Recent developments and challenges in positron emission tomography imaging of gliosis in chronic neuropathic pain.

ABSTRACT: Understanding the mechanisms that underpin the transition from acute to chronic pain is critical for the development of more effective and targeted treatments. There is growing interest in the contribution of glial cells to this process, with cross-sectional preclinical studies demonstrating specific changes in these cell types capturing targeted timepoints from the acute phase and the chronic phase. In vivo longitudinal assessment of the development and evolution of these changes in experimental animals and humans has presented a significant challenge. Recent technological advances in preclinical and clinical positron emission tomography, including the development of specific radiotracers for gliosis, offer great promise for the field. These advances now permit tracking of glial changes over time and provide the ability to relate these changes to pain-relevant symptomology, comorbid psychiatric conditions, and treatment outcomes at both a group and an individual level. In this article, we summarize evidence for gliosis in the transition from acute to chronic pain and provide an overview of the specific radiotracers available to measure this process, highlighting their potential, particularly when combined with ex vivo/in vitro techniques, to understand the pathophysiology of chronic neuropathic pain. These complementary investigations can be used to bridge the existing gap in the field concerning the contribution of gliosis to neuropathic pain and identify potential targets for interventions.

Infraorbital nerve injury triggers sex-specific neuroimmune responses in the peripheral trigeminal pathway and common pain behaviours.

Trigeminal neuropathic pain is emotionally distressing and disabling. It presents with allodynia, hyperalgesia and dysaesthesia. In preclinical models it has been assumed that cephalic nerve constriction injury shows identical molecular, cellular, and sex dependent neuroimmune changes as observed in extra-cephalic injury models. This study sought empirical evidence for such assumptions using the infraorbital nerve chronic constriction model (ION-CCI). We compared the behavioural consequences of nerve constriction with: (i) the temporal patterns of recruitment of macrophages and T-lymphocytes at the site of nerve injury and in the trigeminal ganglion; and (ii) the degree of demyelination and axonal reorganisation in the injured nerve. Our data demonstrated that simply testing for allodynia and hyperalgesia as is done in extra-cephalic neuropathic pain models does not provide access to the range of injury-specific nociceptive responses and behaviours reflective of the experience of trigeminal neuropathic pain. Similarly, trigeminal neuroimmune changes evoked by nerve injury are not the same as those identified in models of extra-cephalic neuropathy. Specifically, the timing, magnitude, and pattern of ION-CCI evoked macrophage and T-lymphocyte activity differs between the sexes.

Serotonin and Dopamine Show Different Response Profiles to Acute Stress in the Nucleus Accumbens and Medial Prefrontal Cortex of Rats with Neuropathic Pain.

The ability to adaptively guide behaviour requires the integration of external information with internal motivational factors. Decision-making capabilities can be impaired by acute stress and is often exacerbated by chronic pain. Chronic neuropathic pain patients often present with cognitive dysfunction, including impaired decision-making. The mechanisms underlying these changes are not well understood but may include altered monoaminergic transmission in the brain. In this study we investigated the relationships between dopamine, serotonin, and their metabolites in key brain regions that regulate motivated behaviour and decision-making. The neurochemical profiles of the medial prefrontal cortex, orbital prefrontal cortex, and nucleus accumbens were analysed using HPLC in rats that received a chronic constriction injury (CCI) of the right sciatic nerve and an acute stress (15-min restraint), prior to an outcome devaluation task. CCI alone significantly decreased dopamine but not serotonin concentrations in the medial prefrontal cortex. By contrast, restraint stress acutely increased dopamine in the medial prefrontal cortex, and the nucleus accumbens; and increased serotonin in the medial prefrontal cortex 2 h later. The sustained dopaminergic and serotonergic responses to acute stress highlight the importance of an animal's ability to mount an effective coping response. In addition, these data suggest that the impact of nerve injury and acute stress on outcome-devaluation occurs independently of dopaminergic and serotonergic transmission in the medial prefrontal cortex, orbital prefrontal cortex and nucleus accumbens of rats.

Nerve injury alters restraint-induced activation of the basolateral amygdala in male rats.

The amygdala is critical for the production of appropriate responses towards emotional or stressful stimuli. It has a characteristic neuronal activation pattern to acute stressors. Chronic pain and acute stress have each been shown to independently modulate the activity of the amygdala. Few studies have investigated the effect of pain or injury, on amygdala activation to acute stress. This study investigated the effects of a neuropathic injury on the activation response of the amygdala to an acute restraint stress. Chronic constriction injury of the right sciatic nerve (CCI) was used to create neuropathic injury and a single brief 15-min acute restraint was used as an emotional/psychological stressor. All rats received cholera toxin B (CTB) retrograde tracer injections into the medial prefrontal cortex (mPFC) to assess if the amygdala to mPFC pathway was specifically regulated by the combination of neuropathic injury and acute stress. To assess differential patterns of activity in amygdala subregions, cFos expression was used as a marker for "acute", restraint triggered neuronal activation, and FosB/ΔFosB expression was used to reveal prolonged neuronal activation/sensitisation triggered by CCI. Restraint resulted in a characteristic increase in cFos expression in the medial amygdala, which was not altered by CCI. Rats with a CCI showed increased cFos expression in the basolateral amygdala (BLA), in response to an acute restraint stress, but not in neurons projecting to the prefrontal cortex. Further, CCI rats showed an increase in FosB/ΔFosB expression which was exclusive to the BLA. This increase likely reflects sensitisation of the BLA as a consequence of nerve injury which may contribute to heightened sensitivity of BLA neurons to acute emotional/ psychological stressors.

Altered monoamine levels in the dorsal striatum of the rat are associated with alterations in behavioural selection and motivation following peripheral nerve injury and acute stress.

Chronic neuropathic pain and psychological stress interact to compromise goal-directed control over behaviour following mild psychological stress. The dorsomedial (DMS) and dorsolateral (DLS) striatum in the rat are crucial for the expression of goal-directed and habitual behaviours, respectively. This study investigated whether changes in monoamine levels in the DMS and DLS following nerve injury and psychological stress reflect these behavioural differences. Neuropathic pain was induced by a chronic constriction injury (CCI) of the sciatic nerve in Sprague-Dawley rats. Acute stress was induced using a 15-min restraint. Behavioural flexibility was assessed using the outcome devaluation paradigm. Noradrenaline, serotonin, dopamine and associated metabolites were measured bilaterally from the DLS and DMS. In uninjured rats, restraint increased dopaminergic markers in the left and serotonergic markers in the right of both the DMS and DLS, indicating a possible left hemisphere-mediated dominance. CCI led to a slightly different lateralised effect, with a larger effect in the DMS than in the DLS. Individual differences in behavioural flexibility following CCI negatively correlated with dopaminergic markers in the right DLS, but positively correlated with these markers in the left DMS. A combination of CCI and restraint reduced behavioural flexibility, which was associated with the loss of the left/DMS dominance. These data suggest that behavioural flexibility following psychological stress or pain is associated with a left hemisphere dominance within the dorsal striatum. The loss of behavioural flexibility following the combined stressors is then associated with a transition from left to right, and DMS to DLS dominance.

Peripheral nerve injury attenuates stress-induced Fos-family expression in the Locus Coeruleus of male Sprague-Dawley rats.

The ability to cope with acute stressors is impaired in people with chronic neuropathic injuries. The regulation of stress coping responses depends critically on several parallel interconnected neural circuits, one of which originates in the Locus Coeruleus. In rats, chronic constriction injury (CCI) and acute stress each modulate noradrenergic activity of the Locus Coeruleus (LC) although with different temporal patterns. This study investigated the effects of CCI on the neuronal activity of the LC to acute restraint stress using the immunohistochemical detection of Fos-family protein expression. Male Sprague-Dawley rats underwent CCI surgery and 11 days later were restrained for 15 min. The number and location of single-labelled neurons (c-Fos, FosB/ΔFosB and tyrosine hydroxylase (TH) immunoreactive) neurons and double labelled neurons (c-Fos, or FosB/ΔFosB with TH) were quantified for the LC and surrounding regions. Comparisons were made with rats that underwent sham surgery or anaesthesia (20 min). Restraint triggered a struggling response in all rats. CCI attenuated restraint-induced Fos expression in LC neurons. A significant proportion (30-50%) of these LC Fos positive neurons did not contain TH. These data suggest that nerve injury might impair the ordinary cellular response of the LC to an acute stress. The association of stress-related disorders in people with neuropathic injuries suggests that the observations made in this study may reflect a part of the mechanism underlying these clinical comorbidities.

PACAP and VIP expression in the periaqueductal grey of the rat following sciatic nerve constriction injury.

Nerve injuries often result in neuropathic pain with co-morbid changes in social behaviours, motivation, sleep-wake cycles and neuroendocrine function. In an animal model of neuropathic injury (CCI) similar co-morbid changes are evoked in a subpopulation (~30%) of injured rats. In addition to anatomical evidence of altered neuronal and glial function, the periaqueductal grey (PAG) of these rats shows evidence of cell death. These changes in the PAG may play a role in the disruption of the normal emotional coping responses triggered by nerve injury. Cell death can occur via a number of mechanisms, including the disruption of neuroprotective mechanisms. Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two endogenous neuropeptides whose activities are tightly regulated by two receptors subtypes, namely the PAC1 and VPAC receptors. These peptides and their receptors exert robust neuroprotective roles. In these studies, we hypothesized that rats expressing disabilities following CCI showed altered expression of PACAP and VIP in the PAG. Rats were categorized as having either Pain alone, Transient or Persistent disability, based on changes in social behaviours pre- and post-CCI. Social interaction behavioural tested (BT), sham-injured and naïve untested rats were also included. For measurements of mRNA and protein expression we utilised micro-dissected PAGs blocks taken from each group. At the mRNA level, VIP was downregulated and PAC1 was upregulated in BT animals, whilst VPAC1 mRNA was specifically increased in the Pain alone group. Interestingly, protein levels of both PACAP and VIP were remarkably increased in the Persistent Disability group. Taken together, sciatic nerve CCI that triggers neuropathic pain and persistent disability results in abnormally increased VIP and PACAP expression in the PAG. Our data also suggest that these effects are likely to be governed by post-transcriptional mechanisms.

Resolving the contributions of anaesthesia, surgery, and nerve injury on brain derived neurotrophic factor expression in the medial prefrontal cortex of male rats in the CCI model of neuropathic pain.

The medial prefrontal cortex (mPFC) is critical for selecting and shaping complex behavioral responses. In rodent models of neuropathic pain there is evidence for both structural and functional changes in the mPFC. Brain derived neurotrophic factor (BDNF) plays a critical role in the normal functioning of the mPFC. It has been suggested that the disruption of complex behaviors and mood seen in some neuropathic pain patients is mediated in part by alterations of BDNF in this cortical region. In Sprague-Dawley rats, mPFC levels of BDNF and TrkB mRNA and protein, were quantified and compared to controls (n = 24) 6 days after either: (a) halothane (1.5%) anaesthesia (n = 12), (b) sham surgery under halothane (n = 12), (c) sciatic nerve chronic constriction injury under halothane (n = 48). The social behaviors of the rats were quantified daily during the experimental period. Halothane anaesthesia increased BDNF and TrkB mRNA bilaterally. These increases were reversed in rats that underwent sham surgical and nerve injury procedures. Further, halothane anaesthesia, surgical procedures, and nerve injury each decreased BDNF protein levels. These results reveal a marked and distinct BDNF expression profile in the mPFC of rats that have undergone each stage of the procedure to produce neuropathic pain by chronic constriction injury of the sciatic nerve. The highly sensitive nature of neurotrophic signalling to general anaesthesia in the mature neuronal circuit of the adult rat brain highlights the importance of careful evaluation and interpretation of data evaluating the effects of experimental procedures on neural substrates.

Peripheral nerve injury impairs the ability to maintain behavioural flexibility following acute stress in the rat.

Chronic neuropathic pain often leads to impaired cognition and reduced behavioural flexibility. This study used a rat model to investigate if a peripheral nerve injury, with or without an additional acute psychological stress, alters behavioural flexibility and goal directed behaviour as measured by sensitivity to devaluation. Neuropathic pain was induced by a chronic constriction injury (CCI) of the sciatic nerve. CCI, sham-injury and naïve rats were trained to press two levers for two rewards. In outcome devaluation tests, one of the rewards was devalued by pre-feeding it to satiety, immediately prior to an extinction test measuring responding on the two levers. The ability to preferentially direct responding toward the action earning the currently-valued reward was taken as evidence of goal-directed behaviour. To test the impact of acute stress, rats were subjected to 15min restraint following pre-feeding and prior to the devaluation test. Neither CCI surgery nor acute stress alone altered sensitivity to devaluation, but in combination CCI and acute stress significantly reduced sensitivity to devaluation. This Study demonstrates that relatively mild stressors that are without effect in uninjured populations can markedly impair cognition under conditions of chronic pain. It further suggests that overlapping neural substrates regulated by nerve injury and/or acute stress are having a cumulative effect on behavioural flexibility.

Recruitment of dorsal midbrain catecholaminergic pathways in the recovery from nerve injury evoked disabilities.

BACKGROUND: The periaqueductal gray region (PAG) is one of several brain areas identified to be vulnerable to structural and functional change following peripheral nerve injury. Sciatic nerve constriction injury (CCI) triggers neuropathic pain and three distinct profiles of changes in complex behaviours, which include altered social and sleep-wake behaviours as well as changes in endocrine function. The PAG encompasses subgroups of the A10 dopaminergic and A6 noradrenergic cell groups; the origins of significant ascending projections to hypothalamic and forebrain regions, which regulate sleep, complex behaviours and endocrine function. We used RT-PCR, western blots and immunohistochemistry for tyrosine hydroxylase to determine whether (1) tyrosine hydroxylase increased in the A10/A6 cells and/or; (2) de novo synthesis of tyrosine hydroxylase, in a 'TH-naïve' population of ventral PAG neurons characterized rats with distinct patterns of behavioural and endocrine change co-morbid with CCI evoked-pain. RESULTS: Evidence for increased tyrosine hydroxylase transcription and translation in the constitutive A10/A6 cells was found in the midbrain of rats that showed an initial 2-3 day post-CCI, behavioural and endocrine change, which recovered by days 5-6 post-CCI. Furthermore these rats showed significant increases in the density of TH-IR fibres in the vPAG. CONCLUSIONS: Our data provide evidence for: (1) potential increases in dopamine and noradrenaline synthesis in vPAG cells; and (2) increased catecholaminergic drive on vPAG neurons in rats in which transient changes in social behavior are seen following CCI. The data suggests a role for dopaminergic and noradrenergic outputs, and catecholaminergic inputs of the vPAG in the expression of one of the profiles of behavioural and endocrine change triggered by nerve injury.

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

BACKGROUND: Chronic 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. METHODS: Sixty-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. RESULTS: All 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-1ß, IL-6 and MCP-1 in the spinal cord. Amongst subgroups, only Pain and disability rats had increased IL-1ß. CONCLUSIONS: This 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.