
Australian Pain Society 35th Annual Scientific Meeting Mar. 2015
Zeynab Alshelh, Flavia Di Pietro, Russell Vickers, Chris C Peck, Greg M Murray and Luke A
Henderson. Altered Neural Oscillations in Neuropathic Pain. Poster selected from abstract. Brisbane
QLD, Australia.
Bosch Young Investigators Symposium Dec. 2015
Zeynab Alshelh, Flavia Di Pietro, Russell Vickers, Chris C Peck, Greg M Murray and Luke A
Henderson. Chronic Neuropathic Pain: It’s all about the rhythm. Poster selected from abstract.
Sydney, NSW, Australia.
Australian Pain Society 36th Annual Scientific Meeting Mar. 2016
Zeynab Alshelh, P. M. Macey, Russell Vickers, Chris C Peck, Greg M Murray and Luke A Henderson.
Reduced T2 relaxation times indicating astrocyte and microglia activation in chronic neuropathic pain. Oral Presentation selected from abstract. Perth, WA, Australia.
International Association for the Study of Pain, 16th World Congress on Pain Sep. 2016
Zeynab Alshelh, P. M. Macey, Russell Vickers, Chris C Peck, Greg M Murray and Luke A Henderson.
Reduced T2 relaxation times indicating glial activation in chronic neuropathic pain. Poster selected from abstract. Yokohama, Japan.
Australian Pain Society 37th Annual Scientific Meeting Mar. 2017
Zeynab Alshelh, F. Di Pietro, E.P. Mills, P.M. Macey, E.R. Vickers, R. Akhter, C.C. Peck, G.M. Murray
xii and L.A. Henderson. Altered infra-slow oscillations and Pain. Oral presentation selected from abstract. Adelaide, SA, Australia.
6th International Congress on Neuropathic pain Jun. 2017
Zeynab Alshelh, K.K. Marciszewski, R. Akhter F. Di Pietro, E.P. Mills, E.R. Vickers, , C.C. Peck, G.M.
Murray and L.A. Henderson. Disruption of default mode network dynamic in acute and chronic pain states. Poster selected from abstract. Gothenburg, Sweden.
Australian Pain Society 38th Annual Scientific Meeting Apr. 2018
Zeynab Alshelh, P.M. Macey, E.R. Vickers, C.C. Peck, G.M. Murray and L.A. Henderson. Decreased brainstem T2 relaxation times in chronic orofacial neuropathic pain indicative or glial activation. Oral
Presentation selected from abstract. Sydney, NSW, Australia.
Awards
Best Poster Prize: Australian Pain Society (800 delegates), assessed based on content and presentation. Awarded 2015.
Postgraduate Research Support Scheme: competitive funding awarded to support attendance of the
International Association for the Study of Pain Conference in Yokohama, Japan (AUD$1037.52),
Sydney Medical School, University of Sydney. Awarded 2016.
xiii IASP Financial Aid Award: competitive grant awarded to cover travel costs for attending the
International Association for the Study of Pain Conference in Yokohama, Japan (USD$750). Awarded
2016.
Postgraduate research support Scheme: Funding to support attendance of the Neuropathic pain
Congress in Sweden (AUD$1221) Sydney Medical School, University of Sydney. Awarded 2017.
Co-authors contributions
This thesis contains selected peer-reviewed and/or submitted manuscripts. Below are descriptions of co-authors contributions to these manuscripts.
Z. Alshelh, F. Di Pietro, A. M. Youssef, J. M. Reeves, P. M. Macey, E.R. Vickers, C.C. Peck G.M. Murray
and L.A. Henderson. Chronic Neuropathic Pain: It’s about the Rhythm. The Journal of Neuroscience,
January 20, 2016, 36(3):1008 –1018.
Flavia Di Pietro: designed and performed research, manuscript editing.
Andrew Youssef and Jenna Reeves: performed research, analysed data.
Paul Macey: designed research, analysed data.
Russell Vickers: designed research, manuscript editing.
Chris Peck and Greg Murray: designed and performed research.
Luke Henderson: designed and performed research, analysed data, manuscript editing.
xiv Z. Alshelh, F. Di Pietro, E.P. Mills, E.R. Vickers, C.C. Peck, G.M. Murray and L.A. Henderson. Altered regional brain T2 relaxation times in individuals with chronic orofacial neuropathic pain. NeuroImage:
Clinical, 2018. Volume 19: 167-173
Emily Mills: assistance with data processing, intellectual input, manuscript editing
Russell Vickers, Chris Peck, Greg Murray, Flavia Di Pietro: intellectual input, manuscript editing.
Luke Henderson: assistance with imaging methods, intellectual input, manuscript editing.
Z. Alshelh, E.P. Mills, D. Kosanovic, F. Di Pietro, P.M. Macey, E.R. Vickers, L.A. Henderson. Effects of
Glial modulator palmitoylethanolamide on chronic pain intensity and brain function. Under review at
The Lancet Neurology.
Emily Mills: assistance with data processing, intellectual input, manuscript editing
Danny Kosanovic: assistance with data processing, manuscript editing
Flavia Di Pietro: intellectual input, manuscript editing
Paul Macey: assistance with imaging methods, intellectual input
Luke Henderson: assistance with imaging methods, intellectual input, manuscript editing.
xv
Co-authors signatures
The co-authors listed below certify that they have contributed to publications and/or submitted manuscripts as outlined previously and they agree to the use of those publications/manuscripts submitted in this thesis.
…………………………………………………………
Flavia Di Pietro
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
Emily P. Mills
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
Luke A. Henderson
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
xvi
…………………………………………………………
Andrew M. Youssef
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
Jenna M. Reeves
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
Danny Kosanovic
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
E. Russell Vickers
xvii
Department of Anatomy and Histology, Faculty of Medicine, The University of Sydney, Australia
…………………………………………………………
Greg M. Murray
Faculty of Dentistry, University of Sydney, Australia
…………………………………………………………
Chris C. Peck
Faculty of Dentistry, University of Sydney, Australia
…………………………………………………………
Paul E. Macey
School of Nursing and Brain Research Institute, David Geffen School of Medicine; University of
California at Los Angeles, Los Angeles, California, USA
xviii
CHAPTER ONE
INTRODUCTION
1 Pain is a biologically important process in life as it serves to protect from potentially harmful surroundings and alerts to tissue damage. It is the most common reason individuals seek medical help, and the likelihood of experiencing pain increases with age for both males and females (Australian
Bureau of statistics, 2011). Pain usually resolves with tissue healing or with the removal of harmful surroundings, however, there are cases where the pain continues for longer than 3 months with no biological advantage, and this is called chronic pain (Merskey et al., 1994).
1. Chronic pain
Chronic pain affects 67% of the Australian population aged 15 years and over, and costs 34.3 billion dollars annually, which includes $11 billion in productivity and $7 billion in direct health care costs.
(ABS, 2011). While acute pain resolves within three months of initial tissue damage whether tissue damage is has resolved or not, and serves to protect by eliciting a change in behaviour to avoid harm or is a by-product of the healing process, chronic pain lasts longer than three months, can be accompanied by tissue damage (such as cancer), appears to serve no biological function (Sessle, 1987), and is recognised as a disease state rather than a symptom of injury (IASP 1986). Although understanding the pathways and mechanisms involved in acute pain is interesting, the central representation of chronic pain appears to be very different (Farrell et al., 2005); Gustin et al., 2014;
Youssef et al., 2014). For example, brain imaging studies have revealed that acute experimental pain evokes increases in blood flow to pain-specific brain regions (Farrell et al., 2005), chronic pain is actually associated with very little change in these areas and indeed in the thalamus it is associated with blood flow decreases (Gustin et al., 2014b; Youssef et al., 2014). Given these differences, chronic- specific pain studies are needed to better understand the underlying mechanisms responsible for chronic pain which could lead to the development of better treatment and prevention regimens.
2 Why the neural mechanism underlying acute and chronic pain are different is yet to be determined, it is interesting that psychological distress has been shown to be a secondary factor or predictor of chronic pain development (Andersson, 1999). Indeed, there are a number of psychological factors that can contribute to the development of chronic pain, such as catastrophizing (Turner et al., 2002). Pain catastrophizing is an individual’s view that their pain state as worse than it actually is and greater catastrophizing levels are associated with greater intensity of on-going pain (Turner et al., 2002).
Similarly, pain anxiety, i.e., the feeling of nervousness about pain and whether it will end, can affect the ability of an individual to cope with their chronic pain and this can alter the pain intensity and overall experience (McCracken and Gross, 1993). Depression, the feeling of severe dejection, has also been associated with the presence of chronic pain and it has been suggested that depression can be a predictor of chronic pain intensity and development, although there is debate whether depression predisposes an individual to chronic pain or that chronic pain predisposes and individual to developing depression (Fishbain et al., 1997). While addressing the psychological aspect of chronic pain can be important in regulating the pain intensity and its effect on social behaviour (Morley et al., 1999), eliminating these factors rarely eliminates or prevents the development of chronic pain itself.
One of the major unresolved issues surrounding chronic pain is exactly what causes acute pain to become chronic in some individuals and not in others. Whilst the precise mechanisms underlying the development and/or maintenance of chronic pain remains largely unknown, some investigations point to a peripheral starting point, whereby changes in the chemical composition in the periphery lead to chronic pain (Voscopoulos and Lema, 2010). Whilst this is likely the case in some individuals, peripheral changes are not necessary for the development of chronic pain as evidenced by the development of chronic pain following strokes encompassing the somatosensory pathways in the brainstem and thalamus (Klit et al., 2009a). Whilst changes in the periphery and central nervous system are likely involved in the development of chronic pain, our lack of understanding is limiting our
3 ability to develop effective chronic pain treatments. Current pharmacological treatments used to effectively treat acute pain are ineffective in treating patients suffering with chronic pain (Dworkin et al., 2003; Finnerup et al., 2005). This attributes to the long-term suffering and physical and economic disadvantages and hence, more research into the mechanisms underlying chronic pain is necessary.
1.1 Chronic neuropathic pain
Whilst there are various forms of chronic pain, the focus of this thesis is to explore the mechanisms underlying chronic neuropathic pain. Neuropathic pain arises due to damage or presumed damage to the nervous system such as that resulting from mechanical trauma, metabolic disease, neurotoxic chemicals, infection or tumour invasion (Campbell and Meyer, 2006). In the United Kingdom, 48% of the population suffer from a chronic pain condition and 8% of those are neuropathic in nature
(Torrance et al., 2006). A review of surveys conducted around the world found that 1 in 14 adults has neuropathic pain and that chronic pain is more common in women and the elderly (Andrew et al.,
2014). Considering that chronic neuropathic pain impedes daily activities and reduces the quality of life, there is a calling for a better understanding and treatment of the condition (Andrew et al., 2014;
Barrett et al., 2007; Tölle et al.).
1.2 Trigeminal neuropathic pain
1.2.1 Background and presentation
Whilst chronic neuropathic pain can occur as a consequence of a brain stroke, diabetic neuropathy, or nerve impingement, to name a few, the focus of this thesis is on neuropathic pain localized to the orofacial region. The reason for this is that orofacial afferents terminate in the caudal brainstem, an area whose structure and function can be explored using modern human brain imaging techniques.
Thus a considerable advantage of using this population of neuropathic pain subjects is that it is
4 possible to explore changes along the entire extent of the ascending pain pathway from the primary afferent synapse to the cerebral cortex.
More specifically, this thesis explores individuals with post-traumatic trigeminal neuropathy which is characterized by mostly constant, unilateral face pain within one or more divisions of the trigeminal nerve (Burchiel, 1993). It can be caused by physical insult from operative procedures such as root canal treatment, traumatic accidents or disease. Patients usually present with sharp or dull continuous pain, often fluctuating throughout the day with shooting pain superimposed (Burchiel, 1993; Gustin et al.,
2011). The pain is often associated with allodynia (pain to a normally non-noxious stimulus), hyperalgesia (an increased response to a noxious stimulus) and sensory loss (Nurmikko and Eldridge,
2001).
1.2.2 Incidence and prognosis
Unlike trigeminal neuralgia, which often results from nerve compression and is often episodic in nature (Love and Coakham, 2001), post-traumatic trigeminal neuropathy cannot be diagnosed using magnetic resonance imaging or other brain imaging techniques. Trigeminal neuropathy has been reported to develop in up to approximately 12% of patients that undergo standard root canal treatment (Polycarpou et al., 2005). Furthermore, since it is usually the result of nerve damage from such endodontic procedures, dentists recommend a clean-up of the wound, antibiotics for infections and possibly performing the procedure again (Yoldas et al.). Essentially, when this does not alleviate the pain and the pain lasts for over 3 months, it is diagnosed as chronic trigeminal neuropathic pain
(Burchiel, 1993; Merskey, 1986).
5 1.2.3 Treatments
Similar to general chronic pain conditions, much of the treatment for trigeminal neuropathic pain has been ineffective due to our limited understanding of the mechanisms underlying the condition. The most common treatments prescribed to neuropathic pain patients, including trigeminal neuropathy, are, but not limited to, tricyclic antidepressants, carbamazepine, gabapentin and tramadol (Sindrup and Jensen, 1999). These treatments, may reduce the intensity of pain in some patients but they usually prove ineffective and have grave short and long term side effects (Bennett and Simpson, 2004;
Killian and Fromm, 1968).
1.3 Models of chronic neuropathic pain
1.3.1 Central sensitization
Over the past few decades, a number of animal models have been developed so that the mechanism response for neuropathic pain can be investigated in experimental animals. Indeed, investigations exploring central sensitization began from the observation that allodynic responses could not be explained by changes in peripheral nociceptor function in animal models of pain. As early as the 1960s,
Iggo (Brown and Iggo, 1967; Iggo, 1960) and Perl (Bessou and Perl, 1969; Burgess and Perl, 1967; Perl,
1968) reported that sensory neurons which respond only to noxious stimuli (Woolf and Ma, 2007) become sensitized after exposure to inflammatory mediators (injury), i.e., their threshold to pain and heat stimuli was reduced (Bishop et al., 2010; Campbell et al., 1988; Kocher et al., 1987). They suggested that whilst peripheral sensitization accounted for pain hypersensitivity following injury, it could not account for dynamic allodynia (pain due to tangential movement across skin), temporal summation of pain or secondary allodynia. This led to studies to determine whether neural receptor sensitization can occur in the central nervous system as well. In 1965, Mendall and Wall found that a repeated low frequency stimulation of a nerve led to a progressive increase in action potentials in the
6 dorsal horn (Mendell and Wall, 1965), and this effect was found to last for multiple hours (Cook et al.,
1986). This effect was termed central sensitization.
It is now well-established that in neuropathic pain conditions, central sensitization occurs. For example, patients with carpal tunnel syndrome in one wrist showed hypersensitivity to cold and heat in the non-affected wrist (De La Llave-Rincón et al., 2009; Fernandez-de-las-Penas et al., 2009).
Furthermore, in a group of patients with chronic neuropathic pain, the increase in pain intensity was concurrent with an increase in pain sensations from an innocuous stimulus (Koltzenburg et al., 1994).
This data suggests that central sensitization is evident in some chronic neuropathic pain conditions and may be important in the development and/or maintenance of pain following nerve injury.
1.3.2 Pain matrix
Numerous investigations have reported functional and anatomical changes in numerous brain regions in multiple chronic pain conditions. For the main part, these changes occur in areas that have been shown to be activated during acute painful stimuli in normally pain-free individuals, a network called the pain matrix. It has been hypothetised that the “flow and integration of information” in this network results in the emergence of pain (Tracey, 2005). This term was adapted from neuromatrix which was proposed by Ronald Melzack, who ironically, proposed this term due to their inability to identify a group of brain regions that were devoted to the processing of pain (Melzack, 1989).
The pain matrix poses a problem when it is viewed as a solid piece of evidence, that is, the areas that make up the pain matrix are specific to pain. Yet, the majority of the regions that make up the pain matrix are also involved in other types of processing such as emotional or motivational processing
(Tracey and Johns, 2010). Electrophysiological studies have identified nociceptive specific neurons in
7 regions of the pain matrix but also sparsely distributed throughout the brain (Kenshalo Jr and Isensee,
1983; Whitsel et al., 1969; Yamamura et al., 1996). Nonetheless, the pain matrix can be useful in imaging studies to identify regions that may be involved in pain processing, and include such areas as the primary somatosensory cortex, thalamus and regions of the cortex encompassing the insula, cingulate and prefrontal cortices (Apkarian et al., 2005; Jones et al., 2003; Lorenz and Casey, 2005;
Schweinhardt and Bushnell, 2010).
A number of investigations throughout the years indicate that painful stimuli activates the cortical regions that make up the pain matrix such as the somatosensory cortices, the insular cortex and the anterior cingulate cortex (Bushnell and Apkarian, 2006; Garcia-Larrea et al., 2003). Furthermore, there are strong correlations between the magnitude of the pain matrix response and the intensity of pain
(Coghill et al., 1999; Derbyshire et al., 1997; Iannetti et al., 2005; Tölle et al., 1999). Therefore, the pain matrix is widely used as a loose guide for the areas that are likely involved in the processing of pain, with the understanding that these regions may also be involved in other processes.
1.3.3 Thalamocortical dysrhythmia
Two regions of the brain, the thalamus and the cortex, are separately involved in processing pain and are in a constant and synchronous loop (Jones, 2001). Recent investigations have demonstrated that whole brain activity changes arise from changes of the connections between these two regions and include changes to overall brain rhythm. Indeed changes in this thalamo-cortical circuitry can alter processing in recurrent circuits resulting in thalamocortical dysrhythmia (Pinault, 2004). There is now considerable evidence that chronic neuropathic pain is characterized by altered thalamocortical rhythm which likely arises from altered function in the thalamus or its cortical targets (Sarnthein et al., 2006; Walton and Llinas, 2010). That is, it is likely that the thalamus can modulate higher cortical regions by setting patterns of activity through ongoing thalamocortical loop. It has been proposed
8 that this altered brain activity pattern may underlie the constant perception of pain, although how this occurs is unknown.
A number of recent human electrophysiological investigations are consistent with this idea that neuropathic pain is associated with altered thalamocortical processing. For example, one study compared baseline brain activity in patients with neuropathic pain and healthy controls. Within specific frequency ranges, there was an increase in power of brain activity in the insula, anterior cingulate, prefrontal and somatosensory cortices. The authors proposed that these increases in power were a result of thalamocortical dysrhythmia, that is, changes in the firing activity of one or more regions within thalamocortical loop (Stern et al., 2006). More recently, an investigation reported altered thalamocortical rhythm, from electrophysiological recordings in patients with type 1 complex regional pain syndrome. This condition was associated with increased spectral power in specific frequency ranges, which were also localized in brain regions including the somatosensory cortex
(Walton and Llinas, 2010). This evidence suggests that changes to the activity to one or more of regions of the thalamocortical circuitry may lead to thalamocortical dysrhythmia which is associated with chronic neuropathic pain.
1.4 Nervous system
1.4.1 Pain Pathways
Whilst there are reported changes in thalamo-cortical loops in individual with chronic pain, what about other regions of the “pain matrix” that also respond to acute painful stimuli? As mentioned above, the pathways that are responsible for acute pain perception have been explored extensively. A number of human and animal investigations have revealed that the sensory and discriminative components of pain originating from the body travel through the spinothalamic tract, and the sensory and
9 discriminative components of pain from the face travel through the trigeminothalamic tract (Nash et al., 2010; Simone et al., 1991). Neurons within these ascending tracts receive inputs from primary afferent neurons in the periphery and they themselves project to a number of brainstem and higher brain areas. Experimental models have established that the spinothalamic tract neurons project to the ventroposterior lateral (VPL) nuclei of the contralateral somatosensory thalamus and trigeminothalamic tract neurons project to the ventroposterior medial (VPM) nuclei of the contralateral somatosensory thalamus (Graziano and Jones, 2004).
While these ascending pathways were mapped using experimental animal models, recent advancement in technology, such as functional magnetic resonance imaging and positron emission tomography, have allowed for the mapping of these pathways in awake humans. The following section will briefly outline what is currently known from experimental animal and human imaging studies with respect to changes within the ascending pain pathway associated with the presence of chronic neuropathic pain.
1.5 Central Nervous System
1.5.1 Dorsal horn
The centre of the spinal cord is composed of gray matter (neuronal cell bodies and axon terminals) and the outer region comprises of white matter (axons). The gray matter has a dorsal section
(posterior), in which sensory information, particularly nociception, first terminates as it enters the central nervous system from the periphery (Brown, 1982) (Figure 1.5.1).
10 Figure 1.5.1: Illustration of the spinal cord showing the inner gray matter and the outer white matter.
The dorsal horn is the posterior section of the gray matter. Adapted from Todd (Todd, 2010).
Damage to the peripheral nerves can result in significant and pronounced changes in the dorsal horn
(McLachlan et al., 1993). For example, animal models of neuropathic pain which involve injury to a peripheral nerve, are associated with neural death at the dorsal horn (Scholz et al., 2005; Sugimoto et al., 1990; Whiteside and Munglani, 2001). Spared nerve injury, chronic constriction injury and spinal nerve ligation models of neuropathic pain are all associated with neural loss in the dorsal horn.
Furthermore, these neural changes are associated with the development of allodynia and hyperalgesia which were prevented when this neural loss was prevented by reducing caspase activity (Scholz et al.,
2005) or treatment with a NMDA receptor antagonist, which reduced the extent of cell death
(Whiteside and Munglani, 2001).
In addition to neural loss in the dorsal horn, animal studies have reported evidence of chemical changes associated with the development of neuropathic pain. For example, following a peripheral
11 nerve injury there is an increase in calcitonin gene-related peptide in the dorsal horn, which has an excitatory effect on postsynaptic neurons (Miki et al., 1997). Furthermore, spared peripheral nerve injury and chronic constriction injury models of neuropathic pain are associated with decreases in presynaptic GABA release in the dorsal horn and a decrease in GABA synthesizing enzymes (Ibuki et al., 1996; Meisner et al., 2010; Moore et al., 2002). In the chronic constriction injury model of neuropathic pain, significant increases in Ca2+ in the dorsal horn occur and these changes are associated with the development of thermal and mechanical hyperalgesia (Kawamata and Omote,
1996). In addition to these chemical changes, other animal neuropathic pain models have reported astrocytic changes in the dorsal horn; these changes will be discussed in detail in a later section.
1.5.1.1 Spinal trigeminal nucleus
Whilst nociceptor afferents innervating the body enter the spinal cord and synapse in the dorsal horn, those innervating the head and oral cavity enter the central nervous system via the trigeminal nerve.
The trigeminal nerve enters the brainstem at the level of the pons and fibres carrying nociceptive information descend in the dorsolateral medulla to synapse onto the spinal trigeminal nucleus on the ipsilateral side (Paxinos et al., 2012). Second order neurons then decussate in the medulla and travel to the thalamus, terminating in the ventral posterior nucleus. The trigeminothalamic projection involved in pain processing, from the subnucleus caudalis of the spinal trigeminal nucleus is well understood. Specifically, neurons in the marginal layer (lamina I) of the subnucelus caudalis project to the ventral posteromedial thalamus and the posterior thalamic nucleus (Shigenaga et al., 1979).
Although the sub nuclei interpolaris and oralis are also involved in processing orofacial pain, their ascending thalamic projects remain less clear (Ralston, 2005; Sessle, 2000) (Figure 1.5.1.1)
12 .
Figure 1.5.1.1: Trigeminothalamic pathway for nociception arising from the peripheral trigeminal nerve in the face. Noxious afferents synapse at the spinal nucleus of the trigeminal complex, where fibres ascend the brain stem to synapse at the ventroposterior medial nucleus of the thalamus, in which they continue to the primary somatosensory cortex. Adapted from Purves (Purves, 2004) .
Investigations have revealed that the spinal trigeminal nucleus is divided into three sub nuclei; the caudalis, interpolaris and the oralis (Olszewski, 1950; Sessle, 2000). The sub nuclei caudalis is the most caudal of the three and lies within the closed medulla. It is generally considered to be the primary relay for orofacial nociceptive information originating in cutaneous and mucosal tissues (Sessle, 2000).
Directly rostral to the sub nuclei caudalis lies the sub nuclei interpolaris which also receives noxious information originating from the orofacial region and in particular from jaw muscles. Finally, the spinal
13 trigeminal sub nucleus oralis lies in the rostral medulla (Dessem et al., 2007). While it is believed that this sub nuclei is involved in the processing of the noxious stimuli from the oral cavity, it has been shown that its lesion does not resolve oral cavity pain perception (Duale et al., 1996; Luccarini et al.,
1998). However, it is suggested that noxious stimuli to the face are processed by both the spinal trigeminal sub nucleus oralis and the sub nucleus interpolaris (Nash et al., 2009).
As the spinal trigeminal nucleus plays a key role in the processing of trigeminal neuropathic pain, this series of investigations will analyse changes in its neural activity in patients compared to controls, its anatomical changes compared to controls and whether these changes can be reversed with therapy. The spinal trigeminal nucleus shows increases in infra-slow oscillatory activity compared to healthy controls (Chapter 2), changes in anatomy (Chapter 3) and a reduction in infra- slow oscillatory activity after PEA (Chapter 4).
1.5.2 Thalamus
Second order neurons originating in SpV project to the ventral posterior (VP) region of the thalamus amongst other thalamic and brainstem targets. The thalamus plays an important role in the processing of chronic neuropathic pain, even in the absence of peripheral nerve injury. This is evidenced by the results from brain stroke studies which have revealed that between 11-55% of patients develop neuropathic pain following a stroke that encompasses the thalamus (Klit et al., 2009b). Since the first description of central post-stroke pain (CPSP) in 1906, research suggests that the development of pain is contingent on the stroke damaging the ascending somatosensory pathway from the medulla to the thalamus or cortex (Leijon et al., 1989). Whilst CPSP can occur after a cerebrovascular lesion at any level of the ascending somatosensory pathway, lesions in the thalamus result in a relatively high occurrence of PSP (17%) (Klit et al., 2009b). Recently, a clinical report described a series of patients with unilateral CPSP following lesions to the ventral posterior lateral/medial thalamus contralateral to
14 their painful side and with no other brain lesions. The authors suggested that for the onset of CPSP, lesions affecting normal synaptic connections to/from the somatosensory thalamus are crucial (Kim et al., 2007). Interestingly, these patients showed clinical characteristics of this CPSP in the thalamus that closely resembled those of other central and peripheral neuropathies (Henry et al., 2008; Klit et al., 2009b).
It is evident that the thalamus plays an important role in the processing of neuropathic pain, and so understanding what role the thalamus plays in generating and/or maintaining pain following nerve injury is important. Numerous human brain imaging investigations have revealed that the thalamus displays altered anatomy in individuals with neuropathic pain. For example, An investigation by
Apkarian and colleagues found decreases in gray matter volume in the thalamus in patients with lower back pain compared to healthy controls (Apkarian et al., 2004). Similarly, in individuals with chronic orofacial neuropathic pain, gray matter volume has shown to be significantly reduced in the ventral posterior thalamus as well as the insula, putamen, somatosensory cortex and the nucleus accumbens
(Gustin et al., 2011; Henderson et al., 2013). Furthermore, chronic pain that occurs following spinal cord injury is associated with significant reductions in mean diffusivity, a marker of anatomical change derived from changes in the motion of free water, in the ventral posterior thalamus (Gustin et al.,
2014b).
As well as structural changes, there are also reports of significant biochemical changes in the thalamus in individuals with chronic neuropathic pain. One investigation found that the concentration of N- acetyl in the thalamus was lower in neuropathic pain patients compared to healthy controls (Pattany et al., 2002). Moreover, the thalamus also had a significant reduction in N-acetyl/creatine ratio in patients with neuropathic pain (Gustin et al., 2011). A reduction in the N-acetyl/creatine ratio is indicative of neural viability (Gustin et al., 2011) and therefore, it appears that the thalamic anatomical
15 changes characterized by altered grey matter volume and mean diffusivity may reflect changes in neural functioning, although changes in other types of cells cannot be ignored. In addition to anatomical and biochemical changes, there is evidence of blood flow changes in the thalamus. One study showed decreased blood flow in the thalamus in patients with central pain compared to healthy controls (Fukui et al., 2002). Similarly, patients with complex regional pain syndrome (sometimes categorized as chronic neuropathic pain) also have decreased blood flow to the thalamus when compared to healthy controls (Hsu and Chang, 2013). Indeed, it has been suggested that the most common resting activity change observed in individuals with various forms of neuropathic pain is that of a decrease in the thalamus (Gustin et al., 2014a; Yen and Lu, 2013).
A number of electrophysiological recordings in individuals with neuropathic pain have revealed that the properties of neurons within the thalamus and thalamic circuitry are altered. Intraoperative studies suggest somatotopic rearrangement, abnormal responsiveness to peripheral stimuli and abnormal spontaneous discharge patterns in thalamic neurons of patients with CPSP (Lenz et al., 1998;
Lenz et al., 1989). These zones have shown changes in neural activity and stimulation causes pain sensations at higher frequency than normally expected (Lenz et al., 1998). Furthermore, lesions to these regions in the medial thalamus have been shown to successfully relieve CPSP (Jeanmonod et al.,
1993).
A number of non-invasive measures in neuropathic pain suggest altered neural activity in the thalamus and in regions of the cortex in which it projects to. Positron emission tomography and functional magnetic resonance imaging studies show that allodynic pain increases activity in the thalamus
(Peyron et al., 1998; Schweinhardt et al., 2006). Whereas, spontaneous pain (at rest) decreases neural activity (Hsieh et al., 1995). Moreover, patients with neuropathic pain revealed thalamic spontaneous activity compared to healthy controls (Sarnthein et al., 2006) and recently altered thalamocortical
16 rhythm is evident in individuals with chronic orofacial neuropathic pain (Di Pietro et al, 2018). These results indicate that, in patients with chronic neuropathic pain, there is a possible change in the pattern of firing in the neurons of the thalamus.
Further to human investigations, experimental animal studies have also demonstrated thalamic activity changes in neuropathic pain models. The spinal cord injury model of neuropathic pain is associated with changes in burst firing within the thalamus in rats that develop neuropathic pain compared to pain-free controls (Hains et al., 2006). Similarly, Gerke and colleagues reported increased burst and oscillatory firing in the thalamus associated with the development of pain following spinal cord injury (Gerke et al., 2003). There is also evidence of functional changes in neurons within the thalamus following lesions within the spinal cord causing neuropathic pain. The investigation showed increases in spontaneous activity and responses that are induced by cutaneous stimuli, and larger receptive field sizes than neurons in the thalamus of primates with intact spinal cords (Weng et al.,
2000). Based on this evidence, neuropathic pain is associated with altered firing in both humans and experimental animal models, which may be transferred to and from other regions of the pain pathway.
Investigations have demonstrated that chronic neuropathic pain is associated with anatomical and biochemical changes in the thalamus as well as changes in the pattern of activity. This series of investigations will analyse changes in the neural activity and anatomical changes in the thalamus in patients with orofacial neuropathic pain, compared to controls, and analyse whether these changes can be reversed with treatment.
17 1.5.2.1 Thalamic reticular nucleus
It is well known that an area of the thalamus, the thalamic reticular nucleus (TRN), regulates all sensory information passing through the thalamus and so it is likely that changes in thalamic activity associated with neuropathic pain are likely regulated by this thalamic region. The thalamic reticular nucleus (TRN) is a layer of cells that insheathes much of the rostral and lateral surfaces of the dorsal thalamus and is bordered laterally by the internal capsule (Jones, 1975). Essentially, all information travelling between the thalamus and the cerebral cortex is modulated by the TRN, and evidence from over four decades ago showed that the cerebral cortex (Carman et al., 1964; Jones, 1975) and thalamic relay nuclei
(Jones, 1975) provide extensive input to the TRN, and the main output of the TRN is to thalamic relay nuclei. The study showed that fibres from a specific nucleus within the thalamus and intended for a particular area of the cortex, are either associated with a particular set of axons that terminate in a specified region of the TRN or give rise to collaterals (Jones, 1975). Furthermore, corticothalamic fibres originating from a specific area of the cerebral cortex passing to a specific thalamic nucleus will also send collaterals to the same portion of the TRN which receives axons from that specific thalamic nucleus. The TRN can be divided into sectors that process specific sensory modalities as there is very little communication between the somatosensory TRN and TRN regions not involved in somatosensation (Beck et al., 1996; Crabtree, 1992; Pinault, 2004). Figure 1.5.2.1 illustrates this circuitry.
18 Figure 1.5.2.1: General organization of the three principal projection neurons. Note that the ascending afferents (green) synapse on ventroposterior (VP) thalamic relay cells (blue), where VP axons pass through the thalamic reticular nucleus (TRN) to synapse in the primary somatosensory cortex.
Corticothalamic neurons (orange) are in layer 6 of the cortex, where they send feedback to both TRN inhibitory neurons (red) and VP neurons. Copied from Guillery and colleagues (Guillery et al., 1998).
The TRN is considered an inhibitory cell of the thalamus as it is the main GABA-producing nucleus
(Spreafico et al., 1994), and any change to the level of GABA in the thalamus is likely a measure of the change in activity of neurons in the TRN. Indeed, magnetic resonance imaging based spectroscopy analysis of GABA levels in the thalamus is likely reflective of TRN activity. As such, Henderson and colleagues found decreased TRN activity in patients with chronic neuropathic pain which was attributed to a decrease in GABA levels in the thalamus. Furthermore, the study also measured resting cerebral blood flow, an indirect measure of neural activity, and found that these patients showed a
19 decrease in neural activity in the TRN (Henderson et al., 2013). These data suggest that the changes in the TRN activity are associated with chronic neuropathic pain through their GABAergic action on the somatosensory thalamus. Through this GABAergic action, the TRN is capable of modulating the thalamocortical circuitry. This process alters thalamocortical dysrhythmia which is associated with chronic neuropathic pain. The phenomenon, thalamocortical dysrhythmia, was discussed earlier.
The thalamus shows changes in pattern of activity in patients compared to healthy controls (Chapter
2) and a reversal with treatment (Chapter 4).
1.5.3 Primary somatosensory cortex
Whilst the role of the dorsal horn/SpV and thalamus have begun to be investigated in greater detail in recent years, there is a also a considerable number of studies that have investigated the role of the primary somatosensory cortex in pain processing. Third order neurons within the thalamus project to the primary somatosensory cortex which is located in the postcentral gyrus of the parietal lobe. This region is composed of Brodmann areas 1, 2, 3a and 3b (Garey, 2006; Nelson and Chen, 2008) and animal and human investigations have revealed that cutaneous input is predominantly received by
Brodmann areas 1 and 3b (Nelson and Chen, 2008; Sur et al., 1980). In the early 20th century, a group of investigators stimulated various parts of the somatosensory cortex and found that according to the region of stimulation, sensations were experienced in particular regions on the contralateral side of the body (Penfield and Rasmussen, 1950). Since then, the somatosensory cortex has become extensively mapped with the leg represented medially in the paracentral lobule and the hand and face represented more laterally. This somatotopic map is often referred to as the sensory homunculus.
20
1.5.3.1 Role in neuropathic pain
Although the function of the somatosensory cortex has been studied extensively, its role in the processing of pain remains unclear. While it is not always activated in experimental pain studies
(Bushnell et al., 1999 199), lesions within the somatosensory cortex increased pain intensity, with only some reporting temporary loss of painful sensations (Head and Holmes, 1911; Holmes, 1927). This has led to the idea that the somatosensory cortex may not be involved in coding painful sensations but it is more likely to code tactile sensibility and localization (Holmes, 1927). Moreover, a study in 1937 demonstrated that deep brain stimulations in the primary somatosensory cortex caused a ‘pins and needles’ sensation rather than a painful sensation (Penfield and Boldrey, 1937). The role of the somatosensory cortex in the processing of pain is also supported in a rat model where painful sensations were recorded after blocking the primary somatosensory cortex(Uhelski et al., 2012).
Whilst these reports do not rule out the prospect that the primary somatosensory cortex is involved in pain processing; indeed, it is generally accepted that it is responsible for the sensory qualities of pain, they do raise the prospect that other brain regions also code these specific aspects of the pain experience.
Whilst it remains unclear what role the primary somatosensory cortex plays in the coding of chronic neuropathic pain, there is emerging evidence that the primary somatosensory cortex displays functional reorganization in individuals with chronic pain states (Elbert et al., 1994; Pleger et al., 2004;
Wrigley et al., 2009). For example, one study measured cortical reorganisation using functional magnetic resonance imaging. An innocuous stimulus was applied to the lip, thumb and little finger to activate their respective cortical representations, and distances from these regions to an anatomical marker were calculated as a measure of cortical organization. All three regions showed changes in cortical organization compared to healthy controls, and in the little finger, the change in the distance
21 was positively correlated to pain intensity (Wrigley et al., 2009). By using a similar technique to determine reorganisation of the primary somatosensory cortex in patients with complex regional pain syndrome, the ulnar nerve and median nerve were stimulated and their cortical representations were mapped. The findings indicate that in these patients, the representation of the arm is smaller than that of healthy controls (Pleger et al., 2004). Furthermore, hand and arm amputees with phantom limb sensations have reorganization of the primary somatosensory cortex in regions that represented the amputated hand/arm (Elbert et al., 1994). Interestingly, some studies show that these cortical reorganisations can be reversed (Maihofner et al., 2004; Pleger et al., 2005).
The primary somatosensory cortex shows changes in neural activity in patients compared to healthy controls in Chapter 2.
1.6 Astrocytes in neuropathic pain
1.6.1 Anatomy and function of astrocytes
Whilst above is a description of studies that have focussed primarily on the impulse-conducting cells of the brain, recent evidence suggests that non-neural cells also play a role in regulating neural activity and maybe a critical role in the development and maintenance of chronic pain. Traditionally, astrocytes were thought to almost exclusively play a role in supporting neural function; however, more recently, astrocytes have been shown to have a more significant role in the central nervous system including the modulation of neural activity (Pekny and Nilsson, 2005; Sofroniew, 2009b). There are two types of astrocytes; astrocytes in gray matter called protoplasmic and astrocytes in white matter called fibrous (Miller and Raff, 1984). Both types of astrocytes make direct contact with blood vessels, form gap junctions with neighbouring astrocytes, and protoplasmic astrocytic processes contact
Nodes of Ranvier and envelop synapses (Butt et al., 1994; Hildebrand, 1971a, b). These contacts
22 suggest that astrocytes may also play a role in modulating neural activity. Indeed, studies indicate that astrocytes can produce and release mediators that control blood vessel diameter and flow in a coordinated matter (Attwell et al., 2010; Zonta et al., 2002). Therefore, an increase in blood flow due to an increase in neural activity (and vice versa) is largely dependent on astrocyte activity.
Furthermore, astrocytes envelop synapses and are responsible for maintaining the fluid (Attwell et al.,
2010; Zonta et al., 2002), ion (O'connor and Kimelberg, 1993; Walz, 1989), pH (Chesler, 2005) and transmitter (Anderson and Swanson, 2000) homeostasis through their foot processes, which are rich in aquaporin and potassium transporters (Newman, 1986; Papadopoulos et al., 2004). These processes also express high levels of transporters for neurotransmitters such as glutamate, GABA and glycine (Auld and Robitaille, 2003; Schousboe et al., 2004). Astrocytes clear neurotransmitters from the neural synapse and release them in response to neural activity, which in turn regulates neural activity.
Although astrocytes express sodium and potassium channels and exhibit an inward current, they do not ‘fire’ like neurons, rather, they exhibit a regulated increase in intracellular calcium which can occur in waves (Pasti et al., 1997). Such astrocytic intracellular calcium waves have functional significance in astrocyte-astrocyte and astrocyte-neuron communication (Pasti et al., 1997). The increase in calcium can occur from internal stores and are triggered by a number of neurotransmitters (Dani et al., 1992).
The calcium waves can themselves elicit the release of neurotransmitters from astrocytes (e.g. glutamate) into the extracellular space and these calcium waves and the subsequent release of gliotransmitters can propagate to neighbouring astrocytes through gap junctions (Kozlov et al., 2006;
Tian et al., 2005).
In mild to moderate astrogliosis (increase in astrocyte activity – usually a result of nerve damage), there is an increase in cell body and process size, namely hypertrophy. However, this occurs within
23 the domain of the individual astrocyte, as, at this stage, astrocytes do not overlap, rather, they tile the entire central nervous system (Sofroniew, 2009a; Sofroniew and Vinters, 2010b). There is also an upregulation of glial fibrillary acidic protein (GFAP), which can give the false impression of proliferation
(Sofroniew and Vinters, 2010b). Mild to moderate astrogliosis can be associated with acute single events, such as nerve damage, and has potential to resolve.
1.6.2 Role of astrocytes in neuropathic pain
Following peripheral nerve injury, such as nerve damage due to root canal treatment, astrocytes can become chronically activated (astrogliosis), a process that is currently not entirely understood.
Nonetheless, in a number of investigations, astrocytes play an important role in the initiation and/or maintenance of neuropathic pain. For example, GFAP (a marker of astrocyte activation) knockout mice showed slower wound healing than wild type, illustrating the importance of astrocytes in the healing process following injury (Pekny et al., 1999). Furthermore, following spinal nerve ligation in rats, there is an immediate activation of both microglia and astrocytes in the dorsal horn. However, on day 21, there is predominant activation of astrocytes, which is associated with persistent allodynia.
Interestingly, blocking astrocyte activity in the dorsal horn reverses allodynia (Zhuang et al., 2005;
Zhuang et al., 2006). Furthermore, hyperactive astrocytes in the dorsal horn increase allodynia responses in rats and blocking this astrocyte activation reverses the allodynic response (Miyoshi et al.,
2008b). In a trigeminal neuropathic pain model, there is an increase in astrocyte activation in the spinal trigeminal nucleus which is associated with hyperalgesia. Inhibition of astrocyte activity prevented hyperalgesia and inflammation in orofacial region of the face (Guo et al., 2007).
Behavioural hyperalgesia correlates with astrocyte activation in a neuropathic pain model. Colburn et al. (1999) measured the effects of different types of nerve injuries on astrocyte activity in the spinal cord. Glial activity and mechanical allodynia were associated with spinal nerve or dorsal root injury.
24 Interestingly, injury to the dorsal root almost exclusively increases astrocyte activity, i.e. no increases in microglial activity (Colburn et al., 1999). Moreover, after nerve injury, astrocyte activity persisted longer than microglial activity. Astrocyte activity can last more than 150 days after nerve injury and although in most cases astrocyte activation follows microglial reaction (Miyoshi et al., 2008a), one investigation showed that bone cancer increased astrocyte activation in the absence of microglia (Hald et al., 2009).
Nerve damage also increases gap junctions between astrocytes which are important for astrocyte- astrocyte communication and the propagation of calcium waves. Following chronic constriction injury of the infra-orbital nerve, there was an increase in gap junction protein (connexion 43) in the trigeminal ganglion cells (Ohara et al., 2008). Moreover, inflammation in the masseter muscle increased astrocytic connexion 43 in the spinal trigeminal nucleus in the rat model (Guo et al., 2007).
These data suggest that astrocytes play an important role in neuropathic pain through processes such as gap junctions and inflammation.
Astrocytes are increasingly being explored for their role in the maintenance of neuropathic pain. In
Chapter 2, astrocytes are speculated to be the key player in the changes in neural activity in the ascending pain pathway.
1.6.2.1 Targeting astrocytes for treatment of neuropathic pain
It is becoming clear that astrocytes play a crucial role in neuropathic pain and therefore modulating astrocyte activity may assist in reducing or preventing neuropathic pain. Indeed, one supplement that has been shown to modulate astrocyte activity, namely, palmitolyethanolamide (PEA), has been used extensively in Europe to reduce neuropathic pain in multiple conditions (Hesselink and Hekker, 2012).
25 PEA is a naturally occurring fatty acid amide that can be found in foods such as meat, eggs, soybeans and peanuts. It belongs to the family of N-acetylethanolamines, which act on cannabinoid receptors and interact with inflammatory cells in the nervous system. It has a neuroprotective, anti- inflammatory, anti-nociceptive and anti-convulsant properties (Calignano et al., 1998; Lambert et al.,
2001; Lo Verme et al., 2005; Skaper et al., 1996). Further, under pathological conditions, such as brain ischemia (Franklin et al., 2003) and neuroinflammation (Darmani et al., 2005), the levels of PEA significantly increase, which suggests neuroprotective activity. However, in chronic conditions, endogenous PEA levels decrease (Petrosino et al., 2007), suggesting that there is an interaction with the chronicity of a disease state and endogenous PEA levels.
Whilst the precise mechanism remains unclear, it is thought that the main target of PEA is the peroxisome proliferator-activated receptor alpha (PPAR-α) (D'Agostino et al., 2009; D'Agostino et al.,
2007; Lo Verme et al., 2005; LoVerme et al., 2006; Raso et al., 2011). PPAR-α is an isotope of the PPARs that can affect the transcription of a number of genes (Brown and Plutzky, 2007; Duval et al., 2007) and is expressed by astrocytes. These astrocytes play a role in the antinociceptive effects of PEA through their PPAR-α pathway (Raso et al., 2011). It is not yet well understood how PEA functions in neuropathic pain, however a number of clinical trials have proven its ability to reduce the pain intensity in a number of neuropathic conditions such as sciatic pain, diabetic neuropathic pain, lower back pain and post-stroke pain (M Keppel Hesselink, 2012). There has also been no documentation of any side effects to the drugs or any drug-drug interactions.
The effects on PEA on neural activity is explored in Chapter 4, where PEA was administered to patients with neuropathic pain and ISOs were measured before and after treatment. Results show that infra-slow oscillations were decreased with PEA in the ascending pain pathway.
26 1.7 Neuroimaging: Magnetic resonance imaging
The overall aim of this thesis is to explore functional and anatomical changes in individuals with trigeminal neuropathic pain with particular focus on the spinal trigeminal nucleus, thalamus and somatosensory cortex. So is it possible to measure changes in brain activity and anatomy in living humans? Magnetic resonance imaging (MRI) is a scanning technique that implements strong magnets, and radiofrequency signals to generate structural and continuous images of the body’s internal organs to measure changes in structure and activity, respectively.
1.7.1 Functional magnetic resonance imaging
One sequence of imaging that can be collected using an MRI that provides an index of neural activity is blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI). The physiological basis of BOLD fMRI relies on the increase in energy required for neurons to fire when activated. This increase in energy increases in blood flow to that area of neurons and an increase in the ratio of oxygenated to deoxygenated haemoglobin in the tissue. Thus, there is a difference in the magnetic properties of oxygenated and deoxygenated haemoglobin, which is what the fMRI measures
(Peyron et al., 2000). FMRI can be used to measure neural activity in response to tasks or resting activity which can then be used to analyse oscillatory and functional network changes between groups.
1.7.1.1 Arterial spin labelling
In chronic neuropathic pain conditions, the pain cannot be switched on and off and hence BOLD cannot be used for investigating blood flow changes in these conditions as it cannot be directly used to compared blood flow levels between groups (Wasan et al., 2011). While, positron emission tomography allows for the measurement of blood flow changes, this technique is invasive and has a poor spatial resolution. Consequently, arterial spin labelling (ASL), pseudo-continuous ASL (pCASL) in
27 particular, which has a higher signal to noise ratio than previous ASL techniques, can be used to measure regional on-going blood flow as an index of on-going levels of neural activity in individuals with chronic neuropathic pain conditions. It allows for the absolute quantification of regional cerebral blood flow by use of magnetically labelled water, similar to the principles of positron emission tomography (Wu et al., 2007).
1.7.1.2 Low frequency oscillations
In addition to measuring evoked or on-going activity levels using fMRI or ASL, the pattern of on-going neural activity can be explored using resting state fMRI. Using a resting state fMRI time series, one can break the signal into its individual component frequencies using Fast Fourier transforms and the power at each frequency can be measured. These low frequency oscillations suggest some connectivity between functionally related regions at rest (Biswal et al., 1995) and it has been shown that infra-slow oscillations (<0.1Hz) are fundamental to cerebral and thalamic function (Hughes et al.,
2011; Lőrincz et al., 2009). Furthermore, as mentioned above, changes in thalamic bursting activity have been shown in chronic neuropathic pain models and as such, this method allows for measurement of low frequency oscillatory activity in regions of the somatosensory pathway between patients and healthy controls.
1.7.2 T2 relaxometry
Whilst it is not possible to measure astrocyte activation directly in a living human, there is evidence that a specific anatomical imaging technique - T2 relaxation time – can be used as an indicator of astrocytic activity (Jackson et al., 1994; Schwarz et al., 1996; Wagner et al., 2012). T2 relaxation times are influenced by the chemical characteristics of the tissue such as static dipolar fields, which increase as the size of structures increase (such as the increase in GFAP levels in astrocyte activity) (Goldman and Shen, 1966; Solomon, 1955). Therefore, an increase in astrocytic release of GFAP (an intermediate
28 filament composed of a variety of proteins), increases the static dipolar field, and reduces the T2 relaxation time (Sofroniew, 2009b; Sofroniew and Vinters, 2010a; Stewart, 1993).
1.8 Overarching aims of the thesis
To investigate altered neural activity in chronic orofacial neuropathic pain and the effects of PEA on neural activity.
1.8.1 Specific aims of the thesis
Investigate infra-slow oscillatory activity using fMRI in patients with orofacial neuropathic pain.
This work is presented in Chapter 2, and was published as Chronic Neuropathic Pain: It’s about
the Rhythm, The Journal of Neuroscience, 2016: 36 (3), p. 1008-1018.
Investigate whether previously identified infra-slow oscillations in orofacial neuropathic pain result from astrogliosis using T2 relaxometry.
This work is presented in Chapter 3, and was published as Altered regional brain T2 relaxation
times in individuals with chronic orofacial neuropathic pain, NeuroImage: Clinical, 2018: 19,
p. 167-173.
To investigate whether an astrocyte modulator can reduce orofacial neuropathic pain and to investigate its effects on infra-slow oscillatory activity and cerebral blood flow.
This work is presented in Chapter 4, and was submitted to Nature Medicine as Effects of the glial modulator, palmitoylethanolamide, on chronic pain intensity and brain function.
29 References
Australian Bureau of Statistics (ABS). (2011). Characteristics of bodily pain in Australia (4841.0).
Retrieved from: http://www.abs.gov.au/ausstats/[email protected]/Lookup/4841.0Chapter12011.
Anderson, C.M., Swanson, R.A., 2000. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32, 1-14. Andersson, G.B.J., 1999. Epidemiological features of chronic low-back pain. The Lancet 354, 581-585. Andrew, R., Derry, S., Taylor, R.S., Straube, S., Phillips, C.J., 2014. The Costs and Consequences of Adequately Managed Chronic Non-Cancer Pain and Chronic Neuropathic Pain. Pain Practice 14, 79- 94. Apkarian, A.V., Bushnell, M.C., Treede, R.-D., Zubieta, J.-K., 2005. Human brain mechanisms of pain perception and regulation in health and disease. European Journal of Pain 9, 463-463. Apkarian, A.V., Sosa, Y., Sonty, S., Levy, R.M., Harden, R.N., Parrish, T.B., Gitelman, D.R., 2004. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. Journal of Neuroscience 24, 10410-10415. Attwell, D., Buchan, A.M., Charpak, S., Lauritzen, M., MacVicar, B.A., Newman, E.A., 2010. Glial and neuronal control of brain blood flow. Nature 468, 232. Auld, D.S., Robitaille, R., 2003. Glial cells and neurotransmission: an inclusive view of synaptic function. Neuron 40, 389-400. Barrett, A.M., Lucero, M.A., Le, T., Robinson, R.L., Dworkin, R.H., Chappell, A.S., 2007. Epidemiology, Public Health Burden, and Treatment of Diabetic Peripheral Neuropathic Pain: A Review. Pain Medicine 8, S50-S62. Beck, P.D., Pospichal, M.W., Kaas, J.H., 1996. Topography, architecture, and connections of somatosensory cortex in opossums: evidence for five somatosensory areas. Journal of Comparative Neurology 366, 109-133. Bennett, M.I., Simpson, K.H., 2004. Gabapentin in the treatment of neuropathic pain. Palliative Medicine 18, 5-11. Bessou, P., Perl, E.R., 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. Journal of Neurophysiology 32, 1025-1043. Bishop, T., Marchand, F., Young, A.R., Lewin, G.R., McMahon, S.B., 2010. Ultraviolet-B-induced mechanical hyperalgesia: A role for peripheral sensitisation. Pain 150, 141-152. Biswal, B., Yetkin, F.Z., Haughton, V.M., Hyde, J.S., 1995. Functional connectivity in the motor cortex of resting human brain using echo-planar MRI. Magn Reson Med 34, 537-541. Brown, A.G., 1982. The dorsal horn of the spinal cord. Q J Exp Physiol 67, 193-212. Brown, A.G., Iggo, A., 1967. A quantitative study of cutaneous receptors and afferent fibres in the cat and rabbit. The Journal of Physiology 193, 707-733. Brown, J.D., Plutzky, J., 2007. Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets. Circulation 115, 518-533. Burchiel, K.J., 1993. Trigeminal Neuropathic Pain. Springer Vienna, Vienna, pp. 145-149. Burgess, P.R., Perl, E.R., 1967. Myelinated afferent fibres responding specifically to noxious stimulation of the skin. The Journal of Physiology 190, 541-562. Bushnell, M., Apkarian, A., 2006. Representation of pain in the brain. Wall and Melzack’s Textbook of Pain, 5th edition. Elsevier. Bushnell, M.C., Duncan, G.H., Hofbauer, R.K., Ha, B., Chen, J.I., Carrier, B., 1999. Pain perception: Is there a role for primary somatosensory cortex? Proc Natl Acad Sci U S A 96, 7705-7709. Butt, A.M., Duncan, A., Berry, M., 1994. Astrocyte associations with nodes of Ranvier: ultrastructural analysis of HRP-filled astrocytes in the mouse optic nerve. Journal of Neurocytology 23, 486-499.
30 Calignano, A., La Rana, G., Giuffrida, A., Piomelli, D., 1998. Control of pain initiation by endogenous cannabinoids. Nature 394, 277-281. Campbell, J.N., Khan, A.A., Meyer, R.A., Raja, S.N., 1988. Responses to heat of C-fiber nociceptors in monkey are altered by injury in the receptive field but not by adjacent injury. Pain 32, 327-332. Campbell, J.N., Meyer, R.A., 2006. Mechanisms of Neuropathic Pain. Neuron 52, 77-92. Carman, J., Cowan, W., Powell, T., 1964. Cortical connexions of the thalamic reticular nucleus. Journal of anatomy 98, 587. Chesler, M., 2005. Failure and function of intracellular pH regulation in acute hypoxic-ischemic injury of astrocytes. Glia 50, 398-406. Coghill, R.C., Sang, C.N., Maisog, J.M., Iadarola, M.J., 1999. Pain intensity processing within the human brain: a bilateral, distributed mechanism. Journal of Neurophysiology 82, 1934-1943. Colburn, R.W., Rickman, A.J., DeLeo, J.A., 1999. The Effect of Site and Type of Nerve Injury on Spinal Glial Activation and Neuropathic Pain Behavior. Experimental Neurology 157, 289-304. Cook, A.J., Woolf, C.J., Wall, P.D., 1986. Prolonged C-fibre mediated facilitation of the flexion reflex in the rat is not due to changes in afferent terminal or motoneurone excitability. Neuroscience Letters 70, 91-96. Crabtree, J.W., 1992. The somatotopic organization within the cat's thalamic reticular nucleus. European Journal of Neuroscience 4, 1352-1361. D'Agostino, G., La Rana, G., Russo, R., Sasso, O., Iacono, A., Esposito, E., Mattace Raso, G., Cuzzocrea, S., Loverme, J., Piomelli, D., Meli, R., Calignano, A., 2009. Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-kappaB nuclear signalling in dorsal root ganglia. Eur J Pharmacol 613, 54-59. D'Agostino, G., La Rana, G., Russo, R., Sasso, O., Iacono, A., Esposito, E., Raso, G.M., Cuzzocrea, S., Lo Verme, J., Piomelli, D., Meli, R., Calignano, A., 2007. Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist, modulates carrageenan-induced paw edema in mice. J Pharmacol Exp Ther 322, 1137-1143. Dani, J.W., Chernjavsky, A., Smith, S.J., 1992. Neuronal activity triggers calcium waves in hippocampal astrocyte networks. Neuron 8, 429-440. Darmani, N.A., Izzo, A.A., Degenhardt, B., Valenti, M., Scaglione, G., Capasso, R., Sorrentini, I., Di Marzo, V., 2005. Involvement of the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflammatory and neuropathic conditions: review of the available pre-clinical data, and first human studies. Neuropharmacology 48, 1154-1163. De La Llave-Rincón, A.I., Fernández-De-Las-Peñas, C., Fernández-Carnero, J., Padua, L., Arendt- Nielsen, L., Pareja, J.A., 2009. Bilateral hand/wrist heat and cold hyperalgesia, but not hypoesthesia, in unilateral carpal tunnel syndrome. Experimental Brain Research 198, 455-463. Derbyshire, S.W., Jones, A.K., Gyulai, F., Clark, S., Townsend, D., Firestone, L.L., 1997. Pain processing during three levels of noxious stimulation produces differential patterns of central activity. Pain 73, 431-445. Dessem, D., Moritani, M., Ambalavanar, R., 2007. Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem. J Neurophysiol 98, 214-223. Duale, C., Luccarini, P., Cadet, R., Woda, A., 1996. Effects of Morphine Microinjections into the Trigeminal Sensory Complex on the Formalin Test in the Rat. Experimental Neurology 142, 331-339. Duval, C., Muller, M., Kersten, S., 2007. PPARalpha and dyslipidemia. Biochim Biophys Acta 1771, 961-971. Dworkin, R.H., Backonja, M., Rowbotham, M.C., et al., 2003. Advances in neuropathic pain: Diagnosis, mechanisms, and treatment recommendations. Archives of Neurology 60, 1524-1534. Elbert, T., Flor, H., Birbaumer, N., Knecht, S., Hampson, S., Larbig, W., 1994. Extensive reorganization of the somatosensory cortex in adult humans after nervous system injury. Neuroreport 5, 2593- 2597. Farrell, M.J., Laird, A.R., Egan, G.F., 2005. Brain activity associated with painfully hot stimuli applied to the upper limb: A meta-analysis. Human Brain Mapping 25, 129-139.
31 Fernandez-de-las-Penas, C., de la Llave-Rincon, A.I., Fernandez-Carnero, J., Cuadrado, M.L., Arendt- Nielsen, L., Pareja, J.A., 2009. Bilateral widespread mechanical pain sensitivity in carpal tunnel syndrome: evidence of central processing in unilateral neuropathy. Brain 132, 1472-1479. Finnerup, N.B., Otto, M., McQuay, H.J., Jensen, T.S., Sindrup, S.H., 2005. Algorithm for neuropathic pain treatment: An evidence based proposal. Pain 118, 289-305. Fishbain, D.A., Cutler, R., Rosomoff, H.L., Rosomoff, R.S., 1997. Chronic pain-associated depression: antecedent or consequence of chronic pain? A review. The Clinical journal of pain 13, 116-137. Franklin, A., Parmentier-Batteur, S., Walter, L., Greenberg, D.A., Stella, N., 2003. Palmitoylethanolamide increases after focal cerebral ischemia and potentiates microglial cell motility. J Neurosci 23, 7767-7775. Fukui, S., Shigemori, S., Nosaka, S., 2002. Central pain associated with low thalamic blood flow treated by electroconvulsive therapy. J Anesth 16, 255-257. Garcia-Larrea, L., Frot, M., Valeriani, M., 2003. Brain generators of laser-evoked potentials: from dipoles to functional significance. Neurophysiologie Clinique/Clinical Neurophysiology 33, 279-292. Garey, L.J., 2006. Brodmann's ‘Localisation in the Cerebral Cortex’. World Scientific. Gerke, M.B., Duggan, A.W., Xu, L., Siddall, P.J., 2003. Thalamic neuronal activity in rats with mechanical allodynia following contusive spinal cord injury. Neuroscience 117, 715-722. Goldman, M., Shen, L., 1966. Spin-Spin Relaxation in La${\mathrm{F}}_{3}$. Physical Review 144, 321-331. Graziano, A., Jones, E.G., 2004. Widespread thalamic terminations of fibers arising in the superficial medullary dorsal horn of monkeys and their relation to calbindin immunoreactivity. J Neurosci 24, 248-256. Guillery, R., Feig, S., Lozsadi, D., 1998. Paying attention to the thalamic reticular nucleus. Trends in Neurosciences 21, 28-32. Guo, W., Wang, H., Watanabe, M., Shimizu, K., Zou, S., LaGraize, S.C., Wei, F., Dubner, R., Ren, K., 2007. Glial–Cytokine–Neuronal Interactions Underlying the Mechanisms of Persistent Pain. The Journal of Neuroscience 27, 6006-6018. Gustin, S.M., Peck, C.C., Wilcox, S.L., Nash, P.G., Murray, G.M., Henderson, L.A., 2011. Different pain, different brain: thalamic anatomy in neuropathic and non-neuropathic chronic pain syndromes. J Neurosci 31, 5956-5964. Gustin, S.M., Wrigley, P.J., Youssef, A.M., McIndoe, L., Wilcox, S.L., Rae, C.D., Edden, R., Siddall, P.J., Henderson, L.A., 2014a. Thalamic activity and biochemical changes in individuals with neuropathic pain following spinal cord injury. Pain 155, 1027-1036. Gustin, S.M., Wrigley, P.J., Youssef, A.M., McIndoe, L., Wilcox, S.L., Rae, C.D., Edden, R.A., Siddall, P.J., Henderson, L.A., 2014b. Thalamic activity and biochemical changes in individuals with neuropathic pain after spinal cord injury. Pain 155, 1027-1036. Hains, B.C., Saab, C.Y., Waxman, S.G., 2006. Alterations in burst firing of thalamic VPL neurons and reversal by Na(v)1.3 antisense after spinal cord injury. J Neurophysiol 95, 3343-3352. Hald, A., Nedergaard, S., Hansen, R.R., Ding, M., Heegaard, A.M., 2009. Differential activation of spinal cord glial cells in murine models of neuropathic and cancer pain. Eur J Pain 13, 138-145. Head, H., Holmes, G., 1911. Sensory disturbances from cerebral lesions. Brain 34, 102-254. Henderson, L.A., Peck, C.C., Petersen, E.T., Rae, C.D., Youssef, A.M., Reeves, J.M., Wilcox, S.L., Akhter, R., Murray, G.M., Gustin, S.M., 2013. Chronic pain: lost inhibition? J Neurosci 33, 7574-7582. Henry, J.L., Lalloo, C., Yashpal, K., 2008. Central poststroke pain: An abstruse outcome. Pain Research & Management : The Journal of the Canadian Pain Society 13, 41-49. Hesselink, J.M., Hekker, T.A., 2012. Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series. J Pain Res 5, 437- 442. Hildebrand, C., 1971a. Ultrastructural and light‐microscopic studies of the developing feline spinal cord white matter. I. The nodes of Ranvier. Acta Physiologica 82, 81-107.
32 Hildebrand, C., 1971b. Ultrastructural and light‐microscopic studies of the nodal region in large myelinated fibres of the adult feline spinal cord white matter. Acta Physiologica 82, 43-79. Holmes, G., 1927. Symposium on sensory disorders in organic disease of the nervous system: III. Disorders of sensation produced by cortical lesions. Brain: A Journal of Neurology. Hsieh, J.C., Belfrage, M., Stone-Elander, S., Hansson, P., Ingvar, M., 1995. Central representation of chronic ongoing neuropathic pain studied by positron emission tomography. Pain 63, 225-236. Hsu, K.C., Chang, S.T., 2013. Correlation of thalamic blood flow redistribution with persistent complex regional pain syndrome in a stroke patient with poor diabetic control. J Diabetes Investig 4, 676-678. Hughes, S.W., Lorincz, M.L., Parri, H.R., Crunelli, V., 2011. Infraslow (<0.1 Hz) oscillations in thalamic relay nuclei basic mechanisms and significance to health and disease states. Prog Brain Res 193, 145- 162. Iannetti, G., Zambreanu, L., Cruccu, G., Tracey, I., 2005. Operculoinsular cortex encodes pain intensity at the earliest stages of cortical processing as indicated by amplitude of laser-evoked potentials in humans. Neuroscience 131, 199-208. Ibuki, T., Hama, A.T., Wang, X.T., Pappas, G.D., Sagen, J., 1996. Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience 76, 845-858. Iggo, A., 1960. Cutaneous mechanoreceptors with afferent C fibres. The Journal of Physiology 152, 337-353. Jackson, G.D., Grunewald, R.A., Connelly, A., Duncan, J.S., 1994. Quantitative MR relaxometry study of effects of vigabatrin on the brains of patients with epilepsy. Epilepsy Res 18, 127-137. Jeanmonod, D., Magnin, M., Morel, A., 1993. Thalamus and neurogenic pain: physiological, anatomical and clinical data. Neuroreport 4, 475-478. Jones, A.K.P., Kulkarni, B., Derbyshire, S.W.G., 2003. Pain mechanisms and their disordersImaging in clinical neuroscience. British Medical Bulletin 65, 83-93. Jones, E., 1975. Some aspects of the organization of the thalamic reticular complex. Journal of Comparative Neurology 162, 285-308. Jones, E.G., 2001. The thalamic matrix and thalamocortical synchrony. Trends in Neurosciences 24, 595-601. Kawamata, M., Omote, K., 1996. Involvement of increased excitatory amino acids and intracellular Ca2+ concentration in the spinal dorsal horn in an animal model of neuropathic pain. Pain 68, 85-96. Kenshalo Jr, D., Isensee, O., 1983. Responses of primate SI cortical neurons to noxious stimuli. Journal of Neurophysiology 50, 1479-1496. Killian, J.M., Fromm, G.H., 1968. Carbamazepine in the treatment of neuralgia: Use and side effects. Archives of Neurology 19, 129-136. Kim, J.H., Greenspan, J.D., Coghill, R.C., Ohara, S., Lenz, F.A., 2007. Lesions limited to the human thalamic principal somatosensory nucleus (ventral caudal) are associated with loss of cold sensations and central pain. J Neurosci 27, 4995-5004. Klit, H., Finnerup, N.B., Jensen, T.S., 2009a. Central post-stroke pain: clinical characteristics, pathophysiology, and management. The Lancet Neurology 8, 857-868. Klit, H., Finnerup, N.B., Jensen, T.S., 2009b. Central post-stroke pain: clinical characteristics, pathophysiology, and management. Lancet Neurol 8, 857-868. Kocher, L., Anton, F., Reeh, P.W., Handwerker, H.O., 1987. The effect of carrageenan-induced inflammation on the sensitivity of unmyelinated skin nociceptors in the rat. Pain 29, 363-373. Koltzenburg, M., Torebjörk, H.E., Wahren, L.K., 1994. Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain. Brain 117, 579- 591. Kozlov, A.S., Angulo, M.C., Audinat, E., Charpak, S., 2006. Target cell-specific modulation of neuronal activity by astrocytes. Proc Natl Acad Sci U S A 103, 10058-10063.
33 Lambert, D.M., Vandevoorde, S., Diependaele, G., Govaerts, S.J., Robert, A.R., 2001. Anticonvulsant activity of N-palmitoylethanolamide, a putative endocannabinoid, in mice. Epilepsia 42, 321-327. Leijon, G., Boivie, J., Johansson, I., 1989. Central post-stroke pain — neurological symptoms and pain characteristics. Pain 36, 13-25. Lenz, F.A., Garonzik, I.M., Zirh, T.A., Dougherty, P.M., 1998. Neuronal activity in the region of the thalamic principal sensory nucleus (ventralis caudalis) in patients with pain following amputations. Neuroscience 86, 1065-1081. Lenz, F.A., Kwan, H.C., Dostrovsky, J.O., Tasker, R.R., 1989. Characteristics of the bursting pattern of action potentials that occurs in the thalamus of patients with central pain. Brain Res 496, 357-360. Lo Verme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A., Piomelli, D., 2005. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol Pharmacol 67, 15-19. Lorenz, J., Casey, K.L., 2005. Imaging of acute versus pathological pain in humans. European Journal of Pain 9, 163-165. Lőrincz, M.L., Geall, F., Bao, Y., Crunelli, V., Hughes, S.W., 2009. ATP-Dependent Infra-Slow (<0.1 Hz) Oscillations in Thalamic Networks. PLoS ONE 4, e4447. Love, S., Coakham, H.B., 2001. Trigeminal neuralgiaPathology and pathogenesis. Brain 124, 2347- 2360. LoVerme, J., Russo, R., La Rana, G., Fu, J., Farthing, J., Mattace-Raso, G., Meli, R., Hohmann, A., Calignano, A., Piomelli, D., 2006. Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-alpha. J Pharmacol Exp Ther 319, 1051-1061. Luccarini, P., Cadet, R., Duale, C., Woda, A., 1998. Effects of lesions in the trigeminal oralis and caudalis subnuclei on different orofacial nociceptive responses in the rat. Brain Res 803, 79-85. M Keppel Hesselink, J., 2012. New targets in pain, non-neuronal cells, and the role of palmitoylethanolamide. The Open Pain Journal 5. Maihofner, C., Handwerker, H.O., Neundorfer, B., Birklein, F., 2004. Cortical reorganization during recovery from complex regional pain syndrome. Neurology 63, 693-701. McCracken, L.M., Gross, R.T., 1993. Does anxiety affect coping with chronic pain? The Clinical journal of pain 9, 253-259. McLachlan, E.M., Jänig, W., Devor, M., Michaelis, M., 1993. Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia. Nature 363, 543. Meisner, J.G., Marsh, A.D., Marsh, D.R., 2010. Loss of GABAergic interneurons in laminae I–III of the spinal cord dorsal horn contributes to reduced GABAergic tone and neuropathic pain after spinal cord injury. Journal of neurotrauma 27, 729-737. Melzack, R., 1989. Phantom limbs, the self and the brain (the DO Hebb Memorial Lecture). Canadian Psychology/Psychologie Canadienne 30, 1. Mendell, L.M., Wall, P.D., 1965. Responses of Single Dorsal Cord Cells to Peripheral Cutaneous Unmyelinated Fibres. Nature 206, 97. Merskey, H., Bogduk, N., Taxonomy, I.A.f.t.S.o.P.T.F.o., 1994. Classification of Chronic Pain: Descriptions of Chronic Pain Syndromes and Definitions of Pain Terms. IASP Press. Merskey, H.E., 1986. Classification of chronic pain: descriptions of chronic pain syndromes and definitions of pain terms. Pain. Miki, K., Fukuoka, T., Tokunaga, A., Noguchi, K., 1997. Calcitonin gene-related peptide increase in the rat spinal dorsal horn and dorsal column nucleus following peripheral nerve injury: Up-regulation in a subpopulation of primary afferent sensory neurons. Neuroscience 82, 1243-1252. Miller, R., Raff, M., 1984. Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. The Journal of Neuroscience 4, 585-592. Miyoshi, K., Obata, K., Kondo, T., Okamura, H., Noguchi, K., 2008a. Interleukin-18-mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain processing after nerve injury. J Neurosci 28, 12775-12787.
34 Miyoshi, K., Obata, K., Kondo, T., Okamura, H., Noguchi, K., 2008b. Interleukin-18-Mediated Microglia/Astrocyte Interaction in the Spinal Cord Enhances Neuropathic Pain Processing after Nerve Injury. The Journal of Neuroscience 28, 12775-12787. Moore, K.A., Kohno, T., Karchewski, L.A., Scholz, J., Baba, H., Woolf, C.J., 2002. Partial Peripheral Nerve Injury Promotes a Selective Loss of GABAergic Inhibition in the Superficial Dorsal Horn of the Spinal Cord. The Journal of Neuroscience 22, 6724-6731. Morley, S., Eccleston, C., Williams, A., 1999. Systematic review and meta-analysis of randomized controlled trials of cognitive behaviour therapy and behaviour therapy for chronic pain in adults, excluding headache. Pain 80, 1-13. Nash, P.G., Macefield, V.G., Klineberg, I.J., Gustin, S.M., Murray, G.M., Henderson, L.A., 2010. Bilateral activation of the trigeminothalamic tract by acute orofacial cutaneous and muscle pain in humans. PAIN® 151, 384-393. Nash, P.G., Macefield, V.G., Klineberg, I.J., Murray, G.M., Henderson, L.A., 2009. Differential activation of the human trigeminal nuclear complex by noxious and non-noxious orofacial stimulation. Human Brain Mapping 30, 3772-3782. Nelson, A.J., Chen, R., 2008. Digit somatotopy within cortical areas of the postcentral gyrus in humans. Cerebral Cortex 18, 2341-2351. Newman, E.A., 1986. High potassium conductance in astrocyte endfeet. Science 233, 453-454. Nurmikko, T.J., Eldridge, P.R., 2001. Trigeminal neuralgia—pathophysiology, diagnosis and current treatment. BJA: British Journal of Anaesthesia 87, 117-132. O'connor, E., Kimelberg, H., 1993. Role of calcium in astrocyte volume regulation and in the release of ions and amino acids. Journal of Neuroscience 13, 2638-2650. Ohara, P.T., Vit, J.P., Bhargava, A., Jasmin, L., 2008. Evidence for a role of connexin 43 in trigeminal pain using RNA interference in vivo. J Neurophysiol 100, 3064-3073. Olszewski, J., 1950. On the anatomical and functional organization of the spinal trigeminal nucleus. Journal of Comparative Neurology 92, 401-413. Papadopoulos, M.C., Manley, G.T., Krishna, S., Verkman, A., 2004. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. The FASEB journal 18, 1291-1293. Pasti, L., Volterra, A., Pozzan, T., Carmignoto, G., 1997. Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. Journal of Neuroscience 17, 7817-7830. Pattany, P.M., Yezierski, R.P., Widerström-Noga, E.G., Bowen, B.C., Martinez-Arizala, A., Garcia, B.R., Quencer, R.M., 2002. Proton magnetic resonance spectroscopy of the thalamus in patients with chronic neuropathic pain after spinal cord injury. American Journal of Neuroradiology 23, 901-905. Paxinos, G., Xu-Feng, H., Sengul, G., Watson, C., 2012. Chapter 8 - Organization of Brainstem Nuclei. The Human Nervous System (Third Edition). Academic Press, San Diego, pp. 260-327. Pekny, M., Johansson, C.B., Eliasson, C., Stakeberg, J., Wallen, A., Perlmann, T., Lendahl, U., Betsholtz, C., Berthold, C.H., Frisen, J., 1999. Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin. J Cell Biol 145, 503-514. Pekny, M., Nilsson, M., 2005. Astrocyte activation and reactive gliosis. Glia 50, 427-434. Penfield, W., Boldrey, E., 1937. Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation. Brain 60, 389-443. Penfield, W., Rasmussen, T., 1950. The cerebral cortex of man; a clinical study of localization of function. Perl, E.R., 1968. Myelinated afferent fibres innervating the primate skin and their response to noxious stimuli. The Journal of Physiology 197, 593-615. Petrosino, S., Palazzo, E., de Novellis, V., Bisogno, T., Rossi, F., Maione, S., Di Marzo, V., 2007. Changes in spinal and supraspinal endocannabinoid levels in neuropathic rats. Neuropharmacology 52, 415-422.
35 Peyron, R., Garcia-Larrea, L., Gregoire, M.C., Convers, P., Lavenne, F., Veyre, L., Froment, J.C., Mauguiere, F., Michel, D., Laurent, B., 1998. Allodynia after lateral-medullary (Wallenberg) infarct. A PET study. Brain 121 ( Pt 2), 345-356. Peyron, R., Laurent, B., Garcia-Larrea, L., 2000. Functional imaging of brain responses to pain. A review and meta-analysis (2000). Neurophysiol Clin 30, 263-288. Pinault, D., 2004. The thalamic reticular nucleus: structure, function and concept. Brain Res Brain Res Rev 46, 1-31. Pleger, B., Tegenthoff, M., Ragert, P., Forster, A.F., Dinse, H.R., Schwenkreis, P., Nicolas, V., Maier, C., 2005. Sensorimotor retuning [corrected] in complex regional pain syndrome parallels pain reduction. Ann Neurol 57, 425-429. Pleger, B., Tegenthoff, M., Schwenkreis, P., Janssen, F., Ragert, P., Dinse, H.R., Völker, B., Zenz, M., Maier, C., 2004. Mean sustained pain levels are linked to hemispherical side-to-side differences of primary somatosensory cortex in the complex regional pain syndrome I. Experimental Brain Research 155, 115-119. Polycarpou, N., Ng, Y.L., Canavan, D., Moles, D.R., Gulabivala, K., 2005. Prevalence of persistent pain after endodontic treatment and factors affecting its occurrence in cases with complete radiographic healing. International Endodontic Journal 38, 169-178. Purves, D., 2004. Neuroscience, Third ed. Sinauer Associates Inc, Sunderland MA. Ralston, H.J., 3rd, 2005. Pain and the primate thalamus. Prog Brain Res 149, 1-10. Raso, G.M., Esposito, E., Vitiello, S., Iacono, A., Santoro, A., D’Agostino, G., Sasso, O., Russo, R., Piazza, P.V., Calignano, A., Meli, R., 2011. Palmitoylethanolamide Stimulation Induces Allopregnanolone Synthesis in C6 Cells and Primary Astrocytes: Involvement of Peroxisome- Proliferator Activated Receptor-α. Journal of Neuroendocrinology 23, 591-600. Sarnthein, J., Stern, J., Aufenberg, C., Rousson, V., Jeanmonod, D., 2006. Increased EEG power and slowed dominant frequency in patients with neurogenic pain. Brain 129, 55-64. Scholz, J., Broom, D.C., Youn, D.-H., Mills, C.D., Kohno, T., Suter, M.R., Moore, K.A., Decosterd, I., Coggeshall, R.E., Woolf, C.J., 2005. Blocking Caspase Activity Prevents Transsynaptic Neuronal Apoptosis and the Loss of Inhibition in Lamina II of the Dorsal Horn after Peripheral Nerve Injury. The Journal of Neuroscience 25, 7317-7323. Schousboe, A., Sarup, A., Bak, L., Waagepetersen, H., Larsson, O., 2004. Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission. Neurochemistry international 45, 521- 527. Schwarz, J., Weis, S., Kraft, E., Tatsch, K., Bandmann, O., Mehraein, P., Vogl, T., Oertel, W.H., 1996. Signal changes on MRI and increases in reactive microgliosis, astrogliosis, and iron in the putamen of two patients with multiple system atrophy. J Neurol Neurosurg Psychiatry 60, 98-101. Schweinhardt, P., Bushnell, M.C., 2010. Pain imaging in health and disease — how far have we come? The Journal of Clinical Investigation 120, 3788-3797. Schweinhardt, P., Glynn, C., Brooks, J., McQuay, H., Jack, T., Chessell, I., Bountra, C., Tracey, I., 2006. An fMRI study of cerebral processing of brush-evoked allodynia in neuropathic pain patients. Neuroimage 32, 256-265. Sessle, B.J., 1987. Invited Review: The Neurobiology of Facial and Dental Pain: Present Knowledge, Future Directions. Journal of Dental Research 66, 962-981. Sessle, B.J., 2000. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit Rev Oral Biol Med 11, 57-91. Shigenaga, Y., Takabatake, M., Sugimoto, T., Sakai, A., 1979. Neurons in marginal layer of trigeminal nucleus caudalis projecting to ventrobasal complex (VB) and posterior nuclear group (PO) demonstrated by retrograde labeling with horseradish peroxidase. Brain research 166, 391-396. Simone, D.A., Sorkin, L.S., Oh, U., Chung, J.M., Owens, C., LaMotte, R.H., Willis, W.D., 1991. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. Journal of Neurophysiology 66, 228-246.
36 Sindrup, S.H., Jensen, T.S., 1999. Efficacy of pharmacological treatments of neuropathic pain: an update and effect related to mechanism of drug action. PAIN® 83, 389-400. Skaper, S.D., Buriani, A., Dal Toso, R., Petrelli, L., Romanello, S., Facci, L., Leon, A., 1996. The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons. Proc Natl Acad Sci U S A 93, 3984-3989. Sofroniew, M.V., 2009a. Molecular dissection of reactive astrogliosis and glial scar formation. Trends in Neurosciences 32, 638-647. Sofroniew, M.V., 2009b. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 32, 638-647. Sofroniew, M.V., Vinters, H.V., 2010a. Astrocytes: biology and pathology. Acta Neuropathol 119, 7- 35. Sofroniew, M.V., Vinters, H.V., 2010b. Astrocytes: biology and pathology. Acta Neuropathol 119, 7- 35. Solomon, I., 1955. Relaxation Processes in a System of Two Spins. Physical Review 99, 559-565. Spreafico, R., Frassoni, C., Arcelli, P., De Biasi, S., 1994. GABAergic interneurons in the somatosensory thalamus of the guinea-pig: a light and ultrastructural immunocytochemical investigation. Neuroscience 59, 961-973. Stern, J., Jeanmonod, D., Sarnthein, J., 2006. Persistent EEG overactivation in the cortical pain matrix of neurogenic pain patients. Neuroimage 31, 721-731. Stewart, M., 1993. Intermediate filament structure and assembly. Current Opinion in Cell Biology 5, 3-11. Sugimoto, T., Bennett, G.J., Kajander, K.C., 1990. Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine. Pain 42, 205-213. Sur, M., Merzenich, M.M., Kaas, J.H., 1980. Magnification, receptive-field area, and" hypercolumn" size in areas 3b and 1 of somatosensory cortex in owl monkeys. Journal of Neurophysiology 44, 295- 311. Tian, G.-F., Azmi, H., Takano, T., Xu, Q., Peng, W., Lin, J., Oberheim, N., Lou, N., Wang, X., Zielke, H.R., 2005. An astrocytic basis of epilepsy. Nature medicine 11, 973. Todd, A.J., 2010. Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews Neuroscience 11, 823. Tölle, T., Xu, X., Sadosky, A.B., Painful diabetic neuropathy: a cross-sectional survey of health state impairment and treatment patterns. Journal of Diabetes and Its Complications 20, 26-33. Tölle, T.R., Kaufmann, T., Siessmeier, T., Lautenbacher, S., Berthele, A., Munz, F., Zieglgänsberger, W., Willoch, F., Schwaiger, M., Conrad, B., 1999. Region‐specific encoding of sensory and affective components of pain in the human brain: a positron emission tomography correlation analysis. Annals of neurology 45, 40-47. Torrance, N., Smith, B.H., Bennett, M.I., Lee, A.J., 2006. The epidemiology of chronic pain of predominantly neuropathic origin. Results from a general population survey. J Pain 7, 281-289. Tracey, I., 2005. Nociceptive processing in the human brain. Current Opinion in Neurobiology 15, 478-487. Tracey, I., Johns, E., 2010. The pain matrix: reloaded or reborn as we image tonic pain using arterial spin labelling. Pain 148, 359-360. Turner, J.A., Jensen, M.P., Warms, C.A., Cardenas, D.D., 2002. Catastrophizing is associated with pain intensity, psychological distress, and pain-related disability among individuals with chronic pain after spinal cord injury. Pain 98, 127-134. Uhelski, M.L., Davis, M.A., Fuchs, P.N., 2012. Pain affect in the absence of pain sensation: evidence of asomaesthesia after somatosensory cortex lesions in the rat. PAIN® 153, 885-892. Voscopoulos, C., Lema, M., 2010. When does acute pain become chronic? Br J Anaesth 105 Suppl 1, i69-85.
37 Wagner, D.C., Deten, A., Hartig, W., Boltze, J., Kranz, A., 2012. Changes in T2 relaxation time after stroke reflect clearing processes. Neuroimage 61, 780-785. Walton, K.D., Llinas, R.R., 2010. Frontiers in Neuroscience Central Pain as a Thalamocortical Dysrhythmia: A Thalamic Efference Disconnection? In: Kruger, L., Light, A.R. (Eds.), Translational Pain Research: From Mouse to Man. CRC Press/Taylor & Francis Llc., Boca Raton, FL. Walz, W., 1989. Role of glial cells in the regulation of the brain ion microenvironment. Progress in neurobiology 33, 309-333. Wasan, A.D., Loggia, M.L., Chen, L.Q., Napadow, V., Kong, J., Gollub, R.L., 2011. Neural correlates of chronic low back pain measured by arterial spin labeling. Anesthesiology 115, 364-374. Weng, H.R., Lee, J.I., Lenz, F.A., Schwartz, A., Vierck, C., Rowland, L., Dougherty, P.M., 2000. Functional plasticity in primate somatosensory thalamus following chronic lesion of the ventral lateral spinal cord. Neuroscience 101, 393-401. Whiteside, G.T., Munglani, R., 2001. Cell death in the superficial dorsal horn in a model of neuropathic pain. Journal of Neuroscience Research 64, 168-173. Whitsel, B.L., Petrucelli, L.M., Werner, G., 1969. Symmetry and connectivity in the map of the body surface in somatosensory area II of primates. Journal of Neurophysiology 32, 170-183. Woolf, C.J., Ma, Q., 2007. Nociceptors-Noxious Stimulus Detectors. Neuron 55, 353-364. Wrigley, P.J., Press, S.R., Gustin, S.M., Macefield, V.G., Gandevia, S.C., Cousins, M.J., Middleton, J.W., Henderson, L.A., Siddall, P.J., 2009. Neuropathic pain and primary somatosensory cortex reorganization following spinal cord injury. PAIN® 141, 52-59. Wu, W.-C., Fernández-Seara, M., Detre, J.A., Wehrli, F.W., Wang, J., 2007. A theoretical and experimental investigation of the tagging efficiency of pseudocontinuous arterial spin labeling. Magn Reson Med 58, 1020-1027. Yamamura, H., Iwata, K., Tsuboi, Y., Toda, K., Kitajima, K., Shimizu, N., Nomura, H., Hibiya, J., Fujita, S., Sumino, R., 1996. Morphological and electrophysiological properties of ACCx nociceptive neurons in rats. Brain research 735, 83-92. Yen, C.-T., Lu, P.-L., 2013. Thalamus and pain. Acta Anaesthesiologica Taiwanica 51, 73-80. Yoldas, O., Topuz, A., Isçi, A.S., Oztunc, H., Postoperative pain after endodontic retreatment: Single- versus two-visit treatment. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology 98, 483- 487. Youssef, A.M., Gustin, S.M., Nash, P.G., Reeves, J.M., Petersen, E.T., Peck, C.C., Murray, G.M., Henderson, L.A., 2014. Differential brain activity in subjects with painful trigeminal neuropathy and painful temporomandibular disorder. PAIN® 155, 467-475. Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114, 149-159. Zhuang, Z.Y., Wen, Y.R., Zhang, D.R., Borsello, T., Bonny, C., Strichartz, G.R., Decosterd, I., Ji, R.R., 2006. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J Neurosci 26, 3551-3560. Zonta, M., Angulo, M.C., Gobbo, S., Rosengarten, B., Hossmann, K.-A., Pozzan, T., Carmignoto, G., 2002. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nature Neuroscience 6, 43.
38 CHAPTER TWO
CHRONIC NEUROPATHIC PAIN: IT’S ABOUT THE RHYTHM
Chapter Two is published as:
Z. Alshelh, F. Di Pietro, A. M. Youssef, J. M. Reeves, P. M. Macey, E.R. Vickers, C.C. Peck G.M.
Murray and L.A. Henderson. Chronic Neuropathic Pain: It’s about the Rhythm. The Journal of
Neuroscience, January 20, 2016, 36(3):1008 –1018.
39 1008 • The Journal of Neuroscience, January 20, 2016 • 36(3):1008–1018
Systems/Circuits Chronic Neuropathic Pain: It’s about the Rhythm
Zeynab Alshelh,1 Flavia Di Pietro,1 Andrew M. Youssef,1 Jenna M. Reeves,1 Paul M. Macey,3 E. Russell Vickers,1 Christopher C. Peck,2 Greg M. Murray,2 and XLuke A. Henderson1 1Department of Anatomy and Histology and 2Faculty of Dentistry, University of Sydney, Sydney, New South Wales 2006, Australia, and 3School of Nursing and Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90095
The neural mechanisms underlying the development and maintenance of chronic neuropathic pain remain unclear. Evidence from human investigations suggests that neuropathic pain is associated with altered thalamic burst firing and thalamocortical dysrhythmia. Additionally, experimental animal investigations show that neuropathic pain is associated with altered infra-slow (Ͻ0.1 Hz) frequency oscillations within the dorsal horn and somatosensory thalamus. The aim of this investigation was to determine whether, in humans, neuropathic pain was also associated with altered infra-slow oscillations within the ascending “pain” pathway. Using resting-state functional magnetic resonance imaging, we found that individuals with orofacial neuropathic pain have increased infra-slow oscillatory activity throughout the ascending pain pathway, including within the spinal trigeminal nucleus, somatosensory thalamus, thalamic reticular nucleus, and primary somatosensory cortex. Furthermore, these infra-slow oscillations were temporally coupled across these multiple sites and occurred at frequencies similar to calcium waves in activated astrocytes. The region encompassing the spinal trigem- inal nucleus also displayed increased regional homogeneity, consistent with a local spread of neural activity by astrocyte activation. In contrast, no increase in oscillatory behavior within the ascending pain pathway occurred during acute noxious stimuli in healthy individuals. These data reveal increased oscillatory activity within the ascending pain pathway that likely underpins increased thalamo- cortical oscillatory activity, a self-sustaining thalamocortical dysrhythmia, and the constant perception of pain. Key words: astrocytes; infra-slow oscillations; orofacial pain; regional homogeneity; spinal trigeminal nucleus; thalamocortical rhythm
Significance Statement Chronic neuropathic pain is associated with altered thalamic firing and thalamocortical dysrhythmia. The mechanisms respon- sible for these changes remain unknown. In this study, we report in individuals with neuropathic pain increased oscillatory neural activity within the ascending pain pathway with evidence that these changes result from altered neural–astrocyte coupling. We propose a series of neural and glial events after nerve injury that result in the generation of altered thalamocortical activity and a persistent neuropathic pain state. Defining the underlying mechanisms responsible for neuropathic pain is critical if we are to develop more effective treatment regimens.
Introduction clinical misdiagnoses as a result of the lack of quantitative and Neuropathic pain (NP) is a disease state with an enormous validated tests, and current treatment regimens are ineffective socioeconomic burden and with unmet needs for the popula- in a significant number of individuals. Hindering our ability to tion. In the United States alone, it is estimated to afflict ϳ10% improve diagnoses and treatment is our poor understanding of the population, costing on average $17,000 per patient of the neural mechanisms responsible for the development yearly (Wang et al., 2009a,c). Additionally, there are frequent and maintenance of chronic NP. Surprisingly, although acute noxious stimuli evoke robust signal increases in a well- described set of neural structures (Farrell et al., 2005) and Received July 21, 2015; revised Nov. 27, 2015; accepted Dec. 14, 2015. individuals with chronic pain display increased activation Author contributions: F.D.P., J.M.R., P.M.M., E.R.V., C.C.P., G.M.M., and L.A.H. designed research; Z.A., F.D.P., during acute noxious stimuli in these same regions (Gracely et A.M.Y., J.M.R., C.C.P., G.M.M., and L.A.H. performed research; Z.A., A.M.Y., and P.M.M. analyzed data; Z.A., F.D.P., E.R.V., and L.A.H. wrote the paper. al., 2002; Giesecke et al., 2004), multiple human brain imaging We are grateful to Dr. Gustin for help with data collection. This work was supported by funding from the Austra- investigations have not found robust increases in ongoing lian National Health and Medical Research Council and the N.W.G. Macintosh Memorial Fund. blood flow in these same regions in individuals with chronic The authors declare no competing financial interests. pain, particularly NP. Indeed, the most consistent differe- CorrespondenceshouldbeaddressedtoLukeA.Henderson,DepartmentofAnatomyandHistology,F13,Univer- sity of Sydney, Sydney, New South Wales 2006, Australia. E-mail: [email protected]. nce is a thalamic blood flow decrease (Hsieh et al., 1995; Iada- DOI:10.1523/JNEUROSCI.2768-15.2016 rola et al., 1995; Moisset and Bouhassira, 2007; Youssef et al., Copyright © 2016 the authors 0270-6474/16/361008-11$15.00/0 2014). 40 Alshelh et al. • Neuropathic Pain and Brain Rhythm J. Neurosci., January 20, 2016 • 36(3):1008–1018 • 1009
Despite the apparent lack of ongoing blood flow increases in Table 1. PTN subject characteristics most of the ascending pain pathway, NP is associated with altered Pain Pain thalamic firing, including increased burst firing in the somato- Age duration intensity sensory thalamus without an overall increase in activity (Lenz et Subject (years) Gender (years) (VAS) Location Analgesic medication BDI PCS al., 1989). Experimental animal investigations confirm that in- 1 51 Male 3.5 2.1 Right None 6 10 creased somatosensory thalamus burst firing is associated with 2 47 Female 5 1.1 Left None 24 47 NP and furthermore have revealed that this altered firing occurs 3 52 Female 1.5 6.9 Bilateral Oxycodone hydrochloride, 18 29 in a rhythmic manner, with burst intensity oscillating at infra- paracetamol slow frequencies (Gerke et al., 2003; Iwata et al., 2011). It is 4 55 Female 2 5.2 Left Docusate sodium; sennosides, 21 25 thought that this altered thalamic firing underpins the thalamo- amitriptyline, gabapentin, cortical dysrhythmia that occurs in individuals with NP and that oxycodone, paracetamol results in the constant perception of pain (Sarnthein et al., 2006; 5 42 Female 11 4.8 Right Carbamazepine 34 36 Walton and Llina´s, 2010). Coincidently, thalamic astrocytes dis- 6 54 Female 6.5 1.6 Right Amitriptyline 9 20 7 44 Female 2 6.4 Left None 11 15 play calcium wave oscillations that modulate neurotransmitter 8 41 Female 3.5 4.0 Left None 6 11 release from adjacent neurons, at approximately the same infra- 9 67 Female 14 8.4 Left None 4 29 slow frequency range as the thalamic neuronal oscillations re- 10 43 Male 16 5.8 Left Desvenlafaxine 5 9 ported in animal models of NP (Crunelli et al., 2002), and it has 11 44 Female 1.5 1.2 Right Ibuprofen, codeine 19 25 been shown that NP is associated with astrocyte activation within phosphate, paracetamol the ascending pain pathway (Okada-Ogawa et al., 2009; Shi et al., 12 65 Female 7 2.5 Left Amitriptyline, paracetamol 6 11 2012; Ji et al., 2013). Thus, it is conceivable that, in individuals 13 50 Female 7 0.5 Right None 6 2 with NP, an increase in oscillatory astrocyte activity within the dor- 14 67 Female 20 4.2 Right Thyroxine sodium, rosuvastatin, 32 18 sal horn and/or somatosensory thalamus results in an increase venlafaxine, pregabalin, thalamocortical oscillatory activity, a self-sustaining thalamocortical phenylethylamine, dysrhythmia, and the constant perception of pain. ergotamine We investigated whether individuals with NP displayed in- 15 67 Female 3 8.4 Left None 6 5 16 43 Male 9 5.8 Left Desvenlafaxine 30 29 creased infra-slow oscillatory activity in ascending pain path- 17 44 Female 7.5 1.2 Right None 5 35 ways, including the level of the primary afferent synapse and whether this activity dominates information flow throughout the brain. Because we propose that pathological processes including 240 ϫ 240 mm; matrix size, 80 ϫ 78; slice thickness, 4 mm; repetition chronic astrocyte activation play a critical role in generating time, 2000 ms; echo time, 30 ms; flip angle, 90°). In each subject, a infra-slow oscillations, we hypothesized that, in healthy individ- high-resolution 3D T1-weighted anatomical image set covering the en- uals, activation of the ascending pain pathway by an acute nox- tire brain was collected (turbo field echo; field of view, 250 ϫ 250 mm; ious stimulus does not increase infra-slow oscillatory activity. matrix size, 288 ϫ 288; slice thickness, 0.87 mm; repetition time, 5600 ms; echo time, 2.5 ms; flip angle, 8°). In addition, in 15 of the control subjects, a catheter connected to a Materials and Methods syringe filled with hypertonic saline (5%) was placed into the right mas- Subjects. Seventeen subjects with chronic orofacial NP (14 females; seter muscle midway between its upper and lower borders. The catheter mean Ϯ SEM age, 50.6 Ϯ 2.8 years) and 44 pain-free controls (33 females; was connected to an infusion pump with a 10 ml syringe placed outside mean age, 45.9 Ϯ 2.0 years) were recruited for the study. There was no the scanner room. An infusion of hypertonic saline (4 ml/h) into the right significant difference in age (t test, p Ͼ 0.05) or gender composition ( 2 masseter muscle was then started, and the subject indicated by pushing a test, p Ͼ 0.05) between the two subject groups. NP subjects were diag- buzzer when the pain intensity reached a score of 5 of 10 on a numerical nosed using the Liverpool criteria as having posttraumatic neuropathy 11 point VAS. While in pain, a series of 180 fMRI image volumes were (Nurmikko and Eldridge, 2001). then collected using the same parameters as described above. Each sub- During the 7 d before the MRI session, each subject kept a pain diary ject was instructed to push a buzzer once if the pain fell below 5 and twice recording the intensity of their ongoing pain, three times a day. Subjects if it increased above 5, and the infusion rate was adjusted accordingly to rated the intensity of their pain using a 10 cm horizontal visual analog maintain the pain intensity at 5 of 10. After the scan, each subject was scale (VAS), with 0 indicating “no pain” and 10 indicating “the most asked to rate the average pain during the scan on a 10 cm horizontal VAS. intense imaginable pain.” These pain intensity scores were then averaged Each NP and control subject was scanned only once, and all data were over the 7 d period to create a mean pain intensity score. On the day of the used for additional analysis. Furthermore, to reduce the potential effects MRI scanning, each NP subject outlined the location of their ongoing of software upgrades and other factors that can alter signal-to-noise ra- pain on a standard drawing of the head and completed a McGill Pain tios, we collected the control and NP subject data randomly throughout Questionnaire, the Beck’s Depression Inventory (BDI; Beck et al., 1961), a 3.5 year period during which this investigation was conducted. and the Pain Catastrophizing Scale (PCS; Sullivan et al., 1995). Table 1 MRI analysis. Using SPM12 (Friston et al., 1995), all functional shows NP subject characteristics, including pain duration, pain location, magnetic resonance images were motion corrected, and global signal 7 d pain intensity, medication, depression, and pain catastrophizing drifts were removed using the detrending method described by Macey et scores. Informed written consent was obtained for all procedures accord- al. (2004), spatially normalized to the Montreal Neurological Institute ing to the Declaration of Helsinki, and the study was approved by our (MNI) template, and spatially smoothed (6 mm full-width half-max- local Institutional Human Research Ethics Committees. Data from 13 of imum Gaussian filter). In no subject was there significant movement the 17 NP subjects were used in previous investigations (Gustin et al., (Ͼ0.5 mm in any direction). In those NP subjects with pain restricted to 2011; Henderson et al., 2013; Wilcox et al., 2015). the left side of the face (n ϭ 9), resting fMRI scans were reflected across MRI acquisition. All NP and control subjects lay supine on the bed of a the midline so that, in all NP subjects, the right side was ipsilateral to 3 tesla MRI scanner (Achieva; Philips) with their head immobilized in a ongoing pain. Given this, the individual analyzing the data was not tight-fitting head coil. With each subject relaxed and at rest, a series of blinded to the subject groups. To assess thalamic oscillatory activity, we 180 gradient echo echo-planar functional MRI (fMRI) image volumes placed a cuboid over the left (contralateral) thalamus, covering 18, 24, using blood oxygen level-dependent contrast were collected. Each image and 15 mm in the x, y, and z directions, respectively. This cube was then volume contained 35 axial slices covering the entire brain (field of view, divided into 240 individual volumes of interest (VOIs) that were 3 ϫ 3 ϫ 41 1010 • J. Neurosci., January 20, 2016 • 36(3):1008–1018 Alshelh et al. • Neuropathic Pain and Brain Rhythm
Figure 1. Infra-slow oscillations within the thalamus. a, A cuboid 18 ϫ 24 ϫ 15 mm consisting of 240 individual VOIs was placed over the left (contralateral to highest pain) thalamus in 44 controls and 16 subjects with orofacial NP. Slice locations are indicated by the horizontal lines on the coronal section and the numbers to the top right of each axial slice in MNI space. At each rostrocaudal level of the thalamus, 48 VOIs were assessed for significant power differences. b, Each gray band beside an axial image consists of a row for each VOI in that slice, with spectral power differences between control and NP subjects (controls Ͼ NP or controls Ͻ NP) at frequencies between 0.006 and 0.25 Hz color coded for significance (p value). Gray represents no significant difference. c, A plot of the percentage of significantly different voxels over the entire frequency spectrum revealed that the frequency band 0.03–0.06 Hz had the highest percentage of significa- ntly different VOIs. This frequency band was used for all subsequent analysis.
3 mm each (Fig. 1a). The individual resting fMRI signal intensity changes we divided these ALFF values by total power over the entire frequency in each of these 240 VOIs were then derived for each of the 44 control and range to obtain fractional ALFF (fALFF) values for each voxel. Both 17 NP subjects from the nonspatially smoothed fMRI image sets. Power ALFFs and fALFFs have been shown to have high test–retest reliability, spectra based on fast Fourier transforms were calculated using MATLAB particularly in gray matter, and reliability is reportedly greater in ALFFs software (MathWorks) for each individual subject’s resting fMRI signal. than fALFFs (Zuo et al., 2010). Furthermore, parcellation-based reliabil- Power values were then averaged over each VOI for the control and NP ity analyses demonstrate highly consistent and reliable rank ordering of groups, and significant differences between power spectra were deter- infra-slow oscillatory power across the brain. These findings are consis- mined at 0.002 Hz intervals from 0.006 to 0.25 Hz (two-sample t tests, tent with ALFFs and fALFFs reflecting neuronal activity. Significant dif- p Ͻ 0.05). Significant differences between control and NP groups were ferences between groups were then determined using a two-sample color coded for significance, and the percentage of significantly different random-effects procedure with age and gender as nuisance variables for VOIs at each frequency were plotted. This analysis revealed that the both ALFF and fALFF values (p Ͻ 0.05, false discovery rate corrected, frequency band with the greatest number of significantly different VOIs minimum cluster size of 10 voxels). For each significant cluster, power was 0.03–0.06 Hz, and this band was used for subsequent analyses. spectra were calculated from resting percentage change fMRI signals, and Low-frequency amplitude analysis. Using the SPM toolbox REST (Song the power at each frequency was calculated. We then plotted the mean et al., 2011), we calculated the sum of amplitudes of low-frequency fluc- power between 0.03 and 0.06 Hz for control and NP subjects. tuations (ALFFs) between 0.03 and 0.06 Hz for each voxel in control and To determine the potential effects of head movement, for each move- NP subjects using the spatially smoothed fMRI image sets. In addition, ment parameter (x, y, z, pitch, roll, yaw), power spectra were calculated, 42 Alshelh et al. • Neuropathic Pain and Brain Rhythm J. Neurosci., January 20, 2016 • 36(3):1008–1018 • 1011
Figure2. NPisassociatedwithincreasedinfra-slowoscillationpowerintheascendingpainpathway.BrainregionsinwhichNPsubjectshadsignificantlyincreased(hotcolor)ordecreased(cool color) ALFFs (a) and fALFFs (b; random effects, p Ͻ 0.05, false discovery rate corrected, minimum cluster size of 10 voxels). Location of each axial slice in MNI space is indicated at the top right. The green shading indicates the thalamic region activated during innocuous brushing of the right lip, i.e., the orofacial somatosensory thalamus. NP is associated with increased infra-slow oscillations in the region of the primary afferent synapse (spinal trigeminal nucleus), as well as other parts of the ascending pain pathway. dlPFC, Dorsolateral prefrontal cortex. c, Power spectrum plots of four clusters in which power in the 0.03–0.06 Hz range was significantly increased in NP subjects (red) compared with controls (black). Plots of the mean Ϯ SEM power between 0.03 and 0.06 Hz are shown for controls and NP subjects. Note that, at frequencies above 0.06 Hz, power was remarkably similar in both control and NP subjects. cont, Controls; ipsi, ipsilateral. and the mean power between 0.03 and 0.06 Hz in control and NP groups r value of 0.25). A binary, undirected adjacency matrix was then ob- were compared (p Ͻ 0.05, two-tailed, two-sample t test). The effects of tained, and degree centrality was calculated by counting the number of BDI and PCS were determined by assessing their relationships with 0.03– significant correlations between each voxel and all other voxels. For NP 0.06 Hz power in significant clusters in NP subjects (p Ͻ 0.05). The effect subjects, voxels with the greatest degree centrality were determined using of medication on infra-slow oscillations was determined by comparing a one-sample t test (p Ͻ 0.05, false discovery rate corrected, minimum 0.03–0.06 Hz power in each significant cluster in NP subjects taking cluster size of 10 voxels). To determine whether the resulting significant analgesic medications (n ϭ 9) with those not taking medication (n ϭ 8; clusters differed between NP and control subjects, degree centrality val- p Ͻ 0.05, two-tailed, two-sample t test). In addition, the effects of later- ues were extracted from each cluster in all subjects and compared using ality were tested by reflecting each significant cluster across the midline two-sample t tests (two-tailed, two-sample t test, p Ͻ 0.05). and comparing mean power between 0.03–0.06 Hz in control and NP Local homogeneity analysis. To determine the role of factors such as the groups (p Ͻ 0.05, two-tailed, two-sample t test). Finally, to determine influence of astrocyte activation on neural function (albeit indirect evi- whether changes in low-frequency oscillations were temporally coupled dence), we measured Kendall’s coefficient of concordance (KCC), which between the resulting significant clusters, cross-correlations were per- evaluates the similarity of the time series within each voxel and its nearest formed between resting fMRI signal intensity changes in each significant neighbors. Using the DPARSF toolbox (Chao-Gan and Yu-Feng, 2010), cluster in control and NP subjects (5 volume moving average applied to the preprocessed images were bandpass filtered (0.03–0.06 Hz), and each cross-correlation curve). voxel-based graphs were generated for each individual subject. For each Degree centrality analysis. To assess the influence of infra-slow fre- voxel, the KCC was computed from the time course of that voxel and its quency oscillations on local network connectivity, we measured degree 19 neighboring voxels. Significant differences in regional homogeneity centrality. Using the DPARSF toolbox (Chao-Gan and Yu-Feng, 2010), between NP and controls subjects were then determined using a two- the preprocessed images were bandpass filtered (0.03–0.06 Hz), and sample random-effects procedure (p Ͻ 0.05, false discovery rate cor- voxel-based graphs were generated for each individual. Correlations be- rected, minimum cluster size of 10 voxels). tween fMRI time series of each voxel with every other voxel in the brain Acute pain analysis. Finally, we determined changes in infra-slow fre- were computed, resulting in a correlation matrix (minimum correlation quency oscillations (0.03–0.06 Hz) in 15 subjects during acute noxious 43 1012 • J. Neurosci., January 20, 2016 • 36(3):1008–1018 Alshelh et al. • Neuropathic Pain and Brain Rhythm
Table 2. MNI coordinates, cluster size, and t score for regions of significant infra-slow oscillation power in the dorsolateral medulla in the difference between control and NP patients region of the ipsilateral (right) spinal trigeminal nucleus subnu- MNI coordinate cleus caudalis/interpolaris (SpV; mean Ϯ SEM, 0.03–0.06 Hz Brain region xyzCluster size t score power; controls, 2.2 Ϯ 0.4; NP, 9.5 Ϯ 3.6; p ϭ 0.0007), contralat- eral medial thalamus encompassing the mediodorsal, centrolat- Amplitudes of low-frequency oscillations Ϯ Ͼ eral, and orofacial somatosensory nuclei (controls, 1.1 0.1; NP, Controls NP Ϯ ϭ Right dorsolateral prefrontal cortex 8 30 20 50 4.41 2.3 0.5; p 0.0004), contralateral lateral thalamus encompass- NP Ͼ controls ing the region of the thalamic reticular nucleus (TRN; controls, Ipsilateral spinal trigeminal nucleus 6 Ϫ40 Ϫ50 31 4.33 0.9 Ϯ 0.1; NP, 1.7 Ϯ 0.3; p ϭ 0.002), and the orofacial represen- Ipsilateral cerebellar cortex 24 Ϫ66 Ϫ26 114 4.09 tation of contralateral primary somatosensory cortex (S1; con- Contralateral somatosensory thalamus Ϫ8 Ϫ24 6 105 5.42 trols, 3.2 Ϯ 0.4; NP, 7.0 Ϯ 1.2; p ϭ 0.0001; Fig. 2c; Table 2, Ϫ Ϫ Contralateral dorsal posterior insula 40 16 20 558 3.62 Amplitudes of low-frequency oscillations and fractional ampli- Contralateral S1 Ϫ64 Ϫ18 18 5 4.98 Fractional amplitudes of low-frequency oscillations tudes of low-frequency oscillations). The location of the con- NP Ͼ controls tralateral somatosensory thalamus was defined by overlaying the Contralateral somatosensory thalamus Ϫ6 Ϫ24 8 31 4.84 change in low-frequency power with functional activation during Contralateral TRN Ϫ12 Ϫ10 6 28 4.40 repeated innocuous brushing of the right side of the mouth as Degree centrality reported previously (Henderson et al., 2013). The location of all Ϫ Ϫ Ipsilateral spinal trigeminal nucleus 10 38 48 26 13.89 other clusters were derived from our previous investigations and Contralateral dorsolateral pons Ϫ8 Ϫ38 Ϫ34 40 13.27 Ipsilateral cerebellar cortex 24 Ϫ70 Ϫ30 199 17.57 human brain atlases (Duvernoy, 1995; Paxinos and Huang, 1995; Contralateral cerebellar cortex Ϫ26 Ϫ88 Ϫ32 66 15.41 Mai et al., 2007), and we are confident that the changes reported Ipsilateral insula 40 8 0 40 16.42 encompass the nuclei described. We confirmed that these infra- 40 Ϫ4 10 17 12.81 slow oscillations were restricted to the frequency range of 0.03– Contralateral medial thalamus Ϫ6 Ϫ24 10 29 12.84 0.06 Hz by extracting and plotting the entire frequency range Contralateral TRN Ϫ12 Ϫ8 4 23 15.18 (0–0.25 Hz; Fig. 2c), and we are confident that movement did not Ϫ Ϫ Contralateral S1 60 12 34 31 13.05 have a significant effect on these results because there was no Ϫ50 Ϫ16 46 53 10.21 Ipsilateral dorsolateral prefrontal cortex 38 38 8 25 17.25 significant difference in movement-related power between con- 38 2 34 61 15.10 trols and NP subjects in any of the six directions (x, p ϭ 0.13; y, 28 22 48 16 13.14 p ϭ 0.30; z, 0.35; pitch, p ϭ 0.57; roll, p ϭ 0.43; yaw, p ϭ 0.59). Contralateral dorsolateral prefrontal cortex Ϫ28 Ϫ20 42 70 14.15 In NP subjects, we found no significant relationship between Mid-cingulate cortex Ϫ2 Ϫ12 28 33 13.19 mean 0.03–0.06 Hz power in any of these ascending pain path- Regional homogeneity ϭ Ͼ way clusters identified above with either BDI (ipsilateral SpV, r NP controls ϭ ϭ ϭ Ipsilateral spinal trigeminal nucleus 6 Ϫ40 Ϫ48 14 5.97 0.35, p 0.19; contralateral medial thalamus, r 0.06, p 0.82; Ipsilateral cerebellar cortex 20 Ϫ52 Ϫ18 21 5.03 contralateral TRN, r ϭϪ0.06, p ϭ 0.82; contralateral S1, r ϭ Contralateral cingulate cortex Ϫ4 Ϫ32 28 64 6.36 0.11, p ϭ 0.67) or PCS (ipsilateral SpV, r ϭ 0.33, p ϭ 0.21; Ϫ6 Ϫ4 46 76 6.19 contralateral medial thalamus, r ϭϪ0.03, p ϭ 0.92; contralateral Ϫ8 20 34 36 5.80 TRN, r ϭϪ0.09, p ϭ 0.72; contralateral S1, r ϭ 0.32, p ϭ 0.23). There were also no significant differences in mean power in these clusters in NP subjects on medications compared with those not stimulus (masseter muscle pain) compared with all 44 controls. We cal- on medications (mean Ϯ SEM power; ipsilateral SpV: medica- culated ALFF and fALFF maps using the spatially smoothed fMRI image Ϯ Ϯ ϭ sets, and significant differences between controls and acute pain were tions, 10.2 6.2; no medications, 8.8 4.0, p 0.43; contralat- Ϯ determined for both ALFF and fALFF values using a two-sample eral medial thalamus: medications, 2.4 0.7; no medications, random-effects procedure with age and gender as nuisance variables 2.2 Ϯ 0.8, p ϭ 0.41; contralateral TRN: medications, 1.8 Ϯ 0.5; no (p Ͻ 0.05, false discovery rate corrected, minimum cluster size of 10 medications, 1.5 Ϯ 0.4, p ϭ 0.329; contralateral S1: medications, voxels). Furthermore, for those clusters that displayed a significant in- 7.1 Ϯ 2.1; no medications, 6.9 Ϯ 1.3, p ϭ 0.46). There were also crease in infra-slow frequency oscillations in NP subjects during the first no significant correlations between pain intensity or pain dura- analysis, signal intensity changes were extracted for each of the subjects tion and mean 0.03–0.06Hz power in any of these ascending pain during acute pain and in controls. For each cluster, power spectra were pathway clusters (pain intensity: ipsilateral SpV, r ϭ 0.05, p ϭ calculated, and significant differences between acute pain and no pain sub- 0.86; contralateral medial thalamus, r ϭϪ0.18, p ϭ 0.51; con- jects were determined in the same process described above (two-sample t ϭϪ ϭ ϭϪ Ͻ tralateral TRN, r 0.09, p 0.74; contralateral S1, r 0.09, test, p 0.05). The anatomical locations of each significant cluster were ϭ ϭϪ ϭ confirmed using the Atlas of the Human Brainstem and Cerebellum (Duver- p 0.74; pain duration: ipsilateral SpV, r 0.07, p 0.81; ϭϪ ϭ noy, 1995) and the Atlas of the Human Brain (Mai et al., 2007). contralateral medial thalamus, r 0.41, p 0.11; contralateral TRN, r ϭϪ0.50, p ϭ 0.06; contralateral S1, r ϭϪ0.45, p ϭ 0.08). Results Furthermore, reflecting these clusters across the midline revealed Altered infra-slow oscillations in ascending pain pathways no significant differences in mean power between controls and Power spectra based on fast Fourier transforms of resting fMRI NP subjects in the ipsilateral SpV (mean Ϯ SEM power; controls, signal intensity fluctuations within the left thalamus (contralat- 8.1 Ϯ 4.7; NP subjects, 8.9 Ϯ 3.7, p ϭ 0.45), contralateral TRN eral to highest ongoing pain) revealed that the greatest number of (controls, 0.9 Ϯ 0.1; NP subjects, 1.6 Ϯ 0.5, p ϭ 0.08), or con- voxels in which power was significantly different in NP subjects tralateral S1 (controls, 4.2 Ϯ 0.6; NP subjects, 4.3 Ϯ 1.1, p ϭ compared with controls occurred within the frequency band 0.47), although there was a significant increase in NP subjects in 0.03–0.06 Hz (Fig. 1b). Using this restricted frequency range the contralateral medial thalamus (controls, 0.9 Ϯ 0.1; NP sub- (0.03–0.06 Hz), a voxel-by-voxel analysis revealed a pattern of jects, 2.4 Ϯ 0.5, p ϭ 0.003). difference that closely overlapped the ascending pain pathway To determine whether infra-slow oscillation power increases in (Fig. 2a,b). That is, NP subjects had a significantly increased the ascending pain pathway were temporally coupled, we performed 44 Alshelh et al. • Neuropathic Pain and Brain Rhythm J. Neurosci., January 20, 2016 • 36(3):1008–1018 • 1013
cross-correlation analyses of resting fMRI signal intensity changes between the SpV, medial thalamus, TRN, and S1. In both con- trol and NP subjects, signal intensity cou- pling occurred between the SpV and medial thalamus and between the medial thalamus and TRN (Fig. 3). However, in NP subjects only, additional correlation peaks occurred at regular time lag intervals of ϳ30 s (0.03 Hz), reflecting the presence of temporally coupled increased infra-slow oscillations within the ascending pain pathway.
Altered network connectivity in ascending pain pathways The influence of these low-frequency os- cillatory differences within the ascending pain pathway on local network connectiv- ity was assessed by using degree centrality. Figure 3. Signal intensity fluctuations in the ascending pain pathway are coupled temporally. Graph shows cross-correlations Network theory methodologies such as of resting signal intensity changes within the ascending pain pathway in controls and NP subjects. Tight temporal coupling with zerotimelagoccurredbetweenthespinaltrigeminalnucleus,somatosensorythalamus,andTRN.Alagofϳ4soccurredbetween degree centrality treat the brain as an in- the somatosensory thalamus and cortex. Also note the, in only NP subjects, correlation peaks (indicated by red asterisks) occurred tegrated network with each voxel consid- at ϳ30 s apart, i.e., indicating ongoing increased infra-slow oscillations in the 0.03 Hz range. ered a node in a network with connections between nodes defined by their resting activity (Bullmore and Sporns, 2009). Degree centrality identifies the most con- nected nodes by counting the number of other voxels that display similar signal intensity fluctuations. It defines central nodes with the highest number of connec- tions and assumes that the importance of a particular node is dictated by the num- ber of other nodes with which it directly interacts (Joyce et al., 2010). In a remark- ably similar pattern to the infra-slow oscillation changes, we found that, in NP subjects, the ascending pain pathway dis- played the greatest degree centrality (Fig. 4a). The number of direct connections was significantly greater in NP subjects in the ipsilateral SpV (mean Ϯ SEM ϫ 10 3 centrality score: controls, 18.7 Ϯ 1.1; NP subjects, 22.2 Ϯ 1.7, p ϭ 0.02), contralat- eral medial thalamus (controls, 28.0 Ϯ 2.1; NP subjects, 33.6 Ϯ 2.3, p ϭ 0.006), contralateral TRN (controls, 22.2 Ϯ 1.6; NP subjects, 27.3 Ϯ 1.4, p ϭ 0.03), and the orofacial representation of the contralat- eral S1 (controls, 22.8 Ϯ 1.4; NP subjects, 29.7 Ϯ 2.0, p ϭ 0.004; Fig. 4b; Table 2, Degree centrality). In addition, we found high degree centrality in the cerebellar Figure4. NPisassociatedwithincreaseddegreecentralityintheascendingpainpathwayandincreasedregionalhomogeneity cortex, contralateral dorsolateral pons, bi- attheprimaryafferentsynapse.a,BrainregionsinwhichNPsubjectsdisplayedthehighest(hotcolor)degreecentrality(pϽ0.05, lateral dorsolateral prefrontal cortex, and falsediscoveryratecorrected,minimumclustersizeof10voxels).Increaseddegreecentralityimpliesagreaterinfluenceofthepain the mid-cingulate cortex. pathway in the overall flow of information through the brain. The location of each axial slice in MNI space is indicated at the top right(z-directioninmillimeters).Thegreenshadingindicatesthethalamicregionactivatedduringinnocuousbrushingoftheright Altered local homogeneity at the lip, i.e., the orofacial somatosensory thalamus. b, Plots of mean Ϯ SEM degree centrality in controls (black) and NP subjects (red) primary afferent synapse infourregionsoftheascendingpainpathway.Theseplotsshowthereisasignificantincreaseindegreecentralityintheascending pain pathway in NP subjects compared with controls. c, NP subjects also displayed greater regional homogeneity than controls, If increased infra-slow oscillation power althoughthisincreasewasrestrictedtotheregionoftheipsilateralspinaltrigeminalnucleus,cerebellarcortex,andmid-cingulate in NP subjects and the concurrent in- cortex(pϽ0.05,falsediscoveryratecorrected,minimumclustersizeof10voxels).Regionalhomogeneityevaluatesthesimilarity crease in local network connectivity are of the time series within each voxel and its nearest neighbors and could result from local astrocyte activation. dlPFC, Dorsolateral associated with increased synchronicity of prefrontal cortex; ipsi, ipsilateral; MCC, mid-cingulate cortex. astrocyte activation and the subsequent 45 1014 • J. Neurosci., January 20, 2016 • 36(3):1008–1018 Alshelh et al. • Neuropathic Pain and Brain Rhythm
Figure 5. Significant changes in infra-slow oscillation power during acute pain compared with pain-free condition in controls. Acute pain did not evoke increased infra-slow oscillation power in anybrainregion,butdidevokedecreasedpower(coolcolorscale)inthecontralateralventralstriatum,bilateralposteriorcingulatecortex(PCC)andprecuneus.Powerspectrumplotsoffourclusters within the ascending pain pathway derived from the analysis between controls and NP subjects are shown in the lower panel. The four clusters chosen were those which in NP subjects displayed increased infra-slow oscillations (red shading). The power spectrum plots as well as plots of mean (ϮSEM) power between 0.03–0.06Hz in controls during acute pain (black) and in controls during nopain(green)showthatpowerwasnotsignificantlyincreasedintheascendingpainpathwayduringacutepain(two-tailed,two-samplettest,pϽ0.05).LocationofeachaxialsliceinMNIspace is indicated at the top right. cont, Controls; ipsi, ipsilateral. recruitment of surrounding astrocytes and neurons, neighboring within the ascending pain pathway. These pain pathway changes voxels should display increased signal intensity synchronization. are also associated with altered whole-brain network connectiv- To determine whether there was such an increase in regional ity, as well as local homogeneity changes at the primary afferent homogeneity, we measured KCC as an index to evaluate the sim- synapse. Importantly, these oscillatory and network alterations ilarity of the time series within each voxel and its 19 nearest do not occur during acute noxious stimuli in healthy individuals. neighbors. Remarkably, this analysis revealed that, within the The oscillatory and network changes that occur in individuals ascending pain pathway, regional homogeneity was increased with chronic NP are consistent with pathological processes that significantly in NP subjects compared with controls within the involve astrocyte activation, synaptic modulation, and the devel- ipsilateral SpV (Fig. 4c; Table 2, Regional homogeneity). In addi- opment of thalamocortical dysrhythmia. tion, increased local homogeneity occurred in the ipsilateral cer- Although individuals with NP display increased sensitivity in ebellar cortex and the contralateral mid-cingulate cortex. ascending pain pathways (Gracely et al., 1992; Petrou et al., 2012), human brain imaging studies report no increase in cere- No increase in infra-slow oscillations during acute pain bral blood flow, an index of neural activity, in these same brain In a final series of experiments, we aimed to provide additional regions (Hsieh et al., 1995; Iadarola et al., 1995; Moisset and evidence that the infra-slow oscillation power increases that oc- Bouhassira, 2007; Youssef et al., 2014). This is curious given that cur in the ascending pain pathway in NP subjects reflected an direct neuronal recordings in the human somatosensory thala- ongoing central pathological process and not constant peripheral mus revealed increased burst firing (Lenz et al., 1989), and elec- nociceptor activation. We found that acute orofacial pain troencephalogram recordings report altered thalamocortical (mean Ϯ SEM intensity, 4.8 Ϯ 0.3 of 10) did not evoke significant rhythm in humans with NP (Sarnthein et al., 2006). Further- increases in infra-slow oscillation power in any brain region using more, thalamocortical dysrhythmia is thought to result from ab- either ALFF or fALFF analysis procedures. However, we did find errant activity in recurrent thalamocortical loops that involve significant ALFF decreases during acute pain in the region of the collaterals to GABAergic neurons in the TRN (Pinault, 2004), ventral striatum, posterior cingulate cortex, and precuneus (Fig. 5). Importantly, in contrast to individuals with chronic NP, acute and we showed recently reduced TRN blood flow and reduced pain did not evoke any change in infra-slow oscillation power in thalamic GABAergic content in individuals with NP (Henderson the ipsilateral SpV (controls, 2.2 Ϯ 0.4; acute pain, 1.9 Ϯ 0.5, p ϭ et al., 2013; Gustin et al., 2014). Our results show that, in individ- 0.36), contralateral medial thalamus (controls, 1.1 Ϯ 0.1; acute uals with NP, the pattern of neural activity is indeed altered at pain, 1.3 Ϯ 0.3, p ϭ 0.22), contralateral TRN (controls, 0.9 Ϯ 0.1; multiple levels of the ascending pain pathway. acute pain, 0.9 Ϯ 0.2, p ϭ 0.49), and contralateral S1 (controls, Infra-slow oscillatory activity is a fundamental property of 3.2 Ϯ 0.4; acute pain, 2.5 Ϯ 0.5, p ϭ 0.15). cerebral and thalamic function and is maintained by adenosine receptor-mediated signaling (Lorincz et al., 2009; Hughes et al., Discussion 2011). Adenosine is likely released by astrocytes because they can We describe here a series of experimental findings that show that display spontaneous intracellular infra-slow calcium oscillations NP is associated with increased infra-slow oscillatory activity (Parri and Crunelli, 2001), are responsive to glutamate and ace- 46 Alshelh et al. • Neuropathic Pain and Brain Rhythm J. Neurosci., January 20, 2016 • 36(3):1008–1018 • 1015
rhythms into paroxysmal events (Hughes et al., 2011) and are coupled to high- frequency power fluctuations in the cor- tex (Vanhatalo et al., 2004; Mantini et al., 2007). These data are consistent with the idea that altered astrocyte function in in- dividuals with NP results in increased tha- lamic infra-slow oscillation power, which in turn evokes increased high-frequency power and the perception of pain (Sarn- thein et al., 2006). Although there are well documented microglial changes within ascending pain pathways in humans withNP(Banati, 2002; Loggia et al., 2015), there is no evidence to suggest that they play a role producing the infra- slow oscillations. Although infra-slow oscillations in individuals with NP might be generated independently at multiple sites, evi- dence suggests that they originate in the primary afferent synapse: (1) there is tight temporal coupling of infra-slow oscillations between regions of the as- cending pain pathway; (2) in experi- mental animal models and human postmortem tissue, NP is associated with astrocyte activation in the dorsal horn/spinal trigeminal nucleus (Garri- son et al., 1991; Okada-Ogawa et al., 2009; Shi et al., 2012); (3) trigeminal neuropathy is associated with decreased ipsilateral SpV mean diffusivity, which is consistent with increased size of acti- vated astrocytes (Wilcox et al., 2015); (4) inhibiting astrocyte activation atten- uates and even reverses nerve injury- Figure 6. Proposed series of events that result in the maintenance of chronic NP. After nerve injury, excess activity within induced mechanical allodynia (Zhuang primaryafferentneuronsresultsinexcessiveneurotransmitterreleaseattheprimaryafferentsynapse.Thisreleaseresultsinneural et al., 2006; Churi et al., 2008; Ji et al., deathandelicitsastrocyteactivation,whichresultsinmoreregularandincreasedmagnitudecalciumwavesresultinginincreased 2009, 2013; Morgenweck et al., 2013); regional homogeneity (RH). This in turn results in oscillatory gliotransmitter release and increased infra-slow neural oscillations, (5) despite no current evidence of a di- which are subsequently transferred to the somatosensory thalamus. Additional neural loss in the somatosensory thalamus results rect relationship between regional in reduced blood flow in the TRN and a subsequent reduction in GABAergic release back onto the somatosensory thalamus. This homogeneity and astrocytic activation, reduced inhibition combined with increased infra-slow oscillations and the recurrent nature of thalamocortical circuits results in we speculate that increased regional ho- alteredthalamocorticalconnectivity,increaseddominanceoftheascendingpainpathwayinoverallbrainfunctionrepresentedby increased degree centrality (DC), altered thalamocortical loop dynamics, a self-sustaining thalamocortical dysrhythmia, and the mogeneity in SpV is consistent with constant perception of pain. infra-slow frequency calcium waves prop- agation among neighboring astrocytes; and (6) in an experimental animal model tylcholine, and a link between adenosine and infra-slow oscilla- of NP, infra-slow oscillatory activity in somatosensory thalamus tory activity has been demonstrated in the cortex (Cunningham was eliminated by severing the connection between the primary et al., 2006). Coincidently, thalamic astrocytes display calcium afferent synapse and the thalamus (Iwata et al., 2011). wave oscillations at approximately the same infra-slow frequency A critical role for astrocyte activation in NP maintenance is range as the signal oscillations in our NP subjects (Crunelli et al., consistent with the finding that NP medications, such as val- 2002), and these infra-slow astrocyte calcium waves can propa- proate, phenytoin, and gabapentin, reduce astrocyte calcium sig- gate among surrounding astrocytes and can elicit large and long- naling (Tian et al., 2005). Furthermore, inhibition of neural lasting NMDA-mediated currents in thalamocortical neurons activity or astrocyte activation reverses the mechanical allodynia- (Crunelli et al., 2002). Indeed, synchronous burst firing has been induced nerve injury in experimental animals (Wang et al., linked to astrocyte calcium signaling and associated glutamate 2009b). Although some of our NP subjects were taking anticon- release (Tian et al., 2005), and, in pathological situations, greater vulsant medications, they showed similar elevations in infra-slow numbers of astrocytes may display enhanced calcium wave syn- oscillations as those not taking medications. We cannot deter- chrony and amplitude and enhanced NMDA receptor function mine whether these levels were reduced compared with before (Parri and Crunelli, 2001; Halassa et al., 2007). Increased infra- medication use and may indeed underlie the fact that we did not slow oscillations are associated with the development of ␣ find significant relationships between pain intensity and duration 47 1016 • J. Neurosci., January 20, 2016 • 36(3):1008–1018 Alshelh et al. • Neuropathic Pain and Brain Rhythm and infra-slow oscillatory changes in NP subjects. In addition to this circularity bias by performing the analysis on separate groups central changes, it has also been reported that ectopic peripheral of subjects because the recruitment of patients with post- afferent discharges contribute to the maintenance of NP (Devor endodontic chronic pain is challenging and a split analysis would and Seltzer, 1999; Liu et al., 2000). Although these investigations have significantly reduced subject numbers in each group and report increased oscillatory firing well above infra-slow levels thus our statistical power. Furthermore, ALFF and fALFF mea- (Ͼ40 Hz), oscillatory input may maintain glial activation and surements are not independent because they are derived from the infra-slow oscillatory activity. same data, although it is recommended that both values are pre- We propose that, after nerve injury, increased activity within sented because they provide slightly different measures (Zuo et primary afferent neurons results in excessive neurotransmitter al., 2010). Third, because we were interested in determining release, likely glutamatergic. This results in neural death, which changes within the ascending pain pathway, it was necessary to likely involves local interneurons because of the following: (1) in reflect the images of some NP subjects across the midline. Our animal models of NP, GABAergic interneuron loss occurs in the result suggest that increased infra-slow oscillation patterns are dorsal horn, which is closely related to glutamate receptor activa- consistent with the contralateral projection of these pain path- tion (de Novellis et al., 2004; Scholz et al., 2005); and (2) orofacial ways because we did not find a significant difference in power NP is associated with reduced gray matter volume and mean when clusters were reflected across the midline, although there diffusivity within SpV (Wilcox et al., 2015). Excess neural dis- remains a potential issue of lateralized function. Despite these charge also elicits astrocyte activation, spreading oscillating cal- limitations, the discrete changes in infra-slow oscillations in pri- cium waves, oscillatory gliotransmitter release characterized by marily pain-related brain regions make us confident that the increased regional homogeneity, and increased infra-slow neural changes reported in this investigation are important in the main- oscillations throughout the ascending pain pathway. Addition- tenance of NP. ally, local interneuron loss within the somatosensory thalamus resulting from a similar mechanism to that which occurs in SpV References Alvarado S, Tajerian M, Millecamps M, Suderman M, Stone LS, Szyf M results in reduced TRN activity, reduced GABAergic output onto (2013) Peripheral nerve injury is accompanied by chronic tra- the somatosensory thalamus (Henderson et al., 2013) and, in nscriptome-wide changes in the mouse prefrontal cortex. Mol Pain 9:21. combination with increased infra-slow oscillatory activity, results CrossRef Medline in altered thalamocortical connectivity, increased dominance of Banati RB (2002) Brain plasticity and microglia: is transsynaptic glial acti- the ascending pain pathway characterized by increased degree vation in the thalamus after limb denervation linked to cortical plasticity centrality, a self-sustaining thalamocortical dysrhythmia, and the and central sensitisation? J Physiol Paris 96:289–299. CrossRef Medline Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J (1961) An inventory constant perception of pain (Fig. 6). for measuring depression. Arch Gen Psychiatry 4:561–571. CrossRef Although this model points to a critical role for the primary Medline afferent synapse in maintaining NP but does not exclude the Bullmore E, Sporns O (2009) Complex brain networks: graph theoretical possibility that, in different individuals and in different NP con- analysis of structural and functional systems. Nat Rev Neurosci 10: ditions, other sites along the ascending pain pathway play a role 186–198. CrossRef Medline in pain maintenance. Furthermore, it is possible that increased Chao-Gan Y, Yu-Feng Z (2010) DPARSF: a MATLAB toolbox for “pipe- line” data analysis of resting-state fMRI. Front Syst Neurosci 4:13. infra-slow oscillations in regions other than the SpV, thalamus, CrossRef Medline and S1 are involved in the maintenance of NP. For example, we Churi SB, Abdel-Aleem OS, Tumber KK, Scuderi-Porter H, Taylor BK found altered infra-slow oscillatory power, degree centrality, (2008) Intrathecal rosiglitazone acts at peroxisome proliferator- and/or regional homogeneity in the cerebellar, dorsolateral pre- activated receptor-gamma to rapidly inhibit neuropathic pain in rats. frontal, mid-cingulate, and insular cortices in NP subjects. In J Pain 9:639–649. CrossRef Medline animal models of NP, astrocyte activation occurs in the cingulate Crunelli V, Blethyn KL, Cope DW, Hughes SW, Parri HR, Turner JP, To`th TI, cortex, and a recent study suggests that this activation may be Williams SR (2002) Novel neuronal and astrocytic mechanisms in thalamocortical loop dynamics. Philos Trans R Soc Lond B Biol Sci 357: related to sleep disturbances (Kuzumaki et al., 2007; Yamashita et 1675–1693. CrossRef Medline al., 2014). Furthermore, increased gene expression indicative of Cunningham MO, Pervouchine DD, Racca C, Kopell NJ, Davies CH, Jones astrocyte activation occurs in the prefrontal cortex of mice with RS, Traub RD, Whittington MA (2006) Neuronal metabolism governs NP that is likely related to pain comorbidities, such as mood cortical network response state. Proc Natl Acad Sci U S A 103:5597–5601. disorders (Alvarado et al., 2013). Although the focus of this in- CrossRef Medline vestigation was on the ascending somatosensory pathway, it is de Novellis V, Siniscalco D, Galderisi U, Fuccio C, Nolano M, Santoro L, Cascino A, Roth KA, Rossi F, Maione S (2004) Blockade of glutamate likely that changes in other brain regions, such as association mGlu5 receptors in a rat model of neuropathic pain prevents early over- cortices, are involved in maintaining the emotional and cognitive expression of pro-apoptotic genes and morphological changes in dorsal aspects of NP. It is also possible that the changes in infra-slow horn lamina II. Neuropharmacology 46:468–479. CrossRef Medline oscillatory power reported here do not result from astrocyte– Devor M, Seltzer Z (1999) Pathophysiology of damaged nerves in relation to neural interactions and may result simply from purely neural chronic pain. In: Textbook of pain, Ed 4 (Wall PD, Melzack R, eds), pp processes, although we suggest this is less likely. 129–164. London: Churchill Livingston. Duvernoy H (1995) The human brainstem and cerebellum. New York: Finally, there are a number of limitations that need to be rec- Springer-Verlag Wien. ognized. First, given the relatively low spatial resolution of fMRI, Farrell MJ, Laird AR, Egan GF (2005) Brain activity associated with pain- it is difficult to accurately localize each cluster to a particular fully hot stimuli applied to the upper limb: a meta-analysis. Hum Brain brain nucleus or region. Although we are confident that the clus- Mapp 25:129–139. CrossRef Medline ter locations described in this study include the regions labeled, Friston KJ, Holmes AP, Worsley KP, Proline JB, Frith CD, Frackowiak RSJ they also spread into other nuclei. Second, there is a potential bias (1995) Statistical parametric maps in functional imaging: a general im- of circularity introduced by first performing an analysis to deter- aging approach. Hum Brain Mapp 2:189–210. Garrison CJ, Dougherty PM, Kajander KC, Carlton SM (1991) Staining of mine the frequency band within the thalamus and then using this glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases fol- band to perform voxel-by-voxel analyses (Kriegeskorte et al., lowing a sciatic nerve constriction injury. Brain Res 565:1–7. CrossRef 2009; Vul et al., 2009). Unfortunately, we were not able to reduce Medline 48 Alshelh et al. • Neuropathic Pain and Brain Rhythm J. Neurosci., January 20, 2016 • 36(3):1008–1018 • 1017
Gerke MB, Duggan AW, Xu L, Siddall PJ (2003) Thalamic neuronal activity Macey PM, Macey KE, Kumar R, Harper RM (2004) A method for removal in rats with mechanical allodynia following contusive spinal cord injury. of global effects from fMRI time series. Neuroimage 22:360–366. Neuroscience 117:715–722. CrossRef Medline CrossRef Medline Giesecke T, Gracely RH, Grant MA, Nachemson A, Petzke F, Williams DA, Mai JK, Paxinos G, Voss T (2007) Atlas of the human brain, Ed 3. San Diego: Clauw DJ (2004) Evidence of augmented central pain processing in id- Academic. iopathic chronic low back pain. Arthritis Rheum 50:613–623. CrossRef Mantini D, Perrucci MG, Del Gratta C, Romani GL, Corbetta M (2007) Medline Electrophysiological signatures of resting state networks in the human Gracely RH, Lynch SA, Bennett GJ (1992) Painful neuropathy: altered cen- brain. Proc Natl Acad Sci U S A 104:13170–13175. CrossRef Medline tral processing maintained dynamically by peripheral input. Pain 51: Moisset X, Bouhassira D (2007) Brain imaging of neuropathic pain. Neuro- 175–194. CrossRef Medline image 37 [Suppl 1]:S80–S88. CrossRef Medline Gracely RH, Petzke F, Wolf JM, Clauw DJ (2002) Functional magnetic res- Morgenweck J, Griggs RB, Donahue RR, Zadina JE, Taylor BK (2013) onance imaging evidence of augmented pain processing in fibromyalgia. PPARgamma activation blocks development and reduces established Arthritis Rheum 46:1333–1343. CrossRef Medline neuropathic pain in rats. Neuropharmacology 70:236–246. CrossRef Gustin SM, Peck CC, Wilcox SL, Nash PG, Murray GM, Henderson LA Medline (2011) Different pain, different brain: thalamic anatomy in neuropathic Nurmikko TJ, Eldridge PR (2001) Trigeminal neuralgia–pathophysiology, and non-neuropathic chronic pain syndromes. J Neurosci 31:5956–5964. diagnosis and current treatment. Br J Anaesth 87:117–132. CrossRef CrossRef Medline Medline Gustin SM, Wrigley PJ, Youssef AM, McIndoe L, Wilcox SL, Rae CD, Edden Okada-Ogawa A, Suzuki I, Sessle BJ, Chiang CY, Salter MW, Dostrovsky JO, RA, Siddall PJ, Henderson LA (2014) Thalamic activity and biochemical Tsuboi Y, Kondo M, Kitagawa J, Kobayashi A, Noma N, Imamura Y, changes in individuals with neuropathic pain after spinal cord injury. Pain Iwata K (2009) Astroglia in medullary dorsal horn (trigeminal spinal 155:1027–1036. CrossRef Medline subnucleus caudalis) are involved in trigeminal neuropathic pain mech- Halassa MM, Fellin T, Haydon PG (2007) The tripartite synapse: roles for anisms. J Neurosci 29:11161–11171. CrossRef Medline gliotransmission in health and disease. Trends Mol Med 13:54–63. Parri HR, Crunelli V (2001) Pacemaker calcium oscillations in thalamic as- CrossRef Medline trocytes in situ. Neuroreport 12:3897–3900. CrossRef Medline Henderson LA, Peck CC, Petersen ET, Rae CD, Youssef AM, Reeves JM, Paxinos G, Huang X (1995) Atlas of the human brainstem, Ed 1. San Diego: Wilcox SL, Akhter R, Murray GM, Gustin SM (2013) Chronic pain: lost Academic. inhibition? J Neurosci 33:7574–7582. CrossRef Medline Petrou M, Pop-Busui R, Foerster BR, Edden RA, Callaghan BC, Harte SE, Hsieh JC, Belfrage M, Stone-Elander S, Hansson P, Ingvar M (1995) Central Harris RE, Clauw DJ, Feldman EL (2012) Altered excitation-inhibition representation of chronic ongoing neuropathic pain studied by positron balance in the brain of patients with diabetic neuropathy. Acad Radiol emission tomography. Pain 63:225–236. CrossRef Medline 19:607–612. CrossRef Medline Hughes SW, Lorincz ML, Parri HR, Crunelli V (2011) Infraslow (Ͻ0.1 Hz) Pinault D (2004) The thalamic reticular nucleus: structure, function and oscillations in thalamic relay nuclei basic mechanisms and significance to concept. Brain Res Brain Res Rev 46:1–31. CrossRef Medline health and disease states. Prog Brain Res 193:145–162. CrossRef Medline Sarnthein J, Stern J, Aufenberg C, Rousson V, Jeanmonod D (2006) In- Iadarola MJ, Max MB, Berman KF, Byas-Smith MG, Coghill RC, Gracely RH, creased EEG power and slowed dominant frequency in patients with neu- Bennett GJ (1995) Unilateral decrease in thalamic activity observed with rogenic pain. Brain 129:55–64. CrossRef Medline positron emission tomography in patients with chronic neuropathic pain. Scholz J, Broom DC, Youn DH, Mills CD, Kohno T, Suter MR, Moore KA, Pain 63:55–64. CrossRef Medline Decosterd I, Coggeshall RE, Woolf CJ (2005) Blocking caspase activity Iwata M, LeBlanc BW, Kadasi LM, Zerah ML, Cosgrove RG, Saab CY (2011) prevents transsynaptic neuronal apoptosis and the loss of inhibition in High-frequency stimulation in the ventral posterolateral thalamus re- lamina II of the dorsal horn after peripheral nerve injury. J Neurosci verses electrophysiologic changes and hyperalgesia in a rat model of pe- 25:7317–7323. CrossRef Medline ripheral neuropathic pain. Pain 152:2505–2513. CrossRef Medline Shi Y, Gelman BB, Lisinicchia JG, Tang SJ (2012) Chronic-pain-associated Ji RR, Gereau RW 4th, Malcangio M, Strichartz GR (2009) MAP kinase and pain. Brain Res Rev 60:135–148. CrossRef Medline astrocytic reaction in the spinal cord dorsal horn of human immunode- Ji XT, Qian NS, Zhang T, Li JM, Li XK, Wang P, Zhao DS, Huang G, Zhang L, ficiency virus-infected patients. J Neurosci 32:10833–10840. CrossRef Fei Z, Jia D, Niu L (2013) Spinal astrocytic activation contributes to Medline mechanical allodynia in a rat chemotherapy-induced neuropathic pain Song XW, Dong ZY, Long XY, Li SF, Zuo XN, Zhu CZ, He Y, Yan CG, Zang model. PLoS One 8:e60733. CrossRef Medline YF (2011) REST: a toolkit for resting-state functional magnetic reso- Joyce KE, Laurienti PJ, Burdette JH, Hayasaka S (2010) A new measure of nance imaging data processing. PLoS One 6:e25031. CrossRef Medline centrality for brain networks. PLoS One 5:e12200. CrossRef Medline Sullivan MJL, Bishop S, Pivik J (1995) The pain catastrophizing scale: devel- Kriegeskorte N, Simmons WK, Bellgowan PS, Baker CI (2009) Circular opment and validation. Psychol Assess 7:432–524. CrossRef analysis in systems neuroscience: the dangers of double dipping. Nat Neu- Tian GF, Azmi H, Takano T, Xu Q, Peng W, Lin J, Oberheim N, Lou N, Wang rosci 12:535–540. CrossRef Medline X, Zielke HR, Kang J, Nedergaard M (2005) An astrocytic basis of epi- Kuzumaki N, Narita M, Hareyama N, Niikura K, Nagumo Y, Nozaki H, lepsy. Nat Med 11:973–981. CrossRef Medline Amano T, Suzuki T (2007) Chronic pain-induced astrocyte activation Vanhatalo S, Palva JM, Holmes MD, Miller JW, Voipio J, Kaila K (2004) in the cingulate cortex with no change in neural or glial differentiation Infraslow oscillations modulate excitability and interictal epileptic activ- from neural stem cells in mice. Neurosci Lett 415:22–27. CrossRef ity in the human cortex during sleep. Proc Natl Acad Sci U S A 101: Medline 5053–5057. CrossRef Medline Lenz FA, Kwan HC, Dostrovsky JO, Tasker RR (1989) Characteristics of the Vul E, Harris C, Winkielman P, Pashler H (2009) Puzzlingly high correla- bursting pattern of action potentials that occurs in the thalamus of pa- tions in fMRI studies of emotion, personality, and social cognition. Per- tients with central pain. Brain Res 496:357–360. CrossRef Medline spect Psychol Sci 4:274–290. CrossRef Medline Liu CN, Michaelis M, Amir R, Devor M (2000) Spinal nerve injury enhances Walton KD, Llina´s RR (2010) Central pain as a thalamocortical dysrhyth- subthreshold membrane potential oscillations in DRG neurons: relation mia: a thalamic efference disconnection? In: Translational pain research: to neuropathic pain. J Neurophysiol 84:205–215. Medline from mouse to man (Kruger L, Light AR, eds). Boca Raton, FL: CRC. Loggia ML, Chonde DB, Akeju O, Arabasz G, Catana C, Edwards RR, Hill E, Wang SH, Liang CT, Liu YW, Huang MC, Huang SC, Hong WF, Su JG (2009a) Hsu S, Izquierdo-Garcia D, Ji RR, Riley M, Wasan AD, Zu¨rcher NR, Crosstalk between activated forms of the aryl hydrocarbon receptor and gluco- Albrecht DS, Vangel MG, Rosen BR, Napadow V, Hooker JM (2015) corticoid receptor. Toxicology 262:87–97. CrossRef Medline Evidence for brain glial activation in chronic pain patients. Brain 138: Wang W, Mei X, Huang J, Wei Y, Wang Y, Wu S, Li Y (2009b) Crosstalk 604–615. CrossRef Medline between spinal astrocytes and neurons in nerve injury-induced neuro- Lo¨rincz ML, Geall F, Bao Y, Crunelli V, Hughes SW (2009) ATP-dependent pathic pain. PLoS One 4:e6973. CrossRef Medline infra-slow (Ͻ0.1 Hz) oscillations in thalamic networks. PLoS One Wang W, Li Q, Yamada T, Matsumoto K, Matsumoto I, Oda M, Watanabe G, 4:e4447. CrossRef Medline Kayano Y, Nishioka Y, Sone S, Yano S (2009c) Crosstalk to stromal 49 1018 • J. Neurosci., January 20, 2016 • 36(3):1008–1018 Alshelh et al. • Neuropathic Pain and Brain Rhythm
fibroblasts induces resistance of lung cancer to epidermal growth factor GM, Henderson LA (2014) Differential brain activity in subjects with receptor tyrosine kinase inhibitors. Clin Cancer Res 15:6630–6638. painful trigeminal neuropathy and painful temporomandibular disorder. CrossRef Medline Pain 155:467–475. CrossRef Medline Wilcox SL, Gustin SM, Macey PM, Peck CC, Murray GM, Henderson LA Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, De- (2015) Anatomical changes at the level of the primary synapse in neuro- costerd I, Ji RR (2006) A peptide c-Jun N-terminal kinase (JNK) inhib- pathic pain: evidence from the spinal trigeminal nucleus. J Neurosci 35: itor blocks mechanical allodynia after spinal nerve ligation: respective 2508–2515. CrossRef Medline roles of JNK activation in primary sensory neurons and spinal astrocytes Yamashita A, Hamada A, Suhara Y, Kawabe R, Yanase M, Kuzumaki N, for neuropathic pain development and maintenance. J Neurosci 26: Narita M, Matsui R, Okano H (2014) Astrocytic activation in the ante- 3551–3560. CrossRef Medline rior cingulate cortex is critical for sleep disorder under neuropathic pain. Zuo XN, Di Martino A, Kelly C, Shehzad ZE, Gee DG, Klein DF, Castellanos Synapse 68:235–247. CrossRef Medline FX, Biswal BB, Milham MP (2010) The oscillating brain: complex and Youssef AM, Gustin SM, Nash PG, Reeves JM, Petersen ET, Peck CC, Murray reliable. Neuroimage 49:1432–1445. CrossRef Medline
50 CHAPTER THREE
ALTERED REGIONAL BRAIN T2 RELAXATION TIMES IN INDIVIDUALS WITH CHRONIC OROFACIAL NEUROPATHIC PAIN
Chapter Three is published as:
Z. Alshelh, F. Di Pietro, E.P. Mills, E.R. Vickers, C.C. Peck, G.M. Murray and L.A. Henderson.
Altered regional brain T2 relaxation times in individuals with chronic orofacial neuropathic pain.
NeuroImage: Clinical, 2018. Volume 19: 167-173
51 NeuroImage: Clinical 19 (2018) 167–173
Contents lists available at ScienceDirect
NeuroImage: Clinical
journal homepage: www.elsevier.com/locate/ynicl
Altered regional brain T2 relaxation times in individuals with chronic T orofacial neuropathic pain ⁎ Z. Alshelha, F. Di Pietroa, E.P. Millsa, E.R. Vickersa, C.C. Peckb, G.M. Murrayb, L.A. Hendersona, a Department of Anatomy and Histology, Sydney Medical School, University of Sydney, 2006, Australia b Faculty of Dentistry, University of Sydney, 2006, Australia
ARTICLE INFO ABSTRACT
Keywords: The neural mechanisms underlying the development and maintenance of chronic pain following nerve injury Spinal trigeminal nucleus remain unclear. There is growing evidence that chronic neuropathic pain is associated with altered thalamic Chronic orofacial pain firing patterns, thalamocortical dysrhythmia and altered infra-slow oscillations in ascending pain pathways. Magnetic resonance imaging Preclinical and post-mortem human studies have revealed that neuropathic pain is associated with prolonged T2 relaxation astrocyte activation in the dorsal horn and we have suggested that this may result in altered gliotransmission, Ascending pain pathway which results in altered resting neural rhythm in the ascending pain pathway. Evidence of astrocyte activation above the level of the dorsal horn in living humans is lacking and direct measurement of astrocyte activation in living humans is not possible, however, there is evidence that regional alterations in T2 relaxation times are indicative of astrogliosis. The aim of this study was to use T2 relaxometry to explore regional brain anatomy of the ascending pain pathway in individuals with chronic orofacial neuropathic pain. We found that in individuals with trigeminal neuropathic pain, decreases in T2 relaxation times occurred in the region of the spinal trigeminal nucleus and primary somatosensory cortex, as well as in higher order processing regions such as the dorsolateral prefrontal, cingulate and hippocampal/parahippocampal cortices. We speculate that these regional changes in T2 relaxation times reflect prolonged astrocyte activation, which results in altered brain rhythm and ultimately the constant perception of pain. Blocking prolonged astrocyte activation may be effective in preventing and even reversing the development of chronic pain following neural injury.
1. Introduction electroencephalography (> 2 Hz) we have previously shown that chronic neuropathic pain is associated with increased infra-slow fre- Chronic neuropathic pain is a complex disease resulting from actual quency (< 0.1 Hz) oscillatory activity throughout the ascending pain or presumed damage to the somatosensory nervous system. The con- pathway, including the region of the primary afferent synapse, ven- stant perception of pain that characterizes neuropathic pain is asso- trocaudal thalamus, thalamic reticular nucleus and primary somato- ciated with increased thalamic bursting activity (Gerke et al., 2003; sensory cortex (Alshelh et al., 2016). Interestingly, these infra-slow Iwata et al., 2011; Lenz et al., 1989), reduced thalamic blood flow oscillation increases occurred at approximately the same frequency (Hsieh et al., 1995; Iadarola et al., 1995; Moisset and Bouhassira, 2007; range as reported calcium waves in astrocytes, i.e. 0.03–0.06 Hz Youssef et al., 2014) and increased cortical power, often termed tha- (Crunelli et al., 2002). Astrocytes are known to modulate synaptic ac- lamocortical dysrhythmia (Di Pietro et al., 2018; Sarnthein et al., 2006; tivity by the release of gliotransmitters and it is well-documented in Walton and Llinás, 2010). The increase in cortical power is associated preclinical animal models that neuropathic pain is associated with with an altered relationship with thalamic GABAergic content (Di prolonged astrocyte activation in the dorsal horn/spinal trigeminal Pietro et al., 2018) and we have proposed that the thalamocortical nucleus (Garrison et al., 1991; Okada-Ogawa et al., 2009). Given these dysrhythmia that occurs in chronic neuropathic pain results from al- data, we have speculated that following nerve injury, prolonged as- tered interactions between the ventrocaudal thalamus, thalamic re- trocyte activation results in increased infra-slow oscillations in the as- ticular nucleus and the cerebral cortex (Henderson and Di Pietro, cending pain pathway which in turn is associated with thalamocortical 2016). dysrhythmia and the constant perception of pain (Henderson and Di In addition to cortical resting activity changes, as read with Pietro, 2016).
Abbreviations: NP, neuropathic pain; PET, Positron Emission Tomography; PCS, Pain Catastrophizing Scale; BDI, Beck Depression Inventory ⁎ Corresponding author at: Department of Anatomy and Histology, F13, University of Sydney, Australia. E-mail address: [email protected] (L.A. Henderson). https://doi.org/10.1016/j.nicl.2018.04.015 Received 15 January 2018; Received in revised form 10 April 2018; Accepted 12 April 2018 Available online 13 April 2018 2213-1582/ Crown Copyright © 2018 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
52 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173
Whilst it is clear from preclinical animal models that neuropathic 2.5 ms; flip angle 8°; raw voxel size 0.87 × 0.87 × 0.87 mm). Following pain is associated with astrocyte activation in the dorsal horn and this, T2-weighted image sets covering the entire brain were collected brainstem (Hsieh et al., 1995; Okada-Ogawa et al., 2009), robust evi- (field of view 250 × 250 mm; matrix size 556 × 556; slice thickness dence is lacking in humans. There is, however, evidence from a post- 5 mm; repetition time 3000 ms; echo times 20, 40, 60, 80, 100 ms; raw mortem study which revealed prolonged astrocyte activation, but no voxel size 0.4 × 0.4 × 5 mm thick). Multiple echo times were used to microglial activation, in the dorsal horn of individuals with neuropathic create multiple image sets for subsequent calculation of T2-relaxation pain associated with HIV (Shi et al., 2012). Indirect measurements of time maps. glial activation using Positron Emission Tomography (PET) have also revealed chronic glial activation in humans with neuropathic pain. 2.3. MRI analysis Banati and colleagues (Banati et al., 2001) reported increases in binding of [11C](R)-PK11195 (a sensitive in vivo marker of glial cell activation) Using SPM12 (Friston et al., 1995) and custom software, the fol- in the contralateral (to injury) thalamus of patients with phantom limb lowing equation was used to calculate T2 relaxation time brain maps: pain. Similarly, Loggia and colleagues recently used the same technique T2 = (TE2 − TE1)/ln(SI1/SI2) where TE1 and TE2 are the echo times for to show increased glial activation in the thalamus and primary soma- proton density and T2-weighted images, and SI1 and SI2 represent tosensory cortex, contralateral to pain, of individuals with chronic low proton density and T2-weighted image signal intensities, respectively. A back pain (Loggia et al., 2015). Whilst these brain imaging studies are ceiling threshold of 500 ms was applied to eliminate cerebrospinal important and suggest chronic glial activation in the thalamus and fluid. In each subject, T2 relaxation maps were co-registered to each above, technical limitations mean that they cannot explore changes at individual's T1-weighted anatomical image set. The T1-weighted ana- the level of the brainstem or, more importantly, the primary afferent tomical image was then spatially normalized to a template in Montreal synapse. Neurological Institute (MNI) space and the parameters applied to the T2 One non-invasive technique that has been used to measure glial relaxation time maps. Finally, the T2 brain maps were spatially activation is T2 relaxometry (Jackson et al., 1994; Wagner et al., 2012), smoothed using an 8 mm full-width-at-half-maximum Gaussian filter. In a magnetic resonance imaging technique that can characterise changes those NP subjects with pain restricted to the left side of the face in the brainstem and above. There is immunohistochemical evidence (n = 17), T2-weighted images were reflected across the midline so that that decreased T2 relaxation times are associated with increased glial T2 differences could be assessed ipsilateral and contralateral to the side activation (Schwarz et al., 1996) and in this investigation we hy- of highest ongoing pain. pothesize that chronic orofacial neuropathic pain is associated with In addition to a wholebrain T2 relaxation analysis, we performed a significantly reduced T2 relaxation time in the ascending pain pathway, brainstem-specific analysis. The T2 relaxation maps were cropped to including the region of the spinal trigeminal nucleus, ventrocaudal (Vc) include only the brainstem and cerebellum, and a mask of these regions thalamus, thalamic reticular nucleus and the orofacial region of the was created using the Spatial Unbiased Infratentorial Template (SUIT) primary somatosensory cortex. toolbox (Diedrichsen, 2006). These brainstem/cerebellum masks were then manually adjusted to accurately encompass the brainstem and 2. Methods cerebellum only. Using this mask, T2 relaxation brainstem images were spatially normalized to the SUIT template in MNI space and spatially 2.1. Subjects smoothed using a 3 mm full-width-at-half-maximum Gaussian filter. Significant differences in T2 relaxation time between controls and Thirty-seven subjects with chronic orofacial neuropathic pain (NP; NP subjects were determined using a voxel-by-voxel analysis of the 28 females, mean age 46.1 ± 2.5 years [ ± SEM]) and 40 healthy wholebrain and the brainstem only (p < 0.05, random effects, false controls without ongoing pain (24 females, mean age 40.6 ± 2.7 discovery rate corrected for multiple comparisons, minimum cluster 10 [ ± SEM]) were recruited for this study. All NP subjects were diagnosed voxels, age and gender as nuisance variables). A brain mask that ex- using the Liverpool criteria as having post-traumatic painful trigeminal cluded CSF as well as the brainstem was applied to the wholebrain neuropathy (Nurmikko and Eldridge, 2001). Written informed consent analysis. For both the wholebrain and brainstem only analyses, mean was obtained for all procedures and the study was approved by In- ( ± SEM) T2 relaxation times of significantly different clusters were stitutional Human Research Ethics Committees, University of Sydney. extracted and plotted. Significant correlations between T2 relaxation For each NP subject, the intensity of their on-going pain was re- times and pain intensity, BDI and PCS scores were also determined corded on a 10 cm horizontal visual analogue scale (VAS) with 0 in- (p < 0.05). In addition, to determine if medication use had a sig- dicating “no pain” and 10 indicating “worst imaginable pain”, three times nificant effect on T2 relaxation times, for each cluster, T2 relaxation a day for the seven days prior to a magnetic resonance imaging (MRI) times were compared between those NP subjects taking acute analgesic session (Carlsson, 1983). These pain intensity scores were averaged medication alone (n = 5) or those taking prophylactic analgesic medi- over the 7-day period to create a mean pain intensity score. On the day cation (n = 11) with those not taking medication (n = 21) (p < 0.05, of the MRI, all subjects were experiencing pain and were asked to draw two-tailed, two-sample t-tests). the distribution of their ongoing pain. Subjects were also asked to complete a McGill Pain Questionnaire (Melzack, 1975), the Beck De- 3. Results pression Inventory (BDI) (Beck et al., 1961) and the Pain Catastro- phizing Questionnaire (PCS) (Sullivan et al., 1995). A subset of the 3.1. Psychophysics control and NP subjects was used in a previous investigation (Alshelh et al., 2016). Individual NP subject characteristics are shown in Table 1, and overall pain distributions and pain descriptors in Fig. 1. In 34 of the NP 2.2. MRI acquisition patients, their on-going pain was unilateral (17 right, 17 left), and the remaining 3 NP subjects reported bilateral pain. In all NP subjects, pain All subjects lay supine on the bed of a 3 Tesla MRI scanner (Philips was located in the 2nd and 3rd trigeminal nerve divisions, with 7 Achieva) with their head immobilized in a tight-fitting 32-channel subjects also reporting pain in the 1st division. The mean ( ± SEM) SENSE head coil. With each subject relaxed and at rest, a high resolu- pain intensity over the 7 days prior to the MRI scanning session was tion T1-weighted anatomical image set covering the entire brain was 3.8 ± 0.4 out of 10 and the mean duration of pain was collected (turbo field echo; field of view 250 × 250 mm; matrix size 4.1 ± 0.9 years. Using the McGill Pain Questionnaire, NP subjects most 288 × 288; slice thickness 0.87 mm; repetition time 5600 ms; echo time frequently described their pain as “throbbing”, “tender” and
168
53 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173
Table 1 NP subject characteristics.
Subject Age (years) Gender Pain duration Site Pain Intensity Acute analgesic medication Prophylactic medication (years) (VAS)
1 27 M 3.4 Right 0.5 – Palmitoylethanolamide 2 21 F 12 Right 1.3 – Palmitoylethanolamide 3 35 M 8 Right 1.4 – Palmitoylethanolamide 4 45 M 14 Left 1.4 – Palmitoylethanolamide 5 33 F 7 Left 2.4 –– 6 53 F 2 Left 2.3 –– 7 21 M 0.3 Left 1.9 – Palmitoylethanolamide 8 64 F 17 Right 4.1 –– 9 76 F 25 Left 5.7 –– 10 67 F 2 Left 6.3 Diazepam – 11 47 F 2.8 Left 4.1 –– 12 40 F 1.5 Right 7.8 Ibuprofen Pregabalin, carbamazepine 13 35 F 2 Left 4.9 –– 14 46 F 0.6 Left 3.6 –– 15 52 F 0.8 Right 2.7 –– 16 58 M 1.8 Left 9.6 Ibuprofen – 17 58 F 10 Right 5.1 –– 18 42 F 2.8 Right 4.5 –– 19 44 M 3.3 Bilateral 7.5 Acetylsalicylic acid, ibuprofen Dextroamphetamine, metaprolol tartrate 20 47 F 0.4 Right 2 –– 21 65 F 10 Left 6.1 Paracetamol – 22 64 F 0.2 Right 1.5 –– 23 24 F 2.6 Left 0 –– 24 28 F 0.2 Bilateral 7.2 Ibuprofen, paracetamol, oxycodone Pregabalin hydrochloride 25 58 F 0.5 Right 2.3 Ibuprofen, paracetamol Pregabalin 26 44 F 2.3 Right 4 – Desvenlafaxine 27 39 F 0.6 Left 3.1 Ibuprofen, paracetamol, codeine 28 55 F 1 Right 0.8 –– 29 41 M 0.5 Right 5.1 –– 30 36 F 0.3 Left 0 –– 31 20 F 0.5 Left 1.7 –– 32 39 F 2.6 Left 5.6 –– 33 74 F 0.4 Bilateral 4.2 –– 34 53 M 0.6 Right 2.1 –– 35 29 M 5.8 Left 4.8 Tramadol – 36 73 F 3 Right 7.5 – Efexor 37 53 F 2.3 Right –– – Mean ± SEM 46.1 ± 2.5 4.1 ± 0.9 3.8 ± 0.4
Fig. 1. Pain distribution and quality of pain in 37 subjects with chronic orofacial neuropathic pain. A) Individual pain distribution patterns in all 37 chronic pain (NP) subjects. B) Frequency (percentage of subjects) of descriptors chosen from the McGill Pain Questionnaire to describe the quality of on-going pain, in NP subjects.
169
54 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173
Fig. 2. Significant differences in T2 relaxation times in individuals with chronic neuropathic pain compared with controls. A) Regions where chronic neuropathic pain (NP) subjects had significantly reduced T2 relaxation times compared with controls. Reductions are indicated by the cool colour scale and overlaid onto a mean T1-weighted anatomical image set derived from all 77 subjects. Note the decreases within the orofacial region of the primary somatosensory cortex (S1), mid-cingulate cortex (MCC), anterior cingulate cortex (ACC), dorsolateral prefrontal cortex (dlPFC) and hippocampus. Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice. B) Plots of mean ± SEM T2 relaxation times (ms) in control (black bars) and NP ⁎ (blue bars) groups for significant clusters. denotes significant differences derived from the voxel-by-voxel random-effects analysis (p < 0.05, false discovery rate corrected). ipsi: ipsilateral to side of highest on-going pain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
“radiating”. There was no significant difference in age (t-test; relaxation times were significantly lower in NP subjects than in controls p > 0.05) or gender composition (chi-squared test, p > 0.05) between in three discrete brainstem regions (Fig. 3, Table 2). Decreased T2 re- the two subject groups. Compared with controls, NP subjects had higher laxation times in NP subjects occurred in the region encompassing the BDI scores (mean ± SEM: controls: 5.0 ± 1.0, NP: 26.6 ± 2.0, right spinal trigeminal nucleus (controls: 106.8 ± 1.1, NP: p < 0.001) and PCS scores (controls: 12.2 ± 1.7, NP: 15.8 ± 1.5, 101.2 ± 1.3), the right spinal trigeminal tract (controls: 118.2 ± 1.7, p < 0.001). NP: 108.9 ± 2.1) and in the region of the right trigeminal nerve entry (controls: 107.3 ± 2.0, NP: 98.1 ± 1.8). In no brainstem region was there a significant increase in T2 relaxation time in NP subjects com- 3.2. T2 relaxation times pared with controls. In NP patients, there were no significant relationships between pain Wholebrain analysis revealed that NP subjects displayed sig- intensity or duration and T2 relaxation times in the left mid-cingulate nificantly decreased T2 relaxation times relative to controls in a number cortex (intensity: r = 0.08, duration: r = 0.08), left anterior cingulate of brain regions (Fig. 2, Table 2). These included the left (contralateral cortex (intensity: r = 0.11, duration: r = 0.36), left primary somato- to highest pain) mid-cingulate cortex (mean ± SEM ms: controls: sensory cortex (intensity: r = 0.05, duration: r = 0.22), left dorsolateral 101.2 ± 1.4, NP: 95.4 ± 1.1), left anterior cingulate cortex (controls: prefrontal cortex (intensity: r = 0.25, duration: r = 0.26), left hippo- 96.8 ± 1.1, NP: 92.7 ± 1.0), left primary somatosensory cortex campus/parahippocampus (intensity: r = 0.23, duration: r = 0.26), (controls: 107.5 ± 1.1, NP: 100.1 ± 1.3), left dorsolateral prefrontal right trigeminal nerve entry (intensity: r = 0.22, duration: r = 0.13), cortex (controls: 122.4 ± 4.6, NP: 105.5 ± 3.2) and the left hippo- right spinal trigeminal tract (intensity: r = 0.11, duration: r = 0.20), campus/parahippocampus (controls: 145.2 ± 2.2, NP: 131.3 ± 1.9). and right spinal trigeminal nucleus (intensity: r = 0.09, duration: Brainstem-specific analysis also revealed regions in which T2 r = 0.14, all p > 0.05). Furthermore, in NP subjects there were no significant linear relationships between PCS or BDI values and T2 re- Table 2 laxation times in the left mid-cingulate cortex (PCS: r = 0.26, BDI: Location, cluster size and significance level of T2 relaxation time decreases in individuals with chronic neuropathic pain compared with controls. Cluster peak r = 0.31), left anterior cingulate cortex (PCS: r = 0.16, BDI: r = 0.06), locations are indicated in Montreal Neurological Institute (MNI) space. left primary somatosensory cortex (PCS: r = 0.38; BDI: r = 0.22), left dorsolateral prefrontal cortex (PCS: r = 0.19, BDI: r = 0.28), left hip- Brain region MNI co-ordinate Cluster size t-score pocampus/parahippocampus (PCS: r = 0.08, BDI: r = 0.01), right tri- Whole brain T2 relaxation time geminal nerve entry (PCS: r = 0.03, BDI: r = 0.26), right spinal tri- Left mid cingulate cortex 6 −11 39 75 4.18 geminal tract (PCS: r = 0.04, BDI: r = 0.24), and right spinal trigeminal −3 −24 38 55 4.31 nucleus (PCS: r = 0.07, BDI: r = 0.11; all p > 0.05). − Left medial prefrontal cortex 6 24 24 10 3.88 Finally, exploring the effects of medication use on T2 relaxation Left primary somatosensory cortex −61 0 21 40 4.40 Left dorsolateral prefrontal cortex −56 24 9 18 4.11 times in NP subjects revealed that in no cluster was T2 relaxation time Left hippocampus/ 20 −32 −17 313 4.42 significantly different in those taking either acute analgesic or pro- parahippocampus phylactic medications compared with those not taking medication: left Brainstem T2 relaxation time mid-cingulate cortex (mean ± SEM ms: acute meds: 96.5 ± 3.4, pro- Right spinal trigeminal tract 9 30 −11 17 4.67 phylactic meds: 89.9 ± 1.4, no meds: 91.6 ± 1.1, acute v control Right trigeminal nerve entry 11 −26 −37 54 5.03 p = 0.08; prophylactic vs controls p = 0.36), left anterior cingulate Right spinal trigeminal nucleus 10 −42 −46 30 4.62 cortex (acute meds: 94.2 ± 0.6, prophylactic meds: 91.0 ± 1.3, no
170
55 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173
Fig. 3. Significant differences in T2 relaxation times in individuals with chronic neuropathic pain com- pared with controls, within the brainstem only. A) Regions in which chronic neuropathic pain (NP) subjects had significantly reduced T2 relaxation times compared with controls. Reductions are in- dicated by the cool colour scale and overlaid onto a brainstem template image set. Note the decreases within the region where orofacial nociceptor affer- ents terminate – the spinal trigeminal nucleus (SpV), as well as the area where the trigeminal nerve (V) enters the pons. Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice. B) Plots of mean ± SEM T2 re- laxation times (ms) in control (black bars) and NP ⁎ (blue bars) groups for significant clusters. denotes significant differences derived from the voxel-by- voxel random-effects analysis (p < 0.05, false dis- covery rate corrected). ipsi: ipsilateral to side of highest on-going pain. (For interpretation of the re- ferences to colour in this figure legend, the reader is referred to the web version of this article.)
meds: 93.3 ± 1.6, p = 0.78, p = 0.36), left primary somatosensory post mortem study of two individuals with multiple system atrophy cortex (acute meds: 99.8 ± 3.6, prophylactic meds: 103.0 ± 0.2.4, no reported reduced T2 relaxation times in the putamen, an area that also meds: 98.5 ± 1.7, p = 0.73, p = 0.12), left dorsolateral prefrontal displayed pronounced reactive astrogliosis and microgliosis (Schwarz cortex (acute meds: 101.4 ± 8.5, prophylactic meds: 101.9 ± 5.4, no et al., 1996). Whilst there are other anatomical changes besides astro- meds: 108.8 ± 4.4, p = 0.46, p = 0.34), left hippocampus/para- gliosis that could have contributed to the change in T2 relaxation times hippocampus (acute meds: 134.6 ± 6.5, prophylactic meds: shown in this investigation, several lines of evidence suggest that as- 130.9 ± 2.8, no meds: 130.6 ± 2.8, p = 0.54, p = 0.95) and right trogliosis is a likely contributing factor. spinal trigeminal nucleus (acute meds: 79.8 ± 5.8, prophylactic meds: We found decreased T2 relaxation time in the region encompassing 87.7 ± 5.4, no meds: 91.4 ± 3.4, p = 0.12, p = 0.53). the ipsilateral (to pain) spinal trigeminal nucleus, the area in which orofacial nociceptor afferents terminate. It is known from both human post-mortem and experimental animal investigations that the presence 4. Discussion of chronic pain following nerve injury is associated with prolonged astrocyte (and not microglial) activation at the level of the primary The results of this investigation reveal significant decreases in T2 afferent synapse (Garrison et al., 1991; Okada-Ogawa et al., 2009; Shi relaxation times in patients with chronic orofacial neuropathic pain at et al., 2012). In a previous investigation, we found an increase in on- multiple points along the ascending pain pathway. Changes occurred in going infra-slow oscillatory activity in the region encompassing the NP subjects in the region of the trigeminal nerve entry, the region of the spinal trigeminal nucleus in individuals with chronic orofacial neuro- ipsilateral (to pain) spinal trigeminal nucleus and in the contralateral pathic pain (Alshelh et al., 2016). Importantly, these increased infra- orofacial representation of the primary somatosensory cortex. In addi- slow oscillations were at similar frequencies as calcium waves in acti- tion, changes occurred in higher processing regions such as the cingu- vated astrocytes, which are associated with the release of glio- late and prefrontal cortices. These anatomical changes are consistent transmitters and the subsequent modulation of neural activity (Parri with pathological processes associated with astrocyte activation. and Crunelli, 2001). We speculated that although chronic neuropathic T2 relaxation times are influenced by the chemical characteristics of pain is not associated with increased on-going activity in the ascending tissue. One factor that will significantly affect T2 relaxation time is the pain pathways, the modulation of this pathway by gliotransmitters re- static dipolar fields created by neighbouring dipoles; in solids and sults in altered activity patterns, thalamocortical dysrhythmia and the polymers the static dipolar field is large. Thus, an increase in the pro- constant perception of pain (Alshelh et al., 2016; Henderson and Di portion of solids and polymers within a region of the brain for example, Pietro, 2016). A decrease in spinal trigeminal nucleus T2 relaxation would result in an increase in T2 relaxation rate, i.e. decrease in T2 time is consistent with this hypothesis, although whether this change in relaxation time, in that region (Goldman and Shen, 1966; Solomon, relaxation time represents astrocyte activation remains debatable. 1955). Such an increase in solids/polymers within a specific brain re- In our previous investigation, we also found altered infra-slow os- gion could result from numerous factors, including astrogliosis. In mild cillation increases associated with chronic neuropathic pain in the re- to moderate astrogliosis, there is an increase in glial fibrillary acidic gion of the ventrocaudal thalamus, thalamic reticular nucleus and in protein, an intermediate filament composed of a variety of proteins, the primary somatosensory cortex (Alshelh et al., 2016). In addition, which would contribute to an increase in static dipolar field and a de- others have reported increased [11C](R)-PK11195 binding, a marker of crease in T2 relaxation time (Sofroniew, 2009; Sofroniew and Vinters, glial activation, in the thalamus and primary somatosensory cortex in 2010; Stewart, 1993). Consistent with this, an experimental animal individuals with NP (Banati et al., 2001; Loggia et al., 2015). Whilst study that investigated changes following stroke reported decreased T2 altered [11C](R)-PK11195 binding may reflect changes in microglial or relaxation times in areas of increased astrocyte and microglial activa- astrocyte activation, surprisingly we found no change in T2 relaxation tion and iron deposition (Justicia et al., 2008). Furthermore, a human
171
56 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173 time in the thalamus in this investigation, despite showing changes in (HPA) axis function (Strange et al., 2014). Consistent with the idea that the spinal trigeminal nucleus and primary somatosensory cortex. Why these T2 relaxation time changes represent astrogliosis, there are mul- no change in T2 relaxation rate occurred in the thalamus is unknown, tiple investigations that report hippocampal volume increases in in- but it might be the case that the thalamus is somehow more resistant to dividuals with chronic pain, although there are some inconsistencies the development of astrogliosis than the primary afferent synapse or (Schweinhardt et al., 2008; Smallwood et al., 2013; Tu et al., 2010; somatosensory cortex, or that recurrent circuits between the thalamus Younger et al., 2010). Since chronic pain is also associated with the and cortex somehow reduces the potential for thalamic astrocyte acti- presence of stress-related disorders and HPA axis dysfunction (Griep vation following nerve injury. Indeed, in contrast to reports of pro- et al., 1998; Korszun et al., 2002; Tennant and Hermann, 2002), it is longed astrocyte activation at the primary afferent synapse, it has been possible that the changes in ventral hippocampal T2 relaxation times reported in the chronic constriction model of neuropathic pain that the are related to changes in HPA axis function in our chronic pain subjects. ventroposterior thalamus displays microglial and not astrocyte activa- We found that neither acute analgesic nor prophylactic medication tion at 7 and 14 days post injury, and that inhibiting microglial acti- use had significant effect on T2 relaxation times in NP subjects, making vation reduces thermal hyperalgesia (LeBlanc et al., 2011). This raises it unlikely that medication itself has a significant effect on T2 relaxation the possibility that increased infra-slow oscillations in the ventrocaudal times and the relationship between T2 relaxation times and pain and thalamus in individuals with NP are driven by such changes at the psychological measures. Whilst the effects of medication use on re- primary afferent synapse. This is consistent with the finding of a pre- gional astrocyte activation and T2 relaxation rate needs to be explored clinical study that shows infra-slow oscillatory activity in the ven- in a longitudinal manner, our hypothesis that neuropathic pain results troposterior thalamus was eliminated by severing the connection be- from chronic astrocyte activation is consistent with finding that neu- tween the primary afferent synapse and the thalamus (Iwata et al., ropathic pain medications such as valproate, phenytoin and gaba- 2011). pentin, reduce astrocyte calcium signalling (Tian et al., 2005). This Whilst the focus of this investigation was on changes in the as- raises the propect that other medications with potential astrocytic cending pain pathway, it is likely that other brain regions are also in- modulatory functions, such as Clozapine, Riluzole, Chromipramine and volved in the maintenance of neuropathic pain. We also found de- Palmitoylethanolamide, may also be effective at treating neuropathic creases in T2 relaxation time in the cingulate, dorsolateral prefrontal pain. and hippocampal cortices in individuals with neuropathic pain. There is In addition to reduced T2 relaxation rates, we have previously evidence from preclinical models of chronic pain of astrocyte activation shown in individuals with chronic orofacial pain, the SpV displays re- in the anterior cingulate cortex and suggestions that these changes are duced gray matter density, reduced mean diffusivity, increased infra- associated with sleep disturbances (Kuzumaki et al., 2007; Yamashita slow oscillations and increased functional connectivity strengths with et al., 2014). Whilst we did not measure sleep patterns in our chronic brainstem endogenous pain modulation circuitry (Alshelh et al., 2016; pain subjects it is possible that the altered T2 relaxation in the cingulate Mills et al., 2018; Wilcox et al., 2015). Despite altered functional con- cortex may reflect changes in sleep patterns that occur in individuals nectivity, we did not observe anatomical or infra-slow oscillatory with various forms of chronic pain (Pilowsky et al., 1985). Alter- changes in brainstem pain modulating regions such as the periaque- natively, there is growing evidence that the cingulate cortex codes the ductal gray, dorsolateral pons, subnucleus reticularis dorsalis or ros- affective dimension of pain and thus cingulate astrocyte activation may troventromedial medulla. Given this, we speculate that the anatomical reflect this aspect of the chronic pain condition, an idea that has sup- changes and resting oscillation changes that occur in SpV reflect as- port from preclinical investigations (Chen et al., 2012; Narita et al., trocyte activation that results from excitotoxic damage and is ex- 2006). acerbated by reduced endogenous analgesic circuitry action at the level Indeed it is well established that neuropathic pain is often asso- of SpV. Future studies are needed to provide solid evidence that such a ciated with psychological changes such as depression and pain cata- chain of events occurs. strophizing (Bair et al., 2003; Keefe et al., 2004) and we found sig- Finally, there are several limitations that need to be noted. Firstly, nificant differences in pain catastrophizing scores and depression scores as mentioned above, altered T2 relaxation times are not a direct index between the healthy controls and NP subjects. We did find significant of astrocyte activation and further combined MRI and histochemical changes in T2 relaxation times in the cingulate cortex, whose function investigations are needed to determine precisely how astrocyte acti- is associated with depression (Fonseka et al., 2017; Guo et al., 2012). vation alters T2 relaxation times. Although PET studies may provide a Although we found no correlation between T2 relaxation in any brain more accurate measure of glial activation, at this stage they are not region and depressive scores, there is evidence of increased infra-slow suited to measure changes at the primary afferent synapse in the oscillation power in the anterior cingulate cortex of individuals with brainstem or spinal cord. Secondly, it is difficult to accurately localize treatment-resistant depression (Guo et al., 2012). Furthermore, these clusters to specific nuclei, particularly those in the brainstem, due to the regions were also not correlated to pain intensity or duration of neu- relatively low spatial resolution and the fine compartmentalization of ropathic pain. Whilst the lack of linear correlations does not exclude the brainstem nuclei. Increasing MRI field strengths and greater accuracy of possibility that the changes in these areas are related to pain and psy- brainstem spatial normalization procedures will improve our con- chological measures, it may be that the underlying mechanisms re- fidence in brainstem-specific analysis in future investigations. Thirdly, sponsible for T2 relaxation time alterations reflect anatomical changes whilst we found that medication use had no effect on T2 relaxation that are simply not linearly coupled to changes in neural activity. times, this finding should be interpreted with caution given the small In contrast to the cingulate cortex, we know of no investigation number of subjects taking acute and preventive medications. Despite exploring astrocyte changes in the dorsolateral prefrontal cortex or these limitations, the discrete localization of decreases in T2 relaxation hippocampus in individuals with neuropathic pain or in preclinical times found in chronic orofacial neuropathic pain subjects in this in- models. Whilst the role of the dorsolateral prefrontal cortex in neuro- vestigation, together with existing human and pre-clinical evidence, pathic pain remains unclear, there is evidence that its anatomy and makes us confident that astrocytes play a critical role in the develop- function is altered in various pain conditions (Apkarian et al., 2004; ment and the maintenance of neuropathic pain following nerve injury. Lorenz et al., 2003; Seminowicz and Moayedi, 2017). Furthermore, there is evidence for a role for the dorsolateral prefrontal cortex in Acknowledgements descending pain modulation, which is altered in individuals with chronic pain (Bingel and Tracey, 2008; Youssef et al., 2016). We also This work was supported by funding from the Australian National found T2 relaxation time differences in the ventral aspect of the hip- Health and Medical Research Council G160279 and G182968 and the pocampus, a region thought to control hypothalamopituitary adrenal NGW Macintosh memorial fund.
172
57 Z. Alshelh et al. NeuroImage: Clinical 19 (2018) 167–173
References glial activation in chronic pain patients. Brain 138, 604–615. Lorenz, J., Minoshima, S., Casey, K.L., 2003. Keeping pain out of mind: the role of the dorsolateral prefrontal cortex in pain modulation. Brain 126, 1079–1091. Alshelh, Z., Di Pietro, F., Youssef, A.M., Reeves, J.M., Macey, P.M., Vickers, E.R., Peck, Mills, E.P., Di Pietro, F., Alshelh, Z., Peck, C.C., Murray, G.M., Vickers, E.R., Henderson, C.C., Murray, G.M., Henderson, L.A., 2016. Chronic neuropathic pain: it's about the L.A., 2018. Brainstem pain-control circuitry connectivity in chronic neuropathic pain. – rhythm. J. Neurosci. 36, 1008 1018. J. Neurosci. 38, 465–473. Apkarian, A.V., Sosa, Y., Sonty, S., Levy, R.M., Harden, R.N., Parrish, T.B., Gitelman, D.R., Moisset, X., Bouhassira, D., 2007. Brain imaging of neuropathic pain. NeuroImage 37 2004. Chronic back pain is associated with decreased prefrontal and thalamic gray (Suppl. 1), S80–88. – matter density. J. Neurosci. 24, 10410 10415. Melzack, R., 1975. The McGill Pain Questionnaire: major properties and scoring methods. Bair, M.J., Robinson, R.L., Katon, W., Kroenke, K., 2003. Depression and pain co- Pain 1, 277–299. – morbidity: a literature review. Arch. Intern. Med. 163, 2433 2445. Narita, M., Kuzumaki, N., Narita, M., Kaneko, C., Hareyama, N., Miyatake, M., Shindo, K., Banati, R.B., Cagnin, A., Brooks, D.J., Gunn, R.N., Myers, R., Jones, T., Birch, R., Anand, Miyoshi, K., Nakajima, M., Nagumo, Y., Sato, F., Wachi, H., Seyama, Y., Suzuki, T., P., 2001. Long-term trans-synaptic glial responses in the human thalamus after per- 2006. Chronic pain-induced emotional dysfunction is associated with astrogliosis due – ipheral nerve injury. Neuroreport 12, 3439 3442. to cortical delta-opioid receptor dysfunction. J. Neurochem. 97, 1369–1378. Beck, A.T., Ward, C.H., Mendelson, M., Mock, J., Erbaugh, J., 1961. An inventory for Nurmikko, T.J., Eldridge, P.R., 2001. Trigeminal neuralgia–pathophysiology, diagnosis – measuring depression. Arch. Gen. Psychiatry 4, 561 571. and current treatment. Br. J. Anaesth. 87, 117–132. Bingel, U., Tracey, I., 2008. Imaging CNS modulation of pain in humans. Physiology Okada-Ogawa, A., Suzuki, I., Sessle, B.J., Chiang, C.Y., Salter, M.W., Dostrovsky, J.O., – (Bethesda) 23, 371 380. Tsuboi, Y., Kondo, M., Kitagawa, J., Kobayashi, A., Noma, N., Imamura, Y., Iwata, K., Carlsson, A.M., 1983. Assessment of chronic pain. I. Aspects of the reliability and validity 2009. Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are – of the visual analogue scale. Pain 16, 87 101. involved in trigeminal neuropathic pain mechanisms. J. Neurosci. 29, 11161–11171. Chen, F.L., Dong, Y.L., Zhang, Z.J., Cao, D.L., Xu, J., Hui, J., Zhu, L., Gao, Y.J., 2012. Parri, H.R., Crunelli, V., 2001. Pacemaker calcium oscillations in thalamic astrocytes in ff Activation of astrocytes in the anterior cingulate cortex contributes to the a ective situ. Neuroreport 12, 3897–3900. fl – component of pain in an in ammatory pain model. Brain Res. Bull. 87, 60 66. Di Pietro, F., Macey, P.M., Rae, C.D., Alshelh, Z., Macefield, V.G., Vickers, E.R., Crunelli, V., Blethyn, K.L., Cope, D.W., Hughes, S.W., Parri, H.R., Turner, J.P., Toth, T.I., Henderson, L.A., 2018. The relationship between thalamic GABA content and resting Williams, S.R., 2002. Novel neuronal and astrocytic mechanisms in thalamocortical cortical rhythm in neuropathic pain. In: Human Brain Mapping, (accepted). – loop dynamics. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 357, 1675 1693. Pilowsky, I., Crettenden, I., Townley, M., 1985. Sleep disturbance in pain clinic patients. Diedrichsen, J., 2006. A spatially unbiased atlas template of the human cerebellum. Pain 23, 27–33. – NeuroImage 33, 127 138. Sarnthein, J., Stern, J., Aufenberg, C., Rousson, V., Jeanmonod, D., 2006. Increased EEG Fonseka, T.M., MacQueen, G.M., Kennedy, S.H., 2017. Neuroimaging biomarkers as power and slowed dominant frequency in patients with neurogenic pain. Brain 129, ff predictors of treatment outcome in Major Depressive Disorder. J. A ect. Disord. 233, 55–64. – 21 35. Schwarz, J., Weis, S., Kraft, E., Tatsch, K., Bandmann, O., Mehraein, P., Vogl, T., Oertel, Friston, K.J., Holmes, A.P., Worsley, K.P., Proline, J.B., Frith, C.D., Frackowiak, R.S.J., W.H., 1996. Signal changes on MRI and increases in reactive microgliosis, astro- 1995. Statistical parametric maps in functional imaging: a general imaging approach. gliosis, and iron in the putamen of two patients with multiple system atrophy. J. – Hum. Brain Mapp. 2, 189 210. Neurol. Neurosurg. Psychiatry 60, 98–101. Garrison, C.J., Dougherty, P.M., Kajander, K.C., Carlton, S.M., 1991. Staining of glial Schweinhardt, P., Kuchinad, A., Pukall, C.F., Bushnell, M.C., 2008. Increased gray matter fi brillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic density in young women with chronic vulvar pain. Pain 140, 411–419. – nerve constriction injury. Brain Res. 565, 1 7. Seminowicz, D.A., Moayedi, M., 2017. The dorsolateral prefrontal cortex in acute and Gerke, M.B., Duggan, A.W., Xu, L., Siddall, P.J., 2003. Thalamic neuronal activity in rats chronic pain. J. Pain 18, 1027–1035. with mechanical allodynia following contusive spinal cord injury. Neuroscience 117, Shi, Y., Gelman, B.B., Lisinicchia, J.G., Tang, S.J., 2012. Chronic-pain-associated astro- – 715 722. cytic reaction in the spinal cord dorsal horn of human immunodeficiency virus-in- – Goldman, M., Shen, L., 1966. Spin-spin relaxation in LaF3. Phys. Rev. 144, 321 331. fected patients. J. Neurosci. 32, 10833–10840. Griep, E.N., Boersma, J.W., Lentjes, E.G., Prins, A.P., van der Korst, J.K., de Kloet, E.R., Smallwood, R.F., Laird, A.R., Ramage, A.E., Parkinson, A.L., Lewis, J., Clauw, D.J., fi 1998. Function of the hypothalamic-pituitary-adrenal axis in patients with - Williams, D.A., Schmidt-Wilcke, T., Farrell, M.J., Eickhoff, S.B., Robin, D.A., 2013. – bromyalgia and low back pain. J. Rheumatol. 25, 1374 1381. Structural brain anomalies and chronic pain: a quantitative meta-analysis of gray Guo, W.-b., Liu, F., Xue, Z.-m., Xu, X.-j., Wu, R.-r., Ma, C.-q., Wooderson, S.C., Tan, C.-l., matter volume. J. Pain 14, 663–675. Sun, X.-l., Chen, J.-d., Liu, Z.-n., Xiao, C.-q., Chen, H.-f., Zhao, J.-p., 2012. Alterations Sofroniew, M.V., 2009. Molecular dissection of reactive astrogliosis and glial scar for- fl of the amplitude of low-frequency uctuations in treatment-resistant and treatment- mation. Trends Neurosci. 32, 638–647. response depression: a resting-state fMRI study. Prog. Neuro-Psychopharmacol. Biol. Sofroniew, M.V., Vinters, H.V., 2010. Astrocytes: biology and pathology. Acta – Psychiatry 37, 153 160. Neuropathol. 119, 7–35. Henderson, L.A., Di Pietro, F., 2016. How do neuroanatomical changes in individuals with Solomon, I., 1955. Relaxation processes in a system of two spins. Phys. Rev. 99, 559–565. – chronic pain result in the constant perception of pain? Pain Manag. Nurs. 6, 147 159. Strange, B.A., Witter, M.P., Lein, E.S., Moser, E.I., 2014. Functional organization of the Hsieh, J.C., Belfrage, M., Stone-Elander, S., Hansson, P., Ingvar, M., 1995. Central re- hippocampal longitudinal axis. Nat. Rev. Neurosci. 15, 655–669. presentation of chronic ongoing neuropathic pain studied by positron emission to- Sullivan, M.J.L., Bishop, S., Pivik, J., 1995. The Pain Catastrophizing Scale: development – mography. Pain 63, 225 236. and validation. Psychol. Assess. 7, 432–524. Iadarola, M.J., Max, M.B., Berman, K.F., Byas-Smith, M.G., Coghill, R.C., Gracely, R.H., Stewart, M., 1993. Intermediate filament structure and assembly. Curr. Opin. Cell Biol. 5, Bennett, G.J., 1995. Unilateral decrease in thalamic activity observed with positron 3–11. – emission tomography in patients with chronic neuropathic pain. Pain 63, 55 64. Tennant, F., Hermann, L., 2002. Normalization of serum cortisol concentration with Iwata, M., LeBlanc, B.W., Kadasi, L.M., Zerah, M.L., Cosgrove, R.G., Saab, C.Y., 2011. opioid treatment of severe chronic pain. Pain Med. 3, 132–134. High-frequency stimulation in the ventral posterolateral thalamus reverses electro- Tian, G.F., Azmi, H., Takano, T., Xu, Q., Peng, W., Lin, J., Oberheim, N., Lou, N., Wang, physiologic changes and hyperalgesia in a rat model of peripheral neuropathic pain. X., Zielke, H.R., Kang, J., Nedergaard, M., 2005. An astrocytic basis of epilepsy. Nat. – Pain 152, 2505 2513. Med. 11, 973–981. Jackson, G.D., Grunewald, R.A., Connelly, A., Duncan, J.S., 1994. Quantitative MR re- Tu, C.H., Niddam, D.M., Chao, H.T., Chen, L.F., Chen, Y.S., Wu, Y.T., Yeh, T.C., Lirng, ff laxometry study of e ects of vigabatrin on the brains of patients with epilepsy. J.F., Hsieh, J.C., 2010. Brain morphological changes associated with cyclic menstrual – Epilepsy Res. 18, 127 137. pain. Pain 150, 462–468. Justicia, C., Ramos-Cabrer, P., Hoehn, M., 2008. MRI detection of secondary damage after Wagner, D.C., Deten, A., Hartig, W., Boltze, J., Kranz, A., 2012. Changes in T2 relaxation stroke: chronic iron accumulation in the thalamus of the rat brain. Stroke 39, time after stroke reflect clearing processes. NeuroImage 61, 780–785. – 1541 1547. Wilcox, S.L., Gustin, S.M., Macey, P.M., Peck, C.C., Murray, G.M., Henderson, L.A., 2015. Keefe, F.J., Rumble, M.E., Scipio, C.D., Giordano, L.A., Perri, L.M., 2004. Psychological Anatomical changes at the level of the primary synapse in neuropathic pain: evidence – aspects of persistent pain: current state of the science. J. Pain 5, 195 211. from the spinal trigeminal nucleus. J. Neurosci. 35, 2508–2515. ff Korszun, A., Young, E.A., Singer, K., Carlson, N.E., Brown, M.B., Cro ord, L., 2002. Basal Walton, K.D., Llinás, R.R., 2010. Central pain as a thalamocortical dysrhythmia: a tha- circadian cortisol secretion in women with temporomandibular disorders. J. Dent. lamic efference disconnection? In: Kruger, L., Light, A.R. (Eds.), Translational Pain – Res. 81, 279 283. Research: From Mouse to Man. CRC Press, Boca Raton, FL. Kuzumaki, N., Narita, M., Narita, M., Hareyama, N., Niikura, K., Nagumo, Y., Nozaki, H., Yamashita, A., Hamada, A., Suhara, Y., Kawabe, R., Yanase, M., Kuzumaki, N., Narita, M., Amano, T., Suzuki, T., 2007. Chronic pain-induced astrocyte activation in the cin- Matsui, R., Okano, H., Narita, M., 2014. Astrocytic activation in the anterior cingu- ff gulate cortex with no change in neural or glial di erentiation from neural stem cells late cortex is critical for sleep disorder under neuropathic pain. Synapse 68, 235–247. – in mice. Neurosci. Lett. 415, 22 27. Younger, J.W., Shen, Y.F., Goddard, G., Mackey, S.C., 2010. Chronic myofascial tem- LeBlanc, B.W., Zerah, M.L., Kadasi, L.M., Chai, N., Saab, C.Y., 2011. Minocycline injection poromandibular pain is associated with neural abnormalities in the trigeminal and in the ventral posterolateral thalamus reverses microglial reactivity and thermal limbic systems. Pain 149, 222–228. – hyperalgesia secondary to sciatic neuropathy. Neurosci. Lett. 498, 138 142. Youssef, A.M., Gustin, S.M., Nash, P.G., Reeves, J.M., Petersen, E.T., Peck, C.C., Murray, Lenz, F.A., Kwan, H.C., Dostrovsky, J.O., Tasker, R.R., 1989. Characteristics of the G.M., Henderson, L.A., 2014. Differential brain activity in subjects with painful tri- bursting pattern of action potentials that occurs in the thalamus of patients with geminal neuropathy and painful temporomandibular disorder. Pain 155, 467–475. – central pain. Brain Res. 496, 357 360. Youssef, A.M., Macefield, V.G., Henderson, L.A., 2016. Cortical influences on brainstem Loggia, M.L., Chonde, D.B., Akeju, O., Arabasz, G., Catana, C., Edwards, R.R., Hill, E., circuitry responsible for conditioned pain modulation in humans. Hum. Brain Mapp. Hsu, S., Izquierdo-Garcia, D., Ji, R.R., Riley, M., Wasan, A.D., Zurcher, N.R., Albrecht, 37, 2630–2644. D.S., Vangel, M.G., Rosen, B.R., Napadow, V., Hooker, J.M., 2015. Evidence for brain
173
58 CHAPTER FOUR
EFFECTS OF GLIAL MODULATOR PALMITOYLETHANOLAMIDE ON CHRONIC PAIN INTENSITY AND BRAIN FUNCTION
Chapter Four was submitted to Nature Medicine
59
Effects of the glial modulator palmitoylethanolamide on chronic pain intensity and brain function
Z. Alshelh1, E.P. Mills1, D. Kosanovic1, F. Di Pietro1, P.M. Macey2, E.R. Vickers1 and L.A. Henderson1
1Department of Anatomy and Histology, University of Sydney, Australia, 2006. 2School of Nursing and Brain Research Institute, David Geffen School of Medicine; University of California at Los Angeles, Los Angeles, California, USA
Corresponding author: Luke A. Henderson, Department of Anatomy and Histology, F13, University of Sydney, Australia. [email protected] (email); +612 9351 7063 (Tel) +612 9351 6556 (Fax)
60 ABSTRACT:
Background: Chronic neuropathic pain is a complex disease that results from damage or presumed damage to the somatosensory nervous system. Current treatment regimens are often ineffective and have significant side-effects. The major impediment in developing more effective treatments is our limited understanding of the underlying mechanisms. Preclinical and post-mortem evidence suggests that glial changes are crucial for the development of neuropathic pain and a recent brain imaging study reported slow oscillatory activity differences within the ascending pain pathway at frequencies similar to that of cyclic gliotransmission in neuropathic pain. Furthermore, there is evidence that glial modifying medications may be effective in treating neuropathic pain. The aim of this phase 1 open-label clinical trial is to determine if the glial modifying medication palmitoylethanolamide (PEA) will reduce neuropathic pain and if this is associated with reductions in oscillatory activity within the pain pathway. Methods: Using resting state functional magnetic resonance imaging and arterial spin labelling, we investigated whether 6 weeks of PEA treatment would reduce on-going pain and infra-slow oscillatory activity within the ascending trigeminal pathway in 22 individuals with chronic orofacial neuropathic pain. Results: PEA reduced pain in 16 (73%) of the 22 subjects, 11 subjects had a pain reduction of over 20%. Those that responded to PEA had an average pain reduction of 2.1±0.3 (out of 10) and non-responders an increase of 0.6±0.5. Whilst both the responders and non-responders showed reductions in infra-slow oscillatory activity where orofacial nociceptor afferents terminate in the brainstem, only responders also displayed reductions in the thalamus. Furthermore, functional connections between the brainstem and thalamus were altered only in PEA responders. Interpretation: PEA is effective at relieving neuropathic pain without side-effects. This reduction is coupled to a reduction in resting oscillations along the ascending pain pathway that are likely driven by rhythmic astrocytic gliotransmission. Funding: Australia NMHRC, G160279 and G182968 and NGW Macintosh memorial fund.
61 Chronic neuropathic pain is a complex disease state that results from damage or presumed damage to the somatosensory nervous system. Current treatment regimens such as non- steroidal anti-inflammatory medication, tricyclic antidepressants and anticonvulsants are often ineffective and have significant side-effects and consequently, many individuals suffer with considerable pain for years or even decades. The major impediment in developing more effective treatment regimens for neuropathic pain is our limited understanding of the mechanisms responsible for the development and maintenance of neuropathic pain following injury.
Critically, neuropathic pain is not associated with prolonged activity increases in the classic “pain pathways” but instead is associated with altered brain rhythm. This altered rhythm is characterized by increased thalamic burst firing, thalamocortical power, and infra-slow oscillations (<0.1Hz) along the ascending pain pathway 1-3. Whilst it is not known how these altered rhythms are generated, the infra-slow oscillation increases occur at approximately the same frequency range as calcium waves in astrocytes, i.e. 0.03-0.06Hz 4. Astrocytes can modulate synaptic activity by the release of gliotransmitters and there is preclinical and post- mortem human evidence of prolonged astrocyte activation in the dorsal horn/spinal trigeminal nucleus in neuropathic pain 5,6. Furthermore, preclinical studies have shown that following nerve injury, increased astrocyte activation is associated with allodynia and hyperalgesia 7,8 and blocking astrocyte activation reverses this allodynia and hyperalgesia 9-11.
Palmitoylethanolamide (PEA) is a naturally occurring fatty acid amide that belongs to the N- acetylethanolamine family. It is thought that PEA targets alpha peroxisome proliferator- activated receptors (PPAR-α) 12-16, which are expressed on neurons and astrocytes 17,18 and are activated as a response to nerve damage 19,20. There have been numerous clinical trials exploring the effects of PEA in various pain states and critically, PEA has reportedly no serious side-effects 21. If PEA can reduce neuropathic pain without any serious side-effects, this would be a major advance in the way in which we treat chronic neuropathic pain and significantly reduce the economic and social burden of this debilitating condition. The aim of this investigation is to provide evidence that PEA reduces the intensity of on-going pain and reverses the increase in infra-slow oscillatory activity within the primary afferent synapse in individuals with chronic neuropathic pain.
62 RESULTS:
Psychophysics:
Prior to PEA treatment, NP subjects had on-going pain of 4.5±0.4 VAS, pain duration of 4.1±1.3 years and 5 NP subjects were taking Serotonin-Noradrenaline Reuptake Inhibitors. PEA treatment resulted in no reported side-effects and an overall reduction in on-going pain intensity of 0.7±0.4 VAS or 21.2±11.4% (Table 1). Overall, PEA treatment reduced pain in 16 (73%) of the 22 NP subjects. Dividing subjects into responders (>20% pain reduction) and non-responders (<20% pain reduction) resulted in 11 subjects as responders and 11 as non- responders (Figure 1A). Responders had an average pain reduction of 2.1±0.3 VAS or 61.9±7.2% and non-responders an overall increase of 0.6±0.5 VAS or 19.6±12.8% (Figure 1B). Cohen’s D showed that as a whole group, the effect size was small (d=0.3). However, in the responders, the effect size is large (d=1.8) and in the non-responders, the effect size is small (d=0.3). Although there was a significant difference in the magnitude of pain intensity reduction between groups, there was no significant change in the area of pain in response to PEA treatment in either group (mean±SEM pixels: responders prePEA 1143±494, postPEA 416±187; non-responders prePEA 7935±4325, postPEA 6890±3841, both p>0.05) (Figure 1C).
In addition to differences in the responsiveness to PEA treatment, responders had a significantly lower pain intensity (3.4±0.3 VAS) compared with non-responders (5.6±0.7 VAS) prior to PEA treatment (p<0.05). There was however, no significant difference in pain duration (responders 3.0±1.5 years, non-responders 5.2±2.1 years, p>0.05) or the area of perceived pain (responders 1143±494 pixels, non-responders 7935±4325 pixels, p>0.05) between groups before PEA treatment.
63
Figure 1: Subject psychophysics. A) daily pain intensity ratings in each of the 22 patients pre- palmitoylethanolamide (PEA) treatment and post-PEA treatment separated into responders (>20% pain reduction) and non-responders (<20% pain reduction). Pain intensity was rated three times daily on a 10cm visual analogue scale (VAS); B) mean (±SEM) pain intensity ratings pre- and post-PEA in the responder (n=11) and non-responder (n=11) groups; C) areas of on-going pain pre- and post-PEA in the responder and non-responder groups.
Infra-slow oscillatory activity and functional connectivity:
PEA treatment had a significant effect on regional resting infra-slow oscillations in both the responder and non-responder groups. Remarkably, in both the responder and non-responder groups, PEA treatment significantly reduced on-going infra-slow oscillatory activity in the region encompassing the ipsilateral (to side of on-going pain) spinal trigeminal nucleus (SpV), i.e., the region where nociceptor afferents from the area of on-going pain terminate (Figure 2, Table 2). Whilst both responders and non-responders displayed reduced oscillatory activity in
64 SpV, only the responders also displayed significant oscillatory activity reductions in the upstream target of SpV, i.e., in the region of the ventroposterior (VP) thalamus (Figure 3). In addition to changes in the ascending trigeminal pathway, the non-responders displayed oscillation decreases in the midbrain including the region of the periaqueductal gray matter (PAG), the anterior and posterior cingulate cortices and increases in the precuneus. Furthermore, in the responders, PEA treatment also resulted in oscillation decreases in the dorsolateral pons, hippocampus, lateral prefrontal cortex and precuneus.
Figure 2: Effects of palmitoylethanolamide (PEA) on infra-slow oscillations. A) Differences in fractional amplitude of low-frequency fluctuations (fALFF) pre-PEA compared with post-PEA treatment on non-responders. Significant increases (hot colour scale) and decreases (cool colour scale) in fALFF are overlaid onto a series of axial and sagittal T1-weighted anatomical slices. Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice. B) Differences in fALFF pre-PEA compared with post-PEA treatment in responders. Note that fALFF decreased in the spinal trigeminal nucleus (SpV) in both responders and non-responders and in the ventroposterior (VP) thalamus only in responders. ACC: anterior cingulate cortex; dlPons: dorsolateral pons; PAG: midbrain periaqueductal gray matter; PCC: posterior cingulate cortex.
The extraction of fALFF values from the SpV and VP thalamus confirmed that PEA treatment resulted in significant reductions in infra-slow oscillations in the SpV in both responders and non-responders and reductions in the VP thalamus in the responder group only (Figure 3). Furthermore, only responders displayed a significant change in resting connectivity between the SpV and VP thalamus in response to PEA treatment. That is, prior to PEA treatment there was a significant signal covariation between the SpV and VP thalamus in responders which was subsequently completely eliminated by PEA treatment (SpV-VP thalamus connectivity:
65 responders prePEA 0.18±0.07 post PEA 0.01±0.04, p<0.05). In contrast, PEA treatment had no effect on SpV-VP thalamus connectivity in the non-responders (prePEA 0.09±0.06 post PEA 0.04±0.05, p>0.05).
Figure 3: Effects of palmitoylethanolamide (PEA) on ascending pain pathway. To the right are plots of mean (±SEM) fractional amplitude of low-frequency fluctuations (fALFF) and cerebral blood flow (CBF) in responders (blue) and non-responders (green), pre-PEA and post-PEA treatment. Note that both the responders and non-responders have a significant reduction in infra-slow oscillations in the spinal trigeminal nucleus, but only the responders have a decrease in the ventroposterior thalamus. In contrast there are no changes in CBF. The regions in which fALFF decreases as a consequence of PEA treatment are colour coded blue (responders) and green (non-responders) and overlaid onto axial T1-weighted anatomical slices. To the left of these overlays are plots of mean (±SEM) resting functional connectivity between the spinal trigeminal nucleus and ventroposterior thalamus. Note that functional connectivity decreases significantly following PEA treatment in the responders only. *p<0.05 derived from voxel-by-voxel analysis; # p<0.05 derived from two-sample t-test.
In addition, the change in infra-slow oscillations within the ascending trigeminal pathway was not associated with changes in overall activity levels as evidenced by no change in resting blood flow in either the SpV (ml/min/100g: responders prePEA 98.7±7.9 post PEA 87.4±10.7, non-responders prePEA 79.9±10.5 post PEA 81.8±9.4, p>0.05) or the VP thalamus
66 (responders prePEA 43.9±6.1 post PEA 54.8±8.5, non-responders prePEA 28.9±5.4 post PEA 45.8±8.6, p<0.05).
Correlations between changes in pain intensity and changes in infra-slow oscillations revealed significant clusters in the SpV and VP thalamus overlapping those significant clusters revealed in the pre versus post fALFF analysis (Figure 4). In responders, the magnitude of the decrease in infra-slow oscillations was significantly positively correlated with a decrease in pain intensity during PEA treatment in both the region of the SpV (r=0.83, p<0.05) and VP thalamus (r=0.93, p<0.05). In contrast, no significant correlations occurred in non-responders in the SpV (r=0.04, p>0.05) or VP thalamus (r=0.28, p>0.05).
Figure 4: Relationships between changes in pain intensity and infra-slow oscillations as a result of palmitoylethanolamide (PEA) treatment. Significant positive linear relationships between changes in on-going pain and fractional amplitude of low-frequency fluctuations (fALFF) in the thalamus and medulla are shown overlaid onto axial T1-weighted anatomical slices for the responder group only (yellow shading). Note no such relationship occurred in the non-responders. Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice. The location of significant decreases in infra-slow oscillations (ISO) pre- versus post-PEA are also shown (light blue shading); note the overlap within the spinal trigeminal nucleus and ventroposterior thalamus. To the right are plots of change in fALFF versus percent change in pain for the SpV and VP thalamus cluster in responders and non-responders pre- versus post-PEA treatment. In responders, the greater the reduction in fALFF the greater the reduction in pain intensity.
67 DISCUSSION:
This investigation describes the effects of PEA on on-going pain and brain function in patients with chronic orofacial neuropathic pain. PEA reduced pain intensity in almost 75% of patients with 50% of all patients exhibiting an overall decrease of over 20% with no recorded side- effects. Additionally, PEA altered regional brain function by reducing infra-slow oscillatory activity in the ascending trigeminal pain pathway. More specifically, our results show that altered activity patterns and connectivity within the trigeminal pathway is associated with an individual’s pain relief responsiveness to PEA treatment. Whilst three previous investigations have reported neuropathic pain relief as a result of PEA treatment 22-24 this is the first to show that the effectiveness of PEA is associated with altered activity patterns in the ascending pain pathway. Critically, like our study, no clinical trial using PEA for pain or any other condition has reported significant side-effects.
We have previously shown that chronic orofacial neuropathic pain is associated with an increase in resting infra-slow oscillations along the ascending trigeminal pain pathway, including the SpV, VP thalamus, thalamic reticular nucleus and primary somatosensory cortex 25,26. Given that there is preclinical and human post-mortem evidence of chronic astrogliosis in the SpV/dorsal horn in individuals with neuropathic pain and that the oscillations were at the same approximate frequencies in which astrocyte calcium and gliotransmission release have been recorded, we speculate that these oscillatory activity increases were the result of chronic astrogliosis. Astrocyte calcium can propagate to neighbouring astrocytes either directly through connecting channels or indirectly through the release of gliotransmitters to produce synchronous oscillatory gliotransmitter release 27,28. This oscillatory astrocyte function can elicit long-lasting NMDA-mediated currents in surrounding neurons 4 and there is recent evidence that astrocytes can elicit long lasting increases in neural firing 29. We have proposed that following nerve injury, excess activity within primary afferent neurons results in neural death and elicits astrocyte activation at the primary afferent synapse. This results in more regular and increased oscillatory gliotransmitter release and increased infra-slow neural oscillations which are transferred along the ascending pain pathway. The recurrent nature of thalamocortical circuits results in self-sustaining thalamocortical dysrhythmia, paroxysmal events and the constant perception of pain 1,30,31.
Consistent with this hypothesis, the present investigation found that PEA administration resulted in a reduction in both pain intensity and oscillatory activity within the ascending trigeminal pain pathway. PEA is abundant in the central nervous system and there is
68 preclinical evidence that neuropathic pain is associated with reductions in PEA levels in the ipsilateral spinal cord and that these levels increase following pain relief 32. PEA is produced by glial cells 33 and has been studied primarily for its anti-inflammatory and neuroprotective effects 34-36. Whilst PEA can exert its effects via multiple mechanisms, critically, it has been shown to blunt astrocyte activation in vitro and improve neuronal survival through selective peroxisome proliferator activated receptor-alpha (PPAR-α) activation 37. Indeed, PEA fails to exert its anti-nociceptive effects in PPAR-α knockout mice 12,14,38-40.
We found that in all patients, oral administration of PEA resulted in significant reductions in infra-slow oscillations specifically at frequencies similar to those of astrocyte calcium waves (0.03-0.06Hz) in the ipsilateral SpV. However, only in individuals in which PEA evoked a significant reduction in pain did a similar reduction in neural oscillations also occur in the VP thalamus. Furthermore, PEA treatment eliminated the resting neural covariation between SpV and VP thalamus only in responders. These results suggest that whilst PEA blunts the increase in resting neural oscillatory activity at the region of the primary afferent synapse in all patients, only when this reduction is then transferred to higher brain centres such as the thalamus does an analgesia result. Indeed, only in responders was the reduction in neural oscillation power significantly correlated to the reduction in pain, i.e. the greater the reduction in oscillations the greater the reduction in on-going pain. This is entirely consistent with the results from an experimental animal model of neuropathic pain in which infra-slow oscillatory activity in the VP thalamus was eliminated by severing the connection between the primary afferent synapse and the thalamus 41.
Whilst we suggest that in individuals with chronic neuropathic pain, PEA produces pain relief via its effects on astrocyte function, PEA may also exert effects via a number of other mechanisms. Within the central nervous system, PPAR-α receptors act via two main mechanisms; a rapid effect which involves activation of calcium dependant potassium channels which has a silencing effect on neuronal firing 15 and a slower effect which is comprised of neurosteroid de novo synthesis which increases allopregnanolone production 42,43. Allopregnanolone is a positive allosteric modulator of GABA action and slows the rate of recovery of GABA receptors 44,45. In addition to a potential action on neuronal GABA receptors, PEA may influence GABA receptors on astrocytes, which when activated can increase intracellular calcium levels 46. Interestingly, astrocyte calcium wave propagation is dependent upon mechanisms which include the removal of excess calcium by mitochondria to prevent IP3 receptor desensitisation 47,48. It is possible that PEA increases calcium concentrations thereby reducing the propagation of astrocyte calcium waves and associated gliotransmission. Interestingly, all of the five patients in this investigation that were also taking SSRIs reported
69 pain relief, although four of these subjects reported reductions that were small and were categorized as nonresponders. Whilst the precise mechanism of SSRIs it not known, it has been shown that they can increase the expression of GABAB receptors and alter astrocyte function directly, evoking astrocyte calcium transients in an often oscillatory manner, even when neural activity is inhibited 49,50. Given this, it is possible that SSRIs partially counteract the mechanism of PEA and thus reduce its overall analgesic effectiveness.
It is also a possibility that PEA may act on other targets in the nervous system such as cannabinoids in neurons and astrocytes 51,52, PPAR-γ 53, or mast cells 54 to reduce pain. It is possible that PEA acts on multiple receptors in a number of sites to attenuate inflammation and pain associated with nerve damage 54. Furthermore, given that this was an open-label clinical trial, we did not control for the potential effects of placebo. However, it is unlikely that the decrease in pain intensity in the responders was solely due to the placebo effect. In fact, a Cochrane meta-analysis showed that over a number of investigations, the effect size of a placebo response, measured as Cohen’s d, is 0.27 55. In this investigation, the effect size as a whole group is 0.3, which may indeed be due to a placebo effect. However, in the responders only, the effect size is 1.8, suggesting that the response in the responders in not a placebo effect. Nonetheless, the decreases in pain intensity correlate with a change in neural activity in the responders as a result of PEA.
70 Online methods:
Subjects: Twenty two subjects with chronic orofacial neuropathic pain (17 females; mean±SEM age, 50.5± 3.0 years) were recruited for the study. Neuropathic pain (NP) subjects were diagnosed using the Liverpool criteria as having posttraumatic neuropathy 56. Subjects were excluded if they had any psychiatric conditions, any other pain conditions or did not meet standard MRI inclusion criteria. Informed written consent was obtained for all procedures according to the Declaration of Helsinki, and our local Institutional Human Research Ethics Committees approved the study.
Study Design: All subjects completed two magnetic resonance imaging (MRI) sessions, 6 weeks apart. During the 7 days prior to the start of PEA treatment and the first MRI session, subjects kept a pain diary in which they recording the intensity of their on-going pain, three times a day. Subjects rated the intensity of their pain using a 10 cm horizontal visual analogue scale (VAS), with 0 indicating “no pain” and 10 indicating “the most intense imaginable pain”. These 21 pain intensity scores were then averaged over the 7 day period to create a mean pain intensity score prior to PEA treatment. On the day of their first MRI scanning session, each NP subject outlined the location of their on-going pain on a standard drawing of the head and completed a McGill Pain Questionnaire. Following their first MRI scanning session, each NP subject began PEA treatment and at six weeks, a second MRI session was performed. During the 7 days prior to the second MRI scan, subjects kept another pain diary recording the intensity of their on-going pain. On the day of the second MRI scan, subjects outlined the location of their on-going pain on a standard drawing of the head and completed a McGill Pain Questionnaire.
Interventions: NP subjects obtained the PEA from Visionary Health Compounding Chemist, Newcastle, Australia in the form of 300mg capsules, with dosage individualised between 600mg to 900mg, 3 times a day.
MRI Acquisition: For each MRI scanning session, NP and control subjects lay supine on the bed of a 3 Tesla MRI scanner (Achieva; Phillips) with their head immobilized in a tight-fitting head coil. With each subject relaxed and at rest, a series of 180 gradient echo echo-planar functional MRI (fMRI) image volumes using blood oxygen level-dependent contrast were collected. Each image volume contained 35 axial slices covering the entire brain (field of view 240x240mm; matrix size 80x78; slice thickness 4mm; repetition time 2000ms; echo time
71 30ms; flip angle 90⁰). Following this, a series of 108 pseudocontinuous arterial spin labeling (pCASL) images containing 50 axial slices covering the entire brain was collected (54 label/control image pairs, field of view 240x240mm; matrix size 100x100; slice thickness 3mm; repetition time 5084ms, echo time 12.7ms, labelling time 1600ms, slice time 36.6ms, labeling efficiency 0.85, blood T1 relaxation time 1667ms). Finally, in each subject, a high-resolution 3D T1-weighted anatomical image set covering the entire brain was also collected (turbo field echo; field of view, 250x250 mm; matrix size 288x288; slice thickness 0.87mm; repetition time 5600ms; echo time 2.5ms; flip angle 8⁰).
Pain intensity changes: At the completion of the 6 week treatment period, NP subjects’ pain intensity changes were assessed. To explore the association between pain intensity reductions and changes in brain function, NP subjects were divided into those that responded to PEA treatment, i.e. responders: greater than 20% reduction in pain intensity, and non- responders: less than 20% reduction in pain intensity. To determine the effect size of changes in pain intensity in responders and non-responders, Cohen’s d was calculated. This is used to determine whether the effect size is small (d=0.2), medium (d=0.5) or large (d=0.8) 57. Changes in pain distributions before and after treatment for each subject in the two groups separately, were determined using a paired t test (p<0.05, Bonferroni corrected).
MRI Analysis; Infra-slow oscillations: Using SPM12 58, all functional magnetic resonance images were motion corrected, global signal drifts removed 59 and co-registered to each individual’s T1-weighted image set. Each subject’s T1-weighted anatomical image was then spatially normalized to a template in Montreal Neurological Institute (MNI) space and the parameters applied to the fMRI image sets. All images were then spatially smoothed (6mm full-width half-maximum [FWHM] Gaussian filter). In those subjects with pain restricted to the left side of the face (n=12), resting fMRI scans were reflected across the midline so that, in all subjects, the right side was ipsilateral to on-going pain. To perform a brainstem specific analysis, the fMRI images were cropped to include only the brainstem and cerebellum and a mask of these regions created automatically using the SUIT toolbox 60. These brainstem/cerebellum masks were then manually adjusted to accurately encompass the brainstem and cerebellum only. Using this mask, fMRI brainstem images were spatially normalized to the Spatial Unbiased Infratentorial Template in MRI space and spatially smoothed using a 3mm FWHM Gaussian filter.
To determine regional changes in infra-slow oscillation (ISO) power, we used the SPM toolbox REST 61 to calculate the sum of amplitudes of low frequency fluctuations (ALFF) in the frequency band 0.03-0.06Hz for the brainstem only and wholebrain fMRI image sets. ALFF
72 values were then divided over the entire frequency range to obtain fractional ALFF (fALFF) value for each voxel. Using the wholebrain and brainstem only images, the effects of PEA treatment on fALFF were determined in all 22 NP subjects using a paired random effects procedure. In addition, the effects of PEA on fALFF values were determined in the responder and non-responder groups separately using paired random effects procedures. Following an initial threshold of p<0.001, small volume corrections were used to account for the effects of multiple comparisons (p<0.05 Bonferonni corrected). For each analysis, the 6 movement parameters derived from the realignment step and signal intensity derived from a 3mm sphere located in the 4th ventricle were included as nuisance variables. For each significant cluster, fALFF values were extracted from each subject and the mean (±SEM) plotted.
We also determined the relationships between changes in fALFF over the treatment period and the associated change in pain intensity. In each voxel, the change in fALFF during PEA treatment (pre-post PEA) were calculated and voxel-by-voxel correlations between this fALFF change and the percent change in pain intensity were performed for the responder and non- responder groups separately (movement and CSF signal included as nuisance variables). Following an initial threshold of p<0.001, small volume corrections were used to account for the effects of multiple comparisons (p<0.05 Bonferonni corrected).
In addition, we also performed a functional connectivity analysis using a seed placed into the region of the spinal trigeminal nucleus (SpV). This seed was based on the results of the fALFF comparison pre compared to post PEA treatment in the responder group. Signal intensity changes from the SpV were extracted in each individual subject and correlations between this signal and every voxel in the brainstem only and wholebrain image sets determined (movement parameters and CSF signal included as nuisance variables). Connectivity values between the SpV and the ventroposterior thalamus (a cluster based on the results from the fALFF analysis) were extracted for each subject and significant differences between pre and post PEA treatment and between responder and non-responder groups determined using paired and two group t tests (p<0.05, Bonferroni corrected).
MRI Analysis; resting blood flow: All pCASL image sets were realigned, co-registered to each individual’s T1-weighted image set, the label and control images averaged and a mean cerebral blood flow (CBF) image created using the subtraction method using the ASL toolbox (Wang et al., 2008). Wholebrain and brainstem only CBF maps were then normalized into MNI space and smoothed in the same manner as the fMRI images sets. For each significant cluster derived from the fALFF analysis, blood flow values were extracted for each subject and the
73 mean values plotted for each subject before and after treatment and significance differences determined using paired t tests (p<0.05, Bonferroni corrected).
REFERENCES:
1. Sarnthein J, Stern J, Aufenberg C, Rousson V, Jeanmonod D. Increased EEG power and slowed dominant frequency in patients with neurogenic pain. Brain 2006; 129(Pt 1): 55-64. 2. Walton KD, Llinás RR. Central Pain as a Thalamocortical Dysrhythmia: A Thalamic Efference Disconnection? In: Kruger L, Light AR, eds. Translational Pain Research: From Mouse to Man. Boca Raton, FL: CRC Press; 2010. 3. Di Pietro F, Macey PM, Rae CD, et al. The relationship between thalamic GABA content and resting cortical rhythm in neuropathic pain. Human Brain Mapping 2018; accepted. 4. Crunelli V, Blethyn KL, Cope DW, et al. Novel neuronal and astrocytic mechanisms in thalamocortical loop dynamics. Philosophical transactions of the Royal Society of London Series B, Biological sciences 2002; 357(1428): 1675-93. 5. Garrison CJ, Dougherty PM, Kajander KC, Carlton SM. Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury. Brain Res 1991; 565(1): 1-7. 6. Okada-Ogawa A, Suzuki I, Sessle BJ, et al. Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms. The Journal of neuroscience : the official journal of the Society for Neuroscience 2009; 29(36): 11161-71. 7. Colburn RW, Deleo JA, Rickman AJ, Yeager MP, Kwon P, Hickey WF. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. Journal of Neuroimmunology 1997; 79(2): 163-75. 8. Aldskogius H, Kozlova EN. Central neuron–glial and glial–glial interactions following axon injury. Progress in Neurobiology 1998; 55(1): 1-26. 9. Miyoshi K, Obata K, Kondo T, Okamura H, Noguchi K. Interleukin-18-Mediated Microglia/Astrocyte Interaction in the Spinal Cord Enhances Neuropathic Pain Processing after Nerve Injury. The Journal of Neuroscience 2008; 28(48): 12775-87. 10. Zhuang ZY, Gerner P, Woolf CJ, Ji RR. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 2005; 114(1-2): 149-59. 11. Zhuang ZY, Wen YR, Zhang DR, et al. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. The Journal of neuroscience : the official journal of the Society for Neuroscience 2006; 26(13): 3551-60. 12. D'Agostino G, La Rana G, Russo R, et al. Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-kappaB nuclear signalling in dorsal root ganglia. European journal of pharmacology 2009; 613(1-3): 54-9. 13. D'Agostino G, La Rana G, Russo R, et al. Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist, modulates carrageenan-induced paw edema in mice. The Journal of pharmacology and experimental therapeutics 2007; 322(3): 1137-43. 14. Lo Verme J, Fu J, Astarita G, et al. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Molecular pharmacology 2005; 67(1): 15-9.
74 15. LoVerme J, Russo R, La Rana G, et al. Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-alpha. The Journal of pharmacology and experimental therapeutics 2006; 319(3): 1051-61. 16. Raso GM, Esposito E, Vitiello S, et al. Palmitoylethanolamide Stimulation Induces Allopregnanolone Synthesis in C6 Cells and Primary Astrocytes: Involvement of Peroxisome- Proliferator Activated Receptor-α. Journal of Neuroendocrinology 2011; 23(7): 591-600. 17. Moreno S, Farioli-Vecchioli S, Cerù MP. Immunolocalization of peroxisome proliferator- activated receptors and retinoid x receptors in the adult rat CNS. Neuroscience 2004; 123(1): 131-45. 18. Bento-Abreu A, Tabernero A, Medina JM. Peroxisome proliferator-activated receptor-alpha is required for the neurotrophic effect of oleic acid in neurons. J Neurochem 2007; 103(3): 871-81. 19. Zhuang Z-Y, Gerner P, Woolf CJ, Ji R-R. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 2005; 114(1): 149-59. 20. Okada-Ogawa A, Suzuki I, Sessle BJ, et al. Astroglia in Medullary Dorsal Horn (Trigeminal Spinal Subnucleus Caudalis) Are Involved in Trigeminal Neuropathic Pain Mechanisms. The Journal of Neuroscience 2009; 29(36): 11161-71. 21. Hesselink JMK, Hekker TAM. Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series. Journal of Pain Research 2012; 5: 437-42. 22. Gabrielsson L, Mattsson S, Fowler CJ. Palmitoylethanolamide for the treatment of pain: pharmacokinetics, safety and efficacy. Br J Clin Pharmacol 2016; 82(4): 932-42. 23. Hesselink JMK. New Targets in Pain, Non-Neuronal Cells, and the Role of Palmitoylethanolamide. The Open Pain Journal 2012; 5: 12-23. 24. P D. Associazione tra pregabalin e palmitoiletanolamide per il trattamento del dolore neuropatico. Pathos 2010; 7(4): 9-14. 25. Alshelh Z, Di Pietro F, Youssef AM, et al. Chronic Neuropathic Pain: It's about the Rhythm. The Journal of Neuroscience 2016; 36(3): 1008-18. 26. Alshelh Z, Di Pietro F, Mills EP, et al. Altered regional brain T2 relaxation times in individuals with chronic orofacial neuropathic pain. NeuroImage: Clinical 2018. 27. Cornell-Bell AH, Finkbeiner SM, Cooper MS, Smith SJ. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 1990; 247(4941): 470-3. 28. Scemes E, Giaume C. Astrocyte Calcium Waves: What They Are and What They Do. Glia 2006; 54(7): 716-25. 29. Deemyad T, Lüthi J, Spruston N. Astrocytes integrate and drive neural activity. bioRxiv 2018. 30. Hughes SW, Lorincz ML, Parri HR, Crunelli V. Infraslow (<0.1 Hz) oscillations in thalamic relay nuclei basic mechanisms and significance to health and disease states. Progress in brain research 2011; 193: 145-62. 31. Walton KD, Llinas RR. Frontiers in Neuroscience Central Pain as a Thalamocortical Dysrhythmia: A Thalamic Efference Disconnection? In: Kruger L, Light AR, eds. Translational Pain Research: From Mouse to Man. Boca Raton, FL: CRC Press/Taylor & Francis Llc.; 2010. 32. Guasti L, Richardson D, Jhaveri M, et al. Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain. Mol Pain 2009; 5: 35. 33. Walter L, Franklin A, Witting A, Moller T, Stella N. Astrocytes in culture produce anandamide and other acylethanolamides. J Biol Chem 2002; 277(23): 20869-76. 34. Hesselink JM, Hekker TA. Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series. Journal of pain research 2012; 5: 437-42. 35. M Keppel Hesselink J. New targets in pain, non-neuronal cells, and the role of palmitoylethanolamide. The Open Pain Journal 2012; 5(1).
75 36. Mattace Raso G, Russo R, Calignano A, Meli R. Palmitoylethanolamide in CNS health and disease. Pharmacological Research 2014; 86: 32-41. 37. Scuderi C, Valenza M, Stecca C, Esposito G, Carratu MR, Steardo L. Palmitoylethanolamide exerts neuroprotective effects in mixed neuroglial cultures and organotypic hippocampal slices via peroxisome proliferator-activated receptor-alpha. J Neuroinflammation 2012; 9: 49. 38. D'Agostino G, La Rana G, Russo R, et al. Acute intracerebroventricular administration of palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-α agonist, modulates carrageenan-induced paw edema in mice. Journal of Pharmacology and Experimental Therapeutics 2007; 322(3): 1137-43. 39. Haller VL, Cichewicz DL, Welch SP. Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice. European journal of pharmacology 2006; 546(1-3): 60-8. 40. LoVerme J, La Rana G, Russo R, Calignano A, Piomelli D. The search for the palmitoylethanolamide receptor. Life sciences 2005; 77(14): 1685-98. 41. Iwata M, LeBlanc BW, Kadasi LM, Zerah ML, Cosgrove RG, Saab CY. High-frequency stimulation in the ventral posterolateral thalamus reverses electrophysiologic changes and hyperalgesia in a rat model of peripheral neuropathic pain. Pain 2011; 152(11): 2505-13. 42. Sasso O, La Rana G, Vitiello S, et al. Palmitoylethanolamide modulates pentobarbital-evoked hypnotic effect in mice: Involvement of allopregnanolone biosynthesis. European Neuropsychopharmacology 2010; 20(3): 195-206. 43. Sasso O, Russo R, Vitiello S, et al. Implication of allopregnanolone in the antinociceptive effect of N-palmitoylethanolamide in acute or persistent pain. Pain 2012; 153(1): 33-41. 44. Neigh GN, Merrill L. Chapter 3 - Sex Differences in Neuroanatomy and Neurophysiology: Implications for Brain Function, Behavior, and Neurological Disease. Sex Differences in Physiology. Boston: Academic Press; 2016: 17-44. 45. Tsutsui K, Haraguchi S. Subchapter 96C - Allopregnanolone. Handbook of Hormones. San Diego: Academic Press; 2016: 544-e96C-3. 46. Meier Silke D, Kafitz Karl W, Rose Christine R. Developmental profile and mechanisms of GABA‐induced calcium signaling in hippocampal astrocytes. Glia 2008; 56(10): 1127-37. 47. Simpson PB, Mehotra S, Langley D, Sheppard CA, Russell JT. Specialized distributions of mitochondria and endoplasmic reticulum proteins define Ca2+ wave amplification sites in cultured astrocytes. J Neurosci Res 1998; 52(6): 672-83. 48. Boitier E, Rea R, Duchen MR. Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes. The Journal of cell biology 1999; 145(4): 795-808. 49. Schipke CG, Heuser I, Peters O. Antidepressants act on glial cells: SSRIs and serotonin elicit astrocyte calcium signaling in the mouse prefrontal cortex. J Psychiatr Res 2011; 45(2): 242-8. 50. Sands SA, McCarson KE, Enna SJ. Relationship between the antinociceptive response to desipramine and changes in GABAB receptor function and subunit expression in the dorsal horn of the rat spinal cord. Biochem Pharmacol 2004; 67(4): 743-9. 51. Re G, Barbero R, Miolo A, Di Marzo V. Palmitoylethanolamide, endocannabinoids and related cannabimimetic compounds in protection against tissue inflammation and pain: Potential use in companion animals. The Veterinary Journal 2007; 173(1): 21-30. 52. Navarrete M, Díez A, Araque A. Astrocytes in endocannabinoid signalling. Philosophical Transactions of the Royal Society B: Biological Sciences 2014; 369(1654): 20130599. 53. Paterniti I, Impellizzeri D, Crupi R, et al. Molecular evidence for the involvement of PPAR-delta and PPAR-gamma in anti-inflammatory and neuroprotective activities of palmitoylethanolamide after spinal cord trauma. J Neuroinflammation 2013; 10: 20. 54. Costa B, Comelli F, Bettoni I, Colleoni M, Giagnoni G. The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: involvement of CB1, TRPV1 and PPARγ receptors and neurotrophic factors. PAIN 2008; 139(3): 541-50.
76 55. Price DD, Hirsh A, Robinson ME. 5.64 - Psychological Modulation of Pain. In: Masland RH, Albright TD, Albright TD, et al., eds. The Senses: A Comprehensive Reference. New York: Academic Press; 2008: 975-1002. 56. Nurmikko TJ, Eldridge P. Trigeminal neuralgia—pathophysiology, diagnosis and current treatment. British Journal of Anaesthesia 2001; 87(1): 117-32. 57. Cohen J. Statistical power analysis for the behavioral sciences. Hillsdale, N.J.: L. Erlbaum Associates; 1988. 58. Friston KJ, Holmes AP, Worsley KJ, Poline JP, Frith CD, Frackowiak RS. Statistical parametric maps in functional imaging: a general linear approach. Human brain mapping 1994; 2(4): 189-210. 59. Macey PM, Macey KE, Kumar R, Harper RM. A method for removal of global effects from fMRI time series. Neuroimage 2004; 22(1): 360-6. 60. Diedrichsen J. A spatially unbiased atlas template of the human cerebellum. Neuroimage 2006; 33(1): 127-38. 61. Song XW, Dong ZY, Long XY, et al. REST: a toolkit for resting-state functional magnetic resonance imaging data processing. PLoS One 2011; 6(9): e25031.
77 Table 1: Subject characteristics: PEA: palmitoylethanolamide; SSRI: Selective Serotonin Reuptake Inhibitors; VAS: visual analogue scale.
subject age gender pain Site pain rating pain rating pain rating medication (years) duration pre PEA post PEA change (not including (years) (VAS) (VAS) (VAS[%]) PEA treatment)
860 33 F 7 left 2.1 0.8 -1.3 [-61.9] - 864 21 M 0.3 left 3.7 1.3 -2.4 [-64.9] - 867 64 F 17 right 3.8 0.3 -3.5 [-92.1] - 869 76 F 25 left 5.5 4.7 -0.8 [-14.5] SSRI 871 47 F 2.8 left 3.6 0.6 -3.0 [-83.3] - 873 40 F 1.5 right 5.1 2.9 -2.2 [-43.1] -
874 46 F 0.6 left 3.2 0.7 -2.5 [-78.1] - 875 52 F 0.8 right 2.7 1.0 -1.7 [-63.0] SSRI 876 58 M 1.8 left 9.6 8.8 -0.8 [-8.3] SSRI 882 44 M 3.3 bilateral 7.3 8.2 +0.9 [+12.3] - 883 47 F 0.4 right 1.9 3.8 +1.9 [+100] - 884 65 F 10 left 6.1 7.6 +1.5 [+24.6] - 890 64 F 0.3 right 1.4 1.1 -0.3 [-21.4] - 898 44 F 2.3 right 3.8 3.2 -0.6 [-15.8] SSRI 945 39 F 2.6 left 5.6 5.8 +0.2 [+3.6] - 947 74 F 0.4 bilateral 4.4 8.7 +4.3 [+97.7] - 950 29 M 5.8 left 6.6 5.6 -1.0 [-15.2] opioid+SSRI 956 46 F 1.0 right 3.6 1.2 -2.3 [-66.7] - 958 55 F 1.1 right 2.6 0.4 -2.3 [-84.6] - 975 57 F 0.7 left 5.2 4.0 -1.2 [-23.1] - 990 65 M 4 left 3.5 4.7 +1.2 [+34.3] - 996 46 F 1.8 left 7.5 7.3 -0.2 [-2.7] - mean 50.5±3.0 4.1±1.3 4.5±0.4 3.8±0.6 -0.7±0.4 ±SEM [-21.1±11.4]
78 Table 2. Montreal Neurological Institute (MNI) coordinates, cluster size and t-score for regions of significant differences in fractional amplitude of low-frequency fluctuations between pre- and post- palmitoylethanolamide treatment.
MNI cluster Brain region t-score Co-ordinate size x y z Responders
pre>post palmitoylethanolamide treatment
6 -30 -19 15 4.62 dorsolateral pons 10 -44 -53 5 5.40 spinal trigeminal nucleus -14 -22 6 10 4.18 left ventroposterior thalamus 18 -22 12 17 4.76 right dorsal thalamus 4 26 32 10 6.16 anterior cingulate cortex -32 -12 -22 19 7.17 hippocampus -10 -44 48 15 5.24 precuneus -4 -66 26 9 3.74
52 -54 24 15 3.93 posterior parietal association cortex -46 30 -10 16 5.70 ventrolateral prefrontal cortex
post>pre palmitoylethanolamide treatment -14 20 0 10 4.32 caudate nucleus -4 -74 -16 23 4.25 cerebellar cortex
Correlations with pain
increases 8 -46 -55 3 3.89 spinal trigeminal nucleus -18 -26 8 18 6.36 ventroposterior thalamus -8 34 20 17 8.78 anterior cingulate cortex
decreases -48 26 14 26 5.95 dorsolateral prefrontal cortex -44 26 30 12 9.34
79 CHAPTER FIVE
CONCLUSIONS
80 The overarching aim of this thesis was to investigate altered neural and glial activity in chronic orofacial neuropathic pain and the effects of palmitoylethanolamide (PEA) on neural activity. This chapter will summarise the main findings of each investigation, discuss the implications of these findings, and finally will provide recommendation for future research based on the questions that were raised by this thesis.
5.1 Main findings
The primary aim of Chapter 2 was to explore infra-slow oscillatory activity using resting state functional magnetic resonance imaging (fMRI) in patients with chronic orofacial neuropathic pain compared to healthy controls. Initially, resting oscillatory activity was explored at frequencies between 0.01 – 0.25 Hz, which was subsequently restricted to a frequency band between 0.03 – 0.06
Hz. Furthermore, Chapter 2 also assessed the effects of the infra-slow oscillatory (ISO) activity on local network connectivity (degree centrality), and local signal covariations (regional homogeneity) in patients and compared to controls.
The results of Chapter 2 revealed a significant increase in resting infra-slow oscillatory activity in patients with orofacial neuropathic pain in areas of the ascending pain pathway, that is, the spinal trigeminal nucleus, the ventral posterior (VP) thalamus, the thalamic reticular nucleus (TRN) and the primary somatosensory cortex (S1). Interestingly, this increase in infra-slow oscillatory activity in patients was restricted to the frequency band of 0.03 – 0.06 Hz in all regions of the ascending pain pathway. The study also revealed an increase in degree centrality in the same regions of the ascending pain pathways in patients and a significant increase in regional homogeneity in the spinal trigeminal nucleus in patients compared with controls. Furthermore, resting infra-slow oscillations within the ascending pain pathway were temporally coupled, suggesting that these signals may be generated at spinal trigeminal nucleus and then transmitted to the thalamus and the S1.
81 To assess whether the increase in infra-slow oscillatory activity was a characteristic of chronic
neuropathic pain or whether similar increases also occurred along the ascending pain pathway in pain-
free controls during an acute orofacial noxious stimulus, the same analysis during acute orofacial pain
in healthy controls was performed. There were no increases in infra-slow oscillatory activity in these
subjects in regions of the ascending pain pathway. There were, however, some decreases in infra-slow oscillatory activity in other regions of the central nervous system (CNS) and this is addressed in another research article in the appendix.
There is growing evidence that astrocytes can modulate neural activity through the release of gliotransmitters and that this release occurs in an oscillatory manner at similar frequencies to that of the increases along the ascending pain pathway identified in Chapter 2. This hypothesis led directly to the research that comprises Chapter 3 of this thesis. Chapter 3 aims to investigate whether the ISO changes observed in Chapter 2 are due to astrogliosis, measured using T2 relaxometry. While T2 relaxometry is not widely used to measure astrocyte activation, research suggests that a decrease in
T2 relaxation time is associated with an increase in astrocyte activation, and can therefore be used as an indirect measure of astrocyte activation. Results of Chapter 3 demonstrated that in patients with chronic orofacial neuropathic pain, there were decreases in T2 relaxation time in specific regions of the ascending pain pathway, that is; the spinal trigeminal nucleus and the primary somatosensory cortex. These decreases in T2 relaxation time are characteristic of an increase in astrocyte activity in these regions of the ascending pain pathway, which is consistent with our hypothesis of increased astrocyte activation along the ascending pain pathway in individuals with chronic orofacial pain.
Together, the results of Chapter 2 and 3 suggest that chronic neuropathic pain is associated with an increase in ISOs due to chronic astrocyte activation. Therefore, it is possible that decreasing astrocyte activity may reduce pain intensity or abolish pain in these patients. As such, Chapter 4 explores the
82 effects of exogenous palmitoylethanolamide (PEA), an astrocyte modulator, on infra-slow oscillatory
activity and pain intensity in patients with orofacial neuropathic pain. The results of Chapter 4 showed
that there is a pain reduction in over 75% of patients and half of all the patients have an overall
decrease in pain intensity of over 20% (responders). Whilst all patients displayed a significant
reduction in ISO within the spinal trigeminal nucleus following PEA treatment, only those patients with
a pain reduction of 20% or more, also displayed ISO reductions in higher regions of the trigeminal
pathway, that is in the VP thalamus. Furthermore, only in these responders did PEA treatment evoke
a significant decrease in resting connectivity between the spinal trigeminal nucleus and thalamus only
suggesting that a decrease in connectivity within regions of the pain pathway is essential for pain
relief.
5.2 Implications
The findings of this thesis indicate that infra-slow oscillatory activity is a fundamental part of pain
processing and may be modulated by astrocytes. The findings indicate that the role of astrocytes in
chronic neuropathic pain is more important than previously suggested, which was to provide
metabolic and physical support to other neural cells (Kıray et al., 2016). While it is currently not possible to non-invasively measure astrocyte activity in patients with neuropathic pain, our results strongly suggest that there in an increase in astrocyte activity associated with increase in ISOs in neuropathic pain and that reducing astrocyte activity and ISOs in certain regions of the ascending pain pathway reduces the intensity of pain in some patients.
The role of ISOs in the brain is fundamental to cerebral and thalamic function and has been studied in several investigations. In the somatosensory thalamus of the cat, ISOs are sustained largely by adenosine receptor mediated signalling, and this can be enabled by the activation of metabotrophic glutamate and/or acetylcholine receptors (Lőrincz et al., 2009). While the underlying mechanisms
83 responsible for ISOs is not yet well understood, it is suggested that astrocytes may play an important
role. As astrocytes can display spontaneous intracellular calcium oscillations at frequency similar to
ISOs, which can then propagate to nearby astrocytes, it is thought that within the thalamus, astrocytes
release adenosine to modulate neural activity (Parri and Crunelli, 2003). Astrocytes are also responsive
to glutamate and acetylcholine, and a link between ATP and ISOs has been demonstrated in the cortex
(Cunningham et al., 2006).
In the somatosensory thalamus of neonatal rats, astrocytes display spontaneous calcium oscillations
that can be regular or irregular in nature (Parri and Crunelli, 2002), and a proportion of astrocytes,
called pacemaker cells, display regular oscillations with a periodicity of approximately 0.01 Hz (Parri
and Crunelli, 2002). A recent investigation showed that astrocytes have the capability of directly
generating action potentials in interneurons, which can last for tens of seconds and this correlated
with calcium wave signalling (Deemyad et al., 2018). These calcium waves can propagate to
surrounding astrocytes and elicit long lasting NMDA-mediated currents in neurons (Crunelli et al.,
2002). It is believed that the mechanisms that underlie the irregular oscillations in astrocytic calcium
waves are the same as the mechanism that generate the calcium wave oscillations. Therefore, it is
possible that astrocytes are capable of transitioning from cells with irregular calcium oscillations to
pacemaker-type cells (Parri and Crunelli, 2001). On the other hand, as astrocytes are capable of activating nearby astrocytes via calcium wave propagation through inositol triphosphate receptors
(IP3) (Scemes and Giaume, 2006), it is also possible that astrocytes cause an increase in ISOs by increasing the number of activated astrocytes in a wave-like pattern to produce waves of glutamate.
These waves of glutamate then activate neurons in an oscillatory pattern and increase astrocytic processes (Lavialle et al., 2011).
84 In our patient population, there is evidence of a change in the pattern of firing within regions of the
ascending pain pathway when compared to healthy controls in the frequency range of 0.03-0.06 Hz.
In this series of investigations, it is suggested that astrocytes play a role in developing these ISOs
through either a transition between spontaneous irregular astrocytes to the pacemaker astrocytes, or
by an increase in the production of calcium waves. It is possible that following nerve damage,
astrocytes are transitioning into pacemaker astrocytes and result in an increase in regular pacemaker-
like activity or that neural damage results in an increase in the production of calcium waves causing
the increase in astrocytic wave-like activity. Additionally, nerve damage may result in an increase in
glutamine or acetylcholine, which results in the increase in adenosine from the activated astrocytes,
causing the increase in ISO power (Parri and Crunelli, 2001).
Importantly, animal studies using the chronic constriction model of neuropathic pain, report an increase in astrocyte activation in the primary afferent synapse of the spinal cord (Garrison et al.,
1991) and an increase in astrocytic processes in the periaqueductal gray (Mor et al., 2010a).
Interestingly, it has been shown that astrocytic, and not microglial, activation contributes to the mechanical allodynia that occurs in a chemotherapy-induced neuropathic pain model (Ji et al., 2013).
While some may argue that microglia also plays an important role in chronic neuropathic pain, it is likely that the initiation may indeed be due to microglia, however the chronicity is due to astrocytic activation in pain related regions such as the dorsal horn (Raghavendra et al., 2003). Another pain related regions that has also shown astrocytic changes is the midbrain periaqueductal grey following chronic constriction injury (Mor et al., 2010b). Remarkably, the increase in astrocyte activity was only evident in rats that showed sensory and behavioural changes, such as social interactions and dominance which suggests that changes in astrocyte activity may reflect sensory and/or behavioural changes. While there were no correlations between ISOs and depression or pain catastrophizing scores in Chapter 2, there is a correlation between reduced pain intensity and ISOs in the primary
85 afferent synapse and the thalamus in Chapter 4 of this investigation. This correlation suggests that the
changes in astrocyte activity likely reflect sensory changes in our patient population rather than other
complex behavioural changes, however, other behavioural changes and non-linear correlations with
ISOs may also be possible.
Consistent with this animal literature, there is evidence of altered anatomy of the primary synapse in patients with orofacial neuropathic pain, that is, a decrease in mean diffusivity which is a measure of free water movement (Wilcox et al., 2015). This decrease is could be attributed to increased astrocytic
processes which occurs in neuropathic pain models in rats (Mor et al., 2010a), causing increased
barriers for water. This investigated led to the suggestion that the increase in astrocyte activity at the
primary synapse are associated with increase in ISOs which may be transferred to the thalamus.
Interestingly, some studies have linked ISOs in the somatosensory system of humans with high
frequency power fluctuations (Mantini et al., 2007). Evidently, an activation of glutamate receptors
have shown to cause an increase in ISOs in the thalamus which causes rhythmic oscillations and
paroxysmal events (Hughes et al., 2011). This result suggests that changes in astrocyte function at the
primary synapse may result in an increase in thalamic ISOs, which underlies an increase in EEG power
(Sarnthein et al., 2006). As the thalamocortical circuits are in a constant loop, this results in a self-
sustaining thalamocortical dysrhythmia and the constant perception of pain.
Astrocytes play an important role in chronic neuropathic pain, therefore it would be ideal to directly
measure astrocyte activity. However, with the current technology, it is impossible to non-invasively
measure astrocytes in humans. Positron emission tomography can measure microglial activity with
the use of radio ligands as markers (Banati et al., 2001; Loggia et al., 2015), however, the brainstem
cannot be imaged using positron emission tomography due to technical limitations. Alternatively, T2
relaxometry (an MRI sequence) can be used to indirectly measure astrocyte activity as it has been
86 associated with astrocyte activity (Jackson et al., 1994; Schwarz et al., 1996; Wagner et al., 2012). In
Chapter 3 of this investigation there is a decrease in T2 relaxation time in regions of the ascending
pain pathway and this is associated with an increase in astrocyte activity (Schwarz et al., 1996).
Specifically, there are decreases in T2 relaxation time in the spinal trigeminal nucleus in patients with
orofacial neuropathic pain. This suggests that, compared to controls, patients have an increase in
astrocyte activity in the spinal trigeminal nucleus, yet no changes in thalamus. Although no changes
were evident in the VP thalamus, it is possible that astrocytic activation in the spinal trigeminal nucleus
may modulate thalamic activity via its direct neural connections. This is supported by Chapter 2, in
which changes in ISOs in the spinal trigeminal nucleus and in the thalamus that were temporally
coupled.
If an increase in astrocyte activity was critical for the development of the increase in ISOs in the
ascending pain pathway and the intensity of on-going pain, then reducing astrocyte activation should reduce ISOs and reduce the intensity of pain. This led to the suggestion that in trigeminal neuropathic pain, reducing astrocyte activity may reduce ISOs in areas of the trigeminal pain pathway and ultimately, reduce the intensity of orofacial pain. This theory was addressed in Chapter 4. An astrocyte modulator, PEA, was given to patients with chronic orofacial neuropathic pain and then these patients were grouped into two groups. Responders had a pain decrease of at least 20% and the remaining patients were classed as non-responders.
In chronic neuropathic pain conditions, there is a decrease in endogenous PEA levels due to an increase in fatty acid amide hydrolase (FAAH) levels. FAAH has been shown to degrade PEA in the spinal cord and regions of the brainstem involved in pain modulation (Petrosino et al., 2007), and an inhibition of FAAH (such as non-steroidal anti-inflammatory (NSAID) drugs) has an analgesic effect by increasing endogenous PEA levels (Jhaveri et al., 2006). While these types of drugs may not entirely
87 be effective in abolishing pain in patients with trigeminal neuropathic pain, it is likely that
administration of exogenous PEA may be necessary to reduce pain intensity by increasing PEA levels
(Guindon and Beaulieu, 2006). Indeed a number of clinical trials have proven the effects of PEA in
reducing pain intensity in a number of disease states (Hesselink and Hekker, 2012), however, none
have measured neural effects of PEA administration.
In Chapter 4, PEA administration decreased ISO activity in patients with orofacial neuropathic pain in
regions of the ascending pain pathway. While the precise mechanism of PEA is unknown, there are
multiple mechanisms of action that may explain its anti-inflammatory and analgesic effects. The most
widely accepted mechanism is through PEA activation of the peroxisome proliferator activated
receptor-alpha (PPAR-α). In PPAR-α knockout mice, PEA failed to exhibit its analgesic properties
(D'Agostino et al., 2009; D'Agostino et al., 2007; Haller et al., 2006; LoVerme et al., 2005a; LoVerme et
al., 2005b) and in the periphery, activation of this receptor can reduce pain and inflammation by
switching off the signalling cascade responsible for the production of pro-inflammatory mediators
(D'Agostino et al., 2009; Mattace Raso et al., 2014). These data suggest that it is likely that PPAR-α is involved in the analgesic and anti-inflammatory effects of PEA.
Activation of PPAR-α reduces astrocyte activation through potentially several mechanisms. In the central nervous system, PPAR-α is expressed on a number of cells, including on astrocytes and neurons
(Cimini et al., 2005) and its activation leads to a fast response and a slow response. The slow response on astrocytes involves a neurosteroidal de novo synthesis of allopregnanolone (ALLO). ALLO is a positive allosteric modulator of GABA. This means that ALLO binds to GABA at a site other than the active site and increases its activity. Through this process, ALLO has demonstrated an ability to exert a neuroprotective effect in the central nervous system (Neigh and Merrill, 2016; Tsutsui and
Haraguchi, 2016). GABA receptors are expressed on astrocytes, and the increase in GABA activity due
88 to ALLO synthesis will elicit an increase in GABA receptor activation in astrocytes. Interestingly,
activation of GABA receptors in astrocytes causes an increase in calcium concentrations which
depolarises the cell (Meier Silke et al., 2008). The increase in calcium concentrations suggests an
increase in calcium wave propagation, an increase in astrocytic processes and an increase in ISOs,
rather than the evident decrease in ISOs.
However, calcium propagation between astrocytes is dependent upon IP3 which are activated by
calcium (Scemes and Giaume, 2006). Indeed, propagation of calcium waves is dependent upon
mitochondrial removal of excess calcium to prevent IP3 receptor desensitisation (Boitier et al., 1999;
Simpson et al., 1998). Sensitivity of this receptor is shaped like a bell curve, i.e. low calcium
concentrations have an excitatory effect, while higher concentrations (up to about 300nM) have an
inhibitory effect on IP3 (Bezprozvanny et al., 1991; Finch et al., 1991). Based on the calcium
concentration-dependant sensitization, it is proposed that the decrease in ISOs evident in the results of Chapter 4 are due to increase calcium concentrations (due to PEA activation of GABA receptors) reaching threshold levels for IP3 receptors to become inhibitory. This prevents calcium waves from propagating to neighbouring astrocytes, decreases astrocytic processes and dampens ISO activity. It is also possible that with PEA administration, calcium concentrations did not reach threshold levels and this causes an increase in calcium propagation between astrocytes. This possibly underlies
increases in pain intensity in a number of patients.
The fast response of activation of PPAR-α does not directly involve astrocyte activity, however, it may also play a role in reducing ISOs. This response involves the activation of calcium dependant potassium channels in neurons (LoVerme et al., 2006). These voltage gated channels are fundamental regulators of neural excitability. Activation of these channels results in hyperpolarisation of the neurons leading to a silencing of neural activity in neighbouring cells (Faber and Sah, 2003). It is possible that in our
89 responders, the increase in PEA increases the hyperpolarisation of the neurons and hence decreases
neural oscillatory firing. Furthermore, the increase in GABA activity through the slow response
reinforces the decrease in neural firing as GABA is the main inhibitory neurotransmitter in the central
nervous system (Chebib and Johnston, 1999).
Another mechanism of action of PEA is through cannabinoid receptors. PEA is an endocannabinoid-
like compound with a low affinity binding property to cannabinoid receptors. There are two main
types of cannabinoid receptors, CB1 which is expressed in the central nervous system and CB2 which
is expressed in the peripheral nervous system (Pertwee, 1997). In the CNS, cannabinoid receptors are expressed on neurons and immune cells, such as astrocytes (Navarrete et al., 2014; Pertwee, 1997) and their activation has shown to produce analgesic effects. Anatomical and electrophysiological studies have shown that activation of CB1 targets presynaptic terminals on neurons to inhibit the release of neurotransmitters and provide analgesic effects (Elphick and Egertová, 2001; Pertwee,
1997). Through the same processes, PEA activation of endocannabinoid receptors could be contributing to the analgesic effects of PEA as evident in Chapter 4. Furthermore, activation of cannabinoid receptors in astrocytes cause an increase in calcium concentration (Navarrete et al.,
2014). This increase in calcium concentration can contribute to the overall increase in calcium levels causing IP3 receptors to become inhibitory and preventing calcium wave oscillations (discussed earlier). Therefore, PEA can act through a number of pathways to increase calcium levels up to threshold levels so that IP3 receptors become inhibitory. This then prevents the propagation of calcium waves and therefore decrease overall astrocyte activity. The decrease in astrocyte activity, along with the increase in GABA concentrations silences neural firing and infra-slow oscillatory activity, which may otherwise be transferred along the ascending pain pathway.
90 Consistent with this idea, in Chapter 4, although PEA decreased ISO activity in all patients, it only produced pain relief when this ISO decrease was transferred to higher brain centres such as the VP thalamus. That is, only patients in which PEA evoked a significant reduction in pain, did PEA also decrease ISOs within the VP thalamus. Furthermore, this Spv-VP thalamus ISO reduction was associated with a significant reduction in resting functional connectivity between the SpV and VP thalamus. This result is consistent with those reported in Chapter 3, whereby a decrease in T2 relaxometry/increase in astrocyte activity was evident only in the SpV, yet Chapter 2 reports increases in infra-slow oscillatory activity in both the SpV and VP thalamus. These results suggest that astrocytic changes in chronic neuropathic pain are confined to the dorsal horn/SpV, causing a change in neural activity locally which are then transferred to higher centres such as the thalamus. This is entirely consistent with an animal investigation which showed that in animals with neuropathic pain, increased
ISOs in the VP thalamus were eliminated by severing the connection between the dorsal horn and the thalamus (Iwata et al., 2011).
In Chapter 1 of this thesis, the role of thalamocortical rhythms in chronic neuropathic pain was discussed such that a change in the circuitry between the thalamus and the cortex is associated with chronic neuropathic pain. Therefore, in chronic neuropathic pain, the changes in pattern of activity that were transferred from the dorsal horn to the thalamus have caused a change in thalamocortical circuity, leading to the constant perception of pain. Yet, with administration of PEA, these changes in the pattern of activity are abolished as the connectivity between the dorsal horn and the thalamus are eliminated and ISOs in the thalamus are dampened. This may reset thalamocortical rhythms and reduce the constant perception of pain.
Overall, the evidence provided in this thesis strongly suggest that chronic neuropathic pain is associated with an increase in infra-slow oscillatory activity in areas of the ascending pain pathway;
91 the spinal trigeminal nucleus, the somatosensory thalamus and the primary somatosensory cortex.
These increases are associated with an increase in astrocyte activity, which was evident through
measuring T2 relaxation which indirectly showed increases in astrocyte activity in regions of the
ascending pain pathway. These increases in astrocyte activity were reduced with PEA treatment at the spinal trigeminal nucleus. PEA also reduced connectivity between the spinal trigeminal nucleus and the thalamus which then prevents the transfer of oscillatory activity along the ascending pain pathway, resulting in significant reductions in the intensity of on-going pain.
5.3 Recommendations for future research
This investigation identified changes in the pattern of activity in patients with trigeminal neuropathic
pain that could be reversed with PEA. However, no placebo group was used in Chapter 4. While the
results strongly suggest that the outcomes of from Chapter 4 were not due to placebo, future
investigations should utilize a randomised double blind, placebo-controlled study on a larger sample size. It would be ideal to extract blood samples from these patients to measure changes pain related protein levels that may affect whether a patient responds to the treatment and for genetic testing
(Andersson et al., 1996; Diatchenko et al., 2005).
To determine the precise mechanisms of action of PEA on astrocyte activity and infra-slow oscillations, it may be useful to use animal investigations using a neuropathic pain model, such as the chronic constriction injury. Such investigation will give the opportunity to address a number of theories that have been proposed in this thesis. Firstly, animal investigations would provide the opportunity to measure the role of endogenous PEA in chronic neuropathic pain and the effect of PEA concentration on calcium oscillations and ISOs. Based on this series of investigations, an increase in PEA concentration should cause an increase in calcium waves, a decrease in calcium oscillations and a decrease in ISOs. Secondly, the model will be able to determine whether abolishing calcium
92 oscillations in the primary synapse will also abolish calcium oscillations in the thalamus and whether
this will reduce pain intensity. Thirdly, such investigations will be able to determine the specific
pathways and mechanisms of action of PEA that allow it to have its analgesic response.
While it is necessary to determine the effects of PEA of pain intensity in animal models of neuropathic
pain, it would be ideal to also determine whether infra-slow oscillations are evident at same
frequencies in regions of the ascending pain pathway. Although this investigation strongly suggests
that the increases in ISOs are due to increases in astrocyte activity, animal investigations will provide
the opportunity to directly measure the relationship between astrocytes and infra-slow oscillations,
such as by testing whether blocking astrocyte activity will abolish infra-slow oscillations. In Chapter 3
of this thesis, T2 relaxation time was associated with astrocyte activity, this was based on physical
properties of the MRI sequence and astrocytes. However, provided the role of glia in pain has become
increasingly studied, for future investigations to use this non-invasive technique to measure astrocyte activity, animal models may be necessary to confirm that a decrease in T2 relaxation time is indeed associated with an increase in astrocyte activity.
93 References
Anderson, C.M., Swanson, R.A., 2000. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32, 1-14.
Andersson, G.B.J., 1999. Epidemiological features of chronic low-back pain. The Lancet 354, 581-585.
Andersson, H.I., Ejlertsson, G., Leden, I., Rosenberg, C., 1996. Characteristics of subjects with chronic pain, in relation to local and widespread pain report. A prospective study of symptoms, clinical findings and blood tests in subgroups of a geographically defined population. Scand J Rheumatol 25,
146-154.
Andrew, R., Derry, S., Taylor, R.S., Straube, S., Phillips, C.J., 2014. The Costs and Consequences of
Adequately Managed Chronic Non-Cancer Pain and Chronic Neuropathic Pain. Pain Practice 14, 79-
94.
Apkarian, A.V., Bushnell, M.C., Treede, R.-D., Zubieta, J.-K., 2005. Human brain mechanisms of pain perception and regulation in health and disease. European Journal of Pain 9, 463-463.
Apkarian, A.V., Sosa, Y., Sonty, S., Levy, R.M., Harden, R.N., Parrish, T.B., Gitelman, D.R., 2004.
Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. Journal of Neuroscience 24, 10410-10415.
Attwell, D., Buchan, A.M., Charpak, S., Lauritzen, M., MacVicar, B.A., Newman, E.A., 2010. Glial and neuronal control of brain blood flow. Nature 468, 232.
Auld, D.S., Robitaille, R., 2003. Glial cells and neurotransmission: an inclusive view of synaptic function. Neuron 40, 389-400.
Banati, R.B., Cagnin, A., Brooks, D.J., Gunn, R.N., Myers, R., Jones, T., Birch, R., Anand, P., 2001. Long- term trans-synaptic glial responses in the human thalamus after peripheral nerve injury.
Neuroreport 12, 3439-3442.
Barrett, A.M., Lucero, M.A., Le, T., Robinson, R.L., Dworkin, R.H., Chappell, A.S., 2007. Epidemiology,
Public Health Burden, and Treatment of Diabetic Peripheral Neuropathic Pain: A Review. Pain
Medicine 8, S50-S62.
94 Beck, P.D., Pospichal, M.W., Kaas, J.H., 1996. Topography, architecture, and connections of somatosensory cortex in opossums: evidence for five somatosensory areas. Journal of Comparative
Neurology 366, 109-133.
Bennett, M.I., Simpson, K.H., 2004. Gabapentin in the treatment of neuropathic pain. Palliative
Medicine 18, 5-11.
Bessou, P., Perl, E.R., 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. Journal of Neurophysiology 32, 1025-1043.
Bezprozvanny, l., Watras, J., Ehrlich, B.E., 1991. Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751.
Bishop, T., Marchand, F., Young, A.R., Lewin, G.R., McMahon, S.B., 2010. Ultraviolet-B-induced mechanical hyperalgesia: A role for peripheral sensitisation. Pain 150, 141-152.
Biswal, B., Yetkin, F.Z., Haughton, V.M., Hyde, J.S., 1995. Functional connectivity in the motor cortex of resting human brain using echo-planar MRI. Magn Reson Med 34, 537-541.
Boitier, E., Rea, R., Duchen, M.R., 1999. Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes. J Cell Biol 145, 795-808.
Brown, A.G., 1982. The dorsal horn of the spinal cord. Q J Exp Physiol 67, 193-212.
Brown, A.G., Iggo, A., 1967. A quantitative study of cutaneous receptors and afferent fibres in the cat and rabbit. The Journal of Physiology 193, 707-733.
Brown, J.D., Plutzky, J., 2007. Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets. Circulation 115, 518-533.
Burchiel, K.J., 1993. Trigeminal Neuropathic Pain. Springer Vienna, Vienna, pp. 145-149.
Burgess, P.R., Perl, E.R., 1967. Myelinated afferent fibres responding specifically to noxious stimulation of the skin. The Journal of Physiology 190, 541-562.
Bushnell, M., Apkarian, A., 2006. Representation of pain in the brain. Wall and Melzack’s Textbook of
Pain, 5th edition. Elsevier.
95 Bushnell, M.C., Duncan, G.H., Hofbauer, R.K., Ha, B., Chen, J.I., Carrier, B., 1999. Pain perception: Is
there a role for primary somatosensory cortex? Proc Natl Acad Sci U S A 96, 7705-7709.
Butt, A.M., Duncan, A., Berry, M., 1994. Astrocyte associations with nodes of Ranvier: ultrastructural
analysis of HRP-filled astrocytes in the mouse optic nerve. Journal of Neurocytology 23, 486-499.
Calignano, A., La Rana, G., Giuffrida, A., Piomelli, D., 1998. Control of pain initiation by endogenous
cannabinoids. Nature 394, 277-281.
Campbell, J.N., Khan, A.A., Meyer, R.A., Raja, S.N., 1988. Responses to heat of C-fiber nociceptors in
monkey are altered by injury in the receptive field but not by adjacent injury. Pain 32, 327-332.
Campbell, J.N., Meyer, R.A., 2006. Mechanisms of Neuropathic Pain. Neuron 52, 77-92.
Carman, J., Cowan, W., Powell, T., 1964. Cortical connexions of the thalamic reticular nucleus.
Journal of anatomy 98, 587.
Chebib, M., Johnston, G.A., 1999. The 'ABC' of GABA receptors: a brief review. Clin Exp Pharmacol
Physiol 26, 937-940.
Chesler, M., 2005. Failure and function of intracellular pH regulation in acute hypoxic-ischemic injury of astrocytes. Glia 50, 398-406.
Cimini, A., Benedetti, E., Cristiano, L., Sebastiani, P., D'Amico, M.A., D'Angelo, B., Di Loreto, S., 2005.
Expression of peroxisome proliferator-activated receptors (PPARs) and retinoic acid receptors (RXRs) in rat cortical neurons. Neuroscience 130, 325-337.
Coghill, R.C., Sang, C.N., Maisog, J.M., Iadarola, M.J., 1999. Pain intensity processing within the human brain: a bilateral, distributed mechanism. Journal of Neurophysiology 82, 1934-1943.
Colburn, R.W., Rickman, A.J., DeLeo, J.A., 1999. The Effect of Site and Type of Nerve Injury on Spinal
Glial Activation and Neuropathic Pain Behavior. Experimental Neurology 157, 289-304.
Cook, A.J., Woolf, C.J., Wall, P.D., 1986. Prolonged C-fibre mediated facilitation of the flexion reflex in the rat is not due to changes in afferent terminal or motoneurone excitability. Neuroscience
Letters 70, 91-96.
96 Crabtree, J.W., 1992. The somatotopic organization within the cat's thalamic reticular nucleus.
European Journal of Neuroscience 4, 1352-1361.
Crunelli, V., Blethyn, K.L., Cope, D.W., Hughes, S.W., Parri, H.R., Turner, J.P., Toth, T.I., Williams, S.R.,
2002. Novel neuronal and astrocytic mechanisms in thalamocortical loop dynamics. Philos Trans R
Soc Lond B Biol Sci 357, 1675-1693.
Cunningham, M.O., Pervouchine, D.D., Racca, C., Kopell, N.J., Davies, C.H., Jones, R.S., Traub, R.D.,
Whittington, M.A., 2006. Neuronal metabolism governs cortical network response state. Proc Natl
Acad Sci U S A 103, 5597-5601.
D'Agostino, G., La Rana, G., Russo, R., Sasso, O., Iacono, A., Esposito, E., Mattace Raso, G., Cuzzocrea,
S., Loverme, J., Piomelli, D., Meli, R., Calignano, A., 2009. Central administration of
palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-kappaB nuclear signalling in
dorsal root ganglia. Eur J Pharmacol 613, 54-59.
D'Agostino, G., La Rana, G., Russo, R., Sasso, O., Iacono, A., Esposito, E., Raso, G.M., Cuzzocrea, S., Lo
Verme, J., Piomelli, D., Meli, R., Calignano, A., 2007. Acute intracerebroventricular administration of
palmitoylethanolamide, an endogenous peroxisome proliferator-activated receptor-alpha agonist,
modulates carrageenan-induced paw edema in mice. J Pharmacol Exp Ther 322, 1137-1143.
Dani, J.W., Chernjavsky, A., Smith, S.J., 1992. Neuronal activity triggers calcium waves in hippocampal astrocyte networks. Neuron 8, 429-440.
Darmani, N.A., Izzo, A.A., Degenhardt, B., Valenti, M., Scaglione, G., Capasso, R., Sorrentini, I., Di
Marzo, V., 2005. Involvement of the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflammatory and neuropathic conditions: review of the available pre-clinical data, and first human studies. Neuropharmacology 48, 1154-1163.
De La Llave-Rincón, A.I., Fernández-De-Las-Peñas, C., Fernández-Carnero, J., Padua, L., Arendt-
Nielsen, L., Pareja, J.A., 2009. Bilateral hand/wrist heat and cold hyperalgesia, but not hypoesthesia, in unilateral carpal tunnel syndrome. Experimental Brain Research 198, 455-463.
Deemyad, T., Lüthi, J., Spruston, N., 2018. Astrocytes integrate and drive neural activity. bioRxiv.
97 Derbyshire, S.W., Jones, A.K., Gyulai, F., Clark, S., Townsend, D., Firestone, L.L., 1997. Pain processing
during three levels of noxious stimulation produces differential patterns of central activity. Pain 73,
431-445.
Dessem, D., Moritani, M., Ambalavanar, R., 2007. Nociceptive craniofacial muscle primary afferent
neurons synapse in both the rostral and caudal brain stem. J Neurophysiol 98, 214-223.
Diatchenko, L., Slade, G.D., Nackley, A.G., Bhalang, K., Sigurdsson, A., Belfer, I., Goldman, D., Xu, K.,
Shabalina, S.A., Shagin, D., Max, M.B., Makarov, S.S., Maixner, W., 2005. Genetic basis for individual variations in pain perception and the development of a chronic pain condition. Human Molecular
Genetics 14, 135-143.
Duale, C., Luccarini, P., Cadet, R., Woda, A., 1996. Effects of Morphine Microinjections into the
Trigeminal Sensory Complex on the Formalin Test in the Rat. Experimental Neurology 142, 331-339.
Duval, C., Muller, M., Kersten, S., 2007. PPARalpha and dyslipidemia. Biochim Biophys Acta 1771,
961-971.
Dworkin, R.H., Backonja, M., Rowbotham, M.C., et al., 2003. Advances in neuropathic pain:
Diagnosis, mechanisms, and treatment recommendations. Archives of Neurology 60, 1524-1534.
Elbert, T., Flor, H., Birbaumer, N., Knecht, S., Hampson, S., Larbig, W., 1994. Extensive reorganization of the somatosensory cortex in adult humans after nervous system injury. Neuroreport 5, 2593-
2597.
Elphick, M.R., Egertová, M., 2001. The neurobiology and evolution of cannabinoid signalling.
Philosophical Transactions of the Royal Society of London. Series B 356, 381-408.
Faber, E.S.L., Sah, P., 2003. Calcium-Activated Potassium Channels: Multiple Contributions to
Neuronal Function. The Neuroscientist 9, 181-194.
Farrell, M.J., Laird, A.R., Egan, G.F., 2005. Brain activity associated with painfully hot stimuli applied to the upper limb: A meta-analysis. Human Brain Mapping 25, 129-139.
98 Fernandez-de-las-Penas, C., de la Llave-Rincon, A.I., Fernandez-Carnero, J., Cuadrado, M.L., Arendt-
Nielsen, L., Pareja, J.A., 2009. Bilateral widespread mechanical pain sensitivity in carpal tunnel syndrome: evidence of central processing in unilateral neuropathy. Brain 132, 1472-1479.
Finch, E.A., Turner, T.J., Goldin, S.M., 1991. Calcium as a coagonist of inositol 1,4,5-trisphosphate- induced calcium release. Science 252, 443-446.
Finnerup, N.B., Otto, M., McQuay, H.J., Jensen, T.S., Sindrup, S.H., 2005. Algorithm for neuropathic pain treatment: An evidence based proposal. Pain 118, 289-305.
Fishbain, D.A., Cutler, R., Rosomoff, H.L., Rosomoff, R.S., 1997. Chronic pain-associated depression: antecedent or consequence of chronic pain? A review. The Clinical journal of pain 13, 116-137.
Franklin, A., Parmentier-Batteur, S., Walter, L., Greenberg, D.A., Stella, N., 2003.
Palmitoylethanolamide increases after focal cerebral ischemia and potentiates microglial cell motility. J Neurosci 23, 7767-7775.
Fukui, S., Shigemori, S., Nosaka, S., 2002. Central pain associated with low thalamic blood flow treated by electroconvulsive therapy. J Anesth 16, 255-257.
Garcia-Larrea, L., Frot, M., Valeriani, M., 2003. Brain generators of laser-evoked potentials: from dipoles to functional significance. Neurophysiologie Clinique/Clinical Neurophysiology 33, 279-292.
Garey, L.J., 2006. Brodmann's ‘Localisation in the Cerebral Cortex’. World Scientific.
Garrison, C.J., Dougherty, P.M., Kajander, K.C., Carlton, S.M., 1991. Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury. Brain Res
565, 1-7.
Gerke, M.B., Duggan, A.W., Xu, L., Siddall, P.J., 2003. Thalamic neuronal activity in rats with mechanical allodynia following contusive spinal cord injury. Neuroscience 117, 715-722.
Goldman, M., Shen, L., 1966. Spin-Spin Relaxation in La${\mathrm{F}}_{3}$. Physical Review 144,
321-331.
99 Graziano, A., Jones, E.G., 2004. Widespread thalamic terminations of fibers arising in the superficial
medullary dorsal horn of monkeys and their relation to calbindin immunoreactivity. J Neurosci 24,
248-256.
Guillery, R., Feig, S., Lozsadi, D., 1998. Paying attention to the thalamic reticular nucleus. Trends in
Neurosciences 21, 28-32.
Guindon, J., Beaulieu, P., 2006. Antihyperalgesic effects of local injections of anandamide, ibuprofen, rofecoxib and their combinations in a model of neuropathic pain. Neuropharmacology 50, 814-823.
Guo, W., Wang, H., Watanabe, M., Shimizu, K., Zou, S., LaGraize, S.C., Wei, F., Dubner, R., Ren, K.,
2007. Glial–Cytokine–Neuronal Interactions Underlying the Mechanisms of Persistent Pain. The
Journal of Neuroscience 27, 6006-6018.
Gustin, S.M., Peck, C.C., Wilcox, S.L., Nash, P.G., Murray, G.M., Henderson, L.A., 2011. Different pain, different brain: thalamic anatomy in neuropathic and non-neuropathic chronic pain syndromes. J
Neurosci 31, 5956-5964.
Gustin, S.M., Wrigley, P.J., Youssef, A.M., McIndoe, L., Wilcox, S.L., Rae, C.D., Edden, R., Siddall, P.J.,
Henderson, L.A., 2014a. Thalamic activity and biochemical changes in individuals with neuropathic pain following spinal cord injury. Pain 155, 1027-1036.
Gustin, S.M., Wrigley, P.J., Youssef, A.M., McIndoe, L., Wilcox, S.L., Rae, C.D., Edden, R.A., Siddall,
P.J., Henderson, L.A., 2014b. Thalamic activity and biochemical changes in individuals with neuropathic pain after spinal cord injury. Pain 155, 1027-1036.
Hains, B.C., Saab, C.Y., Waxman, S.G., 2006. Alterations in burst firing of thalamic VPL neurons and reversal by Na(v)1.3 antisense after spinal cord injury. J Neurophysiol 95, 3343-3352.
Hald, A., Nedergaard, S., Hansen, R.R., Ding, M., Heegaard, A.M., 2009. Differential activation of spinal cord glial cells in murine models of neuropathic and cancer pain. Eur J Pain 13, 138-145.
Haller, V.L., Cichewicz, D.L., Welch, S.P., 2006. Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice. Eur J Pharmacol
546, 60-68.
100 Head, H., Holmes, G., 1911. Sensory disturbances from cerebral lesions. Brain 34, 102-254.
Henderson, L.A., Peck, C.C., Petersen, E.T., Rae, C.D., Youssef, A.M., Reeves, J.M., Wilcox, S.L.,
Akhter, R., Murray, G.M., Gustin, S.M., 2013. Chronic pain: lost inhibition? J Neurosci 33, 7574-7582.
Henry, J.L., Lalloo, C., Yashpal, K., 2008. Central poststroke pain: An abstruse outcome. Pain Research
& Management : The Journal of the Canadian Pain Society 13, 41-49.
Hesselink, J.M., Hekker, T.A., 2012. Therapeutic utility of palmitoylethanolamide in the treatment of
neuropathic pain associated with various pathological conditions: a case series. J Pain Res 5, 437-
442.
Hildebrand, C., 1971a. Ultrastructural and light‐microscopic studies of the developing feline spinal
cord white matter. I. The nodes of Ranvier. Acta Physiologica 82, 81-107.
Hildebrand, C., 1971b. Ultrastructural and light‐microscopic studies of the nodal region in large myelinated fibres of the adult feline spinal cord white matter. Acta Physiologica 82, 43-79.
Holmes, G., 1927. Symposium on sensory disorders in organic disease of the nervous system: III.
Disorders of sensation produced by cortical lesions. Brain: A Journal of Neurology.
Hsieh, J.C., Belfrage, M., Stone-Elander, S., Hansson, P., Ingvar, M., 1995. Central representation of chronic ongoing neuropathic pain studied by positron emission tomography. Pain 63, 225-236.
Hsu, K.C., Chang, S.T., 2013. Correlation of thalamic blood flow redistribution with persistent complex regional pain syndrome in a stroke patient with poor diabetic control. J Diabetes Investig 4,
676-678.
Hughes, S.W., Lorincz, M.L., Parri, H.R., Crunelli, V., 2011. Infraslow (<0.1 Hz) oscillations in thalamic relay nuclei basic mechanisms and significance to health and disease states. Prog Brain Res 193, 145-
162.
Iannetti, G., Zambreanu, L., Cruccu, G., Tracey, I., 2005. Operculoinsular cortex encodes pain intensity at the earliest stages of cortical processing as indicated by amplitude of laser-evoked potentials in humans. Neuroscience 131, 199-208.
101 Ibuki, T., Hama, A.T., Wang, X.T., Pappas, G.D., Sagen, J., 1996. Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience 76, 845-858.
Iggo, A., 1960. Cutaneous mechanoreceptors with afferent C fibres. The Journal of Physiology 152,
337-353.
Iwata, M., LeBlanc, B.W., Kadasi, L.M., Zerah, M.L., Cosgrove, R.G., Saab, C.Y., 2011. High-frequency stimulation in the ventral posterolateral thalamus reverses electrophysiologic changes and hyperalgesia in a rat model of peripheral neuropathic pain. Pain 152, 2505-2513.
Jackson, G.D., Grunewald, R.A., Connelly, A., Duncan, J.S., 1994. Quantitative MR relaxometry study of effects of vigabatrin on the brains of patients with epilepsy. Epilepsy Res 18, 127-137.
Jeanmonod, D., Magnin, M., Morel, A., 1993. Thalamus and neurogenic pain: physiological, anatomical and clinical data. Neuroreport 4, 475-478.
Jhaveri, M.D., Richardson, D., Kendall, D.A., Barrett, D.A., Chapman, V., 2006. Analgesic Effects of
Fatty Acid Amide Hydrolase Inhibition in a Rat Model of Neuropathic Pain. The Journal of
Neuroscience 26, 13318-13327.
Ji, R.R., Berta, T., Nedergaard, M., 2013. Glia and pain: is chronic pain a gliopathy? Pain 154 Suppl 1,
S10-28.
Jones, A.K.P., Kulkarni, B., Derbyshire, S.W.G., 2003. Pain mechanisms and their disordersImaging in clinical neuroscience. British Medical Bulletin 65, 83-93.
Jones, E., 1975. Some aspects of the organization of the thalamic reticular complex. Journal of
Comparative Neurology 162, 285-308.
Jones, E.G., 2001. The thalamic matrix and thalamocortical synchrony. Trends in Neurosciences 24,
595-601.
Kawamata, M., Omote, K., 1996. Involvement of increased excitatory amino acids and intracellular
Ca2+ concentration in the spinal dorsal horn in an animal model of neuropathic pain. Pain 68, 85-96.
102 Kenshalo Jr, D., Isensee, O., 1983. Responses of primate SI cortical neurons to noxious stimuli.
Journal of Neurophysiology 50, 1479-1496.
Killian, J.M., Fromm, G.H., 1968. Carbamazepine in the treatment of neuralgia: Use and side effects.
Archives of Neurology 19, 129-136.
Kim, J.H., Greenspan, J.D., Coghill, R.C., Ohara, S., Lenz, F.A., 2007. Lesions limited to the human
thalamic principal somatosensory nucleus (ventral caudal) are associated with loss of cold sensations
and central pain. J Neurosci 27, 4995-5004.
Kıray, H., Lindsay, S.L., Hosseinzadeh, S., Barnett, S.C., 2016. The multifaceted role of astrocytes in regulating myelination. Experimental Neurology 283, 541-549.
Klit, H., Finnerup, N.B., Jensen, T.S., 2009a. Central post-stroke pain: clinical characteristics, pathophysiology, and management. Lancet Neurol 8, 857-868.
Klit, H., Finnerup, N.B., Jensen, T.S., 2009b. Central post-stroke pain: clinical characteristics, pathophysiology, and management. The Lancet Neurology 8, 857-868.
Kocher, L., Anton, F., Reeh, P.W., Handwerker, H.O., 1987. The effect of carrageenan-induced inflammation on the sensitivity of unmyelinated skin nociceptors in the rat. Pain 29, 363-373.
Koltzenburg, M., Torebjörk, H.E., Wahren, L.K., 1994. Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain. Brain 117, 579-
591.
Kozlov, A.S., Angulo, M.C., Audinat, E., Charpak, S., 2006. Target cell-specific modulation of neuronal activity by astrocytes. Proc Natl Acad Sci U S A 103, 10058-10063.
Lambert, D.M., Vandevoorde, S., Diependaele, G., Govaerts, S.J., Robert, A.R., 2001. Anticonvulsant activity of N-palmitoylethanolamide, a putative endocannabinoid, in mice. Epilepsia 42, 321-327.
Lavialle, M., Aumann, G., Anlauf, E., Pröls, F., Arpin, M., Derouiche, A., 2011. Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors. Proceedings of the National Academy of Sciences 108, 12915-12919.
103 Leijon, G., Boivie, J., Johansson, I., 1989. Central post-stroke pain — neurological symptoms and pain characteristics. Pain 36, 13-25.
Lenz, F.A., Garonzik, I.M., Zirh, T.A., Dougherty, P.M., 1998. Neuronal activity in the region of the thalamic principal sensory nucleus (ventralis caudalis) in patients with pain following amputations.
Neuroscience 86, 1065-1081.
Lenz, F.A., Kwan, H.C., Dostrovsky, J.O., Tasker, R.R., 1989. Characteristics of the bursting pattern of action potentials that occurs in the thalamus of patients with central pain. Brain Res 496, 357-360.
Lo Verme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A., Piomelli, D., 2005. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol Pharmacol 67, 15-19.
Loggia, M.L., Chonde, D.B., Akeju, O., Arabasz, G., Catana, C., Edwards, R.R., Hill, E., Hsu, S.,
Izquierdo-Garcia, D., Ji, R.R., Riley, M., Wasan, A.D., Zurcher, N.R., Albrecht, D.S., Vangel, M.G.,
Rosen, B.R., Napadow, V., Hooker, J.M., 2015. Evidence for brain glial activation in chronic pain patients. Brain 138, 604-615.
Lorenz, J., Casey, K.L., 2005. Imaging of acute versus pathological pain in humans. European Journal of Pain 9, 163-165.
Lőrincz, M.L., Geall, F., Bao, Y., Crunelli, V., Hughes, S.W., 2009. ATP-Dependent Infra-Slow (<0.1 Hz)
Oscillations in Thalamic Networks. PLoS ONE 4, e4447.
Love, S., Coakham, H.B., 2001. Trigeminal neuralgiaPathology and pathogenesis. Brain 124, 2347-
2360.
LoVerme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A., Piomelli, D., 2005a. The nuclear receptor peroxisome proliferator-activated receptor-alpha mediates the anti-inflammatory actions of palmitoylethanolamide. Mol Pharmacol 67, 15-19.
LoVerme, J., La Rana, G., Russo, R., Calignano, A., Piomelli, D., 2005b. The search for the palmitoylethanolamide receptor. Life Sci 77, 1685-1698.
104 LoVerme, J., Russo, R., La Rana, G., Fu, J., Farthing, J., Mattace-Raso, G., Meli, R., Hohmann, A.,
Calignano, A., Piomelli, D., 2006. Rapid broad-spectrum analgesia through activation of peroxisome
proliferator-activated receptor-alpha. J Pharmacol Exp Ther 319, 1051-1061.
Luccarini, P., Cadet, R., Duale, C., Woda, A., 1998. Effects of lesions in the trigeminal oralis and caudalis subnuclei on different orofacial nociceptive responses in the rat. Brain Res 803, 79-85.
M Keppel Hesselink, J., 2012. New targets in pain, non-neuronal cells, and the role of palmitoylethanolamide. The Open Pain Journal 5.
Maihofner, C., Handwerker, H.O., Neundorfer, B., Birklein, F., 2004. Cortical reorganization during recovery from complex regional pain syndrome. Neurology 63, 693-701.
Mantini, D., Perrucci, M.G., Del Gratta, C., Romani, G.L., Corbetta, M., 2007. Electrophysiological signatures of resting state networks in the human brain. Proc Natl Acad Sci U S A 104, 13170-13175.
Mattace Raso, G., Russo, R., Calignano, A., Meli, R., 2014. Palmitoylethanolamide in CNS health and disease. Pharmacological Research 86, 32-41.
McCracken, L.M., Gross, R.T., 1993. Does anxiety affect coping with chronic pain? The Clinical journal of pain 9, 253-259.
McLachlan, E.M., Jänig, W., Devor, M., Michaelis, M., 1993. Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia. Nature 363, 543.
Meier Silke, D., Kafitz Karl, W., Rose Christine, R., 2008. Developmental profile and mechanisms of
GABA‐induced calcium signaling in hippocampal astrocytes. Glia 56, 1127-1137.
Meisner, J.G., Marsh, A.D., Marsh, D.R., 2010. Loss of GABAergic interneurons in laminae I–III of the spinal cord dorsal horn contributes to reduced GABAergic tone and neuropathic pain after spinal cord injury. Journal of neurotrauma 27, 729-737.
Melzack, R., 1989. Phantom limbs, the self and the brain (the DO Hebb Memorial Lecture). Canadian
Psychology/Psychologie Canadienne 30, 1.
Mendell, L.M., Wall, P.D., 1965. Responses of Single Dorsal Cord Cells to Peripheral Cutaneous
Unmyelinated Fibres. Nature 206, 97.
105 Merskey, H., Bogduk, N., Taxonomy, I.A.f.t.S.o.P.T.F.o., 1994. Classification of Chronic Pain:
Descriptions of Chronic Pain Syndromes and Definitions of Pain Terms. IASP Press.
Merskey, H.E., 1986. Classification of chronic pain: descriptions of chronic pain syndromes and
definitions of pain terms. Pain.
Miki, K., Fukuoka, T., Tokunaga, A., Noguchi, K., 1997. Calcitonin gene-related peptide increase in the
rat spinal dorsal horn and dorsal column nucleus following peripheral nerve injury: Up-regulation in a subpopulation of primary afferent sensory neurons. Neuroscience 82, 1243-1252.
Miller, R., Raff, M., 1984. Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. The Journal of Neuroscience 4, 585-592.
Miyoshi, K., Obata, K., Kondo, T., Okamura, H., Noguchi, K., 2008a. Interleukin-18-mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain processing after nerve injury. J Neurosci 28, 12775-12787.
Miyoshi, K., Obata, K., Kondo, T., Okamura, H., Noguchi, K., 2008b. Interleukin-18-Mediated
Microglia/Astrocyte Interaction in the Spinal Cord Enhances Neuropathic Pain Processing after Nerve
Injury. The Journal of Neuroscience 28, 12775-12787.
Moore, K.A., Kohno, T., Karchewski, L.A., Scholz, J., Baba, H., Woolf, C.J., 2002. Partial Peripheral
Nerve Injury Promotes a Selective Loss of GABAergic Inhibition in the Superficial Dorsal Horn of the
Spinal Cord. The Journal of Neuroscience 22, 6724-6731.
Mor, D., Bembrick, A.L., Austin, P.J., Wyllie, P.M., Creber, N.J., Denyer, G.S., Keay, K.A., 2010a.
Anatomically specific patterns of glial activation in the periaqueductal gray of the sub-population of rats showing pain and disability following chronic constriction injury of the sciatic nerve.
Neuroscience 166, 1167-1184.
Mor, D., Bembrick, A.L., Austin, P.J., Wyllie, P.M., Creber, N.J., Denyer, G.S., Keay, K.A., 2010b.
Anatomically specific patterns of glial activation in the periaqueductal gray of the sub-population of rats showing pain and disability following chronic constriction injury of the sciatic nerve.
Neuroscience 166, 1167-1184.
106 Morley, S., Eccleston, C., Williams, A., 1999. Systematic review and meta-analysis of randomized
controlled trials of cognitive behaviour therapy and behaviour therapy for chronic pain in adults,
excluding headache. Pain 80, 1-13.
Nash, P.G., Macefield, V.G., Klineberg, I.J., Gustin, S.M., Murray, G.M., Henderson, L.A., 2010.
Bilateral activation of the trigeminothalamic tract by acute orofacial cutaneous and muscle pain in
humans. PAIN® 151, 384-393.
Nash, P.G., Macefield, V.G., Klineberg, I.J., Murray, G.M., Henderson, L.A., 2009. Differential
activation of the human trigeminal nuclear complex by noxious and non-noxious orofacial
stimulation. Human Brain Mapping 30, 3772-3782.
Navarrete, M., Díez, A., Araque, A., 2014. Astrocytes in endocannabinoid signalling. Philosophical
Transactions of the Royal Society B: Biological Sciences 369, 20130599.
Neigh, G.N., Merrill, L., 2016. Chapter 3 - Sex Differences in Neuroanatomy and Neurophysiology:
Implications for Brain Function, Behavior, and Neurological Disease. Sex Differences in Physiology.
Academic Press, Boston, pp. 17-44.
Nelson, A.J., Chen, R., 2008. Digit somatotopy within cortical areas of the postcentral gyrus in humans. Cerebral Cortex 18, 2341-2351.
Newman, E.A., 1986. High potassium conductance in astrocyte endfeet. Science 233, 453-454.
Nurmikko, T.J., Eldridge, P.R., 2001. Trigeminal neuralgia—pathophysiology, diagnosis and current treatment. BJA: British Journal of Anaesthesia 87, 117-132.
O'connor, E., Kimelberg, H., 1993. Role of calcium in astrocyte volume regulation and in the release of ions and amino acids. Journal of Neuroscience 13, 2638-2650.
Ohara, P.T., Vit, J.P., Bhargava, A., Jasmin, L., 2008. Evidence for a role of connexin 43 in trigeminal pain using RNA interference in vivo. J Neurophysiol 100, 3064-3073.
Olszewski, J., 1950. On the anatomical and functional organization of the spinal trigeminal nucleus.
Journal of Comparative Neurology 92, 401-413.
107 Papadopoulos, M.C., Manley, G.T., Krishna, S., Verkman, A., 2004. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. The FASEB journal 18, 1291-1293.
Parri, H.R., Crunelli, V., 2001. Pacemaker calcium oscillations in thalamic astrocytes in situ.
Neuroreport 12, 3897-3900.
Parri, H.R., Crunelli, V., 2002. Astrocytes, spontaneity, and the developing thalamus. J Physiol Paris
96, 221-230.
Parri, H.R., Crunelli, V., 2003. The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations. Neuroscience 120, 979-992.
Pasti, L., Volterra, A., Pozzan, T., Carmignoto, G., 1997. Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ.
Journal of Neuroscience 17, 7817-7830.
Pattany, P.M., Yezierski, R.P., Widerström-Noga, E.G., Bowen, B.C., Martinez-Arizala, A., Garcia, B.R.,
Quencer, R.M., 2002. Proton magnetic resonance spectroscopy of the thalamus in patients with chronic neuropathic pain after spinal cord injury. American Journal of Neuroradiology 23, 901-905.
Paxinos, G., Xu-Feng, H., Sengul, G., Watson, C., 2012. Chapter 8 - Organization of Brainstem Nuclei.
The Human Nervous System (Third Edition). Academic Press, San Diego, pp. 260-327.
Pekny, M., Johansson, C.B., Eliasson, C., Stakeberg, J., Wallen, A., Perlmann, T., Lendahl, U.,
Betsholtz, C., Berthold, C.H., Frisen, J., 1999. Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin. J Cell Biol 145, 503-514.
Pekny, M., Nilsson, M., 2005. Astrocyte activation and reactive gliosis. Glia 50, 427-434.
Penfield, W., Boldrey, E., 1937. Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation. Brain 60, 389-443.
Penfield, W., Rasmussen, T., 1950. The cerebral cortex of man; a clinical study of localization of function.
Perl, E.R., 1968. Myelinated afferent fibres innervating the primate skin and their response to noxious stimuli. The Journal of Physiology 197, 593-615.
108 Pertwee, R.G., 1997. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 74, 129-
180.
Petrosino, S., Palazzo, E., de Novellis, V., Bisogno, T., Rossi, F., Maione, S., Di Marzo, V., 2007.
Changes in spinal and supraspinal endocannabinoid levels in neuropathic rats. Neuropharmacology
52, 415-422.
Peyron, R., Garcia-Larrea, L., Gregoire, M.C., Convers, P., Lavenne, F., Veyre, L., Froment, J.C.,
Mauguiere, F., Michel, D., Laurent, B., 1998. Allodynia after lateral-medullary (Wallenberg) infarct. A
PET study. Brain 121 ( Pt 2), 345-356.
Peyron, R., Laurent, B., Garcia-Larrea, L., 2000. Functional imaging of brain responses to pain. A review and meta-analysis (2000). Neurophysiol Clin 30, 263-288.
Pinault, D., 2004. The thalamic reticular nucleus: structure, function and concept. Brain Res Brain Res
Rev 46, 1-31.
Pleger, B., Tegenthoff, M., Ragert, P., Forster, A.F., Dinse, H.R., Schwenkreis, P., Nicolas, V., Maier, C.,
2005. Sensorimotor retuning [corrected] in complex regional pain syndrome parallels pain reduction.
Ann Neurol 57, 425-429.
Pleger, B., Tegenthoff, M., Schwenkreis, P., Janssen, F., Ragert, P., Dinse, H.R., Völker, B., Zenz, M.,
Maier, C., 2004. Mean sustained pain levels are linked to hemispherical side-to-side differences of primary somatosensory cortex in the complex regional pain syndrome I. Experimental Brain
Research 155, 115-119.
Polycarpou, N., Ng, Y.L., Canavan, D., Moles, D.R., Gulabivala, K., 2005. Prevalence of persistent pain after endodontic treatment and factors affecting its occurrence in cases with complete radiographic healing. International Endodontic Journal 38, 169-178.
Purves, D., 2004. Neuroscience, Third ed. Sinauer Associates Inc, Sunderland MA.
Raghavendra, V., Tanga, F., DeLeo, J.A., 2003. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther
306, 624-630.
109 Ralston, H.J., 3rd, 2005. Pain and the primate thalamus. Prog Brain Res 149, 1-10.
Raso, G.M., Esposito, E., Vitiello, S., Iacono, A., Santoro, A., D’Agostino, G., Sasso, O., Russo, R.,
Piazza, P.V., Calignano, A., Meli, R., 2011. Palmitoylethanolamide Stimulation Induces
Allopregnanolone Synthesis in C6 Cells and Primary Astrocytes: Involvement of Peroxisome-
Proliferator Activated Receptor-α. Journal of Neuroendocrinology 23, 591-600.
Sarnthein, J., Stern, J., Aufenberg, C., Rousson, V., Jeanmonod, D., 2006. Increased EEG power and slowed dominant frequency in patients with neurogenic pain. Brain 129, 55-64.
Scemes, E., Giaume, C., 2006. Astrocyte Calcium Waves: What They Are and What They Do. Glia 54,
716-725.
Scholz, J., Broom, D.C., Youn, D.-H., Mills, C.D., Kohno, T., Suter, M.R., Moore, K.A., Decosterd, I.,
Coggeshall, R.E., Woolf, C.J., 2005. Blocking Caspase Activity Prevents Transsynaptic Neuronal
Apoptosis and the Loss of Inhibition in Lamina II of the Dorsal Horn after Peripheral Nerve Injury. The
Journal of Neuroscience 25, 7317-7323.
Schousboe, A., Sarup, A., Bak, L., Waagepetersen, H., Larsson, O., 2004. Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission. Neurochemistry international 45, 521-
527.
Schwarz, J., Weis, S., Kraft, E., Tatsch, K., Bandmann, O., Mehraein, P., Vogl, T., Oertel, W.H., 1996.
Signal changes on MRI and increases in reactive microgliosis, astrogliosis, and iron in the putamen of two patients with multiple system atrophy. J Neurol Neurosurg Psychiatry 60, 98-101.
Schweinhardt, P., Bushnell, M.C., 2010. Pain imaging in health and disease — how far have we come? The Journal of Clinical Investigation 120, 3788-3797.
Schweinhardt, P., Glynn, C., Brooks, J., McQuay, H., Jack, T., Chessell, I., Bountra, C., Tracey, I., 2006.
An fMRI study of cerebral processing of brush-evoked allodynia in neuropathic pain patients.
Neuroimage 32, 256-265.
Sessle, B.J., 1987. Invited Review: The Neurobiology of Facial and Dental Pain: Present Knowledge,
Future Directions. Journal of Dental Research 66, 962-981.
110 Sessle, B.J., 2000. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive
transmission and neuroplasticity, and their clinical correlates. Crit Rev Oral Biol Med 11, 57-91.
Shigenaga, Y., Takabatake, M., Sugimoto, T., Sakai, A., 1979. Neurons in marginal layer of trigeminal nucleus caudalis projecting to ventrobasal complex (VB) and posterior nuclear group (PO) demonstrated by retrograde labeling with horseradish peroxidase. Brain research 166, 391-396.
Simone, D.A., Sorkin, L.S., Oh, U., Chung, J.M., Owens, C., LaMotte, R.H., Willis, W.D., 1991.
Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons.
Journal of Neurophysiology 66, 228-246.
Simpson, P.B., Mehotra, S., Langley, D., Sheppard, C.A., Russell, J.T., 1998. Specialized distributions of mitochondria and endoplasmic reticulum proteins define Ca2+ wave amplification sites in cultured astrocytes. J Neurosci Res 52, 672-683.
Sindrup, S.H., Jensen, T.S., 1999. Efficacy of pharmacological treatments of neuropathic pain: an update and effect related to mechanism of drug action. PAIN® 83, 389-400.
Skaper, S.D., Buriani, A., Dal Toso, R., Petrelli, L., Romanello, S., Facci, L., Leon, A., 1996. The
ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons. Proc Natl Acad Sci U S A
93, 3984-3989.
Sofroniew, M.V., 2009a. Molecular dissection of reactive astrogliosis and glial scar formation. Trends in Neurosciences 32, 638-647.
Sofroniew, M.V., 2009b. Molecular dissection of reactive astrogliosis and glial scar formation. Trends
Neurosci 32, 638-647.
Sofroniew, M.V., Vinters, H.V., 2010a. Astrocytes: biology and pathology. Acta Neuropathol 119, 7-
35.
Sofroniew, M.V., Vinters, H.V., 2010b. Astrocytes: biology and pathology. Acta Neuropathol 119, 7-
35.
Solomon, I., 1955. Relaxation Processes in a System of Two Spins. Physical Review 99, 559-565.
111 Spreafico, R., Frassoni, C., Arcelli, P., De Biasi, S., 1994. GABAergic interneurons in the
somatosensory thalamus of the guinea-pig: a light and ultrastructural immunocytochemical investigation. Neuroscience 59, 961-973.
Stern, J., Jeanmonod, D., Sarnthein, J., 2006. Persistent EEG overactivation in the cortical pain matrix of neurogenic pain patients. Neuroimage 31, 721-731.
Stewart, M., 1993. Intermediate filament structure and assembly. Current Opinion in Cell Biology 5,
3-11.
Sugimoto, T., Bennett, G.J., Kajander, K.C., 1990. Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine.
Pain 42, 205-213.
Sur, M., Merzenich, M.M., Kaas, J.H., 1980. Magnification, receptive-field area, and" hypercolumn" size in areas 3b and 1 of somatosensory cortex in owl monkeys. Journal of Neurophysiology 44, 295-
311.
Tian, G.-F., Azmi, H., Takano, T., Xu, Q., Peng, W., Lin, J., Oberheim, N., Lou, N., Wang, X., Zielke, H.R.,
2005. An astrocytic basis of epilepsy. Nature medicine 11, 973.
Todd, A.J., 2010. Neuronal circuitry for pain processing in the dorsal horn. Nature Reviews
Neuroscience 11, 823.
Tölle, T., Xu, X., Sadosky, A.B., Painful diabetic neuropathy: a cross-sectional survey of health state impairment and treatment patterns. Journal of Diabetes and Its Complications 20, 26-33.
Tölle, T.R., Kaufmann, T., Siessmeier, T., Lautenbacher, S., Berthele, A., Munz, F., Zieglgänsberger,
W., Willoch, F., Schwaiger, M., Conrad, B., 1999. Region‐specific encoding of sensory and affective components of pain in the human brain: a positron emission tomography correlation analysis.
Annals of neurology 45, 40-47.
Torrance, N., Smith, B.H., Bennett, M.I., Lee, A.J., 2006. The epidemiology of chronic pain of predominantly neuropathic origin. Results from a general population survey. J Pain 7, 281-289.
112 Tracey, I., 2005. Nociceptive processing in the human brain. Current Opinion in Neurobiology 15,
478-487.
Tracey, I., Johns, E., 2010. The pain matrix: reloaded or reborn as we image tonic pain using arterial
spin labelling. Pain 148, 359-360.
Tsutsui, K., Haraguchi, S., 2016. Subchapter 96C - Allopregnanolone. Handbook of Hormones.
Academic Press, San Diego, pp. 544-e596C-543.
Turner, J.A., Jensen, M.P., Warms, C.A., Cardenas, D.D., 2002. Catastrophizing is associated with pain intensity, psychological distress, and pain-related disability among individuals with chronic pain after spinal cord injury. Pain 98, 127-134.
Uhelski, M.L., Davis, M.A., Fuchs, P.N., 2012. Pain affect in the absence of pain sensation: evidence of asomaesthesia after somatosensory cortex lesions in the rat. PAIN® 153, 885-892.
Voscopoulos, C., Lema, M., 2010. When does acute pain become chronic? Br J Anaesth 105 Suppl 1, i69-85.
Wagner, D.C., Deten, A., Hartig, W., Boltze, J., Kranz, A., 2012. Changes in T2 relaxation time after stroke reflect clearing processes. Neuroimage 61, 780-785.
Walton, K.D., Llinas, R.R., 2010. Frontiers in Neuroscience
Central Pain as a Thalamocortical Dysrhythmia: A Thalamic Efference Disconnection? In: Kruger, L.,
Light, A.R. (Eds.), Translational Pain Research: From Mouse to Man. CRC Press/Taylor & Francis
Llc., Boca Raton, FL.
Walz, W., 1989. Role of glial cells in the regulation of the brain ion microenvironment. Progress in neurobiology 33, 309-333.
Wasan, A.D., Loggia, M.L., Chen, L.Q., Napadow, V., Kong, J., Gollub, R.L., 2011. Neural correlates of chronic low back pain measured by arterial spin labeling. Anesthesiology 115, 364-374.
113 Weng, H.R., Lee, J.I., Lenz, F.A., Schwartz, A., Vierck, C., Rowland, L., Dougherty, P.M., 2000.
Functional plasticity in primate somatosensory thalamus following chronic lesion of the ventral
lateral spinal cord. Neuroscience 101, 393-401.
Whiteside, G.T., Munglani, R., 2001. Cell death in the superficial dorsal horn in a model of
neuropathic pain. Journal of Neuroscience Research 64, 168-173.
Whitsel, B.L., Petrucelli, L.M., Werner, G., 1969. Symmetry and connectivity in the map of the body surface in somatosensory area II of primates. Journal of Neurophysiology 32, 170-183.
Wilcox, S.L., Gustin, S.M., Macey, P.M., Peck, C.C., Murray, G.M., Henderson, L.A., 2015. Anatomical
Changes at the Level of the Primary Synapse in Neuropathic Pain: Evidence from the Spinal
Trigeminal Nucleus. The Journal of Neuroscience 35, 2508-2515.
Woolf, C.J., Ma, Q., 2007. Nociceptors-Noxious Stimulus Detectors. Neuron 55, 353-364.
Wrigley, P.J., Press, S.R., Gustin, S.M., Macefield, V.G., Gandevia, S.C., Cousins, M.J., Middleton, J.W.,
Henderson, L.A., Siddall, P.J., 2009. Neuropathic pain and primary somatosensory cortex reorganization following spinal cord injury. PAIN® 141, 52-59.
Wu, W.-C., Fernández-Seara, M., Detre, J.A., Wehrli, F.W., Wang, J., 2007. A theoretical and experimental investigation of the tagging efficiency of pseudocontinuous arterial spin labeling. Magn
Reson Med 58, 1020-1027.
Yamamura, H., Iwata, K., Tsuboi, Y., Toda, K., Kitajima, K., Shimizu, N., Nomura, H., Hibiya, J., Fujita,
S., Sumino, R., 1996. Morphological and electrophysiological properties of ACCx nociceptive neurons in rats. Brain research 735, 83-92.
Yen, C.-T., Lu, P.-L., 2013. Thalamus and pain. Acta Anaesthesiologica Taiwanica 51, 73-80.
Yoldas, O., Topuz, A., Isçi, A.S., Oztunc, H., Postoperative pain after endodontic retreatment: Single- versus two-visit treatment. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology 98, 483-
487.
114 Youssef, A.M., Gustin, S.M., Nash, P.G., Reeves, J.M., Petersen, E.T., Peck, C.C., Murray, G.M.,
Henderson, L.A., 2014. Differential brain activity in subjects with painful trigeminal neuropathy and
painful temporomandibular disorder. PAIN® 155, 467-475.
Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is sequentially activated in neurons, microglia,
and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic
pain model. Pain 114, 149-159.
Zhuang, Z.Y., Wen, Y.R., Zhang, D.R., Borsello, T., Bonny, C., Strichartz, G.R., Decosterd, I., Ji, R.R.,
2006. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J Neurosci 26, 3551-3560.
Zonta, M., Angulo, M.C., Gobbo, S., Rosengarten, B., Hossmann, K.-A., Pozzan, T., Carmignoto, G.,
2002. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.
Nature Neuroscience 6, 43.
115
APPENDIX
116 NeuroImage: Clinical 17 (2018) 222–231
Contents lists available at ScienceDirect
NeuroImage: Clinical
journal homepage: www.elsevier.com/locate/ynicl
Disruption of default mode network dynamics in acute and chronic pain MARK states
Z. Alshelha, K.K. Marciszewskia, R. Akhterb, F. Di Pietroa, E.P. Millsa, E.R. Vickersa, C.C. Peckb, ⁎ G.M. Murrayb, L.A. Hendersona, a Department of Anatomy and Histology, University of Sydney, 2006, Australia b Faculty of Dentistry, University of Sydney, 2006, Australia
ARTICLE INFO ABSTRACT
Keywords: It has been proposed that pain competes with other attention-demanding stimuli for cognitive resources, and Precuneus many chronic pain patients display significant attention and mental flexibility deficits. These alterations may Posterior cingulate cortex result from disruptions in the functioning of the default mode network (DMN) which plays a critical role in Attention attention, memory, prospection and self-processing, and recent investigations have found alterations in DMN Prefrontal cortex function in multiple chronic pain conditions. Whilst it has been proposed that these DMN alterations are a Chronic orofacial pain characteristic of pain that is chronic in nature, we recently reported altered oscillatory activity in the DMN Functional magnetic resonance imaging during an acute, 5 minute noxious stimulus in healthy control subjects. We therefore hypothesize that altered DMN activity patterns will not be restricted to those in chronic pain but instead will also occur in healthy individuals during tonic noxious stimuli. We used functional magnetic resonance imaging to measure resting state infra-slow oscillatory activity and functional connectivity in patients with chronic orofacial pain at rest and in healthy controls during a 20-minute tonic pain stimulus. We found decreases in oscillatory activity in key regions of the DMN in patients with chronic pain, as well as in healthy controls during tonic pain in addition to changes in functional connectivity between the posterior cingulate cortex and areas of the DMN in both groups. The results show that similar alterations in DMN function occur in healthy individuals during acute noxious stimuli as well as in individuals with chronic pain. These DMN changes may reflect the presence of pain per se and may underlie alterations in attentional processes that occur in the presence of pain.
1. Introduction One cortical network that is thought to be involved in higher order functions is the default mode network (DMN). This network consists of Chronic pain is a disease state with an enormous socioeconomic a set of regions that are functionally highly connected and include the burden. Whilst most investigations focus on understanding the under- medial prefrontal cortex (mPFC), posterior cingulate cortex (PCC), in- lying pathophysiology of chronic pain largely center on sensory aspects, ferior parietal cortex (IPC) and precuneus (Raichle et al., 2001; many chronic pain patients also report significant alterations in their Shulman et al., 1997). This network was firstly shown to be more active sleep-wake cycle, family and social relations and cognitive function during passive task conditions than during numerous goal-directed (Iverson and McCracken, 1997; Smith-Seemiller et al., 2003). It has tasks. More recently it has been hypothesized that in addition to a role been proposed that chronic pain is attention demanding, competing in memory, prospection and self-processing (Cavanna and Trimble, with other attention-demanding stimuli for cognitive resources 2006; Spreng and Grady, 2010), all of which require a continuous level (Eccleston and Crombez, 1999). Indeed, attention deficits are displayed of background activity, the DMN also may act as a sentinel by mon- in individuals with chronic pain conditions (Eccleston, 1994; Grisart itoring the external environment (Buckner et al., 2008). and Van der Linden, 2001; Van Damme et al., 2010), and significant Some recent investigations using resting state functional magnetic associations between chronic pain and sustained attention and mental resonance imaging (fMRI) have reported significant changes in DMN flexibility have been reported (Karp et al., 2006; Sjogren et al., 2000; function in many chronic pain conditions (Baliki et al., 2008; Baliki Verdejo-Garcia et al., 2009). et al., 2014; Letzen et al., 2013; Napadow et al., 2012; Tagliazucchi
Abbreviations: ALFF, amplitude of low-frequency fluctuations; fALFF, fractional amplitude of low-frequency fluctuations; DMN, default mode network; IPC, inferior parietal cortex; ISO, infra-slow oscillations; mPFC, medial prefrontal cortex; PCC, posterior cingulate cortex ⁎ Corresponding author at: Department of Anatomy and Histology, F13, University of Sydney, Australia. E-mail address: [email protected] (L.A. Henderson). http://dx.doi.org/10.1016/j.nicl.2017.10.019 Received 7 July 2017; Received in revised form 8 September 2017; Accepted 18 October 2017 Available online 19 October 2017 2213-1582/ © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
117 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231 et al., 2010). These studies clearly show that chronic pain is associated planar functional MRI image volumes using BOLD contrast were col- with altered DMN dynamics, and it has been strongly suggested that lected. Each image volume contained 35 axial slices covering the entire chronic pain reorganizes “the dynamics of the DMN and as such reflect brain (repetition time = 2000 ms; echo time = 30 ms, flip the maladaptive physiology of different types of chronic pain” (Baliki angle = 90°, raw voxel size = 3 × 3 × 4 mm). In each subject, a high- et al., 2008). Whilst this may be true, acute pain in a healthy, non- resolution 3D T1-weighted anatomical image set, covering the entire chronic pain individual is unquestionably attention demanding, com- brain, was collected (turbo field echo; repetition time = 5600 ms; echo peting with other attention-demanding stimuli for cognitive resources. time = 2.5 ms, flip angle = 8°, raw voxel size 0.87 mm3). Though Seminowicz and Davis (2007a) found little effect of acute pain In 16 of the 57 control subjects (5 females, age 24.9 ± 1.2 years) a on cognitive performance, this lack of effect might be due to the short tonic pain stimulus was delivered. A catheter connected to a syringe stimulus duration (14 s). Indeed, there is considerable brain imaging filled with hypertonic saline (5%) was placed into the right masseter evidence showing that pain and cognitive-related brain activities in- muscle midway between its upper and lower borders. The catheter was teract, possibly due to similar neural circuitries (Bantick et al., 2002; attached to an infusion pump with a 10 ml syringe placed outside the Petrovic et al., 2000; Remy et al., 2003; Valet et al., 2004). scanner room. With the subject relaxed, 500 gradient echo echo-planar In a separate investigation, Seminowicz and Davis also found that fMRI image volumes using BOLD contrast were collected over a 25- whilst performing an attention-demanding cognitive task during acute minute period. Each image volume contained 42 axial slices covering noxious stimuli, activity in the DMN was anti-correlated (one signal the entire brain (repetition time = 3000 ms, echo time = 30 ms, flip increased whilst the other decreased) to activity in a task-positive angle = 90°, raw voxel size = 1.5 × 1.5 × 4 mm3). Following a 5- network and that these networks were operating at a frequency be- minute baseline period (100 fMRI volumes), a 0.2 ml bolus of hyper- tween 0.03 and 0.08 Hz (Seminowicz and Davis, 2007b). Curiously, tonic saline was injected into the masseter muscle followed by a con- although not the focus of the investigation, we recently reported sig- tinuous infusion for the remaining 20 min. During the infusion we nificant decreases in oscillatory power in areas of the DMN during a 6 aimed to keep a moderate pain intensity of approximately 5 out of 10. minute muscle pain stimulus in healthy control subjects (Alshelh et al., This was achieved by instructing each subject to push a buzzer once if 2016). Although we did not explore these oscillation decreases in detail, the pain fell below 5 and twice if it increased above 5, and the infusion given the evidence presented above, it is possible that altered DMN rate was adjusted accordingly to maintain the pain intensity at 5 out of dynamics also occur during acute pain stimuli in healthy controls; these 10. Following the scan, each subject was asked to rate the average pain acute pain stimuli simply need to be of long enough duration to allow during the scan on a 10 cm horizontal VAS, outline the location of the for reliable detection. The aim of this study is to use resting state fMRI pain on a standard drawing of the head and complete a McGill Pain to investigate infra-slow oscillations and connectivity within the DMN Questionnaire describing the qualities of the pain. in individuals with chronic orofacial neuropathic pain and in healthy individuals during tonic orofacial pain. We hypothesize that altered 2.3. MRI analysis DMN activity patterns will not be restricted to those in chronic pain but will also occur in healthy individuals during tonic noxious stimuli. 2.3.1. Overall changes in infra-slow oscillatory power Using SPM12 (Friston et al., 1995), all fMRI images were motion 2. Materials and methods corrected, global signal drifts removed using the detrending method described by Macey et al. (2004), spatially normalized to the Montreal 2.1. Subjects Neurological Institute (MNI) template, and spatially smoothed using a 6 mm full-width-half-maximum Gaussian filter. In no subject was there Forty-three subjects with chronic orofacial neuropathic pain (32 significant movement (> 0.5 mm in any direction). Furthermore, females; mean age 47.3 ± 2.3 years [ ± SEM]) and 57 pain-free con- comparison of the mean movement in 6 directions (X, Y, Z, roll, yaw, trols (35 females; mean age 43.4 ± 1.7 years) were recruited for the tilt) revealed no significant differences between chronic pain and con- study. There was no significant difference in age (t-test; p = 0.08) or trol groups (p > 0.05, two-sample t-test, Bonferroni corrected for gender composition (chi-squared test, p = 0.25) between groups. All multiple comparisons), or between the baseline and pain periods in the chronic pain subjects were diagnosed using the Liverpool criteria as tonic pain group (p > 0.05, paired t-test, Bonferroni corrected for having post-traumatic trigeminal neuropathy (Nurmikko and Eldridge, multiple comparisons). 2001). During the seven days prior to the MRI session, each chronic pain 2.3.2. Identifying the default mode network using independent components subject kept a pain diary recording the intensity of their on-going pain, analysis three times a day using a 10 cm horizontal visual analogue scale (VAS) To identify the DMN we used ICA on the control (n = 57), chronic with 0 indicating “no pain” and 10 indicating “the most intense imagin- pain (n = 43) and tonic pain (n = 16) groups using the Group ICA able pain”. These pain intensity scores were then averaged over the 7- toolbox (Calhoun et al., 2001). This technique can extract independent day period to create a mean diary pain intensity score. On the day of the sources of activity within a recorded mixture of sources and can MRI scanning, each chronic pain subject outlined the location of their therefore detect various networks (Brown et al., 2001; Frigyesi et al., on-going pain on a standard drawing of the head, completed a McGill 2006). Using the Infomax ICA algorithm, we estimated 20 independent Pain Questionnaire and listed their current medication use. Informed component maps in which signal intensity within multiple regions written consent was obtained for all procedures according to the covaried. All components were displayed (p < 0.05, false discovery Declaration of Helsinki and the study was approved by our local rate corrected for multiple comparisons with a minimum of 10 con- Institutional Human Research Ethics Committees. Data from 27 of the tiguous voxels) and for each group, the component map that included 43 chronic pain subjects were used in previous investigations (Alshelh the major regions that define the DMN such as the medial prefrontal et al., 2016; Gustin et al., 2011; Henderson et al., 2013; Wilcox et al., cortex, inferior parietal cortex, precuneus and posterior cingulate 2015). cortex was selected. The DMN component map for each of the three groups was then added together to create a single DMN mask, which 2.2. MRI acquisition was used in further analyses.
All subjects lay supine on the bed of a 3 Tesla MRI scanner (Philips, 2.3.3. Voxel-by-voxel infra-slow frequency amplitude analysis Achieva) with their head immobilized in a tight-fitting head coil. With To explore regional differences in infra-slow oscillation (ISO) power each subject relaxed and at rest, a series of 180 gradient echo echo- we used the SPM toolbox REST (Song et al., 2011) to calculate the sum
223
118 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Fig. 1. Pain distribution and quality of pain in chronic pain subjects and in individuals during tonic pain. A) Individual pain distribution patterns in 43 chronic pain subjects and 16 healthy controls during experimentally induced tonic pain. B) Frequency (percentage of subjects) of descriptors chosen from the McGill Pain Questionnaire to describe the on-going pain in chronic pain subjects (white bars) and in healthy controls during tonic pain (grey bars).
of amplitudes of low frequency fluctuations (ALFF) in the frequency relationship with 0.03–0.06 Hz power in significant clusters in chronic band 0.03–0.06 Hz. We also divided these ALFF values by total power pain subjects (p < 0.05, Bonferroni corrected for the number of cor- over the entire frequency range to obtain fractional ALFF (fALFF) va- relations examined). The effects of medication on infra-slow oscillations lues for each voxel. Significant differences within the DMN between were determined by comparing 0.03–0.06 Hz power in each significant control and chronic pain subjects were then determined using a two- cluster in chronic pain subjects taking analgesic medications (n = 16) sample random-effects procedure with age and gender added as nui- with those not taking medication (n = 27) (p < 0.05, 2-tailed, 2- sance variables (p < 0.05, false discovery rate corrected for multiple sample t-test). comparisons, minimum 10 contiguous voxels). For each of the com- For the tonic pain group, ALFF power was calculated for the base- parisons, ALFF and fALFF values from significant clusters were ex- line and tonic pain periods and significant differences within the DMN tracted from the control and chronic pain groups and plotted. The effect were determined using a paired random effects procedure (p < 0.05, of pain intensity and pain duration was determined by comparing its false discovery rate corrected for multiple comparisons, minimum 10
224
119 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Fig. 2. The default mode network in controls, chronic pain and tonic pain groups as defined by an independent component analysis. Areas displaying significant signal intensity covariation are indicated by the hot colour scale and overlaid onto an individual's T1-weighted anatomical image set. The default mode network consists of the precuneus, posterior cingulate cortex (PCC), inferior parietal cortex and the medial prefrontal cortex (mPFC). Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice in the top panel. contiguous voxels). These comparisons were made between the baseline DMN was determined. Cerebrospinal fluid signal intensity, calculated as period (100 volumes) and each of the four 5-minute periods (100 vo- the average signal intensity of a 4 mm diameter sphere placed in the 4th lumes each) during the tonic pain period. Using the significant clusters ventricle in each individual subject, in addition to the 6-direction derived from the baseline versus the first 5-minute pain period, ALFF movement parameters derived from the realignment step, were in- values were extracted from the baseline and each of the 5-minute pain cluded as nuisance variables. Comparisons of PCC resting connectivity periods and plotted. Significant differences between baseline and pain strength between controls and chronic pain subjects were determined periods were then determined (paired t-test, p < 0.05, Bonferroni using a two-group random effects analysis. For the tonic pain group, corrected for multiple comparisons). In addition, for each significant differences in PCC connectivity strength between the baseline and each cluster, the percentage change in signal intensity relative to the baseline of the tonic pain periods were determined using paired random effects period was calculated for each of the four 5-minute pain periods in each analysis. For each of these analyses, following an initial threshold of subject and plotted. Significant differences between signal intensity p < 0.001 with a minimum cluster extent of 10 contiguous voxels, we during the baseline and each of the pain periods were determined used cluster correction to correct for multiple comparisons (p < 0.05, (paired t-test, p < 0.05, Bonferroni corrected). Bonferroni corrected for the number of voxels in each cluster). For the chronic pain analysis, functional connectivity values were extracted from each significant cluster and plotted. The effect of pain intensity 2.3.4. Voxel-by-voxel functional connectivity analysis and pain duration was determined by comparing its relationship with Results from the infra-slow frequency analysis revealed an area of PCC connectivity strengths in significant clusters in chronic pain sub- the posterior cingulate cortex (PCC) in which both chronic and tonic jects (p < 0.05, Bonferroni corrected for the number of correlations fi pain groups displayed signi cant decreases in infra-slow frequency examined). For the tonic pain analysis, PCC connectivity strength va- amplitude relative to controls and baseline, respectively. This PCC re- lues were extracted from each significant cluster for the baseline and “ ” ff gion was used as a seeding area to assess the e ects of pain on PCC each of the 5-minute pain periods and plotted. Significant differences functional connectivity within the DMN. For the control and chronic between baseline and each pain period were then determined (paired t- pain groups, PCC signal intensity was extracted for each subject and the test, p < 0.05, Bonferroni corrected). strength of covariation between this signal and each voxel within the
225
120 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Fig. 3. Significant changes in infra-slow oscillatory power (ISO: 0.03–0.06 Hz) in individuals with chronic pain. A) Regions where chronic pain sub- jects have significantly reduced amplitude of low- frequency fluctuations (ALFF) and fractional ALFF (fALFF) compared with controls within the default mode network. Power reductions are indicated by the cool colour scale and overlaid onto an in- dividual's T1-weighted anatomical image set. Note the decreases within the precuneus, posterior cin- gulate cortex (PCC), inferior parietal cortex and medial prefrontal cortex (mPFC). Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice in the top panel. B) Plots of mean ± SEM ALFF and C) mean ± SEM fALFF in controls and chronic pain groups for sig- nificant clusters. ‡ significant differences derived from the voxel-by-voxel random effects analysis (p < 0.05, false discovery rate corrected).
3. Results 3.2. Voxel-by-voxel infra-slow frequency amplitude analysis
3.1. Subject characteristics 3.2.1. Chronic pain A comparison of infra-slow oscillation power between 0.03 and The mean ( ± SEM) diary pain of the chronic pain group was 0.06 Hz revealed significantly reduced power in chronic pain subjects 3.8 ± 0.4 and the mean VAS scores for the tonic pain group was in multiple regions of the DMN (Fig. 3A, Table 1A). That is, decreased 4.8 ± 0.3. Both chronic and tonic pain groups reported pain encom- power occurred in the region encompassing the precuneus (mean ± passing the maxillary and mandibular distributions of the trigeminal SEM 0.03–0.06 Hz ALFF: controls: 1.52 ± 0.04, chronic pain: nerve, although the chronic pain subjects generally reported a wider 1.26 ± 0.05), PCC (controls: 1.64 ± 0.05, chronic pain: 1.37 ± 0.06), spread of on-going pain that often included the ophthalmic trigeminal left and right IPC (left: controls: 1.12 ± 0.02, chronic pain: division (Fig. 1A). Furthermore, 29 of the chronic pain subjects re- 0.91 ± 0.03; right: controls: 1.28 ± 0.04, chronic pain: 1.01 ± 0.05) ported left sided only pain, and 14 right side only. Chronic pain subjects and the mPFC (controls: 1.28 ± 0.05, chronic pain: 1.05 ± 0.05) most often described their on-going pain as “shooting”, “throbbing” and (Fig. 3B). Similarly, comparison of infra-slow oscillation power relative “nagging”, and similarly the tonic pain was described as “throbbing”, to an individual's total power (fALFF) also revealed significantly re- “annoying” and “nagging” (Fig. 1B). The mean pain duration of the duced power in chronic pain subjects compared with controls in the chronic pain group was 6.0 ± 0.9 years and 16 of the chronic pain precuneus (controls: 1.22 ± 0.02 chronic pain: 1.10 ± 0.03), PCC subjects were taking some form of daily analgesic medication. ICA re- (controls: 1.23 ± 0.02, chronic pain: 1.10 ± 0.03), left and right IPC vealed a component comprising of the DMN in the control, chronic pain (left: controls: 1.21 ± 0.02, chronic pain: 1.07 ± 0.02; right: controls: and tonic pain groups. This component encompassed vast regions of the 1.30 ± 0.02, chronic pain: 1.16 ± 0.02) and the mPFC (controls: precuneus, PCC, anterior cingulate cortex, medial prefrontal cortex 1.14 ± 0.02, chronic pain: 1.03 ± 0.03) (Fig. 3C). In no region of the (mPFC) and the inferior parietal cortex (IPC) (Fig. 2). DMN was power greater in chronic pain subjects compared with
226
121 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Table 1 1.17 ± 0.07,4th 5 min: 1.13 ± 0.05) and the mPFC (baseline: Location of significant infra-slow oscillation power decreases within the default mode 1.23 ± 0.06, 1st 5 min tonic pain: 1.02 ± 0.05, 2nd 5 min: network in individuals with chronic pain (A) and during tonic pain (B). Locations are in 1.08 ± 0.07, 3rd 5 min: 1.22 ± 0.05, 4th 5 min: 1.14 ± 0.07). In the Montreal Neurological Institute (MNI) space. precuneus and PCC, the significant reduction in power was sustained Cluster size t value MNI co-ordinate for the tonic pain period, whereas in the IPC and mPFC the power re- ductions were transient and returned to baseline by the 3rd 5-minute XYZ pain period.
(A) Chronic pain The reduction in infra-slow oscillation power within the precuneus ALFF and PCC was accompanied by significant signal intensity increases that Precuneus 16 3.49 −4 −66 26 were sustained for the tonic pain period (mean ± SEM %signal in- 10 3.44 8 −68 38 tensity increase: precuneus: 1st 5 min tonic pain: 0.47 ± 0.15, 2nd Posterior cingulate cortex 32 3.26 4 −48 28 5 min: 0.60 ± 0.16, 3rd 5 min: 0.69 ± 0.16, 4th 5 min: 0.78 ± 0.16; Inferior parietal cortex Left 51 4.26 −30 −76 32 PCC: 1st 5 min tonic pain: 0.62 ± 0.15, 2nd 5 min: 0.69 ± 0.14, 3rd Right 53 3.73 −42 −58 22 5 min: 0.68 ± 0.14, 4th 5 min: 0.71 ± 0.14: all p < 0.05). In con- 36 3.88 34 −66 36 trast, within the IPC and mPFC, signal intensity did not change sig- 40 3.80 46 −60 42 fi − ni cantly during the tonic pain period (IPC: 1st 5 min tonic pain: Medial prefrontal cortex 38 4.94 63812 − − 25 4.62 −12 38 42 0.19 ± 0.17, 2nd 5 min: 0.06 ± 0.19, 3rd 5 min: 0.15 ± 0.19, fALFF 4th 5 min: 0.35 ± 019; mPFC: 1st 5 min tonic pain: −0.05 ± 0.23, 2nd Precuneus 663 4.75 −8 −54 20 5 min: 0.28 ± 0.24, 3rd 5 min: 0.54 ± 0.22, 4th 5 min: 0.71 ± 0.23: Posterior cingulate cortex 144 3.86 4 −28 32 all p > 0.05). Inferior parietal cortex Left 75 4.68 −42 −54 18 94 4.10 −32 −76 32 3.3. Functional connectivity of PCC Right 139 4.26 44 −60 42 77 4.15 36 −70 28 The PCC region that displayed decreased infra-slow oscillation Medial prefrontal cortex 12 3.39 2 66 8 power in both the chronic and tonic pain groups and also a sustained (B) Tonic pain increase in signal intensity during tonic pain was used to explore con- ALFF nectivity changes within the DMN. Similar to the infra-slow oscillation Precuneus 585 4.32 −6 −70 38 power changes, chronic pain subjects displayed significantly reduced Posterior cingulate cortex 24 4.78 −10 −54 10 12 4.58 −4 −22 36 PCC connectivity strengths compared with controls in the precuneus 68 4.55 −6 −44 44 (mean ± SEM PCC connectivity strength: controls: 0.42 ± 0.02, Inferior parietal cortex chronic pain: 0.32 ± 0.02), left and right IPC (left: controls: − − Left 411 6.63 38 74 36 0.39 ± 0.02, chronic pain: 0.29 ± 0.03; right: controls: 0.29 ± 0.02, Right 18 3.91 −48 −60 46 29 4.89 46 −72 26 chronic pain: 0.18 ± 0.03) and the mPFC (controls: 0.37 ± 0.02, Medial prefrontal cortex 12 4.11 8 60 6 chronic pain: 0.26 ± 0.02) (Fig. 5A, Table 2A). In no DMN region was PCC connectivity strength greater in the chronic pain subjects com- pared with controls. controls. In no region was PCC connectivity strength significantly correlated fi In no region was either ALFF or fALFF power signi cantly corre- to diary pain (precuneus r = −0.01, left IPC r = 0.08, right IPC lated to diary pain (ALFF: precuneus r = −0.13, PCC r = −0.17, left r = 0.01, mPFC r = −0.09; all p > 0.05) or pain duration (precuneus − IPC r = 0.14, right IPC r = 0.01, mPFC r = 0.11; fALFF: precuneus r = −0.12, left IPC r = 0.18, right IPC r = −0.11, mPFC r = 0.01; all r=−0.17, PCC r = −0.18, left IPC r = −0.11, right IPC r = −0.07, p > 0.05). In addition, in no region was there a significant difference mPFC r = 0.06; all p > 0.05) or pain duration (ALFF: precuneus in PCC connectivity strength between those taking medication com- r = 0.17, PCC r = 0.13, left IPC r = 0.01, right IPC r = −0.04, mPFC pared with those that were not (mean ± SEM PCC connectivity r = 0.03; fALFF: precuneus r = 0.22, PCC r = 0.23, left IPC r = 0.01, strength medication versus no medication: precuneus 0.30 ± 0.03 vs right IPC r = 0.03, mPFC r = 0.32; all p > 0.05). In addition, in no 0.33 ± 0.03, left IPC 0.28 ± 0.04 vs 0.29 ± 0.04, right IPC fi ff region was there a signi cant di erence in power between those taking 0.13 ± 0.06 vs 0.22 ± 0.03, mPFC 0.25 ± 0.04 vs 0.27 ± 0.03; all medication compared with those that were not (mean ± SEM power p > 0.05). medication versus no medication: ALFF: precuneus 1.22 ± 0.06 vs The tonic pain group displayed reduced PCC connectivity strength 1.29 ± 0.04, PCC 1.31 ± 0.05 vs 1.40 ± 0.06, left IPC 0.90 ± 0.03 during pain compared to baseline in the right IPC (baseline: vs 0.92 ± 0.03, right IPC 0.97 ± 0.04 vs 1.03 ± 0.04, mPFC 0.18 ± 0.05; 1st 5 min tonic pain: 0.07 ± 0.05, 2nd 5 min: 1.14 ± 0.06 vs 0.99 ± 0.03; fALFF: precuneus 1.16 ± 0.03 vs 0.08 ± 0.06, 3rd 5 min: 0.07 ± 0.04, 4th 5 min: 0.08 ± 0.04), left 1.07 ± 0.02, PCC 1.16 ± 0.03 vs 1.07 ± 0.02, left IPC 1.10 ± 0.02 mPFC (baseline: 0.26 ± 0.04; 1st 5 min tonic pain: 0.04 ± 0.05, 2nd vs 1.06 ± 0.02, right IPC 1.13 ± 0.02 vs 1.17 ± 0.02, mPFC 5 min: 0.12 ± 0.05, 3rd 5 min: 0.12 ± 0.04, 4th 5 min: 0.12 ± 0.05) 1.01 ± 0.03 vs 1.04 ± 0.02; all p > 0.05). and right mPFC (baseline: 0.27 ± 0.06; 1st 5 min tonic pain: 0.07 ± 0.05, 2nd 5 min: 0.14 ± 0.06, 3rd 5 min: 0.14 ± 0.04, 4th 3.2.2. Tonic pain 5 min: 0.10 ± 0.0.05) (Fig. 5B, Table 1B). These significant reductions A comparison of infra-slow oscillation power during tonic pain with in PCC connectivity were sustained for the entire tonic pain period. In baseline revealed significant reductions in the same DMN regions as no DMN region was PCC connectivity strength greater during tonic pain those that occurred in individuals with chronic pain (Fig. 4A, Table 1B). compared with baseline. That is, decreased power occurred in the region of the precuneus (mean ± SEM 0.03–0.06 Hz ALFF: baseline: 1.59 ± 0.07, 1st 5 min 4. Discussion tonic pain: 1.24 ± 0.06, 2nd 5 min: 1.44 ± 0.07, 3rd 5 min: 1.43 ± 0.07, 4th 5 min: 1.49 ± 0.07), PCC (baseline: 1.10 ± 0.05, 1st Consistent with our overall hypothesis, we found that DMN func- 5 min tonic pain: 0.89 ± 0.05, 2nd 5 min: 0.98 ± 0.05, 3rd 5 min: tional properties were significantly altered in both subjects with chronic 0.98 ± 0.06, 4th 5 min: 1.00 ± 0.06), left IPC (baseline: 1.15 ± 0.05, pain and in healthy individuals during tonic painful stimuli. Indeed, we 1st 5 min tonic pain: 0.94 ± 0.05, 2nd 5 min: 1.11 ± 0.07, 3rd 5 min: found decreased ISO power in both chronic pain and tonic pain groups
227
122 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Fig. 4. Significant changes in infra-slow oscillatory power (ISO: 0.03–0.06 Hz) during tonic pain. A) Regions of the default mode network where the amplitude of low-frequency fluctuations (ALFF) are significantly reduced during the 1st, 2nd, 3rd and 4th 5 minute periods of tonic pain compared with a baseline period. Power reductions are indicated by the cool colour scale and overlaid onto an in- dividual's T1-weighted anatomical image set. Note the decreases within the precuneus, posterior cin- gulate cortex (PCC), inferior parietal cortex and medial prefrontal cortex (mPFC). Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice in panel A. B) Plots of mean ± SEM ALFF during baseline and the four 5 minute pain periods for significant clusters. C) Plots of mean ± SEM % signal intensity changes re- lative to baseline for the four 5 minute pain per- iods for clusters with significantly reduced ALFF power. Note that only the precuneus and PCC dis- play both reduced ALFF and increased signal in- tensity changes during all of the tonic pain periods. The grey shading indicates the tonic pain period. * p < 0.05, 2-tailed paired t-test.
over most of the DMN, including in the precuneus, PCC, IPC and mPFC. eliminate the opportunity of finding changes within discrete regions of Additionally, in healthy controls, tonic pain evoked sustained ISO de- the DMN. More importantly, we found that similar DMN changes to creases in the precuneus and PCC which were associated with sustained those in chronic pain subjects also occurred in healthy individuals overall signal intensity increases. Finally, both chronic and tonic pain during a tonic noxious stimulus. Both chronic and tonic pain were as- groups displayed significant reductions in PCC connectivity within the sociated with reduced ISOs within multiple regions of the DMN, in- DMN, and these changes were sustained for the entire pain period in the cluding the precuneus, PCC, mPFC and IPC. This clearly indicates that tonic pain group. DMN functional changes are not a unique characteristic of chronic pain Consistent with previous investigations we found altered DMN dy- but instead likely represent the presence of pain itself. It has been namics in individuals with chronic pain (Baliki et al., 2008; Baliki et al., proposed that ISO activity is a fundamental property of brain function 2014; Letzen et al., 2013; Loggia et al., 2013; Napadow et al., 2012; that is maintained by adenosine receptor-mediated signalling (Hughes Tagliazucchi et al., 2010). Although in one of these previous studies, et al., 2011; Lorincz et al., 2009). Adenosine is likely released by as- ISOs were investigated and no significant differences at low frequencies trocytes since they can display spontaneous intracellular infra-slow (0.01–0.05 Hz) were found within the DMN (Baliki et al., 2014), ISO calcium oscillations (Parri and Crunelli, 2001), are responsive to glu- power was averaged over the entire DMN network, which would tamate and acetylcholine, and a link between adenosine and infra-slow
228
123 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Fig. 5. Significant changes in posterior cingulate cortex (PCC) functional connectivity in chronic and tonic pain groups. A) Regions where PCC con- nectivity strength within the default mode network is significantly reduced in chronic pain subjects compared with controls. Connectivity strength re- ductions are indicated by the cool colour scale and overlaid onto an individual's T1-weighted anato- mical image set. Note decreases in the precuneus, inferior parietal cortex (IPC) and medial prefrontal cortex (mPFC). Slice locations in Montreal Neurological Institute space are indicated at the top right of each slice. Plots of mean ± SEM PCC connectivity strength are also shown. ‡ significant differences derived from the voxel-by-voxel random effects analysis (p < 0.05, false discovery rate corrected). B) Regions where PCC connectivity strength is significantly reduced during tonic pain compared with the baseline period. Connectivity strength reductions are indicated by the cool colour scale and overlaid onto an individual's T1-weighted anatomical image set. Note the decreases within the IPC and mPFC. Plots of mean ± SEM PCC con- nectivity strength are also shown for the baseline and each of the 5 min tonic pain periods. The grey shading indicates the tonic pain period. * p < 0.05, 2-tailed paired t-test.
oscillatory activity has been demonstrated in the cortex (Cunningham overall signal intensity. In contrast, ISOs within the inferior parietal et al., 2006). Whilst we have previously speculated that increased ISO cortex and mPFC were transient, and overall signal intensity in these activity in the ascending pain pathways of chronic orofacial pain pa- two regions did not change. Similarly, we have previously shown that tients results from astrocyte activation (Alshelh et al., 2016; Henderson chronic orofacial pain is associated with increased on-going activity in and Di Pietro, 2016), what ISO power reductions represent is unclear. the DMN as indicated by increased cerebral blood flow in the DMN, The rapid nature of the ISO decreases during tonic pain suggests particularly within the precuneus (Youssef et al., 2014). These data that they do not result from astrogliosis. During tonic pain, decreased suggest that within the DMN, decreased ISOs are coupled with overall ISOs were sustained for the entire 20 min pain period in the precuneus activity increases which together may result from increased afferent and PCC, two regions which also displayed sustained increases in drive rather than longer-term changes mediated by mechanisms such as
229
124 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Table 2 specific type of pain investigated, the modality and/or analysis of the Location of significant posterior cingulate cortex connectivity decreases within the de- acquired data or the specific circuitry examined. Our study does how- fault mode network in individuals with chronic pain (A) and during tonic pain (B). ever, show that DMN changes occur during experimentally applied Locations are in Montreal Neurological Institute (MNI) space. painful stimuli in healthy controls, suggesting that it is the presence of Cluster size t value MNI co-ordinate pain per se that is more likely responsible for such DMN changes. Furthermore, it is curious that this decreased DMN connectivity XYZ strength is coupled to decreased ISO power, raising the prospect that fi fl (A) Chronic pain DMN functional coupling at rest is signi cantly in uenced by signal Precuneus 48 3.87 0 −72 40 coupling within the 0.03–0.06 Hz range. Inferior parietal cortex Whilst we have clearly shown that altered DMN dynamics occur in Left 24 3.41 −46 −68 36 healthy individuals during experimentally applied painful stimuli, the Right 10 2.90 50 −68 40 interpretation of what these changes represent remain unknown. Medial prefrontal cortex 29 3.76 12 40 50 10 3.48 6 66 8 One limitation of this study is that during the tonic pain experiment, we asked each subject to indicate when deviations from the target pain (B) Tonic pain Right inferior parietal cortex 21 4.05 48 −68 30 intensity percept occurred. Although this could have introduced a po- Medial prefrontal cortex tential motor confound, given that most subjects indicated a deviation Left 16 3.91 −258 −4 no more than 5 times over the course of the entire 20 min pain period, − Right 18 3.00 2 56 6 we suggest that this potential for such a confound is negligible. Furthermore, we did not measure behavioural changes such as changes in cognitive or attention-demanding tasks, particularly in the tonic pain astrogliosis. As mentioned above, Seminowicz and Davis found that group. Future investigations could explore the effect of changes in DMN during an attention-demanding cognitive task in the presence of acute function evoked by both tonic and chronic pain. pain, the DMN and task-positive networks displayed anti-correlated ISO fluctuations within a similar frequency range as those explored in this 5. Conclusions study (Seminowicz and Davis, 2007b). This further supports the notion that changes in ISO power may represent a fundamental property of We have found that altered DMN dynamics is not unique to chronic brain function. pain but also occurs during tonic pain in healthy controls. Both chronic In addition to altered ISO and overall activity levels, we found that and tonic pain are attention demanding stimuli competing for cognitive both chronic and tonic pain groups displayed decreased PCC con- resources, and functional changes within the DMN may underlie some nectivity strengths with other DMN regions. Decreased PCC con- of the attentional and cognitive alterations that occur in the presence of nectivity with the mPFC and IPC occurred in both chronic and tonic pain. pain groups, and also with the precuneus in chronic pain subjects. These decreases are consistent with previous studies reporting de- Acknowledgements creased DMN connectivity in individuals with chronic back pain, complex regional pain syndrome and osteoarthritis (Baliki et al., 2014), This work was supported by funding from the Australian National although in individuals with temporomandibular disorder, DMN con- Health and Medical Research Council (G160279 and G182968) and the nectivity strengths reportedly increase (Kucyi et al., 2014). The PCC is a NGW Macintosh Memorial Fund. key neural hub in the DMN (Fransson and Marrelec, 2008) and is ac- tivated during the regulation of attention (Gusnard et al., 2001; Hahn References et al., 2007; Small et al., 2003) and displays hypometabolism in in- dividuals with cognitive impairment (Liang et al., 2008; Minoshima et al., 1997; Valla et al., 2001). Since there is evidence of decreased Alshelh, Z., Di Pietro, F., Youssef, A.M., Reeves, J.M., Macey, P.M., Vickers, E.R., Peck, C.C., Murray, G.M., Henderson, L.A., 2016. Chronic neuropathic pain: It's about the connectivity between the PCC and other DMN areas, such as the pre- rhythm. J. Neurosci. 36, 1008–1018. cuneus, in attention deficit hyperactivity disorder, a condition char- Baliki, M.N., Geha, P.Y., Apkarian, A.V., Chialvo, D.R., 2008. Beyond feeling: chronic pain acterised by a reduced ability in paying attention (Castellanos et al., hurts the brain, disrupting the default-mode network dynamics. J. Neurosci. 28, 1398–1403. 2008; Rubia et al., 2005; Uddin et al., 2008), it is possible that reduced Baliki, M.N., Mansour, A.R., Baria, A.T., Apkarian, A.V., 2014. Functional reorganization PCC connectivity during pain also underlies an altered ability to attend of the default mode network across chronic pain conditions. PLoS One 9, e106133. to particular tasks. Bantick, S.J., Wise, R.G., Ploghaus, A., Clare, S., Smith, S.M., Tracey, I., 2002. Imaging how attention modulates pain in humans using functional MRI. Brain 125, 310–319. If as hypothesized, the DMN is acting as a sentinel by monitoring the Brown, G.D., Yamada, S., Sejnowski, T.J., 2001. Independent component analysis at the external environment (Buckner et al., 2008), ISO and connectivity neural cocktail party. Trends Neurosci. 24, 54–63. changes within the DMN may underpin such monitoring during pain Buckner, R.L., Andrews-Hanna, J.R., Schacter, D.L., 2008. The brain's default network: anatomy, function, and relevance to disease. Ann. N. Y. Acad. Sci. 1124, 1–38. and reduce the ability of other stimuli to attract an individual's atten- Calhoun, V.D., Adali, T., Pearlson, G.D., Pekar, J.J., 2001. A method for making group tion away from the pain. Indeed, connectivity strength within the DMN inferences from functional MRI data using independent component analysis. Hum. in chronic pain subjects is correlated to pain rumination, a measure of Brain Mapp. 14, 140–151. Castellanos, F.X., Margulies, D.S., Kelly, C., Uddin, L.Q., Ghaffari, M., Kirsch, A., Shaw, D., an individual's continual focus on his/her pain and its potential nega- Shehzad, Z., Di Martino, A., Biswal, B., Sonuga-Barke, E.J.S., Rotrosen, J., Adler, L.A., tive outcomes (Kucyi et al., 2014). Consistent with this previous study, Milham, M.P., 2008. Cingulate-precuneus interactions: a new locus of dysfunction in we did not find any significant relationships between either ISO power adult attention-deficit/hyperactivity disorder. Biol. Psychiatry 63, 332–337. or PCC connectivity strengths with either on-going pain intensity or Cavanna, A.E., Trimble, M.R., 2006. The precuneus: a review of its functional anatomy and behavioural correlates. Brain 129, 564–583. pain duration in chronic pain subjects. However, others have reported Cunningham, M.O., Pervouchine, D.D., Racca, C., Kopell, N.J., Davies, C.H., Jones, R.S., relationships between DMN connectivity and ongoing pain intensity Traub, R.D., Whittington, M.A., 2006. Neuronal metabolism governs cortical network – and duration and there is evidence that connectivity strengths between response state. Proc. Natl. Acad. Sci. U. S. A. 103, 5597 5601. Eccleston, C., 1994. Chronic pain and attention: a cognitive approach. Br J Clin Psychol the DMN and areas such as insular cortex are related to the on-going 33 (Pt 4), 535–547. pain intensity in individuals with chronic lower back pain (Loggia et al., Eccleston, C., Crombez, G., 1999. Pain demands attention: a cognitive-affective model of – 2013) and also in those with fibromyalgia (Napadow et al., 2012). the interruptive function of pain. Psychol. Bull. 125, 356 366. ff Fransson, P., Marrelec, G., 2008. The precuneus/posterior cingulate cortex plays a pivotal These data raise the prospect that di erences in the relationships be- role in the default mode network: evidence from a partial correlation network ana- tween DMN connectivity and clinical pain variables may depend on the lysis. NeuroImage 42, 1178–1184.
230
125 Z. Alshelh et al. NeuroImage: Clinical 17 (2018) 222–231
Frigyesi, A., Veerla, S., Lindgren, D., Hoglund, M., 2006. Independent component analysis situ. Neuroreport 12, 3897–3900. reveals new and biologically significant structures in micro array data. BMC Petrovic, P., Petersson, K.M., Ghatan, P.H., Stone-Elander, S., Ingvar, M., 2000. Pain- Bioinformatics 7, 290. related cerebral activation is altered by a distracting cognitive task. Pain 85, 19–30. Friston, K.J., Holmes, A.P., Worsley, K.P., Proline, J.B., Frith, C.D., Frackowiak, R.S.J., Raichle, M.E., MacLeod, A.M., Snyder, A.Z., Powers, W.J., Gusnard, D.A., Shulman, G.L., 1995. Statistical parametric maps in functional imaging: a general imaging approach. 2001. A default mode of brain function. Proc. Natl. Acad. Sci. U. S. A. 98, 676–682. Journal of Human Brain Mapping 2, 189–210. Remy, F., Frankenstein, U.N., Mincic, A., Tomanek, B., Stroman, P.W., 2003. Pain mod- Grisart, J.M., Van der Linden, M., 2001. Conscious and automatic uses of memory in ulates cerebral activity during cognitive performance. NeuroImage 19, 655–664. chronic pain patients. Pain 94, 305–313. Rubia, Katya, Smith, Anna B., Brammer, Michael J., Toone, Brian, Taylor, Eric, 2005. Gusnard, D.A., Raichle, M.E., Raichle, M.E., 2001. Searching for a baseline: functional Abnormal brain activation during inhibition and error detection in medication-naive imaging and the resting human brain. Nat. Rev. Neurosci. 2, 685–694. adolescents with ADHD. Am. J. Psychiatr. 162, 1067–1075. Gustin, S.M., Peck, C.C., Wilcox, S.L., Nash, P.G., Murray, G.M., Henderson, L.A., 2011. Seminowicz, D.A., Davis, K.D., 2007a. Interactions of pain intensity and cognitive load: Different pain, different brain: thalamic anatomy in neuropathic and non-neuropathic the brain stays on task. Cereb. Cortex 17, 1412–1422. chronic pain syndromes. J. Neurosci. 31, 5956–5964. Seminowicz, D.A., Davis, K.D., 2007b. Pain enhances functional connectivity of a brain Hahn, B., Ross, T.J., Stein, E.A., 2007. Cingulate activation increases dynamically with network evoked by performance of a cognitive task. J. Neurophysiol. 97, 3651–3659. response speed under stimulus unpredictability. Cereb. Cortex 17, 1664–1671. Shulman, G.L., Corbetta, M., Buckner, R.L., Fiez, J.A., Miezin, F.M., Raichle, M.E., Henderson, L.A., Di Pietro, F., 2016. How do neuroanatomical changes in individuals with Petersen, S.E., 1997. Common blood flow changes across visual tasks: I. Increases in chronic pain result in the constant perception of pain? Pain Manag 6, 147–159. subcortical structures and cerebellum but not in nonvisual cortex. J. Cogn. Neurosci. Henderson, L.A., Peck, C.C., Petersen, E.T., Rae, C.D., Youssef, A.M., Reeves, J.M., Wilcox, 9, 624–647. S.L., Akhter, R., Murray, G.M., Gustin, S.M., 2013. Chronic pain: lost inhibition? J. Sjogren, P., Thomsen, A.B., Olsen, A.K., 2000. Impaired neuropsychological performance Neurosci. 33, 7574–7582. in chronic nonmalignant pain patients receiving long-term oral opioid therapy. J. Hughes, S.W., Lorincz, M.L., Parri, H.R., Crunelli, V., 2011. Infraslow (< 0.1 Hz) oscil- Pain Symptom Manag. 19, 100–108. lations in thalamic relay nuclei basic mechanisms and significance to health and Small, D.M., Gitelman, D.R., Gregory, M.D., Nobre, A.C., Parrish, T.B., Mesulam, M.M., disease states. Prog. Brain Res. 193, 145–162. 2003. The posterior cingulate and medial prefrontal cortex mediate the anticipatory Iverson, G.L., McCracken, L.M., 1997. 'Postconcussive' symptoms in persons with chronic allocation of spatial attention. NeuroImage 18, 633–641. pain. Brain Inj. 11, 783–790. Smith-Seemiller, L., Fow, N.R., Kant, R., Franzen, M.D., 2003. Presence of post-concussion Karp, J.F., Reynolds 3rd, C.F., Butters, M.A., Dew, M.A., Mazumdar, S., Begley, A.E., syndrome symptoms in patients with chronic pain vs mild traumatic brain injury. Lenze, E., Weiner, D.K., 2006. The relationship between pain and mental flexibility in Brain Inj. 17, 199–206. older adult pain clinic patients. Pain Med. 7, 444–452. Song, X.W., Dong, Z.Y., Long, X.Y., Li, S.F., Zuo, X.N., Zhu, C.Z., He, Y., Yan, C.G., Zang, Kucyi, A., Moayedi, M., Weissman-Fogel, I., Goldberg, M.B., Freeman, B.V., Tenenbaum, Y.F., 2011. REST: a toolkit for resting-state functional magnetic resonance imaging H.C., Davis, K.D., 2014. Enhanced medial prefrontal-default mode network functional data processing. PLoS One 6, e25031. connectivity in chronic pain and its association with pain rumination. J. Neurosci. 34, Spreng, R.N., Grady, C.L., 2010. Patterns of brain activity supporting autobiographical 3969–3975. memory, prospection, and theory of mind, and their relationship to the default mode Letzen, J.E., Craggs, J.G., Perlstein, W.M., Price, D.D., Robinson, M.E., 2013. Functional network. J. Cogn. Neurosci. 22, 1112–1123. connectivity of the default mode network and its association with pain networks in Tagliazucchi, E., Balenzuela, P., Fraiman, D., Chialvo, D.R., 2010. Brain resting state is irritable bowel patients assessed via lidocaine treatment. J. Pain 14, 1077–1087. disrupted in chronic back pain patients. Neurosci. Lett. 485, 26–31. Liang, W.S., Reiman, E.M., Valla, J., Dunckley, T., Beach, T.G., Grover, A., Niedzielko, Uddin, L.Q., Kelly, A.M.C., Biswal, B.B., Margulies, D.S., Shehzad, Z., Shaw, D., Ghaffari, T.L., Schneider, L.E., Mastroeni, D., Caselli, R., Kukull, W., Morris, J.C., Hulette, C.M., M., Rotrosen, J., Adler, L.A., Castellanos, F.X., Milham, M.P., 2008. Network Schmechel, D., Rogers, J., Stephan, D.A., 2008. Alzheimer's disease is associated with homogeneity reveals decreased integrity of default-mode network in ADHD. J. reduced expression of energy metabolism genes in posterior cingulate neurons. Proc. Neurosci. Methods 169, 249–254. Natl. Acad. Sci. U. S. A. 105, 4441–4446. Valet, M., Sprenger, T., Boecker, H., Willoch, F., Rummeny, E., Conrad, B., Erhard, P., Loggia, M.L., Kim, J., Gollub, R.L., Vangel, M.G., Kirsch, I., Kong, J., Wasan, A.D., Tolle, T.R., 2004. Distraction modulates connectivity of the cingulo-frontal cortex Napadow, V., 2013. Default mode network connectivity encodes clinical pain: an and the midbrain during pain–an fMRI analysis. Pain 109, 399–408. arterial spin labeling study. Pain 154, 24–33. Valla, J., Berndt, J.D., Gonzalez-Lima, F., 2001. Energy Hypometabolism in posterior Lorincz, M.L., Geall, F., Bao, Y., Crunelli, V., Hughes, S.W., 2009. ATP-dependent infra- cingulate cortex of Alzheimer's patients: superficial laminar cytochrome oxidase as- slow (< 0.1 Hz) oscillations in thalamic networks. PLoS One 4, e4447. sociated with disease duration. J. Neurosci. 21, 4923–4930. Macey, P.M., Macey, K.E., Kumar, R., Harper, R.M., 2004. A method for removal of global Van Damme, S., Legrain, V., Vogt, J., Crombez, G., 2010. Keeping pain in mind: a mo- effects from fMRI time series. NeuroImage 22, 360–366. tivational account of attention to pain. Neurosci. Biobehav. Rev. 34, 204–213. Minoshima, S., Giordani, B., Berent, S., Frey, K.A., Foster, N.L., Kuhl, D.E., 1997. Verdejo-Garcia, A., Lopez-Torrecillas, F., Calandre, E.P., Delgado-Rodriguez, A., Bechara, Metabolic reduction in the posterior cingulate cortex in very early Alzheimer's dis- A., 2009. Executive function and decision-making in women with fibromyalgia. Arch. ease. Ann. Neurol. 42, 85–94. Clin. Neuropsychol. 24, 113–122. Napadow, V., Kim, J., Clauw, D.J., Harris, R.E., 2012. Decreased intrinsic brain con- Wilcox, S.L., Gustin, S.M., Macey, P.M., Peck, C.C., Murray, G.M., Henderson, L.A., 2015. nectivity is associated with reduced clinical pain in fibromyalgia. Arthritis Rheum. Anatomical changes at the level of the primary synapse in neuropathic pain: evidence 64, 2398–2403. from the spinal trigeminal nucleus. J. Neurosci. 35, 2508–2515. Nurmikko, T.J., Eldridge, P.R., 2001. Trigeminal neuralgia–pathophysiology, diagnosis Youssef, A.M., Gustin, S.M., Nash, P.G., Reeves, J.M., Petersen, E.T., Peck, C.C., Murray, and current treatment. Br. J. Anaesth. 87, 117–132. G.M., Henderson, L.A., 2014. Differential brain activity in subjects with painful tri- Parri, H.R., Crunelli, V., 2001. Pacemaker calcium oscillations in thalamic astrocytes in geminal neuropathy and painful temporomandibular disorder. Pain 155, 467–475.
231
126 The Journal of Neuroscience, January 10, 2018 • 38(2):465–473 • 465
Neurobiology of Disease Brainstem Pain-Control Circuitry Connectivity in Chronic Neuropathic Pain
Emily P. Mills,1 Flavia Di Pietro,1 Zeynab Alshelh,1 Chris C. Peck,2 Greg M. Murray,2 E. Russell Vickers,1 and X Luke A. Henderson1 1Department of Anatomy and Histology and 2Faculty of Dentistry, University of Sydney, Sydney 2006, New South Wales, Australia
Preclinical investigations have suggested that altered functioning of brainstem pain-modulation circuits may be crucial for the mainte- nance of some chronic pain conditions. While some human psychophysical studies show that patients with chronic pain display altered pain-modulation efficacy, it remains unknown whether brainstem pain-modulation circuits are altered in individuals with chronic pain. The aim of the present investigation was to determine whether, in humans, chronic pain following nerve injury is associated with altered ongoing functioning of the brainstem descending modulation systems. Using resting-state functional magnetic resonance imaging, we found that male and female patients with chronic neuropathic orofacial pain show increased functional connectivity between the rostral ventromedial medulla (RVM) and other brainstem pain-modulatory regions, including the ventrolateral periaqueductal gray (vlPAG) and locus ceruleus (LC). We also identified an increase in RVM functional connectivity with the region that receives orofacial nociceptor afferents, the spinal trigeminal nucleus. In addition, the vlPAG and LC displayed increased functional connectivity strengths with higher brain regions, including the hippocampus, nucleus accumbens, and anterior cingulate cortex, in individuals with chronic pain. These data reveal that chronic pain is associated with altered ongoing functioning within the endogenous pain-modulation network. These changes may underlie enhanced descending facilitation of processing at the primary synapse, resulting in increased nociceptive trans- mission to higher brain centers. Further, our findings show that higher brain regions interact with the brainstem modulation system differently in chronic pain, possibly reflecting top–down engagement of the circuitry alongside altered reward processing in pain conditions. Key words: analgesia; chronic pain; locus ceruleus; midbrain periaqueductal gray; rostral ventromedial medulla; spinal trigeminal nucleus
Significance Statement Experimental animal models and human psychophysical studies suggest that altered functioning of brainstem pain-modulation systems contributes to the maintenance of chronic pain. However, the function of this circuitry has not yet been explored in humans with chronic pain. In this study, we report that individuals with orofacial neuropathic pain show altered functional connectivitybetweenregionswithinthebrainstempain-modulationnetwork.Wesuggestthatthesechangesreflectlargelycentral mechanisms that feed back onto the primary nociceptive synapse and enhance the transfer of noxious information to higher brain regions, thus contributing to the constant perception of pain. Identifying the mechanisms responsible for the maintenance of neuropathic pain is imperative for the development of more efficacious therapies.
Introduction facilitate incoming nociceptive information at the level of the Experimental animal studies have revealed that the brainstem primary afferent synapse. These descending modulatory circuits contains multiple neural circuits that can profoundly inhibit or include direct and indirect descending projections to the dorsal horn and spinal trigeminal nucleus (SpV) from such regions as the midbrain periaqueductal gray matter (PAG), the rostral ven- Received June 13, 2017; revised Oct. 23, 2017; accepted Nov. 12, 2017. tromedial medulla (RVM), the locus ceruleus (LC), and the Author contributions: C.C.P., G.M.M., E.R.V., and L.A.H. designed research; E.P.M., F.D.P., Z.A., E.R.V., and L.A.H. subnucleus reticularis dorsalis (SRD; Basbaum and Fields, 1984; performed research; E.P.M. analyzed data; E.P.M., F.D.P., Z.A., C.C.P., G.M.M., E.R.V., and L.A.H. wrote the paper. This work was supported by the National Health and Medical Research Council of Australia, Grants 1032072 and Heinricher et al., 2009; Ossipov et al., 2010; Mason, 2012). In 1059182. We thank the many volunteers in this study. The authors declare no competing financial interests. CorrespondenceshouldbeaddressedtoLukeA.Henderson,DepartmentofAnatomyandHistology,F13,Univer- DOI:10.1523/JNEUROSCI.1647-17.2017 sity of Sydney, Sydney 2006, NSW, Australia. E-mail: [email protected] 127 Copyright © 2018 the authors 0270-6474/18/380465-09$15.00/0 466 • J. Neurosci., January 10, 2018 • 38(2):465–473 Mills et al. • Brainstem Connectivity and Neuropathic Pain addition to a presumed role in modulating acute noxious stimuli, these circuits might be involved in the generation and mainte- nance of chronic pain, particularly that following nerve damage or presumed damage (i.e., chronic neuropathic pain), as sug- gested by recent preclinical data (Burgess et al., 2002; Pertovaara, 2013; Wang et al., 2013). While it remains unknown whether the function of these brainstem modulatory circuits is altered in humans with chronic pain, multiple psychophysical investigations have suggested that they are. For example, some studies using conditioned pain mod- ulation, in which the perceived pain intensity of one noxious stimulus is modified by another at a distant site, have provided evidence that the effectiveness of these pain-modulation circuits is significantly altered in individuals with various chronic pain conditions (Yarnitsky, 2010; Garrett et al., 2013; Nasri-Heir et al., 2015). There is evidence from experimental studies in both ani- mals and humans that the effects of these pain-modulatory sys- tems are mediated by structures in the brainstem, in particular the area of the SRD (Le Bars et al., 1979; De Broucker et al., 1990; Youssef et al., 2016). These studies strongly suggest that altera- tions in the ongoing functioning of brainstem nociceptive mod- ulation systems may play a critical role in the initiation and/or the maintenance of various human chronic pain conditions. To date, no study has focused on the ongoing function of brainstem nociceptive modulation circuits, including the PAG, RVM, LC, and SRD in humans with chronic pain. The aim of the Figure 1. Locations of functional connectivity seeds overlaid onto axial slices of a wet spec- present study was to use resting-state functional magnetic reso- imen, a representative T1-weighted anatomical image, and a representative brainstem fMRI nance imaging (fMRI) to explore the ongoing function of these image normalized to the SUIT template. Slice locations in MNI space are indicated at the top brainstem circuits in individuals with painful trigeminal neurop- right.Thepresentstudyfocusedonthefollowingregionsofinterest:themidbrainvlPAG,theLC, athy (PTN), a chronic orofacial neuropathic pain disorder. We the RVM, and the SpV. hypothesize that individuals with PTN would display enhanced fMRI signal covariation between regions involved in endogenous pain modulation, such as the ventrolateral PAG (vlPAG), RVM, intensity. Subjects also described their pain distribution by outlining the LC, and SRD, and the region where orofacial nociceptive infor- area of their chronic pain on a standard drawing of the face and described the quality of their ongoing pain by completing a McGill Pain Question- mation enters the CNS, i.e., the SpV. Such changes may result in naire (MPQ). greater facilitation of ongoing activity within the SpV, enhanced noxious information transfer to higher brain centers, and the MRI scans constant perception of pain. Furthermore, these brainstem cir- Each subject was positioned supine onto the MRI scanner bed and placed cuits can be engaged by higher brain regions, such as the orbito- into a 3 tesla MRI scanner (Achieva, Philips Medical Systems), with the frontal cortex and ventral striatum (Bandler et al., 2000; Floyd et head immobilized in a 32-channel head coil to which padding was added al., 2000) and so we hypothesize that PTN subjects will display to prevent head movement. A series of 180 gradient echo echo-planar altered functional connectivity strengths between these rostral image sets with blood oxygen level-dependent contrast were collected. brain regions and the PAG and LC. Each image volume was positioned to cover the entire brain and extend caudally to include the caudal brainstem (37 axial slices; repetition time ϭ 2000 ms; echo time ϭ 30 ms; raw voxel size, 3.0 ϫ 3.0 ϫ 4.0 mm Materials and Methods thick). In all 70 subjects, a high-resolution T1-weighted anatomical im- Subjects and pain measures age of the entire brain was also collected (288 axial slices; repetition Twenty-four subjects with PTN [eight males; mean (ϮSEM) age, 46 Ϯ 3 time ϭ 5600 ms; raw voxel size, 0.87 ϫ 0.87 ϫ 0.87 mm thick). years; range, 19–71 years] and 46 pain-free subjects (17 males; age, 42 Ϯ 2 years; range, 22–66 years) were recruited for the study between 2011 MRI and statistical analysis and 2017. There was no significant difference in age (t test; p Ͼ 0.05) or Image preprocessing gender distribution ( 2 test, p Ͼ 0.05) between the two subject groups. Using SPM12 (Friston et al., 1995) and custom software, fMRI images PTN subjects were recruited and diagnosed in accordance with the were realigned and movement parameters examined to ensure no subject Liverpool criteria (Nurmikko and Eldridge, 2001) by clinicians (C.C.P, displayed Ͼ1 mm volume-to-volume movement in the X, Y, and Z E.R.V, G.M.M) in the research group. Control participants were re- planes and 0.05 radians in the pitch, roll, and yaw directions. Physiolog- cruited by word of mouth. Informed written consent was obtained for all ical (i.e., cardiovascular and respiratory) noise was modeled and re- procedures, which were conducted with the approval of local institu- moved using the drifter toolbox ( Sa¨rkka¨et al., 2012). The fMRI images tional human research ethics committees and are consistent with the were detrended to remove global signal-intensity changes and then Declaration of Helsinki. Other data from several subjects used in this coregistered to the same subject’s T1-weighted anatomical image. The investigation have been used in previous studies (Gustin et al., 2011; T1-weighted image was then spatially normalized to the Montreal Neu- Henderson et al., 2013; Wilcox et al., 2015a; Alshelh et al., 2016). rological Institute (MNI) template. The normalization parameters were The PTN subjects assessed the intensity of their ongoing pain using a then applied to the fMRI images. This process resulted in the fMRI im- visual analog scale (0, no pain, to 10, worst pain imaginable) three times ages being resliced into 2 ϫ 2 ϫ 2 mm voxels. To improve brainstem per day for 7 consecutive days during the week of the scanning session. normalization accuracy, the brainstems of the fMRI image sets were The average of these pain ratings was taken as a measure of current pain128 isolated using the SUIT (A Spatially Unbiased Atlas Template of the Mills et al. • Brainstem Connectivity and Neuropathic Pain J. Neurosci., January 10, 2018 • 38(2):465–473 • 467
Table 1. PTN subject characteristics Age Pain intensity Pain duration Subject (years) Gender Site (visual analog scale) (months) Medications 1 56 Male Right lower jaw 2.4 72 Amitriptyline 2 45 Female Bilateral (left jaw and temple) 4.8 132 Carbamazepine 3 45 Female Left upper jaw 4.5 42 None 4 42 Female Bilateral lower jaw 4.0 42 Paracetamol, ibuprofen 5 67 Female Left upper jaw and temple 8.4 168 None 6 43 Male Left upper jaw and temple 5.8 192 Desvenlafaxine 7 44 Female Bilateral upper and lower jaw 1.2 18 Ibuprofen, codeine phosphate, paracetamol 8 65 Female Left face and neck 2.5 84 Amitriptyline, paracetamol 9 50 Female Right lower jaw 0.1 84 None 10 67 Female Right upper jaw 3.9 240 Thyroxine sodium, rosuvastatin, venlafaxine, pregabalin, phenylethylamine, ergotamine 11 27 Female Left upper jaw 6.9 29 Ibuprofen, duloxetine 12 65 Male Bilateral: left lower and right upper jaw 0.4 26 Amitriptyline 13 38 Female Left lower jaw 3.4 96 Gabapentin, amitriptyline 14 45 Female Bilateral jaw 2.8 17 Ibuprofen 15 19 Male Right upper jaw 5.0 88 None 16 59 Female Left lower jaw 4.4 115 None 17 59 Female Left upper jaw 4.4 120 None 18 27 Male Left upper jaw and under eye 3.6 8 Palmitoylethanolamide, paracetemol, benzodiazepines 19 71 Female Left upper jaw 5.1 9 Meloxicam, paracetamol, gabapentin, nortriptyline 20 37 Male Left upper jaw and under eye 2.0 84 Oxycodone, gabapentin, paracetamol 21 35 Female Bilateral upper jaw 6.1 27 Flavonoids, paracetamol 22 27 Male Right upper and lower jaw 0.5 41 Palmitoylethanolamide, sertraline 23 21 Female Right upper jaw 1.3 144 Palmitoylethanolamide 24 35 Male Right upper jaw 1.5 96 Palmitoylethanolamide
Table 2. Location of clusters in MNI space, t values, and sizes of clusters that had significantly different resting RVM and LC connectivity strengths in subjects with PTN compared to control subjects MNI coordinates XY Z t value Cluster size RVM connectivity PTN Ͼ Control Right vlPAG 4 Ϫ32 Ϫ9 2.72 9 LC Right 4 Ϫ38 Ϫ23 3.92 24 Left Ϫ2 Ϫ36 Ϫ21 3.67 Right SRD 6 Ϫ44 Ϫ41 2.81 2 Left SpV Ϫ6 Ϫ42 Ϫ53 3.22 6 Right LC connectivity PTN Ͼ Control Left SRD 4 Ϫ44 Ϫ55 2.71 1 RVM 0 Ϫ36 Ϫ47 3.11 6 Control Ͼ PTN Right vlPAG 6 Ϫ28 Ϫ5 3.10 28
Cerebellum and Brainstem) toolbox in native space (Diedrichsen, 2006). Binary masks of the brainstem were created and these masks were then used to spatially normalize each image set to the SUIT brainstem tem- plate in MNI space. The resulting normalized fMRI images remained Figure 2. Regions within the brainstem endogenous analgesic pathways that show signifi- unsmoothed so as to maintain spatial accuracy of small brainstem cantly greater RVM functional connectivity in subjects with PTN compared with pain-free con- structures. trols. Left, Diagram of the endogenous analgesic pathways that modulate nociceptive transmission at the spinal dorsal horn (DH) and SpV. Yellow, Nociceptor afferent projection; Resting functional connectivity analysis blue, efferent projections from vlPAG; red, efferent projections from LC; green, efferent projec- Voxel-by-voxel analysis. We performed brainstem-only functional con- tions from RVM. Middle, Regions with enhanced RVM functional connectivity in subjects with nectivity analyses using the RVM region as the seed. Our previous inves- PTN (hot color scale) overlaid onto template axial T1-weighted images. Slice locations in MNI tigations have shown that the nucleus raphe magnus within the RVM space are indicated at the top right of each slice. The location of the RVM seed is indicated in region displays anatomical changes in individuals with chronic pain green. Subjects with PTN show an increase in RVM connectivity with the right vlPAG, bilateral (Wilcox et al., 2015b). The RVM seed comprised six contiguous voxels: LC,rightSRD,andleftSpV.Right,Bargraphsofmean(ϮSEM)RVMconnectivitystrengthswith two voxels each at three rostrocaudal levels from z coordinate Ϫ53 to significant clusters identified in the voxel-by-voxel analysis. *, Significant difference deter- Ϫ49 in MNI space (Fig. 1). In each subject, signal intensity changes were mined in the voxel-by-voxel analysis. Connectivity strength in arbitrary units. 129 extracted from this RVM seed and voxel-by-voxel analyses were per- 468 • J. Neurosci., January 10, 2018 • 38(2):465–473 Mills et al. • Brainstem Connectivity and Neuropathic Pain formed to determine which brainstem areas displayed significant signal-intensity cova- riations with the RVM. The movement para- meters derived from the realignment step were included as nuisance variables. The connectiv- ity maps were smoothed usinga2mmfull- width half-maximum Gaussian filter and then placed into a second-level random-effects pro- cedure to determine significant differences in RVM connectivity strength in arbitrary units, (A.U.), between PTN and control subjects. Fol- lowing an initial uncorrected threshold of p Ͻ 0.001, small-volume corrections were applied on the PAG, RVM, LC, SRD, and SpV using 40 mm 3 hyper-rectangles centered at the location of each region based on the Duvernoy Brains- tem Atlas (Naidich et al., 2009). The resulting clusters of significant differ- ence were used to extract connectivity strength values in each subject, and the mean (ϮSEM) values were plotted to provide a measure of connectivity direction. In PTN subjects, we in- vestigated the significance of relationships be- tween RVM connectivity strengths and pain characteristics (i.e., pain intensity and dura- Figure 3. Subjects with PTN show increased RVM functional connectivity in discrete regions within the SpV. Middle, Locations tion) using regression analyses (Pearson’s r, of rostrocaudal vlPAG and SpV volumes of interest are indicated in green, with slice locations in MNI space indicated at the top left Ͻ p 0.05). ofeachslice.Top,Mean(ϮSEM)RVMconnectivitystrengthsalongtherostrocaudalaxisoftheleftandrightvlPAGinsubjectswith The brainstem RVM connectivity analysis PTN and healthy controls. Compared with controls, PTN subjects show a trend toward increased RVM connectivity with the vlPAG. resulted in a significant cluster in the region of However, this did not reach statistical significance. Bottom, RVM connectivity strengths in PTN and control subjects with the left the LC. Since this region is known to be in- andrightSpV.Comparedwithcontrols,subjectswithPTNshowenhancedRVMconnectivitywithintheleftSpVi(zlevelϪ53)and volved in nociceptive modulation (Taylor and SpVo (z level Ϫ51), and no connectivity differences within the SpV caudalis (SpVc). *p Ͻ 0.05, Bonferroni corrected for multiple Westlund, 2017), we also explored the resting comparisons. Connectivity strength in arbitrary units. connectivity of this brainstem region. The LC seed comprised six contiguous voxels: two vox- rostrally to z coordinate Ϫ5(Fig. 1). Similarly, for the SpV, 14 four-voxel els each at three rostrocaudal levels from z coordinate Ϫ23 to Ϫ19 in volumes of interest were created along the rostrocaudal axis on the left MNI space (Fig. 1). We performed a brainstem resting connectivity anal- and right sides, extending from the caudal SpV at z coordinate Ϫ61, ysis of the right LC in the same way as that described above for the RVM. rostrally to z coordinate Ϫ35. RVM and LC connectivity strengths of Additionally, in PTN subjects, the significance of relationships between these vlPAG and SpV volumes of interest were extracted and differences LC connectivity strengths and pain characteristics (i.e., pain intensity between controls and PTN subjects determined (p Ͻ 0.05, two-sample t and duration) were determined for significant clusters using regression test, Bonferroni corrected for multiple comparisons). Two PTN subjects analyses (Pearson’s r, p Ͻ 0.05). were excluded from the SpV subdivision analyses due to their fMRI In addition to brainstem-specific analyses, resting connectivity strengths images not covering the entire caudal brainstem at z level Ϫ61. from the PAG and LC to higher brain regions were explored. Resting signal intensities from the right LC seed used for the brainstem analysis and from a right PAG seed (six voxels in size, derived from RVM con- Results nectivity analysis and spread across z coordinates Ϫ11, Ϫ9, Ϫ7) were Pain characteristics extracted and voxel-by-voxel analyses performed to determine connec- Individual PTN subject characteristics are shown in Table 1. All tivity strengths with higher brain regions. Movement parameters were 24 PTN subjects were classified as having post-traumatic neurop- included as nuisance variables and the resultant connectivity maps were athy. In 19 PTN patients, chronic pain was unilateral (7 right, 12 smoothed usinga6mmfull-width half-maximum Gaussian filter. These left), and the remaining five PTN subjects reported bilateral pain. connectivity maps were placed into a second-level random-effects pro- cedure to determine significant differences in connectivity strengths In all PTN subjects, pain was located in the second and third between PTN and control subjects. Following an initial uncorrected trigeminal nerve divisions, with one patient also reporting pain in threshold of p Ͻ 0.001, we applied small-volume corrections (familywise the first division. Their mean (ϮSEM) pain intensity was 3.5 Ϯ0.4 of error, p Ͻ 0.05). The resulting clusters of significant difference were used 10 and their mean duration of pain was 82.2 Ϯ 12.5 months. Using to extract connectivity strength values in each subject and the mean the MPQ, PTN subjects most frequently described their pain as (ϮSEM) values plotted to provide a measure of connectivity direction. In throbbing (58%), shooting (54%), and nagging (67%). PTN subjects, the significance of relationships between LC and PAG connectivity strengths and pain characteristics (i.e., pain intensity and Brainstem resting functional connectivity duration) were determined for significant clusters using regression anal- RVM voxel-by-voxel analysis Ͻ yses (Pearson’s r, p 0.05). The brainstem voxel-by-voxel analysis revealed several key areas Subdivision analysis. To determine whether there are variations in ros- that display altered resting RVM functional connectivity in PTN trocaudal connectivity strengths within the vlPAG and SpV, we exam- subjects compared with controls (Fig. 2, Table 2). PTN subjects ined resting functional connectivity between these regions and both the showed a significant increase in RVM connectivity strength in the RVM and LC. Rostrocaudal subdivisions of the vlPAG and SpV were Ϯ created based on our voxel-by-voxel analyses and the Duvernoy Human region encompassing the right vlPAG (mean SEM functional Brainstem Atlas (Naidich et al., 2009). For the vlPAG, five four-voxel connectivity, a.u.: control, Ϫ0.19 Ϯ 0.05; PTN, 0.11 Ϯ 0.09), the volumes of interest were created along the rostrocaudal axis on the left region of the LC bilaterally (control, Ϫ0.12 Ϯ 0.04; PTN, 0.18 Ϯ Ϫ and right sides, extending from the caudal vlPAG at z coordinate 13,130 0.07), the right dorsomedial medulla in the area of the SRD (con- Mills et al. • Brainstem Connectivity and Neuropathic Pain J. Neurosci., January 10, 2018 • 38(2):465–473 • 469 trol, 0.11 Ϯ 0.03; PTN, 0.29 Ϯ 0.06), and in the region encom- passing the left SpV (control, 0.23 Ϯ 0.03; PTN, 0.42 Ϯ 0.05). In no brainstem region did controls show significantly greater RVM connectivity than PTN patients. In PTN subjects, there were no significant correlations between RVM connectivity strength in these brainstem regions and pain intensity (vlPAG: r ϭ 0.21; LC: r ϭ 0.11; SRD: r ϭ 0.15; SpV: r ϭ 0.03, all p’s Ͼ 0.05) or pain duration (vlPAG: r ϭ 0.18; LC: r ϭ 0.05; SRD: r ϭ 0.19; SpV: r ϭ 0.38, all p’s Ͼ 0.05).
RVM to vlPAG and SpV subdivision analysis The subdivision analysis of RVM connectivity over the rostro- caudal extent of the bilateral vlPAG revealed that PTN subjects showed a trend toward higher RVM connectivity. However, this did not reach significance at the threshold we set (Fig. 3). In no vlPAG subdivision did controls show greater RVM connectivity than PTN subjects. In contrast, RVM–SpV subdivision analysis revealed that PTN subjects displayed greater connectivity strength with the left SpV (Fig. 3). This significantly greater RVM connec- tivity strength was restricted to the SpV within the region of the subnucleus interpolaris (SpVi; z Ϫ53: control, 0.24 Ϯ 0.03; PTN, 0.46 Ϯ 0.06, p Ͻ 0.001) and subnucleus oralis (SpVo; z Ϫ51: control, 0.27 Ϯ 0.03; PTN, 0.46 Ϯ 0.06, p ϭ 0.002). There were no differences between groups in RVM–SpV connectivity strength on the right side and, again, in no SpV region did controls show greater connectivity than PTN subjects.
LC voxel-by-voxel analysis Figure4. Regionswithinthebrainstempain-modulationsysteminwhichsubjectswithPTN The brainstem voxel-by-voxel analysis revealed two brainstem showalteredLCfunctionalconnectivitycomparedwithpain-freecontrols.Areaswithenhanced regions in which PTN subjects displayed greater LC connectivity (hot color scale) and reduced (cool color scale) LC connectivity in PTN subjects are overlaid onto compared with controls (Fig. 4, Table 2). Significantly greater LC templateaxialT1-weightedimages.SlicelocationsinMNIspaceareindicatedatthetoprightof connectivity in PTN subjects occurred within the left caudal SRD each slice, with the location of the LC seed indicated in green. Subjects with PTN showed an Ϯ Ϯ increase in LC connectivity with the left caudal SRD and the RVM, as well as a decrease in LC (control, 0.02 0.03; PTN, 0.21 0.06) and in the region of the Ϯ Ϫ Ϯ Ϯ connectivitywiththerightvlPAG.Right,Bargraphsofmean( SEM)LCconnectivitystrengths RVM (control, 0.06 0.05; PTN, 0.21 0.06). In contrast, with significant clusters identified in the voxel-by-voxel analysis. *, Significant difference de- PTN subjects showed significantly reduced LC connectivity termined in the voxel-by-voxel analysis. Connectivity strength in arbitrary units. strength with the right rostral vlPAG (control, 0.21 Ϯ 0.04; PTN, Ϫ0.03 Ϯ 0.05). In PTN subjects, there were no significant corre- hippocampus (left: control, Ϫ0.01 Ϯ 0.03; PTN, 0.18 Ϯ 0.05; lations between LC connectivity strength in these three brainstem Ϯ Ϯ regions and pain intensity (SRD: r ϭ 0.26; RVM: r ϭ 0.06; vlPAG: right: control, 0.003 0.02; PTN, 0.17 0.06), the bilateral ϭ Ͼ ϭ thalamus in the region of the mediodorsal nucleus (left: control, r 0.33; all p’s 0.05) or pain duration (SRD: r 0.11; RVM: Ϯ Ϯ Ϫ Ϯ r ϭ 0.12; vlPAG: r ϭ 0.21; all p’s Ͼ 0.05). 0.02 0.04; PTN, 0.26 0.06; right: control, 0.03 0.03; PTN, 0.17 Ϯ 0.06), and in the bilateral anterior hippocampus (left: control, Ϫ0.01 Ϯ 0.03; PTN, 0.24 Ϯ 0.06; right: control, Ϫ0.01 Ϯ LC to vlPAG and SpV subdivision analysis 0.02; PTN, 0.18 Ϯ 0.04). In PTN subjects, there were no signifi- The subdivision analysis revealed that, in direct contrast to the cant correlations between vlPAG connectivity strength in these RVM, LC connectivity strength with the right vlPAG was signif- brain regions and pain intensity (NAc, r ϭ 0.09; left posterior icantly reduced in PTN subjects (Fig. 5). PTN subjects showed hippocampus, r ϭ 0.32; right posterior hippocampus, r ϭ 0.027; significantly reduced LC connectivity strength in a discrete re- left thalamus, r ϭ 0.04; right thalamus, r ϭ 0.27; left anterior gion of the rostral vlPAG (z Ϫ5: control, 0.25 Ϯ 0.05; PTN, hippocampus, r ϭ 0.15; right anterior hippocampus, r ϭ 0.08; all Ϫ0.04 Ϯ 0.07, p Ͻ 0.001). There were no differences between p’s Ͼ 0.05) or pain duration (NAc, r ϭ 0.31; left posterior hip- groups in LC–vlPAG connectivity strength on the left side and in pocampus, r ϭ 0.21; right posterior hippocampus, r ϭ 0.20; left no vlPAG region did controls show greater LC connectivity than thalamus, r ϭ 0.14; right thalamus, r ϭ 0.09; left anterior hip- PTN subjects. In contrast, the LC–SpV subdivision analysis re- pocampus, r ϭ 0.17; right anterior hippocampus, r ϭ 0.06; all vealed that PTNs did not differ from controls in LC connectivity p’s Ͼ 0.05). strength at any region of the SpV. LC voxel-by-voxel analysis Whole-brain resting functional connectivity The whole-brain voxel-by-voxel analysis revealed several regions PAG voxel-by-voxel analysis in which PTN subjects displayed greater LC connectivity com- The whole-brain voxel-by-voxel analysis revealed several key ar- pared with controls (Fig. 6, Table 3). Significantly greater LC eas that display altered resting vlPAG functional connectivity connectivity in PTN subjects occurred within the left insula (con- in PTN subjects compared with controls (Fig. 6, Table 3). PTN trol, Ϫ0.001 Ϯ 0.02; PTN, 0.11 Ϯ 0.04), the left NAc (control, subjects showed a significant increase in vlPAG connectivity 0.003 Ϯ 0.03; PTN, 0.16 Ϯ 0.04), the right anterior cingulate strength in the region of the left nucleus accumbens (NAc; con- cortex (ACC; control, 0.02 Ϯ 0.01; PTN, 0.08 Ϯ 0.02), and within Ϫ Ϯ Ϯ Ϯ Ϯ trol, 0.02 0.03; PTN, 0.16 0.04), the bilateral posterior131 the bilateral frontal cortex (left: control, 0.02 0.02; PTN, 0.15 470 • J. Neurosci., January 10, 2018 • 38(2):465–473 Mills et al. • Brainstem Connectivity and Neuropathic Pain
0.03; right: control, 0.002 Ϯ 0.02; PTN, 0.13 Ϯ 0.04). In PTN subjects, there was a significant positive correlation between LC–ACC connectivity strength and pain intensity (r ϭ 0.42, p ϭ 0.047). That is, the greater the pain intensity, the greater the LC–ACC connectivity strength. No other significant correlations were observed. Discussion The results of this study demonstrate that PTN, a chronic neuropathic pain condi- tion, is associated with ongoing functional alterations within the brainstem endog- enous pain-modulation circuitry. Spe- cifically, PTN patients showed increased functional connectivity between the RVM and the vlPAG, SRD, and LC, as well as with the brainstem region that receives orofacial nociceptor afferents, the SpV. Furthermore, PTN subjects showed in- creased functional connectivity strengths between these brainstem circuits and higher Figure 5. Mean (ϮSEM) LC connectivity strengths along the rostrocaudal axis of the vlPAG and SpV in subjects with PTN brain regions, such as the hippocampus, comparedwithpain-freecontrols.Middle,LocationsofrostrocaudalvlPAGandSpVvolumesofinterestareindicatedingreen,with NAc, and ACC. These data show that slicelocationsinMNIspaceindicatedatthetopleftofeachslice.Top,SubjectswithPTNshowreducedLCconnectivitywiththeright chronic neuropathic pain is associated rostral vlPAG at z level Ϫ5. Bottom, LC connectivity with the left and right SpV does not differ between subjects with PTN and with ongoing functional changes in brain- controls at any point along the SpVc, SpVi, or SpVo subdivisions. *p Ͻ 0.05, Bonferroni corrected for multiple comparisons. stem pain-modulation circuits that are Connectivity strength in arbitrary units. likely involved in maintaining pain fol- lowing nerve injury. SpV. This was expected given that the majority of our PTN sub- It is well known from experimental animal investigations that jects reported left-sided pain and the location of these functional the vlPAG can evoke an opioid-mediated analgesia and that this coupling changes overlaps with our previous reports of structural effect is mediated by a projection to the spinal dorsal horn and changes at the SpVi and SpVo in PTN patients (Wilcox et al., SpV via the RVM (Basbaum and Fields, 1984; Fields and Hei- 2015a). nricher, 1985; Bandler and Shipley, 1994). While it is well known Alongside the vlPAG and RVM, the LC can also directly and that the RVM can inhibit incoming noxious information, it can indirectly modulate nociceptive transmission at the dorsal horn also facilitate it and it has been suggested that opposing RVM and SpV (Sasa et al., 1979; Jones and Gebhart, 1986; Tsuruoka et inhibitory and facilitatory effects are finely balanced at rest in al., 2003; Pertovaara, 2006; Kaushal et al., 2016). The LC has pain-free individuals (Fields and Heinricher, 1985; Heinricher et reciprocal connections with the vlPAG and the SpV (Sasa and al., 2009; Ossipov et al., 2010). In contrast, in individuals with Takaori, 1973; Jones and Moore, 1977; Cedarbaum and Aghaja- neuropathic pain, it has been proposed that this brainstem cir- nian, 1978; Mantyh, 1983), and indeed we found a decrease in cuitry shifts to favor pronociceptive processes that contribute right LC connectivity strength with the rostral region of the ipsilat- significantly to the maintenance of chronic pain (Pertovaara et eral vlPAG. However, we found no difference in LC connectivity al., 1996; Burgess et al., 2002; Vera-Portocarrero et al., 2006; strength with the SpV in the PTN subjects. This is surprising, since Wang et al., 2013). Indeed, in rodents, deactivation of the PAG or facilitation by the noradrenergic system contributes to mechan- RVM eliminates the hyperalgesia, allodynia, and spontaneous ical hypersensitivity in animal models of neuropathic pain pain evoked by spinal nerve ligation (Pertovaara et al., 1996; Bur- (Brightwell and Taylor, 2009; Kaushal et al., 2016). However, it gess et al., 2002; Wang et al., 2013). This functional shift appears has been proposed that the LC inhibitory effects are engaged to influence only the primary synapse ipsilateral to the side of through a direct connection to the dorsal horn and SpV (Sasa et nerve injury, since these analgesic effects are produced following al., 1974), whereas LC pronociceptive effects may be mediated via interruption of the ipsilateral but not the contralateral RVM– the SRD (Martins et al., 2013; Taylor and Westlund, 2017). Our dorsal horn pathway (Wang et al., 2013). Furthermore, this pro- results are consistent with this idea, as PTN subjects displayed nociceptive shift in RVM function appears to be strictly related to enhanced LC–SRD connectivity strength and no change in LC–SpV the presence of pain behaviors and not to the nerve injury itself connectivity strength. Alongside a decrease in LC–vlPAG con- (De Felice et al., 2011). nectivity, we found that PTN subjects display greater LC–RVM The increased resting functional coupling between between connectivity strength than controls, raising the prospect that the the PAG and RVM and between the RVM and SpV reported in endogenous opioid and noradrenergic pain modulation systems neuropathic pain subjects in this study is consistent with these interact differently in individuals with neuropathic pain. Al- preclinical findings and may reflect a state of continuous de- though tract-tracing studies have shown that direct connections scending facilitation of nociceptive transmission at the SpV between the RVM and LC are modest (Clark and Proudfit, 1991), driven by the vlPAG via the RVM. Interestingly, in PTN subjects it has been demonstrated that the vlPAG–RVM and noradrener- we found increased RVM connectivity specifically within the gic pain systems interact in experimental pain states to produce transition between the interpolaris and oralis regions of the left132 inhibitory effects at the dorsal horn (Aimone et al., 1987). Mills et al. • Brainstem Connectivity and Neuropathic Pain J. Neurosci., January 10, 2018 • 38(2):465–473 • 471
We cannot infer directionality from functional connectivity analyses, and thus ascending signal transmission may also contribute to the alterations in fMRI sig- nal coupling observed in PTN patients. Indeed, there are ascending projections from the SpV to the PAG, RVM, and LC (Craig, 1992; Bandler and Shipley, 1994; Sugiyo et al., 2005). While these ascending influences may be important, we suggest that the changes in brainstem connectivi- ties that occur in PTN subjects most likely represent changes in descending modula- tion of the SpV since previous investiga- tions suggest that neuropathic pain can be maintained solely by central mechanisms, even if initiated by a peripheral nerve in- jury (Burgess et al., 2002; Henderson et al., 2013; Wang et al., 2013). While the brainstem contains the neu- ral circuitry necessary for pain modula- tion, this system can be engaged by higher brain regions (Heinricher et al., 2009). In- terestingly, we found increased resting connectivity strengths between the PAG, the mediodorsal thalamus, and the hip- pocampus in PTN subjects compared with controls. While it is possible that these re- gions represent top–down modulation of brainstem circuits, it has been suggested that the midline thalamic nuclei, such as the mediodorsal nuclei, receive ascending Figure6. RostralbrainregionsinwhichsubjectswithPTNshowincreasedvlPAGandLCfunctionalconnectivitycomparedwith healthy controls. Areas in which subjects with PTN display enhanced vlPAG or LC connectivity (hot color scale) are overlaid onto communication from the PAG, which is template axial and coronal T1-weighted images. Slice locations in MNI space are indicated at the top right of each slice, with the transferred to the ventral hippocampus to locationofvlPAGandLCseedsindicatedingreenonthefarleft.Top,SubjectswithPTNshowenhancedvlPAGconnectivitywiththe influence pain-related behaviors (Balles- left NAc, bilateral posterior hippocampus (pHipp), bilateral anterior hippocampus (aHipp), and bilateral thalamus. Bottom, Sub- teros et al., 2014). Furthermore, the hip- jectswithPTNshowincreasedLCconnectivitywiththeleftinsulaandNAc,rightACC,andbilateralfrontalcortex.Belowbrainslices, pocampus sends extensive projections to plots of mean (ϮSEM) vlPAG and LC connectivity strengths in significant clusters identified in the voxel-by-voxel analysis. *, the NAc (Viard et al., 2011). The NAc is Significant difference determined in the voxel-by-voxel analyses. Connectivity strength in arbitrary units. involved in mediating reward processing and goal-directed behavior, two processes Table 3. Location of whole-brain clusters in MNI space, t values, and sizes of that are disrupted in individuals with chronic pain (Navratilova clusters that had significantly different resting vlPAG and LC connectivity strengths in subjects with painful PTN compared to control subjects and Porreca, 2014; Navratilova et al., 2016). Our PTN patients also displayed enhanced LC connectivity with the NAc and ACC, MNI coordinates two regions involved in the rewarding effects of pain relief XYZt value Cluster size (Navratilova et al., 2016). Indeed, the only significant linear rela- Right vlPAG connectivity tionship occurred between LC–ACC connectivity strength and PTN Ͼ Control pain intensity in PTN subjects. Left NAc Ϫ18 2 Ϫ16 3.44 25 Several limitations are worth noting. First, given the small size Posterior hippocampus Ϫ14 Ϫ36 6 4.29 43 of brainstem nuclei and the limited spatial resolution of current Ϫ Left 22 36 4 3.29 3 functional human brain-imaging techniques, it is difficult to as- Right 30 Ϫ30 Ϫ12 3.98 14 Thalamus sign significant clusters to specific brainstem nuclei. This does Left Ϫ6 Ϫ12 0 3.30 9 not, however, negate the validity of our results when placed into Right 6 Ϫ12 2 3.10 3 context. That is, our brainstem clusters overlap with regions that Anterior hippocampus Ϫ24 Ϫ14 Ϫ24 4.17 67 have been shown in preclinical studies to be critical for the ex- Left 36 Ϫ16 Ϫ22 4.17 50 pression of analgesia and our a priori hypothesis was that the Ϫ Ϫ Right 26 12 20 function of these regions would be altered in PTN subjects. Right LC connectivity PTN Ͼ Control Furthermore, we did not smooth our images so that we could Left insula Ϫ24 20 Ϫ12 3.26 8 maintain spatial accuracy, though all these factors do not elimi- Left NAc Ϫ18 8 Ϫ16 3.47 17 nate the possibility that closely neighboring nuclei are responsi- Right ACC 12 32 16 3.41 2 ble for the reported differences. Second, the brainstem is Frontal cortex susceptible to physiological noise and movement-related arti- Left Ϫ14 16 62 3.11 7 facts. To limit the potential influence of these factors, we applied Right 16 12 62 3.37 13 133 a physiological noise correction and included movement param- 472 • J. Neurosci., January 10, 2018 • 38(2):465–473 Mills et al. • Brainstem Connectivity and Neuropathic Pain eters as nuisance variables. A further potential limitation is the medulla to pontine catecholamine cell groups involved in the modulation use of unflipped brain images for the PTN subjects. Most subjects of nociception. Brain Res 540:105–115. CrossRef Medline reported unilateral pain, making it unlikely in most cases to observe Craig AD (1992) Spinal and trigeminal lamina I input to the locus coeruleus anterogradely labeled with Phaseolus vulgaris leucoagglutinin (PHA-L) connectivity changes bilaterally. Finally, a possible limitation is that in the cat and the monkey. Brain Res 584:325–328. CrossRef Medline when reporting between-group differences, we used a statistical De Broucker T, Cesaro P, Willer JC, Le Bars D (1990) Diffuse noxious in- threshold that was not corrected for multiple comparisons. Al- hibitory controls in man. Involvement of the spinoreticular tract. Brain though this may introduce type-II errors, we limited this possi- 113:1223–1234. CrossRef Medline bility by applying small-volume corrections. As detailed above, De Felice M, Sanoja R, Wang R, Vera-Portocarrero L, Oyarzo J, King T, since numerous experimental animal investigations have re- Ossipov MH, Vanderah TW, Lai J, Dussor GO, Fields HL, Price TJ, Por- ported that the RVM, PAG, LC, and SRD are critically involved in reca F (2011) Engagement of descending inhibition from the rostral ventromedial medulla protects against chronic neuropathic pain. Pain analgesic responses, we strongly suggest that small-volume cor- 152:2701–2709. CrossRef Medline rections of these brainstem regions are entirely appropriate. De- Diedrichsen J (2006) A spatially unbiased atlas template of the human cer- spite these limitations, we are confident that the altered ebellum. Neuroimage 33:127–138. CrossRef Medline brainstem connectivity presented here represents a functional Fields HL, Heinricher MM (1985) Anatomy and physiology of a nociceptive difference associated with the presence of chronic neuropathic modulatory system. Philos Trans R Soc Lond B Biol Sci 308:361–374. pain. CrossRef Medline Overall, our findings show that chronic neuropathic pain in Floyd NS, Price JL, Ferry AT, Keay KA, Bandler R (2000) Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the humans is associated with ongoing functional alterations in the periaqueductal gray in the rat. J Comp Neurol 422:556–578. CrossRef brainstem endogenous pain-modulation system. These findings Medline complement a wealth of experimental animal data suggesting Friston KJ, Holmes AP, Worsley KP, Proline JB, Frith CD, Frackowiak RSJ that the functioning of brainstem modulation systems shifts to (1995) Statistical parametric maps in functional imaging: a general im- promote the maintenance of pain behaviors following nerve in- aging approach. J Hum Brain Mapp 2:189–210. CrossRef jury (Burgess et al., 2002; Ossipov et al., 2010; Wang et al., 2013). Garrett PH, Sarlani E, Grace EG, Greenspan JD (2013) Chronic temporo- Furthermore, it is likely that the resting functioning of these mandibular disorders are not necessarily associated with a compromised endogenous analgesic system. J Orofac Pain 27:142–150. CrossRef Medline brainstem circuits sets the ongoing gain of nociceptive process- Granovsky Y (2013) Conditioned pain modulation: a predictor for develop- ing, and may promote maladaptive responses to experimental ment and treatment of neuropathic pain. Curr Pain Headache Rep 17: noxious stimuli observed in various pain conditions (King et al., 361. CrossRef Medline 2009; Yarnitsky, 2010; Granovsky, 2013). Our findings also sug- Gustin SM, Peck CC, Wilcox SL, Nash PG, Murray GM, Henderson LA gest that higher brain regions, such as the hippocampus, NAc, (2011) Different pain, different brain: thalamic anatomy in neuropathic and ACC, interact with the brainstem modulation circuitry dif- and non-neuropathic chronic pain syndromes. J Neurosci 31:5956–5964. ferently in patients with neuropathic pain, possibly reflecting a CrossRef Medline Heinricher MM, Tavares I, Leith JL, Lumb BM (2009) Descending control top–down engagement of the circuitry alongside altered reward- of nociception: specificity, recruitment and plasticity. Brain Res Rev 60: circuit functioning in chronic pain states. 214–225. CrossRef Medline Henderson LA, Peck CC, Petersen ET, Rae CD, Youssef AM, Reeves JM, References Wilcox SL, Akhter R, Murray GM, Gustin SM (2013) Chronic pain: lost Aimone LD, Jones SL, Gebhart GF (1987) Stimulation-produced descend- inhibition? J Neurosci 33:7574–7582. CrossRef Medline ing inhibition from the periaqueductal gray and nucleus raphe magnus in Jones BE, Moore RY (1977) Ascending projections of the locus coeruleus in the rat: mediation by spinal monoamines but not opioids. Pain 31:123– the rat. II. Autoradiographic study. Brain Res 127:23–53. CrossRef Medline 136. CrossRef Medline Jones SL, Gebhart GF (1986) Quantitative characterization of ceruleospinal Alshelh Z, Di Pietro F, Youssef AM, Reeves JM, Macey PM, Vickers ER, Peck inhibition of nociceptive transmission in the rat. J Neurophysiol 56:1397– CC, Murray GM, Henderson LA (2016) Chronic neuropathic pain: it’s 1410. Medline about the rhythm. J Neurosci 36:1008–1018. CrossRef Medline Kaushal R, Taylor BK, Jamal AB, Zhang L, Ma F, Donahue R, Westlund KN Ballesteros CI, de Oliveira Galva˜o B, Maisonette S, Landeira-Fernandez J (2016) GABA-A receptor activity in the noradrenergic locus coeruleus ␣ (2014) Effect of dorsal and ventral hippocampal lesions on contextual drives trigeminal neuropathic pain in the rat; contribution of NA 1 fear conditioning and unconditioned defensive behavior induced by elec- receptors in the medial prefrontal cortex. Neuroscience 334:148–159. trical stimulation of the dorsal periaqueductal gray. PLoS One 9:e83342. CrossRef Medline CrossRef Medline King CD, Wong F, Currie T, Mauderli AP, Fillingim RB, Riley JL 3rd (2009) Bandler R, Shipley MT (1994) Columnar organization in the midbrain peri- Deficiency in endogenous modulation of prolonged heat pain in patients aqueductal gray: modules for emotional expression? Trends Neurosci 17: with irritable bowel syndrome and temporomandibular disorder. Pain 379–389. CrossRef Medline 143:172–178. CrossRef Medline Bandler R, Keay KA, Floyd N, Price J (2000) Central circuits mediating pat- Le Bars D, Dickenson AH, Besson JM (1979) Diffuse noxious inhibitory terned autonomic activity during active vs passive emotional coping. controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat. Brain Res Bull 53:95–104. CrossRef Medline Pain 6:283–304. CrossRef Medline Basbaum AI, Fields HL (1984) Endogenous pain control systems: brainstem Mantyh PW (1983) Connections of midbrain periaqueductal gray in the spinal pathways and endorphin circuitry. Annu Rev Neurosci 7:309–338. monkey. II. Descending efferent projections. J Neurophysiol 49:582–594. CrossRef Medline Medline Brightwell JJ, Taylor BK (2009) Noradrenergic neurons in the locus coeruleus Martins I, de Vries MG, Teixeira-Pinto A, Fadel J, Wilson SP, Westerink BH, contribute to neuropathic pain. Neuroscience 160:174–185. CrossRef Tavares I (2013) Noradrenaline increases pain facilitation from the brain Medline during inflammatory pain. Neuropharmacology 71:299–307. CrossRef Burgess SE, Gardell LR, Ossipov MH, Malan TP Jr, Vanderah TW, Lai J, Medline Porreca F (2002) Time-dependent descending facilitation from the ros- Mason P (2012) Medullary circuits for nociceptive modulation. Curr Opin tral ventromedial medulla maintains, but does not initiate, neuropathic Neurobiol 22:640–645. CrossRef Medline pain. J Neurosci 22:5129–5136. Medline Naidich TP, Duvernoy H. M., Delman B. N., Sorensen A. G., Kollias SS, M. Cedarbaum JM, Aghajanian GK (1978) Afferent projections to the rat locus HE (2009) Duvernoy’s atlas of the human brain stem and cerebellum. coeruleus as determined by a retrograde tracing technique. J Comp Neu- Vienna: Springer/Wien. rol 178:1–16. CrossRef Medline Nasri-Heir C, Khan J, Benoliel R, Feng C, Yarnitsky D, Kuo F, Hirschberg C, Clark FM, Proudfit HK (1991) Projections of neurons in the ventromedial134 Hartwell G, Huang CY, Heir G, Korczeniewska O, Diehl SR, Eliav E Mills et al. • Brainstem Connectivity and Neuropathic Pain J. Neurosci., January 10, 2018 • 38(2):465–473 • 473
(2015) Altered pain modulation in patients with persistent postendo- zone/rostral ventromedial medulla connections and facilitation of orofa- dontic pain. Pain 156:2032–2041. CrossRef Medline cial hyperalgesia after masseter inflammation in rats. J Comp Neurol Navratilova E, Porreca F (2014) Reward and motivation in pain and pain 493:510–523. CrossRef Medline relief. Nat Neurosci 17:1304–1312. CrossRef Medline Taylor BK, Westlund KN (2017) The noradrenergic locus coeruleus as a Navratilova E, Morimura K, Xie JY, Atcherley CW, Ossipov MH, Porreca F chronic pain generator. J Neurosci Res 95:1336–1346. CrossRef Medline (2016) Positive emotions and brain reward circuits in chronic pain. Tsuruoka M, Matsutani K, Inoue T (2003) Coeruleospinal inhibition of no- J Comp Neurol 524:1646–1652. CrossRef Medline ciceptive processing in the dorsal horn during unilateral hindpaw inflam- Nurmikko TJ, Eldridge PR (2001) Trigeminal neuralgia–pathophysiology, mation in the rat. Pain 104:353–361. CrossRef Medline diagnosis and current treatment. Br J Anaesth 87:117–132. CrossRef Medline Vera-Portocarrero LP, Xie JY, Yie JX, Kowal J, Ossipov MH, King T, Porreca Ossipov MH, Dussor GO, Porreca F (2010) Central modulation of pain. F (2006) Descending facilitation from the rostral ventromedial medulla J Clin Invest 120:3779–3787. CrossRef Medline maintains visceral pain in rats with experimental pancreatitis. Gastroen- Pertovaara A (2006) Noradrenergic pain modulation. Prog Neurobiol 80: terology 130:2155–2164. CrossRef Medline 53–83. CrossRef Medline Viard A, Doeller CF, Hartley T, Bird CM, Burgess N (2011) Anterior hip- Pertovaara A (2013) The noradrenergic pain regulation system: a potential pocampus and goal-directed spatial decision making. J Neurosci 31:4613– target for pain therapy. Eur J Pharmacol 716:2–7. CrossRef Medline 4621. CrossRef Medline Pertovaara A, Wei H, Ha¨ma¨la¨inen MM (1996) Lidocaine in the rostroven- Wang R, King T, De Felice M, Guo W, Ossipov MH, Porreca F (2013) De- tromedial medulla and the periaqueductal gray attenuates allodynia in scending facilitation maintains long-term spontaneous neuropathic pain. neuropathic rats. Neurosci Lett 218:127–130. CrossRef Medline J Pain 14:845–853. CrossRef Medline Sa¨ rkka¨S, Solin A, Nummenmaa A, Vehtari A, Auranen T, Vanni S, Lin FH (2012) Dynamic retrospective filtering of physiological noise in BOLD Wilcox SL, Gustin SM, Macey PM, Peck CC, Murray GM, Henderson LA fMRI: DRIFTER. Neuroimage 60:1517–1527. CrossRef Medline (2015a) Anatomical changes at the level of the primary synapse in neu- Sasa M, Takaori S (1973) Influence of the locus coeruleus on transmission in ropathic pain: evidence from the spinal trigeminal nucleus. J Neurosci the spinal trigeminal nucleus neurons. Brain Res 55:203–208. CrossRef 35:2508–2515. CrossRef Medline Medline Wilcox SL, Gustin SM, Macey PM, Peck CC, Murray GM, Henderson LA Sasa M, Munekiyo K, Ikeda H, Takaori S (1974) Noradrenaline-mediated (2015b) Anatomical changes within the medullary dorsal horn in chronic inhibition by locus coeruleus of spinal trigeminal neurons. Brain Res temporomandibular disorder pain. Neuroimage 117:258–266. CrossRef 80:443–460. CrossRef Medline Medline Sasa M, Fujimoto S, Igarashi S, Munekiyo K, Takaori S (1979) Microionto- Yarnitsky D (2010) Conditioned pain modulation (the diffuse noxious in- phoretic studies on noradrenergic inhibition from locus coeruleus of hibitory control-like effect): its relevance for acute and chronic pain spinal trigeminal nucleus neurons. J Pharmacol Exp Ther 210:311–315. states. Curr Opin Anaesthesiol 23:611–615. CrossRef Medline Medline Youssef AM, Macefield VG, Henderson LA (2016) Pain inhibits pain; hu- Sugiyo S, Takemura M, Dubner R, Ren K (2005) Trigeminal transition man brainstem mechanisms. Neuroimage 124:54–62. CrossRef Medline
135 Received: 10 September 2017 | Revised: 14 December 2017 | Accepted: 5 January 2018 DOI: 10.1002/hbm.23973
RESEARCH ARTICLE
The relationship between thalamic GABA content and resting cortical rhythm in neuropathic pain
Flavia Di Pietro1 | Paul M. Macey2 | Caroline D. Rae3 | Zeynab Alshelh1 | Vaughan G. Macefield3,4 | E. Russell Vickers1 | Luke A. Henderson1
1Department of Anatomy and Histology, Sydney Medical School, University of Abstract Sydney, Sydney, Australia Recurrent thalamocortical connections are integral to the generation of brain rhythms and it is 2UCLA School of Nursing and Brain thought that the inhibitory action of the thalamic reticular nucleus is critical in setting these Research Institute, University of California, rhythms. Our work and others’ has suggested that chronic pain that develops following nerve Los Angeles, California injury, that is, neuropathic pain, results from altered thalamocortical rhythm, although whether this 3Neuroscience Research Australia, Sydney, dysrhythmia is associated with thalamic inhibitory function remains unknown. In this investigation, Australia we used electroencephalography and magnetic resonance spectroscopy to investigate cortical 4College of Medicine, Mohammed Bin Rashid University of Medicine & Health power and thalamic GABAergic concentration in 20 patients with neuropathic pain and 20 Sciences, Dubai, United Arab Emirates pain-free controls. First, we found thalamocortical dysrhythmia in chronic orofacial neuropathic pain; patients displayed greater power than controls over the 4–25 Hz frequency range, most Correspondence marked in the theta and low alpha bands. Furthermore, sensorimotor cortex displayed a strong Luke A. Henderson, Department of Anatomy and Histology, F13, University of positive correlation between cortical power and pain intensity. Interestingly, we found no differ- Sydney, Australia. ence in thalamic GABA concentration between pain subjects and control subjects. However, we Email: [email protected] demonstrated significant linear relationships between thalamic GABA concentration and enhanced
Funding information cortical power in pain subjects but not controls. Whilst the difference in relationship between National Health and Medical Research thalamic GABA concentration and resting brain rhythm between chronic pain and control subjects Council of Australia, Grant/Award Number: does not prove a cause and effect link, it is consistent with a role for thalamic inhibitory neuro- G160279; National Health and Medical transmitter release, possibly from the thalamic reticular nucleus, in altered brain rhythms in Research Council of Australia, Grant/Award Number: 1091415; Human Capital and individuals with chronic neuropathic pain. Mobility, Grant/Award Number: CHRX-CT94-0432; Training and Mobility of KEYWORDS Researchers, Grant/Award Number: chronic pain, electroencephalography, GABA, thalamocortical rhythm ERB-FMRXCT970160
1 | INTRODUCTION proposed than an aberration in this circuitry, that is, thalamocortical dysrhythmia, is a characteristic of several central nervous system disor- There is a body of evidence that the thalamus plays a key role in the ders, and may be critical for the development and maintenance of development or maintenance of some forms of chronic pain. Direct some forms of chronic pain (Jones, 2010; Sarnthein, Stern, Aufenberg, electrical stimulation of the thalamus can evoke pain (Lenz et al., 1993); Rousson, & Jeanmonod, 2006; Walton & Llin as, 2010). lesions in the somatosensory thalamus can lead to persistent pain (Kim, A number of investigations have shown that chronic neuropathic Greenspan, Coghill, Ohara, & Lenz, 2007); and there are abnormal firing pain, that is, chronic pain that arises from damage to the nervous sys- patterns of thalamic neurons in the pain state (Gerke, Duggan, Xu, & tem, is associated with thalamocortical dysrhythmia (Llinas, Ribary, Siddall, 2003; Lenz, Kwan, Dostrovsky, & Tasker, 1989). Furthermore, Jeanmonod, Kronberg, & Mitra, 1999; Sarnthein et al., 2006; Schulman, the thalamus and its recurrent connections with the cortex play an Zonenshayn, Ramirez, Ribary, & Llinas, 2005; Stern, Jeanmonod, & integral role in the generation and sustenance of the brain rhythms Sarnthein, 2006). While these previous studies used both electroence- that underlie brain function (Buzsaki, 2006; Hughes & Crunelli, 2005). phalography and magnetoencephalography techniques to explore rest- Given that our perceptions arise from on-going activity within recur- ing brain rhythms in individuals with neuropathic pain in different body rent thalamocortical circuits, that is, thalamocortical rhythm, it has been locations, they consistently found increases in resting low-frequency
Hum Brain Mapp. 2018;39:1945–1956. wileyonlinelibrary.com/journal/hbm VC 2018 Wiley Periodicals, Inc. | 1945 136 1946 | DI PIETRO ET AL. cortical power in individuals with neuropathic pain. Similarly, we used and EEG safety considerations (history of head injury or seizures) were resting-state functional magnetic resonance imaging to demonstrate applied and potential participants were also excluded if they suffered significantly increased infra-slow oscillation power (0.03–0.06 Hz) from psychiatric or neurological disorders. within the ascending pain pathway, including the thalamus, in individu- As well as giving information on their relevant clinical history, each als with chronic orofacial neuropathic pain (Alshelh et al., 2016). We chronic pain subject kept a pain diary during the seven days prior to or demonstrated that increased infra-slow oscillations were temporally following the EEG/MRI session. They recorded the intensity of their coupled across specific regions in the ascending pain pathway, includ- on-going pain three times a day using a 10 cm horizontal visual ana- ing the ventroposterior medial thalamus, the thalamic reticular nucleus logue scale (VAS), with 0 indicating “no pain” and 10 indicating “the (TRN), and the orofacial representation in the primary somatosensory most intense imaginable pain.” These pain intensity scores were then cortex. averaged over the 7-day period to create a mean pain intensity score. We also previously showed that neuropathic pain is associated Each chronic pain subject also outlined the location of their on-going with significantly reduced on-going activity in the region of the TRN pain on a standard drawing of the head, listed their current medication (Gustin et al., 2014; Henderson et al., 2013). The TRN surrounds the use and completed the McGill Pain Questionnaire (Melzack, 1975), rostral and lateral surface of the dorsal thalamus, contains exclusively which directs respondents to select sensory, emotional, and cognitive GABAergic neurons and, via extensive inhibitory outputs, modulates all qualitative descriptors of their pain. Informed written consent was incoming sensory information on its way to the cerebral cortex (Buz- obtained for all procedures according to the Declaration of Helsinki saki, 2006; Guillery, Feig, & Lozsadi, 1998; Krause, Hoffmann, & Hajos, and the study was approved by our local Institutional Human Research 2003; McAlonan, Cavanaugh, & Wurtz, 2008; Pinault, 2004; Zikopou- Ethics Committees. A subset of the neuroimaging data (not spectros- los & Barbas, 2006). The TRN therefore plays a critical role in control- copy or EEG data) from 11 of the 20 chronic pain subjects was used in ling the firing patterns of ventroposterior thalamic neurons and is a previous investigation (Alshelh et al., 2016). thought to play a critical role in controlling thalamocortical rhythm (Fuentealba & Steriade, 2005). Indeed, preclinical studies have shown 2.2 | Electroencephalography (EEG) that inhibition of the TRN increases power in the same low-frequency range as that seen in individuals with neuropathic pain (Marini, Giglio, The EEG assessment was carried out at approximately the same time Macchi, & Mancia, 1995; Marini, Ceccarelli, & Mancia, 2002). This is of day, in the morning, for all participants, to control for natural diurnal consistent with our previous finding of significantly reduced thalamic changes in brain rhythms (Sarnthein et al., 2006). A 32-channel EEG (TRN) GABAergic content in individuals with neuropathic pain (Gustin cap (NEURO PRAX, neuroCare Group, Germany) containing premeas- – et al., 2014; Henderson et al., 2013) and raises the prospect that fol- ured electrode sites according to the 10 20 system was fitted to each lowing nerve injury, reduced GABAergic output from the TRN results participant. Chest and eye electrodes were also fitted to monitor car- in thalamocortical dysrhythmia. diac activity, and eye/facial muscle contractions, respectively. EEG data In this study, we used electroencephalography and magnetic reso- were acquired in a quiet, dimly-lit room for a continuous 5-min period nance spectroscopy to determine the relationship between thalamo- with the subject relaxed and with their eyes closed. EEG recordings cortical rhythm and thalamic GABA concentration in individuals with were sampled at a rate of 500 Hz and a band filter of 0.3–70 Hz with a chronic orofacial neuropathic pain compared to healthy pain-free notch filter at 50 Hz was applied. controls. We hypothesise that individuals with chronic orofacial neuro- pathic pain would (a) display altered thalamocortical rhythm character- 2.3 | Magnetic resonance spectroscopy (MRS) ized by increased low frequency power, (b) display reduced thalamic MRS measurements were acquired immediately prior to the EEG and on GABA content, and (c) show a magnitude of thalamocortical dysrhyth- the same morning. All subjects lay supine on the bed of a 3 T MRI scan- mia correlated with a reduction in thalamic GABA. ner (Philips Achieva TX) with their head immobilized in a close-fitting 32-channel head coil. With each subject relaxed and at rest, a high | 2 MATERIALS AND METHODS resolution T1-weighted anatomical scan was acquired (repetition time 5 5600 ms; echo time 5 2.5 ms; flip angle 5 88; voxel size 5 2.1 | Participants 0.87 mm3). Using this T1-weighted anatomical scan as a guide, a Twenty subjects with chronic orofacial neuropathic pain (7 males; 20 3 20 3 20 mm voxel was placed on the thalamus contralateral to mean 6 SEM age 50.1 6 4.4 years), and 20 healthy pain-free control the side of the on-going pain in the chronic pain subjects and on the subjects (7 males, mean age 42.2 6 2.9 years) were recruited. The two right thalamus of the control subjects. GABA-edited MEGA-PRESS groups were gender-matched and there was no significant difference in spectroscopy was then performed on this voxel (repetition age between the groups (t test; p > .05). Subjects with chronic (>3 time 5 2000 ms; echo time 5 68 ms; 1024 data points) (Mullins et al., months duration) orofacial neuropathic pain were referred by an oral 2014). One hundred averages were acquired with the MEGA-PRESS maxillofacial surgeon (author E.R.V.) after a diagnosis of post-traumatic editing pulse centered at 1.9 parts per million (ppm) (“on” spectra) and trigeminal neuropathy (Nurmikko & Eldridge, 2001). Standard MRI 100 averages were acquired with the pulse centered at 7.46 ppm (“off” safety contraindications (cardiac pacemaker, pregnancy, metal implants) spectra). Following this, a single-voxel short-echo PRESS sequence was
137 DI PIETRO ET AL. | 1947 performed in the same voxel location in order to measure concentration the GABA peak as a Gaussian singlet. GABA concentration was of other metabolites, including creatine for the relative quantification of expressed relative to creatine concentration (GABA:Cr) as per recent thalamic GABA (repetition time 5 2000 ms; echo time 5 28 ms; 1024 technical guidelines (Mullins et al., 2014). After the same process for data points). removal of the dominant water peak as for the GABA concentration, creatine concentration was quantified using QUEST, a time-domain 2.4 | Data analysis algorithm that fits a weighted metabolite basis set to the spectra acquired (Ratiney et al., 2005). Creatine concentration was measured at 2.4.1 | Electroencephalography 3.02 ppm. Significant differences between chronic pain and control Using SPM12 software (Wellcome Trust Centre for Neuroimaging, Uni- groups were then determined (p < .05, one-tailed two-sample t test). versity College London), EEG data were referenced to the average ref- For the chronic pain group, significant linear relationships between tha- erence montage; filtered 0.5–30 Hz; downsampled to 200 Hz; and lamic GABA:Cr and pain intensity and duration were determined manually inspected for artefacts in 2-s epochs (Boord et al., 2008; Jen- (p < .05). sen et al., 2013; Stern et al., 2006). Channels or trials of data were removed if the underlying EEG signal contained artefacts such that the 2.4.3 | Electroencephalography versus magnetic resonance underlying signal was not clear. Noise was found across multiple data- spectroscopy sets at frequencies <4 Hz, and for this reason we discarded these data To explore the relationship between thalamic GABA levels and EEG for all subjects. The remaining EEG signal between 4 and 25 Hz was power, we performed regression analyses of linear relationships then Fourier time–frequency transformed and power values at each between total theta, alpha and beta power and an individual’s thalamic frequency were determined. These power values were logarithmically GABA:Cr levels (p < .05). In addition, to explore regional relationships, transformed (log 10) to achieve normality. Power values at each fre- we performed regression analyses of linear relationships between quency were then plotted for the control and chronic pain groups and power at each of the 27 EEG electrodes and an individual’s thalamic significant differences in power between groups over the entire fre- GABA:Cr levels. The rho values of these relationships for each elec- quency range, 4–25 Hz, and between groups within the theta (4–8Hz), trode were then plotted onto a surface representation of the head for alpha (9–12 Hz), and beta (13–25 Hz) ranges were determined (p < .05, the control and chronic pain groups and any significant relationships one-way ANOVA, one-tailed). determined (p < .05). To explore the spatial distribution of these power differences, we compared the logged power of the chronic pain subjects with that of 3 | RESULTS controls at each frequency across each EEG channel (two-sample t tests). We plotted the t values at each frequency for each of the 27 3.1 | Participants EEG channels. We also calculated t values for power differences The pain subjects’ mean (6 SEM) pain intensity over a week was 3.7 6 between controls and chronic pain subjects in the theta, alpha, and beta 0.4 and their median duration of pain was 66 months (range 4–302 ranges for each EEG electrode, plotted these values onto a surface rep- months). Chronic pain subjects reported on-going pain encompassing resentation of the head, and determined electrodes with significant the maxillary and mandibular distributions of the trigeminal nerve (Fig- power differences (two-sample t tests). In addition to power differences ure 1a). Fifteen of the chronic pain subjects reported left-sided pain, between control and chronic pain groups, we explored linear relation- and five reported right-sided pain. (Two subjects reported bilateral ships between power at each EEG electrode and an individual’son- pain; however, the pain was only mild on one side therefore the pre- going pain intensity (Pearson’s correlation coefficient) and pain duration dominantly painful side was used here.) Chronic pain subjects most (Spearman’s rank correlation coefficient). The rho values of these rela- often described their on-going pain as “shooting,”“throbbing,” or “nag- tionships for each electrode were then plotted onto a surface represen- ging.” Eighteen of the 20 participants in the patient group were taking tation of the head and any significant relationships determined (p < .05). some medication for pain. See Table 1 for other patient characteristics ’ 2.4.2 | Magnetic resonance spectroscopy and Figure 1a for the distribution of patients pain. The acquired spectra were analysed with the Java-based magnetic res- 3.2 | Cortical EEG power onance users’ interface version 3 (jMRUI 3.0, MRUI Consortium). First, the dominant water peak was removed using the Hankel Lanczos Sin- Power spectra over the entire frequency range (4–25 Hz) for both groups gular Values Decomposition Filter tool, then the “on” and “off” spectral are depicted in Figure 1b. Chronic pain subjects displayed significantly subsets were summed. The 68 ms “on” and “off” subspectra were then greater power than controls over the entire frequency range 4–25 Hz 2 subtracted, resulting in GABA-edited difference spectra to measure (mean 6 SEM log10[mv /Hz]: controls 22.43 6 0.10, chronic pain GABA at the resonance of 3.01 ppm. GABA concentration was quanti- 22.20 6 0.085, p 5 .04), in the theta (controls 22.03 6 0.12, chronic pain fied using AMARES, an Advanced Method for Accurate, Robust and 21.73 6 0.11, p 5 .04) and alpha (controls 22.20 6 0.12, chronic pain Efficient Spectral fitting (Vanhamme, van den Boogaart, & Van Huffel, 21.89 6 0.10, p 5 .03) ranges, but the two groups were not significantly 1997). Peak fitting for GABA was performed after manually defining different in the beta range (controls 22.66 6 0.09, chronic pain 22.48 6 the center frequency and line width of the GABA peak, and modelling 0.07, p 5 .07). In addition, chronic pain subjects exhibited greater power
138 1948 | DI PIETRO ET AL.
FIGURE 1 Pain distribution and EEG power: (a) Individual pain distribution patterns in 20 chronic orofacial pain subjects. (b) Plots of mean (6SEM) resting EEG power between 4 and 25 Hz in controls (black) and chronic orofacial pain (grey) subjects, over all 27 cortical EEG electrodes. Note that overall power is greater in chronic pain subjects than in controls over most frequencies
TABLE 1 Pain subject characteristics. Pro re nata (as needed)
Primary site of pain Pain (if bilateral worse Pain diary duration Subject Age Sex side is in bold) (ave/10) (months) Medications in last 24 h
01 67 F Right side, upper jaw 4.2 240 Palmitoylethanolamide, Lyrica, Efexor, Oroxine, Crestor, Ergotamine, Diphenhydramine
02 27 F Left side, upper jaw 6.9 29 Nurofen PRN
03 65 M Bilateral (left side lower 0.4 26 Endep jaw worse than right side upper jaw)
04 73 M Right side lower jaw 2.9 264 Monopril, Aspirin PRN, Norvasc, Crestor, Lithicarb
05 37 F Left lower jaw 3.4 96 Gabapentin, Endep
06 43 F Bilateral (right upper and 2.8 24 Mobic lower jaw worse than left-upper only)
07 66 F Left upper jaw 3.7 22 Lyrica
08 59 F Left lower jaw 7.1 115 Aspirin RPN
09 25 M Left side upper jaw 3.6 8 Palmitoylethanolamide, Panadeine PRN
10 71 F Left side upper jaw 5.1 8.5 Mobic, Panadol PRN, Gabapentin, Nortriptyline
11 36 M Left side upper jaw - 84 Oxycontin, Gabapentin, Panadol PRN
12 19 M Right side upper jaw 5 84 None
13 59 F Left side upper jaw 4.4 120 None
14 27 F Left side upper and lower jaw 2.7 8 Palmitoylethanolamide
15 57 F Left side upper jaw 2.4 6 Palmitoylethanolamide, Coversyl
16 69 F Left upper and lower jaw 4.8 108 Palmitoylethanolamide
17 64 M Right side upper and lower jaw 1.2 66 Gabapentin, Endep, Zyloprim
18 21 M Left side lower jaw 1.9 4 Palmitoylethanolamide
19 33 F Left side upper jaw 2.3 88 Palmitoylethanolamide, Dienogest
20 76 F Left side upper jaw 4.8 302 Palmitoylethanolamide, Panadol PRN, Prestiq, Somac, Alprazolam
139 DI PIETRO ET AL. | 1949
FIGURE 2 Spatial distribution of power differences: (a) Plots of t values representing power differences between controls and chronic pain subjects at each of the 27 EEG channels, across the 4–25 Hz frequency range. Note that power is greater in chronic pain subjects than in controls. To the right is an outline showing the approximate location of each of the 27 EEG electrodes. (b) Plotted on a surface representation of the head, t values representing power differences between controls and chronic pain subjects separately within the theta, alpha, and beta bands at the dominant frequency (controls 21.71 6 0.14, chronic pain 21.33 6 15 Hz and were strong between 8 and 10 Hz. These power increases 0.13, p 5 .03). Although the mean dominant frequency was lower in in chronic pain subjects occurred in areas of the medial prefrontal cor- patients than controls, this result was not significant (patients 6.75 6 0.58 tex (EEG electrodes FP1 and FP2), and the dorsolateral prefrontal cor- Hz, controls 7.80 6 0.94 Hz, p 5 .18). In the chronic pain group there tex (F8), primary sensorimotor and temporal cortices (FC5 and T3) and were no significant linear relationships between pain intensity and theta parietal association cortex (P3, PZ, P4, and CP6). Plots of power differ- power (r 5 .21, p 5 .40), alpha power (r 5 .25, p 5 .31), or beta power ences separately within the theta, alpha and beta ranges mapped onto (r 5 .14, p 5 .56), or between pain duration and theta power (Spearman’s a surface representation of the head again showed that although rho 52.05, p 5 .85), alpha power (rho 5 .33, p 5 .15), or beta power power differences occurred primarily in the alpha range, differences (rho 5 .19, p 5 .42). Although only two of the chronic pain subjects were also occurred in both the theta and beta ranges (Figure 2b). Within the not taking any medications, they both had greater power than control alpha range, significant power increases in chronic pain subjects subjects in the theta (20.60, 20.86), alpha (21.18, 21.41), and beta occurred in the frontal (FP2, F4, and F8), primary sensorimotor (FC5), (21.91, 22.02) ranges. temporal (T3), and parietal (CP1, P3, PZ, and P4) cortices. A similar pat- tern of differences between groups, although less widespread, also | 3.2.1 Spatial distribution of EEG power differences occurred in the theta and beta ranges (theta: F7, F8, FC5, T3, T5; beta: Consistent with the evidence of an overall increase in power, an explo- F8, FC5, T3, CP2, T5, P3, PZ, O2). Importantly, in theta, alpha and beta ration of the difference in power between groups at each EEG elec- ranges, at no EEG electrode was power significantly greater in controls trode at each frequency revealed significant power increases in chronic compared to chronic pain subjects. pain subjects compared with controls over several brain regions (Figure While there were significant power differences between controls 2a). Power increases were particularly evident at frequencies below and chronic pain subjects over multiple areas of the cerebral cortex, in
140 1950 | DI PIETRO ET AL.
FIGURE 3 The relationship between EEG power and pain: (a) Plots of the relationship between pain intensity and EEG power in the theta, alpha and beta bands at each of the 27 channels, on a surface representation of the head. (b) Plots of the relationship between pain duration and EEG power in the theta, alpha, and beta bands at each of the 27 channels, on a surface representation of the head. (c) Plots of the correlation between pain intensity and beta power in electrodes FC6 and CP6, and the correlation between pain duration and alpha power in electrode CP5 only a few regions was power significantly linearly correlated to either pain duration in the frontal (FP2: r 5 .52, p 5 .02) and parietal cortices pain intensity (Figure 3a) or duration (Figure 3b) in the chronic pain sub- (CP5: r 5 .50, p 5 .02, P4: r 5 .66, p 5 .002) (Figure 3c). jects. In the theta range no region displayed a significant relationship 5 between power and pain intensity. In the alpha range, only at T4 (r .60, 3.3 | Thalamic GABA concentration p 5 .007) was there a significant positive correlation. Within the beta range, significant positive relationships occurred between power and pain GABA-edited MEGA-PRESS spectroscopy was not acquired in two of intensity in the sensorimotor (FC6: r 5 .53, p 5 .02), parietal (CP6: r 5 .57, the chronic pain subjects (patients declined MRI), and we excluded a p 5 .01) (Figure 3c) and occipital (O1: r 5 .57, p 5 .01) cortices. With further two chronic pain and two control subjects due to either outly- respect to pain duration, there were no significant relationships between ing GABA:Cr values or inadequate signal-to-noise for the spectra analy- theta or beta power at any EEG electrode and pain duration. However, sis. This resulted in data from 15 chronic pain subjects and 18 controls. there were significant positive relationships between alpha power and In direct contrast to our previous work (Henderson et al., 2013), we
141 DI PIETRO ET AL. | 1951
FIGURE 4 Thalamic GABA concentration: (a) Axial MR-image representing the voxel placement in the thalamus contralateral to the side of on- going chronic pain in the pain subjects, together with a diagram depicting the chemical shift axis. Note GABA concentration was measured at 3.01 parts per million (ppm). (b) Distribution of GABA:Cr ratios for chronic pain (grey circles) and control (black squares) subjects, demonstrating the lack of difference between groups. Plots of the correlations between pain intensity and pain duration with thalamic GABA concentration, demonstrating a lack of correlation with these measures. (c) Plots demonstrating the correlation between EEG power and thalamic GABA concen- tration in both chronic pain and control subjects in each of the theta, alpha, and beta ranges. Note the moderate to strong correlations in all bands in the pain subjects, while no such relationship exists in the control group. Glx, glutamate; NAA, N-Acetylaspartic acid found no significant thalamic GABA difference between chronic pain thalamic GABA and cortical power was remarkably different between the and control subjects (mean 6 SEM GABA:Cr: chronic pain: 0.18 6 0.01, two groups (Figure 4c). In controls, there was no significant relationship controls: 0.17 6 0.01, p 5 .27) (Figure 4a,b). Additionally, there was no between thalamic GABA and overall theta power (r 5 .1, p 5 .70), alpha significant relationship between thalamic GABA and on-going pain inten- power (r 52.01, p 5 .98), or beta power (r 52.05, p 5 .84). In contrast, sity (r 5 .14, p 5 .62) or pain duration (Spearman’srho5 .05, p 5 .85). chronic pain subjects displayed significant positive relationships between thalamic GABA and overall theta power (r 5 .61, p 5 .02), alpha power 3.3.1 | Thalamic GABA concentration and its association (r 5 .52, p 5 .04), and beta power (r 5 .68, p 5 .006). Furthermore, the with power relationships between thalamic GABA and overall theta and beta power Although there was no difference in thalamic GABA content between were significantly different between control and chronic pain groups control and chronic pain subjects, we found that the relationship between (all p < .05, two-tailed, Fisher r-to-z transformation).
142 1952 | DI PIETRO ET AL.
FIGURE 5 Spatial distribution of thalamic GABA versus power: (a) Plots of the relationship between thalamic GABA and EEG power in control subjects, and (b) chronic pain subjects, mapped onto a surface representation of the head, in theta, alpha, and beta bands. Note the lack of relationship in control subjects in all bands, and the relationship between GABA and power in pain subjects in all three frequency bands. (c) Plots of the correlation between thalamic GABA and theta power in EEG electrodes CP1, FC6, and FP1; note the striking difference in this relationship between groups
3.3.2 | Spatial distribution of relationships between between power and thalamic GABA occurred in the frontal (FP1 thalamic GABA and power r 5 .66, FP2 r 5 .59, F3, r 5 .61, FZ r 5 .61, F4 r 5 .57, F8 r 5 .62, FC1 5 5 5 5 In addition to the overall differences between groups in the relation- r .57), sensorimotor (FC6 r .61, CZ r .55), temporal (T3 r .67), ship between thalamic GABA and EEG power, there were striking dif- parietal (CP1 r 5 .78), and occipital cortices (O1 r 5 .72)(all p < 0.05; ferences in the spatial distributions of the relationships. In control Figure 5c). More restricted significant correlations occurred between subjects, at none of the EEG electrodes was there a significant relation- thalamic GABA and alpha power (F8 r 5 .57, T3 r 5 .60, CP5 r 5 .55, ship between thalamic GABA and theta, alpha or beta power (Figure CP1 r 5 .73, O1 r 5 .54) and thalamic GABA and beta power (F7 5a). In contrast, in chronic pain subjects, multiple electrodes showed r 5 .62, F3 r 5 .57, F8 r 5 .58, T3 r 5 .66, CZ r 5 .66, CP5, r 5 .78, positive correlations between thalamic GABA and theta, alpha, or beta CP1 r 5 .86, O1 r 5 .76). At no EEG electrode was there a significant power (Figure 5b). Within the theta range, significant correlations negative relationship between thalamic GABA and power.
143 DI PIETRO ET AL. | 1953
4 | DISCUSSION Jones, 2010). Given this, we were surprised we found no difference in thalamic GABA concentration between pain subjects and control sub- Consistent with our hypothesis, we found that chronic orofacial neuro- jects. Indeed, one would predict that neuropathic pain would be associ- pathic pain was associated with significantly increased cortical power, ated with increased TRN GABAergic output, which is contrary to the particularly in theta and alpha frequency bands, compared to healthy results of our previous studies in which we found reduced thalamic controls. Furthermore, the region encompassing the sensorimotor GABA content and reduced TRN blood flow in individuals with neuro- cortex displayed a strong positive correlation between power and pathic pain (Gustin et al., 2014; Henderson et al., 2013). Although we on-going pain intensity. Although we found no significant difference in found remarkable differences in the relationships between thalamic thalamic GABA concentration between the two groups, which was in GABA and cortical power between subject groups, and a relationship in contrast to our previous findings and our hypothesis, there was a strik- pain subjects suggestive of increased TRN GABAergic output, we can- ing difference in the relationship between thalamic GABA and cortical not draw the conclusion that the TRN is solely responsible for the power between pain subjects and controls. In the chronic pain group, altered cortical rhythm. Indeed, we found no evidence of a difference significant positive linear relationships occurred between thalamic in GABA content between groups, which raises the possibility of other GABA and power, particularly in regions of the sensorimotor, frontal, mechanisms, for instance intracortical and corticothalamic projections, and parietal association cortices. That is, the greater the thalamic which include influences on both thalamic relay and possibly TRN neu- GABA concentration, the greater the resting cortical power. In striking rons, and possibly affecting the hyperpolarization of thalamocortical contrast, we detected no significant relationships between thalamic relay cells. GABA and theta, alpha, or beta power in any region in the control Why thalamic GABA was reduced in our previous study but not in group. the present one is not known, though it is unlikely due to technical dif- Consistent with much of the literature, the thalamocortical dys- ferences since similar collection and analysis techniques were used. It is rhythmia that occurred here in individuals with orofacial neuropathic possible that differences in thalamic GABA results from differences in pain was characterized by a significant enhancement of activity in the patient characteristics and while pain duration and intensity were simi- theta and alpha ranges and a significant increase in power at the domi- lar across studies, medication regimens were not (Henderson et al., nant spectral power peak. The enhanced power in pain subjects relative 2013). In our previous investigation, 10 of the 23 pain subjects were to controls was most marked between 8 and 10 Hz. These changes not taking any medication compared with only 2 of 20 in this investiga- are consistent with many previous studies demonstrating thalamocorti- tion. Furthermore, in our previous study, those taking medication were cal dysrhythmia in chronic neuropathic pain (Sarnthein et al., 2006; on relatively standard treatment regimens such as gabapentin and ami- Schulman et al., 2005; Stern et al., 2006; Walton & Llin as, 2010). Some triptyline. In direct contrast, in this study, in addition to these standard studies demonstrated reduced or absent power in the alpha band (Jen- medications, 8 of the 20 pain subjects were taking palmitoylethanola- sen et al., 2013; Llin as et al., 1999) and enhanced power in the beta mide (PEA), which has been reported to target the peroxisome band in pain subjects (Stern et al., 2006), however these samples dif- proliferator-activated receptor alpha as well as cannabinoid-like G- fered from ours and none comprised exclusively orofacial neuropathic coupled receptors (Godlewski, Offertaler, Wagner, & Kunos, 2009; Lo pain. That we found a distinct increase in power at 8Hzissimilarto Verme et al., 2005). While we do not have a sample large enough to that found in neuropathic pain in the past (Sarnthein et al., 2006; Schul- explore the effects of PEA on thalamic GABA and brain activity, there is man et al., 2005). Interestingly though, in their neuropathic pain investi- some evidence to suggest PEA may reduce neuropathic pain and thus gation, Schulman et al. (2005) termed this 7–9 Hz range the “high theta this could have affected the results of this study (Keppel Hesselink & range.” There are indeed differences across the literature with regard Kopsky, 2015). The effects of medications on thalamic GABA and rest- to the grouping of frequencies, and different definitions of frequency ing brain activity require further detailed and directed investigations. bands would go some of the way to explaining some of the apparent, Although we found no reduction in thalamic GABA in this study, slight discrepancies across the EEG literature. our finding of significant positive relationships between thalamic GABA The thalamus plays a key role in the generation and maintenance and cortical power in individuals with neuropathic pain, but not con- of brain rhythms (Buzsaki, 2006; Hughes & Crunelli, 2005). It is thought trols, is consistent with the idea that increased GABAergic inhibition of that increases in low-frequency cortical power are due to a shift in tha- specific thalamic nuclei such as Vc by the TRN results in increased tha- lamic neuron activity from a state dominated by tonic firing to one in lamocortical power. Furthermore, this is consistent with our previous which there is an increase in low-threshold spike burst firing (Hughes & study in which we reported significant positive relationships between Crunelli, 2005). This theory is supported by the finding that neuro- thalamic GABA and resting Vc-S1 connectivity strength in neuropathic pathic pain is associated with an increase in burst firing in the ventro- pain subjects but not in controls (Henderson et al., 2013). The lack of caudal (Vc) thalamus and an associated increase in theta power (Lenz relationship between Vc-S1 connectivity strength and thalamic GABA et al., 1989). Low-threshold calcium bursts occur when thalamocortical in controls is congruous with the lack of significant relationships relay cells are in a state of hyperpolarisation; there is evidence that the between thalamic GABA and power at any of the 27 electrode loca- TRN is capable of entraining this ‘burst firing mode’ (Golshani, Liu, & tions in controls in the current study. However, this was surprising Jones, 2001), and it is argued that the TRN serves to maintain the low given the proposed role of the TRN and its GABAergic output in frequency thalamocortical oscillations (4–10 Hz) (Buzsaki, 1991; setting cortical rhythms. This, together with the lack of difference in
144 1954 | DI PIETRO ET AL. thalamic GABA content between groups found here, leads us to ques- responsible for the findings we report on; however, we think it less tion somewhat the importance of the role of the TRN in setting cortical likely to be these sources than the TRN. As well as our previous finding rhythms particularly in individuals with chronic neuropathic pain. of decreased cerebral blood flow in the TRN in neuropathic pain (Hen- While there are variations in the spatial distribution, or topography, derson et al., 2013), extrathalamic inhibitory nuclei do not exhibit of the enhanced EEG/MEG power in pain groups across the literature, recurrent circuits with the thalamus (i.e., they have no thalamic inputs), most studies report enhanced low frequency power in somatosensory nor do they directly innervate the primary somatosensory thalamus regions in patients relative to controls (Stern et al., 2006; Walton, (Bartho et al., 2002; Bokor et al., 2005; Halassa & Acsady, 2016). Dubois, & Llinas, 2010). Our findings demonstrate the presence of Several limitations influence the interpretation of the results. First, more widespread activation in the patient group. Indeed, low- while it is common for EEG and MEG studies to discount the spectral frequency brain rhythms (<10 Hz) show long-range cortical coherence data at and below 2Hz(Llin as et al., 1999; Sarnthein et al., 2006), we and can span the entire cortex, whereas higher frequency oscillations omitted spectral data below 4 Hz because of high noise levels, and > ( 30 Hz) tend to be associated with more restricted cortical activity thus we may have excluded some interesting differences within the (Contreras & Llinas, 2001). Furthermore, electrical stimulation immedi- delta range. Furthermore, we did not collect MRS and EEG data con- ately below the cortical surface at low frequencies activates a signifi- currently, due to practical constraints and inherent technical difficulties. cantly greater cortical area than that produced by higher frequency Whilst it is possible that this may have altered the relationships stimulation, a difference thought to result from lateral inhibition (Llinas, between thalamic GABA and cortical power, we collected MRS and Urbano, Leznik, Ramírez, & van Marle, 2005). This is consistent with EEG data on the same day, directly after one another and at a similar our finding of increased low frequency power over a relatively large time of day for all participants. Last, because the voxel size in MRS is region of the cerebral cortex in individuals with neuropathic pain. Inter- large, it is possible we may have collected chemical spectra from other estingly, however, only a restricted part of this power increase was cor- thalamic nuclei as well as the TRN. However, we are confident we related to on-going pain intensity, namely the sensorimotor and acquired spectra in the TRN in all participants, and the GABAergic con- parietal association cortices. While we did not extend our analyses tent of the TRN is indeed well documented. here to investigation of rhythms in the gamma band (>30 Hz), interest- The results of this study raise important questions about mecha- ing questions remain as to whether the patches of lower and higher nisms of altered resting brain rhythm in chronic neuropathic pain. Given frequency cortical activity that we report on are evidence of the “edge its GABAergic output, it follows that the TRN is an integral part of thala- effect,” whereby GABAergic inhibition of lateral cortical areas is dimin- mocortical circuitry; however, we found no direct evidence of altered ished in disease states (Llin as et al., 2005). thalamic GABA concentration in the pain state here, and the relationship We have previously shown that orofacial neuropathic pain is asso- between GABA concentration and cortical rhythms is not fully under- ciated with increased infra-slow oscillations in the ascending pain path- stood. While the thalamus has long been suggested to play a key role in way, including in the spinal trigeminal nucleus, Vc, TRN, and S1, and the maintenance of pain, this too has not been fully elucidated. That we we have provided anatomical evidence that these changes may result found evidence of an association between GABA concentration in the from chronic astrocyte activation and changes in oscillatory gliotrans- TRN and thalamocortical rhythm in pain subjects, but no such associa- mission (Alshelh et al., 2016). It is possible that following nerve injury, tion in controls, is intriguing and offers a new avenue for investigation altered Vc thalamus infra-slow oscillatory activity, in addition to altered into the mechanisms possibly underpinning chronic neuropathic pain. GABAergic influences of TRN outputs to the Vc thalamus, results in increased low frequency (theta and alpha) thalamocortical rhythm in Vc-S1 recurrent loops. Increased low frequency oscillations in this rela- ACKNOWLEDGMENTS tively restricted thalamocortical network could then feasibly evoke The authors would like to thank many generous subjects that partici- increased oscillatory changes over a relatively wide area of the cerebral pated in this investigation. They would also like to thank Segar Sup- cortex, which in turn feeds back to the thalamus and so on, driving a piah for his assistance with all the MRI scanning. The authors loop of altered activity. All that said however, in this study, we were declare that they have no conflicts of interest. not able to demonstrate the altered GABAergic influence of the TRN— and so, while feasible, this prospect is somewhat speculative. It is also important to consider the alternative sources of thalamic GABAergic ORCID inhibition. Extrathalamic inhibitory structures, such as the zona incerta Flavia Di Pietro http://orcid.org/0000-0002-9642-0805 (Bartho, Freund, & Acsady, 2002), the anterior pretectal nucleus (Bokor Paul M. Macey http://orcid.org/0000-0003-4093-7458 et al., 2005), the basal ganglia (Bodor, Giber, Rovo, Ulbert, & Acsady, Luke A. Henderson http://orcid.org/0000-0002-1026-0151 2008), and the pontine reticular formation (Giber et al., 2015) also tar- get the thalamus. Furthermore the thalamus contains a proportion of local GABAergic interneurons (Jones & Hendry, 1989; Smith, Seguela, REFERENCES & Parent, 1987), although the TRN contains exclusively GABAergic Alshelh, Z., Di Pietro, F., Youssef, A. M., Reeves, J. M., Macey, P. M., cells (Houser, Vaughn, Barber, & Roberts, 1980; Smith et al., 1987). It is Vickers, E. R., ... Henderson, L. A. (2016). Chronic neuropathic pain: possible that mechanisms other than the circuit with the TRN could be It’s about the rhythm. The Journal of Neuroscience, 36, 1008–1018.
145 DI PIETRO ET AL. | 1955
Bartho, P., Freund, T. F., & Acsady, L. (2002). Selective GABAergic Jones, E. G., & Hendry, S. H. (1989). Differential calcium binding protein innervation of thalamic nuclei from zona incerta. European Journal of immunoreactivity distinguishes classes of relay neurons in monkey Neuroscience, 16, 999–1014. thalamic nuclei. European Journal of Neuroscience, 1, 222–246. Bodor, A. L., Giber, K., Rovo, Z., Ulbert, I., & Acsady, L. (2008). Structural Keppel Hesselink, J. M., & Kopsky, D. J. (2015). Palmitoylethanolamide, a correlates of efficient GABAergic transmission in the basal ganglia- neutraceutical, in nerve compression syndromes: Efficacy and safety thalamus pathway. Journal of Neuroscience, 28, 3090–3102. in sciatic pain and carpal tunnel syndrome. Journal of Pain Research, – Bokor, H., Frere, S. G., Eyre, M. D., Slezia, A., Ulbert, I., Luthi, A., & 8, 729 734. Acsady, L. (2005). Selective GABAergic control of higher-order tha- Kim, J. H., Greenspan, J. D., Coghill, R. C., Ohara, S., & Lenz, F. A. (2007). lamic relays. Neuron, 45, 929–940. Lesions limited to the human thalamic principal somatosensory Boord, P., Siddall, P. J., Tran, Y., Herbert, D., Middleton, J., & Craig, A. nucleus (ventral caudal) are associated with loss of cold sensations and central pain. The Journal of Neuroscience, 27, 4995–5004. (2008). Electroencephalographic slowing and reduced reactivity in neu- ropathic pain following spinal cord injury. Spinal Cord, 46, 118–123. Krause, M., Hoffmann, W. E., & Hajos, M. (2003). Auditory sensory gat- ing in hippocampus and reticular thalamic neurons in anesthetized Buzsaki, G. (1991). The thalamic clock: Emergent network properties. rats. Biological Psychiatry, 53, 244–253. Neuroscience, 41, 351–364. Lenz, F. A., Kwan, H. C., Dostrovsky, J. O., & Tasker, R. R. (1989). Character- Buzsaki, G. (2006). Rhythms of the brain. New York: Oxford University istics of the bursting pattern of action potentials that occurs in the Press. thalamus of patients with central pain. Brain Research, 496, 357–360. Contreras, D., & Llinas, R. (2001). Voltage-sensitive dye imaging of neo- Lenz, F. A., Seike, M., Richardson, R. T., Lin, Y. C., Baker, F. H., Khoja, I., cortical spatiotemporal dynamics to afferent activation frequency. ... Gracely, R. H. (1993). Thermal and pain sensations evoked by Journal of Neuroscience, 21, 9403–9413. microstimulation in the area of human ventrocaudal nucleus. Journal Fuentealba, P., & Steriade, M. (2005). The reticular nucleus revisited: of Neurophysiology, 70, 200–212. Intrinsic and network properties of a thalamic pacemaker. Progress in Llin as,R.,Urbano,F.J.,Leznik,E.,Ramírez,R.R.,&vanMarle,H.J. Neurobiology, 75, 125–141. F. (2005). Rhythmic and dysrhythmic thalamocortical dynamics: Gerke, M. B., Duggan, A. W., Xu, L., & Siddall, P. J. (2003). Thalamic neu- GABA systems and the edge effect. Trends in Neurosciences, 28, ronal activity in rats with mechanical allodynia following contusive 325–333. spinal cord injury. Neuroscience, 117, 715–722. Llin as, R. R., Ribary, U., Jeanmonod, D., Kronberg, E., & Mitra, P. P. Giber, K., Diana, M. A., Plattner, V., Dugue, G. P., Bokor, H., Rousseau, C. (1999). Thalamocortical dysrhythmia: A neurological and neuropsychi- ... V., Acsady, L. (2015). A subcortical inhibitory signal for behavioral atric syndrome characterized by magnetoencephalography. Proceed- – arrest in the thalamus. Nature Neuroscience, 18, 562 568. ings of the National Academy of Sciences, 96, 15222–15227. Godlewski, G., Offertaler, L., Wagner, J. A., & Kunos, G. (2009). Recep- Lo Verme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A., & tors for acylethanolamides-GPR55 and GPR119. Prostaglandins & Piomelli, D. (2005). The nuclear receptor peroxisome proliferator- Other Lipid Mediators, 89, 105–111. activated receptor-alpha mediates the anti-inflammatory actions of Golshani, P., Liu, X. B., & Jones, E. G. (2001). Differences in quantal palmitoylethanolamide. Molecular Pharmacology, 67,15–19. amplitude reflect GluR4- subunit number at corticothalamic synapses Marini, G., Ceccarelli, P., & Mancia, M. (2002). Thalamocortical dysrhyth- on two populations of thalamic neurons. Proceedings of the National mia and the thalamic reticular nucleus in behaving rats. Clinical Neuro- Academy of Sciences of the United States of America, 98, 4172–4177. physiology, 113, 1152–1164. Guillery, R. W., Feig, S. L., & Lozsadi, D. A. (1998). Paying attention to Marini, G., Giglio, R., Macchi, G., & Mancia, M. (1995). Nucleus reticularis the thalamic reticular nucleus. Trends in Neurosciences, 21,28–32. thalami and neocortical paroxysms in the rat. The European Journal of Gustin, S. M., Wrigley, P. J., Youssef, A. M., McIndoe, L., Wilcox, S. L., Neuroscience, 7, 2301–2307. Rae, C. D., ... Henderson, L. A. (2014). Thalamic activity and bio- McAlonan, K., Cavanaugh, J., & Wurtz, R. H. (2008). Guarding the gateway chemical changes in individuals with neuropathic pain after spinal to cortex with attention in visual thalamus. Nature, 456, 391–394. cord injury. Pain, 155, 1027–1036. Melzack, R. (1975). The McGill Pain Questionnaire: Major properties and Halassa, M. M., & Acsady, L. (2016). Thalamic inhibition: Diverse sources, scoring methods. Pain, 1, 277–299. diverse scales. Trends in Neurosciences, 39, 680–693. Mullins, P. G., McGonigle, D. J., O’gorman, R. L., Puts, N. A., Vidyasagar, Henderson, L. A., Peck, C. C., Petersen, E. T., Rae, C. D., Youssef, A. M., R., Evans, C. J., & Edden, R. A. (2014). Current practice in the use of Reeves, J. M., ... Gustin, S. M. (2013). Chronic pain: Lost inhibition? MEGA-PRESS spectroscopy for the detection of GABA. NeuroImage, The Journal of Neuroscience:, 33, 7574–7582. 86,43–52. Houser, C. R., Vaughn, J. E., Barber, R. P., & Roberts, E. (1980). GABA Nurmikko, T. J., & Eldridge, P. R. (2001). Trigeminal neuralgia–pathophys- neurons are the major cell type of the nucleus reticularis thalami. iology, diagnosis and current treatment. British Journal of Anaesthesia, Brain Research, 200, 341–354. 87, 117–132. Hughes, S. W., & Crunelli, V. (2005). Thalamic mechanisms of EEG alpha Pinault, D. (2004). The thalamic reticular nucleus: Structure, function and rhythms and their pathological implications. The Neuroscientist:, 11, concept. Brain Research. Brain Research Reviews, 46,1–31. – 357 372. Ratiney, H., Sdika, M., Coenradie, Y., Cavassila, S., van Ormondt, D., & Jensen, M. P., Sherlin, L. H., Gertz, K. J., Braden, A. L., Kupper, A. E., Gia- Graveron-Demilly, D. (2005). Time-domain semi-parametric estima- nas, A., ... Hakimian, S. (2013). Brain EEG activity correlates of tion based on a metabolite basis set. NMR in Biomedicine, 18,1– chronic pain in persons with spinal cord injury: Clinical implications. 13. – Spinal Cord, 51,55 58. Sarnthein, J., Stern, J., Aufenberg, C., Rousson, V., & Jeanmonod, D. (2006). Jones, E. G. (2010). Thalamocortical dysrhythmia and chronic pain. Pain, Increased EEG power and slowed dominant frequency in patients with 150,4–5. neurogenic pain. Brain:, 129,55–64.
146 1956 | DI PIETRO ET AL.
Schulman, J. J., Zonenshayn, M., Ramirez, R. R., Ribary, U., & Llinas, R. Walton, K. D., & Llin as, R. R. (2010). Central pain as a thalamocortical (2005). Thalamocortical dysrhythmia syndrome: MEG imaging of dysrhythmia: A thalamic efference disconnection? In L. Kruger, A.R. neuropathic pain. Thalamus and Related Systems, 3,33–39. Light (Eds.), Translational pain research: From mouse to man. Boca Smith, Y., Seguela, P., & Parent, A. (1987). Distribution of GABA- Raton, FL: CRC Press. immunoreactive neurons in the thalamus of the squirrel monkey (Sai- Zikopoulos, B., & Barbas, H. (2006). Prefrontal projections to the – miri sciureus). Neuroscience, 22, 579 591. thalamic reticular nucleus form a unique circuit for attentional Stern, J., Jeanmonod, D., & Sarnthein, J. (2006). Persistent EEG overacti- mechanisms. Journal of Neuroscience, 26, 7348–7361. vation in the cortical pain matrix of neurogenic pain patients. Neuro- Image, 31, 721–731.
Vanhamme, L., van den Boogaart, A., & Van Huffel, S. (1997). Improved How to cite this article: Di Pietro F, Macey PM, Rae CD, et al. method for accurate and efficient quantification of MRS data with use The relationship between thalamic GABA content and resting of prior knowledge. Journal of Magnetic Resonance, 129,35–43. cortical rhythm in neuropathic pain. Hum Brain Mapp. Walton, K. D., Dubois, M., & Llinas, R. R. (2010). Abnormal thalamocorti- – cal activity in patients with Complex Regional Pain Syndrome (CRPS) 2018;39:1945 1956. https://doi.org/10.1002/hbm.23973 type I. Pain, 150,41–51.
147