Paul J. Austin

The neuro-immune balance in neuropathic pain: Involvement of inflammatory immune cells, immune-like glial cells and cytokines

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.

Regulatory T cells attenuate neuropathic pain following peripheral nerve injury and experimental autoimmune neuritis.

Summary Increasing the number of regulatory T cells (Tregs) reduces neuroinflammation and alleviates mechanical pain hypersensitivity, while depleting Tregs potentiates pain in animal models of neuropathy. Abstract 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.

Symptomatic and neuroprotective effects following activation of nigral group III metabotropic glutamate receptors in rodent models of Parkinson's disease

Background and purpose:  Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinsons disease (PD). This study aimed to establish whether activation of pre‐synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD.

Antiparkinsonian potential of targeting group III metabotropic glutamate receptor subtypes in the rodent substantia nigra pars reticulata.

Increased firing of the glutamatergic pathway between the subthalamic nucleus and substantia nigra pars reticulata (SNpr) contributes to the abnormal firing of motor circuits and subsequent motor deficits seen in Parkinsons disease. Broad spectrum agonist‐induced activation of presynaptic group III metabotropic glutamate (mGlu) receptors within the SNpr reduced glutamate release and reversed akinesia in the reserpine‐treated rat model of Parkinsons disease. Here, we have sought to identify which subtypes of group III mGlu receptor in the SNpr were responsible for these beneficial effects.

Chronic constriction of the sciatic nerve and pain hypersensitivity testing in rats.

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 Xies (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.

PERIPHERAL NERVE INJURY DIFFERENTIALLY REGULATES DOPAMINERGIC PATHWAYS IN THE NUCLEUS ACCUMBENS OF RATS WITH EITHER 'PAIN ALONE' OR 'PAIN AND DISABILITY'

Following unilateral chronic constriction injury (CCI) of the sciatic nerve, histochemical and gene expression changes were examined in the rat nucleus accumbens (NAcc), a region critical to affective-motivational regulation. Rats were categorised as having Pain alone (45%) or Pain and Disability (30%), on the basis of either unaltered or decreased dominance behaviour in the resident-intruder paradigm, respectively. Tyrosine hydroxylase (TH) expression was significantly increased bilaterally, throughout the rostrocaudal extent of the NAcc in Pain alone animals. Increased TH likely reflects increased dopamine levels in the Pain alone group, which may modulate dopamine receptor subtype 2 (D2) receptor expression. Stereological analyses of D2 receptor immunoreactive (D2-IR) cells revealed lateralised changes which correlated significantly with dominance behaviour. In the contralateral NAcc, D2-IR negatively correlated with post-CCI dominance behaviour (i.e. Pain alone animals have decreased D2-IR), whereas ipsilaterally there was a positive correlation between D2-IR and post-CCI dominance behaviour (i.e. Pain and Disability animals have decreased D2-IR). Western blots for D2 protein expression confirmed these correlations. Additionally, D2 mRNA expression within the NAcc showed lateralised and group specific changes. In the ipsilateral NAcc D2 mRNA was increased in Pain alone animals. It is hypothesised that increased D2 mRNA in the ipsilateral NAcc of Pain alone animals may be a protective mechanism, maintaining D2-IR despite increased dopamine, which may otherwise induce receptor desensitisation. D2 mRNA is not altered in the ipsilateral NAcc of Pain and Disability animals, therefore loss of D2-IR is likely, albeit by an alternate mechanism. In summary, unilateral CCI in rats induces specific and lateralised adaptations in the dopaminergic circuitry of the NAcc. These distinct neural adaptations correlate with changes in social behaviour, and likely underlie some of the affective-motivational state changes associated with neuropathic pain in a subset of rats (i.e. Pain and Disability group).

Evidence for a distinct neuro-immune signature in rats that develop behavioural disability after nerve injury

BackgroundChronic neuropathic pain is a neuro-immune disorder, characterised by allodynia, hyperalgesia and spontaneous pain, as well as debilitating affective-motivational disturbances (e.g., reduced social interactions, sleep-wake cycle disruption, anhedonia, and depression). The role of the immune system in altered sensation following nerve injury is well documented. However, its role in the development of affective-motivational disturbances remains largely unknown. Here, we aimed to characterise changes in the immune response at peripheral and spinal sites in a rat model of neuropathic pain and disability.MethodsSixty-two rats underwent sciatic nerve chronic constriction injury (CCI) and were characterised as either Pain and disability, Pain and transient disability or Pain alone on the basis of sensory threshold testing and changes in post-CCI dominance behaviour in resident-intruder interactions. Nerve ultrastructure was assessed and the number of T lymphocytes and macrophages were quantified at the site of injury on day six post-CCI. ATF3 expression was quantified in the dorsal root ganglia (DRG). Using a multiplex assay, eight cytokines were quantified in the sciatic nerve, DRG and spinal cord.ResultsAll CCI rats displayed equal levels of mechanical allodynia, structural nerve damage, and reorganisation. All CCI rats had significant infiltration of macrophages and T lymphocytes to both the injury site and the DRG. Pain and disability rats had significantly greater numbers of T lymphocytes. CCI increased IL-6 and MCP-1 in the sciatic nerve. Examination of disability subgroups revealed increases in IL-6 and MCP-1 were restricted to Pain and disability rats. Conversely, CCI led to a decrease in IL-17, which was restricted to Pain and transient disability and Pain alone rats. CCI significantly increased IL-6 and MCP-1 in the DRG, with IL-6 restricted to Pain and disability rats. CCI rats had increased IL-1β, IL-6 and MCP-1 in the spinal cord. Amongst subgroups, only Pain and disability rats had increased IL-1β.ConclusionsThis study has defined individual differences in the immune response at peripheral and spinal sites following CCI in rats. These changes correlated with the degree of disability. Our data suggest that individual immune signatures play a significant role in the different behavioural trajectories following nerve injury, and in some cases may lead to persistent affective-motivational disturbances.

Evidence for cellular injury in the midbrain of rats following chronic constriction injury of the sciatic nerve

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.

Are the emergence of affective disturbances in neuropathic pain states contingent on supraspinal neuroinflammation

Neuro-immune interactions contribute to the pathogenesis of neuropathic pain due to peripheral nerve injury. A large body of preclinical evidence supports the idea that the immune system acts to modulate the sensory symptoms of neuropathy at both peripheral and central nervous system sites. The potential involvement of neuro-immune interactions in the highly debilitating affective disturbances of neuropathic pain, such as depression, anhedonia, impaired cognition and reduced motivation has received little attention. This is surprising given the widely accepted view that sickness behaviour, depression, cognitive impairment and other neuropsychiatric conditions can arise from inflammatory mechanisms. Moreover, there is a set of well-described immune-to-brain transmission mechanisms that explain how peripheral inflammation can lead to supraspinal neuroinflammation. In the last 5years increasing evidence has emerged that peripheral nerve injury induces supraspinal changes in cytokine or chemokine expression and alters glial cell activity. In this systematic review, based on strong preclinical evidence, we advance the argument that the emergence of affective disturbances in neuropathic pain states are contingent on pro-inflammatory mediators in the interconnected hippocampal-medial prefrontal circuitry that subserve affective behaviours. We explore how dysregulation of inflammatory mediators in these networks may result in affective disturbances through a wide variety of neuromodulatory mechanisms. There are also promising results from clinical trials showing that anti-inflammatory agents have efficacy in the treatment of a variety of neuropsychiatric conditions including depression and appear suited to sub-groups of patients with elevated pro-inflammatory profiles. Thus, although further research is required, aggressively targeting supraspinal pro-inflammatory mediators at critical time-points in appropriate clinical populations is likely to be a novel avenue to treat debilitating affective disturbances in neuropathic conditions.

Neuropathic Pain: Pathophysiology of neuropathic pain: inflammatory mediators

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.