Mild Traumatic Brain Injury-Induced Augmented Postsurgical Pain Is Driven by Central Serotonergic Pain-Facilitatory Signaling.

BACKGROUND: Individuals recovering from mild traumatic brain injury (mTBI) have increased rates of acute and chronic pain. However, the mechanism through which mTBI triggers heightened pain responses and the link between mTBI and postsurgical pain remain elusive. Recent data suggest that dysregulated serotonergic pain-modulating circuits could be involved. We hypothesized that mTBI triggers dysfunction in descending serotonergic pain modulation, which exacerbates acute pain and delays pain-related recovery after surgery. METHODS: Using mouse models of mTBI and hindpaw incision for postsurgical pain in C57BL/6J mice, mechanical withdrawal thresholds were assessed throughout the postsurgical period. To determine whether mTBI leads to persistent alteration of endogenous opioid tone, mu-opioid receptors (MORs) were blocked with naloxone. Finally, the role of descending serotonergic signaling on postsurgical allodynia in animals with mTBI was examined using ondansetron (5-HT 3 receptor antagonist) or a serotonin-specific neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), to ablate descending serotonergic fibers. The treatment effects on withdrawal thresholds were normalized to baseline (percentage of maximum possible effect, MPE%), and analyzed using paired t -test or 2-way repeated-measures ANOVA with post hoc multiple comparisons. RESULTS: Post-mTBI mice demonstrated transient allodynia in hindpaws contralateral to mTBI, while no nociceptive changes were observed in sham-mTBI animals (mean difference, MD, MPE%, post-mTBI day 3: -60.9; 95% CI, -88.7 to -35.0; P < .001). After hindpaw incision, animals without mTBI exhibited transient allodynia, while mice with prior mTBI demonstrated prolonged postsurgical allodynia (MD-MPE% postsurgical day 14: -65.0; 95% CI, -125.4 to -4.5; P = .04). Blockade of MORs using naloxone transiently reinstated allodynia in mTBI animals but not in sham-mTBI mice (MD-MPE% post-naloxone: -69.9; 95% CI, -94.8 to -45.1; P < .001). Intrathecal administration of ondansetron reversed the allodynia observed post-mTBI and postincision in mTBI mice (compared to vehicle-treated mTBI mice, MD-MPE% post-mTBI day 3: 82.7; 95% CI, 58.5-106.9; P < .001; postsurgical day 17: 62.5; 95% CI, 38.3-86.7; P < .001). Both the acute allodynia after TBI and the period of prolonged allodynia after incision in mTBI mice were blocked by pretreatment with 5,7-DHT (compared to sham-mTBI mice, MD-MPE% post-mTBI day 3: 0.5; 95% CI, -18.5 to 19.5; P = .99; postsurgical day 14: -14.6; 95% CI, -16.7 to 45.9; P = .48). Similar behavioral patterns were observed in hindpaw ipsilateral to mTBI. CONCLUSIONS: Collectively, our results show that descending serotoninergic pain-facilitating signaling is responsible for nociceptive sensitization after mTBI and that central endogenous opioid tone opposes serotonin's effects. Understanding brain injury-related changes in endogenous pain modulation may lead to improved pain control for those with TBI undergoing surgery.

Functional bias of morphine and oliceridine under conditions of minor injury.

Recent reports suggest pain from surgical injury may influence the risks associated with exposure to opioids. In mice, hind-paw incision attenuates morphine-primed reinstatement due to kappa opioid receptor activation by dynorphin. In this focused group of studies, we examined the hypotheses that kappa-opioid receptor activation in the nucleus accumbens mediates attenuated drug- primed reinstatement after incisional surgery, and the G-protein biased mu-opioid agonist, oliceridine, leads to less priming of the dynorphin effect in comparison to morphine. To address these hypotheses, adult C57BL/6 male mice underwent intracranial cannulation for administration of the selective kappa-opioid antagonist norBNI directly into the nucleus accumbens. After recovery, they were conditioned with morphine or oliceridine after hind-paw incisional injury, then underwent extinction followed by opioid-primed reinstatement. Intra-accumbal administration of norBNI was carried out prior to testing. The nucleus accumbens and medial prefrontal cortex were extracted and analyzed for expression of prodynorphin. We observed that animals conditioned with morphine in the setting of incisional injury demonstrated blunted responses to opioid-primed reinstatement, and that the blunted responses were reversed with intra-accumbal norBNI administration. Persistently elevated levels of prodynorphin expression in the medial prefrontal cortex and nucleus accumbens were observed in the incised morphine-treated animals. However, both behavioral and molecular changes were absent in animals with incisional injury conditioned with oliceridine. These findings suggest a role for prodynorphin expression in the nucleus accumbens with exposure to morphine after surgery that may protect individuals from relapse not shared with biased mu- opioid receptor agonists.

IL-6 signaling mediates the germinal center response, IgM production and nociceptive sensitization in male mice after tibia fracture.

BACKGROUND: Up-regulated interleukin 6 (IL-6) signaling, immune system activation, and pronociceptive autoantibodies are characteristic of complex regional pain syndrome (CRPS). IL-6 is known to promote B cell differentiation, thus we hypothesized that IL-6 signaling plays a crucial role in the development of adaptive immune responses and nociceptive sensitization in a murine tibia fracture model of CRPS. METHODS: Mice deficient in IL-6 expression (IL-6-/-) or B cell deficient (muMT) underwent tibia fracture and 3 weeks of cast immobilization or sham injury. The deposition of IgM in fractured limbs was followed using Western blotting, and passive serum transfer to muMT fracture mice was used to detect nociception-supporting autoantibodies. Lymph nodes were assessed for hypertrophy, IL-6 expression was measured using qPCR and ELISA, and germinal center formation was evaluated using FACS and immunohistochemistry. The therapeutic effects of exogenous neutralizing anti-IL-6 antibodies were also evaluated in the CRPS fracture model. RESULTS: Functional IL-6 signaling was required for the post fracture development of nociceptive sensitization, vascular changes, and IgM immune complex deposition in the skin of injured limbs. Passive transfer of sera from wild-type, but not IL-6-/- fracture mice into muMT fracture mice caused enhanced allodynia and postural unweighting. IL-6-/- fracture mice displayed reduced popliteal lymphadenopathy after fracture. Germinal center responses were detected in the popliteal lymph nodes of wild-type, but not in IL-6-/- fracture mice. We observed that IL-6 expression was dramatically enhanced in popliteal lymph node tissue after fracture. Conversely, administration of anti-IL-6 antibodies reduced nociceptive and vascular changes after fracture and inhibited lymphadenopathy. CONCLUSIONS: Collectively, these data support the hypothesis that IL-6 signaling in the fracture limb of mice is required for germinal center formation, IgM autoantibody production and nociceptive sensitization. Anti-IL-6 therapies might, therefore, reduce pain after limb fracture or in the setting of CRPS.

Morphine Exacerbates Postfracture Nociceptive Sensitization, Functional Impairment, and Microglial Activation in Mice.

BACKGROUND: Emerging evidence suggests that opioid use immediately after surgery and trauma may worsen outcomes. In these studies, the authors aimed to determine whether morphine administered for a clinically relevant time period (7 days) in a tibia fracture orthopedic surgery model had adverse effects on postoperative recovery. METHODS: Mice were given morphine twice daily for 7 days after unilateral tibial fracture and intramedullary pin fixation to model orthopedic surgery and limb trauma. Mechanical allodynia, limb-specific weight bearing, gait changes, memory, and anxiety were measured after injury. In addition, spinal cord gene expression changes as well as glial activation were measured. Finally, the authors assessed the effects of a selective Toll-like receptor 4 antagonist, TAK-242, on nociceptive and functional changes after injury. RESULTS: Tibial fracture caused several weeks of mechanical nociceptive sensitization (F(1, 216) = 573.38, P < 0.001, fracture + vehicle vs. sham + vehicle, n = 10 per group), and this change was exacerbated by the perioperative administration of morphine (F(1, 216) = 71.61, P < 0.001, fracture + morphine vs. fracture + vehicle, n = 10 per group). In additional testing, injured limb weight bearing, gait, and object location memory were worse in morphine-treated fracture mice than in untreated fracture mice. Postfracture expression levels of several genes previously associated with opioid-induced hyperalgesia, including brain-derived neurotrophic factor and prodynorphin, were unchanged, but neuroinflammation involving Toll-like receptor 4 receptor-expressing microglia was observed (6.8 ± 1.5 [mean ± SD] cells per high-power field for fracture + vehicle vs. 12 ± 2.8 fracture + morphine, P < 0.001, n = 8 per /group). Treatment with a Toll-like receptor 4 antagonist TAK242 improved nociceptive sensitization for about 2 weeks in morphine-treated fracture mice (F(1, 198) = 73.36, P < 0.001, fracture + morphine + TAK242 vs. fracture + morphine, n = 10 per group). CONCLUSIONS: Morphine treatment beginning at the time of injury impairs nociceptive recovery and other outcomes. Measures preventing glial activation through Toll-like receptor 4 signaling may reduce the adverse consequences of postoperative opioid administration.

Exercise Reverses Nociceptive Sensitization, Upregulated Neuropeptide Signaling, Inflammatory Changes, Anxiety, and Memory Impairment in a Mouse Tibia Fracture Model.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: This study tested the hypothesis that ad lib running wheel exercise in a tibia fracture model of complex regional pain syndrome can reverse hindlimb nociceptive sensitization and inflammation in mice. METHODS: Three weeks after tibia fracture, the cast was removed and hindlimb von Frey thresholds and unweighting were tested; the mice were then randomized to either ad lib access to a running wheel for 4 weeks or no wheel access. After 4 weeks the behavioral testing was repeated and then skin, sciatic nerve, and spinal cord tissues collected for polymerase chain reaction and enzyme immunoassay measurements of neuropeptide and inflammatory mediator levels. A similar protocol was used in fracture mice treated with exercise for 4 weeks, and then the running wheel was removed for 2 weeks. Memory and anxiety were measured in both groups with use of open-field, zero-maze, and novel-objects recognition assays. RESULTS: At 7 weeks postfracture the mice with no wheel access exhibited hindlimb allodynia and unweighting, anxiety, memory loss, upregulated spinal neuropeptide signaling, and increased hind paw and spinal inflammatory mediator expression, but the postfracture mice allowed to exercise for 4 weeks exhibited none of these changes (n = 12/cohort). When exercise was stopped for 2 weeks after 4 weeks of running, hindlimb allodynia and unweighting were rekindled, and this nociceptive sensitization was associated with increased sciatic nerve neuropeptide levels and hind paw skin interleukin 6 and nerve growth factor expression (n = 12/cohort). CONCLUSIONS: Daily exercise reversed nociceptive sensitization, inflammation, anxiety, and memory loss after tibia fracture.

The Chemokine Receptor CXCR2 Supports Nociceptive Sensitization after Traumatic Brain Injury.

Abstract: Chronic pain after traumatic brain injury (TBI) is very common, but the mechanisms linking TBI to pain and the pain-related interactions of TBI with peripheral injuries are poorly understood. Chemokine receptors play an important role in both pain and brain injury. In the current work, we pursued the hypothesis that the epigenetically regulated CXC chemokine receptor 2 (CXCR2) is a crucial modulator of nociceptive sensitization induced by TBI. For these studies, we used the rat lateral fluid percussion model of TBI. Histone actyltransferase activity was blocked using anacardic acid beginning immediately following injury, or delayed for seven days prior to administration. The selective CXCR2 antagonist SCH527123 administered systemically or intrathecally was used to probe the role of chemokine signaling on mechanical hindpaw sensitization after TBI. The expression of the CXCR2 receptor was accomplished using real-time PCR, immunohistochemistry, and Western blotting, while epigenetic regulation was assessed using chromatin immunoprecipitation assay. The spinal levels of several pain-related mediators including CXCL1, an endogenous ligand for CXCR2, as well as brain-derived neurotrophic factor and prodynorphin were measured by enzyme-linked immunosorbent assay. We observed that anacardic acid potently blocked and reversed mechanical hindpaw sensitization after TBI. The same drug was able to prevent the upregulation of CXCR2 after TBI, but did not affect the spinal expression of other pain mediators. On the other hand, both systemically and intrathecally administered SCH527123 reversed hindpaw allodynia after TBI. Most of the spinal CXCR2 appeared to be expressed by spinal cord neurons. Chromatin immunoprecipitation experiments demonstrated TBI-enhanced association of the CXCR2 promoter with acetylated-H3K9 histone protein that was also reversible using anacardic acid. Taken together, our findings suggested that TBI causes the upregulation of spinal CXCR2 through an epigenetic mechanism ultimately supporting nociceptive sensitization. The use of CXCR2 antagonists may, therefore, be useful in pain resulting from TBI.

The multiple PDZ domain protein Mpdz/MUPP1 regulates opioid tolerance and opioid-induced hyperalgesia.

BACKGROUND: Opioids are a mainstay for the treatment of chronic pain. Unfortunately, therapy-limiting maladaptations such as loss of treatment effect (tolerance), and paradoxical opioid-induced hyperalgesia (OIH) can occur. The objective of this study was to identify genes responsible for opioid tolerance and OIH. RESULTS: These studies used a well-established model of ascending morphine administration to induce tolerance, OIH and other opioid maladaptations in 23 strains of inbred mice. Genome-wide computational genetic mapping was then applied to the data in combination with a false discovery rate filter. Transgenic mice, gene expression experiments and immunoprecipitation assays were used to confirm the functional roles of the most strongly linked gene. The behavioral data processed using computational genetic mapping and false discovery rate filtering provided several strongly linked biologically plausible gene associations. The strongest of these was the highly polymorphic Mpdz gene coding for the post-synaptic scaffolding protein Mpdz/MUPP1. Heterozygous Mpdz +/- mice displayed reduced opioid tolerance and OIH. Mpdz gene expression and Mpdz/MUPP1 protein levels were lower in the spinal cords of low-adapting 129S1/Svlm mice than in high-adapting C57BL/6 mice. Morphine did not alter Mpdz expression levels. In addition, association of Mpdz/MUPP1 with its known binding partner CaMKII did not differ between these high- and low-adapting strains. CONCLUSIONS: The degrees of maladaptive changes in response to repeated administration of morphine vary greatly across inbred strains of mice. Variants of the multiple PDZ domain gene Mpdz may contribute to the observed inter-strain variability in tolerance and OIH by virtue of changes in the level of their expression.

Epigenetic regulation of spinal cord gene expression contributes to enhanced postoperative pain and analgesic tolerance subsequent to continuous opioid exposure.

BACKGROUND: Opioids have become the mainstay for treatment of moderate to severe pain and are commonly used to treat surgical pain. While opioid administration has been shown to cause opioid-induced hyperalgesia and tolerance, interactions between opioid administration and surgery with respect to these problematic adaptations have scarcely been addressed. Accumulating evidence suggests opioids and nociceptive signaling may converge on epigenetic mechanisms in spinal cord to enhance or prolong neuroplastic changes. Epigenetic regulation of Bdnf (brain-derived neurotrophic factor) and Pdyn (prodynorphin) genes may be involved. RESULTS: Four days of ascending doses of morphine treatment caused opioid-induced hyperalgesia and reduced opioid analgesic efficacy in mice. Both opioid-induced hyperalgesia and the reduced opioid analgesic efficacy were enhanced in mice that received hindpaw incisions. The expression of Bdnf and Pdyn (qPCR) was increased after morphine treatment and incision. Chromatin immunoprecipitation assays demonstrated that the Pdyn and Bdnf promoters were more strongly associated with acetylated H3K9 after morphine plus incision than in the morphine or incision alone groups. Selective tropomyosin-related kinase B (ANA-12) and κ-opioid receptor (nor-binaltorphimine) antagonists were administered intrathecally, both reduced hyperalgesia one or three days after surgery. Administration of ANA-12 or nor-binaltorphimine attenuated the decreased morphine analgesic efficacy on day 1, but only nor-binaltorphimine was effective on day 3 after incision in opioid-exposed group. Coadministration of histone acetyltransferase inhibitor anacardic acid daily with morphine blocked the development of opioid-induced hyperalgesia and attenuated incision-enhanced hyperalgesia in morphine-treated mice. Anacardic acid had similar effects on analgesic tolerance, showing the involvement of histone acetylation in the interactions detected. CONCLUSIONS: Spinal epigenetic changes involving Bdnf and Pdyn may contribute to the enhanced postoperative nociceptive sensitization and analgesic tolerance observed after continuous opioid exposure. Treatments blocking the epigenetically mediated up-regulation of these genes or administration of TrkB or κ-opioid receptor antagonists may improve the clinical utility of opioids, particularly after surgery.

Sex differences in a Murine Model of Complex Regional Pain Syndrome.

Complex Regional Pain Syndrome (CRPS) is a major cause of chronic pain after surgery or trauma to the limbs. Despite evidence showing that the prevalence and severity of many forms of chronic pain, including CRPS, differ between males and females, laboratory studies on sex-related differences in animal models of CRPS are not available, and the impact of sex on the transition from acute to chronic CRPS pain and disability are unexplored. Here we make use of a tibia fracture/cast mouse model that recapitulates the nociceptive, functional, vascular, trophic, inflammatory and immune aspects of CRPS. Our aim is to describe the chronic time course of nociceptive, motor and memory changes associated with fracture/cast in male and female mice, in addition to exploring their underlying spinal mechanisms. Our behavioral data shows that, compared to males, female mice display lower nociceptive thresholds following fracture in the absence of any differences in ongoing or spontaneous pain. Furthermore, female mice show exaggerated signs of motor dysfunction, deficits in fear memory, and latent sensitization that manifests long after the normalization of nociceptive thresholds. Our biochemical data show differences in the spinal cord levels of the glutamate receptor NR2b, suggesting sex differences in mechanisms of central sensitization that could account for differences in duration and severity of CRPS symptoms between the two groups.

Extracellular superoxide dismutase is important for hippocampal neurogenesis and preservation of cognitive functions after irradiation.

Cranial irradiation is widely used in cancer therapy, but it often causes cognitive defects in cancer survivors. Oxidative stress is considered a major cause of tissue injury from irradiation. However, in an earlier study mice deficient in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD KO) showed reduced sensitivity to radiation-induced defects in hippocampal functions. To further dissect the role of EC-SOD in neurogenesis and in response to irradiation, we generated a bigenic EC-SOD mouse model (OE mice) that expressed high levels of EC-SOD in mature neurons in an otherwise EC-SOD-deficient environment. EC-SOD deficiency was associated with reduced progenitor cell proliferation in the subgranular zone of dentate gyrus in KO and OE mice. However, high levels of EC-SOD in the granule cell layer supported normal maturation of newborn neurons in OE mice. Following irradiation, wild-type mice showed reduced hippocampal neurogenesis, reduced dendritic spine densities, and defects in cognitive functions. OE and KO mice, on the other hand, were largely unaffected, and the mice performed normally in neurocognitive tests. Although the resulting hippocampal-related functions were similar in OE and KO mice following cranial irradiation, molecular analyses suggested that they may be governed by different mechanisms: whereas neurotrophic factors may influence radiation responses in OE mice, dendritic maintenance may be important in the KO environment. Taken together, our data suggest that EC-SOD plays an important role in all stages of hippocampal neurogenesis and its associated cognitive functions, and that high-level EC-SOD may provide protection against irradiation-related defects in hippocampal functions.

The Netrin-1 receptor DCC is a regulator of maladaptive responses to chronic morphine administration.

BACKGROUND: Opioids are the cornerstone of treatment for moderate to severe pain, but chronic use leads to maladaptations that include: tolerance, dependence and opioid-induced hyperalgesia (OIH). These responses limit the utility of opioids, as well as our ability to control chronic pain. Despite decades of research, we have no therapies or proven strategies to overcome this problem. However, murine haplotype based computational genetic mapping and a SNP data base generated from analysis of whole-genome sequence data (whole-genome HBCGM), provides a hypothesis-free method for discovering novel genes affecting opioid maladaptive responses. RESULTS: Whole genome-HBCGM was used to analyze phenotypic data on morphine-induced tolerance, dependence and hyperalgesia obtained from 23 inbred strains. The robustness of the genetic mapping results was analyzed using strain subsets. In addition, the results of analyzing all of the opioid-related traits together were examined. To characterize the functional role of the leading candidate gene, we analyzed transgenic animals, mRNA and protein expression in behaviorally divergent mouse strains, and immunohistochemistry in spinal cord tissue. Our mapping procedure identified the allelic pattern within the netrin-1 receptor gene (Dcc) as most robustly associated with OIH, and it was also strongly associated with the combination of the other maladaptive opioid traits analyzed. Adult mice heterozygous for the Dcc gene had significantly less tendency to develop OIH, become tolerant or show evidence of dependence after chronic exposure to morphine. The difference in opiate responses was shown not to be due to basal or morphine-stimulated differences in the level of Dcc expression in spinal cord tissue, and was not associated with nociceptive neurochemical or anatomical alterations in the spinal cord or dorsal root ganglia in adult animals. CONCLUSIONS: Whole-genome HBCGM is a powerful tool for identifying genes affecting biomedical traits such as opioid maladaptations. We demonstrate that Dcc affects tolerance, dependence and OIH after chronic opioid exposure, though not through simple differences in expression in the adult spinal cord.

Epigenetic regulation of spinal cord gene expression controls opioid-induced hyperalgesia.

BACKGROUND: The long term use of opioids for the treatment of pain leads to a group of maladaptations which includes opioid-induced hyperalgesia (OIH). OIH typically resolves within few days after cessation of morphine treatment in mice but is prolonged for weeks if histone deacetylase (HDAC) activity is inhibited during opioid treatment. The present work seeks to identify gene targets supporting the epigenetic effects responsible for OIH prolongation. RESULTS: Mice were treated with morphine according to an ascending dose protocol. Some mice also received the selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) additionally. Chronic morphine treatment with simultaneous HDAC inhibition enhanced OIH, and several spinal cord genes were up-regulated. The expression of Bdnf (Brain-derived neurotrophic factor) and Pdyn (Prodynorphin) were most closely related to the observed behavioral changes. ChIP (Chromatin immuoprecipation) assays demonstrated that promoter regions of Pdyn and Bdnf were strongly associated with aceH3K9 (Acetylated histone H3 Lysine9) after morphine and SAHA treatment. Furthermore, morphine treatment caused an increase in spinal BDNF and dynorphin levels, and these levels were further increased in SAHA treated mice. The selective TrkB (tropomyosin-receptor-kinase) antagonist ANA-12 reduced OIH when given one or seven days after cessation of morphine. Treatment with the selective kappa opioid receptor antagonist nor-BNI also reduced established OIH. The co-administration of either receptor antagonist agent daily with morphine resulted in attenuation of hyperalgesia present one day after cessation of treatment. Additionally, repeated morphine exposure induced a rise in BDNF expression that was associated with an increased number of BDNF+ cells in the spinal cord dorsal horn, showing strong co-localization with aceH3K9 in neuronal cells. Lastly, spinal application of low dose BDNF or Dynorphin A after resolution of OIH produced mechanical hypersensitivity, with no effect in controls. CONCLUSIONS: The present study identified two genes whose expression is regulated by epigenetic mechanisms during morphine exposure. Treatments aimed at preventing the acetylation of histones or blocking BDNF and dynorphin signaling may reduce OIH and improve long-term pain using opioids.

Brain neuroplastic changes accompany anxiety and memory deficits in a model of complex regional pain syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) is a painful condition with approximately 50,000 annual new cases in the United States. It is a major cause of work-related disability, chronic pain after limb fractures, and persistent pain after extremity surgery. Additionally, CRPS patients often experience cognitive changes, anxiety, and depression. The supraspinal mechanisms linked to these CRPS-related comorbidities remain poorly understood. METHODS: The authors used a previously characterized mouse model of tibia fracture/cast immobilization showing the principal stigmata of CRPS (n = 8 to 20 per group) observed in humans. The central hypothesis was that fracture/cast mice manifest changes in measures of thigmotaxis (indicative of anxiety) and working memory reflected in neuroplastic changes in amygdala, perirhinal cortex, and hippocampus. RESULTS: The authors demonstrate that nociceptive sensitization in these mice is accompanied by altered thigmotactic behaviors in the zero maze but not open field assay, and working memory dysfunction in novel object recognition and social memory but not in novel location recognition. Furthermore, the authors found evidence of structural changes and synaptic plasticity including changes in dendritic architecture and decreased levels of synaptophysin and brain-derived neurotrophic factor in specific brain regions. CONCLUSIONS: The study findings provide novel observations regarding behavioral changes and brain plasticity in a mouse model of CRPS. In addition to elucidating some of the supraspinal correlates of the syndrome, this work supports the potential use of therapeutic interventions that not only directly target sensory input and other peripheral mechanisms, but also attempt to ameliorate the broader pain experience by modifying its associated cognitive and emotional comorbidities.

Curcumin treatment attenuates pain and enhances functional recovery after incision.

BACKGROUND: Acute pain after surgery remains moderate to severe for 20% to 30% of patients despite advancements in the use of opioids, adjuvant drugs, and regional anesthesia. Depending on the type of surgery, 10% to 50% of patients experience persistent pain postoperatively, and there are no established methods for its prevention. Curcumin (diferuloylmethane) is one of the phenolic constituents of turmeric that has been used in Eastern traditional medicine as an antiseptic, antioxidant, anti-inflammatory, and analgesic agent. It may be effective for treating postoperative pain. METHODS: We used the hindpaw incision model with C57BL/6 mice. Sensitization to mechanical and thermal stimuli as well as effects on edema and temperature were measured up to 7 days after surgery. Spontaneous pain after incision was assessed by using conditioned place preference (CPP), and alterations in gait function were assessed using multiparameter digital gait analysis. RESULTS: Curcumin (50 mg/kg) significantly reduced the intensity of mechanical and heat sensitization after hindpaw incision in mice. No effects of curcumin on baseline nociceptive thresholds were observed. Curcumin also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. In addition, perioperative curcumin treatment attenuated hyperalgesic priming due to incision when mice were subsequently challenged with hindpaw prostaglandin E2 application. Furthermore, while vehicle-treated mice had evidence of spontaneous pain 48 hours after incision in the CPP paradigm, no evidence of ongoing pain was observed in the mice treated with curcumin. Likewise, hindpaw incision caused changes in several gait-related indices, but most of these were normalized in the curcumin-treated animals. The peri-incisional levels of several pronociceptive immune mediators including interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and macrophage inflammatory protein-1α were either not reduced or were even augmented 1 and 3 days after incision in curcumin-treated mice. The anti-inflammatory cytokine IL-10 was unchanged, while transforming growth factor-ß levels were enhanced under the same conditions. CONCLUSIONS: Our studies suggest that curcumin treatment is effective in alleviating incision-induced inflammation, nociceptive sensitization, spontaneous pain, and functional gait abnormalities. Augmented transforming growth factor-ß production provides one possible mechanism. These preclinical findings demonstrate curcumin's potential as a preventative strategy in postoperative pain treatment.

Brain Expression of CPB2 and Effects of Cpb2 Deficiency in Mouse Models of Behavior.

BACKGROUND: Procarboxypeptidase B2 (proCPB2 or TAFI) is a zymogen that after activation cleaves C-terminal basic residues from peptides or proteins with many identified targets. A splice variant of CPB2 has been found in the brain lacking essential residues for its carboxypeptidase function. The aim was to determine CPB2 expression in the brain and effects of CPB2 deficiency (Cpb2 -/-) on behavior. MATERIALS AND METHODS: Behavioral effects were tested by comparing Cpb2 -/- mice in short-term (open field and elevated zero maze tests) and long-term (Phenotyper) observations with wild-type (WT) controls. RESULTS: Long-term observation compared day 1 (acclimatizing to novel environment) to day 4 (fully acclimatized) with the inactive (day) and active (night) periods analyzed separately. Brain expression of CPB2 mRNA and protein was interrogated in publicly available databases. Long-term observation demonstrated differences between WT and Cpb2 -/- mice in several parameters. For example, Cpb2 -/- mice moved more frequently on both days 1 and 4, especially in the normally inactive periods. Cpb2 -/- mice spent more time on the shelter and less time in it. Differences were more pronounced on day 4 after the mice had fully acclimatized. In short-term observations, no differences were observed between Cpb2 -/- mice and WT mice. Brain expression of CBP2 was not detectable in the human protein atlas. Databases of single-cell RNAseq did not show expression of CPB2 mRNA in either human or mouse brain. CONCLUSION: Continuous observation of home-cage behavior suggests that Cpb2 -/- mice are more active than WT mice, show different day-night activity levels, and might have a different way of processing information.

Epigenetic regulation of spinal CXCR2 signaling in incisional hypersensitivity in mice.

BACKGROUND: The regulation of gene expression in nociceptive pathways contributes to the induction and maintenance of pain sensitization. Histone acetylation is a key epigenetic mechanism controlling chromatin structure and gene expression. Chemokine CC motif receptor 2 (CXCR2) is a proinflammatory receptor implicated in neuropathic and inflammatory pain and is known to be regulated by histone acetylation in some settings. The authors sought to investigate the role of histone acetylation on spinal CXCR2 signaling after incision. METHODS: Groups of 5-8 mice underwent hind paw incision. Suberoylanilide hydroxamic acid and anacardic acid were used to inhibit histone deacetylase and histone acetyltransferase, respectively. Behavioral measures of thermal and mechanical sensitization as well as hyperalgesic priming were used. Both message RNA quantification and chromatin immunoprecipitation analysis were used to study the regulation of CXCR2 and ligand expression. Finally, the selective CXCR2 antagonist SB225002 was administered intrathecally to reveal the function of spinal CXCR2 receptors after hind paw incision. RESULTS: Suberoylanilide hydroxamic acid significantly exacerbated mechanical sensitization after incision. Conversely, anacardic acid reduced incisional sensitization and also attenuated incision-induced hyperalgesic priming. Overall, acetylated histone H3 at lysine 9 was increased in spinal cord tissues after incision, and enhanced association of acetylated histone H3 at lysine 9 with the promoter regions of CXCR2 and keratinocyte-derived chemokine (CXCL1) was observed as well. Blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision. CONCLUSIONS: Histone modification is an important epigenetic mechanism regulating incision-induced nociceptive sensitization. The spinal CXCR2 signaling pathway is one epigenetically regulated pathway controlling early and latent sensitization after incision.

Preclinical characterization of the efficacy and safety of biologic N-001 as a novel pain analgesic for post-operative acute pain treatment.

Inhibition of actin remodeling in nerves modulates action potential propagation and therefore could be used to treat acute pain. N-001 is a novel protein analgesic engineered from several C. Botulinum toxins. N-001 targets sensory neurons through ganglioside GT1b binding and ADP-ribosylates G-actin reducing actin remodeling. The activity and efficacy of N-001 was evaluated previously in vitro and in a mouse inflammatory pain model. To assess the relevance of N-001 for treatment of acute post-surgical pain, the current study evaluated the efficacy of N-001 in a mouse hind-paw incision model by peri-incisional and popliteal nerve block administration combined with mechanical testing. N-001 provided relief of pain-like behavior over 3 days and 2 days longer than the conventional long-acting anesthetic bupivacaine. Preclinical safety studies of N-001 indicated the drug produced no toxic or adverse immunological reactions over multiple doses in mice. These results combined with past targeting results encourage further investigation of N-001 as an analgesic for post-operative pain management with the potential to function as a differential nociceptor-specific nerve block.

Effect of Voluntary Exercise on Endogenous Pain Control Systems and Post-traumatic Headache in Mice.

Traumatic brain injury (TBI) can cause acute and chronic pain along with motor, cognitive, and emotional problems. Although the mechanisms are poorly understood, previous studies suggest disruptions in endogenous pain modulation may be involved. Voluntary exercise after a TBI has been shown to reduce some consequences of injury including cognitive impairment. We hypothesized, therefore, that voluntary exercise could augment endogenous pain control systems in a rodent model of TBI. For these studies, we used a closed-head impact procedure in male mice modeling mild TBI. We investigated the effect of voluntary exercise on TBI-induced hindpaw nociceptive sensitization, diffuse noxious inhibitory control failure, and periorbital sensitization after bright light stress, a model of post-traumatic headache. Furthermore, we investigated the effects of exercise on memory, circulating markers of brain injury, neuroinflammation, and spinal cord gene expression. We observed that exercise significantly reduced TBI-induced hindpaw allodynia and periorbital allodynia in the first week following TBI. We also showed that exercise improved the deficits associated with diffuse noxious inhibitory control and reduced bright light stress-induced allodynia up to 2 months after TBI. In addition, exercise preserved memory and reduced TBI-induced increases in spinal BDNF, CXCL1, CXCL2, and prodynorphin expression, all genes previously linked to TBI-induced nociceptive sensitization. Taken together, our observations suggest that voluntary exercise may reduce pain after TBI by reducing TBI-induced changes in nociceptive signaling and preserving endogenous pain control systems. PERSPECTIVE: This article evaluates the effects of exercise on pain-related behaviors in a preclinical model of traumatic brain injury (TBI). The findings show that exercise reduces nociceptive sensitization, loss of diffuse noxious inhibitory control, memory deficits, and spinal nociception-related gene expression after TBI. Exercise may reduce or prevent pain after TBI.

Pronociceptive autoantibodies in the spinal cord mediate nociceptive sensitization, loss of function, and spontaneous pain in the lumbar disk puncture model of chronic back pain.

ABSTRACT: Previously, we observed that B cells and autoantibodies mediated chronic nociceptive sensitization in the mouse tibia fracture model of complex regional pain syndrome and that complex regional pain syndrome patient antibodies were pronociceptive in fracture mice lacking mature B cells and antibodies (muMT). The current study used a lumbar spinal disk puncture (DP) model of low back pain in wild-type (WT) and muMT mice to evaluate pronociceptive adaptive immune responses. Spinal disks and cords were collected 3 weeks after DP for polymerase chain reaction and immunohistochemistry analyses. Wild-type DP mice developed 24 weeks of hindpaw mechanical allodynia and hyperalgesia, grip weakness, and a conditioned place preference response indicative of spontaneous pain, but pain responses were attenuated or absent in muMT DP mice. Spinal cord expression of inflammatory cytokines, immune cell markers, and complement components were increased in WT DP mice and in muMT DP mice. Dorsal horn immunostaining in WT DP mice demonstrated glial activation and increased complement 5a receptor expressionin spinal neurons. Serum collected from WT DP mice and injected into muMT DP mice caused nociceptive sensitization, as did intrathecal injection of IgM collected from WT DP mice, and IgM immune complexes were observed in lumbar spinal disks and cord of WT DP mice. Serum from WT tibia fracture mice was not pronociceptive in muMT DP mice and vice versa, evidence that each type of tissue trauma chronically generates its own unique antibodies and targeted antigens. These data further support the pronociceptive autoimmunity hypothesis for the transition from tissue injury to chronic musculoskeletal pain state.

Roles of Gr-1+ leukocytes in postincisional nociceptive sensitization and inflammation.

BACKGROUND: Neutrophils are one of the predominant immune cells initially migrating to surgical wound edges. They produce mediators both associated with supporting (interleukin [IL]-1ß, C5a) and reducing (opioid peptides) pain. Studies demonstrate neutrophil depletion/blockade reduces nociceptive sensitization after nerve injury and carrageenan administration, but enhance sensitization in complete Freund's adjuvant inflammation. This research identifies the contribution of infiltrating neutrophils to incisional pain and inflammation. METHODS: Antibody-mediated Gr1 neutrophil depletion preceded hind paw incisions. Sensitization to mechanical and thermal stimuli, effects on edema and local levels of IL-1ß and C5a were measured. Local effects of C5a or IL-1 receptor antagonists PMX-53 and anakinra on sensitization after neutrophil depletion were examined. Groups of 4-8 mice were used. RESULTS: Anti-Gr1 antibody depleted more than 90% of circulating and infiltrating skin neutrophils after incision. Neutrophil depletion did not change magnitude or duration of mechanical hypersensitivity in incised mice. However, paw edema was significantly reduced and heat hypersensitivity was slightly increased in depleted animals. In depleted animals IL-1ß levels were half of controls 24 h after incision, whereas C5a levels were increased in both. Prominent IL-1ß immunohistochemical staining of epidermis was seen in both groups. PMX-53 and anakinra reduced incisional mechanical and heat nociceptive sensitization to the same extent, regardless of neutrophil depletion. CONCLUSIONS: Neutrophil-derived IL-1ß and C5a do not appear to contribute critically to peri-incisional nociceptive signaling. Other sources of mediators, such as epidermal cells, may need to be considered. Controlling inflammatory activation of resident cells in epidermis/deeper structures may show therapeutic efficacy in reducing pain from surgical incisions.