Priming of Adult Incision Response by Early-Life Injury: Neonatal Microglial Inhibition Has Persistent But Sexually Dimorphic Effects in Adult Rats.

Neonatal hindpaw incision primes developing spinal nociceptive circuitry, resulting in enhanced hyperalgesia following reinjury in adulthood. Spinal microglia contribute to this persistent effect, and microglial inhibition at the time of adult reincision blocks the enhanced hyperalgesia. Here, we pharmacologically inhibited microglial function with systemic minocycline or intrathecal SB203580 at the time of neonatal incision and evaluated sex-dependent differences following adult reincision. Incision in adult male and female rats induced equivalent hyperalgesia and spinal dorsal horn expression of genes associated with microglial proliferation (Emr1) and transformation to a reactive phenotype (Irf8). In control adults with prior neonatal incision, the enhanced degree and duration of incision-induced hyperalgesia and spinal microglial responses to reincision were equivalent in males and females. However, microglial inhibition at the time of the neonatal incision revealed sex-dependent effects: the persistent mechanical and thermal hyperalgesia following reincision in adulthood was prevented in males but unaffected in females. Similarly, reincision induced Emr1 and Irf8 gene expression was downregulated in males, but not in females, following neonatal incision with minocycline. To evaluate the distribution of reincision hyperalgesia, prior neonatal incision was performed at different body sites. Hyperalgesia was maximal when the same paw was reincised, and was increased following prior incision at ipsilateral, but not contralateral, sites, supporting a segmentally restricted spinal mechanism. These data highlight the contribution of spinal microglial mechanisms to persistent effects of early-life injury in males, and sex-dependent differences in the ability of microglial inhibition to prevent the transition to a persistent pain state span developmental stages.SIGNIFICANCE STATEMENT Following the same surgery, some patients develop persistent pain. Contributory mechanisms are not fully understood, but early-life experience and sex/gender may influence the transition to chronic pain. Surgery and painful procedural interventions in vulnerable preterm neonates are associated with long-term alterations in somatosensory function and pain that differ in males and females. Surgical injury in neonatal rodents primes the developing nociceptive system and enhances reinjury response in adulthood. Neuroimmune interactions are critical mediators of persistent pain, but sex-dependent differences in spinal neuroglial signaling influence the efficacy of microglial inhibitors following adult injury. Neonatal microglial inhibition has beneficial long-term effects on reinjury response in adult males only, emphasizing the importance of evaluating sex-dependent differences at all ages in preclinical studies.

Microglial P2X4R-evoked pain hypersensitivity is sexually dimorphic in rats.

Microglia-neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury-induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury-induced hypersensitivity in males differ from those in females, with microglial P2X4 receptors (P2X4Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X4Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X4Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X4Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation-qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X4R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.

Cognitive Impairment in Patients with Chronic Neuropathic or Radicular Pain: An Interaction of Pain and Age.

A growing body of empirical research has confirmed an association between chronic pain and cognitive dysfunction. The aim of the present study was to determine whether cognitive function is affected in patients with a diagnosis of chronic neuropathic or radicular pain relative to healthy control participants matched by age, gender, and years of education. We also examined the interaction of pain with age in terms of cognitive performance. Some limitations of previous clinical research investigating the effects of chronic pain on cognitive function include differences in the pain and cognitive scale materials used, and the heterogeneity of patient participants, both in terms of their demographics and pathological conditions. To address these potential confounds, we have used a relatively homogenous patient group and included both experimental and statistical controls. We have also specifically investigated the interaction effect of pain and age on cognitive performance. Patients (n = 38) and controls (n = 38) were administered a battery of cognitive tests measuring IQ, spatial and verbal memory, attention, and executive function. Educational level, depressive symptoms, and state anxiety were assessed as were medication usage, caffeine, and nicotine consumption to control for possible confounding effects. Both the level of depressive symptoms and the state anxiety score were higher in chronic pain patients than in matched control participants. Chronic pain patients had a lower estimated IQ than controls, and showed impairments on measures of spatial and verbal memory. Attentional responding was altered in the patient group, possibly indicative of impaired inhibitory control. There were significant interactions between chronic pain condition and age on a number of cognitive outcome variables, such that older patients with chronic pain were more impaired than both age-matched controls and younger patients with chronic pain. Chronic pain did not appear to predict performance on the Wisconsin Card Sorting Task, which was used a measure of executive function. This study supports and extends previous research indicating that chronic pain is associated with impaired memory and attention. Perspective: Compared to healthy control participants, patients with chronic neuropathic or radicular pain showed cognitive deficits which were most pronounced in older pain patients.

A novel therapeutic with two SNAP-25 inactivating proteases shows long-lasting anti-hyperalgesic activity in a rat model of neuropathic pain.

A pressing need exists for long-acting, non-addictive medicines to treat chronic pain, a major societal burden. Botulinum neurotoxin type A (BoNT/A) complex - a potent, specific and prolonged inhibitor of neuro-exocytosis - gives some relief in several pain disorders, but not for all patients. Our study objective was to modify BoNT/A to overcome its inability to block transmitter release elicited by high [Ca2+]i and increase its limited analgesic effects. This was achieved by fusing a BoNT/A gene to that for the light chain (LC) of type/E. The resultant purified protein, LC/E-BoNT/A, entered cultured sensory neurons and, unlike BoNT/A, inhibited release of calcitonin gene-related peptide evoked by capsaicin. Western blotting revealed that this improvement could be due to a more extensive truncation by LC/E of synaptosomal-associated protein of Mr = 25 k, essential for neuro-exocytosis. When tested in a rat spared nerve injury (SNI) model, a single intra-plantar (IPL) injection of LC/E-BoNT/A alleviated for ∼2 weeks mechanical and cold hyper-sensitivities, in a dose-dependent manner. The highest non-paralytic dose (75 U/Kg, IPL) proved significantly more efficacious than BoNT/A (15 U/Kg, IPL) or repeated systemic pregabalin (10 mg/Kg, intraperitoneal), a clinically-used pain modulator. Effects of repeated or delayed injections of this fusion protein highlighted its analgesic potential. Attenuation of mechanical hyperalgesia was extended by a second administration when the effect of the first had diminished. When injected 5 weeks after injury, LC/E-BoNT/A also reversed fully-established mechanical and cold hyper-sensitivity. Thus, combining advantageous features of BoNT/E and/A yields an efficacious, locally-applied and long-acting anti-hyperalgesic.

Impaired cued and spatial learning performance and altered cannabinoid CB1 receptor functionality in the substantia nigra in a rat model of diabetic neuropathy.

Diabetes, and associated diabetic neuropathic pain, impact negatively on cognitive function. However, the underlying mechanisms remain poorly understood. This study investigated neuropathic pain-related behaviour and cognitive function in the rat streptozotocin (STZ) model of diabetes, and assessed cannabinoid1 (CB1) receptor functionality in discrete brain regions. Male Lister-Hooded rats received STZ (60 mg/kgs.c.) or vehicle. Sensory responses were assessed in von Frey and Hargreaves tests. Cognitive, motor and sensorimotor functions were assessed using novel object recognition and Morris water maze tasks. CB1 receptor functionality was assessed by [(35)S]GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) autoradiography. STZ treatment was associated with mechanical allodynia and thermal hypoalgesia. Novel object recognition was unaltered in diabetic rats. STZ treatment was associated with impaired performance in the Morris water maze acquisition phase, but there were no differences in memory retrieval in the probe trial. Stimulus-response learning in the water maze cued trial was also disrupted in STZ-treated rats, possibly indicating sensorimotor deficits. CB1 receptor agonist-stimulated [(35)S]GTPγS binding was attenuated in the substantia nigra of STZ-treated rats but unaltered in the hippocampus. In conclusion, STZ treatment as a model of diabetic neuropathy was associated with specific functional deficits in the Morris water maze, effects which may be related to altered CB1 receptor functionality in the substantia nigra.

Impaired recognition memory and cognitive flexibility in the rat L5-L6 spinal nerve ligation model of neuropathic pain.

BACKGROUND AND AIMS: Although neuropathic pain is known to negatively affect cognition, the neural mechanisms involved are poorly understood. Chronic pain is associated with changes in synaptic plasticity in the brain which may impact on cognitive functioning. The aim of this study was to model neuropathic pain in mid-aged rats using spinal nerve ligation (SNL). Following establishment of allodynia and hyperalgesia, behaviour was assessed in a battery of cognitive tests. Expression of the presynaptic protein, synaptophysin, and its colocalisation with the vesicular GABA and glutamate transporters (vGAT and vGLUT, respectively), was investigated in the medial prefrontal cortex (mPFC) and hippocampus. METHODS: Nine month old male Sprague Dawley rats underwent L5-L6 spinal nerve ligation or a sham procedure. Mechanical and cold allodynia and thermal hyperalgesia were assessed using von Frey, acetone and Hargreaves tests, respectively. Cognition was assessed in the novel-object recognition, air-puff passive avoidance and Morris water maze behavioural tasks. Immunohistochemistry was used to examine the expression of synaptophysin in the mPFC and CA1 region of the hippocampus and double labelling of synaptophysin and the vesicular transporters vGAT and vGlut was used to investigate the distribution of synaptophysin on GABAergic and glutamatergic neurons. RESULTS: SNL rats displayed impaired performance in the novel-object recognition task. Passive-avoidance responding, and spatial learning and memory in the Morris water maze, were unaffected by SNL surgery. However, in the water maze reversal task, pain-related impairments were evident during training and probe trials. SNL surgery was not associated with any differences in the expression of synaptophysin or its colocalisation with vGAT or vGLUT in the mPFC or the hippocampal CA1 region. CONCLUSIONS: These results suggest that the SNL model of neuropathic pain is associated with deficits in recognition memory and cognitive flexibility, but these deficits are not associated with altered synaptophysin expression or distribution in the mPFC and CA1. IMPLICATIONS: Cognitive complaints are common amongst chronic pain patients. Here we modelled cognitive impairment in a well-established animal model of neuropathic pain and investigated the neural mechanisms involved. A better understanding of this phenomenon is an important prerequisite for the development of improved treatment of patients affected.

Characterization of the Affective Component of Acute Postoperative Pain Associated with a Novel Rat Model of Inguinal Hernia Repair Pain.

AIMS: Acute postoperative pain remains a significant healthcare issue. Historically, the assessment of postoperative pain in rodents has relied on evoked withdrawal or reflexive measures. Using a recently developed, anatomically relevant rat model of acute postoperative pain (J Pain, 16, 2015, 421), the present experiments sought to investigate the affective component of acute postoperative pain associated with inguinal hernia repair. METHODS: Male Lister hooded rats underwent surgery to model Lichtenstein inguinal hernia repair (without hernia induction), or a sham procedure. Postsurgical characterization involved a modified place escape/avoidance paradigm (mPEAP), as well as home cage and open field locomotor activity monitoring. In pharmacological validation studies, rats received either morphine or carprofen prior to mPEAP testing. RESULTS: Surgery was associated with a significantly increased proportion of the trial duration in the light compartment of the mPEAP arena, in avoidance of the noxious stimulus, compared with sham animals. When retested in the mPEAP at day 7 postsurgery, there was no difference between sham and surgery animals for time spent in either compartment, but surgery animals displayed a persistent increase in the percentage response to noxious stimulation. Morphine and carprofen treatment in surgery animals reduced escape/avoidance behavior at discrete time points over the trial. Surgery-induced reductions in home cage and open field locomotor activity were also observed. CONCLUSION: The present studies report for the first time the characterization of the affective component of acute postoperative pain using the mPEAP in a rodent model, which may facilitate development of improved understanding and treatment of postoperative pain.

Development and characterization of a novel, anatomically relevant rat model of acute postoperative pain.

UNLABELLED: Acute postoperative pain remains a significant health care issue. Development of anatomically relevant animal models of postoperative pain, with improved predictive validity, would advance understanding of postoperative pain mechanisms and improve treatment outcomes. This study aimed to develop, characterize, and validate a rat model of acute postoperative pain associated with inguinal hernia repair based on the Lichtenstein inguinal hernia repair procedure (without hernia induction). We hypothesized that the surgery would result in reduced spontaneous locomotor activity, which would represent a pain-related phenotype. Postsurgical characterization involved extensive monitoring of home cage and open field locomotor activity, as well as mechanical hypersensitivity and assessment of c-Fos expression in the dorsal horn of the spinal cord. In pharmacologic validation studies, rats received morphine, carprofen, or paracetamol 1 hour before, and/or immediately after, surgery. Rats that underwent hernia repair surgery exhibited significantly lower horizontal and vertical activities in the home cage and open field in the early postsurgical period, compared with sham rats or rats that underwent skin incision only. Morphine, carprofen, and paracetamol attenuated the surgery-induced reductions in locomotor activity, to varying degrees. Surgery was associated with significantly increased c-Fos expression in the ipsilateral dorsal horn of the spinal cord, an effect attenuated by carprofen treatment. These results support the development and characterization of a novel, anatomically relevant animal model of acute postoperative pain that may facilitate development of improved treatment regimens. PERSPECTIVE: Acute pain following inguinal hernia repair can be difficult to treat. Here we report, for the first time, the development of a novel, anatomically relevant rat model to facilitate improved understanding and treatment of acute postoperative pain following inguinal hernia repair.

Minocycline modulates neuropathic pain behaviour and cortical M1-M2 microglial gene expression in a rat model of depression.

There is a paucity of data on the role of microglia and neuroinflammatory processes in the association between chronic pain and depression. The current study examined the effect of the microglial inhibitor minocycline on depressive-like behaviour, spinal nerve ligation (SNL)-induced mechanical and cold allodynia and associated changes in the expression of genes encoding microglial markers (M1 vs. M2 polarisation) and inflammatory mediators in the prefrontal cortex in the olfactory bulbectomised (OB) rat model of depression. Acute minocycline administration did not alter OB-induced depressive-like behaviour but prevented SNL-induced mechanical allodynia in both OB and sham rats. In comparison, chronic minocycline attenuated OB-induced depressive-like behaviour and prevented the development of SNL-induced mechanical allodynia in OB, but not sham, rats. Further analysis revealed that SNL-induced mechanical allodynia in OB rats was attenuated by chronic minocycline at almost all time-points over a 2week testing period, an effect observed only from day 10 post-SNL in sham rats. Chronic administration of minocycline reduced the expression of CD11b, a marker of microglial activation, and the M1 pro-inflammatory cytokine IL-1ß, in the prefrontal cortex of sham-SNL animals. In comparison, the expression of the M2 microglia marker (MRC2) and anti-inflammatory cytokine IL-10 was increased, as were IL-1ß, IL-6 and SOCS3, in the prefrontal cortex of OB-SNL animals following chronic minocycline. Thus, chronic minocycline attenuates neuropathic pain behaviour and modulates microglial activation and the central expression of inflammatory mediators in a manner dependent on the presence or absence of a depressive-like phenotype.

Cognition and pain.

PURPOSE OF REVIEW: Pain and cognition share common neural substrates and are known to interact reciprocally. This has implications for treatment and management of pain conditions; pain can negatively affect cognitive performance, whereas cognitively demanding tasks may reduce pain perception. This article will review recent research investigating the impact of pain on cognition and the cognitive modulation of pain. RECENT FINDINGS: Recent clinical and preclinical studies have provided new evidence for impairment of cognition in pain with a focus on the type of cognitive construct affected and the influence of factors such as age and pain localization. Reduced connectivity between important brain structures has emerged as a possible underlying mechanism. Imaging studies have continued to identify neuroanatomical structures involved in different types of cognitive pain modulation, and attempts have been made to delineate the descending pathways by which pain relief is achieved. New and established methods to investigate cognitive modulation of pain in animal models have revealed insights into the molecular and neurochemical mechanisms involved. SUMMARY: Progress has been made in understanding the complex relationship between pain and cognitive function. However, both synthesis of current research findings and further novel research studies are required to maximize the therapeutic potential.

Validation of an air-puff passive-avoidance paradigm for assessment of aversive learning and memory in rat models of chronic pain.

Chronic pain is associated with cognitive deficits. Considerable overlap in brain regions involved in pain and aversion suggests that aversive learning and memory may be affected during chronic pain. Passive-avoidance paradigms traditionally use foot-shock to induce context-conditioned avoidance and may be unsuitable for use in animal models of chronic pain, which are commonly associated with hypersensitivity of the hind-paws. The aim of the present study was to develop and validate a novel passive-avoidance paradigm in rats, employing air-puff as the aversive stimulus, and to use this paradigm to assess aversive learning and memory in rat models of chronic inflammatory and neuropathic pain. Air-puff exposure produced a significant passive-avoidance and this response was attenuated following administration of scopolamine. Nerve-ligated rats and rats injected with complete Freund's adjuvant developed allodynia and hyperalgesia. Air-puff produced a significant passive-avoidance response in both chronic pain models. However, there was no difference in the response between either model and its respective control group. Thus, air-puff can be used as an alternative to foot-shock to induce a passive-avoidance response. The data generated using this model suggest that aversive learning and memory remain intact in the rat spinal nerve ligation and complete Freund's adjuvant models of chronic neuropathic and inflammatory pain, respectively.

The effect of pain on cognitive function: a review of clinical and preclinical research.

Cognitive impairment is commonly associated with the pain experience. This impairment represents a major obstacle to daily activities and rehabilitation, especially in the chronic pain population. Here we review clinical and preclinical studies that have investigated pain-related alterations in cognition. These include impaired attentional, executive and general cognitive functioning. We describe the anatomical, neurochemical and molecular substrates common to both cognitive processing and supraspinal pain processing, and present the evidence for their involvement in pain-related cognitive impairment. We also examine the added complexity of cognitive impairment caused by analgesic medications and how this can further impact on morbidity in chronic pain patients. The need for a better understanding of the mechanisms of both pain-induced and treatment-related cognitive impairment is highlighted. Further research in this area will aid our understanding of patient symptoms and their underlying pathophysiology, ultimately leading to increased provision of guided therapy.

Investigating the effects of distracting stimuli on nociceptive behaviour and associated alterations in brain monoamines in rats.

Distraction interventions are used clinically to relieve pain. Exposure to distracting stimuli causes withdrawal of attention from the painful stimulus and reduces perceived pain. However, the neurobiological mechanisms mediating distraction-induced analgesia are poorly understood due, in part, to a paucity of animal studies modelling this phenomenon. The present study investigated the effects of three distracting stimuli on formalin-evoked nociceptive behaviour and brain tissue monoamine levels in rats. The three distractors were: exposure to a novel environment, exposure to a novel object, and exposure, without contact, to another rat (conspecific). A control group, habituated to the test arena, was also included. Formalin-evoked nociceptive behaviour was significantly reduced in rats exposed to the novel object or novel arena, but not those exposed to the conspecific, compared with controls. Antinociception resulting from exposure to the novel object was of longer duration than that resulting from exposure to the novel arena. Failure to detect any distractor-induced effects on plasma corticosterone levels or aversive behaviours suggests that the stimuli used were non-stressful. HPLC analysis revealed that there was a significant reduction in serotonin and dopamine metabolites in the medial prefrontal cortex in animals exposed to the novel object. These results indicate that exposure to a novel object or arena reduces nociceptive behaviour in rats, effects accompanied by discrete alterations in serotonin and dopamine metabolites in the medial prefrontal cortex.