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.

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.

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.

Monoamine control of descending pain modulation after mild traumatic brain injury.

Traumatic brain injury (TBI) is a significant public health concern, with the majority of injuries being mild. Many TBI victims experience chronic pain. Unfortunately, the mechanisms underlying pain after TBI are poorly understood. Here we examined the contribution of spinal monoamine signaling to dysfunctional descending pain modulation after TBI. For these studies we used a well-characterized concussive model of mild TBI. Measurements included mechanical allodynia, the efficacy of diffuse noxious inhibitory control (DNIC) endogenous pain control pathways and lumber norepinephrine and serotonin levels. We observed that DNIC is strongly reduced in both male and female mice after mild TBI for at least 12 weeks. In naïve mice, DNIC was mediated through α2 adrenoceptors, but sensitivity to α2 adrenoceptor agonists was reduced after TBI, and reboxetine failed to restore DNIC in these mice. The intrathecal injection of ondansetron showed that loss of DNIC was not due to excess serotonergic signaling through 5-HT3 receptors. On the other hand, the serotonin-norepinephrine reuptake inhibitor, duloxetine and the serotonin selective reuptake inhibitor escitalopram both effectively restored DNIC after TBI in both male and female mice. Therefore, enhancing serotonergic signaling as opposed to noradrenergic signaling alone may be an effective pain treatment strategy after TBI.

Autonomic Regulation of Nociceptive and Immunologic Changes in a Mouse Model of Complex Regional Pain Syndrome.

Chronic pain frequently develops after limb injuries, and its pathogenesis is poorly understood. We explored the hypothesis that the autonomic nervous system regulates adaptive immune system activation and nociceptive sensitization in a mouse model of chronic post-traumatic pain with features of complex regional pain syndrome (CRPS). In studies sympathetic signaling was reduced using 6-hydroxydopamine (6-OHDA) or lofexidine, while parasympathetic signaling was augmented by nicotine administration. Hindpaw allodynia, unweighting, skin temperature, and edema were measured at 3 and 7 weeks after fracture. Hypertrophy of regional lymph nodes and IgM deposition in the skin of injured limbs were followed as indices of adaptive immune system activation. Passive transfer of serum from fracture mice to recipient B cell deficient (muMT) mice was used to assess the formation of pain-related autoantibodies. We observed that 6-OHDA or lofexidine reduced fracture-induced hindpaw nociceptive sensitization and unweighting. Nicotine had similar effects. These treatments also prevented IgM deposition, hypertrophy of popliteal lymph nodes, and the development of pronociceptive serum transfer effects. We conclude that inhibiting sympathetic or augmenting parasympathetic signaling inhibits pro-nociceptive immunological changes accompanying limb fracture. These translational results support the use of similar approaches in trials potentially alleviating persistent post-traumatic pain and, possibly, CRPS. PERSPECTIVE: Selective treatments aimed at autonomic nervous system modulation reduce fracture-related nociceptive and functional sequelae. The same treatment strategies limit pain-supporting immune system activation and the production of pro-nociceptive antibodies. Thus, the therapeutic regulation of autonomic activity after limb injury may reduce the incidence of chronic pain.

SNAP25 mutation disrupts metabolic homeostasis, steroid hormone production and central neurobehavior.

OBJECTIVE: SNAP-25 is one of the key proteins involved in formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that are at the core of hormonal secretion and synaptic transmission. Altered expression or function of SNAP-25 can contribute to the development of neuropsychiatric and metabolic disease. A dominant negative (DN) I67T missense mutation in the b-isoform of SNAP-25 (DN-SNAP25mut) mice leads to abnormal interactions within the SNARE complex and impaired exocytotic vesicle recycling, yet the significance of this mutation to any association between the central nervous system and metabolic homeostasis is unknown. METHODS: Here we explored aspects of metabolism, steroid hormone production and neurobehavior of DN-SNAP25mut mice. RESULTS: DN-SNAP25mut mice displayed enhanced insulin function through increased Akt phosphorylation, alongside increased adrenal and gonadal hormone production. In addition, increased anxiety behavior and beigeing of white adipose tissue with increased energy expenditure were observed in mutants. CONCLUSIONS: Our results show that SNAP25 plays an important role in bridging central neurological systems with peripheral metabolic homeostasis, and provide potential insights between metabolic disease and neuropsychiatric disorders in humans.

Circulating microRNAs from the mouse tibia fracture model reflect the signature from patients with complex regional pain syndrome.

INTRODUCTION: Complex regional pain syndrome (CRPS) often results from an initial trauma that later produces a disproportionate amount of pain. The mechanisms underlying CRPS have been studied using a tibia fracture model (TFM) in rodents because this model closely mimics symptoms and has several molecular correlates observed in patients with CRPS. OBJECTIVE: Here, we determined whether the TFM has alterations in circulating microRNAs (miRNAs) and cytokines transported by small extracellular vesicles (sEVs) that faithfully model previously reported miRNA alterations from patients with CRPS. METHODS: We isolated and characterized serum-derived sEVs from mice 3 weeks after fracture when symptoms such as pain hypersensitivity develop. Whole-transcriptome profiling was used to determine sEV miRNAs, and Bio-Plex Pro Mouse Cytokine 23-plex assay was used to measure cytokines. Differentially expressed miRNAs from TFM were compared with previously reported circulating miRNA alterations from patients with CRPS. RESULTS: Although sEV cytokine levels were unchanged, there were significant changes in sEV miRNA profiles. Differentially expressed miRNAs from TFM sEVs significantly overlapped with those previously reported in patients with CRPS. Of the 57 sEV miRNAs dysregulated in the TFM, 30 were previously reported in patients with CRPS compared with healthy control donors both in sEVs and 23 in whole blood. CONCLUSIONS: These findings enhance the validity of TFM as a model for CRPS and suggest that specific miRNA dysregulation may be a shared feature of CRPS and the TFM. These dysregulated miRNAs could help identify mechanistic targets or serve as biomarker candidates for both diagnosis and treatment responses in clinical trials.

Exposure to Gulf War Illness-related agents leads to the development of chronic pain and fatigue.

AIMS: A substantial contingent of veterans from the first Gulf War continues to suffer from a number of Gulf War-related illnesses (GWI) affecting the neurological and musculoskeletal systems; the most common symptoms include chronic pain and fatigue. Although animal models have recapitulated several aspects of cognitive impairments in GWI, the pain and fatigue symptoms have not been well documented to allow examination of potential pathogenic mechanisms. MAIN METHODS: We used a mouse model of GWI by exposing mice repeatedly to a combination of Gulf War chemicals (pyridostigmine bromide, permethrin, DEET, and chlorpyrifos) and mild immobilization stress, followed by investigating their pain susceptibilities and fatigue symptoms. To assess whether enhanced antioxidant capacity can counter the effects of GW agents, transgenic mice overexpressing extracellular superoxide dismutase (SOD3OE) were also examined. KEY FINDINGS: The mouse model recapitulated several aspects of the human illness, including hyperalgesia, impaired descending inhibition of pain, and increased tonic pain. There is a close association between chronic pain and fatigue in GWI patients. Consistent with this observation, the mouse model showed a significant reduction in physical endurance on the treadmill. Examination of skeletal muscles suggested reduction in mitochondrial functions may have contributed to the fatigue symptoms. Furthermore, the negative impacts of GW agents in pain susceptibilities were largely diminished in SOD3OE mice, suggesting that increased oxidative stress was associated with the emergence of these Gulf War symptoms. SIGNIFICANCE: the mouse model will be suitable for delineating specific defects in the pain pathways and mechanisms of fatigue in GWI.

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.

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.

Loss of diffuse noxious inhibitory control after traumatic brain injury in rats: A chronic issue.

Chronic pain is one of the most challenging and debilitating symptoms to manage after traumatic brain injury (TBI), yet the underlying mechanisms remain elusive. The disruption of normal endogenous pain control mechanisms has been linked to several forms of chronic pain and may play a role in pain after TBI. We hypothesized therefore that dysfunctional descending noradrenergic and serotonergic pain control circuits may contribute to the loss of diffuse noxious inhibitory control (DNIC), a critical endogenous pain control mechanism, weeks to months after TBI. For these studies, the rat lateral fluid percussion model of mild TBI was used along with a DNIC paradigm involving a capsaicin-conditioning stimulus. We observed sustained failure of the DNIC response up to 180-days post injury. We confirmed, that descending α2 adrenoceptor-mediated noradrenergic signaling was critical for endogenous pain inhibition in uninjured rats. However, augmenting descending noradrenergic signaling using reboxetine, a selective noradrenaline reuptake inhibitor, failed to restore DNIC after TBI. Furthermore, blocking serotonin-mediated descending signaling using selective spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine was also unsuccessful at restoring endogenous pain modulation after TBI. Unexpectedly, increasing descending serotonergic signaling using the selective serotonin reuptake inhibitor escitalopram and the serotonin-norepinephrine reuptake inhibitor duloxetine restored the DNIC response in TBI rats at both 49- and 180- days post injury. Consistent with these observations, spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine eliminated the effects of escitalopram. Intact α2 adrenoceptor signaling, however, was not required for the serotonin-mediated restoration of DNIC after TBI. These results suggest that TBI causes maladaptation of descending nociceptive signaling mechanisms and changes in the function of both adrenergic and serotonergic circuits. Such changes could predispose those with TBI to chronic pain.

Complex regional pain syndrome patient immunoglobulin M has pronociceptive effects in the skin and spinal cord of tibia fracture mice.

It has been proposed that complex regional pain syndrome (CRPS) is a post-traumatic autoimmune disease. Previously, we observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture model and that serum immunoglobulin M (IgM) antibodies from fracture mice have pronociceptive effects in muMT fracture mice lacking B cells. The current study evaluated the pronociceptive effects of injecting CRPS patient serum or antibodies into muMT fracture mice by measuring hind paw allodynia and unweighting changes. Complex regional pain syndrome serum binding was measured against autoantigens previously identified in the fracture mouse model. Both CRPS patient serum or IgM antibodies had pronociceptive effects in the fracture limb when injected systemically in muMT fracture mice, but normal subject serum and CRPS patient IgG antibodies had no effect. Furthermore, CRPS serum IgM antibodies had pronociceptive effects when injected into the fracture limb hind paw skin or intrathecally in the muMT fracture mice. Early (1-12 months after injury) CRPS patient (n = 20) sera were always pronociceptive after systemic injection, and chronic (>12 months after injury) CRPS sera were rarely pronociceptive (2/20 patients), while sera from normal subjects (n = 20) and from patients with uncomplicated recoveries from orthopedic surgery and/or fracture (n = 15) were never pronociceptive. Increased CRPS serum IgM binding was observed for keratin 16, histone 3.2, gamma actin, and alpha enolase autoantigens. We postulate that CRPS patient IgM antibodies bind to neoantigens in the fracture mouse skin and spinal cord to initiate a regionally restricted pronociceptive complement response potentially contributing to the CRPS disease process.

Mild Traumatic Brain Injury Causes Nociceptive Sensitization through Spinal Chemokine Upregulation.

High rates of acute and chronic pain are associated with traumatic brain injury (TBI), but mechanisms responsible for the association remain elusive. Recent data suggest dysregulated descending pain modulation circuitry could be involved. Based on these and other observations, we hypothesized that serotonin (5-HT)-dependent activation of spinal CXC Motif Chemokine Receptor 2 (CXCR2) may support TBI-related nociceptive sensitization in a mouse model of mild TBI (mTBI). We observed that systemic 5-HT depletion with p-chlorophenylalanine attenuated mechanical hypersensitivity seen after mTBI. Likewise, selective spinal 5-HT fiber depletion with 5,7-dihydroxytryptamine (5,7-DHT) reduced hypersensitivity after mTBI. Consistent with a role for spinal 5-HT3 serotonin receptors, intrathecal ondansetron administration after TBI dose-dependently attenuated nociceptive sensitization. Also, selective CXCR2 antagonist SCH527123 treatment attenuated mechanical hypersensitivity after mTBI. Furthermore, spinal CXCL1 and CXCL2 mRNA and protein levels were increased after mTBI as were GFAP and IBA-1 markers. Spinal 5,7-DHT application reduced both chemokine expression and glial activation. Our results suggest dual pathways for nociceptive sensitization after mTBI, direct 5-HT effect through 5-HT3 receptors and indirectly through upregulation of chemokine signaling. Designing novel clinical interventions against either the 5-HT3 mediated component or chemokine pathway may be beneficial in treating pain frequently seen in patients after mTBI.

Enhanced descending pain facilitation in acute traumatic brain injury.

Acute and persistent pain are recognized consequences of TBI that can enhance suffering and significantly impair rehabilitative efforts. Both experimental models and clinical studies suggest that TBI may result in an imbalance between descending pain facilitatory and inhibitory pathways. The aim of this study was to assess the role of enhanced descending serotonin-mediated pain facilitation in a rat TBI model using selective spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine (DHT). We observed significant hindpaw allodynia in TBI rats that was reduced after DHT but not vehicle treatment. Immunohistochemical studies demonstrated profound spinal serotonin depletion in DHT-treated rats. Furthermore, lumbar intrathecal administration of the 5-HT3 receptor antagonist ondansetron at 7 days post-injury (DPI), when hindpaw allodynia was maximal, also attenuated nociceptive sensitization. Additional immunohistochemical analyses of the lumbar spinal cord at 7 DPI revealed a robust bilateral microglial response in the superficial dorsal horns that was significantly reduced with DHT treatment. Furthermore, serotonin depletion also prevented the TBI-induced bilateral increase in c-Fos positive cells within the Rexed laminae I and II of the dorsal horns. These results indicate that in the weeks following TBI, pain may be responsive to 5-HT3 receptor antagonists or other measures which rebalance descending pain modulation.

Pre-treatment with microRNA-181a Antagomir Prevents Loss of Parvalbumin Expression and Preserves Novel Object Recognition Following Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) can result in permanent impairment in memory and learning and may be a precursor to other neurological sequelae. Clinical treatments to ameliorate the effects of mTBI are lacking. Inhibition of microRNA-181a (miR-181a) is protective in several models of cerebral injury, but its role in mTBI has not been investigated. In the present study, miR-181a-5p antagomir was injected intracerebroventricularly 24 h prior to closed-skull cortical impact in young adult male mice. Paw withdrawal, open field, zero maze, Y maze, object location and novel object recognition tests were performed to assess neurocognitive dysfunction. Brains were assessed immunohistologically for the neuronal marker NeuN, the perineuronal net marker wisteria floribunda lectin (WFA), cFos, and the interneuron marker parvalbumin. Protein quantification was performed with immunoblots for synaptophysin and postsynaptic density 95 (PSD95). Fluorescent in situ hybridization was utilized to localize hippocampal miR-181a expression. MiR-181a antagomir treatment reduced neuronal miR-181a expression after mTBI, restored deficits in novel object recognition and increased hippocampal parvalbumin expression in the dentate gyrus. These changes were associated with decreased dentate gyrus hyperactivity indicated by a relative reduction in PSD95 and cFos expression. These results suggest that miR-181a inhibition may be a therapeutic approach to reduce hippocampal excitotoxicity and prevent cognitive dysfunction following mTBI.

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.

Nociceptive and Cognitive Changes in a Murine Model of Polytrauma.

Polytrauma commonly involves concussion (mild traumatic brain injury [mTBI]) and peripheral trauma including limb fractures. Interactions between mTBI and peripheral injuries are poorly understood, both leading to chronic pain and neurobehavioral impairments. To elucidate these interactions, a murine polytrauma model was developed. mTBI alone resulted in similar increased mechanical allodynia in male and female mice. Female fracture and polytrauma groups displayed greater increases in hind paw tactile hypersensitivity for weeks after injury than did the respective male groups. Capsaicin-evoked spontaneous pain behaviors were greater in fracture and polytrauma female mice compared with male mice. The mTBI and polytrauma male mice displayed significant deficits in spatial working memory. All fracture, mTBI, or polytrauma groups had deficits in object recognition memory. Only male mTBI or polytrauma mice showed greater agitation and increased risk-taking behavior in open field testing as well as zero maze tests. Additionally, impaired diffuse noxious inhibitory control was observed in all mTBI and polytrauma mice. The model presented offers clinically relevant features useful for studying persistent pain as well as cognitive and other behavioral changes after TBI including polytrauma. A better understanding of nervous system dysfunction after TBI and polytrauma might help prevent or reduce persistent pain and disability in these patients. PERSPECTIVE: The polytrauma model presented has relevant features of chronic pain and neurobehavioral impairments useful for studying mechanisms involved in their development. This model may have special value in understanding altered descending pain modulation after TBI and polytrauma.

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.