Maladaptive changes to Cholecystokinergic systems in the
Periaqueductal grey in rats experiencing persistent behavioural disability in the wake of Neuropathic pain
Manuel Alfonso Argueta Dominguez
A thesis submitted in fulfillment of the requirements for the degree of
Master of Philosophy
Faculty of Medicine
Discipline of Anatomy & Histology
The University of Sydney
Aug 2020
2
Thesis statement of Originality
This is to certify that, to the best of my knowledge, the body of work contained in this thesis is my own work. This thesis has not been submitted for any other degree or purposes.
3
Abstract
In 2018, it was estimated that 3.24 million Australians were living with chronic pain, with the total estimated cost to the economy of $73.2 billion (Painaustralia, 2019).
People with chronic neuropathic pain report substantial reductions in their quality of life, with disturbances to social relations; alterations to sleep, appetite, metabolic endocrine and sexual dysfunction; a loss of interest in external events; and moderate to severe depression. More often, it is these disabilities rather than the sensory disturbances of allodynia, hyperalgesia, and spontaneous pain, which are deemed by sufferers to be the most debilitating.
The neural adaptations underlying the sensory changes of neuropathic pain have been studied in rats and, using a sciatic chronic constriction injury (CCI) model, in combination with resident-intruder behavioural testing, it has been elucidated that a subgroup of animals undergoes complex behavioural and physiological dysfunction despite all animals experiencing similar levels of pain (Kilburn-Watt et al., 2010, Monassi et al., 2003, Mor et al., 2011). The general aim of this thesis is to further characterise the complex behavioural and physiological disturbances (i.e. disability) in a subgroup of animals marked as having pain and persistent disability.
Work from our group has focussed on a midbrain region, the Periaqueductal grey (PAG), as a site with the potential to contribute to behavioural disability. Some of this work used a molecular approach to assess gene expression patterns in PAG and identified a significant upregulation of mRNA encoding
Cholecystokinin (CCK), a peptide neurotransmitter.
Several brainstem and hypothalamic nuclei contain CCK-ergic cell populations, and these areas are also known to have reciprocal connectivity with the PAG. A pilot study (chapter 3) aimed to identify if rats which undergo stereotaxic surgery for retrograde tracing are compatible with the existing laboratory 4 resident-intruder paradigm. We observed that, in animals that had undergone stereotaxic surgery and
CCI, there was an increase in non-social behaviours compared with CCI-alone, though a larger sample size may negate this effect. Importantly, given the long time-frame of these experiments, we were able to successfully stain for both Cholera Toxin B (CTB) and CCK in both the brainstem and hypothalamus and found co-localization in the nucleus tractus solitarius and the hypothalamus.
Populations of neurons within the PAG have distinct functions, as such it was an important step to determine where in the PAG these changes are taking place. With the use of in situ hybridization techniques Chapter 4 of this thesis showed significant up-regulation of CCK mRNA in cells of the ventrolateral PAG (vlPAG) and the adjacent nucleus of the dorsal raphe selectively in rats with pain and persistent disability, with these animals not only showing and increase in CCK mRNA expressing cells, but also indicating higher levels of CCK mRNA expression in these cell populations, with mean densities of silver grain labelling correlated strongly with the degree of behavioural change (decreased dominance) in resident-intruder interactions.
This anatomical specific pattern of increased CCK expression in a functionally distinct subregion of the midbrain leads us to consider if similar changes were occurring in this region for the cognate receptors of CCK, the CCK1 and CCK2 receptors. Thus, in chapter 5, we aimed to see if the neurons of the midbrain PAG expressed these receptors, and to observe any differences in the expression of these receptors following CCI. Our findings showed that, following CCI, two distinct, behaviourally categorised groups of animals- non-affected and transient disability- display increased CCK receptor immunoreactivity, indicative of an adaptive response, whilst animals with pain and persistent disability showed significantly lower expressions of CCK receptors in functionally relevant regions of the PAG despite all animals showing identical levels of sensory disturbance characteristic of neuropathic pain.
The PAG is an important integrator of pain and behaviour. A selective upregulation of CCK in animals displaying pain and persistent disability is of particular significance given that we have shown the juxtaaqueductal gray and the vlPAG receive inputs from areas of the brainstem and hypothalamus that
5 are important not only in exerting descending inhibition of pain transmission, but also in the affective components of pain.
6 Abbreviations
Chronic Constriction injury (CCI)
Periaqueductal grey (PAG)
Cholecystokinin (CCK)
Ventrolateral PAG (vlPAG)
International Association for the Study of Pain (IASP)
World Health Organisation (WHO)
Central Nervous System (CNS)
Peripheral Nervous System (PNS)
Magnetic Resonance Imaging (MRI)
McGill pain questionnaire (MPQ)
Neuropathic Pain Scale (NPS)
Sickness Impact Profile (SIP)
Wide Dynamic Range (WDR)
Health-related quality of life (HR-QOL)
Multidimensional Pain Inventory (MPI)
Functional magnetic resonance imaging (FMRI)
Blood oxygen level dependent (BOLD)
Pre-frontal cortices (PFC)
Voxel-based morphometry (VBM)
7 Glial fibrilliary acidic protein (GFAP)
Single point mutations (SPNs)
N-methy-D-aspartate (NMDA)
Selective serotonin and noradrenaline re-uptake inhibitors (SNRIs)
Non-steroidal anti-inflammatory drugs (NSAIDs)
Partial sciatic ligation (PSL)
Spinal nerve ligation (SNL)
Spared nerve injury (SNI)
Hypothalamic-pituitary-adrenal (HPA)
Slow wave sleep (SWS)
Rapid eye movement sleep (REM)
Hypothalamo-pituitary gonadal (HPG)
Dorsal root ganglia (DRG)
Calcitonin-gene related peptide (CGRP)
Nerve growth factor (NGF)
Glial-derived neurotrophic factor (GDNF)
Projection neurons (PN)
Intersegmental propriospinal neurons (IPN)
Interneurons-inhibitory (INI)
Interneurons-excitatory (INE)
Ventral Posterior nucleus of thalamus (VP) 8 Posterior part of ventral medial nucleus (VMpo)
Ventral posterior inferior nucleus (VPI)
Ventral caudal division of the medial dorsal nucleus (MDvc)
Primary somatosensory cortex (S1)
Secondary somatosensory cortex (S2)
Positron emission tomography (PET)
Electroencephalography (EEG)
Magnetoencephalography (MEG)
Rostroventral medulla (RVM)
Nucleus tractus solitarius (NTS)
Parabrachial Nucleus (PBN)
Dorsal reticular nucleus (DRT)
Intermediolateral cell column (IML)
Excitatory postsynaptic currents (EPSCs)
Long term potentiation (LTP)
Type-1 interferons (IFN-1)
Nerve growth factor (NGF)
Leukaemia inhibitory factor (LIF)
Chemoattractant protein-1 (MCP-1)
Nitric oxide (NO)
Fractalkine (CX3CL1) 9 Interdisciplinary pain rehabilitation programs (IPRPs)
Heat shock protein 60 (HSP60)
Dorsomedial periaqueductal grey (dmPAG) dorsolateral periaqueductal grey (dlPAG) lateral periaqueductal grey (lPAG) ventrolateral periaqueductal grey (vlPAG)
Excitatory amino acid (EAA)
Ventromedial medulla (VMM)
Caudal ventrolateral medulla (CVLM)
Paraventricular nucleus of the hypothalamus (PVN)
Adrenocorticotrophic hormone (ACTH)
Corticotrophin releasing factor (CRF)
Anterior hypothalamic nucleus (AHN)
Dorsal pre-mammillary nucleus (PMd)
Dorsomedial hypothalamus (DMH)
Orbital and medial prefrontal cortex (OMPFC)
Tyrosine hydroxylase (TH)
TH-immunoreactive (TH-ir)
Ventrolateral preoptic area (VLPOe)
Cholera toxin subunit B (CTB)
Analysis of variance (ANOVA) 10 Protected least significance difference (PLSD)
Cholecystokinin mRNA (CCK mRNA)
Dorsal raphe nucleus (DRN)
Edinger-Westphal nucleus (EW)
Saline citrate (SCC)
Beta-mercaptoethanol (b-ME) phosphate buffered saline (PBS) diethyl pyrocarbonate (DEPC)
CTB-immunoreactivity (CTB-IR)
11 Table of Contents
CHAPTER1 INTRODUCTION ...... 20
1.1 DEFINITION OF PAIN STATES ...... 22
1.2 NEUROPATHIC PAIN ...... 24
1.2.1 Clinical presentation of neuropathic pain ...... 25
1.2.2 Clinical assessment of the patient ...... 26
1.2.3 Clinical signs and symptoms in neuropathic pain ...... 27
1.2.4 Negative signs and symptoms ...... 27
1.2.5 Stimulus-independent symptoms ...... 28
1.2.6 Stimulus-dependent symptoms ...... 28
1.2.7 Complex behavioural and physiological disturbances ...... 30
1.3 IMAGING PAIN ...... 32
1.4 PAIN GENETICS ...... 33
1.5 TREATMENT OPTIONS FOR CHRONIC PAIN ...... 34
1.5.1 Pharmacological treatment ...... 34
1.5.2 Interventional pain management ...... 35
1.5.3 Physical, rehabilitation, and psychological approaches ...... 35
1.6 ANIMAL MODELS OF PAIN ...... 36
1.6.1 Chronic constriction injury of the Sciatic Nerve (CCI) ...... 37
1.6.2 Behavioural and emotional changes post-CCI ...... 38
1.6.3 Social Interactions in rats ...... 39
1.6.4 Sleep-wake cycle changes ...... 42
1.6.5 Resident-Intruder CCI model ...... 42
1.7 PAIN PATHWAYS ...... 44
1.7.1 Peripheral receptors ...... 44
1.7.2 Peripheral afferent fibres ...... 45
1.7.3 Spinal Cord ...... 48
1.7.4 Afferent pathways from the spinal cord to supraspinal sites ...... 49 12 1.7.5 Supraspinal nuclei ...... 51
1.8 DESCENDING CONTROL OF PAIN ...... 54
1.9 PERIPHERAL SENSITIZATION ...... 55
1.10 CENTRAL SENSITIZATION ...... 56
1.10.1 Glutamatergic systems ...... 56
1.10.2 GABAergic tonic inhibitory control ...... 56
1.10.3 Glial-neuronal interactions ...... 57
1.11 NEURO-IMMUNE INTERATIONS AND NEUROPATHIC PAIN ...... 58
1.11.1 Peripheral immune mechanisms ...... 58
1.11.2 Central immune mechanisms ...... 59
1.12 THE PERIAQUEDUCTAL GREY (PAG) OF THE MIDBRAIN ...... 60
1.12.1 Functional PAG columns mediating emotional-coping behaviours ...... 61
1.12.2 Inputs and outputs of the PAG ...... 62
1.12.3 The PAG in pain modulation ...... 64
1.12.4 Autonomic nervous system modulation ...... 65
1.12.5 PAG modulation of sleep and arousal ...... 65
1.13 CHOLECYSTOKININ ...... 66
1.13.1 CNS CCK synthesis ...... 66
1.13.2 CCK expression in the CNS ...... 66
1.13.3 Neuronal actions of CCK ...... 67
1.14 THESIS AIMS ...... 68
CHAPTER2 METHODS ...... 71
2.1 ETHICS ...... 71
2.2 ANIMALS ...... 71
2.3 SURGICAL PROCEDURES ...... 71
2.3.1 Chronic constriction injury of the sciatic nerve ...... 71
2.3.2 Retrograde tracing experiments ...... 72
2.4 BEHAVIOURAL TESTING ...... 73 13 2.4.1 Resident/intruder testing general procedure ...... 73
2.4.2 Analysis of social interactions ...... 74
2.4.3 Categorization of animals ...... 74
2.4.4 Statistical analysis of behavioural groups ...... 75
2.5 SENSORY TESTING FOR ALLODYNIA AND HYPERALGESIA ...... 75
2.5.1 Tactile (mechanical) sensitivity testing ...... 75
2.5.2 Thermal sensitivity testing ...... 76
2.5.3 Statistical analysis of sensory tests ...... 76
2.6 SACRIFICE, PERFUSION, AND TISSUE REMOVAL ...... 76
2.7 EXPERIMENTAL PROCEDURES- IN SITU HYBRIDIZATION ...... 77
2.7.1 Perfusion procedures ...... 77
2.7.2 Blocking and cutting ...... 77
2.7.3 In situ hybridization ...... 77
2.7.4 Tissue analysis of in situ hybridization animals ...... 78
2.8 IMMUNOHISTOCHEMISTRY PROCEDURES ...... 79
2.8.1 General perfusion procedures ...... 79
2.8.2 Blocking and cutting ...... 80
2.9 IMMUNOHISTOCHEMISTRY LABELLING WITH IN SITU HYBRIDIZATION (CHAPTER 4) ...... 80
2.9.1 Tissue analysis ...... 81
2.10 LABELLING FOR CCK1- AND CCK2-RECEPTOR IN THE MIDBRAIN PAG (CHAPTER 5) ...... 81
2.10.1 Analysis of CCK-receptor fluorescent immunohistochemistry ...... 82
CHAPTER3 CHANGES TO BEHAVIOUR FOLLOWING CCI ...... 83
3.1 INTRODUCTION ...... 83
3.2 METHODS ...... 84
3.3 RESULTS ...... 85
3.3.1 Social interactions ...... 85
3.3.2 Comparison of experimental groups ...... 89
3.3.3 Sensory testing ...... 91 14 3.3.4 CTB microinjections ...... 92
3.35 Distribution of retrograde labelled cells in the brainstem following CTB injection in the
VLPAG/juxtaaqueductal PAG ...... 95
3.3.7 Distribution of retrograde labelled cells in the hypothalamus following CTB injection in the
VLPAG/juxtaaqueductal PAG ...... 99
3.4 DISCUSSION ...... 101
3.4.2 Methodological considerations ...... 102
CHAPTER4 SELECTIVE UP-REGULATION OF CCK MRNA IN THE VLPAG AND DRN ...... 107
4.1 INTRODUCTION ...... 107
4.2 METHODS ...... 108
4.3 RESULTS ...... 109
4.4 DISCUSSION ...... 119
4.4.1Midbrain localisation of CCK mRNA and CCK immunoreactivity ...... 120
4.4.2 Selectivity of patterns of mRNA up-regulation ...... 122
4.4.3 Possible functional consequences of transcription and translation of CCK mRNA in the vlPAG and
DRN ...... 123
4.4.4Methodological considerations ...... 125
4.5COMPARISON TO PREVIOUS IMMUNOHISTOCHEMICAL STUDIES OF CCK-8 ...... 126
4.5.1 Is CCK expression related to behaviour? ...... 126
4.5.2 Technical consideration of mRNA findings ...... 127
4.5.3 Increased peptide levels within the PAG ...... 127
CHAPTER5 THE PRESENCE AND ALTERATION OF CCK1 & CCK2 RECEPTORS FOLLOWING CCI IN THE PAG .. 130
5.1 INTRODUCTION ...... 130
5.1.1 CCK receptors ...... 130
5.2 METHODS ...... 131
5.3 RESULTS ...... 132
5.3.1 Behavioural changes following CCI ...... 132
15 5.3.2 Distribution of CCK1 receptors ...... 132
5.3.3 CCK2 receptors ...... 139
5.4 DISCUSSION ...... 144
5.4.1Summary of findings ...... 144
5.4.2 Comparison to previous studies ...... 146
5.4.3 Functional considerations of alterations to CCK receptors following CCI ...... 147
5.4.4 Functional anatomical pathways of the PAG ...... 151
5.5 METHODOLOGICAL CONSIDERATIONS ...... 153
5.6 SUMMARY ...... 154
CHAPTER6 GENERAL DISCUSSION AND SUMMARY ...... 155
6.1 THESIS AIMS ...... 155
6.2 DESCRIPTIVE FINDINGS OF TWO SUPRASPINAL CCKERGIC NEURONAL POPULATIONS WITH PROJECTIONS TO THE MIDBRAIN PAG
...... 156
6.3 POPULATIONS OF CCK EXPRESSING NEURONS IN THE MIDBRAIN ...... 158
6.4 DISTRIBUTION OF CCK RECEPTORS ALONG THE ROSTROCAUDAL EXTENT OF THE PAG ...... 160
16 Table of Figures
FIGURE 1.1: RESPONSES TO CHEMICAL, TACTILE, AND SENSORY TESTS AS REPORTED BY MONASSI AND
COLLEAGUES (2003). 39
FIGURE 1.2 VIDEO STILL SHOWING EXAMPLES OF DOMINANT BEHAVIOUR DISPLAYED BY RESIDENT RAT
TOWARDS INTRUDER RAT IN RESIDENT-INTRUDER PARADIGM. 41
FIGURE 1.3 SCHEMATIC DEPICTION OF NOCICEPTOR SUBTYPES. 47
FIGURE 1.4 SCHEMATIC SPINAL CORD. 48
FIGURE 1.5 SUPRASPINAL NUCELI OF THE PAIN PATHWAY. 52
FIGURE 1.6 SCHEMATIC DIAGRAM DEPICTING THE FUNCTIONAL COLUMNS OF THE PAG THROUGH ITS
ROSTROCAUDAL EXTENT. 60
FIGURE 3.1 GRAPHS COMPARING THE MEAN DURATION IN SECONDS (±SEM) OF DOMINANCE BEHAVIOURS,
SOCIAL BEHAVIOURS, NON-SOCIAL BEHAVIOURS, AND SUBMISSIVE BEHAVIOURS. 86
FIGURE 3.2 LINE GRAPHS COMPARING THE MEAN DURATION IN SECONDS (±SEM) OF DOMINANCE
BEHAVIOURS, SOCIAL BEHAVIOURS, NON-SOCIAL BEHAVIOURS, AND SUBMISSIVE BEHAVIOURS. 88
FIGURE 3.3 SUMMARY OF DIFFERENCE IN BEHAVIOUR PRE-CCI. 90
FIGURE 3.4 LINE GRAPHS COMPARING A) RESPONSES TO TACTILE (VON FREY) SENSORY B) THERMAL (ICE
PLATE) SENSORY TESTS IN RATS WITH EITHER PAIN ALONE (N=7), PAIN & TRANSIENT DISABILITY (N=5),
AND PAIN & PERSISTENT DISABILITY (N=6) RATS. 92
FIGURE 3.5 CAMERA LUCIDA DRAWINGS. 93
FIGURE 3.6 PHOTOMICROGRAPHS SHOWING RETROGRADE TRACER INJECTION SITES. 94
FIGURE 3.7 COLUMN GRAPH. 96
FIGURE 3.8 COLUMN GRAPHS DEPICTING DISTRIBUTION OF CHOLERA TOXIN. 98
FIGURE 3.9. COLUMN GRAPHS DEPICTING TOTAL CELL COUNTS. 100
FIGURE 4.1 PHOTOMICROGRAPHS ILLUSTRATING THE PRESENCE OF CCK MRNA. 110
FIGURE 4.2 SCHEMATIC DIAGRAM SHOWING THE LOCATION OF CCK MRNA EXPRESSION. 111
FIGURE 4.3 BARGRAPH DEPICTING THE DISTRIBUTION OF CCK MRNA POSITIVE CELLS. 112
FIGURE 4.4. RELATIONSHIP BETWEEN CCK MRNA IN CELLS OF THE VLPAG AND BEHAVIOURAL DISABILITY
FOLLOWING INJURY. 113 17 FIGURE 4.5 BARGRAPHS DEPICTING THE DISTRIBUTION OF CCK MRNA POSITIVE CELLS. 114
FIGURE 4.6 PHOTOMICROGRAPHS ILLUSTRATING THE PRESENCE OF CCK MRNA. 115
FIGURE 4.7 FIGURES DEPICTING THE DISTRIBUTION AND RELATIVE CHANGE OF EXPRESSION OF CCK MRNA
POSITIVE CELLS. 116
FIGURE 4.8 PHOTOMICROGRAPHS ILLUSTRATING THE PRESENCE OF 5HT-IR CELLS AND CCK MRNA. 117
FIGURE 4.9 BARGRAPHS DEPICTING 5HT-IR CELLS. 118
FIGURE 5.1 PHOTOMICROGRAPHS ILLUSTRATING THE PRESENCE OF CCK1-IR CELLS AS VISUALISED BY
FLUORESCENT IMMUNOHISTOCHEMISTRY. 133
FIGURE 5.2 IDEALIZED DIAGRAM SHOWING SIX EQUIDISTANT SECTIONS OF THE MIDBRAIN PAG WITH DEFINED
ANATOMICAL BORDERS FOR THE FUNCTIONAL COLUMNS OF THE PAG. 133
FIGURE 5.3 DISTRIBUTION OF CCK1 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE VENTROLATERAL
PERIAQUEDUCTAL GRAY (VLPAG). 134
FIGURE 5.4 DISTRIBUTION OF CCK1 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE LATERAL
PERIAQUEDUCTAL GRAY (LPAG). 136
FIGURE 5.5 DISTRIBUTION OF CCK1 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE DORSOMEDIAL
PERIAQUEDUCTAL GRAY (DMPAG). 137
FIGURE 5.6 DISTRIBUTION OF CCK1 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE DORSOLATERAL
PERIAQUEDUCTAL GRAY (DLPAG). 138
FIGURE 5.7 PHOTOMICROGRAPHS ILLUSTRATING THE PRESENCE OF CCK2-IR CELLS. 140
FIGURE 5.8 DISTRIBUTION OF CCK2 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE VENTROLATERAL
PERIAQUEDUCTAL GRAY (VLPAG). 141
FIGURE 5.9 DISTRIBUTION OF CCK2 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE LATERAL
PERIAQUEDUCTAL GRAY (LPAG). 142
FIGURE 5.10 DISTRIBUTION OF CCK2 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE DORSOMEDIAL
PERIAQUEDUCTAL GRAY (DMPAG). 143
FIGURE 5.11 DISTRIBUTION OF CCK2 RECEPTOR THROUGH ROSTROCAUDAL EXTENT OF THE DORSOLATERAL
PERIAQUEDUCTAL GRAY (DLPAG). 144
18
19 Chapter1 Introduction
Sir Charles Sherrington, in 1904, delivered a series of remarkable lectures at Yale College in New
Haven, Connecticut, based on his seminal textbook, The integrative action of the nervous system
(Breathnach, 2004). In his outstanding Silliman lecture VII, after drawing on the results of spinal lesion experiments to explain nociceptive pathways, Sherrington concluded, “…the visceral expression of emotion is secondary to the cerebral action occurring with the psychical state” (Burke, 2007). At the outset of his final lecture, on sensation and perception, he commenced with,
We may agree that if such sensations and feelings or anything at all closely like them do
accompany the reactions we have studied, the neural machinery to whose working they are
adjunct lies not confined in the nervous arcs we have so far traced but in fields of nervous
apparatus that, though connected with those arcs, lie beyond them, in the cerebral hemispheres.
Thus, as far back as the early 20th century, Sherrington attempted to represent pain as a complex multidimensional experience comprising sensory-discriminatory and affective-motivational components.
The International Association for the Study of Pain (1986) defined pain as an “unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”. The perception and experience of pain is affected by complex higher-order brain processing of the sensory stimuli, which can be significantly influenced by factors external to the stimulus itself, such as genetic, developmental and cultural factors (Almeida et al., 2004, Millan, 1999, Mogil, 2012).
This highlights the subjective nature of the pain experience, arising from individual variability that exists in the interpretation of the painful stimulus, and which can drastically alter responses to pain.
It is important that the body be aware of potentially damaging stimuli to guard against tissue injury. In most cases then, pain is biologically protective, signaling against actual or impending tissue damage, and acting as an indication of the physiological condition of the tissue itself whilst generating an inseparable affect which includes both physiological and emotional responses (Almeida et al., 2004,
20 Costigan et al., 2009, Craig, 2002). The unpleasant, aversive qualities of pain provoke a drive to escape or further avoid noxious sensory stimuli. Thus, pain may be considered a “motivational state” similar to hunger and thirst (Craig, 2002, Fields, 1999, Scholz and Woolf, 2002).
The experience of pain can therefore be thought of as a complex sensory modality accompanied by affective, motivational and cognitive aspects associated with neurological responses to stimuli. While the sensory-discriminative aspect of pain permits the perception of the quality, duration, intensity and location of pain, the motivational-affective component is responsible for the emotional responses to pain (e.g. anxiety, suffering, depression), as well as engaging the mechanisms of attention and arousal
(Almeida et al., 2004).
It is also worth considering that several types of pain have been distinguished, the location and intensity of which can lead to differing physiological and behavioural responses (Woolf, 2004). Whilst pain is important for the development and survival of the individual, in the absence of an adequate stimulus pain can become maladaptive, having no functional benefit, and becoming a deleterious process for the sufferer. These maladaptive responses can often persist, leading to a chronic pain state, the biochemical processes of which the following chapters will attempt to better elucidate.
The World Health Organization (WHO) estimates that 20% of individuals worldwide have some degree of chronic pain, the presence of which has both direct healthcare and associated indirect costs, which include disability, loss of productivity, and associated costs to the state and general economy. In
Australia alone, prevalence studies have shown that 17% of males and 20% of females were reported as suffering chronic pain (Blyth et al., 2001, Turk et al., 2011). Furthermore, chronic pain is a pervasive health issue that exerts a substantial economic burden. Health studies conducted in England suggest that up to a staggering 8% of the UK population suffers from chronic neuropathic pain (Torrance et al.,
2006), a figure which is expected to rise given the increase in the prevalence of co-morbid chronic conditions and an ageing population. In Australia, the total financial costs of chronic pain were estimated as costing $73.2 billion in 2018, with reduction in quality of life for 3.24 million Australian sufferers estimated at a further $66.1 billion (Painaustralia, 2019). These costs are expected to rise to
21 $215.6 billion by 2050 in real dollars, highlighting that chronic pain is a pervasive health issue that exerts a substantial social and economic burden on both the affected individual and society in general.
The introduction to thesis begins with a definition of pain, and the various pain states, with a focus on neuropathic pain in particular; the aetiology and clinical presentation of neuropathic pain; the mechanisms involved in the induction and maintenance of neuropathic pain, and pain genetics; pharmacological and non-pharmacological treatments of neuropathic pain and their limitations; animal models of neuropathic pain, particularly the use of the chronic constriction injury (CCI) of the sciatic nerve as a model of peripheral nerve injury and neuropathic pain; and an introduction to the midbrain periaqueductal gray (PAG).
1.1 Definition of pain states
Pain states are broadly classified as being either acute or chronic, with acute and chronic pain typically differing in clinical terms as to their affective and cognitive components, where symptoms such as depression, anxiety, and physical and social dysfunction are co-morbid for the chronic pain state
(Becker et al., 1997). Acute pain is self-limiting (usually lasting less than three months), and characterised by relatively discrete neuroanatomical pathways mediating the effects of somatic injury.
The transmission of this information has, at its essence, a survival value that initiates protective physiological mechanisms against further tissue injury, and signals the need for corrective action to promote healing. The time course of acute pain is limited to the time taken for the body to correct pathological processes, i.e. tissue injury, and there is a relative absence of marked psychosocial/behavioural changes disproportionate to pain intensity (Hart et al., 2000). Its principal aim is to guard against tissue injury, and most acute pain is mediated via nociceptive pathways mediated by high-threshold unmyelinated C or thinly myelinated A primary sensory neurons (Woolf and Ma, 2007).
This stimulus can be external, such as a pinprick or burning heat, or internal, such as in the case of myocardial ischaemia.
Nociceptive pain is a noxious stimulus-detecting sensory system mediated by high-threshold unmyelinated C or thinly myelinaed A primary sensory neurons, whose peripheral terminals are located 22 in skin, bone, connective tissue, muscle, and viscera, that feed into nociceptive pathways of the central nervous system (CNS). These nociceptor neurons express specialised transducer ion receptors capable of responding not just to intense thermal or mechanical stimuli, but also exogenous and endogenous chemical mediators. Thus, nociceptive pain sensation serves a protective function by providing an unpleasant stimulus, generally as a result of tissue damage. Typically, it continues only in the maintained presence of the noxious stimulus, whether that is an external or internal stimulus. Certain disease states, such as osteoarthritis, may generate recurrent noxious stimuli to produce chronic nociceptive pain. Depending on the location of the insult (deep or cutaneous) there will be characteristic affective responses; for example, deep pain (muscle, joints, viscera) will typically be described by the sufferer as dull and aching in nature, and will be poorly localised. Behaviourally, passive emotional coping will be observed, characterised by quiescence and decreased responsiveness to the external environment. Conversely, cutaneous pain tends to be well localised, oft described as being sharp, pricking, and throbbing in nature. Behaviourally, it is associated with an active emotional coping response, i.e. “fight or flight”.
As the name implies, inflammatory pain occurs in response to the subsequent inflammatory response to tissue injury. Here, the stimulus serves to address the consequences of tissue damage by attempting to limit further damage (Costigan et al., 2009). At the site of injury, plasticity of the local sensory nervous system results in a profound change in its responsiveness, whereby normally innocuous stimuli now produces pain (allodynia), and responses to noxious stimuli are both exaggerated and prolonged
(hyperalgesia). This increased responsiveness and reduced threshold of nociceptors to stimulation of their receptive fields, defined as peripheral sensitization, helps to aid in the healing and repair of the injured body part, and is usually coupled with reduced movement and guarding behaviours towards the injured body part (Sandkuhler, 2009). Typically, inflammatory pain resolves following healing of the initial tissue injury, although in autoimmune disorders, such as rheumatoid arthritis, chronicity develops.
23 1.2 Neuropathic pain
Neuropathic pain is not a single entity, but rather a heterogeneous group of conditions differing in aetiology and anatomical location that, nevertheless, result in a clinical presentation defined by the
IASP as “Pain arising as a direct consequence of a lesion or disease affecting the somatosensory system”
(Jensen et al., 2001, Woolf and Mannion, 1999). Whilst traditionally the classification of neuropathic pain is based on the underlying aetiology or location of nerve injury, it is important to note that the common principal manifestation of neuropathic pain states, regardless of the basis of the underlying disease, is a maladaptive plasticity in the nervous system that lead to and sustain the neuropathic pain state (Jensen et al., 2001, Smith et al., 2013).
Common peripheral injuries resulting in neuropathic pain states include neuropathies resulting from metabolic conditions including diabetes, alcoholism, and vasculitis; neuralgia following viral infections such as herpes zoster; trigeminal neuralgia; neurotoxicity from drugs or toxins; mechanical nerve injury, such as carpal tunnel syndrome, vertebral disc herniation, and lumbar radiculopathy; post-operative nerve damage; inflammation; and cancer infiltration. At the level of the spinal cord and brain, central neuropathic pain can develop following spinal cord injury; multiple sclerosis; neoplasms; cerebrovascular lesions; and in neurodegenerative disorders, such as Parkinson’s disease. Due to the complex interconnectivity between the CNS and the peripheral nervous system (PNS), as well as the considerable plasticity of the nociceptive system, different mechanisms may be involved and account for various symptoms, which may partly explain why pharmacological measures have shown limited success thus far (Jensen et al., 2001, Woolf and Mannion, 1999).
The difficulty in classifying neuropathic pain based on an etiological basis is that there is no direct relationship between aetiology, mechanism, and symptoms. The development of neuropathic pain itself is variable, and often dynamic and unpredictable in nature. Chronic neuropathic pain sufferers can often have normal clinical signs, and there is no diagnostic test that can confirm or refute nerve lesion or dysfunction.
24 1.2.1 Clinical presentation of neuropathic pain
Clinicians have traditionally been taught to examine and classify patients on the basis of lesion topography and underlying disorder. In chronic pain conditions however, a classification based on anatomy and disease is insufficient, mainly because despite the obvious differences in aetiology, shared clinical phenomena are observed amongst patients. The limitation of using a traditional approach is further emphasised by the fact that different signs and symptoms may be present in the same disease, and the neuroplastic changes following nervous system lesions often give rise to sensory and pain distributions that do not respect nerve, root, segmental, or cortical territories.
The diagnosis of neuropathic pain is based on medical history, review of systems, physical and neurological examination, and appropriate laboratory studies which include blood and serologic tests, magnetic resonance imaging (MRI), and electrophysiological studies. In some instances nerve or skin biopsy is necessary to directly visualise nerve fibres. Once elucidated, classification of neuropathic pain is an important clinical consideration, but due to the complex relation between aetiology, mechanisms, and symptoms, aetiology does not typically dictate treatment options or outcomes. Whilst it is common clinical practice to classify neuropathic pain according to disease and anatomical site, pain which manifests in diverse diseases often shares common pathways, and the pain mechanism itself is typically independent of the disease process, as evidenced by the fact that only a fraction of patients are affected, and there are no predictors to determine susceptibility to neuropathic pain. Due to the plasticity and complexity of the nervous system common or disparate mechanisms may result in similarly presenting symptoms, which may change over time. Thus, in patients with neuropathic pain it is difficult to predict the mechanism of symptoms based on only the aetiology of the neuropathy or on the distribution and nature of the symptoms. Despite this heterogeneity in aetiology, anatomical location, and affected neurological pathways, many conditions resulting in neuropathic pain share common clinical characteristics, namely sensory abnormalities.
25 1.2.2 Clinical assessment of the patient
A thorough clinical assessment of pain and other symptoms is typical in the clinical setting. When obtaining the patient’s medical history it is important to assess the intensity, quality, and duration of spontaneous pain and abnormal sensations, as well as triggering and relieving factors, previous treatments, and effects of the pain on quality of life. The topographical distribution of positive and negative sensory signs and symptoms is especially helpful in guiding the neurological examination. A thorough physical and neurological examination can determine the site of the lesion, as well as help to identify non-neuropathic contributions to the patient’s pain, most commonly musculoskeletal, inflammatory, myofascial, and psychological processes.
Pain may be evoked by everyday environmental stimuli such as: gentle touch, pressure of clothing, wind, posture, and hot and cold temperatures. Common neurological examination tools, including a cotton wisp, a foam brush, tuning fork, and cold and warm water-filled tubes can be used to mimic these stimuli. Pain intensity should be rated with any of several reliable and validated scales, including the
McGill pain questionnaire (MPQ) (Melzack, 1975), and the short form MPQ (Melzack, 1987), both of which have been shown to distinguish between chronic neuropathic pain and other chronic pain types, and to describe changes in the quality of the pain over time (Dworkin, 2002). The neuropathic pain scale (NPS) (Galer and Jensen, 1997) and the self-completed Leeds assessment of neuropathic symptoms and signs (Bennett, 2001) are also used to screen and evaluate signs and symptoms specific to neuropathic pain. Finally, several measures of the quality of life have been validated; among them the sickness impact profile (SIP), the Nottingham Health Profile, and the SF-36. Assessment of psychological comorbidity, sleep disturbance, work-related issues, treatment expectations, rehabilitative needs, and the availability of social support from family and friends should not be overlooked (Haythornthwaite and Benrud-Larson, 2000).
Paradoxically, patients may have sensory deficits with one modality, such as pinprick sensitivity, and hyperalgesia to another, such as light touch, in the same nerve distribution. This is particularly unsettling for the patient, who may be confused by the complexity of their sensory experience and have trouble
26 describing the unusual nature of their symptoms, or otherwise fear not being believed by the clinician.
It is for this reason that thorough stimulation of both affected and non-affected regions is vital in elucidating symptoms and signs of any particular case.
As there is no single diagnostic test for neuropathic pain, ancillary studies can confirm, or exclude, underlying causes and suggest disease-specific treatment. Nerve conduction tests and electromyography assess peripheral nerve function; however, whilst providing information about large myelinated fibres they do not test smaller, myelinated or unmyelinated nerve fibres, which are usually the principal culprits in nerve dysfunction. MRI assesses the anatomical integrity of thermonociceptive sensory- processing regions of the CNS, but its role in clinical practice remains limited.
1.2.3 Clinical signs and symptoms in neuropathic pain
Clinically, the cardinal diagnostic signs of chronic neuropathic pain are the presence of positive and negative sensory changes, which include allodynia, hyperalgesia, and spontaneous pain (Dworkin et al., 2003, Zimmermann, 2001). These disturbances in sensation, which are the principal diagnostic features of the neuropathic pain state, are typically accompanied by an array of disabilities that patients often describe as being more deleterious to quality of life than the sensory disturbances.
These changes include disturbances to social and familial interactions; disrupted sleep-wake patterns and associated lethargy; autonomic nervous system dysfunction; metabolic, endocrine, and sexual dysfunction; loss of interest and pleasure in previously enjoyable activities; and, often, moderate to severe depression.
1.2.4 Negative signs and symptoms
Negative signs and symptoms associated with neuropathic pain, defined by reduced perception and numbness, are commonly seen in patients. The consequences of sensory disturbances should not be underestimated, as they can lead to significant tissue damage as a result of loss of pain and temperature sensibility leading to burns, pressure ulcerations, lack of adequate tissue oxygenation, and amputation. The sensory loss may involve all sensory modalities, but curiously, it appears that loss of spinothalamic function (temperature and touch) is crucial (Jensen and Lenz, 1995). Negative 27 signs and symptoms include phenomena such as a reduced sensation to non-painful stimuli
(hypoesthesia); reduced sensation to vibration (pallhypoesthesia); reduced sensation to painful stimuli
(hypoalgesia); and reduced sensation to cold or warm stimuli (thermohypoesthesia).
1.2.5 Stimulus-independent symptoms
Neuropathic pain sufferers often experience sensation or pain in the absence of stimulus. Paraesthesia is defined as an uncomfortable, non-painful, ongoing sensation, commonly described by patients as feeling like “pins and needles”. Stimulus-independent pains are spontaneous and may be continuous or paroxysmal. The character differs but is typically described as being shooting, shock-like, aching, cramping, crushing, and burning in quality. Episodic, paroxysmal pain is typically described as shooting, electric, shock-like or stabbing in character (Baron, 2006, Dworkin et al., 2003, Jensen et al., 2001). Spontaneous and paroxysmal pain can occur alone or be accompanied by dysesthesia (an uncomfortable absence of sensation) and/or paraesthesia.
1.2.6 Stimulus-dependent symptoms
Stimulus-evoked symptoms reflect a state of hyperexcitability in the nervous system. Stimulus- evoked pain is classified according to the stimulus type that provokes it, typically being: mechanical, thermal, or chemical. Onset, severity, and duration of evoked pains are variable, and patients have variable triggers. Evoked pains are usually brief, lasting for the duration of stimulation, but may persist after cessation of stimulation (Baron, 2006, Dworkin et al., 2003, Jensen et al., 2001). Forms of evoked pain include:
i) Mechanical dynamic allodynia: normally non-painful light-pressure moving stimuli on skin
evokes pain
ii) Mechanical static allodynia: normally non-painful gentle static pressure stimuli on skin
evokes pain
iii) Mechanical punctate or pinprick hyperalgesia: normally stinging-but-not-painful stimuli
28 evokes pain
iv) Temporal summation: repetitive application of identical single noxious stimuli is perceived
as increasing pain sensation
v) Cold allodynia: normally non-painful cold stimuli evokes pain
vi) Heat allodynia: normally non-painful heat stimuli evokes pain
vii) Mechanical deep somatic allodynia: normally non-painful pressure on deep somatic tissue
evokes pain
viii) Hyperpathia: prototypic disorder in neuropathic pain, which is observed whenever there is
fibre loss, or peripheral or central deafferentiation, leading to an elevation of threshold on
one limb and a central hyperexcitability due to loss of afferent or abnormal input from
affected limb. Hyperpathia is associated with explosive pain evoked from affected
cutaneous areas.
Referred pain and abnormal pain radiation are also seen following peripheral and central lesions.
Patients may experience spreading sensations of pain following punctate stimulation, and a relationship has been observed between pain intensity, spread, and pain referral. Most cases report cutaneous pain referral from deep to cutaneous structures, rarely the inverse. Abnormal radiation can be attributed to changes in spinal Wide Dynamic Range (WDR) neurons encoding noxious information. In WDR neurons, non-noxious stimuli normally evoke smaller receptive fields than do noxious stimuli. Thus, with dysfunction, larger numbers of WDR neurons are activated disproportionate to stimulus, and pain spread seen in neuropathic patients may be a reflection of a progressive, disproportionate recruitment of WDR neurons along the spinal cord (Jensen et al., 2001).
29 1.2.7 Complex behavioural and physiological disturbances
In addition to these sensory dysfunctions, chronic neuropathic pain patients experience complex behavioural and pathophysiological changes which include profound disturbances of familial and social relations; altered sleep-wake cycles; decreased appetite; metabolic, endocrine, and sexual dysfunction; reduced ability to concentrate; loss of interest and pleasure in external events; and moderate to severe depression (Harding et al., 1994, Jensen et al., 2001, Menefee et al., 2000, Monassi et al., 2003,
Sternbach, 1974, Timmermans and Sternbach, 1974). Several measures of health status have been developed in order to quantify changes in health-related quality of life (HR-QOL), with neuropathic pain being shown to reduce quality of life in all parameters of quality of life in comprehensive, reproducible health surveys such as the SF-36. A systematic review of such studies (Jensen et al., 2007) showed that in various conditions which feature chronic neuropathic pain, significant subsets of patients reported that pain had a negative impact on HR-QOL in regard to physical function, emotional function, and sleep. Furthermore, neuropathic pain patients show greater affectation to HR-QOL compared to non-neuropathic chronic pain patients in all domains of the SF-36, indicating poorer health and greater disability. Of the effects on emotional functioning, depression and anxiety are reported as having the most negative impacts on quality of life, with sleep interference also being amongst the most common pain-related problems reported by studies (Jensen et al., 2007).
A study of disease burden on patients with painful diabetic peripheral neuropathy showed that 59% reported decreased home productivity, and 85% reported activity limitations, with 64% of employed patients reporting missing work/decreased work productivity over a three month period due to pain
(Gore et al., 2007). 60% of patients had sought >2 consults with health professionals, with 87% taking pain medications, highlighting that burden of disease falls not just on patients, but on the taxpayer.
A common finding in these studies is the individual differences in presentation of symptoms in patients suffering from chronic neuropathic pain conditions. That is, not all neuropathic pain sufferers will develop debilitating comorbidities, nor will they be equally affected. This conundrum not only highlights our limited knowledge of the complex neurocircuitry involved, but also presents the
30 limitations of current clinical efficacy of treatments. In order to direct patients towards better clinical outcomes Jamison and colleagues (1994) refined earlier studies made by Turk & Rudy using an empirically derived pain patient taxonomy, which focused on psychopathological elements via use of the Multidimensional Pain Inventory (MPI), and grouped 1954 patients based on 4 constructs (activity interference, emotional distress, pain intensity, and perceived support)(Jamison et al., 1994). Strong evidence was found for a taxonomy of 3 chronic patient groups labelled as dysfunctional, interpersonally distressed, and adaptive copers. These patient groups were characterized by distinct coping patterns.
(i) The dysfunctional group reported high levels of pain, activity interference and emotional distress.
They reported greater use of prescription analgesic medication, greater number of physician treatments, more sleep disturbances, and higher physician-rated emotionality than the Adaptive Coper group, coupled with higher unemployment, a greater number of hospitalizations, and greater limitation due to their pain.
(ii) The interpersonally distressed group exhibited similar behaviours to the dysfunctional group, with the distinguishing features being they felt socially isolated; clinically exhibited more pain in comparison with the dysfunctional group; and had greater marital disharmony. They had greater difficulties in pain management due to their perceived lack of social support.
(iii) The adaptive coper group, in contrast, reported better management of their condition. They relied less on medical treatments or services and showed greater emotional stability. Additionally, they were more likely to be working, despite clinical findings showing they had similar organic pathology to the other two groups.
Since then, studies have shown that greater pain levels do not predict the level of disability and social functioning (Gauntlett-Gilbert and Eccleston, 2007).
Pharmacological studies have attempted to target sensory disturbances as a way of reversing emotional/behavioural dysfunction. Drug trials which have shown significant reductions in allodyina and sleep disturbances failed to improve anxiety, depression, and social dysfunction (Berman et al., 31 2004, Nurmikko et al., 2007), indicating that the complex neural systems mediating dysfunction are processed independently of each other.
Whilst a wide range of research has been devoted to the sensory disturbances observed in neuropathic pain states, relatively less emphasis has been placed on studying the specific neural maladaptations underlying the complex behavioural and physiological changes. The importance of understanding these complex behavioural changes is underscored by the fact that, relative to the sensory disturbances, these complex disabilities are described by neuropathic pain sufferers to be the most disabling and debilitating
(Blyth et al., 2001, Harding et al., 1994, Jensen et al., 2001, Meyer-Rosberg et al., 2001, Zimmermann,
2001).
1.3 Imaging pain
Functional magnetic resonance imaging (FMRI) enables blood oxygen level dependent (BOLD) signals to detect changes in cerebral activity in patients with neuropathic pain, and this technique allows the study of discriminative sensory, emotional, motivational, and modulatory responses in particular regions of the brain and brain stem. New modelling approaches can explore the dynamic processes influencing pain perception, with the effects of personality and expectancy on pain perception and analgesia of particular interest (Lee and Tracey, 2013).
Prefrontal-limbic control of pain has been a focus of imaging studies, with numerous studies demonstrating that the pre-frontal cortices (PFC), which are reciprocally connected to the “limbic” regions of the brain, form the connection through which motivational-emotional aspects of pain can be regulated. Various regions of the PFC have shown reciprocal connectivity with regions of the brain including the NAcc, the amygdala, the para-hippocampus, and the PAG (Ochsner et al., 2009, Ploghaus et al., 2001, Wager et al., 2008). Early FMRI studies have shown altered functional activation of PFC by noxious stimuli in patients with chronic pain (Apkarian et al., 2001, Mayer et al., 2005).
Numerous investigators have demonstrated subtle alterations in grey-matter densities in the brains of patients with chronic pain using voxel-based morphometry (VBM) (Apkarian et al., 2004, Draganski et
32 al., 2006), which longitudinal studies have shown can be resolved with improvement in symptoms
(Obermann et al., 2009). Baliki and colleagues also published a longitudinal imaging study involving
12 acute versus 12 chronic back pain patients where the persistent group showed decreased grey matter density (Baliki et al., 2012). Of particular interest, was data derived from the initial “acute phase” imaging data, where it was found that greater functional connectivity between the NAcc and the PFC predicted pain persistence by more than 80%, implying the importance of corticostriatal pathways in the transition from acute to chronic pain.
Powerful modulatory input transmitting information between the spinal cord and frontal-limbic regions is integrated within the RVM and the PAG, with these brainstem regions co-ordinating the inhibition or facilitation of nociception (Keltner et al., 2006).
1.4 Pain genetics
Whilst inflammatory and/or nerve injuries are known or suspected to be the aetiology of most chronic pain syndromes, only a small minority of individuals go on to develop chronic pain, suggestive of gene x environment interactions possibly playing a role in this highly variable response. Despite the prevalence and cost to society that chronic pain poses, human pain genetics has lagged for reasons including: subjective quantification, low funding levels, and low numbers of familial aggregation and twin studies. There is also the issue that, in contrast with rare Mendelian conditions, the study of pain genetics presents a more complex picture, with heritability estimates from twin studies ranging between
13–60%, depending on cohort and pain phenotype examined (Crow et al., 2013). Loci for which a positive association has been established are involved in neurotransmitter systems (COMT, OPRM1,
GCH1, 5HTR2A, ADRB2), ion channel function (KCNS1, CACNA2D3), and immune function (IL-1,
TNF) (Mogil, 2012). In addition, genechip, RT-PCR, and western blotting studies reveal select up- regulation in translation and transcription of glial fibrilliary acidic protein (GFAP) and vimentin, in the midbrain PAG of a subgroup of rats exhibiting behavioural dysfunction following CCI (Mor et al.,
2010). Be that as it may, mechanistic steps by which single point mutations (SPNs) in these genes confer risk towards chronic pain are poorly understood.
33 1.5 Treatment options for chronic pain
Currently, there is a broad array of pharmaceutical, surgical, neuro-augmentative, somatic, behavioural, rehabilitative, and complementary and alternative treatment options available for the management of patients with chronic pain. In spite of this, overall treatment effectiveness remains inconsistent and fairly poor, as even treatments that have been shown to effectively reduce pain do not provide concomitant improvements in the physical and emotional function of patients.
1.5.1 Pharmacological treatment
Opioids are the most common class of drug prescribed for the treatment of chronic pain, but remain a controversial option, both with respect to efficacy and adverse physical effects and to aberrant behaviours (Chou et al., 2009a, Stein et al., 2010). A meta-analysis of 41 randomised controlled trials evaluating the effectiveness of opioids for the treatment of various forms of chronic pain, including osteoarthritis, diabetic painful neuropathy, low back pain, and rheumatoid arthritis, concluded that, on average, opioids result in a small improvement in pain severity and functional improvement compared with placebo; and similar reductions in pain, but less improvement in function compared with other analgesic drugs (Furlan et al., 2006). Furthermore, various studies investigating the physical side-effects associated with opioids, which are significant and include opioid-induced hyperalgesia, have observed low long-term compliance in patients, with 44% of patients abandoning treatment within 7–24 months
(Chu et al., 2008, Noble et al., 2010). Opioids carry a substantial risk of misuse due to their addictive nature, and are the leading cause of accidental overdose and mortality for prescription drugs.
Antidepressants have diverse effects that might contribute to their effect, including effects on N-methy-
D-aspartate (NMDA) and adenosine receptors, sodium channels, and serotonin, noradrenaline, and opioid systems. Meta-analyses suggest that antidepressants are superior to placebo for the treatment of chronic pain, resulting in moderate symptom reduction (Kroenke et al., 2009). Most recently, trials focusing on the new selective serotonin and noradrenaline re-uptake inhibitors (SNRIs) have lead to duloxetine being recommended as a first-line treatment for patients with neuropathic pain (Attal et al.,
2006). 34 Other drug classes, such as non-steroidal anti-inflammatory drugs (NSAIDs) and anti-convulsants, have not shown to be effective, or, in the case of skeletal muscle relaxants and topical agents, are seen only as adjuvant therapy for short-term relief.
1.5.2 Interventional pain management
Interventional pain management involves the application of various techniques, which include injection therapy, surgery, and implantable devices, and is most commonly used when a specific region of the spine is thought to contribute to a patient’s chronic pain. Epidural steroid injections are the most commonly performed pain management procedure; however, evidence for their effectiveness is mixed, and the decision to use injection needs to address the patient’s specific benefits versus serious adverse events and costs. Surgery for lower back pain is a controversial option, with studies finding diminished benefits over time, with as many as 41% of patients reporting no change or a worsened quality of life up to 4.5 years after surgery. High complication rates and repeat procedures are the realities of spinal surgery, with an estimated 30% of patients suffering from failed back surgery syndrome, the resolution of which is not guaranteed with subsequent surgery (Chou et al., 2009b, Manchikanti et al., 2008).
1.5.3 Physical, rehabilitation, and psychological approaches
Physical and psychological approaches are commonly included as components of interdisciplinary pain rehabilitation programs (IPRPs). Physical, rehabilitation and psychological approaches have been shown to decrease pain and improve physical and emotional functioning, though improvements are small to moderate. Whilst there are varying psychological treatments employed, all emphasise patient coping, adaptation, self-management, and reduction of disability associated with symptoms, rather than elimination of pain per se. Although there is no single format for IPRPs, their integrated approach involving close coordination between physicians, psychologists, physical therapists, and other health care providers have shown significant pain reduction in several meta-analyses, with investigators also identifying reductions in prescription pain treatment, as well as significant reductions in social costs
(welfare benefits, sickness benefits, and pensions) (Hoffman et al., 2007, Morley et al., 1999).
35 1.6 Animal models of pain
Studying the central and peripheral processes underlying the development and maintenance of chronic neuropathic pain using human tissue has proved a challenging process for researchers, often limited by technological advances and ethics. Additionally, due to the limited usefulness of non-invasive techniques, human tissue has traditionally been limited mostly to autopsy material, or biopsy, making results difficult to interpret, as it is difficult to discern whether biochemical changes are due to pathophysiology or long periods of treatment. Though recent advances in imaging techniques have made it a useful tool for visualising structural vulnerabilities, their use is limited, as it does not provide mechanistic or causal correlations. Using animal models to study the relationship between anatomical and biochemical changes, though having its limitations, has enabled much of the progress made in the field (Wang and Wang, 2003).
Wall and colleagues (1979) developed the first model for painful neuropathy, which involved a 5 mm resection of sciatic and saphenous nerves at the level of the mid-thigh, tightly ligated at the proximal stump. This neuroma model was shown to lead to complete denervation of the hindpaw. Later models attempted to mimic partial nerve damage, and these methods included the partial sciatic ligation (PSL) model, whereby a portion of the sciatic nerve is tightly ligated (Seltzer et al., 1990); the spinal nerve ligation (SNL) model, where one or more spinal nerves going to the foot are ligated and cut (Kim and
Chung, 1992); the spared nerve injury (SNI) model, involving cutting of the common peroneal and tibial nerves (Decosterd and Woolf, 2000); and the method employed in this thesis, the chronic constriction injury (CCI) model, developed by Bennett and Xie (1988), which involves placement of four loose chromic gut ligatures around the sciatic nerve.
These animal models are only relevant as to the extent to which they mimic the human clinical picture of neuropathic pain, both in its formation, in the development and maintenance of central and peripheral signs and symptoms, as well as the impact of treatment. Several methods have allowed identification of sensory disturbances in animal models, and these include the observation of paw guarding, limping, excessive grooming and biting, changes in exploratory behaviour, alterations in weight bearing, and
36 autotomy, all of which have been suggested as indicators of spontaneous pain (Bennett and Xie, 1988,
Wall et al., 1979). Tactile allodynia can be quantified using von Frey filaments to apply a quantitative force on specific cutaneous sites, and measure changes in tactile threshold following nerve injury
(Chaplan et al., 1994). Both modalities of thermal hyperalgesia following nerve injury can also be tested with Hargreave’s test using a radiant heat source positioned underneath the paw (Hargreaves et al.,
1988), and similarly, cold allodynia using an ice plate underneath the injured paw (Bennett and Xie,
1988).
1.6.1 Chronic constriction injury of the Sciatic Nerve (CCI)
Developed by Bennett and Xie (1988), and used in subsequent experimental chapters of this thesis, the
CCI model uses a set of four chromic gut ligatures placed loosely along the sciatic nerve, medial to the trifurcation point, at intervals of 1 mm, to ensure circulation through the superficial epineural vasculature is restricted, but not arrested. Subsequent pressure from the ligatures causes intraneural oedema, resulting in translucent, demyelinated constriction of 25–75% of the original diameter of the sciatic nerve, with swelling observed proximal to the constricted area. The presence of the chromic gut itself leads to a local immune reaction, further damaging the nerve. Demyelination occurs through the ligated region, with near-total loss of large, myelinated Aβ fibres, and substantial loss of small, myelinated Ad fibres (Munger et al., 1992).
The CCI model was developed to trigger observable changes that mirror the sensory signs and symptoms, which characterise the chronic neuropathic conditions clinically observed in human patients.
Following CCI, rats typically display thermal and mechanical hyperalgesia, as quantitatively shown by the use of von Frey filaments; thermal and tactile allodynia, as shown by behavioural changes post-CCI in Hargreave’s test and ice plate; behaviours indicating spontaneous pain, which include limping, guarding of the injured limb, altered weight bearing, partial autotomy, and excessive grooming (Bennett and Xie, 1988).
37 1.6.2 Behavioural and emotional changes post-CCI
While positive and negative sensory symptoms in neuropathic pain have been well demonstrated in the literature, far fewer studies have attempted to study the development of complex behavioural disabilities observed in the human clinical picture of neuropathic pain. Monassi and colleagues (2003) used the
CCI model to observe whether—in addition to the development of allodynia, hyperalgesia, and spontaneous pain—CCI triggers complex behavioural dysfunction in Sprague-Dawley rats such as is observed in human patients.
Consistent with previous studies, and as depicted in fig 1.1, all rats in the study showed reduced thresholds for mechanical pressure (hyperalgesia) following CCI, as well as thermal allodynia, as observed by changes in behaviour to thermal (cold) stimuli (Attal et al., 1990, Bennett, 1993, Bennett and Xie, 1988, Zimmermann, 2001).
38
Figure 1.1: Responses to chemical, tactile, and sensory tests as reported by Monassi and colleagues (2003).
A: Depicts withdrawal responses following application of five acetone droplets to either the injured or uninjured hindpaws. B: The number of paw withdrawal responses from a cold surface over a six minute period in injured and uninjured hindpaws. C: Changes in threshold force (using von frey filaments) required to evoke paw withdrawal. In all animals CCI results in sensory changes to affected hindpaw but not to uninjured contralateral hindpaw.
1.6.3 Social Interactions in rats
The issue of whether animal models provide an accurate comparison with humans in regards to emotional and psychological processing has long been debated. One of the key observations noted in human neuropathic pain patients suffering from behavioural dysfunction (i.e. depression and anxiety) is that of social and familial withdrawal, with sufferers unwilling to engage in perceived potentially stressful social situations. A number of different social stress situations are used in laboratory studies aimed at mimicking psychosocial stressors observed in human patients (Blanchard et al., 2001), with the activity of the hypothalamic-pituitary-adrenal (HPA) axis, using the prototypical marker cortisol as a marker of stress (either by change in plasma levels, saliva, or faeces), being well established and 39 consistent in male rodent species. Monassi and colleagues adapted a resident-intruder paradigm aimed at exposing CCI injured rats to chronic social stress (Blanchard and Blanchard, 1990, Blanchard et al.,
1975).
Monassi and colleagues exposed a “resident” male rat, situated in its home cage, to a weight-matched, uninjured, same-age, male “intruder”, over a five-minute period for five days pre-CCI (to establish mean behavioural patterns), and for six days post-CCI, to observe behavioural changes subsequent to injury. No resident was tested with the same intruder more than twice, and never met the same intruder on consecutive days. In addition to videotape recordings of the social interactions, using a low-light infrared camera, both sonic and ultrasonic vocalizations were recorded (22–28kHz range) by means of a bat detector. Subsequently, recordings of each resident-intruder interaction were analysed and coded into four main behavioural categories identical to those used previously by DePaulis and colleagues
(1992, 1994), which were based on the earlier descriptions of Grant and MacKintosh (1963).
Behaviours were classified as being:
i) Dominant: standing above, or leaning on, the supine partner; offensive sideways (lateral
attack) posture; biting targeted at the neck or back of the partner; chasing. For examples
see fig 1.2.
ii) Social: investigation or sniffing of the partner; often focused around the ano-genital region.
iii) Non-Social: cage exploration, self-grooming.
iv) Submissive: defensive alerting or freezing; defensive sideways or supine posture to the
approach/attack of intruder (often accompanied by 22–28kHz vocalization).
40
Figure 1.2 Video still showing examples of dominant behaviour displayed by resident rat towards intruder rat in resident-intruder paradigm.
A: Shows an aggressive resident posture on top of a submissive intruder. B: Upright defensive posture displayed by intruder towards dominant resident behaviour. C: Biting targeted towards the neck/back of the intruder. D: Resident animal chasing intruder rat.
Monassi and colleagues (2003) reported three clear behavioural patterns emerging from analysis of social interaction.
i) The first group, comprising around 50% rats, labelled as the no-effect group, showed
remarkably unchanged behaviour to the intruder post-CCI.
ii) A second group, comprising around 30% of the cohort, which Monassi coined the
persistent disability group, showed a large and significant reduction in dominance
behaviours, and an increase in non-social behaviour post-CCI.
iii) A third group, referred to as the recovery group, showed transient reductions in dominance,
initially similar to the persistent group, which had reverted to pre-injury levels by day four
of post-CCI testing.
41 1.6.4 Sleep-wake cycle changes
Monassi and colleagues (2003) further tested if nerve injury leads to similar sleep disturbances in the rat, as seen in chronic neuropathic pain patients (Meyer-Rosberg et al., 2001). Using electroencephalographic (EEG) and electromyographic (EMG) electrodes, slow wave sleep (SWS) and rapid eye movement (REM) sleep were measured during a 24-hour cycle consisting of 12 hours light,
12 hours dark. Three distinct sleeping patterns emerged:
i) The no-effect group, compared to their pre-surgery patterns, showed no change in the
amount of time spent awake, or in SWS or REM sleep.
ii) Persistent change animals spent a significantly greater period of time awake during both
dark and light phases. These changes were accompanied by a significant reduction in time
spent in SWS. REM sleep was unaffected.
Recovery animals showed a significant and persistent increase in awake time during the light phase, and a significant reduction in time spent in SWS during the light phase only.
1.6.5 Resident-Intruder CCI model
The data illustrated by Monassi and colleagues (2003) using a CCI model provide an animal model of chronic neuropathic pain showing strikingly similar disabilities to those shown by patients in a clinical setting. The CCI model not only shows the positive and negative clinical signs which are the hallmark feature of neuropathic pain, but a subgroup of animals also display the physiological, and behavioural disabilities observed in human patients. That all rats show similar levels of sensory disturbance, but differ in behavioural and sleeping dysfunction, indicates that the two types of disturbances associated with neuropathic pain, the sensory-affective component and the emotional-behavioural component, are most likely independent of each other.
Since this pivotal investigation, several other findings of significance, using the CCI model of neuropathic pain, have further highlighted the level of behavioural dysfunction observed in this group
42 of rats. Hypothalamic-pituitary thyroid, hypothalamo-pituitary adrenal (HPA), and hypothalamo- pituitary gonadal axes are selectively affected in persistent disability animals, in spite of all experimental animals showing similar levels of sensory disturbances following CCI (Kilburn-Watt et al., 2010).
Similarly, distinct and lateralised changes in the mesolimbic dopaminergic system have been observed following CCI, with rats with pain alone (i.e. no-effect rats) displaying high TH expression, and those with pain and persistent disabilities (i.e. persistent animals) displaying low TH expression (Austin et al., 2010). Levels of dopamine are thought to be reflective of coping strategies to a chronic stressor.
Increased dopamine in the NAcc may act to maintain the pre-injury affective-motivational behavioural repertoire of injured rats.
Following CCI of the sciatic nerve, there is also evidence for cellular injury in the midbrain of pain and persistent disability rats, which show astrocyte activation, a hallmark of cell death. Specifically, rats with pain and persistent disability following CCI showed pro-apoptotic ratios of Bax:Bcl-2 mRNAs; decreased heat shock protein 60 (HSP60) mRNA; increased levels of induced nitrous oxide mRNA; increased levels of MEK2 mRNA; and TUNEL-positive profiles in the lateral and ventrolateral PAG; as well as caspase-3 immunoreactive neurons in the mesencephalic nucleus of the trigeminal nerve
(Mor et al., 2011). These markers of cellular injury and death, specific to a subgroup of animals showing behavioural dysfunction in the presence of neuropathic pain highlight the importance of the midbrain
PAG in the maladaptive processes observed in neuropathic pain.
This ever-growing body of data strongly indicates that supraspinal sites that receive input from sciatic nerve recipient segments of the spinal cord are regions likely to play a role in modulating the neural circuits mediating maladaptive behavioural changes following CCI. The midbrain PAG, which receives significant direct spinal inputs, and whose functionally organised columns play a significant role in regulating social interactions (Keay and Bandler, 2001), sleep-wake cycle behaviour (Lu et al., 2006,
Sastre et al., 1996), activity of the HPA (Floyd et al., 1996) and HPG axes (Lakoski and Gebhart, 1982), and which connects to regions of the brain associated with affective-motivational changes following
43 the onset of neuropathic pain, as well as being a region that modulates descending inhibition of pain, is of particular interest.
1.7 Pain pathways
In order to understand the pathological processes at play in maladaptive neuropathic pain states, it is important to understand the neural architecture that signals noxious stimuli in the non-pathological setting. The pain signal begins peripherally, at the site of injury, synapsing in the dorsal horn of the spinal cord, where it is then interpreted locally via an interplay of interneurons, and also via supraspinal
(or descending) mechanisms from many brain regions (Heinricher et al., 2009). The resultant conscious experience of pain is thus not necessarily related linearly to the initial nociceptive input, but comes about as a result of a complex, nuanced matrix that takes into account external threats, competing behavioural needs, and homeostatic drive.
Pseudo-unipolar neurons, whose cell bodies are located in the dorsal root ganglia (DRG) have peripheral terminals acting as nociceptors in body tissues, as well as a central terminal which synapses in the spinal dorsal horn. In peripheral tissue, insult(s)/noxious stimuli are detected primarily by nociceptors, fibres of which lie in the dorsal root ganglia (DRG) and synapse in the dorsal horn of the spinal cord. At the level of the spinal cord these afferents synapse to form reflex loops; process information at spinal cord level via populations of interneurons; as well as projecting to spinal and supra-spinal areas (Almeida et al., 2004, Basbaum et al., 2009, Millan, 1999).
1.7.1 Peripheral receptors
Pain propagation commences with the activation of specialised receptors called nociceptors, which are widely found in the skin, mucosa, membranes, deep fascia, connective tissue of visceral organs, ligaments and articular capsules, periosteum, muscles, tendons, and arterial vessels. Nociceptors are free nerve endings constituting the distal ends of first-order afferent neurons of small diameter fibre A- d (myelinated) and C type (unmyelinated), the cell bodies of which are located in the DRG, and the trigeminal ganglion in the face (Basbaum et al., 2009). Activation of nociceptors is based on specific
44 sensory modality, e.g. mechanical, thermal, or chemical stimulus, and receptor fields vary depending on anatomical location.
1.7.2 Peripheral afferent fibres
These fibres project to the spinal cord, relaying information to the CNS on nociceptor activation by mechanical, thermal, or chemical stimulus. Peripheral afferent fibres are classified in terms of structure, diameter, and conduction velocity in humans as follows:
i) C fibres: 04.1.2 µM in diameter. Unmyelinated, slow conducting (0.5–2.0 m sec-1),
ii) Ad fibers: 2–6 µM in diameter, lightly myelinated, intermediate (12–30 m sec-1),
iii) Ab fibers: large > µM10, myelinated, fast conducting (30–100 m sec-1), typically respond
only to light touch.
Differentiated patterns of propagation are observed following nociceptor activation by noxious stimulus.
Ad propagate modally specific information, with marked intensity and short latency. They promote a quick sensation of first phase (acute) pain, triggering withdrawal actions. Ad fibres are classified into two groups, depending on responsiveness to mechanical and thermal stimulus (Almeida et al., 2004,
Basbaum et al., 2009, Millan, 1999). Type I respond to both mechanical and chemical stimuli but have relatively high heat thresholds (>50°). Type I Ad nociceptors will respond to heat stimuli if it is maintained; however, it may also become sensitised during tissue injury. Type II Ad have a much lower heat threshold but a very high mechanical threshold. C fibres propagate information in a slow manner, with prolonged potentials undergoing summation and inducing the manifestations of dull pain. C fibres are also heterogeneous, may respond to various sensory modalities (polymodal), and contain peptidergic and non-peptidergic subpopulations capable of releasing chemical mediators upon stimulation (Dong et al., 2001, Julius and Basbaum, 2001). Peptidergic nociceptors release the neuropeptides Substance P and calcitonin-gene related peptide (CGRP), and express the TrkA neurotophin receptor, which responds to nerve growth factor (NGF). Non-peptidergic nociceptors 45 express the c-Ret neurotrophin receptor that is targeted by glial-derived neurotrophic factor (GDNF), neurturin, and artemin (Dong et al., 2001, Julius and Basbaum, 2001). Functionally, nociceptors are also distinguished according to channel expression (fig 1.3), which confers sensitivity to distinct pain modalities such as heat (TRPV1), cold (TRPM8), acidic milieu (ASICs), and chemical irritants (TRPA1)
(Julius and Basbaum, 2001).
46
Figure 1.3 Schematic depiction of nociceptor subtypes.
Heat sensitive afferents express TRPV1; Cold sensitive afferents TRPM8; Polymodal nociceptors express chemoreceptors, mechanotransduction channels, a host of sodium channels, as well as potassium channels.
In skeletal muscles and joints, noxious stimuli in both Ad and C fibre activation produce: an aching, poorly differentiated, and less localised sensation than their cutaneous counterparts, fibres typically
47 display polymodal characteristics and are specialised to detect ischaemia/hypoxia and muscle fatigue.
In visceral organs both noxious and innocuous stimuli are propagated by Ad and C fibres. Stimuli is thought to be intensity related, with high intensity stimuli eliciting pain. The sparse organization and poor differentiation of receptors also means pain is poorly localised and may be referred to distant regions from the affected area.
1.7.3 Spinal Cord
The dorsal horn of the spinal cord is organised into anatomically distinct laminae, as depicted in fig 1.3.
B
A I II III IV C V
D
Figure 1.4 Schematic spinal cord.
Section depicting A: distribution of Spinal cord laminae I-V, where sensory afferents synapse. B: dorsal column fibres. C: fibres of the lateral funiculus. D: fibres of the anterolateral funiculus.
Sensory afferents entering the CNS do so via the DRG or cranial nerves V, VII, IX, and X. Within the spinal cord, nociceptive inputs enter into laminae I, II, V, and VI forming synapses in the dorsal horn with one or more types of neurons:
i) Projection neurons (PN)- which transmit information to supraspinal sites,
48 ii) Intersegmental propriospinal neurons (IPN)- integrate information between spinal cord
layers, as well as ipsi- and contralaterally,
iii) Interneurons-inhibitory (INI), or excitatory (INE)- which either diminish, or enhance,
sensory inputs. Interneurons are also divided into interlaminar and intrasegmental
intralaminar types.
More specifically, Ad fibres project primarily to lamina I, as well as lamina V, whilst C-nociceptors project more superficially in lamina I and II. Ab fibres, that are responsive light touch, project to laminae III, IV, and V. Furthermore, stratification occurs within laminae, with peptidergic C fibres terminating within lamina I and the dorsal aspect of lamina II, whilst non-peptidergic afferents converge around the mid-region of lamina II, with the ventral aspect of lamina II being dominated by INE
(Basbaum et al., 2009, Malmberg et al., 1997). Anatomical and electrophysiological studies have shown that lamina I spinal cord neurons, receiving inputs from Ad and C fibres, are responsive to noxious stimulation; laminae III and IV neurons preferentially respond to innocuous stimulation; and lamina V neurons receive mixed inputs. Populations of WDR neurons, mainly located in laminae IV, V, and VI, but also found in laminae I, II, X, and the ventral horn, incorporate cutaneous, muscle and visceral inputs, and code stimulus intensity. It is widely thought that the convergence of somatic and visceral inputs by WDR neurons likely contribute to the phenomenon of referred pain (Basbaum et al., 2009).
1.7.4 Afferent pathways from the spinal cord to supraspinal sites
Following direct or indirect interactions with the PN of the dorsal horn, the axons of second order neurons form discrete afferent bundles in the dorsal column, lateral funiculus, and anterolateral funiculus that transmit nociceptive impulses to brain structures which include the thalamus, the PAG, parabrachial region, reticular formation of the medulla, amygdala, septal nucleus, and hypothalamus
(Almeida et al., 2004, Millan, 1999, Sandkuhler, 2009).
The spinothalamic tract contains the axons of neurons carrying innocuous and noxious stimuli relating to pain, temperature, and touch. The spinothalamic tract can be further subdivided into three functional 49 components: the ventral spinothalamic tract, which projects to the nuclei of the lateral complex of the thalamus, carrying the sensory-discriminative component of pain; the dorsal spinothalamic tract, that projects to the posterior medial and intralaminar complex of the thalamus, and is involved in the motivational-affective aspects of pain; and the monosynaptic spinothalamic tract which projects directly to the medial nucleus of the thalamus, and is involved in the affective component of pain (Almeida et al., 2004, Basbaum et al., 2009, Tracey and Mantyh, 2007).
The spinoreticular tract consists of two projection components in the brain stem, one of them directed at the precerebellar nucleus in the lateral reticular formation, involved in motor control, and the other directed to the medial pontobulbar reticular formation involved in the mechanisms of nociception. The spinoreticular tracts are involved in the motivational affective and neurovegetative responses to pain
(Almeida et al., 2004, Millan, 1999).
The spinomesencephalic tract consists of two different components. The spinoannular bundle projects to the PAG and can be subcategorised into the portion that terminates dorsally (excitatory) and the spinotectal bundle, which terminates ventrally (inhibitory). Stimulation of these regions provokes aversive behaviours. The spinotectal bundle that reaches the superior colliculus is primarily involved in control of head movements in response to visual stimuli. The stimulation of regions innervated by the spinomesencephalic tract produces different responses, namely aversive behaviours in the presence of noxious stimuli, coupled with motor (visual) responses, autonomic, cardiovascular, motivational, and affective responses (Hylden et al., 1986, Yezierski and Mendez, 1991). the parabrachial nucleus receives direct afferents emanating from laminae I and II, as well as a mixture of collaterals forming indirect pathways. These fibres ascend contralaterally in the dorsolateral funiculus up to the brainstem, where they terminate in the mesencephalic and pontine portions of the parabrachial nucleus. At this level these pathways are involved in the propagation of visceral pain due to inflammatory processes and noxious thermal stimuli. Additionally, this tract gives origin to the spinoparabrachial amygdaloid pathway and the spinoparabrachial hypothalamic, which are thought to
50 influence the function of autonomic, motivational, and affective responses, as well as mediating neuroendocrine responses to pain (Almeida et al., 2004, Millan, 1999). the spinohypothalamic tract originates from laminae I, V, X, the lateral spinal nucleus, nucleus caudalis, and the central grey region of the spinal cord. It is composed of a heterogeneous population of spinal cord cells responding to both noxious and innocuous stimulation coming from muscles, tendons, joints, skin, and viscera. After projecting to the lateral hypothalamus, half of its fibres travel in the supraoptic decussation and go on to innervate the thalamus, amygdala, septum, and striatum. The afferent projections of this tract suggest that its projections contribute to the neuroendocrine autonomic, motivational-affective, and alert responses of somatic and visceral origin of pain (Almeida et al., 2004,
Millan, 1999).
The spinocervical tract, thought to have a vestigial role in humans, originates primarily from laminae
III and IV, and also receives inputs from laminae I, II, and V. The spinocervical tract reaches the lateral cervical nucleus in the medulla, whereupon it crosses the midline, ultimately terminating in the posterior and medial complex of the thalamus. Collaterals also exit into the midbrain PAG and superior colliculus.
The functions related to this tract concern the sensory-discriminative, motivational-affective, and autonomic characteristics of pain (Almeida et al., 2004, Ericson et al., 1996).
The postsynaptic dorsal column pathway originates mainly from laminae III and V, as well as VI and
VII. It is organised into a multi-synaptic pathway running ipsilaterally along the spinal cord up to the nucleus of the spinal column, whereupon it projects to the lateral thalamus and superior colliculus.
Additionally, it sends extensive direct and indirect projections along the gracile nucleus, playing an important role in the integration of sensory inputs from abdominal organs and from the skin (Almeida et al., 2004).
1.7.5 Supraspinal nuclei
What we experience as pain results from the activation and interplay of various neural structures, some of which are more associated with sensory-discriminative processes, commonly called the lateral pain system (Bushnell et al., 1999, Kanda et al., 2000), and others which are involved with the affective 51 component of pain, known as the medial pain system (Brooks and Tracey, 2005, Ingvar, 1999). It is the interaction and interpretation of nociceptive input at these sites (see fig 1.5) which gives us pain as a conscious experience, with the overall perception not necessarily related linearly to its nociceptive input, but influenced by memories, emotional, pathological, genetic, and cognitive factors (Tracey and
Mantyh, 2007).
Spinal projections to the brainstem are particularly important for integrating nociceptive activity with homeostatic, arousal, and autonomic processes, as well as providing a means to indirectly convey nociceptive information to forebrain regions following brainstem processing (Craig, 2003). These brainstem regions include nuclei that are vital for homeostatic function and include the solitary nucleus, the caudal and rostral ventrolateral medulla, and the A1-2 catecholamine cell groups (Craig, 2003).
There are also strong projections to the cerebellum and the brainstem reticular formation, regions that deal with somatomotor coordination, behavioural states, and postural states, and are capable of influencing descending modulatory systems (Craig, 2003, Verburgh et al., 1990).
Figure 1.5 Supraspinal nuceli of the pain pathway.
Projection neurons in the spinal cord convey noxious information to various brain nuclei which play different roles in processing information about the location and intensity of the stimulus; providing affective components to the pain experience; exerting descending feedback systems to regulate spinal cord neurons.
The PAG is a region of the brain best known for its roles in the descending modulation of pain, and its role as an important integrator of behavioural responses to physical and psychological stressors
(Bandler et al., 1991, Bandler and Depaulis, 1988, Behbehani, 1995, Keay and Bandler, 2001).
Structurally it is composed of anatomically distinct columns, which mediate active and passive emotional coping strategies to stressors and painful stimuli. In the PAG ascending pain stimuli are 52 integrated with descending influences from the diencephalon and the limbic forebrain regions such as the hypothalamus, amygdala, rostral components of the anterior cingulate cortex, insula, and orbitofrontal cortex. Due to the ability of its circuitry to either facilitate or inhibit pain it has been postulated that sustained activation of pronociceptive PAG circuits may be responsible for some chronic pain states (Gebhart, 2004, Porreca et al., 2002, Suzuki et al., 2004).
The thalamus is the main relay structure for sensory information destined to the cortex, and is intricately involved in the reception, integration, and transfer of nociceptive signalling. Distinct spinal laminae project to functionally and anatomically distinct divisions of the thalamus (Craig, 2003, Craig and
Blomqvist, 2002), and the pain literature generally divides these inputs and their projections into two parallel circuits, the lateral pain system, primarily dealing with the sensory discriminative aspect of pain, and the medial system, involved in the affective (cognitive-evaluative) component of pain. Lamina I neurons principally project to the ventral posterior nucleus (VP), the posterior part of the ventral medial nucleus (VMpo), the ventral posterior inferior nucleus (VPI), and the ventral caudal division of the medial dorsal nucleus (MDvc). Lamina V axons terminate in VP, VPI, ventral lateral nucleus, and intralaminar nuclei (Craig, 2003). The thalamus has been implicated in chronic pain, with studies showing decreased thalamic blood flow contralateral to the site of pain in cancer patients (Di Piero et al., 1991). Thalamic hypoperfusion has been shown in patients experiencing pain following PNS and
CNS lesions, reflective of either a decrease in neural activity or deafferentiation (Tracey and Mantyh,
2007).
From the thalamus, fibres of the lateral pain system project primarily to the primary (S1) and secondary
(S2) somatosensory cortex, which is primarily thought to encode location and intensity of painful stimuli (Bushnell et al., 1999, Kanda et al., 2000), whilst fibres of the medial system, encoding affective components of pain, project to the anterior cingulate and the insula cortex (Rainville et al., 1997, Vogt et al., 2003). The insula itself has been shown to be an intermediary between the medial and lateral systems, as it has been shown to encode intensity and localization of painful and non-painful thermal stimuli (Coghill et al., 1999, Craig et al., 2000) as well as being active during the affective experience of pain (Singer et al., 2004). Furthermore, meta-analysis of human data from positron emission 53 tomography (PET), MRI, electroencephalography (EEG), and magnetoencephalography (MEG) studies confirm that, during acute pain episodes, the commonest regions activated are S1, S2, the insula cortex, cingulate, and prefrontal cortices (Apkarian et al., 2005).
1.8 Descending control of pain
Supraspinal modulation of pain signalling is a complex process involving multiple roles of transmitter systems, which may either inhibit or facilitate signalling via heterogeneous receptor subtypes and complex interaction of interneurons. Briefly, terminals of descending pathways originating in the rostroventral medulla (RVM), the nucleus tractus solitarius (NTS), the parabrachial nucleus (PBN), the dorsal reticular nucleus (DRT), the PAG, the hypothalamus and the cortex interact with afferent fibres, interneurons and projection neurons in the dorsal horn. Actions at these sites, as a function of the influence of individual receptors upon cellular excitability, either suppress or enhance the passage of nociceptive information to the PAG, thalamus, hypothalamus, PBN, NTS, amygdala, and other cerebral structures involved in its secondary processing. These structures transfer nociceptive information to cortico-limbic regions and interact with other centres to modulate the activity of descending pathways themselves. Descending pathways modulate sympathetic outflow by actions at preganglionic sympathetic neurons in the intermediolateral cell column (IML). Following injury, sympathetic efferents modify nociceptive input to the dorsal horn via actions in the DRG. Within the ventral horn, actions of descending pathways at motor neurons may indirectly modify nociceptive input by the initiation of appropriate motor behaviours (Millan, 2002). The PAG in particular, is involved in many interconnecting pathways, and plays a pivotal role in the modulation of nociceptive processing. In addition, synaptic and chemical transmission, volume transmission, as well as the activation of glial and immunocompetent cells, also play a role in the interactions of descending pathways (Millan, 2002).
We can thus see how this integrated neural circuitry allows for a dynamic balance between inhibition and facilitation of nociceptive signalling. Furthermore, different behavioural, emotional, and pathological states are capable of attenuating or amplifying pain signals. An ever-increasing body of literature has elucidated the PAG, which is heavily interconnected with the hypothalamus and limbic
54 forebrain structures, as well as critical descending brainstem regions, plays a pivotal role in the functioning of this circuitry. Whilst we can identify the brain regions involved, we currently lack an understanding of how these distinct areas of the brain interact at cellular, molecular and systems level.
1.9 Peripheral sensitization
In order to understand the maladaptive processes that ultimately result in neuropathic pain, we recognise that living organisms recognize and react to harmful stimuli to avoid them. Nociceptors then, react to stimuli which can potentially or actually damage tissue, with peripheral sensitization representing a form of stimulus-evoked functional plasticity (Woolf and Ma, 2007).
Inflammatory mediators activate intracellular signal transduction pathways in the nociceptor terminal.
This prompts the cell machinery to increase production, transport, and membrane insertion of transducer channels and voltage-gated ion channels. Thus, functionally, the threshold for activation is reduced and membrane excitability increases, resulting in a heightened and erroneous response to stimuli. A reduction in thermal and mechanical thresholds also occurs in some patients with peripheral nerve lesions, which might reflect nociceptor sensitization owing to increased membrane excitability without inflammation (Fields et al., 1998).
Phenotypic switches occur in nociceptors in response to inflammation and axonal injury by virtue of exposure to retrogradely-transported signal molecules or absence of target-derived signals (Woolf and
Ma, 2007). If the peripheral axon is injured, or severed, disrupting contact of the cell body with its terminal, then profound changes in transcription occur. Some of these represent attempts by the neurons to survive the physical or metabolic insult, whilst others are attempts at axonal regrowth. Many axotomy-induced transcriptional changes are maladaptive and produce alterations that in function that can drive neuropathic pain. This can involve changes in receptor expression/activation; channel expression, function, and sensitivity; change in the molecular identity of the nociceptor (Tegeder et al.,
2006, Woolf and Ma, 2007).
55 1.10 Central Sensitization
Central sensitization refers to the process through which, following injury or inflammation, nociceptive neurons in the CNS show an increased responsiveness, leading to an enhanced processing of nociception (Sandkuhler, 2009, Woolf, 1983). The major factors leading to this state of hyperexcitability are alterations in glutamatergic systems; loss of GABAergic tonic inhibitory control resulting in disinhibition; and glial-neuronal interactions (Basbaum et al., 2009).
Hyperalgesia in areas adjacent to, or remote from, the site of injury (secondary hyperalgesia) also occur as a result of heterosynaptic facilitation, whereby non-noxious inputs from Ab afferents, normally responding to light touch, now activate pain transmission circuits, resulting in mechanical allodynia
(Basbaum et al., 2009, Campbell et al., 1988, Sandkuhler, 2009).
1.10.1 Glutamatergic systems
Transmission of acute pain signalling in the spinal cord occurs via glutamate release from nociceptor central terminals generating excitatory postsynaptic currents (EPSCs) in second order dorsal horn neurons. This occurs primarily via postsynaptic AMPA and kainate ionotropic glutamate receptors, with a summation of subthreshold EPSCs eventually resulting in action potential firing and transmission of nociceptive messaging to higher order neurons. Normally the NMDA subtype of glutamate channel will not be activated however, following injury, increased glutamate release from nociceptor terminals will activate NMDA receptors on postsynaptic neurons. The consequent increase in Calcium influx has the effect of causing downstream activation of numerous signalling pathways and second messenger systems which result in long term potentiation of synaptic strength (LTP) occurring in dorsal horn pain transmission neurons. The net effect of these cellular changes are exacerbated responses to noxious stimuli (hyperalgesia) (Basbaum et al., 2009, Sandkuhler, 2009).
1.10.2 GABAergic tonic inhibitory control
GABAergic inhibitory interneurons are densely distributed in the superficial dorsal horn where they regulate transmission of nociceptive signalling. Loss of function of these inhibitory interneurons
56 (disinhibition) in chronic pain states has been widely noted in the literature. Whilst the aetiology of this disinhibition is contested, peripheral injury leads to a decrease in inhibitory postsynaptic current in superficial dorsal horn neurons, leading to a marked decreased in tonic inhibition and resulting in enhanced depolarization and excitation of dorsal horn projection neurons (Basbaum et al., 2009).
1.10.3 Glial-neuronal interactions
Glial cells- microglia and astrocytes- also contribute to the central sensitization process that occurs in the setting of injury. Important differences exist between these two cell types in the context of pain. For example, microglia are activated after nerve, but not inflammatory, injury, whereas astrocytes contribute to nearly all chronic pain conditions. Both the time and duration of gliosis following nerve injury also differs between microglia and astrocytes, with microglial activation generally preceding and subsiding prior to astrocyte activation (Ji et al., 2019).
Homogeneously distributed within the grey matter of the spinal cord under normal conditions, following nerve injury microglia accumulate in the superficial dorsal horn within the termination zone of injured nerve fibres. Microglia also surround the cell bodies of ventral horn motoneurons, whose peripheral axons are concurrently damaged. These activated microglia release signalling molecules, including cytokines, which enhance neuronal central sensitization and neuropathic pain (Watkins and
Maier, 2005). Whilst the mechanism of activation of microglia is disputed, the net result is sensitization of lamina I neurons (Basbaum et al., 2009).
Astrocytes account for 20-40% of glial cells in the CNS, occupying non-overlapping territories, and are physically coupled to one another via gap junction protein complexes which directly link the cytosol of adjoining cells to allow free exchange of ions and small cytosolic components. Astrocytes provide metabolic support to neurons and contribute to the maintenance, regulation, and clearance of physiological levels of extracellular ions, glutamate, water, and proteinaceous waste products.
Astrocytes also form a structural barrier around synapses, thereby insulating them from glutamate spillover, while also providing metabolic support. Astrocytes play an important role in the control of
57 the formation, maturation, elimination, and maintenance of synapses and support synaptic function through a variety of diffusible and contact-mediated signals.
Under normal physiological conditions spinal cord astrocytes supress nociception via adenosine, receptors of which are expressed by sensory neurons (Goldman et al., 2010, Zhang et al., 2003), as well as via production of anti-inflammatory cytokines like type-1 interferons (IFN-1), which bind to cognate receptors on primary afferent presynaptic terminals (Liu et al., 2016).
Astrocytes have been implicated in the pathogenesis of neuropathic pain, with a clear correlation in animal models of peripheral nerve injury between astrocyte hypertrophy and pain hypersensitivity
(Garrison et al., 1991). Astrogliosis, the reactive response of astrocytes which is characterized by morphological, molecular, and functional changes, has been consistently observed in animal models of neuropathic pain (Garrison et al., 1991, Nesic et al., 2005), with a time course that is induced early after injury and sustained for considerable periods of time- indicative of a role in both the acute response to pain, and in the maintenance of chronic pain (Gwak et al., 2012).
1.11 Neuro-immune interations and neuropathic pain
The immune system appears to play a role in initiating and maintaining neuropathic pain, both at the
PNS and CNS. Inhibition of immune function represents a major avenue for therapeutic intervention for neuropathic pain.
1.11.1 Peripheral immune mechanisms
There is mounting evidence for the role of immune mechanisms playing a role in the initiation, but not the maintenance, of neuropathic pain. Nociceptors respond to a rich variety of immune receptors, with the interleukins IL-1b and IL-6, TNFa, bradykinin, and prostanoids possibly being the most important in activating and perpetuating the cascade of immune responses that occur following nerve damage.
This cascade ultimately results in macrophage infiltration, T cell activation, and increased expression of pro-inflammatory cytokines. Circulating levels of IL-1b lead to increased expression of nerve growth
58 factor (NGF), and NGF may sensitise nociceptors (Kanaan et al., 1998). A knockout model of IL-1 showed decreased hyperalgesia, with the effect observed most likely being peripheral (Wolf et al., 2006).
TNFa has been widely implicated as playing a role in neuropathic pain, and has been shown to initiate activity in nociceptors (Sorkin et al., 1997). Endoneural administration of TNFa induces hyperalgesia
(Wagner and Myers, 1996), and pre-emptive, but not post-injury, administration of the TNF- sequestering drug etanercept decreases hyperalgesia (Sommer et al., 2001). Inhibition of TNFa blocks phosphorylation of the MAP kinase p38 in DRG, and hyperalgesia, but again, only when administered pre-emptively (Schafers et al., 2003).
Additionally, Schwann cell injury also recruits macrophages via leukaemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 (MCP-1) (Sugiura et al., 2000), and MCP-1 inactivation in knockout mice has found to ablate mechanical hyperalgesia following a partial nerve ligation injury
(Abbadie et al., 2003).
1.11.2 Central immune mechanisms
There is mounting evidence for the role of central immune mechanisms playing a role in the initiation, but not the maintenance, of neuropathic pain. Microglia serves as the principal macrophage of the CNS, and interthecal delivery of the antibiotic minocycline, an inhibitor of microglial activation, attenuates neuropathic pain (Raghavendra et al., 2003). The mechanisms for activation are far from clear, and are likely multifactorial, but evidence exists for a role of the ATP receptors P2X4 and P2X7, both of which are expressed on microglia.
Activation of microglia produces expression of cytokines IL-1, IL-6, and TNFa, as well as nitric oxide
(NO), excitatory amino acids, ATP, and prostaglandins (Inoue, 2006). The chemokine fractalkine
(CX3CL1), expressed by neurons and astrocytes, may also be involved, with intrathecal administration of CX3CL1 producing pain in naïve animals (Abbadie, 2005), and blockade of its receptor (CX3CR1) attenuating hyperalgesia in neuropathic pain models (Milligan et al., 2005).
59 Toll-like receptors are a family of pattern recognition receptors that mediate innate immune responses to stimuli from pathogens or endogenous signals. The toll-like receptors TLR-2, TLR-3, and TLR-4 are involved in immune-mediated pain signalling in the dorsal horn (DeLeo et al., 2004, Guo and
Schluesener, 2007).
1.12 The periaqueductal grey (PAG) of the midbrain
The PAG comprises the cell-dense grey matter located around the cerebral aqueduct within the tegmentum of the midbrain, which is bordered laterally by descending tectospinal fibres, and is composed of predominantly small- to medium-sized fusiform-, triangular-, and stellate-shaped neurons that are functionally separated along distinct longitudinal columns that run across its rostro-caudal extent, as depicted schematically in fig.
Figure 1.6 Schematic diagram depicting the functional columns of the PAG through its rostrocaudal extent.
The dorsolateral and lateral columns are involved in active emotional coping, whilst the ventrolateral column mediates passive coping behaviours.
60 At its most rostral extent, the PAG runs in continuity with the periventricular grey matter of the hypothalamus, whilst at its most caudal, as the cerebral aqueduct expands to become the fourth ventricle, the PAG progressively narrows and disappears. The PAG excludes, by definition, functionally distinct nuclei, which lie within its boundaries, including the dorsal raphe nucleus, the Edinger-Westphal nucleus, and the oculomotor and trochlear nuclei. Primarily, it is involved in the descending modulation of pain and is an important integrator for a range of physical and psychological stressors, vocalization, lordosis, and cardiovascular control, with the columns facilitating different types of emotional coping strategies to different types of stressors (Bandler et al., 1991, Behbehani, 1995, Keay and Bandler,
2001). The PAG receives distinct sets of connections with forebrain, brainstem, and spinal regions
(Keay and Bandler, 2001).
1.12.1 Functional PAG columns mediating emotional-coping behaviours
The cytoarchitecture of the PAG allow for anatomically distinct columns to mediate active and passive emotional coping strategies appropriate to the stress, threat, or pain experienced (Keay and Bandler,
2001). In the coronal plane along the dorsal-ventral axis, the PAG contains four longitudinal columns, the dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG), and ventrolateral columns (vlPAG), which are differentiated not only by neural substrates and the behaviours they elicit (Bandler et al.,
1991, Bandler and Keay, 1996, Bandler and Shipley, 1994), but also by the distinct innervations they receive. Studies carried out using microinjection of excitatory amino acid (EAA) or electrical stimulation have shown that active coping strategies, characterised by vigorous motor reactions appropriate to a threatening environment, are organised by lateral and dorsolateral columns of the PAG
(Bandler et al., 2000). Furthermore, functional columns of the PAG are also organised on a rostro- caudal basis, with EAA injections into rostral areas of the lPAG and dlPAG eliciting a confrontational style of defensive response to stimuli, whilst EAA microinjection caudally within the lPAG/dlPAG evokes flight, with the animal turning and running from, rather than confronting, the potential threat
(Bandler et al., 2000, Keay and Bandler, 2001). These responses are also accompanied with appropriate, patterned increases in arterial pressure and heart rate. That is, after caudal microinjection of EAA into the lPAG/dlPAG increased blood flow to skeletal muscle, accompanied by decreased blood flow to 61 visceral and extracranial beds is observed; whilst following rostral lPAG/dlPAG activation increased blood flow to extracranial vasculature is noted (Bandler et al., 2000). Stimulation of the lPAG/dlPAG also evokes a short, lasting, non-opioid mediated analgesia (Fig 1.6).
Conversely, injections of EAA into the vlPAG evokes a passive coping reaction, dominated by quiescence, hyporeactivity, and non-responsiveness to the external environment, with associated hypotension and bradycardia (Bandler and Depaulis, 1988, Keay and Bandler, 2001). In addition, excitation of the vlPAG evokes an opioid-mediated analgesia, which is long lasting and may serve a protective function.
These patterned somatomotor and autonomic adjustments characteristic of active and passive emotional coping can readily be evoked in rats and cats in which forebrain structures, including the hypothalamus, have been severed (pre-collicular decerebrate), indicating that PAG connections with lower brainstem structures is sufficient to enable these responses.
1.12.2 Inputs and outputs of the PAG
Interestingly, the dlPAG does not receive spinal inputs, but both the lPAG and vlPAG are major spinal- recipient regions. Spinal projections to both the lPAG and vlPAG arise contra-laterally from superficial dorsal horn, deep dorsal horn, and the lateral spinal nucleus. The vlPAG also receives input from the upper cervical spinal cord, which includes a projection arising from the ventral horn (Bandler and Keay,
1996, Keay and Bandler, 2001). The PAG receives spinal inputs from cutaneous, deep somatic, and visceral primary afferent recipient areas, and various studies indicate that anatomically and physiologically identified PAG-projecting spinal neurons are activated readily by noxious stimulation
(Clement et al., 2000, Dougherty et al., 1999, Hylden et al., 1986, Yezierski and Broton, 1991). No topographical organization is apparent in the termination of spinal afferents within the PAG.
In addition to spinal afferents, the vlPAG receives direct input from general, visceral afferent recipient portions of the nucleus of the solitary tract (NTS), whilst the lPAG also receives sparse input from the
NTS (Herbert and Saper, 1992). The lPAG also receives strong projections from the laminar portion of the spinal trigeminal nerve, which appears to be topographically organised along the rostro-caudal 62 gradient of the lPAG (Wiberg et al., 1986). Both the vlPAG and lPAG project extensively to medullary regions, including the raphe nuclei and adjacent paramedian reticular formation in the ventromedial medulla (VMM), the rostral “vasopressor” (RVLM) and caudal “vasodepressor” (CVLM) regions of the ventrolateral medulla (Abols and Basbaum, 1981, Cameron et al., 1995, Carrive and Bandler, 1991,
Chen and Aston-Jones, 1996, Henderson et al., 1998). Whilst the vlPAG and lPAG broadly target the same cardio-regulatory medullary regions, they do so via different neurotransmitters and neural populations; consistent with evidence their activation has functionally opposed physiological effects
(Lovick, 1985, Lovick, 1992, Lovick, 1996, Verberne and Struyker Boudier, 1991).
The PAG also shares reciprocal connections with the hypothalamus, with early studies showing significant inputs to the PAG stemming from the hypothalamus, in particular the ventromedial nucleus of the hypothalamus, lateral hypothalamus, posterior hypothalamus, anterior hypothalamus, and the perifornical nucleus (Beitz, 1982). Of particular importance, the vlPAG projects to endocrine regulatory areas of the paraventricular nucleus of the hypothalamus (PVN) (Bhatnagar et al., 2000, Floyd et al.,
1996), an important site for the release of adrenocorticotrophic hormone (ACTH), and corticotrophin releasing factor (CRF), both of which are important in response to stress, as well as behavioural and cardiovascular changes. Further studies identified connectivity with three established areas recognised to be involved in the expression of defense responses and conditioned fear.
i) The anterior hypothalamic nucleus (AHN), which projects to all PAG divisions.
ii) The dorsomedial aspect of the ventromedial hypothalamic nucleus, which projects
mostly to the rostral dlPAG.
iii) The dorsal pre-mammillary nucleus (PMd), which projects to the dlPAG, and
rostral portions of the PAG.
The dlPAG projects into the AHN and the vlPAG into the dorsomedial hypothalamus (DMH), both predominantly from rostral areas (Canteras, 2002).
63 Amygdaloid projections to the PAG arise from the central nucleus and terminate in all PAG regions, with the exception of the dlPAG (Rizvi et al., 1991).
In higher order mammals the regions of the cortex that deal with emotional processing are the orbital and medial prefrontal cortex (OMPFC). In macaques, distinct PAG columnar patterns of innervation with areas of the OMPFC have been established. The dlPAG receives by far the densest OMPFC inputs, arising from the medial PFC wall. The vlPAG column receives selective input from orbital and anterior insular areas, as well as a weaker input from medial and dorsomedial PFC areas, whilst the lPAG receives input from the dorsomedial PFC. Parallel circuits from these areas of the OMPFC also innervate the hypothalamus (Floyd et al., 2000).
The reciprocal connectivity with various forebrain and brainstem regions known to be involved in emotional processing, as well as its direct innervation from spinal nociceptive input, suggests that the
PAG plays an important role in driving emotional-affective responses. In the context of chronic neuropathic pain, this makes it a region of strong interest.
1.12.3 The PAG in pain modulation
The PAG has been well established as an essential part of CNS circuitry that integrates and controls bidirectional nociceptive modulation. In a seminal study on the subject Reynolds (1969) reported that stimulation of the PAG produced such a profound analgesia that exploratory laparotomy could be performed without the use of chemical anaesthetics. Further work elucidated that PAG output is associated with both analgesia and pain facilitation, through stimulation of either the vlPAG or dPAG/lPAG, with vlPAG stimulation being associated with opioid-mediated analgesia, which is long lasting, whereas dlPAG/lPAG mediated analgesia is mediated via non-opioidergic mechanisms (Keay and Bandler, 2001). This spinally-mediated analgesia (inhibition) is largely achieved via relays to the
RVM, which then inhibits pain signal transmission at the dorsal horn level.
PAG output to the spinal cord can also facilitate hyperalgesic states such as those associated with inflammation, nerve injury, and opioid dependence (Ren and Dubner, 2002, Vanegas and Schaible,
2004). This is due to the RVM having a bidirectional switch, mediated by two types of neurons, those 64 that discharge just prior to the occurrence of withdrawal from stimulus (ON cells), and those that stop firing just prior to a withdrawal reflex (OFF cells). Selective activation of ON cells results in enhanced sensitivity to noxious stimulation (Neubert et al., 2004), while recruitment of OFF cells produces behavioural antinociception (Heinricher and Tortorici, 1994).
1.12.4 Autonomic nervous system modulation
The neuronal networks that control respiration, blood pressure and heart rate are located in the medulla.
That events such as fear, anxiety, anger, and pain can significantly alter blood pressure and heart rate gives us some insight into the involvement of PAG areas in modulating this brainstem circuitry.
Activities of nearly all hypothalamic nuclei can influence heart rate, and the insular cortex, amygdala, and prefrontal cortex have regulatory actions on the cardiovascular system. One of the key hypothalamic nuclei involved in cardiovascular regulation is the PVN, which via extensive afferents to the VLM and the NTS regulates medullary regions that control blood pressure and heart rate. Due to interconnectivity with these same nuclei, the PAG may also modulate, or mediate, the emotional aspects of cardiovascular regulation, and PAG neurons that modulate cardiovascular output appear to be viscerotopically organised (Behbehani, 1995, Carrive and Bandler, 1991).
1.12.5 PAG modulation of sleep and arousal
Sastre and colleagues (1996) showed that microinjections of the GABAA agonist muscimol into the vlPAG resulted in a significant increase in REM sleep. Furthermore, Lu and colleagues (2006) identified wake-active dopaminergic neurons by combining immunohistochemical staining for Fos and tyrosine hydroxylase (TH) in awake and sleeping rats, where they discovered a subpopulation of TH- immunoreactive (TH-ir) cells in the vlPAG express Fos protein during wakefulness, but not during sleep. Injections of 6-hydroxydopamine, which selectively kills TH-ir cells increased total daily sleep by ~20%. Furthermore, using tracing studies, they showed that this population of cells have extensive connections with the sleep-wake regulatory system, namely the ventrolateral preoptic area (VLPOe), which contains REM-active neurons, and the lateral hypothalamus, which contain orexinergic neurons, which cease firing during REM sleep.
65 1.13 Cholecystokinin
Cholecystokinin (CCK), derived from the Greek, bile-sack-move, was discovered early in the 20th century as a gastrin-like molecule of the gastrointestinal tract, where it plays an important role in the release of pancreatic enzymes, gall bladder contraction, and gastric motility. Whilst originally identified as a hormone in the gut, in the CNS CCK acts as a neurotransmitter (Larsson and Rehfeld, 1979,
Palkovits et al., 1982), acting on a broad spectrum of brain functions such as memory consolidation, satiety, anxiety, nociception, and lordosis (Lovick, 2008, Micevych and Sinchak, 2001, Wang et al.,
2005). CCK has often been found to be co-localised with other substrates in many neurotransmitter systems in the brain, including the dopaminergic neurons of the mesolimbic system (Hokfelt et al.,
1980), cardiovascular modulating circuits (Benkelfat et al., 1995, Gaw et al., 1995), and the interneurons of the hippocampus (Freund and Buzsaki, 1996).
1.13.1 CNS CCK synthesis
The synthesis and processing of CCK is cell-type specific, and commences with the complex processing of the precursor molecule preprocholecystokinin (Deschenes et al., 1984), which is then subject to tissue-specific post-translational processing. Prohormone convertases cleave the initial proCCK into several different active forms, the endocrine cell containing a mixture of CCK-33 and CCK-22, whilst the predominant form of CCK in neurons is sulfated CCK-8, with the smaller CCK-4 peptide also being widely distributed (Cain et al., 2003, Dockray, 1980, Rehfeld et al., 2007). Whilst aminopeptidase A and tripeptidyl peptidase II have shown to cleave CCK-8 in vivo, other clearing mechanisms are thought to play a role in its metabolism in the CNS (Migaud et al., 1995).
1.13.2 CCK expression in the CNS
Since discovery of its synthesis in the CNS, CCK has been shown to be one of the most abundant neuropeptides present in the brain, expressed in especially high levels in the hippocampus, amygdala, septum, olfactory tubercles, caudate nucleus, and the hypothalamus (Beinfeld et al., 1981, Crawley,
1985). In humans, radioimmunoassay analysis, in situ hybridization, and northern blot techniques have
66 been used to show that CCK is expressed in high amounts throughout the brain, with high levels in the neocortex, particularly in frontal regions, hippocampus, and subiculum; intermediate amounts in the caudate nucleus, putamen, nucleus accumbens, thalamus, hypothalamus, PAG, and substantia nigra pars compacta; low amounts in globus pallidus, lateral thalamic nuclei, mesencephalic, and metencephalic nuclei (Crawley, 1985, Lindefors et al., 1993, Savasta et al., 1990).
1.13.3 Neuronal actions of CCK
Peripherally, the actions of CCK involve satiety signalling, whereby CCK stimulation, due to the presence of fatty acids and proteins in the small intestine, acts directly on vagal afferent neurons that terminate in the NTS and activate ascending pathways that control ingestive behaviour, primarily via
CCK1 receptors (Dockray, 2012). Cholecystokinin is also thought to mediate satiety signalling via the hypothalamus, with neurons of the PVN being activated by peripherally administered CCK, though its role here is unknown (Lembke et al., 2011, Peter et al., 2010, Pirnik et al., 2010).
In the hippocampus, studies have shown that CCK:
i) A direct excitation of parvalbumin-expressing basket cells, thought to primarily generate
precisely timed, synchronised membrane potential oscillations in hippocampal circuits
(Freund, 2003, Freund and Katona, 2007, Glickfeld and Scanziani, 2006).
ii) A CCKc-receptor mediated an increase in inhibition of CA1 pyramidal cells, and dentate
gyrus granular cells, via cannabinoid mediated pathways (Karson et al., 2008).
CCK, therefore, acts as a molecular switch for processes affecting hippocampal principal cells
(Glickfeld and Scanziani, 2006, Hefft and Jonas, 2005), processes that may be affected in mood-related behavioural disorders, with Chhatwal and colleagues (2009) having shown that extinction learning in the amygdala is mediated by endocannabinoid and CCK neurotransmitter systems.
The HPA response to acute stress is mediated by the PVN of the hypothalamus, with a wide body of research showing that neuronal activity in the PVN is increased by exposure to stressors (Cullinan et
67 al., 1996, Imaki et al., 1993, Sharp et al., 1991). Interestingly, the response of the PVN is modulated by previous experience with stressors (Bhatnagar et al., 2000), and chronic stress is associated with enhanced HPA responsiveness to acute stressors, despite negative feedback mechanisms of circulating glucocorticoids (Akana et al., 1996, Bhatnagar and Dallman, 1998, Young et al., 1990). The posterior portion of the PVN in particular, has been shown to express a moderate density of CCK2 receptors, which when blocked, produce a marked increase in ACTH in response to chronic stressors in rats
(Bhatnagar et al., 2000). Neurons in this same region of the brain were shown to project to midbrain nuclei expressing CCK, chiefly, the vlPAG, the dorsal raphe, and the parabrachial nucleus.
In the periphery, CCK affects the gut vasculature via CCK1-receptor mediated vagal afferents within the intestinal mucosa (Gieroba and Blessing, 1992, Sartor and Verberne, 2006, Sartor and Verberne,
2008), through inputs to the brainstem RVLM, and consequent spinal relays (Guyenet, 2006, Sartor and
Verberne, 2003). Whilst originally thought to occur strictly via peripheral mechanisms, CCK has been extensively shown to evoke centrally-mediated effects following systemic administration both in humans and in rats (Eser et al., 2007, Hernandez-Gomez et al., 2002, Khan et al., 2008, Wang et al.,
2005). Panic-like symptoms evoked by systemic administration of CCK2-receptor agonists (de
Montigny, 1989) can be reproduced by stimulation of the amygdala, hypothalamus, and PAG (Hilton and Redfern, 1986, Schenberg et al., 2001), with imaging studies in humans verifying the involvement of these brain regions (Benkelfat et al., 1995, Eser et al., 2007). These symptoms are accompanied by corresponding cardiovascular changes including tachycardia, hypertension, cutaneous vasoconstriction, and skeletal muscle vasodilation (Lovick et al., 2000).
1.14 Thesis aims
Monassi and colleagues were the first to show that chronic constriction injury of the sciatic nerve leads to behavioural disabilities in a subset of rats despite all rats experiencing identical levels of pain. Since this pivotal study, others have also shown that, following CCI, rats can be categorized into (1) Pain alone; (2) Pain and transient disability; and (3) Pain and persistent disability (Keay et al., 2004,
Kilburn-Watt et al., 2010, Monassi et al., 2003, Mor et al., 2010). Animals which experience disability
68 in addition to pain also show altered sleep behaviour, as well as altered sensitivity of the hypothalamo- pituitary adrenal (HPA), hypothalamo-pituitary gonadal (HPG) and hypothalamo-pituitary thyroid
(HPT) axes (Kilburn-Watt et al., 2010). This is distinctive from animals which experience Pain Alone, which show no alterations in circuitry associated with sleep and stress centres of the brain.
Given that the complex behavioural and physiological disabilities resulting from CCI involve alterations in social behaviours; sleep disturbances; and changes in the activity of stress regions of the brain in a specific subset of animals, despite identical sensory changes, it is most likely that supra-spinal areas are involved. Of specific interest in this study was the midbrain PAG, which receives significant direct spinal inputs and whose longitudinally organised, functionally distinct columns play significant roles in regulating social interactions (Keay and Bandler, 2001); the activity of the HPA axis (Bereiter and Gann, 1990, Floyd et al., 1996) and the HPG axis (Barone et al., 1981, Lakoski and Gebhart, 1982); and sleep-wake cycle behaviours (Lu et al., 2006, Sastre et al., 1996).
This thesis first examines the effects of CCI on behaviour, as a subpopulation of rats that undergo complex behavioural changes following CCI is identified, in addition to showing that all rats experience similar levels of the clinical sensory hallmarks of neuropathic pain (Chapter 3). Additionally, retrograde tracing techniques are useful in allowing for the assessment of neuronal connections from a population of neurons to their various targets throughout the nervous system. This is a stressful surgical procedure for rats as it involves stereotaxic surgery and recovery. In the context of CNS studies the surgical procedure includes exposure and careful drilling of the skull, penetration of the meningeal membranes, and injection into the desired brain region with a glass micropippette. This surgery is a considerable stressor and may have behavioural consequences for the animal. Therefore, can retrograde tracing studies be used in conjunction with CCI in rats without altering the reproducibility of social behaviours observed in animals?
Using Affymetrix Gene-Chip technologies and qRT-PCR, Mor and colleagues (2010) have reported that six days following sciatic nerve CCI, the time at which rats with transient disability have recovered and returned to their pre-CCI patterns of social behaviour, rats with pain and persistent disability
69 showed a select and significant up-regulation in 13 mRNAs in the PAG, which included the mRNA of the neuropeptide cholecystokinin (CCK). Given the columnar organization of the PAG, and its various functions, it is necessary to investigate where CCK mRNA- expressing neurons in the PAG are located, if we are to elucidate their function. As such, the second experimental chapter of this thesis (Chapter 4) deals with a further examination of CCK mRNA up-regulation in the midbrain, using in situ hybridization techniques to identify anatomically specific sites within the midbrain. Additionally, using in situ hybridization techniques in combination with fluorescence immunohistochemistry, we also attempt to identify specific neuronal populations that may be co-expressing these changes.
Two major subtypes of CCK receptor have been identified: CCK1 and CCK2. Whilst formerly thought to be restricted to the periphery (where they are the predominant form), CCK1-receptors have been shown to be widely distributed in the CNS, particularly the brainstem (Mercer and Beart, 1997, Mercer et al., 2000). The CCK2-receptor is, however, the most common isoform in the CNS, especially in forebrain structures. Data expressed in Chapter 5 attempts to identify whether the cognate receptors of
CCK, the CCK1- and CCK2 –receptors, are expressed in the PAG, their patterns of distribution, and changes that occur to the expression of these receptors following CCI.
70 Chapter2 Methods
2.1 Ethics
All experimental procedures were conducted in accordance with the guidelines of the National Health and Medical Research Council’s (NHMRC) Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes (8th edition, 2005). All procedures were approved by the University of
Sydney Animal Care and Ethics Committee.
2.2 Animals
Male Sprague-Dawley rats were obtained from Gore Hill Animal Facility, St Leonards, NSW. Rats weighed ~200g on arrival, were housed in groups of eight, and were provided with food and water ad libitum. Animals were kept on a 12-hour day cycle, with lights in the animal house turned on from 7 pm to 7 am. Animals were between 240–350g at time of experimentation.
2.3 Surgical procedures
All surgical procedures were undertaken under sterile conditions. All surgical instruments were washed in a sonicator and sterilised by immersion in 50% ethanol in distilled water during surgery. An antiseptic solution (Betadine) was applied to surgical instruments, as well as all cutaneous areas of the animal exposed for surgery. Vital signs such as respiration and heart rate were monitored, and body temperature monitored via rectal probe, and maintained between 36°C and 37.5°C (homeothermic blanket, Hadland photonics).
2.3.1 Chronic constriction injury of the sciatic nerve
On day five of the resident-intruder paradigm, following the six-minute intruder interaction, each resident animal underwent a chronic constriction injury (CCI) of the right sciatic nerve as described by
Bennett and Xie (1988).
71 Rats were initially anaesthetised in an airtight chamber, where general anaesthesia was induced (5% halothane 2L O2). Once induced, a subcutaneous injection of atropine sulphate (0.25 ml, 0.65 mg/ml) was administered to assist respiration. Anaesthesia was maintained via use of a customised face-mask during the entirety of the surgical procedure (2% halothane 1L O2). Adequate anaesthesia levels were indicated by a lack of response to toe pinch, and the absence of the blink reflex, and were monitored throughout all surgical procedures.
The right leg was shaved and placed perpendicular to the body. Foot pinch and blink reflex were observed to ensure each rat was under an adequate level of anaesthesia. An incision was then made, just below the femur, and extended by blunt dissection through biceps femoris to expose the sciatic nerve.
A section of the sciatic nerve medial to the sciatic nerve trifurcation was exposed, isolated, and tied with four chromic gut ligatures (5–0, 1 mm apart) in such a way as to ensure circulation through superficial epineural vasculature was retarded but not arrested. The incision was then sutured using interrupted sutures (silk 3–0), Povidone-Iodine (Orion Laboratories Pty. Ltd., Australia) and triple antibiotic powder (Tricin, Jurox, Australia) were administered topically to prevent infection of the wound. Quinine was also topically administered to ensure the rats would not bite at their sutures. Each rat was allowed to recover from anaesthesia in a clean plastic cage resting on a heat pad (Quickheat
Industries, NZ), and recovery of each animal was closely monitored. Observations were documented, specifically regarding: (i) the animal’s gait, most notably in relation to post-injury changes in walking and cage exploration, the degree of limping or favouring of the opposite hind limb; (ii) resting postures of the hind limb, in particular protective “guarding” postures; and (iii) the condition of the hindpaw, including signs of excessive grooming or autotomy (Wall et al., 1979). Resident/intruder testing resumed the following day and continued for six days post-CCI.
2.3.2 Retrograde tracing experiments
Rats were anaesthetised with an intraperitoneal injection of ketamine and xylazine (7.5 ml ketamine, 2 ml xylazil, 0.5 ml saline, 1 ml/kg) before being placed in a stereotaxic frame in the flat skull position
(Paxinos & Watson, 1997). Foot pinch and blink reflex were observed to ensure each rat was under an
72 adequate level of anaesthesia. The skull was exposed, a surgical window created, and a sterile dental drill used to bore through the skull. Following boring of the skull the dura was retracted to expose brain surface.
Injections of the retrograde tracer cholera toxin subunit B (CTB, 1%) were made using a single barrel glass micropipette (tip diameter 10–20 µm), which was lowered and positioned relative to Bregma -12 mm, midline +0.7 mm, surface -6 mm in order to inject into the ventrolateral PAG at a 35° caudo-rostral angle to avoid leakage of the tracer into dorsally adjacent columns of the PAG. Using an automated air pressure system (pneumatic picopump), microinjections (50 nl) of CTB were applied via brief air pulses
(10 ms) to the pipette barrel. To determine the volume of injectate, the level of the meniscus in the micropipette was monitored via a calibrated graticule in a dissecting microscope. Each injection was made over a period of 10 minutes after which time the pipette remained in situ for a further 10 minutes to avoid leakage of the tracer along the pipette track dorsal to the injection.
Animals were then given a 2 ml intraperitoneal injection of physiological saline before being returned to their home cage. Recovery was monitored. Animals were allowed to recover seven days post-surgery, to allow transport of the retrograde tracer, before resident/intruder paradigms and CCI were implemented.
Data from 44 injections, in which there was no apparent spread of the tracer, were analysed.
2.4 Behavioural testing
2.4.1 Resident/intruder testing general procedure
The resident/intruder paradigm was conducted over a period of 11 days. Following habituation,
“resident” animals (n=95) were individually housed in home cages throughout the testing period. At approximately the same time on each day an age, weight and sex-matched animal, known as an
“intruder”, taken from a group-cage was introduced to the home cage of the resident animal for a period of six minutes. The same intruder was never presented to the same resident on consecutive days nor
73 more than twice throughout the testing paradigm. Each six-minute interaction was video recorded using a low light infrared camera for subsequent analysis.
2.4.2 Analysis of social interactions
Following video recording on each day of the paradigm, the behaviour of each animal was analysed over the six-minute test period using a signal box attached to a MacLab and Chart3.6 software in order to record the duration of certain behaviours (criteria previously established by Monassi et al., 2003; descriptive terms for behaviour based on Grant & Mackintosh, 1963). Behaviours were classified as being:
• Dominant: standing on top of intruder, biting to the back, neck or tail, lateral attack or chasing
of the intruder
• Social: general sniffing and investigation of the intruder, often particularly focused around the
ano-genital region
• Non-social: exploration of the cage and self-grooming, behaviour not directed towards the
intruder.
• Submissive: supine posture, defensive retreat movement, “freezing”.
The four behaviours were mutually exclusive and the animal always displayed one of the four behaviours at any one time. To ensure comparability between all studies in the laboratory all scorers were pre-trained to 90% reliability using training sessions consisting of random six-minute interactions.
2.4.3 Categorization of animals
Each resident animal was classified into one of the three groups according to the level of dominance displayed in the six-days following CCI relative to the mean of the three days directly preceding CCI.
Three distinct behavioural groups were defined in the following way:
74 • Pain and persistent disability: a decrease of at least 30% in the duration of dominance
behaviour displayed for at least 75% of the six days following CCI (at least five of the six post-
CCI days).
• Pain and transient disability: transient reduction (three or four days) of at least 30% in the
duration of dominant behaviour relative to pre-CCI levels. These animals then return to a
minimum of 70% of their pre-CCI levels of dominance in the final two to three days of testing.
• Pain alone: minimal or no change between pre- and post-CCI dominance levels
2.4.4 Statistical analysis of behavioural groups
Results are presented as means ± (SEM) with comparisons between groups being made using a two- way analysis of variance (ANOVA), followed by a one-way analysis of variance (ANOVA) for each experimental day. A repeated measure ANOVA was employed for analysis within each experimental group. Fischer’s protected least significance difference (PLSD) and Bonferroni’s correction tests were both utilised for post-hoc analysis.
2.5 Sensory testing for allodynia and hyperalgesia
Sensory testing was conducted on 36 resident animals ~1.5 h after resident-intruder testing, throughout the duration of the resident/intruder paradigm. Sensory testing for both tactile and thermal sensitivity was tested on alternate days and consisted of the following:
2.5.1 Tactile (mechanical) sensitivity testing
The threshold force, which evoked a withdrawal response, was determined as follows. Each rat was placed inside a test cage, with a mesh floor that allowed access to the plantar surface of the paws. After a 20-minute period of adaptation von Frey filaments with bending force corresponding to 0.3, 0.4, 0.8,
1.2, 2, 4, 6, 7, 12, 15, 20g were applied perpendicular to the plantar surface of both hindpaws (Cui et al., 1996). The test commenced with assessment of the withdrawal threshold in the intact paw. The
75 filament was pressed against the paw for 1-2 sec until it bent. Each filament was applied 10 times, starting with the softest and continuing in ascending order of stiffness. A brisk withdrawal of the paw was considered a positive response, with a 50% withdrawal rate designated as the threshold value for withdrawal. Allodynia was considered to be present when there was a 50% withdrawal response evoked by filaments corresponding to 6g or less (Cui et al., 1996).
2.5.2 Thermal sensitivity testing
Each rat was placed on a glass plate that sat inside an open-topped plexiglass cylinder for a six-minute period. The glass plate had been cooled to a temperature of 11±1°C by placement on freezer bricks and temperature monitored with a thermometer. The frequency and latency of hindpaw withdrawals during the six-minute test period were recorded.
2.5.3 Statistical analysis of sensory tests
Results for sensory tests are presented as means ± (SEM) for injured paws. No changes were observed in uninjured paws. Pre- and post-CCI thresholds for each hindpaw were compared for each of the three behavioural groups. Comparisons were also made between animals that had only undergone CCI (n =4 ) and four animals that had undergone stereotaxic surgery and CCI, in order to observe if stereotaxic surgery affected sensory thresholds pre- or post-CCI. Statistical analysis was made using two-way
ANOVA, using a Bonferroni post-hoc analysis.
2.6 Sacrifice, perfusion, and tissue removal
Following behavioural testing on the sixth day post-CCI, each animal was placed in a clean perspex chamber and briefly exposed to CO2 before being deeply anaesthetised via i.p injection of 0.15 ml sodium pentobarbitone (Lethabarb 130 mg/kg), made up to 1 ml volume with physiological saline. Once complete absence of reflexes occurred the animal was placed in a supine position, a midline incision of the skin was made posterior to the xyphoid process and whilst holding the xyphoid process, bilateral incisions were made across the ribcage to expose the diaphragm and abdominal visceral structures. The diaphragm was severed to expose the thoracic cavity, whilst the ventral aspect of the ribcage was
76 retracted to expose the heart. A perfusion needle (19G x 1.5”) connected to a motorised pump (Cole
Palmer, USA) was inserted into the apex of the heart (left ventricle), and the right atrium cut, to allow outflow of blood. This was followed by fixation with either: (i) paraformaldehyde in borate buffer (pH
9.6; 4˚C) for in situ hybridization and retrograde labelling experiments, including double-labelling studies, or (ii) 4% paraformaldehyde in phosphate buffer (pH 7.4; 4˚C) for CCK1- and CCK2-receptor immunohistochemistry. The portion of the injured sciatic nerve, brain, and spinal cord tissue were removed for experimental procedures and analysis.
2.7 Experimental procedures- In situ hybridization
2.7.1 Perfusion procedures
Following resident/intruder testing 36 rats were sacrificed, with the animal’s circulation cleared of blood by flushing through 500 ml of heparinised physiological saline, followed by 500 ml of 4% paraformaldehyde in borax (4°C, pH 9.6) to fix the tissue. The brain and injured sciatic nerve were removed and stored in 4% paraformaldehyde to post-fix overnight. Brains were then placed in 30% sucrose (in 4% paraformaldehyde) and stored at 4°C. All glassware was cleaned using an autoclave prior to use and milli-Q water was used in all solutions to ensure no RNA contamination occurred. All solutions were autoclaved prior to use.
2.7.2 Blocking and cutting
Midbrain sections of tissue were blocked, frozen in tissue-mounting medium (Tissue-Tek) and stored at -80°C, prior to use. Brains were mounted onto the freezing stage (-20°C) of a cryostat (Leica,
Germany) and cut coronally at 12 µm in 12 series dry mounted straight onto slides. Slides were stored in cryoprotectant at -20°C until reacted.
2.7.3 In situ hybridization
Three µl 35S dATP (1200 Ci/mmol, Dupont/NEN #NEG-034H) was aliquoted and added to 5 x tailing
buffer (Promega), 39µl ultrapure H2O (to give total volume of 50 µl), 4 µl oligonucleotide (4 µl of 1
77 pmol/µl stock) and 4 µl terminal deozynucleotidyl transferase (Promega). Contents were briefly centrifuged and incubated at 37°C for one to two hours before being separated on a Pharmacia sephadex
G50 DNA grade “Nick” column (GE Healthcare 17-0855-02). The specific activity of 2/µl of oligonucleotide probe was measured before the probe was freeze-dried for later use.
The probe was resuspended in 2 ml of hybridization and heated at 65°C for five minutes, then put on ice. Approximately 80 µl of probe was applied to each slide before carefully applying a coverslip. Slides were then incubated in a humid chamber at 37°C overnight. Coverslips were removed using 2 x standard saline citrate (SCC) with beta-mercaptoethanol (b-ME). This was followed by another two washes of
2x SCC with b-ME at room temperature and two washes of 1 x SCC in a water bath at 50°C. A 15- minute high stringency wash of 0.2 SCC at 50°C was followed by two washes in SCC at room temperature. Slides were quickly dipped in ascending concentrations of alcohol and allowed to air dry.
Slides were dipped in Amersham LM-1 emulsion (43°C) and allowed to drip off vertically for about 10 seconds. The slides were cooled on an aluminium plate on ice for 10 minutes and placed horizontally to set the emulsion. Once emulsion was set slides were placed vertically and allowed to dry in absolute darkness overnight. Slides were then placed in light-tight boxes, stored in the fridge away from radiation and allowed to develop for two to three weeks. Following this incubation, slides were dipped in Kodak
D19 developer for approximately 2.5 minutes and then dipped briefly in 0.5% acetic acid stop solution.
This was followed by two five-minute washes in 30% sodium thiosulfate solution before slides were thoroughly rinsed out in water changes until salts were removed. Slides were consequently counterstained using Toledeine Blue and passed through ascending alcohol series before being coverslipped with neutral mounting medium DPX.
2.7.4 Tissue analysis of in situ hybridization animals
For each animal analysis of cholecystokinin mRNA (CCK mRNA) was performed for the four columns of the periaqueductal gray (PAG): dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG), and ventrolateral (vlPAG) as well as the dorsal raphe nucleus (DRN) and the Edinger-Westphal nucleus
78 (EW, accessory III). Boundaries of nuclei where taken according to the rat brain atlas of Paxinos and
Watson (5th edition). CCK mRNA positive cells were identified using both darkfield (Olympus, Vanox) and light microscope (Olympus BX51), and were characterised by silver deposition throughout the cell body, which was indicative of individual CCK mRNA. A cell was considered CCK mRNA positive if the density of silver deposition was at least four times stronger than background levels. For each animal, six equidistant sections of the midbrain encompassing the rostrocaudal extent of the PAG (Bregma -8.8 to -6.3) were selected for analysis. Using a light microscope CCK mRNA positive cells in the PAG,
DRN and EW were counted bilaterally using a 1 mm x 1 mm graticule. The mean number (±SEM) of
CCK mRNA positive cells within each nucleus was compared between the pain and persistent disability, pain and transient disability, and pain alone groups of rats. Two-way ANOVAs with post-hoc
Bonferroni tests were performed on the data. Additionally, for each animal individual grains were counted per cell along six equidistant regions and the mean number (±SEM) of individual silver grains representative of CCK mRNA were compared between the pain and persistent disability, pain and transient disability, and pain alone groups of rats. Photomicrographs of representative sections of the midbrain were taken using a light microscope (Olympus BX51) with a digital camera attachment at x200, x400, and x1000 magnification.
2.8 Immunohistochemistry procedures
2.8.1 General perfusion procedures
Following resident/intruder testing on the sixth day post-CCI rats were sacrificed and the circulation cleared of blood using 500 ml of heparinised physiological saline, followed by fixation with either: (i) paraformaldehyde in borate buffer (pH 9.6; 4˚C) for in situ hybridization and retrograde labelling experiments, including double-labelling studies, or (ii) 4% paraformaldehyde in phosphate buffer (pH
7.4; 4˚C) for CCK1- and CCK2-receptor immunohistochemistry.
Brain, spinal cord, and injured sciatic nerves were removed and post-fixed in the same fixative for two hours post-fixation before being placed in 30% sucrose (0.1 M phosphate buffer, pH 7.4) for at least two days at 4˚C. 79 2.8.2 Blocking and cutting
Brains were taken out of sucrose and blocked into brainstem, midbrain, and forebrain sections. Blocks of brain tissue were mounted onto the freezing stage of a sledge microtome (Leica), or cryostat (Leica), using a tissue mounting media (Tissue-tek). Coronal sections were cut and collected serially as follows:
i) In situ hybridization studies: brains were mounted onto the freezing stage (-20°C) of a
cryostat (Leica, Germany) and cut coronally at 12 µm in 12 series dry mounted straight
onto slides. Slides were stored in cryoprotectant at -20°C until reacted.
ii) CCK receptor studies: brains were mounted onto the freezing stage of a sledge
microtome (Leica, Germany), and cut coronally at 25 µm in five series into plastic vials
containing 0.1 M phosphate buffered saline (PBS, pH 7.4).
iii) Retrograde tracer studies: brains were mounted onto the freezing stage (-20°C) of a
cryostat (Leica, Germany) and cut coronally. Midbrain PAG sections, for verification
of injection sites were cut at 40 µm in five series; brainstem sections, for projections
originating from the NTS were cut at; forebrain sections containing the hypothalamus
were cut at. All brain tissue was placed into plastic vials containing 0.1 M phosphate
buffered saline (PBS, pH 7.4) if tissue was to be reacted immediately, or placed in
cryoprotectant, and stored at -4°C.
2.9 Immunohistochemistry labelling with in situ hybridization (Chapter 4)
In experiments combining immunohistochemistry techniques with in situ hybridization to determine phenotype of cholecystokinin MRNA expressing cells, all immunohistochemical procedures were performed prior to in situ hybridization techniques.
Slides were rinsed three times in phosphate buffered saline (PBS) containing diethyl pyrocarbonate
(DEPC) for 10 minutes each time, followed by a 30-minute wash in a blocking/permeability buffer.
80 Finally, sections were incubated in DEPC PBS in the presence of 5-HT (rabbit) at a dilution of 1:2000, at room temperature for 24 hours.
Following the incubation period, slides were thoroughly washed using DEPC PBS before being incubated with anti-rabbit biotin (1:400) made up in DEPC PBS for two hours. Following thorough washing, incubation in ABC reagent (1:5) for one hour took place, before the visualization reaction with avidin Cy-3 (1:600) took place.
Visualization was promptly followed by in situ hybridization techniques.
2.9.1 Tissue analysis
5-HT-positive cells were counted along five equidistant regions and the mean number (±SEM) were compared between the pain and persistent disability, pain and transient disability, and pain alone groups of rats using a Cy3-specific filter on an Olympus BX51 microscope. Boundaries of nuclei where taken according to the rat brain atlas of Paxinos and Watson (5th edition). Additionally, silver grains representative of CCK mRNA were also concurrently observed in all animals using both an Olympus
BX51 and a confocal microscope (Leica) with a digital camera attachment at x200, x400, and x1000 magnification to observe if any 5_HT positive cells were co-localised with CCK mRNA.
2.10 Labelling for CCK1- and CCK2-receptor in the midbrain PAG
(Chapter 5)
Immediately after sectioning, free-floating sections were washed thoroughly in PBS (3 x 10minutes).
Sections were then washed in PBS containing sodium borohydride (1 mg/ml) before once again being washed thoroughly in PBS. Sections were then treated in a blocking/permeability buffer (PBS containing 10% horse serum/0.3% triton x100) before being incubated in PBS containing either CCK1- or CCK2- receptor antibody (1:3000, rabbit) (Beart et al., 2004, 2000, 1997) at 4°C overnight.
Following the overnight incubation period sections were washed three times in PBS before being incubated in the presence of a secondary antibody (Biotinylated donkey anti-rabbit, 1:500) in PBS
81 containing 1% horse serum for two hours at room temperature. Following consequent washing in PBS, visualization occurred by incubating in Cy3-conjugated streptavidin (1:500 in PBS) for 30 minutes.
Sections were then mounted, coverslipped in gelmount media, and kept out of light prior to analysis.
2.10.1 Analysis of CCK-receptor fluorescent immunohistochemistry
For each animal analysis of CCK1- and CCK2-receptor immunoreactivity was performed along equidistant sections encompassing the rostrocaudal extent of the four columns of the PAG, dorsomedial
(dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG). CCK-receptor positive cells were identified using a Cy3-specific filter on an Olympus BX51 microscope and were characterised by bright, punctate labelling of the cell body. The mean number (±SEM) of CCK-receptor positive cells within each column of the PAG was compared between the pain and persistent disability, pain and transient disability, and pain alone groups of rats. Two-way ANOVAs with post-hoc Bonferroni tests were performed on the data.
82 Chapter3 Changes to behaviour following CCI
3.1 Introduction
Monassi and colleagues (2003) have previously described varying levels in behavioural changes following CCI, despite all animals showing similar changes in pain sensitivity, i.e. allodynia and hyperalgesia. In humans, it has often been reported that it is this complex behavioural and physiological dysfunction, which includes disturbances to familial and social interactions, disturbance of sleep patterns, loss of libido, loss of appetite, reduced attention and cognitive function, and moderate to severe depression (Harding et al., 1994, Jensen et al., 2001, Menefee et al., 2000, Murray and Lopez, 2013,
Sternbach, 1974, Timmermans and Sternbach, 1974), which is more detrimental to the quality of life in chronic neuropathic pain sufferers than sensory disturbances.
The aim of this study was to evaluate the sensory and behavioural responses of rats following CCI.
Additionally, retrograde tracing studies are an important functional anatomical tool. The surgical procedure is, however, significant. It is therefore worth observing the feasibility of employing stereotaxic surgery and CCI; to observe if stereotaxic surgery affects the behavioural responses observed in the CCI model; and, given the time interval between the stereotaxic surgery and perfusion of animals, if reproducible retrograde labelling is possible. Of note in these pilot studies is that future experiments will have to take variability of recovery time into account, given the significance of stereotaxic surgery, and how this could influence behavioural results.
Two supraspinal regions that have strong projections to the PAG and are functionally involved in pain pathways are the solitary tract nucleus (NTS) of the brainstem and the hypothalamus.
The solitary tract nucleus (NTS) in the dorsal medulla integrates multiple viscerosensory processes and is organised in a roughly topographical manner that incorporates visceral afferent neurons transmitting gustatory, gastrointestinal, hepatic, renal, and cardiopulmonary afferent signals. The PAG has been shown to receive a dense, noradrenergic fibre plexus from the noradrenergic A2 neurons of the nucleus of the solitary tract (Clement et al., 1998, Herbert and Saper, 1992). This same population of neurons
83 in the NTS is activated in response to deep pain (Palkovits et al., 1995), as well as peripheral infusion of CCK (Viltart et al., 2006), thus making this region an obvious candidate for the source of increased
CCKergic transmission following sciatic CCI.
The largest number of afferents to the PAG arises from the hypothalamus (Behbehani, 1995), with hypothalamic afferents to the PAG terminating in distinct subregions of the PAG and showing discrete patterns of innervation. The lateral hypothalamus has a strong connection to the PAG (Behbehani et al.,
1988, Beitz, 1982, Veening et al., 1987), and stimulation of this hypothalamic region has not only shown to increase the activity of vlPAG neurons, but lateral hypothalamic stimulation has also been shown to inhibit dorsal horn nociceptive neurons (Carstens et al., 1983).
As part of this study the pattern of distribution of CTB-labelled neurons through two supraspinal regions following stereotaxic surgery and CCI will be descriptively described.
3.2 Methods
Results provided in this chapter are derived from all behaviourally tested animals used in this thesis (n
= 95). Protocols and procedures for resident-intruder testing, and sensory testing are as described in the previous chapter, with all rats having undergone CCI as described. Briefly, following a habituation period in its home cage “resident” male rats were subjected to daily resident-intruder social interactions with an age, weight, sex matched rat for five days pre- and six days post-CCI. In one group of rats (n =
36) sensory tests were conducted concurrently with social interactions in order to evaluate changes in mechanical and thermal sensitivity produced by CCI.
Additionally, a subgroup of animals also underwent stereotaxic surgery, and their behaviours pre-CCI compared to other CCI animals, with an unpaired t-test used for analysis of data points.
Rats used in this study (n = 40) underwent stereotaxic surgery for microinjection of the retrograde tracer cholera toxin subunit-B (CTB) in the midbrain PAG, whereupon they were allowed to recover for seven days before commencing resident-intruder testing and undergoing CCI. Following sacrifice, midbrain blocks were cut and analysed for location and spread of injection sites. Injection sites that were
84 restricted to either the vlPAG or the juxtaaqueductal grey were selected for analysis of brainstem and hypothalamic tissue. Brainstem and hypothalamic tissue were cut frozen at 30 and 50 µm, respectively, using a cryostat (Leica) and reacted using fluorescence immunohistochemistry techniques for the presence of CTB-immunoreactivity (CTB-IR). CTB-IR cells were counted along equidistant regions of the nucleus of the solitary tract (NTS) in the brainstem and labelled hypothalamic subnuclei.
3.3 Results
3.3.1 Social interactions
Ninety-five rats underwent sciatic nerve CCI and resident-intruder social interactions testing. As described in the previous chapter, each rat was categorised as either: pain and persistent disability; pain and transient disability; or pain alone; based on altered dominance behaviours in the resident-intruder test (Monassi et al., 2003). For the experimental chapters described in Chapters 4 and 5 of this thesis, a total of 11 pain and persistent disability, 10 pain and transient disability, and 12 pain alone rats, underwent CCI. Graphs summarising the resident-intruder behaviours of these rats are shown in Figure
3.1 (described below). These line graphs show the mean levels (±SEM) of dominance, social behaviour, non-social behaviour, and submissive behaviour, pre- and post-sciatic CCI.
85
Figure 3.1 Graphs comparing the mean duration in seconds (±SEM) of Dominance Behaviours, Social Behaviours, Non-Social Behaviours, and Submissive Behaviours.
On each day of the test period in rats with either Pain Alone (n=12), Pain & Transient Disability (n=10), and Pain & Behavioural Disability (n=11) rats. Significance between Pain & Behav- ioural Disability rats on each post CCI day with respect to Pain Alone animals is shown by * P<0.05, and with respect between Pain & Transient Disability rats and Pain Alone rats is shown by # P<0.05 (two-way ANOVA, Bonferoni test).
For the three days before sciatic nerve CCI (i.e. pre-CCI days 3–5), in response to the introduction of an intruder, the amount of time spent in each of the behavioural categories did not differ between any of the subsequently defined “disability” sub-groups. Dominance accounted for approximately 30%,
86 social behaviour 30%, non-social behaviour 40%, and submissive behaviour <5% of the residents’ behaviour during the six-minute observation period. Intruder rats typically displayed submissive behaviours, and often emitted ultrasonic vocalisations, consistent with earlier reports (Monassi et al.,
2003).
Rats in the pain alone category maintained their pre-CCI dominance levels on each of the six days of post-CCI resident intruder testing (n.s. one-way ANOVA). In contrast, the pain and transient disability rats showed significant decreases in dominance between post CCI days 2–4 (post-CCI days 2–3, p<0.01 one-way ANOVA; post-CCI day 4, p<0.05 one-way ANOVA) and the pain and persistent disability rats showed significant and ongoing reductions in dominance (post-CCI days 2–6, p<0.01 one-way
ANOVA). The reductions in dominance behaviour were due largely to decreased lateral and back- attacks, which when they did occur, were less intense. As well, the striking ‘standing on top’ behaviour of the resident (with the intruder often assuming a submissive position) was rarely observed in post-
CCI, pain and persistent disability rats.
The reduced dominance was predominantly replaced by significant increases in non-social behaviour.
The increased non-social behaviour usually consisted of self-grooming focused on the CCI-denervated hind-paw. We noted also occasional submissive behaviours in pain and persistent disability rats.
Of the 40 rats that had undergone stereotaxic surgery, CCI, and resident-intruder testing, there were 10 pain and persistent disability, 10 pain and transient disability, and 12 pain alone rats (n = 32). Graphs summarising the resident-intruder behaviours of these rats are shown in Figure 3.2.
87
Figure 3.2 Line Graphs comparing the mean duration in seconds (±SEM) of Dominance Behaviours, Social Behaviours, Non-Social Behaviours, and Submissive Behaviours.
On each day of the test period in rats with either Pain alone (n=17), Pain & Transient Disability (n=8), and Pain & Behavioural Disability (n=7) rats. Significance between Pain & Behavioural Disability rats on each post CCI day with respect to Pain alone rats is shown by * P<0.05, and with respect to Transient Disability rats is shown by # P<0.05 (two-way ANOVA, Bonferoni post hoc test).
88 In rats that had undergone stereotaxic surgery, for the three days before sciatic nerve CCI (i.e. pre-CCI days 3–5), in response to the introduction of an intruder, the amount of time spent in each of the behavioural categories did not differ between any of the subsequently defined “disability” sub-groups.
Dominance accounted for approximately 30%, social behaviour 25%, non-social behaviour 45%, and submissive behaviour <5% of the residents’ behaviour during the six-minute observation period.
Intruder rats typically displayed submissive behaviours and often emitted ultrasonic vocalisations, consistent with earlier reports (Monassi et al., 2003).
Rats in the pain alone category maintained their pre-CCI dominance levels on each of the six days of post-CCI resident intruder testing (n.s. one-way ANOVA). In contrast, the pain and transient disability rats showed significant decreases in dominance on post CCI days two and four (post-CCI days 2–3, p<0.05 one-way ANOVA; post-CCI day 4, p<0.05 one-way ANOVA) and the pain and persistent disability rats showed significant and ongoing reductions in dominance (post-CCI days 26, p<0.01 one- way ANOVA). The reductions in dominance behaviour were due largely to decreased lateral and back- attacks, which when they did occur, were less intense. As well, the striking ‘standing on top’ behaviour of the resident (with the intruder often assuming a submissive position) was rarely observed in post-
CCI, pain and persistent disability rats.
The reduced dominance was predominantly replaced by significant increases in non-social behaviour.
The increased non-social behaviour usually consisted of self-grooming focused on the CCI-denervated hind-paw. We also noted occasional submissive behaviours in pain and persistent disability rats.
3.3.2 Comparison of experimental groups
Given the invasive nature of the stereotaxic surgery, and the subsequent stress that it could pose on the animals, we strived to ensure that rats that had undergone stereotaxic surgery still exhibited comparable behaviours to other experimental animals. Of particular importance, given that the basis of classification of animals was into disability subgroups, was ensuring that no significant changes in dominance behaviours prior to CCI occurred between the different groups. Column graphs summarising the differences in behaviour pre-CCI are shown in Figure 3.3.
89
Figure 3.3 Summary of difference in behaviour pre-CCI.
Column Graphs comparing the mean duration in seconds (±SEM) of Dominance Behaviours, Social Behav- iours, Non-Social Behaviours, and Submissive Behaviours on each day of the test period in rats with either CCI only (n=34) and rats that had undergone stereotaxic surgery (n=32). Significance between groups is shown by * P<0.05 (unpaired t-test).
90 As shown by performing unpaired t-tests on rats which had undergone CCI only (n = 34) versus rats that had undergone stereotaxic surgery seven days before commencing resident-intruder testing and
CCI (n = 32), there were no changes observed in either dominance or social behaviours. Significant differences (P<0.01) were observed in non-social and submissive behaviours, with stereotaxic surgery animals showing higher time spent in non-social behaviours, offset by lower time spent in submissive behaviours. Conversely, CCI only animals, whilst spending lower time in non-social activities, had higher time spent in submissive behaviours.
3.3.3 Sensory testing
Sensory testing using von Frey filaments (mechanical) or ice plates (thermal) reveal evidence of allodynia, hyperalgesia, and spontaneous pain behaviours in all rats subsequent to CCI. However, all animals experienced similar levels of allodynia and hyperalgesia irrespective of changes in behaviour post-CCI.
Prior to CCI, in a group of animals having undergone CCI only (n = 36), the application of a 15.1g von
Frey filament was required, on average, to elicit hindpaw withdrawal in rats. Following CCI, the threshold filament required to evoke withdrawal on the injured hindpaw only dropped significantly, and by post-CCI days three and five had plateaued to 0.407g, as depicted in Figure 3.4a. Tactile sensitivity did not differ between pain alone, pain and transient disability, and pain and persistent disability groups.
Ice plate (thermal sensitivity) also resulted in no significant differences between behaviourally categorised animals (Figure 3.4b), either pre- or post-CCI. Prior to CCI, lifting of the hindpaw was negligible, whilst thermal testing post-CCI resulted in a significant increase in the frequency of paw lifts in the injured hindpaw in all rats, specifically rising from 8–10 paw withdrawals on post-CCI day two, 14–18 paw withdrawals on day four, and 17–20 paw withdrawals on post-CCI day 6. Lifting of the contralateral uninjured hindpaw was rarely observed, beyond the shifting of position within the perspex cage.
91
Figure 3.4 Line Graphs comparing a) responses to tactile (von frey) sensory b) thermal (ice plate) sensory tests in rats with either Pain alone (n=7), Pain & Transient Disability (n=5), and Pain & Persistent Disability (n=6) rats.
No significantly different changes were observed in groups post CCI.
3.3.4 CTB microinjections
Three microinjections of the retrograde tracer cholera toxin subunit B (CTB) were centred on the ventrolateral column of the PAG (P20, P25, P11, Figures 6.1A, 6.2A), and were made into intermediate
92 sections of the vlPAG (-7.8 to -6.8 relative to Bregma). In four other cases, the injection site was predominantly centred round the immediate vicinity of the aqueduct, a region known as the juxtaaqueductal grey (P35, P29, P34, P37, Figures 6.1B, 6.2B), with spread into adjacent vlPAG and lPAG. These injections were also made into intermediate areas of the PAG, (-7.8 to -6.8 relative to
Bregma).
Figure 3.5 Camera Lucida drawings.
Coronal sections through the midbrain with the location of retrograde tracer Cholera Toxin B indicated A: CTB injections predominantly in the ventrolateral column of the periaqueductal gray (vlPAG). B: CTB injections predominantly restricted through the juxtaaqueductal gray.
93
Figure 3.6 Photomicrographs showing retrograde tracer injection sites.
A: Low magnification, light field image of the PAG, approximately 7.3mm caudal to Bregma, showing a CTB injction in the ventrolateral PAG (vlPAG). B: Low magnification, light field image of the PAG approximately 7.8mm caudal to bregma, showing a CTB injection site encompassing the juxtaaqueductal region of the PAG.
Following microinjection into either the vlPAG or the juxtaaqueductal grey, retrogradely labelled neurons (Figure 3.6) were observed in the brainstem nucleus of the solitary tract (NTS), the caudal and rostral ventrolateral medulla, the dorsomedial medulla, and in the medullary raphe nucleus. In the hypothalamus, labelling was noted in the suprachiasmatic nucleus, lateral mammillary nucleus, arcuate nucleus, lateral hypothalamus, posterior hypothalamus, tuberal magnocellular, dorsal, dorsomedial, and ventromedial nuclei of the hypothalamus. We will first describe the distribution of retrograde labelled cells in the nucleus of the solitary tract (NTS).
94 3.35 Distribution of retrograde labelled cells in the brainstem following CTB injection in the
VLPAG/juxtaaqueductal PAG
Following injection of CTB in cases in which the injection is restricted to the ventrolateral periaqueductal grey (n = 3, P20, P25, P11), we observed CTB-labelled cells in the nucleus of the solitary tract (NTS), caudal and rostral ventrolateral medulla, the dorsomedial medulla, and in the medullary raphe nucleus. In this study we will concentrate on describing the distribution of the numerous retrogradely labelled neurons that were observed in seven equidistant regions of the solitary tract, commencing rostrally at -12.3 relative to Bregma and ending caudally at -14.2 relative to Bregma.
In the NTS distribution of retrogradely labelled neurons was mostly observed in the commissural,
Medius, and Intermedius subnuclei, with only rare, scattered retrograde labelling observed in the ventrolateral subnuclei of the NTS. The total number of retrogradely labelled neurons was about twice as high on the ipsilateral side compared to the contralateral side (Figure 3.7A), with the number of CTB-
IR neurons at caudal regions of the NTS, consistent with A2/C2 catecholamine neuron populations, being more numerous than CTB-IR neurons at more rostral sites.
In rats that received CTB injections into the vlPAG (n = 3, P20, P25, P11) and were subsequently stained for the octapeptide CCK-8 (Immunostar), the distribution of CCK-8 in the brainstem was observed in the NTS, the area postrema, and the caudal and rostral ventrolateral medulla. In this study we focused only on describing the distribution of neurons that expressed both CCK- and CTB-IR.
CTB-IR neurons were more numerous in the NTS ipsilateral to the CTB microinjection site, with a higher distribution of cells at caudal areas of the NTS compared to rostral areas. Throughout the rostro-caudal extent of the NTS, the expression of double-labelled neurons reflects this pattern of distribution, with a high proportion of CCK- and CTB-IR cells in the commissural, medial, and intermedius subnuclei of the NTS.
95
Figure 3.7 Column graph.
Depicting A: The distribution of CTB-IR neurons in specific subnuclei of the NTS in rats that were microinjected in the vlPAG (n=3). B: Distribution of CTB-CCK double labelled neurons.
In rats that received CTB injections into the juxtaaqueductal gray (n = 4, P35, P29, P34, P37) and were subsequently stained for the octapeptide CCK-8 (Immunostar), the distribution of CCK-8 in the
96 brainstem was observed in the NTS, the area postrema, and the caudal and rostral ventrolateral medulla.
In this study we focused only on describing the distribution of neurons that expressed both CCK- and
CTB-IR. CTB-IR neurons were more numerous in the NTS ipsilateral to the CTB microinjection site, with a higher distribution of cells at caudal areas of the NTS compared to rostral areas. Throughout the rostro-caudal extent of the NTS, the expression of double-labelled neurons reflects this pattern of distribution, with a high proportion of CCK- and CTB-IR cells in the commissural, medial, and intermedius subnuclei of the NTS.
97
Figure 3.8 Column graphs depicting distribution of Cholera Toxin.
A: Cholera Toxin subunit B-immunoreactive (CTB-IR)neurons in specific subnuclei of the nucleus of the solitary tract (NTS) in rats that were microinjected in the juxtaaqueductal gray region (n=4). B: Distribution of CTB-CCK double-labelled neurons in the NTS.
98 3.3.7 Distribution of retrograde labelled cells in the hypothalamus following CTB injection in
the VLPAG/juxtaaqueductal PAG
Following identification of injection sites restricted to the vlPAG (P20, P25, P11) or juxtaaqueduct (n
= 4, P35, P29, P34, P37), hypothalamic tissue was cut coronally at 50 µm, and analysed along five equidistant regions encompassing the levels -4.8 to -2.8 from Bregma. CTB-IR cells were located extensively throughout various subnuclei of the hypothalamus, including the dorsal premammillary nucleus, arcuate, lateral hypothalamus, posterior hypothalamus, tuberal magnocellular, dorsal hypothalamus, dorsomedial hypothalamus, and the ventromedial hypothalamus, with most nuclei showing clearly lateralised distribution, with the exception of tuberal magnocellular cells, and cells observed in the lateral mammillary nucleus. In particular, the strongest CTB-IR was located in the lateral hypothalamus, with the arcuate nucleus, and the dorsomedial nucleus also showing large numbers of CTB-IR cells.
99
Figure 3.9. Column graphs depicting total cell counts.
(A-D): CTB-IR neurons in the lateral hypothalamus in rats that were either microinjected in the vlPAG (n=3) or juxtaaqueductal gray region (n=4). (B-E): CTB-IR neurons in the arcuate nucleus of the hypothalamus in rats that were either microinjected in the vlPAG (n=3) or juxtaaqueductal gray region (n=4). (C-F): CTB-IR neurons in the Dorsomedial hypothalamus in rats that were either microinjected in the vlPAG (n=3) or juxtaaqueductal gray region (n=4). For all animals, CTB-CCK co-localised cell numbers appear in column next to total CTB-IR neurons, coloured black.
100 3.4 Discussion
Following CCI, animals could be classified into three distinct behavioural subgroups, pain alone, pain and transient disability, and pain and persistent disability, based on alterations to dominance behaviours. Pain alone animals showed no significant changes to dominance behaviours following injury; pain and transient disability animals showed initial reductions in dominance behaviours following CCI, but by the end of the testing period were indistinguishable from pain alone animals; and pain and persistent disability animals showed significantly reduced post-CCI dominance behaviours throughout the testing period. The distribution of CCI animals into each behavioural group is comparable to those described in earlier studies (Austin et al., 2010, Keay et al., 2004, Kilburn-Watt et al., 2010, Monassi et al., 2003, Mor et al., 2010), thus validating the consistency of this model.
Of novelty, animals which had undergone stereotaxic surgery seven days prior to resident-intruder testing, whilst showing some differences in non-social and submissive behaviours, showed no significant changes in dominance levels pre-CCI, and were also able to be classified into disability subgroups post-CCI in a manner comparable to CCI animals used in chapters 4 and 5 of this thesis, and to the literature.
It has previously been reported in the literature that interactions between a resident and intruder rat remain stable, even in the long-term (Blanchard and Blanchard, 1990, Blanchard et al., 1978, Monassi et al., 2003), and as such, given the stable nature of dominance behaviour observed pre-injury, we would not logically expect a natural variation in behavioural patterns. We can only assume, then, that the changes that occur, if they occur, are reflective of the animal’s ability to cope with CCI.
Consistent with previous studies using an animal model of CCI, the presence of hyperalgesia, allodynia, and spontaneous pain behaviours, in addition to alterations in gait, marked grooming and licking of injured hindpaw, and guarding behaviours, were observed in all animals (Austin et al., 2010, Bennett and Xie, 1988, Monassi et al., 2003).
101 In our study we observed the distribution of CTB-IR neurons through the rostro-caudal extent of the
NTS was predominantly ipsilateral, with a significantly higher number of neurons located in more caudal regions of the NTS. Previous studies report a similar pattern of retrograde label following injection into the PAG (Clement et al., 1998, Herbert and Saper, 1992, Viltart et al., 2006), and is characteristic of the distribution of the A2 population of noradrenergic cells located in the NTS.
Retrograde labelling observed in the hypothalamus also followed a predominantly ipsilateral pattern, and was particularly strong in the lateral hypothalamus, and the dorsomedial hypothalamus. This too is in keeping with patterns of retrograde labelling observed in previous studies (Behbehani et al., 1988,
Beitz, 1982, Veening et al., 1987).
3.4.2 Methodological considerations
As an ethical consideration, given the stressful nature of stereotaxic surgery, this group of animals were not sensory tested. Given the similarities in their behavioural responses both pre- and post-CCI we would not assume that their sensory threshold would be significantly different to other animals used in this study; however, we do recognise that may be a limitation of this study.
The data of eight rats that had undergone sciatic CCI is presented in this chapter. Due to the spread of
CTB injection sites observed in other stereotaxic surgery animals, cell counts would not have been a true reflection of cell populations projecting to the PAG and, as such, the data of those animals had to be omitted from this study. Similar issues were had with control animals, which had significant spread to the adjacent dorsal raphe, and tegmental nuclei. Further studies would focus on getting enough animals from each of the behaviourally categorised groups described in the previous chapters of this thesis to observe whether any significant differences in the number of CTB-CCK projecting neurons from the NTS or the hypothalamus are evident.
The vlPAG, where the up-regulation observed in this study appears to be most pronounced, receives inputs from the forebrain, brainstem and spinal cord. The potential ascending inputs will be addressed first, and specifically inputs from the lumbar regions of the spinal cord (those segments receiving input from the injured sciatic nerve). 102 There have been several studies investigating ascending input to the vlPAG. Yezierski (1988) used anterograde tracer injected into the lumbo-sacral enlargement to demonstrate the extensive connections of this section of the spinal cord to the midbrain in rat, cat and monkey. It was found that terminal axon labelling was present in the caudal vlPAG although decreased labelling intensity in this region was observed in more rostral sections.
Using retrograde and anterograde tracer techniques, Keay et al. (1997) showed that the vlPAG receives convergent afferents from lumbar and cervical enlargements as well as a strong projection from the nucleus of the solitary tract. Their anterograde experiments showed that after injecting tracer specifically into the superficial dorsal horn of the lumbar spinal cord, there was moderate anterograde labelling within the caudal lPAG whereas injections restricted to the deep dorsal horn of the lumbar spinal produced terminal anterograde label in the juxtaaqueductal region.
In the experiments conducted in the present study, each of these regions (vlPAG, lPAG and juxtaaqueductal PAG) was found to contain CCK-ir terminals with the vlPAG being especially well labelled at caudal levels. More specifically from their retrograde experiments, Keay et al. (1997) found that the vlPAG receives input from neurons located in multiple laminae of the dorsal horn and also the dorsal lateral funiculus.
Descending inputs to the PAG from higher cortical areas have also been defined (Floyd et al., 2000).
Specifically, the dorsolateral orbital, agranular insular and medial prefrontal cortical areas (including prelimbic, infralimbic, and anterior cingulate cortices) project quite selectively to the vlPAG and CCK mRNA has been identified throughout the medial pre-frontal cortex (Hebb et al., 2003). Inputs to the
PAG have also been demonstrated originating from the central nucleus of the amygdala (Rizvi et al.,
1991), which has also been shown to contain CCK mRNA (Schiffmann and Vanderhaeghen, 1991).
Thus there are several sources of descending input that are both CCKergic and terminate in the areas of the PAG where there is an observed up-regulation.
It is possible that the differences in CCK expression seen in the PAG are the end result of an ascending pathway such as the spinothalamic initially causing higher cortical effects, which in turn send efferents
103 back to the PAG via the pathways outlined above. In that case the signal would be initiated in the injured peripheral nerve before synapsing in the thalamus and processed in the forebrain, before finally causing differential tonic expression of CCK in the PAG.
Alternatively, the sciatic CCI could be modifying the processing of stimuli only during more complex activities, such as during a social interaction in the resident/intruder paradigm. During social interaction, the animal receives a complex array of visual, olfactory and tactile stimuli from the intruder, all of which contribute to the behavioural response. The sciatic CCI could potentially alter the processing of these stimuli at the level of the cortex, the result of which is altered output from the forebrain to the
PAG during the interaction.
3.4.3 Comparison to previous studies and functional considerations
In our study we observed the distribution of CTB-IR neurons through the rostro-caudal extent of the
NTS was predominantly ipsilateral, with a significantly higher number of neurons located in more caudal regions of the NTS. Previous studies report a similar pattern of retrograde label following injection into the PAG (Clement et al., 1998, Herbert and Saper, 1992, Viltart et al., 2006), and is characteristic of the distribution of the A2 population of noradrenergic cells located in the NTS.
Retrograde labelling observed in the hypothalamus also followed a predominantly ipsilateral pattern, and was particularly strong in the lateral hypothalamus, and the dorsomedial hypothalamus. This too is in keeping with patterns of retrograde labelling observed in previous studies.
Both CCK mRNA and peptide levels are increased in response to a range of stressful stimuli. Aversive stimuli such as exposure to the odour of a predator, or inescapable stressors such as repeated restraint or social isolation have been shown to increase CCK levels and/or expression in the PAG and also regions such as the amygdala and hippocampus. Repeated electroconvulsive shock has also been shown to increase CCK mRNA expression throughout the PAG with a particularly high level in the Edinger-
Westphal nucleus (Lindefors et al., 1993), and acutely administered morphine has been shown to increase CCK levels in the PAG (Rattray and de Belleroche, 1987, Rosen and Brodin, 1989). The CCI
104 could be classed as an inescapable physical stressor, which has the potential to evoke similar changes in the expression of CCK.
Several other studies have reported the existence of CCK immunoreactive fibres and terminals within the PAG of uninjured animals. Kubota et al. (1983) showed that CCK-LI was present in the PAG in both cell bodies and fibres, however pre-treatment with colchicine was required in order to reveal the presence of the cell bodies. This would seem to indicate that the CCK turnover in the cell body is high and that it is being transported out of the cell body (presumably to the terminal) and/or released immediately following synthesis. They reported a moderate to low density of CCK-ir fibres distributed heterogeneously throughout the PAG and Edinger-Westphal nucleus and CCK-ir cell bodies primarily in the ventral part of the PAG and Edinger-Westphal(Kubota et al., 1983).
Liu et al. (1994) showed that whilst CCK-ir fibres and varicosities were present at all rostro-caudal levels of the PAG, the greatest density was observed in the caudal two-thirds. Furthermore, they found that whilst CCK-ir was heterogeneously distributed throughout the caudal two-thirds, there was a selective concentration in a wedge-shaped region in the dorsolateral area of the PAG. In addition, within the most caudal third, CCK-ir was concentrated in the ventrolateral region, especially laterally, extending from the PAG as the nucleus cuneiformis(Liu et al., 1994).
This heterogeneous distribution of CCK-ir fibres in the PAG coupled with the fact that the PAG is comprised of distinct functional columns suggests that CCK potentially has multiple roles in the PAG.
More specifically, CCK may be responsible for eliciting distinctive behavioural responses to stressors according to its concentration and specific site of action within the PAG. CCKergic inputs from one region of the CNS may make selective synapses with a certain functional column of the PAG, or at least preferentially synapse there, whereas inputs from another region may synapse in another column. In this way, different stimuli could evoke different responses while using the same neuropeptide accounting for the heterogeneous distribution of terminals containing that peptide. Similarly, the same stimulus in two animals, for example a certain odour or a certain type of painful injury, could send more
105 or less collaterals to a specific area of the PAG, and these small adjustments in connectivity could elicit different responses, accounting for individual reactions to the same stimulus.
106 Chapter4 Selective up-regulation of CCK mRNA in the vlPAG and DRN
4.1 Introduction
In addition to the debilitating sensory disabilities associated with chronic neuropathic pain, namely allodynia and hyperalgesia, sufferers commonly report disturbances to familial and social relations; altered sleep habits; decreased appetite; social withdrawal; metabolic, endocrine, and sexual dysfunction; and clinical depression. In fact, it is often these disabilities which are reported as being more debilitating to quality of life of sufferers than sensory disturbances.
It has been reported that chronic constriction injury (CCI) of the sciatic nerve leads to altered behaviours in some rats despite all animals experiencing identical levels of allodynia and hyperalgesia (Keay et al.,
2004, Monassi et al., 2003). Altered behaviours previously reported include reductions in dominance behaviours in resident-intruder social interactions; reduced sleep; and altered sensitivity of the HPA and HPG axes (Monassi et al., 2003). Thus, it has been shown that rats which undergo CCI of the sciatic nerve present with similar levels of disturbances to humans experiencing chronic neuropathic pain. In chapter 3 of this thesis these behavioural changes first observed by Monassi and colleagues were replicated.
Given the complex behavioural changes observed in animals that develop disability following CCI it is unlikely that neural changes specific to damage at the peripheral nerve and its spinal recipient regions is sufficient to drive these maladaptive changes. Thus, supraspinal sites which receive inputs from sciatic nerve recipient segments of the spinal cord are likely to play a key role in mediating these behavioural and physiological maladpations.
Recently, using Affymetrix Gene-Chip technologies, Mor and colleagues (2010) have reported that six days following sciatic nerve CCI, rats with pain and persistent disability showed significant up- regulation in thirteen mRNAs in the PAG and immediately adjacent regions. One of the most significant changes observed was a selective increase in the expression of the peptide cholecystokinin (CCK). 107 Selective increases in CCK mRNA within midbrain tissue of rats with pain and persistent disability were confirmed using qRT-PCR techniques (unpublished).
The midbrain PAG could be a region playing a pivotal role in the expression of maladaptive behaviours observed in rats with pain and persistent disability given that it is an important mediator in regulating social behaviours (Keay and Bandler, 2001); is involved in sleep circuitry (Lu et al., 2006, Sastre et al.,
1996); and helps regulate the activity of the HPA and HPG axes (Floyd et al., 1996, Lakoski and
Gebhart, 1982).
Our aim then was to anatomically localise neural cells showing observed changes in CCK mRNA using
Gene-chip and qRT-PCR techniques using single and double-label in-situ hybridisation techniques in order to locate where up-regulation of CCK mRNA was occurring within the midbrain of these rats.
The importance of anatomically localising these cell populations stems from the columnar organization of the PAG, which contains neuronal populations that communicate with discrete nuclei.
4.2 Methods
All experimental procedures were conducted in accordance with the guidelines of the National Health and Medical Research Council’s (NHMRC) Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes (2005). All procedures were approved by the University of Sydney,
Animal Care and Ethics Committee.
Seventy-two male Sprague-Dawley rats were used in the study (210–250g, Gore Hill Animal Facility,
St Leonards, NSW), 36 of which underwent CCI and behavioural testing in these experiments. Rats were maintained on a 12 hour light-dark cycle, (lights off 7 am – 7 pm). Rats were housed in groups of eight for at least two weeks to habituate to new housing and were provided with food and water ad libitum. As described in the methods chapter (Chapter 2) and behavioural chapter (Chapter 3) of this thesis, rats which underwent behavioural testing and CCI were categorised into, pain and persistent disability, pain and transient disability, or pain alone groups, based on changes to dominance behaviours, with the incident rates being comparable to previous literature. The data of six pain and
108 persistent disability, five pain and transient disability, and seven pain alone animals is presented in this chapter. Rats underwent behavioural testing and CCI, before tissue was prepared for in situ hybridisation, and in situ hybridisation and immunohistochemistry techniques.
For each animal, analysis of cholecystokinin mRNA (CCK mRNA) was performed for the four columns of the PAG: dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG), and ventrolateral (vlPAG), as well as the dorsal raphe nucleus (DRN) and the Edinger-Westphal nucleus (EW, accessory III).
Boundaries of nuclei where taken according to the rat brain atlas of Paxinos and Watson (5th edition).
CCK mRNA positive cells were identified using both darkfield (Olympus Vanox) and light microscope
(Olympus BX51) and were characterised by silver deposition throughout the cell body, which was indicative of individual CCK mRNA. A cell was considered CCK mRNA positive if the density of silver deposition was at least four times stronger than background levels. For each animal six equidistant sections of the midbrain encompassing the rostrocaudal extent of the PAG (Bregma –8.8 to –6.3) were selected for analysis. Using a light microscope CCK mRNA positive cells in the PAG, DRN and EW were counted bilaterally using a 1 mm x 1 mm graticule. The mean number (±SEM) of CCK mRNA positive cells within each nucleus was compared between the persistent change, recovery and no-effect groups of rats. ANOVAs with post-hoc Bonferroni tests were performed on the data. Additionally, for each animal individual grains were counted per cell along six equidistant regions and the mean number
(±SEM) of individual silver grains representative of CCK mRNA were compared between the persistent change, recovery and no-effect groups of rats. Photomicrographs of representative sections of the midbrain were taken from persistent change, recovery, and no-effect were taken using a light microscope (Olympus BX51) with a digital camera attachment at x200, x400, and x1000 magnification.
4.3 Results
Midbrain neurons expressing CCK mRNA were identified by in situ hybridisation using an oligonucleotide probe to detect pre-pro-cholecystokinin mRNA. Positive cells were indicated by clusters of silver grains, with specific labelling >3 times above background (see Figure 4.1).
109
Figure 4.1 Photomicrographs illustrating the presence of CCK mRNA.
mRNA (silver grains) in neurons of the ventrolateral periaqueductal gray as revealed by in situ hybridization. Top panel a) Dark field photomicrograph depicting CCK mRNA (silver grains) on neuronal cell bodies with schematic section showing location within the ventrolateral periaqueductal gray (vlPAG) at which the image was taken b) Pain Alone animal c) Pain & Transient Disability animal d) Pain & Persistent Disability animal.
In all rats, CCK mRNA positive cells were identified bilaterally in: (i) the ventrolateral and lateral columns of the PAG; the Edinger-Westphal nucleus, and the dorsal raphe nucleus. However, there were clear differences in the number and distribution of CCK mRNA positive cells between each of the behaviourally-categorised, nerve-injured groups (Figure 4.2).
110
Figure 4.2 Schematic diagram showing the location of CCK mRNA expression.
CCK mRNA expression throughout the rostrocaudal extent of the dorsal midbrain in rats with Pain Alone; rats with Pain & Transient Disability; and rats with Pain & Persistent Disability. Each schematic diagram represents one 12um thick coronal section, each dot represents one CCK mRNA positive cell.
Ventrolateral periaqueductal grey (vlPAG): in the six coronal sections analysed, the mean total number of CCK mRNA expressing cells in the vlPAG of rats with pain and persistent disability (55 ±4 cells) was significantly greater than either rats with pain and transient disability (28 ±3 cells) or pain alone
(27 ±2) (P<0.01) (Figure 4.5a). Specifically, there were twice the numbers of CCK mRNA positive cells in the rostral portion of this column (-7.3 to -7.8mm bregma) in rats with pain and persistent disability when compared with the other injured rats (see Figures 4.1 and 4.3(a)).
111
Figure 4.3 Bargraph depicting the distribution of CCK mRNA positive cells.
Distribution of CCK mRNA positive cells throughout six equidistant sections of the vlPAG. a) Depicts total CCK mRNA positive cell counts in behaviourally categorised groups whilst b) illustrates the bilateral distribution of CCK mRNA positive cells throughout six equidistant sections of the vlPAG in animals with Pain & Behavioural Disability following CCI. Significant changes in Pain & Persistent Disability animals with respect to Pain & Transient Disability and Pain Alone groups is shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test).
Despite a unilateral sciatic nerve injury, there were no differences in the mean number of CCK mRNA positive cells either ipsilateral or contralateral to the side of injury; the data for pain and persistent disability rats is illustrated in Figure 4.3(b). In addition to an ~90% increase in the number of CCK mRNA expressing cells in the vlPAG of pain and persistent disability rats, the mean number of silver grains identifying each neuron (i.e. within the Nissl stained boundaries) was also greater. Specifically, rats with pain and persistent disability, had a mean number of 28.2 ±2.7 grains per cell, compared with
24.5 ±2.8 grains in rats with pain and transient disability and 21.5 ±3.1 grain in rats with pain alone.
There was a strong correlation between the degree and pattern of disability with the number of silver grains in vlPAG neurons (Figure 4.4)
112
Figure 4.4. Relationship between CCK mRNA in cells of the vlPAG and behavioural disability following injury.
Linegraph depicting the relationship between CCK mRNA expression (individual silver grains) and changes in dominance following chronic constriction injury (CCI). Individual silver grains in CCK mRNA positive cells, representative of individual CCK mRNA, were counted along the rostrocaudal extent of the vlPAG for each behaviourally categorised CCI rat. Pearson’s value significant at the 0.01 level.
113
Figure 4.5 Bargraphs depicting the distribution of CCK mRNA positive cells.
CCK mRNA positive cells throughout six equidistant sections of the lateral periaqueductal gray (lPAG) and three equidistant sections of the Edinger Westphal nucleus (III acc). a) Depicts total CCK mRNA positive cell counts throughout six equidistant coronal sections of the lPAG; b) Depicts total CCK mRNA positive cell counts throughout three equidistant coronal sections of the Edinger Westphal nucleus; in rats with pain & behavioural disability, pain & transient disability, and pain alone following CCI. Significant changes in Pain & disability animals with respect to Transient Disability and pain alone groups is shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test). No significant changes in CCK mRNA positive cells between behavioural groups were observed in the Edinger Westphal nucleus.
Lateral periaqueductal grey (lPAG): in contrast to the vlPAG, there were few CCK mRNA positive cells in the lPAG, and there were no differences between behaviourally categorised rats in either their number, distribution or silver grain content (Figure 4.5a).
Edinger-Westphal nucleus: similar to the lPAG, there were no differences in either the number, distribution nor silver grain content of cells in the Edinger-Westphal nucleus (accessory c.n. III), between behaviourally categorised rats (Figure 4.5b).
114
Figure 4.6 Photomicrographs illustrating the presence of CCK mRNA. mRNA (silver grains) in neurons of the Dorsal Raphe as revealed by in situ hybridization. Top panel a) Dark field photomicrograph depicting CCK mRNA (silver grains) on neuronal cell bodies with schematic section showing location within the Dorsal Raphe at which the image was taken b) Pain and Persistent Disability animal c) Pain and Transient Disability animal d) Pain Alone animal.
Dorsal Raphe nucleus: CCK mRNA expressing cells were also observed in the dorsal raphe nucleus.
Examples of the hybridisation signal in this region are shown in Figure 4.6, silver grains were located on cells located in the dorsal raphe dorsalis, and the dorsal raphe ventralis, but not in the so called
“wings”, or dorsal raphe ventrolateral. In rats with pain and persistent disability there were three times the number of CCK mRNA positive cells observed in rats with pain alone; and in rats with pain and transient disability there were almost twice the number of CCK mRNA positive cells observed in the rats with pain alone (i.e. pain and persistent disability: mean total 71 ±3 cells, vs., pain and transient disability: mean total 45 ±3 cells, vs., pain alone: mean total 28 ±5 cells) (Figure 4.7a).
115
Figure 4.7 Figures depicting the distribution and relative change of expression of CCK mRNA positive cells.
CCK mRNA positive cells throughout four equidistant sections of the Dorsal Raphe Nuclei. a) Bargraph depicts total CCK mRNA positive cell counts b) Linegraph depicting the relationship between CCK mRNA expression (individual silver grains) and changes in dominance following chronic constriction injury (CCI). Individual silver grains in CCK mRNA positive cells, representative of individual CCK mRNA, were counted along the rostrocaudal extent of the DRN for each behaviourally categorised CCI rat. Significant changes in Pain & Persistent Disability animals with respect to Pain & Transient Disability and Pain Alone groups is shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test). Pearson’s value significant at the 0.01 level.
Thus, in the dorsal raphe overall, there was an ~70% increase in the number of cells expressing CCK mRNA in rats with pain and persistent disability compared with other CCI rats. In addition to increased numbers of cells expressing CCK mRNA in rats with pain and persistent disability, the silver grain numbers in each cell were also larger than those in the other two behavioural groups. The mean silver grain numbers in dorsal raphe cells correlated strongly with the degree of disability as indicated by the magnitude of the decrease in dominance behaviours after CCI (Figure 4.7b).
116
Figure 4.8 Photomicrographs illustrating the presence of 5HT-IR cells and CCK mRNA.
CCK mRNA (silver grains) in cells of the dorsal raphe as revealed by fluorescence immunohistochemistry and in situ hybridization techniques. Top panel a) fluorescence immunohistochemistry depicting 5HT-IR cells b) CCK mRNA c) Overlay. Top right panel shows a schematic section of the coronal level at which the images above are taken, the red square illustrates the location within the dorsal raphe at which the image was taken.
Double label studies for CCK mRNA and 5HT did not reveal a single double-labelled cell in any section analysed. In double-labelled sections, single labelled cells were clearly visible from either the hybridisation signal or the immuno-fluoresence (Figure 4.8)
117
Figure 4.9 Bargraphs depicting 5HT-IR cells.
5HT-IR cells throughout five equidistant sections of the Dorsal Raphe Nuclei in behaviourally categorised CCI rats. a) Bargraph depicts the distribution of 5HT-IR cells throughout the ventral dorsal raphe; b) Bargraph depicts the distribution of 5HT-IR cells throughout Dorsal Raphe Dorsalis; c) Bargraph depicts the distribution of 5HT-IR cells throughout the ventrolateral Dorsal Raphe (ipsilateral to injury); d) Bargraph depicts the distribution of 5HT-IR cells throughout the ventrolateral Dorsal Raphe (contralateral to injury); in rats with Pain & Persistent Disability, Pain & Transient Disability, and Pain Alone following CCI. Significant changes in Pain & Persistent Disability animals with respect to Pain & Transient Disability animals is shown by * P<0.05 (ANOVA, Bonferoni post-hoc test). Significant changes in Pain & Persistent Disability animals with respect to Pain Alone animals is shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test).Significant changes in Pain & disability animals with respect to Pain & Transient Disability and Pain Alone groups is shown by *** P<0.05 (ANOVA, Bonferoni post-hoc test).
118 An unexpected observation in these double-labelled procedures was that in rats with pain and persistent disability there were fewer 5-HT immunoreactive neurons in the rostral portion of the dorsal raphe ventrolateral subregion compared with the other behavioural groups (Figure 4.9).
4.4 Discussion
In this study it was found that, following sciatic CCI, approximately one-third of the animals (35%) showed persistent reductions in dominance behaviour. About half of the animals (46%) showed no significant change in their behaviour while one-fifth (19%) showed transient reductions in dominance behaviours. Both the pre-injury durations of each behavioural category (i.e. dominance, social, non- social and submissive behaviours) and the patterns of CCI-evoked changes in dominance behaviours are comparable to those described in earlier studies (Keay et al., 2004, Monassi et al., 2003).
In agreement with earlier Gene-Chip data (Mor et al., 2010), rats with pain and persistent disability following sciatic nerve CCI, showed a significant up-regulation of CCK mRNA in the dorsal midbrain.
In situ hybridisation studies revealed greater numbers of CCK mRNA containing cells in the vlPAG and dorsal raphe (dorsalis and ventralis) in rats with pain and persistent disability. In addition, CCK mRNA positive cells of the vlPAG and dorsal raphe (dorsalis and ventralis) in rats with pain and persistent disability had a greater density of silver grains, indicating higher levels of CCK mRNA expression. The mean densities of silver grain labelling correlated strongly with the degree of behavioural change (decreased dominance) in resident-intruder interactions. CCK mRNA was not expressed in the 5-HT-IR cells of the dorsal raphe nucleus. Although not a specific focus of this study, we also revealed a significantly smaller number of 5-HT-immunoreactive cells in the lateral wings of the dorsal raphe in rats with pain and persistent disability. The reduction in 5-HT observed in these results also begs the question as to whether lower numbers of 5-HT are caused by CCI, or whether the injury unmasks a pre-existing deficit in that population of cells. Further work may gleam light on this.
This chapter focuses on: (i) specific anatomical location of CCK mRNA up-regulation, (ii) why of all the CCK cells in this region do only vlPAG and DRN change? (iii) What normally regulates CCK mRNA regulation, and (iv) consequences of increased translation. 119 4.4.1Midbrain localisation of CCK mRNA and CCK immunoreactivity
There are few reports of CCK-IR neurons in the midbrain of the rat, which is most probably due to technical difficulties with this specific approach. These difficulties include: (i) the cross-reactivity of many CCK antibodies, with gastrin, and (ii) the need for the use of colchicine pre-perfusion to allow the accumulation of sufficient peptide in the cell soma to be detected by the available antibodies. The impact of the former has been reduced somewhat by designing antibodies to the precursor sequences of
CCK (pre-pro-CCK), the latter impacts significantly on most functional-anatomical studies of this peptide. By far the most reliable and replicable means for describing the anatomical localisation of CCK containing neural cell groups has been the use of in-situ hybridisation techniques.
The area dissected out for analysis in this study consisted of the PAG but also the Edinger-Westphal nucleus and dorsal raphe. Several researchers have reported the presence of CCK mRNA throughout the PAG, Edinger-Westphal and dorsal raphe in uninjured animals. Intense labelling has been reported in the Edinger-Westphal nucleus using in situ hybridisation (Lanaud et al., 1989, Schiffmann and
Vanderhaeghen, 1991) while De Belleroche et al. (1990) showed the presence of CCK mRNA in the
PAG during rat development (De Belleroche et al., 1990). Also using in situ hybridisation, Rattray et al. (1992) later reported two distinct populations of cells expressing pre-pro-CCK mRNA in a similar area. One of these populations confirmed the presence of large heavily labelled cells in the Edinger-
Westphal nucleus and the other population consisted of small lightly labelled cells in the vlPAG
(Rattray et al., 1992). More recently, Del Bel et al. (1997) showed the presence of mRNA coding for
CCK in the dorsal raphe amongst several other regions of the brain using autoradiography.
Thus the presence of CCK mRNA has been consistently demonstrated in all of the areas analysed in the present study, with intense labelling in the Edinger-Westphal and a separate population in the vlPAG of particular note.
Repeated electroconvulsive shock has also been shown to increase CCK mRNA expression throughout the PAG with a particularly high level in the Edinger-Westphal nucleus (Lindefors et al., 1991), and acutely administered morphine has been shown to increase CCK levels in the PAG (Rattray and de
120 Belleroche, 1987, Rosen and Brodin, 1989). CCI could be classed as an inescapable physical stressor that has the potential to evoke similar changes in the expression of CCK. These data indicate clearly that it is not only possible to trigger mRNA up-regulation in neuronal cell populations (increased constitutive expression), but also that it is possible to alter the chemical phenotypes of cells (increased de novo expression). Ours is the first study to systematically describe anatomically specific, constitutive and de novo changes in CCK mRNA expression in the midbrain of nerve-injured rats. Moreover, the patterns of change correlate well with both the presence, and intensity of, altered social interactions
(decreased dominance and increased non-social behaviours i.e. disability) following peripheral nerve injury. The patterns of CCK mRNA expression do not however, correlate with the expression of either allodynia or hyperalgesia, which was triggered equally in all injured rats.
The up-regulation of CCK mRNA observed in the vlPAG and dorsal raphe of rats with pain and persistent disability suggests an “anatomically specific”, rather than a “systemic” mechanism for these changes. It is likely therefore that altered activity of discrete anatomical inputs to the vlPAG and dorsal raphe nucleus may trigger the select patterns of up-regulation in these dorsal midbrain regions.
Consistent with this suggestion are earlier reports from this laboratory of the up-regulation of synaptojanin-2 mRNA (Mor et al., 2010), which is known to be up-regulated during periods of high synaptic activity, sustaining neurotransmitter release. The vlPAG and dorsal raphe receive significant afferent inputs from sciatic nerve recipient regions of the spinal cord via the spino-mesencephalic tract
(Keay et al., 1997, Yezierski and Mendez, 1991). In the rat, the sciatic nerve projects directly into spinal cord segments T10–L5 and synapses directly on neurons in laminae I, III, IV, V, X and the lateral spinal nucleus (Swett and Woolf, 1985). In addition, following sciatic nerve CCI, neurons in these laminae increase their spontaneous firing rates, have lowered thresholds for activation (Kohno et al., 2003,
Moore et al., 2002, Somers and Clemente, 2002), and express the immediate-early gene c-Fos, an accepted marker of neuronal activation (Catheline et al., 1999, Delander et al., 1997, Jergova and
Cizkova, 2005, Kajander et al., 1996, Yamazaki et al., 2001). The spinal inputs to the PAG terminate selectively in the lateral and ventrolateral columns. The lateral PAG receives a topographically organised input with the lumbar enlargement projecting most strongly to the caudal half of the column
121 (Keay and Bandler, 2001, Keay et al., 1997), and the ventrolateral column receives spinal inputs with little obvious topographical specificity. The sciatic nerve recipient regions of the spinal cord thus project to both the lPAG and vlPAG columns. The spino-PAG pathway is reported to have a significant glutamatergic component (Azkue et al., 1998, Yezierski et al., 1993), while PAG projections arising from the lateral spinal nucleus are known to contain substance P (Leah et al., 1988).
4.4.2 Selectivity of patterns of mRNA up-regulation
It has been previously suggested that increased activity in the spino-mesencephalic pathway may be an important trigger for the activation of astrocytes in the lPAG and vlPAG of rats with pain and persistent disability (Mor et al., 2010). The select activation of astrocytes in the vlPAG of rats with pain and persistent disability (Mor et al., 2010) raises the possibility that FGF-2 expressed in midbrain astrocytes, may contribute in part to the select pattern of CCK mRNA up-regulation observed in this study.
In addition to inputs from sciatic nerve recipient spinal segments, the vlPAG column receives significant inputs from the nucleus tractus solitariius (NTS), including the A2 (noradrenergic) and C2
(adrenergic) cell groups. As well, the vlPAG also receives substantial projections from noradrenergic neurons in the caudal ventrolateral medulla (A1 cells) and adrenergic neurons of the rostral ventrolateral medulla (C1). These catecholaminergic cell groups have each, been shown to be activated by injurious stimuli (Herbert and Saper, 1992), and the cognate receptors in the midbrain are all G protein coupled.
In addition to ascending inputs, the vlPAG and dorsal raphe nucleus receive significant inputs from the hypothalamus, specifically the arcuate nucleus (beta-endorphinergic neurons) and the lateral sub-nuclei, and from specific cortical regions including the pre-limbic, infralimbic and anterior cingulate cortices.
The descending projections highlighted are known to be critical in the integration of the responses to physical and psychological challenges, from which escape is not possible and for which control cannot be established.
The dorsal raphe, which was also shown to have CCK-mRNA, displays a topographical organisation along its rostrocaudal axis, and its neurons are known to target several functionally related targets through branched fibres. The DRN constitutes the majority of serotonergic drive to the brain and
122 receives inputs from a plethora of forebrain regions including the cingulate, insular and orbital cortices, central nucleus of the amygdala, the basal forebrain, hypothalamus, and the thalamus.
The circuitry, and functional anatomy, of the dorsal raphe suggests that the increase in CCK mRNA observed in rats with pain and transient disability may be due to differences in higher regions of the brain, or indirectly through regions of the brain that receive ascending inputs.
4.4.3 Possible functional consequences of transcription and translation of CCK mRNA in the
vlPAG and DRN
These experiments have shown that, in a subgroup of CCI rats experiencing pain and persistent disability, there is an anatomically specific up-regulation of CCK mRNA in the vlPAG and DRN. Given the complexity of the behavioural changes noted in this group of rats, the implications of this up- regulation could be at least partially responsible for the autonomic changes, sleep disturbances, as well as desensitisation of the HPA axis seen in the group of animals.
The vlPAG receives both ascending and descending inputs from CCKergic brain nuclei that could be considered strong candidates for the anatomically specific changes observed, given their functional role.
It is, however, also worth noting that the PAG itself contains a population of interneurons, which may of themselves be driving these changes in CCK expression, perhaps as a result of altered transmission into this region from the aforementioned structures.
Sciatic nerve recipient regions of the spinal cord are the first logical place to look for changes in CCK expression. The vlPAG receives projections from sciatic recipient regions of the spinals cord (Keay et al., 1997), namely between S1–S3, as well as lumbar and thoracic (T10–12) regions. Within the spinal cord, CCK immunoreactivity has been reported, particularly in lamina I–III, and lamina X. The vlPAG also contains a strong projection from the caudal nucleus of the solitary tract (A2 region), which conveys visceral afferent signals and contains CCK-responsive cells, in addition to providing strong catecholaminergic inputs.
123 Given that both the vlPAG and the DRN contain cell groups known to modulate the orexin-containing population of neurons in the lateral hypothalamus responsible for arousal, it is plausible that upregulated
CCK mRNA in these cell groups may be driving sleep disturbances in these rats, most likely via modulation of dopaminergic cell groups. Previous studies have shown that CCK is often co-localised with dopamine, thus a logical next step would be to investigate the phenotype of CCK mRNA expressing cells in the vlPAG and DRN.
CCK has previously been shown to modulate pituitary-adrenal activation in the brain both by peripheral administration as well as centrally, via receptor specific agonists and antagonists. Hypothalamic- pituitary adrenal response to intravenous injection of cholecystokinin-2 receptor agonist pentagastrin has been shown to produce a dose-dependant release of adrenocorticotropin (ACTH) and cortisol, with the ACTH response being found to be resistant to cortisol feedback inhibition. In an animal model of social defeat, a chronic blockade of CCK2 receptors by the specific antagonist CI-988 was found to prevent HPA axis hyperactivity, reduction of hippocampal volume, and decreased sucrose intake normally evoked by repeated social defeat, an inescapable chronic stressor. Studies of chronically stressed rats have shown that the paraventricular nucleus of the thalamus (PVTh) receives inputs from
CCK mRNA containing cells in the PAG and DRN, and that stimulation of PVTh neurons via CCK-B receptors inhibits ACTH responses, subsequently altering HPA activity (Bhatnagar et al., 2000).
Additionally, intravenous injection of CCK-B receptor agonist pentagastrin produces robust, dose- dependant releases of adrenocorticotropin (ACTH) and cortisol, with the ACTH response being relatively resistant to cortisol feedback inhibition, raising the possibility that CCK could contribute to acute activation of the HPA axis even in the face of elevated basal cortisol levels. Because of its well- defined connections to limbic and sensory regions the PVTh is a good candidate as a modifier of stress- sensitive systems of the brain, and studies have shown that it receives strong input from CCK expressing cells of the PAG and DRN, suggesting a plausible mechanism via which desensitisation of the HPA axis might be occurring.
124 4.4.4Methodological considerations
In this study, external stressors on the resident animals were kept to a minimum to allow any observed changes in the level of CCK mRNA to be more closely attributed to the CCI. Two features of the experimental paradigm had the potential to alter the levels of gene expression of animals in this study:
(i) sacrifice order, and (ii) social isolation, applicable to the controls and the residents respectively.
On a short-term scale, it is possible that the successive removal from a grouped cage preceding sacrifice could alter the expression of certain genes, such as CCK, that have been shown to be up-regulated in response to stress. This was eliminated in the experimental resident animals by the fact that all residents were housed individually. Notwithstanding the potential of this stressor to increase levels of CCK expression in the controls, the persistent change group of animals still showed a significant increase in
CCK mRNA, whereas those in the no-effect and recovery groups did not. This suggests that the injury itself has a more significant effect on levels of CCK mRNA than any other potential factor.
The only potential stressor on the residents during the testing procedure—aside from the nerve injury itself—that potentially could have contributed to the observed changes in gene expression was the effect of social isolation on the residents. Del Bel and colleagues (1997) showed that individually housing animals just after weaning and leaving them isolated for a period of 30 days significantly increased
CCK mRNA expression in several brain regions including the dorsal raphe. All residents in this study were individually housed at an age of six weeks and were maintained that way for one week prior to testing and for the duration of the paradigm (a total of 18 days). This factor could have contributed to the observed CCK mRNA levels to a certain extent. However, all experimental animals were treated identically and notwithstanding this possible effect of social isolation, the pain and transient disability group were the only animals to show a significant increase of CCK mRNA in the PAG.
Thus six days after sciatic CCI, it is clear that those animals that displayed persistent decreased levels of dominant behaviour (pain and transient disability animals) also show a significantly elevated (+1.67 fold) amount of mRNA coding for CCK, a neuropeptide that has been shown to be elevated in response to a variety of stressors. It is also evident that this elevation is in specific response to the CCI over any
125 other potential factor. Furthermore, this increase is seen in the PAG, an area of the brain known to be associated with the co-ordination of behavioural responses to stressful stimuli. Finally, the most probable location of the increase is in cell bodies, suggesting that the CCK peptide synthesised from the mRNA observed here is having its effect in an area to which the PAG projects.
4.5Comparison to previous immunohistochemical studies of CCK-8
4.5.1 Is CCK expression related to behaviour?
To gain an understanding of the potential effects of increased CCK mRNA expression in the PAG, it is necessary to examine those areas of the CNS that receive CCK-ergic input from the PAG that could potentially be responsible for controlling the specific behavioural changes displayed by the persistent change group. The decreased dominance shown by some of these animals post-CCI is manifested by increased quiescence and reduced somatomotor activity (as defined by their increased non-social activity and decreased dominance, refer Figure 3.2). They also display altered HPA-axis activity and an altered sleep/wake cycle. There are several areas of the brain with efferents from the PAG that would appear to be likely candidates for the generating of such disabilities.
The brain stem is an area that controls many functions such as somatomotor, cardiovascular, and antinociceptive activity, and also a region that receives efferent projections from the PAG. Both the vlPAG and lPAG project extensively to ventrolateral and ventromedial medullary regions (VLM, VMM) responsible for these functions (Bernard et al., 1998; Bandler et al., 1991). In addition, the dorsomedial
PAG also projects to the VLM and VMM. It is possible that these areas are targeted by that cell population in the PAG that experience an up-regulation of CCK mRNA in the persistent change animals.
There are also higher areas of the brain that receive PAG efferents. The diencephalon is a region of the brain that plays a crucial role in the co-ordination of many stress responses. The hypothalamus, for example, controls the primary hormonal response to stressful stimuli, the HPA axis. The paraventricular nucleus receives input from the PAG and is a crucial regulator of the pituitary gland and HPA axis
126 function in general. It is clear that there is high potential for input to such an area to have control over the animals’ responses to a variety of stressors, including a peripheral nerve injury.
The lateral hypothalamic area, which receives selective vlPAG input, is associated with hypotension and bradycardia, effects that could potentially be associated with the observed behavioural changes seen after CCI.
These are all possible areas that could be both receiving CCK-ergic input from the PAG and affecting the observed behaviour. The PAG is set up in such a way as to have interconnections with a range of crucial control centres and thus up-regulation of CCK in the PAG of a subset of animals has the potential to affect a wide array of other brain areas in those animals.
Another distinct possibility is that the CCK mRNA up-regulation occurs in interneurons and this, in turn, is responsible for the observed terminal up-regulation.
4.5.2 Technical consideration of mRNA findings
When analysing real time PCR data it is important to recognise that changes in mRNA levels do not necessarily equate to changes in levels of the protein. Some studies show CCK mRNA-containing neurons to be considerably more numerous than CCK-ir neurons. Schiffmann et al. (1991) report that
CCK mRNA levels in thalamo-cortical and thalamo-striatal projection neurons do not correlate with peptide expression as shown by immunohistochemistry. This could indicate that not all of the mRNA is undergoing translation.
Thus, with respect to the findings reported here, it is essential to confirm that the mRNA levels correlate with protein levels by conducting further experiments using western blotting and in situ hybridisation.
It could then be ascertained whether or not any transportation of the mRNA has occurred prior to translation and if the up-regulation of the mRNA results in biologically active protein.
4.5.3 Increased peptide levels within the PAG
It is clear that the potential effect of increasing CCK in the vlPAG is to elicit the passive emotional coping response due to the well-documented role of this column of the PAG. This response entails 127 quiescence and an increased disengagement from the environment and thus it presents a possible conclusion to explain certain aspects of the persistent disability animals such as their reductions in dominant behaviour relative to the no-effect animals.
However, the conclusion that the behaviour displayed by the persistent change animals is merely reflective of a passive coping style is overly simplistic and does not take into account several trends in the behaviour, such as increased approach-avoidance activity. Is it a passive coping style, or a combination of that and other effects (which perhaps are also elicited from the PAG)?
One possible scenario involves the concept of volume transmission in the brain and the control of the responses to a neuropeptide via the distribution and concentration of its specific receptors. Volume transmission is the hypothesised mode of intercellular communication involving the diffusion of transmitters via extracellular fluid pathways from nerve cells selectively capable of producing the signal
(signal source) to nerve and glial cells selectively capable of recognising it (signal target) (Cintra et al.,
1994). The relevance of the proposal to this study is that CCK peptide is potentially released from nerve terminals selectively in the vlPAG and then diffuses over time to affect regions that are spatially removed from there, such as the lateral or dorsal regions of the PAG. Due to the presence of fast-acting endopeptidases, the detection of individual CCK molecules undergoing volume transmission with immunohistochemical techniques would be difficult and could account for the absence of CCK-ir observed in this study outside of the concentrated vesicles at the site of release (vlPAG).
The question would then arise as to the distribution of CCK receptors throughout the PAG as a means of controlling the response. CCK-2 receptors (the most abundant CCK receptor in mammalian brains) have been shown to exist widely throughout the brain (Dietl et al., 1987) and specifically throughout the PAG, pineal gland and superior colliculus (Mercer and Beart, 2004, Mercer et al., 2000).
Thus, it is possible that an individual animal’s response to CCK release is contingent upon the up or down regulation of CCK receptors in various regions of the PAG. Animals expressing a greater concentration of receptors in the vlPAG, for example, may be more quiescent, whereas animals with more receptors in the dlPAG, an area associated with fight and flight type responses, may exhibit more
128 rapid and erratic types of behaviour given the confined setting of the interaction. This could be represented by increased approach-avoidance type behaviour. Thus, effects in both columns of the PAG could be contributing to the overall behaviour that a persistent change animal displays with the underlying influence being an increased input of CCK.
In situ hybridisation to determine the extent of the up or down regulation of receptors in the disabled rats versus non-disabled rats would be essential to gauge whether or not this is the process being used.
One could also conduct experiments involving injection of CCK-8 into discrete areas of the PAG in uninjured animals and examine which areas elicit behaviour reminiscent of that displayed by a persistent change animal after CCI.
129 Chapter5 The Presence and Alteration of CCK1 & CCK2
Receptors Following CCI in the PAG
5.1 Introduction
In humans, CCK occurs predominantly as a COOH-terminal amidated 58-, 33- and 8- amino acid peptide product of the gene residing on chromosome 3, which is expressed in neurons throughout the peripheral and central nervous system, and in intestinal neuroendocrine cells and neurons of the enteric nervous system (Dufresne et al., 2006, Larsson and Rehfeld, 1979, Wank, 1995). In the CNS, several molecular forms of the peptide have been isolated, with the sulphated octapeptide (CCK-8) being the most abundant, although other forms, like CCK-4 and CCK-5, also exist in the brain (Zwanzger et al.,
2012).
In the previous chapter of this thesis we have shown for the first time that, following CCI, significant increases in neuronal expression of CCK were found, using in situ hybridisation techniques, to be anatomically restricted to the vlPAG, lPAG, and the dorsal raphe in a subgroup of animals experiencing pain and transient disability. This anatomically specific pattern of increased CCK expression suggests activation of neurons by select neural pathways, which likely include afferents of spinal, brainstem, and forebrain regions. The presence of The PAG columns in which these populations of neurons are activated play significant roles in modulating social interactions, responses to stressors, and the sleep- wake cycle (Bhatnagar et al., 2000, Keay and Bandler, 2001, Lu et al., 2006). These selective changes observed in the synthesis of CCK mRNA for the peptide lead us to consider if there are similar changes occurring in this region for the receptors of CCK.
5.1.1 CCK receptors
The physiological effects of cholecystokinin in the brain are mediated by two specific G-protein coupled receptor subtypes (GPCRs), CCK1R, originally thought to only be expressed in the periphery, and
CCK2R, both of which have been found to be widely distributed in the brain using electrophysiological
(Boden and Woodruff, 1994, Woodruff et al., 1991), in situ hybridisation (Honda et al., 1993), receptor 130 autoradiography (Carlberg et al., 1992, Hill et al., 1987, Hill et al., 1988) and immunohistochemistry techniques (Mercer and Beart, 1997, Mercer and Beart, 2004, Mercer et al., 2000) in a pattern that parallels the distribution of CCK immunoreactivity and mRNA (Lindefors et al., 1991, Lindefors et al.,
1993, Rattray et al., 1992, Savasta et al., 1990). The genes encoding the CCK1 and CCK2 receptors in humans exceeds 8kb in length, and are organised in a similar manner consisting of five exons and four introns of 164–1177bp (Song et al., 1993), with the rat and mouse genes organised similarly (Wang et al., 2005).
Both CCK receptor subtypes have equally high affinity for CCK-8S, while they differ substantially for unsulphated CCK peptides, gastrin, and amidated peptides shorter than CCK-7, i.e. CCK-4. The CCK1 receptors are relatively specific for CCK8S than its unsulfated form with up to 1000-fold higher affinity than for CCK-4 and gastrin, with the difference in affinity involving a Met and an Arg in the second extracellular loop (Gigoux et al., 1998, Gigoux et al., 1999).
Given that CCI causes a selective increase in CCK mRNA levels in rats with persistent behavioural disability and pain, which has been verified by in situ hybridisation techniques, we aimed to see if: (i) the neurons of the midbrain PAG contained cognate CCK receptors, and (ii) to observe the differences, if any, on the expression of these receptors following CCI.
5.2 Methods
Rats used in this study were either behaviourally characterised using the resident-intruder behavioural paradigm, with CCI surgery occurring following behavioural testing on day five (n = 15); or control rats (n = 6) group-housed for similar time duration, as well as being age, weight, sex matched to experimental animals. Both groups of animals were sacrificed by trans-cardial perfusion for immunohistochemistry reaction and analysis. As described in Chapter 3 of this thesis, CCI rats were categorised into disability groups, based on changes in behaviour following CCI. In this chapter the data of five pain alone, five pain and transient disability, and five pain and persistent disability rats is presented. Following sacrifice and perfusion, midbrain sections containing the PAG were blocked, and midbrain tissue was frozen and cut by microtome (Leica) into 25 µm sections and analysed for 131 expression and distribution of CCK1 and CCK2 receptors using primary antibodies with known specificity (Mercer & Beart, 2004; Mercer et al., 2000; Mercer & Beart, 1997), reacted using fluorescent immunohistochemistry techniques described in Chapter 2. CCK1- & CCK2-receptor immuno-reactive cells were counted along six equidistant PAG sections, and their distribution along the rostro-caudal columns of the PAG analysed using a fluorescence microscope with graticule (Olympus BX51).
The mean number (±SEM) of CCK-IR cells within each functional column of the PAG was compared between the pain and persistent disability, pain and transient disability, and pain alone animals, and an uninjured control group of rats. ANOVAs with post-hoc Bonferroni tests were performed on the data.
5.3 Results
5.3.1 Behavioural changes following CCI
As described in an earlier section of this thesis (see Chapter 3, Figure 3.1), each rat was categorised as either: pain and persistent disability, pain and transient disability, or pain alone; based on altered dominance behaviours in the resident-intruder test (Monassi et al., 2003). In the experiments described in this study: five pain and persistent disability, five pain and transient disability, five pain alone rats, and six uninjured control rats were used.
5.3.2 Distribution of CCK1 receptors
Midbrain neurons expressing CCK1 receptor immuno-reactivity (CCK1-IR) were identified by fluorescence immunohistochemistry using a specific antipeptide antibody directed against an extracellular portion of the amino terminal sequence of the CCK1 receptor. CCK1-IR neurons were identified bilaterally along the rostro-caudal gradient of all the columns of the PAG in all rats, as shown in Figure 5.1. Additionally, CCKa-IR was observed, though not quantified in the colliculli of the midbrain, the dorsal raphe nucleus, and the substantia nigra in all animals studied, regardless of behavioural classification or CCI. CCK1-IR was quantified through six equidistant sections of the PAG in all animals (n = 21). A stylised diagram (Figure 5.2) shows the boundaries of the functional columns of the PAG.
132
Figure 5.1 Photomicrographs illustrating the presence of CCK1-IR cells as visualised by fluorescent immunohistochemistry.
Top panel a) Low magnification photomicrograph showing CCK1-IR through a coronal section of the midbrain PAG in a CCI animal, with labelling of colliculi also evident (-7.8 from Bregma, 4x magnification) b) CCK1-IR taken at -7.8 Bregma in a Pain Alone CCI animal c) CCK1-IR taken at -7.8 Bregma in a Pain and Transient Disability animal d) CCK1-IR taken at -7.8 Bregma in a Pain and Disability animal.
Figure 5.2 Idealized diagram showing six equidistant sections of the midbrain PAG with defined anatomical borders for the functional columns of the PAG.
133
Figure 5.3 Distribution of CCK1 receptor through rostrocaudal extent of the Ventrolateral Periaqueductal gray (vlPAG).
Distribution of CCK1 receptor immunoreactive cells through five equidistant regions of the ventrolateral column of the Periaqueductal gray (vlPAG) in three behaviourally categorized CCI rat groups (Pain & Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 6). Significant changes were noted between Pain and Disability animals and other CCI animals as shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test). Significant changes between Pain and transient disability animals and Pain alone animals compared to Pain and Disability and uninjured controls is shown by #P<0.05 (ANOVA, Bonferoni post-hoc test). Significant changes were noted between Pain and Disability animals, other CCI animals, and uninjured controls as shown by * P<0.05 (ANOVA, Bonferoni post-hoc test).
Morphology and distribution of CCK1-IR expressing cells throughout the rostro-caudal extent of the
PAG, and differences between behaviourally classified animal groups and controls are described below.
Qualitatively, there was variability in the size of neurons expressing CCK1-IR throughout the midbrain.
In particular, in the PAG, there was a size distribution ranging from very small (~10 µm) to large-sized neurons (~40 µm). CCK1-IR neurons were also expressed ubiquitously throughout the columns of the
PAG and its neighbouring nuclei. The colliculi also showed prominent CCK1-IR (Figure 5.1).
Ventrolateral periaqueductal gray (vlPAG): in the five equidistant coronal sections analysed in each animal, the mean total number of CCK1-IR cells in the vlPAG of rats with pain and persistent disability
134 (2937 ± 107 cells) was significantly lower than either rats with pain and transient disability (3685 ±
170 cells), or pain alone (3486 ± 40 cells), and more akin to control (3152 ± 119 cells) rats (p<0.05 one-way ANOVA, Bonferroni post-hoc) (Figure 5.3a). The distribution of these CCK1-IR cells throughout the five equidistant regions of the vlPAG (Figure 5.3b) also showed significant differences, with pain and persistent disability animals having significantly lower CCK1-IR cells compared to other
CCI rats at -7.8 from Bregma (*p<0.01 two-way ANOVA, Bonferroni post-hoc). At more rostral levels
(-6.8 from Bregma) the number of CCK1-IR cells in pain and transient disability rats and pain alone rats is significantly higher compared to non-injured control rats and pain and persistent disability
(p<0.05 two-way ANOVA, Bonferroni post-hoc).
Lateral periaqueductal gray (lPAG): in the five coronal sections analysed, the mean total number of
CCK1-IR neurons in the lPAG of rats with pain and persistent disability (2824 ± 96 cells) was significantly lower than either rats with pain and transient disability (3971 ± 157 cells), or rats with pain alone (3707 ± 200 cells), and more akin to control (3013 ± 124 cells) rats, which were also significantly lower when compared to pain alone and pain and transient disability animals (Figure 5.4a).
At caudal levels (-8.3 and -7.8 mm relative to Bregma) pain and persistent disability animals have fewer
CCK1-IR cells than other experimental groups (p<0.05 two-way ANOVA, Bonferroni post-hoc). At rostral levels (-6.8 & -6.3 relative to Bregma) the number of CCK1-IR cells in all CCI groups is significantly higher compared to uninjured control rats (p<0.05 two-way ANOVA, Bonferroni post- hoc) (Figure 5.4b).
135
Figure 5.4 Distribution of CCK1 receptor through rostrocaudal extent of the lateral Periaqueductal gray (lPAG).
Distribution of CCK1 receptor immunoreactive cells through five equidistant regions of the lateral column of the Periaqueductal gray (lPAG) in three behaviourally categorized CCI rat groups (Pain & Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n= 6). Significant changes were noted between Pain and Disability animals and all other animals as shown by * P<0.05 (ANOVA, Bonferoni post-hoc test). Significant changes between CCI animals compared to uninjured controls is shown by # P<0.05 (ANOVA, Bonferoni post-hoc test).
136
Figure 5.5 Distribution of CCK1 receptor through rostrocaudal extent of the Dorsomedial Periaqueductal gray (dmPAG).
Distribution of CCK1 receptor immunoreactive cells through five equidistant regions of the dorsomedial column of the Periaqueductal gray (dmPAG) in three behaviourally categorized CCI rat groups (Pain & Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 6). No significant changes were observed.
Dorsomedial periaqueductal gray (dmPAG): in the six coronal sections analysed, the mean total number of CCK1-IR neurons in the dmPAG of rats with pain and persistent disability was (2354 ± 63 cells), rats with pain and transient disability (2470 ± 125 cells), pain alone (2413 ± 80 cells), and uninjured control rats (2213 ± 64 cells). No significant changes in the distribution of CCK1 receptors along the rostrocaudal extent of the dmPAG were observed (Figure 5.5).
137
Figure 5.6 Distribution of CCK1 receptor through rostrocaudal extent of the Dorsolateral Periaqueductal gray (dlPAG).
Distribution of CCK1 receptor immunoreactive cells through four equidistant regions of the dorsolateral column of the periaqueductal gray (dlPAG) in three behaviourally categorized CCI rat groups (Pain & Disability, n=5; Pain & transient Disability, n=5); Pain Alone, n=5), and an uninjured control group (n=6). CCI animals had higher CCK1IR cells compared to uninjured controls *** P<0.05 (ANOVA, Bonferoni post-hoc test). Pain & Transient Disability and Pain Alone animals had significantly higher CCK1IR compared to uninjured controls as shown by ** P<0.05 (ANOVA, Bonferoni post-hoc test).
Dorsolateral periaqueductal gray (dlPAG): in the four coronal sections analysed, the mean total number of CCK1-IR neurons in the dlPAG of experimental CCI rats with pain and persistent disability
(900 ± 32 cells), rats with pain and transient disability (1178 ± 67 cells), or pain alone (1163 ± 86 cells), were significantly higher than for uninjured control rats (646 ± 26 cells) (Figure 5.7a). Additionally, rats with pain and persistent disability had significantly lower CCK1-IR neurons than pain and transient disability and pain alone groups. Compared to other CCI animals, rats with pain and persistent disability had significantly lower numbers of CCK1-IR cells at -6.8 relative to Bregma, and differed to uninjured controls only at rostral levels (-6.3 relative to Bregma) (p<0.05 two-way ANOVA, Bonferroni post-hoc). Conversely, both pain alone and pain and transient disability animals show significantly
138 increased CCK1-IR cells compared to uninjured controls, with one exception (-7.3 relative to Bregma)
(p<0.05 two-way ANOVA, Bonferroni post-hoc) (Figure 5.6b).
5.3.3 CCK2 receptors
Midbrain neurons expressing CCK2 receptor immunoreactivity (CCKb-IR) were identified by fluorescence immunohistochemistry using a specific antipeptide antibody directed against the carboxyl tail of the amino terminal sequence of the CCKb receptor. Much like CCK1-IR (Figure 5.1), CCK2-IR cells were identified bilaterally along the rostro-caudal gradient of all the columns of the PAG in all rats, showing a size distribution ranging from very small (~10um) to large-sized neurons (~40um)
(Figure 5.7. Additionally, CCK2-IR was observed, though not quantified in the colliculli of the midbrain, the dorsal raphe nucleus, and the substantia nigra in all animals studied, regardless of behavioural classification or CCI.
Distribution of CCK2-IR expressing cells throughout the rostro-caudal extent of the PAG, and differences between behaviourally classified animal groups and controls, are described below.
139
Figure 5.7 Photomicrographs illustrating the presence of CCK2-IR cells.
CCK2-IR cells as visualised by fluorescent immunohistochemistry. Top panel a) Low magnification photomicrograph showing CCK2-IR through a coronal section of the midbrain PAG in a CCI animal, with labelling of colliculi also evident (-7.3 from Bregma, 4x magnification) b) CCK2-IR taken at -7.3 Bregma in a Pain Alone CCI animal c) CCK2-IR taken at -7.3 Bregma in a Pain and Transient Disability animal d) CCK2-IR taken at -7.3 Bregma in a Pain and Disability animal.
Ventrolateral periaqueductal gray (vlPAG): in the five equidistant coronal sections analysed, the mean total number of CCK2-IR neurons in the vlPAG of rats with pain and persistent disability (1596 ± 106 cells), rats with pain and transient disability (1516 ± 90 cells), and rats experiencing pain alone (1582
± 19 cells), were not found to be different to those of uninjured control rats (1806 ± 84 cells) (ANOVA,
Bonferroni post-hoc). The distribution of these CCK2-IR cells throughout five equidistant regions of the vlPAG showed significant differences between uninjured control animals and rats with pain and transient disability and rats with pain alone at the level of Bregma -7.8 (p<0.01 two-way ANOVA,
Bonferroni post-hoc)(Figure 5.8).
140
Figure 5.8 Distribution of CCK2 receptor through rostrocaudal extent of the Ventrolateral Periaqueductal gray (vlPAG).
Distribution of CCK2 receptor immunoreactive cells through five equidistant regions of the ventrolateral column of the Periaqueductal gray (vlPAG) in three behaviourally categorized CCI rat groups (Pain & Behavioural Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 5). Significant changes were noted between Pain and Transient Disability and Pain Alone animals compared to uninjured control rats as shown by * P<0.05 (ANOVA, Bonferoni post-hoc test).
Lateral periaqueductal gray grey (lPAG): In the five coronal sections analysed, the mean total number of CCK2-IR neurons in the lPAG of rats with pain and persistent disability (1873 ± 71 cells), rats with pain and transient disability (1748 ± 81 cells), and rats with pain alone (1846 ± 40 cells) were not significantly different to uninjured control rats (1973 ± 62 cells). The distribution of these receptors throughout the rostrocaudal extent of the lPAG is significantly different between Pain and Transient disability and uninjured control animals at -7.8 relative to Bregma (p<0.05 two-way ANOVA,
Bonferroni post-hoc) (Figure 5.9).
141
Figure 5.9 Distribution of CCK2 receptor through rostrocaudal extent of the Lateral Periaqueductal gray (lPAG).
Distribution of CCK2 receptor immunoreactive cells through five equidistant regions of the lateral column of the Periaqueductal gray (lPAG) in three behaviourally categorized CCI rat groups (Pain & Behavioural Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 5). Significant changes were noted between Pain and Transient Disability animals compared to uninjured control rats as shown by * P<0.05 (ANOVA, Bonferoni post-hoc test).
Dorsomedial periaqueductal gray grey (dmPAG): in the six coronal sections analysed, the mean total number of CCK2-IR cells in the dmPAG of rats with pain and persistent disability was (824 ± 36 cells), rats with pain and transient disability (742 ± 27 cells), pain alone (728 ± 26 cells), and uninjured control rats (827 ± 71 cells). A significant change was observed in the distribution of CCK2-IR cells along the rostro-caudal extent of the dmPAG, at the level of -7.3mm Bregma, between uninjured controls and pain alone CCI rats (*p<0.05 two-way ANOVA, Bonferroni post-hoc) (Figure 5.10).
142
Figure 5.10 Distribution of CCK2 receptor through rostrocaudal extent of the Dorsomedial Periaqueductal gray (dmPAG).
Distribution of CCK2 receptor immunoreactive cells through five equidistant regions of the dorsomedial column of the Periaqueductal gray (dmPAG) in three behaviourally categorized CCI rat groups (Pain & Behavioural Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 5). Significant changes were noted between Pain Alone animals compared to uninjured control rats as shown by * P<0.05 (ANOVA, Bonferoni post-hoc test).
Dorsolateral periaqueductal gray (dlPAG): in the four equidistant coronal sections of the dlPAG analysed, the mean total number of CCK2IR cells in uninjured control rats (323 ± 17 cells) were no different to experimental CCI rats with pain and persistent disability (319 ± 18 cells), rats with pain and transient disability (307 ± 20 cells), and pain alone (356 ± 15 cells). The distribution of these
CCK2IR cells in the uninjured control rats is also the same as that of experimental CCI animals throughout the rostro-caudal extent of the dlPAG (Figure 5.11).
143
Figure 5.11 Distribution of CCK2 receptor through rostrocaudal extent of the Dorsolateral Periaqueductal gray (dlPAG).
Distribution of CCK2 receptor immunoreactive cells through four equidistant regions of the dorsolateral column of the Periaqueductal gray (dlPAG) in three behaviourally categorized CCI rat groups (Pain & Behavioural Disability, n = 5; Pain & Transient Disability n = 5; Pain Alone, n = 5), and an uninjured control group (n = 5). No significant changes were noted between CCI animals and uninjured control rats.
The number of CCK1IR cells within the columns of the PAG exceeds that of CCK2IR cells by two-fold.
Both receptor subtypes are found abundantly throughout the rostro-caudal extent of the functional columns of the PAG in both uninjured control rats and experimental CCI rats.
5.4 Discussion
5.4.1Summary of findings
In this study we aimed to see if: (i) the neurons of the midbrain periaqueductal gray contained cognate
CCK receptors, which have been previously identified on a pharmacological basis as being of two subtypes, CCK1 and CCK2; and (ii) to determine the differences, if any, that CCI has on the expression of these receptors, and whether all animals which undergo CCI have similar changes, or whether behaviourally sub-categorised CCI animals show distinct patterns of CCK1- and/or CCK2-receptor expression. Following sciatic nerve CCI and resident-intruder testing animals were categorised on the basis of changes in dominance behaviours as having pain alone, pain and transient disability, or pain 144 and persistent disability. The data presented in this chapter has revealed distinct patterns of CCK receptor immunoreactivity in functionally distinct columns of the midbrain PAG in subcategorised CCI animals.
Both CCK1 and CCK2 receptor subtypes are present within the functional columns of the PAG in both uninjured control rats and behaviourally categorised CCI rats, with the expression of the CCK1 receptor being found in twice as many cells as that of the CCK1IR receptor.
With regards to CCK1 receptors, following CCI rats showed:
(i) an increase in the expression of CCK1IR cells in the dlPAG, with pain and transient
disability and pain alone groups, in particular, having generalised changes
throughout the rostro-caudal extent of this nucleus,
(ii) an increase in the expression of CCK1IR cells in caudal regions of the lPAG (-8.3
& -7.8 relative to Bregma) in rats with pain alone and rats with pain and transient
disability, compared with rats with pain and persistent disability and uninjured
controls, coupled with an increase in the expression of CCK1IR cells in rostral
regions of the lPAG (-6.8 & -6.3 relative to Bregma) in all CCI rats compared to
uninjured control rats,
(iii) a decrease in the expression of CCK1IR cells in an intermediate region of the
vlPAG (-7.8 relative to Bregma) in rats with pain and persistent disability
compared to other CCI rats and uninjured controls, coupled with an increase in the
expression of CCK1IR cells in rostral regions of the vlPAG (-6.8 relative to Bregma)
in all CCI rats compared to uninjured control rats. Generally speaking, pain alone
and pain and transient disability animals showed increased CCK1IR cells
compared to uninjured controls, whilst CCK1IR in pain and persistent disability
animals largely resembled uninjured controls.
Thus, it would seem that pain alone and pain and transient disability animals show an
increase in CCK1IR as a consequence of sciatic CCI. 145 With regards to CCK2 receptors, expression of CCK2IR cells throughout the rostrocaudal extent of the midbrain PAG was lower when compared to CCK1IR. Following CCI:
(i) significant changes were noted between pain and transient disability animals compared to
uninjured control animals in an intermediate region of the lPAG (-7.8 relative to Bregma),
(ii) significant changes were observed between pain alone and pain and transient disability animals
compared to uninjured control rats at an intermediate level of the vlPAG (-7.8 relative to
Bregma),
(iii) significant changes were noted between pain alone animals compared to uninjured control rats
at an intermediate region of the dmPAG (-7.3 relative to Bregma).
Thus, following sciatic CCI no relative increase in CCK2IR is noted.
5.4.2 Comparison to previous studies
Similar to findings by Mercer and colleagues (2004, 2000), using the same antibodies directed at the rat CCK1 and CCK2 receptors, labelling was, in general, exclusively cytoplasmic in cell bodies, with some examples of punctate immunoreactivity in neuronal processes, presumed to be axons and dendrites, and suggestive of the trafficking of CCK receptors. Furthermore, though not quantified, other nuclei reported to be labelled by previous studies, such as the colliculi and the dorsal raphe, were also extensively labelled in our studies. The distribution of these receptors in the midbrain PAG is also in keeping with results obtained by electrophysiology (Crawley and Corwin, 1994), in-situ hybridisation
(Hinks et al., 1995, Honda et al., 1993), and autoradiography studies (Carlberg et al., 1992, Hill and
Woodruff, 1990).
Thus, the presence of CCK receptors has been consistently demonstrated in previous studies, with cells displaying a comparative morphology to that described in the literature.
146 5.4.3 Functional considerations of alterations to CCK receptors following CCI
The data presented in this chapter suggests that complex changes are occurring predominantly in CCK1- receptor, but also in CCK2-receptor, expression following CCI in animals. Previously, Bhatnagar and colleagues (2000) described how a novel acute stressor given to chronically stressed animals triggers activation of CCK2 receptor mRNA in cells of the hypothalamus, via cholecystokinin-mediated pathways, emphasising that CCK receptors can be induced in the face of physical and psychological stressors. In this study we have also shown that, following sciatic CCI, two behaviourally categorised subgroups of animals show increased CCK receptor synthesis, despite all animals showing similar levels of sensory dysfunction following injury.
Sciatic nerve CCI, which could be described as an inescapable chronic physical stressor, in combination with a novel psychological stressor, such as what the interaction with an “intruder” rat represents in the resident-intruder paradigm, has been shown in this study to alter the pattern of CCK receptor expression in the midbrain PAG of CCI animals. In particular, the dorsolateral, lateral, and ventrolateral columns of the PAG underwent significant changes, with pain and persistent disability animals, in general, showing unchanged numbers of CCK receptor immunoreactivity whilst pain and transient disability and pain alone animals show an increase in CCK1-IR compared to uninjured control animals.
Furthermore, these overall patterns in CCKr-IR were attributed to specific changes in regions along the rostro-caudal extent of the functional columns of the PAG, which have been previously shown to have distinct functions. Previously, Keay and Bandler (2001) have described that the lateral PAG has been shown to be critical in the expression of active emotional coping behaviours triggered in response to escapable, physical stress, threat, or pain. In contrast, the dorsolateral PAG is critical for the expression of active emotional coping strategies to primarily “psychological” threats or stressors. In our study we report that rats with pain and persistent disability show significantly lower levels of CCK1-IR compared to pain alone, pain and transient disability, and uninjured control animals at caudal levels of the lPAG
(-8.3 and -7.8 relative to Bregma), suggesting that some of the behavioural effects previously observed in this subgroup of animals, which includes ongoing reductions in dominance in resident-intruder social
147 interactions and altered sensitivity of the hypothalamo-pituitary adrenal (HPA), hypothalamo-pituitary gonadal (HPG), and hypothalamo-pituitary thyroid (HPT) axes may be reflective or a functional consequence of this. Recent work by Mor and colleagues (2010) has shown reactive astrocytes are present in the lateral PAG of pain and persistent disability animals, in addition to other markers of cell death, including pro-apoptotic markers of Bax:Bcl-2 mRNAs, decreased heat shock protein 60 (HSP60), increased iNOS and MEK2 mRNAs, and TUNEL-positive profiles. Thus, the lower number of CCK1-
IR cells observed in pain and persistent disability animals, compared to other CCI animals may be reflective of apoptosis in the lPAG. On the other hand, the fact that CCK1-IR levels are similar to controls suggests a de novo increase in other groups.
The dorsolateral PAG is critical for the expression of active emotional coping strategies to primarily
“psychological” threats or stressors, and is a region widely implicated in conditioned fear behavioural studies (Vianna et al., 2001), and anxiety, which is often co-morbid with depression in human patients.
In this study we found that rats with pain and persistent disability had significantly lower numbers of
CCK1-IR cells compared to pain alone and pain and transient disability animals, a consequence of which may involve dysfunction of the circuits mediated by the dlPAG. A CCK1R knock-out model in the rat has been reported to produce significant anxiogenic-like effects, a condition often found to be co-morbid in depression, and selective pharmacological antagonism of CCK1 receptors has been shown to exert anxiolytic-like activity in several animal models (Ballaz et al., 1997, Hendrie et al., 1993,
Yamamoto et al., 2000).
Patterns of cell apoptosis reported by Mor and colleagues (2011) in the lPAG were also observed in the vlPAG. The vlPAG has been shown to play a critical role in the expression of sleep-wake regulatory circuits (Lu et al., 2006), and it has previously been shown that rats with pain and persistent disability experience alterations to sleep-wake cycles (Monassi et al., 2003). The expression of social behaviours, which have clearly been shown to be dysregulated in rats with pain and persistent disability, is also critically regulated by the vlPAG, which has extensive connectivity with brain regions also critical in regulating the expression of social interactions and neuroendocrine stress responses, such as the
148 orbitomedial prefrontal cortex, central amygdala, the nucleus accumbens, and the hypothalamus, (Floyd et al., 1996, Floyd et al., 2000, Hasue and Shammah-Lagnado, 2002). In this study, significant changes between pain and transient disability and pain alone animals compared to pain and persistent disability and uninjured control rats is observed at an intermediate level of the vlPAG (-7.8 from Bregma), with overall numbers of CCK1-IR cells in pain and persistent disability animals being significantly lower than other CCI animals, and more akin to uninjured controls. This set of results implies that significant cellular damage in the vlPAG, as described by Mor and colleagues (2010) could be leading to functional alterations in neuronal systems crucial in the expression of adaptive behaviours in the wake of a stressor such as CCI represents, with lowered CCK1-IR cells in pain and persistent disability likely being reflective of a behavioural dysfunction characteristic of this subgroup of animals.
CCK1 and CCK2 receptors belong to the class I, rhodopsin receptor family (Dufresne et al., 2006), and are typical of G-protein coupled receptors in having seven hydrophobic segments representing transmembrane helices that form a helicle bundle domain (Kolakowski, 1994). Other notable features are sites of glycosylation, on external loop and tail regions, a conserved disulfide bond linking the predicted first and second extracellular loop regions, a site of palmitoylation on vicinal cysteines on the intracellular side of the seventh segment, and multiple potential sites for serine and threonine phosphorylation in internal loop and tail regions, thought to be critical for proper folding and trafficking to the cell surface (Hadac et al., 1996), and for which structural differences are observed between the two subtypes.
During activation, G protein coupled receptors undergo conformational changes involving movements of several transmembrane helices and of intracellular loops leading to activation of the cognate G protein and other effector proteins regulating receptor activity. As such, the activation of CCK1 receptors can trigger a multitude of signaling pathways, including:
(i) Stimulation of classical second messenger pathways which include phospholipase C (PLC),
phosphatidynositol 4,5-bisphosphate (PIP2), and protein kinase C (PKC)/inositol 3, 4, 5-
triphosphate (IP3) ultimately result in the release of intracellular calcium.
149 (ii) Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPr)
pathways, which release calcium.
(iii) Nitric oxide (NO), which can be released via a NO/cGMP pathway in CCK1 receptor
pathways.
(iv) Class I phosphatidylinositol 3-kinase (PI3K) pathway involving Akt and stimulation of
mammalian target of rapamycin (mTOR) that results in downstream activation of
transcriptions factors, the NF-kB pathway, as well as the Gs-initiated adenylate cyclase
(AC) pathway. PI3K pathways play a role in numerous cell processes, including cell
survival, protein synthesis, motility, and adhesion.
(v) Mitogen-activated protein kinase (MAPK) pathways have been shown to be stimulated by
CCK receptor activation, including p38-MAPK, extracellular-regulated kinase (ERK) and
c-Jun amino-terminal kinase (JNK) pathways.
(vi) The activation of the CCK1 receptor has also been shown to stimulate the phosphorylation
of epidermal growth factor receptor (EGFR), resulting in activation of Ras that in turn has
been shown to be important in the activation of the JNK and ERK pathways.
Thus, activation of CCK receptors can potentially activate gene transcription and alter functionality of cells. Excessive intracellular calcium can potentially result in excitoxicity in cells, which has been shown to occur at the level of the dorsal horn in CCI studies, typically associated with glutamate transmission (Whiteside and Munglani, 2001), and increased intracellular calcium levels activate multiple calcium-dependent enzymes which contribute to neuronal death and dysfunction (Yuan, 2009) as well as activating intrinsic pathways of apoptosis in conditions of cellular stress (Broughton et al.,
2009, Galluzzi et al., 2009).
Inducible nitric oxide synthase (iNOS) has been shown to be up-regulated in both pain and transient disability and pain and persistent disability animals (Mor et al., 2011), and has also been shown to lead to either apoptosis or necrosis in several neurodegenerative and neuroinflammatory conditions (Pannu and Singh, 2006). Increased NO, a viable product of activated CCK receptor pathways, can produce extensive cellular damage by oxidising DNA, proteins, and lipids, as well as being capable of increasing 150 mitochondrial permeability and triggering the release of apoptotic factors into the cytoplasm (Pacher et al., 2007, Vieira et al., 2001).
MAPKs have been shown to regulate a number of important cellular processes, such as gene transcription, protein translation, vesicle trafficking, metabolism, and cytoskeletal function (Cawston and Miller, 2010, Vanhaesebroeck et al., 2001), with JNK pathways, in particular, also being associated with apoptosis (Ip and Davis, 1998, Xia et al., 1995). In light of evidence indicating cells in the midbrain
PAG in CCI animals show hallmarks of cellular stress and cell death (Mor et al., 2011) it is worth highlighting that JNK and p38-MAPK pathways have been implicated in mediating cellular stress induced by pro-inflammatory shock (Gutkind, 2000), with p38-MAPK pathways, in particular, being inducible by physiological doses of CCK (Schafer et al., 1998).
In light of evidence reported by Mor and colleagues (2011, 2010), which showed reactive astrocytes present in the lateral and ventrolateral PAG of pain and persistent disability animals, in addition to other markers of cell death, including pro-apoptotic markers of Bax:Bcl-2 mRNAs, decreased heat shock protein 60 (HSP60), increased iNOS and MEK2 mRNAs, and TUNEL-positive profiles, as well as evidence of altered CCK mRNA expression in the vlPAG and dorsal raphe of pain and persistent disability animals presented in Chapter 4 of this thesis, the findings of reduced CCK1-IR in pain and persistent disability animals shown in this study, in comparison to two other behaviourally categorised subgroups of CCI animals (pain alone and pain and transient disability), add further evidence to a drastically altered neurological environment within the midbrain PAG, indicative of the behavioural dysfunction seen in these animals despite the experience of similar levels of sensory disturbance with other CCI rats.
5.4.4 Functional anatomical pathways of the PAG
The inherent differences observed in CCK receptor expression in pain and persistent disability animals compared to other CCI animals in this study, namely, reduced CCK1IR in caudal regions of the lPAG, the vlPAG, and the dlPAG, is indicative of alterations to CCKergic transmission in the CNS of these animals. That the pain and persistent disability group of animals resembles labelling patterns of
151 uninjured controls in a lot of respects indicates that the differences in pain and transient disability and pain alone groups are adaptive to CCI. Thus, changes in expression of CCK receptors observed in this study suggest activation of CCKergic neuronal populations that likely include afferents of spinal, brainstem, and forebrain regions.
The dlPAG is a region of the brain critical for the expression of active emotional coping strategies to primarily “psychological” threats or stressors that has been shown to undergo complex changes in
CCK1-IR in this study, with rats with pain and persistent disability showing significantly lower CCK1-
IR compared to other CCI animals. Both the dlPAG (Sewards and Sewards, 2003, Vianna et al., 2001) and CCK have been unequivocally shown to play a role in severe anxiety and panic induction (Abelson et al., 2005, Bradwejn and Koszycki, 2001, Strohle et al., 2000), prompting inference that variations in brainstem and spinal nuclei associated with respiratory and cardiopulmonary regulation contribute to some of the behavioural symptoms (Bystritsky and Shapiro, 1992, Gardner, 1996). The dlPAG does not receive spinal inputs and whilst it does not directly project to cardio-, respiratory-, and pain- modulatory medullary regions, it can influence them via projections to the cuneiform nucleus and the parabrachial nucleus (Keay and Bandler, 2001, Krout et al., 1998). The dlPAG also projects to dorsal and medial hypothalamic areas important in cardiomodulation and somatomotor activation (Bhatnagar et al., 2000, Keay and Bandler, 2001), and cortex regions important in regulating behaviours (Floyd et al., 2000). Furthermore, c-Fos studies have shown, following electrical stimulation of the dlPAG, increases in c-Fos immunoreactivity in behaviour-regulatory regions of the brain including medial prefrontal and orbital cortex, somatosensory cortex, hypothalamus, and the amygdala (Lim et al., 2009), all regions which have been shown to have extensive CCKergic cell populations.
In the lPAG and vlPAG, rats with pain alone and rats with pain and transient disability showed an increase in CCK1-IR consequent to sciatic CCI, with pain and persistent disability animals resembling uninjured controls. The lateral PAG has been shown to be critical in the expression of active emotional coping behaviours triggered in response to escapable, physical stress, threat, or pain, whilst the vlPAG has been shown to also be involved in the expression of behaviours, whilst also playing key roles in regulating sleep-wake arousal circuits and nociception, as well as having extensive connectivity with 152 regions of the brain involved in neuroendocrine, and autonomic function. Both the lPAG and vlPAG are major spinal-recipient regions. Spinal projections to both the lPAG and vlPAG arise contra-laterally from superficial dorsal horn, deep dorsal horn, and the lateral spinal nucleus, with PAG-projecting spinal neurons readily activated by noxious stimulation (Clement et al., 2000, Dougherty et al., 1999,
Hylden et al., 1986, Yezierski and Broton, 1991). Additionally, both the vlPAG and the lPAG receive inputs from general visceral afferent pathway-recipient regions of the nucleus of the solitary tract, and project to cardio-, respiratory-, and pain-modulatory regions of the medulla (Keay and Bandler, 2001).
Of particular importance, studies have shown c-fos protein expression in the NTS and circumventricular organ in response to CCK infusion (Viltart et al., 2006, Zittel et al., 1999), with Viltart and colleagues
(2006) intriguingly show a significant portion of these CCK-reactive NTS neurons projecting to the
PAG.
The vlPAG column receives selective input from orbital and anterior insular areas, as well as a weaker input from medial and dorsomedial PFC areas, whilst the lPAG receives input from the dorsomedial
PFC. Parallel circuits from these areas of the OMPFC also innervate the hypothalamus (Floyd et al.,
2000). The PAG also shares reciprocal connections with the hypothalamus, with early studies showing significant inputs to the PAG stemming from the hypothalamus, in particular the ventromedial nucleus of the hypothalamus, lateral hypothalamus, posterior hypothalamus, anterior hypothalamus, and the perifornical nucleus (Beitz, 1982). Of particular importance, the vlPAG projects to endocrine regulatory areas of the paraventricular nucleus of the hypothalamus (PVN) (Bhatnagar et al., 2000, Floyd et al.,
1996), an important site for the release of adrenocorticotrophic hormone (ACTH), and corticotrophin releasing factor (CRF), both of which are important in responses to stress, as well as behavioural and cardiovascular changes.
5.5 Methodological considerations
It was not possible to complete double-labelling studies of CCK1 and CCK2 receptors in the midbrain, in order to elucidate whether these receptors are co-localised in cells, due to tissue scarcity and the possibility of cross-labelling due to antibodies being raised in the same animal (rabbit). The antibodies
153 used in this study have been used in published data (Mercer and Beart, 1997, Mercer et al., 2000) and both positive and negative reactions showed specificity of primary and secondary antibodies. Whilst counting of cell labelling was done in a blinded manner, to reduce scorer bias, it was not possible to counterstain the functional columns of the PAG in the tissue sections examined. As such, all counting was performed based on the boundaries according to the rat brain atlas of Paxinos and Watson (5th edition) using a microscope with specific fluorescent filters (Olympus BX51) and a counting graticule.
In this study, external stressors on the resident animals were kept to a minimum. Whilst we acknowledge that the features of the experimental paradigm such as sacrifice order and social isolation may potentially alter the levels of protein expression observed in the controls and the “resident” CCI animals, the differing levels of both CCK1- and CCK2-IR between the behaviourally categorised CCI groups suggest that the injury itself has a significant effect on the levels of receptor expression than any other potential factor.
5.6 Summary
Following CCI, two distinct, behaviourally categorised groups of animals pain alone and pain and transient disability animals, show increased CCK-receptor immunoreactivity, indicative of an adaptive response. Whilst a third behaviourally categorised group of animals exhibiting pain and persistent disability post-CCI, and which have previously been shown to have extensive dysfunction, including altered sensitivity of HPA, HPG, and HPT stress axes; and altered sleep behaviours (Kilburn-Watt et al., 2010; Mor et al., 2010) shows significantly lower expression of CCK receptors in functionally relevant regions of the PAG, despite all animals showing identical levels of the hallmark sensory disturbances of neuropathic pain. We postulate that these significant differences in CCK receptor expression are reflective of the maladaptive responses observed in pain and persistent disability rats, which bear a striking resemblance to the human clinical picture of neuropathic pain.
154 Chapter6 General Discussion and summary
6.1 Thesis aims
The aims of each of the studies reported in this thesis are revisited in this chapter, including a summary of the major findings of each experiment, followed by a comment on the impact of the data in the context of our current knowledge of chronic neuropathic pain.
People with chronic neuropathic pain report disturbances of social relations, sleep and appetite; metabolic, endocrine and sexual dysfunction; a loss of interest in external events and moderate to severe depression. More often, it is these disabilities, rather than the allodynia and hyperalgesia, which are the most debilitating. The neural adaptations underlying the sensory changes of neuropathic pain have been studied experimentally in rats. In these studies pain is defined by allodynia and hyperalgesia, however the usefulness and adequacy of animal models depends critically on the extent to which they mimic all principal features of chronic neuropathic pain in humans. It is surprising given clinical observations that few systematic attempts have been made to assess whether similar disabilities occur after nerve injury in animals.
Chronic constriction injury (CCI) of the sciatic nerve leads to disabilities in some rats (Keay et al., 2004,
Monassi et al., 2003), despite identical levels of pain (i.e., allodynia and hyperalgesia), three distinct patterns of disability are triggered by CCI: (1) Persistent Disability: a pattern shown by ~30% of rats is characterised by: (i) reductions in dominance in resident-intruder social interactions; (ii) reduced sleep and; (iii) altered sensitivity of the hypothalamo-pituitary adrenal (HPA), hypothalamo-pituitary gonadal (HPG) and hypothalamo-pituitary thyroid (HPT) axes (Kilburn-Watt et al., 2010) (2) Transient Disability: a pattern shown by ~ 20% of CCI rats comprising: (i) reductions in dominance on post-injury days 2-4; (ii) reduced sleep during light phase; or (3) No Disability: 50% of
CCI rats with sensory changes only. That all rats showed similar degrees of “pain”, also fits well with clinical studies which suggest that the degree of sensory dysfunction resulting from surgical nerve damage predicts neither the development of chronic pain nor the degree of disability (Price, 2000). 155 The general aim of this thesis was to further characterise the complex behavioural and physiological consequences of sciatic CCI in this subpopulation of rats which display pain and persistent disability.
6.2 Descriptive findings of two supraspinal CCKergic neuronal populations with projections to the midbrain PAG
Unpublished work in the Laboratory of Neural structure and function identified the presence of CCK-
8-IR varicosities clustered around arborisations that are strongly indicative of labelling restricted to terminals and ‘en passant’ fibres. Specifically, in sciatic CCI animals, rats with pain and persistent disability showed increased overall expression of this terminal labelling compared to pain alone rats, with labelling being particularly pronounced in the vlPAG. Given the changes in the vlPAG specific to rats with pain and persistent disability presented in this thesis, namely, increases in CCK mRNA expression and a decrease in CCK1-receptor expression in an intermediate region of the vlPAG, the question arises, what is the anatomical source of these CCK-expressing terminal fibres?
In the case of this study, due to technical constraints, it was not possible to generate enough tissue from behaviourally categorized CCI groups. This would be an important first step, as it would make it possible to observe any inherent differences in the number of CCKergic inputs between pain alone, pain and transient disability, and pain and persistent disability animals compared to an uninjured control group. Further characterization as to the phenotype of these cells projecting on to the PAG would then be possible, as a wide body of literature has reported that CCK co- localizes with a diverse number of different neurotransmitters in the brain. Furthermore, the PAG also receives ascending spinal projections, and descending cortical projections, which may also contain CCKergic cells, and be contributing to the altered levels of CCK transmission originally indicated by CCK terminal labelling.
Perhaps the most obvious site to look for changes in CCK expression would be in Sciatic nerve recipient regions of the Spinal cord. The PAG receives significant Spinal cord afferents and changes to these projections may underlie some of the observable differences noted in this thesis. Experimental analysis of the spinal cord would thus be a priority in the future.
156
Focusing on the two supraspinal regions focused in this study; (i) the brainstem nucleus of the solitary tract (NTS); and (ii) the hypothalamus, we show that there are significant CCKergic projections from both of these regions into the vlPAG of sciatic CCI animals.
The brainstem NTS plays an important role in the supraspinal modulation of pain, where it interacts with midbrain and brainstem structures to supress or enhance nociceptive signalling. The NTS also contains a noradrenergic cell group (A2) responsive to stressors, which has been previously shown to project densely to the PAG.
In this study a majority of CTB-IR cells, whose pattern strongly resembles that of the A2 cells known to project to the PAG, were found to be co-localised with CCK. The solitary tract nucleus (NTS) in the dorsal medulla integrates multiple viscerosensory processes and has been shown to be activated in response to deep pain (Palkovits et al., 1995), and to mediate cardiovascular responses to stress (Daubert et al., 2012). Thus, the possibility that afferents from this brainstem region are mediating the physiological and behavioural disturbances observed in pain and persistent disability animals is of interest in the future.
Similarly, cells originating from three hypothalamic subnuclei, the Lateral hypothalamus, the arcuate nucleus, and the dorsomedial hypothalamus, also contained a significant number of co-localised CTB-
CCK-IR cells. Of particular interest,
(i) The lateral hypothalamus contains neurons important in regulating sleep-wake arousal systems
of the brain, which have previously been shown to be dysfunctional in pain and persistent
disability animals (Monassi et al., 2003).
(ii) Altered HPT axis regulation has been observed in a sub-population of rats experiencing
behavioural disability following CCI (Kilburn-Watt et al., 2010). The arcuate nucleus of the
hypothalamus is an important site of Thyrotropin releasing hormone (TRH) distribution.
Alterations to CCKergic systems may be responsible for these observed changes. 157 (iii) Circadian rhythm disruption is a hallmark of dysfunction in human patients suffering chronic
pain and is observable in animal models. A significant number of co-localised CTB-CCK-IR
cells were observed in the dorsomedial hypothalamus, a region involved in feeding and
drinking behaviours, body weight regulation, and circadian activity.
Identifying differences, if any, in our behaviourally categorised animals would be of experimental importance.
Additionally, an experimental group that undergoes brain injection following CCI may also be required to capture neuroplastic changes associated with injury. This would be a valuable group to study given the possibilities of sprouting, increased ramification, and novel inputs into the PAG from spinal afferents or supraspinal inputs following injury.
The emotional state has been shown to have a significant impact on pain perception and the ability to cope. Critical regions involved in amplifying/exacerbating pain include the entorhinal complex, amygdala, anterior insula, and prefrontal cortices. These regions would thus also be of interest in future studies.
6.3 Populations of CCK expressing neurons in the midbrain
Work by Mor and colleagues (2011, 2010) has described multiple changes occurring at the molecular level in the midbrain PAG of pain and persistent disability rats indicative of cellular stress and apoptosis in these animals. One of these findings was of increased expression of cholecystokinin mRNA. Given the multiple, often opposing, actions that different regions of the PAG regulate, the aim of chapter 4 was to anatomically localise these changes using single and double-label in-situ hybridization techniques.
Results showed that rats with pain and persistent disability, following sciatic nerve CCI, showed a significant up-regulation of CCK mRNA in the dorsal midbrain. In situ hybridization studies revealed greater numbers of CCK mRNA containing cells in the vlPAG and dorsal raphe (dorsalis and ventralis)
158 in rats with Pain and Disability. In addition, CCK mRNA positive cells of the vlPAG and dorsal raphe
(dorsalis and ventralis) in rats with Pain and Disability had a greater density of silver grains, indicating higher levels of CCK mRNA expression. The mean densities of silver grain labelling correlated strongly with the degree of behavioural change (decreased dominance) in resident-intruder interactions. CCK mRNA was not expressed in the 5- HT-IR cells of the dorsal raphe nucleus. Although not a specific focus of this study, we also revealed a significantly smaller number of 5-HT-immunoreactive cells in the lateral wings of the dorsal raphe in rats with Pain and Disability.
The selectivity of patterns of up-regulation suggests that changes are occurring in specific neural circuits.
The PAG receives afferents from spinal, brainstem, and forebrain regions all of which could be responsible for triggering these changes. Whilst spinal afferents project directly to the PAG, the complexity of behavioural and physiological disturbances observed in these animals indicates that supraspinal regions of the brain may be driving these changes. It is, however, also worth noting that the
PAG itself contains a population of interneurons, which may of themselves be driving these changes in
CCK expression, either as a result of changes within the local cellular environment, which has been shown to be altered in these animals, or as a result of altered transmission into these regions as previously mentioned. The next logical question to ask is whether these are intrinsic cells, which communicate exclusively to other cells within the PAG, or whether these cells send outputs to different regions of the brain.
CCK mRNA expressing cells in this study were not co-localised with serotonin, a neurotransmitter often implicated as playing an important role in “limbic” systems of the brain. Further work will need to focus on identifying the phenotype of these cells. Given their location, and the fact that previous studies have observed that (i) rats with pain and persistent disability have altered sleep-wake patterns
(Monassi et al., 2003), and; (ii) Wake-active dopaminergic neurons are located in the ventrolateral PAG
(Lu et al., 2006), performing double label in situ hybridization studies with tyrosine hydroxylase (TH), the enzyme responsible for catalysing dopamine, would be a focus for future studies.
Furthermore, given that we have observed increased CCK mRNA in the vlPAG and Dorsal Raphe 159 selectively in animals with pain and persistent disability compared to other CCI animals it would be worthwhile to see if the behavioural and physiological changes observed in this subgroup of animals can be reversed by antagonism of CCK in this region of the brain. That is, would microinjection of
CCK antagonists/agonists into the midbrain vlPAG change behavioural and physiological patterns in rats with pain and persistent disability?
6.4 Distribution of CCK receptors along the rostrocaudal extent of the PAG
Given that the results obtained in chapter 4 showed an anatomically specific pattern of increased CCK expression, suggesting activation of neurons by select neural pathways, we next considered if there are similar changes occurring in this region for the receptors of CCK. CCK1- and CCK2-receptors are specific G-protein coupled receptor subtypes whose distribution throughout the CNS has been previously reported. Using antibodies reported to be specific for these receptors we aimed to see if: (i) the neurons of the midbrain PAG contained cognate CCK receptors; and (ii) to observe the differences, if any, on the expression of these receptors following CCI.
With regards to CCK receptors, following CCI rats showed:
. (i) an increase in the expression of CCK1IR cells in the dlPAG, with pain and transient disability
and pain alone groups, in particular, having generalised changes throughout the rostro-caudal
extent of this nucleus,
. (ii) an increase in the expression of CCK1IR cells in caudal regions of the lPAG (-8.3 & -7.8 relative
to Bregma) in rats with pain alone and rats with pain and transient disability, compared with
rats with pain and persistent disability and uninjured controls, coupled with an increase in the
expression of CCK1IR cells in rostral regions of the lPAG (-6.8 & -6.3 relative to Bregma) in
all CCI rats compared to uninjured control rats,
. (iii) a decrease in the expression of CCK1IR cells in an intermediate region of the vlPAG (-7.8
relative to Bregma) in rats with pain and persistent disability compared to other CCI rats and 160 uninjured controls, coupled with an increase in the expression of CCK1IR cells in rostral
regions of the vlPAG (-6.8 relative to Bregma) in all CCI rats compared to uninjured control
rats. Generally speaking, pain alone and pain and transient disability animals showed
increased CCK1IR cells compared to uninjured controls, whilst CCK1IR in pain and
persistent disability animals largely resembled uninjured controls.
Thus, it would seem that pain alone and pain and transient disability animals show an increase in
CCK1IR as a consequence of sciatic CCI, highly indicative of an adaptive response that fails in the pain and persistent disability animals.
Altered levels of CCK mRNA expression may be related to altered levels of its receptors. If the pain and persistent disability animals fail to compensate with up-regulation of receptors, a second strategy may be to increase neurotransmitter levels, as reflected by higher CCK mRNA expression in these animals.
The functional implications of these changes need to be considered based on the circuits the affected regions of the PAG mediate. The dorsolateral PAG is critical for the expression of active emotional coping strategies to primarily psychological threats or stressors and, as such, is a region widely implicated in anxiety, which is commonly found co-morbid with depression in humans, and fear. The lateral PAG has been shown to be critical in the expression of active emotional coping behaviours triggered in response to escapable, physical stress, threat, or pain, and thus differences between pain and persistent disability animals compared to other CCI animals may be accounting for some of the behavioural dysfunction observed in these animals. Similarly, changes in the vlPAG may be driving some of the behavioural and physiological maladaptions previously described.
Given the results obtained in our study future work may focus on the effect that agonists and antagonists of CCK1- and CCK2-receptors may have on these functionally specific regions of the PAG in CCI injured animals, with a mind to observe if the behavioural and physiological dysfunctions observed in pain and persistent disability animals can be reversed via pharmacological application of such
161 compounds. Given that activation of these receptors can trigger multiple molecular pathways that can drastically alter cellular metabolism, protein synthesis, and viability of the cell, it would also be important to elucidate which pathways are being activated in the cells of the PAG.
162
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