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The neural circuits underling sensory, affective, and cognitive dimensions of pain are well-defined, and there is strong evidence that these circuits are compromised by the activation of glial cells and the release of immune mediators in chronic pain states. Immune mediators can modulate glutamatergic and GABAergic signaling, synaptic plasticity, neurogenesis, and neurotrophic factors. Recent neural imaging studies highlight astrocyte and microglial activation within the sensory-discriminative circuits in chronic pain patients. There is also emerging pre-clinical evidence that individuals with neuropathic pain that are ‘susceptible’ to co-morbid affective disturbances have neuroinflammation in the interconnected prefrontal cortex-ventral hippocampal circuitry. The therapeutic potential of anti-inflammatory agents to mitigate these detrimental supraspinal neuroimmune interactions should be considered in chronic pain patients, particularly those with elevated peripheral immune biomarkers and co-morbid affective disturbances.

Diabetic neuropathic pain is a common and devastating complication of type 1 diabetes, but the mechanism by which it develops and persists is yet to be fully elucidated. This study utilised high-dimensional suspension mass cytometry in a pilot cohort to investigate differences in peripheral blood immunophenotypes between type 1 diabetes patients with (n ​= ​9) and without (n ​= ​9) peripheral neuropathic pain. The abundance and activation of several leukocyte subsets were investigated with unsupervised clustering approaches FlowSOM and SPADE, as well as by manual gating. Major findings included a proportional increase in CD4+ central memory T cells and an absolute increase in classical monocytes, non-classical monocytes, and mature natural killer cells in type 1 diabetes patients with pain compared to those without pain. The expression of CD27, CD127, and CD39 was upregulated on select T cell populations, and the phosphorylated form of pro-inflammatory transcription factor MK2 was upregulated across most populations. These results provide evidence that distinct immunological signatures are associated with painful neuropathy in type 1 diabetes patients. Further research may link these changes to mechanisms by which pain in type 1 diabetes is initiated and maintained, paving the way for much needed targeted treatments.

Neuropathic pain is a common complication of diabetes with high morbidity and poor treatment outcomes. Accumulating evidence suggests the immune system is involved in the development of diabetic neuropathy, whilst neuro-immune interactions involving the kynurenine (KYN) and tetrahydrobiopterin (BH4) pathways have been linked to neuropathic pain pre-clinically and in several chronic pain conditions. Here, using a multiplex assay, we quantified serum levels of 14 cytokines in 21 participants with type 1 diabetes mellitus, 13 of which were classified as having neuropathic pain. In addition, using high performance liquid chromatography and gas chromatography-mass spectrometry, all major KYN and BH4 pathway metabolites were quantified in serum from the same cohort. Our results show increases in GM-CSF and IL-8, suggesting immune cell involvement. We demonstrated increases in two inflammatory biomarkers: neopterin and the KYN/TRP ratio, a marker of indoleamine 2,3-dioxygenase activity. Moreover, the KYN/TRP ratio positively correlated with pain intensity. Total kynurenine aminotransferase activity was also higher in the diabetic neuropathic pain group, indicating there may be increased production of the KYN metabolite, xanthurenic acid. Overall, this study supports the idea that inflammatory activation of the KYN and BH4 pathways occurs due to elevated inflammatory cytokines, which might be involved in the pathogenesis of neuropathic pain in type 1 diabetes mellitus. Further studies should be carried out to investigate the role of KYN and BH4 pathways, which could strengthen the case for therapeutically targeting them in neuropathic pain conditions.

Complex regional pain syndrome (CRPS) is a debilitating condition where trauma to a limb results in devastating persistent pain that is disproportionate to the initial injury. The pathophysiology of CRPS remains unknown; however, accumulating evidence suggests it is an immunoneurological disorder, especially in light of evidence of auto-antibodies in ~ 30% of patients. Despite this, a systematic assessment of all circulating leukocyte populations in CRPS has never been performed.We characterised 14 participants as meeting the Budapest clinical criteria for CRPS and assessed their pain ratings and psychological state using a series of questionnaires. Next, we performed immunophenotyping on blood samples from the 14 CRPS participants as well as 14 healthy pain-free controls using mass cytometry. Using a panel of 38 phenotypic and activation markers, we characterised the numbers and intracellular activation status of all major leukocyte populations using manual gating strategies and unsupervised cluster analysis.We have shown expansion and activation of several distinct populations of central memory T lymphocytes in CRPS. The number of central memory CD8These findings represent the first mass cytometry immunophenotyping study in any chronic pain state and provide preliminary evidence of an antigen-mediated T lymphocyte response in CRPS. In particular, the presence of increased numbers of long-lived central memory CD4

We investigated serum levels of 29 cytokines and immune-activated kynurenine and tetrahydrobiopterin pathway metabolites in 15 complex regional pain syndrome (CRPS) subjects and 14 healthy controls. Significant reductions in interleukin-37 and tryptophan were found in CRPS subjects, along with positive correlations between kynurenine/tryptophan ratio and TNF-α levels with kinesiophobia, tetrahydrobiopterin levels with McGill pain score, sRAGE, and xanthurenic acid and neopterin levels with depression, anxiety and stress scores. Using machine learning, we identified a set of binary variables, including IL-37 and GM-CSF, capable of distinguishing controls from established CRPS subjects. These results suggest possible involvement of various inflammatory markers in CRPS pathogenesis.

Background Complex regional pain syndrome (CRPS) is a debilitating condition where trauma to a limb results in devastating persistent pain that is disproportionate to the initial injury. The pathophysiology of CRPS remains unknown; however, accumulating evidence suggests it is an immunoneurological disorder, especially in light of evidence of auto-antibodies in ~ 30% of patients. Despite this, a systematic assessment of all circulating leukocyte populations in CRPS has never been performed. Methods We characterised 14 participants as meeting the Budapest clinical criteria for CRPS and assessed their pain ratings and psychological state using a series of questionnaires. Next, we performed immunophenotyping on blood samples from the 14 CRPS participants as well as 14 healthy pain-free controls using mass cytometry. Using a panel of 38 phenotypic and activation markers, we characterised the numbers and intracellular activation status of all major leukocyte populations using manual gating strategies and unsupervised cluster analysis. Results We have shown expansion and activation of several distinct populations of central memory T lymphocytes in CRPS. The number of central memory CD8+ T cells was increased 2.15-fold; furthermore, this cell group had increased phosphorylation of NFkB and STAT1 compared to controls. Regarding central memory CD4+ T lymphocytes, the number of Th1 and Treg cells was increased 4.98-fold and 2.18-fold respectively, with increased phosphorylation of NFkB in both populations. We also found decreased numbers of CD1c+ myeloid dendritic cells, although with increased p38 phosphorylation. These changes could indicate dendritic cell tissue trafficking, as well as their involvement in lymphocyte activation. Conclusions These findings represent the first mass cytometry immunophenotyping study in any chronic pain state and provide preliminary evidence of an antigen-mediated T lymphocyte response in CRPS. In particular, the presence of increased numbers of long-lived central memory CD4+ and CD8+ T lymphocytes with increased activation of pro-inflammatory signalling pathways may indicate ongoing inflammation and cellular damage in CRPS.

Nerve damage leads to the development of disabling neuropathic pain in susceptible individuals, where patients present with pain as well as co-morbid affective behavioural disturbances, such as anhedonia, decreased motivation and depression. In this study we aimed to characterise changes in neuroinflammation in the medial prefrontal cortex (mPFC) and hippocampus (HP) in a rat model of neuropathic pain (NP) and behavioural changes. 53 rats underwent sciatic nerve chronic constriction injury (CCI) and were characterised as either, No effect, Acute effect or Lasting effect on the basis of changes in exploration behaviour in a radial-arm maze. Microglial and astrocyte morphology, as well as IL-1β, IL-6, IL-10, MCP-1, p38 MAPK and BDNF expression was quantified throughout the mPFC and HP using protein multiplex assays and immunofluorescence. All behavioural groups of CCI rats displayed equal levels of mechanical allodynia; however, the characteristic withdrawal from pellet-seeking observed in Lasting effect rats was accompanied by neuroimmune activation within the contralateral ventral HP and mPFC. This includes increased expression of IL-1β, IL-6 and MCP-1, increased phospho-p38 MAPK expression in neurons and microglia, and a shift to a reactive microglial morphology in the caudal PL and IL, ventral CA1 and DG. Therefore, neuroinflammation in the mPFC and ventral HP may influence individual differences in radial-arm maze behaviour following CCI. Our data provide further evidence that individual differences in neuroimmune activation in the interconnected ventral HP-mPFC circuitry may play a role in the divergent behavioural trajectories following nerve injury, with neuroinflammation being coincident with affective behavioural changes in susceptible individuals.

Neuroimmune crosstalk in neuropathic pain is a key contributor to pain hypersensitivity following nervous system injury. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are endogenous immune suppressors, reducing T-cell proliferation and proinflammatory cytokine production. Currently, the role of Tregs in neuropathic pain is unknown. In this study, we tested the effects of expanding Tregs on pain hypersensitivity and neuroinflammation in 2 models of neuropathy; sciatic nerve chronic constriction injury and experimental autoimmune neuritis in rats. Following chronic constriction injury, treatment with CD28 superagonist (CD28SupA), a Treg population expander, significantly increased Tregs in the lymphoid tissues, injured sciatic nerve, and lumbar spinal cord of rats. CD28SupA treatment led to a significant reduction in mechanical pain hypersensitivity, alongside a decrease in the numbers of infiltrating T cells, macrophages, and antigen-presenting cells in the sciatic nerve and dorsal root ganglia. In experimental autoimmune neuritis-affected rats, CD28SupA treatment resulted in a significant improvement in disease severity and in mechanical pain hypersensitivity. This was associated with a reduction in the numbers of T cells, macrophages, and antigen-presenting cells in the sciatic nerve and dorsal root ganglia, and reduced activation of microglia and infiltration of T cells in the spinal cord. Furthermore, depletion of Tregs by a CD25 antibody in mice with a partial sciatic nerve ligation resulted in prolonged mechanical pain hypersensitivity. These findings suggest that Tregs play a role in endogenous recovery from neuropathy-induced pain. Thus, this T-cell subset may be specifically targeted to alleviate chronic neuropathic pain.

In a large proportion of individuals nervous system damage may lead to a debilitating chronic neuropathic pain. Such pain may now be considered a neuro-immune disorder, since recent data indicate a critical involvement of innate and adaptive immune responses following nerve injury. Activation of immune and immune-like glial cells in the injured nerve, dorsal root ganglia and spinal cord results in the release of both pro- and anti-inflammatory cytokines, as well as algesic and analgesic mediators, the balance of which determines whether pain chronicity is established. This review will critically examine the role of the immune system in modulating chronic pain in animal models of nervous system injury, and highlight the possible therapeutic opportunities to intervene in the development and maintenance of neuropathic pain.

This chapter summarizes a standard approach to identifying neuropathic pain for the clinician. For neuropathic pain, and for the condition of complex regional pain syndrome (CRPS) especially, the six Ss should be queried when obtaining details regarding the affected region. A useful tool to rapidly and accurately localize sources of chronic pain and assist in the diagnosis of causes of neuropathic pain is a pain diagram. The examination of a chronic pain patient should start with an appropriate and directed general examination including a neurological examination. Quantitative sensory testing (QST) provides indirect information used to evaluate underlying sensory function abnormalities using only small, portable tools and with less time requirement than protocols developed by the German Neuropathic Research Network. In the future, bedside QST is expected to continue to play a role in determining potential pain mechanisms to help direct further evaluation and treatment.

Complex behavioural disabilities, as well as pain, characterise neuropathic pain conditions for which clinical treatment is sought. In rats, chronic constriction injury (CCI) of the sciatic nerve evokes, allodynia and hyperalgesia as well as three distinct patterns of disability, characterised by changes in social and sleep-wake behaviours: (i) Pain & Disability; (ii) Pain & Transient Disability and (iii) Pain alone. Importantly, the degree of allodynia and hyperalgesia is identical for each of these groups. Social-interactions and sleep-wake behaviours are regulated by neural networks, which converge on the periaqueductal grey (PAG). Rats with Pain & Disability show astrocyte activation restricted to the lateral and ventrolateral PAG. Reactive astrocytes are a hallmark of cell death (apoptosis and necrosis). Quantitative real-time RT-PCR for the mRNAs encoding Bax, Bcl-2, heat shock protein 60 (HSP60), mitogen activated kinase kinase (MEK2) and iNOS was performed on the dorsal midbrains of individual, disability characterised rats, extending our earlier Gene-Chip data, showing a select up-regulation of Bax and MEK2 mRNA, and a down-regulation of HSP60 mRNA, in Pain & Disability rats. The anatomical location of TUNEL and cleaved-caspase-3 immunoreactive profiles in the midbrain was also identified. Rats with Pain & Disability showed: (i) pro-apoptotic ratios of Bax:Bcl-2 mRNAs; (ii) decreased HSP60 mRNA; (iii) increased iNOS and MEK2 mRNAs; (iv) TUNEL-positive profiles in the lateral and ventrolateral PAG; and (v) caspase-3 immunoreactive neurons in the mesencephalic nucleus of the trigeminal nerve. Cell death in these specific midbrain regions may underlie the disabilities characterising this subgroup of nerve-injured rats.

Allodynia, hyperalgesia and spontaneous pain are cardinal sensory signs of neuropathic pain. Clinically, many neuropathic pain patients experience affective-motivational state changes, including reduced familial and social interactions, decreased motivation, anhedonia and depression which are severely debilitating. In earlier studies we have shown that sciatic nerve chronic constriction injury (CCI) disrupts social interactions, sleep-wake-cycle and endocrine function in one third of rats, a subgroup reliably identified six days after injury. CCI consistently produces allodynia and hyperalgesia, the intensity of which was unrelated either to the altered social interactions, sleep-wake-cycle or endocrine changes. This decoupling of the sensory consequences of nerve injury from the affective-motivational changes is reported in both animal experiments and human clinical data. The sensory changes triggered by CCI are mediated primarily by functional changes in the lumbar dorsal horn, however, whether lumbar spinal changes may drive different affective-motivational states has never been considered. In these studies, we used microarrays to identify the unique transcriptomes of rats with altered social behaviours following sciatic CCI to determine whether specific patterns of lumbar spinal adaptations characterised this subgroup. Rats underwent CCI and on the basis of reductions in dominance behaviour in resident-intruder social interactions were categorised as having Pain & Disability, Pain & Transient Disability or Pain alone. We examined the lumbar spinal transcriptomes two and six days after CCI. Fifty-four 'disability-specific' genes were identified. Sixty-five percent were unique to Pain & Disability rats, two-thirds of which were associated with neurotransmission, inflammation and/or cellular stress. In contrast, 40% of genes differentially regulated in rats without disabilities were involved with more general homeostatic processes (cellular structure, transcription or translation). We suggest that these patterns of gene expression lead to either the expression of disability, or to resilience and recovery, by modifying local spinal circuitry at the origin of ascending supraspinal pathways.

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population(1,2). Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)(3) (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity. CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins. For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs(4). The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter(5,6). The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.