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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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

Chronic opioid consumption increases postoperative pain. Epigenetic changes related to chronic opioid use and surgical incision may be partially responsible for this enhancement. The CXCL1/CXCR2 signaling pathway, implicated in several pain models, is known to be epigenetically regulated via histone acetylation. The current study was designed to investigate the role of CXCL1/CXCR2 signaling in opioid-enhanced incisional sensitization and to elucidate the possible epigenetic mechanism underlying CXCL1/CXCR2 pathway-mediated regulation of nociceptive sensitization in mice. Chronic morphine treatment generated mechanical and thermal nociceptive sensitization and also significantly exacerbated incision-induced mechanical allodynia. Peripheral but not central messenger RNA levels of CXCL1 and CXCR2 were increased after incision. The source of peripheral CXCL1 appeared to be wound area neutrophils. Histone H3 subunit acetylated at the lysine 9 position (AcH3K9) was increased in infiltrating dermal neutrophils after incision and was further increased in mice with chronic morphine treatment. The association of AcH3K9 with the promoter region of CXCL1 was enhanced in mice after chronic morphine treatment. The increase in CXCL1 near wounds caused by chronic morphine pretreatment was mimicked by pharmacologic inhibition of histone deacetylation. Finally, local injection of CXCL1 induced mechanical sensitivity in naive mice, whereas blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision. Perspective Peripheral CXCL1/CXCR2 signaling helps to control nociceptive sensitization after incision, and epigenetic regulation of CXCL1 expression explains in part opioid-enhanced incisional allodynia in mice. These results suggest that targeting CXCL1/CXCR2 signaling may be useful in treating nociceptive sensitization, particularly for postoperative pain in chronic opioid-consuming patients. [ABSTRACT FROM AUTHOR]

Abstract: The preprotachykinin A gene (ppt-A) codes for Substance P (SP), supports nociceptive sensitization, and modulates inflammatory responses after incision. Repeated opioid use produces paradoxical pain sensitization—termed opioid-induced hyperalgesia (OIH) —which can exacerbate pain after incision. Here the contribution of SP to peri-incisional nociceptive sensitization and nociceptive mediator production after opioid treatment was examined utilizing ppt-A knockout (−/−) mice and the neurokinin (NK1) receptor antagonist LY303870. Less mechanical allodynia was observed in ppt-A−/− mice compared to wild types (wt) after morphine treatment both before and after incision. Moreover, LY303870 administered with morphine reduced incisional hyperalgesia in wt mice. Incision after saline or escalating morphine treatment upregulated skin IL-1β, IL-6, G-CSF and MIP-1α levels in ppt-A−/− and wt mice similarly. However, chronic morphine treatment greatly exacerbated increases in skin nerve growth factor levels after incision, an effect entirely dependent upon intact SP signaling. Additionally, SP dependent upregulation of prodynorphin, NMDA1 and NK1 receptor expression in spinal cord was seen after morphine treatment and incision. A similar pattern was seen for 5-HT3 receptor expression in tissue from dorsal root ganglia. Therefore, SP may work at both central and peripheral sites to enhance nociceptive sensitization after morphine treatment and incision. Perspective: These studies show that SP signaling modulates enhanced nerve growth factor production and changes in neuronal gene expression seen after incision in mice previously exposed to morphine. [Copyright &y& Elsevier]

Background: Neutrophils are one of the predominant immune cells initially migrating to surgical wound edges. They produce mediators both associated with supporting (interleukin [IL]-1β, 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-1β 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-1β levels were half of controls 24 h after incision, whereas C5a levels were increased in both. Prominent IL-1β 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-1β 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. [ABSTRACT FROM AUTHOR]

Background & Aims: Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time. Methods: Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified. Results: Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 ± 0.09 μm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 ± 3.4 μm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001). Conclusions: Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation. [Copyright &y& Elsevier]

Background & Aims: Barrett’s esophagus is a precursor of esophageal adenocarcinoma. DNA microarrays that enable a genome-wide assessment of gene expression enhance the identification of specific genes as well as gene expression patterns that are expressed by Barrett’s esophagus and adenocarcinoma compared with normal tissues. Barrett’s esophagus length has also been identified as a risk factor for progression to adenocarcinoma, but whether there are intrinsic biological differences between short-segment and long-segment Barrett’s esophagus can be explored with microarrays. Methods: Gene expression profiles for endoscopically obtained biopsy specimens of Barrett’s esophagus or esophageal adenocarcinoma and associated normal esophagus and duodenum were identified for 17 patients using DNA microarrays. Unsupervised and supervised approaches for data analysis defined similarities and differences between the tissues as well as correlations with clinical phenotypes. Results: Each tissue displays a unique expression profile that distinguishes it from others. Barrett’s esophagus and esophageal adenocarcinoma express a unique set of stromal genes that is distinct from normal tissues but similar to other cancers. Adenocarcinoma also showed lower and higher expression for many genes compared with Barrett’s esophagus. No difference in gene expression was found between short-segment and long-segment Barrett’s esophagus. Conclusions: The genome-wide assessment provided by current DNA microarrays reveals many candidate genes and patterns not previously identified. Stromal gene expression in Barrett’s esophagus and adenocarcinoma is similar, indicating that these changes precede malignant transformation. [Copyright &y& Elsevier]

Background: It has been suggested that Barrett''s esophagus (BE) is associated with an increased risk of developing colorectal neoplasia, but this has not been reported consistently. Aim: To study whether BE is associated with an increased risk of colorectal neoplasia, and if it is, whether it is dependent on use of proton-pump inhibitors (PPIs) or aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs). Design: Case-control study. Setting: Endoscopic database of the Palo Alto Veterans Affairs Health Care System. Population: 268 veterans with BE were matched with 268 controls without BE. Intervention: Controls had undergone upper GI endoscopy within 14 days of the corresponding case. Colonoscopy was performed within 6 months in cases and controls. Main Outcome Measure: Development of colorectal neoplasia. Results: Colorectal neoplasia was present in 162 of 268 (60%) BE patients and in 105 of 268 (40%) controls (p < 0.001). The presence of BE (odds ratio [OR] 2.02: 95% CI [1.35, 3.04]), but also increasing age (OR 1.24 per decade: 95% CI [1.04, 1.48]) and alcohol use (OR 1.70: 95% CI [1.16, 2.50]) were associated with an increased risk of colorectal neoplasia in multivariable logistic regression analysis, whereas PPIs (OR 0.99: 95% CI [0.66, 1.48]) and aspirin/NSAIDs (OR 0.90: 95% CI [0.61, 1.33]) had no meaningful effect. Limitations: This was a retrospective study in mostly male veterans. Conclusions: Veterans with BE are at an increased risk of developing colorectal neoplasia. This association is independent from the use of PPIs or aspirin/NSAIDs. [Copyright &y& Elsevier]

Abstract Background Formalin injection into rodent hind paws is one of the most commonly employed pain assays. The resulting nocifensive behaviors can be divided into two phases differing in timing, duration and underlying mechanisms. Spinal sensitization has long been felt to participate in the second phase of this response, although this sensitization is incompletely understood. By using correlative analysis between spinal gene expression and mouse strain-dependent intensity of late phase behavior, we hypothesized genes participating in variability of the response could be identified. Results Late phase formalin behavior scores among 10 inbred mouse strains were correlated with a spinal cord gene expression database constructed using expression arrays. Messenger RNA levels for several genes were highly correlated with the late phase behavioral responses. Most of these genes had already been implicated in mechanisms regulating pain and analgesia. One of the most strongly correlated genes, Mapk8 coding for c-Jun N-terminal kinase 1 (JNK1), was chosen for further analysis. Studies using additional strains of mice confirmed that spinal cord mRNA expression levels of Mapk8 followed the pattern predicted by strain-specific levels of formalin behavior. Interestingly, spinal cord JNK1 protein levels displayed an inverse relationship with mRNA measurements. Finally, intrathecal injections of the selective JNK inhibitor, SP600125, selectively reduced late phase licking behavior. Conclusions Wide differences in pain behaviors, including those resulting from the injection of formalin, can be observed in inbred strains of mice suggesting strong genetic influences. Correlating levels of gene expression in tissues established to be mechanistically implicated in the expression of specific behaviors can identify genes involved in the behaviors of interest. Comparing formalin late phase behavior levels with spinal cord gene expression yielded several plausible gene candidates, including the Mapk8 gene. Additional molecular and pharmacologic evidence confirmed a functional role for this gene in supporting formalin late phase responses.

Abstract Background A Stanford University study reported that in asymptomatic GERD patients who were being treated with a proton pump inhibitor (PPI), 50% had pathologic esophageal acid exposure. Aim We considered the possibility that the high prevalence of pathologic esophageal reflux might simply have resulted from calculating acidity as time pH < 4. Methods We calculated integrated acidity and time pH < 4 from the 49 recordings of 24-hour gastric and esophageal pH from the Stanford study as well as from another study of 57 GERD subjects, 26 of whom were treated for 8 days with 20 mg omeprazole or 20 mg rabeprazole in a 2-way crossover fashion. Results The prevalence of pathologic 24-hour esophageal reflux in both studies was significantly higher when measured as time pH < 4 than when measured as integrated acidity. This difference was entirely attributable to a difference between the two measures during the nocturnal period. Nocturnal gastric acid breakthrough was not a useful predictor of pathologic nocturnal esophageal reflux. Conclusion In GERD subjects treated with a PPI, measuring time esophageal pH < 4 will significantly overestimate the prevalence of pathologic esophageal acid exposure over 24 hours and during the nocturnal period.

Abstract Background Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. Methods Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. Results The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. Conclusion Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.

Background: A Stanford University study reported that in asymptomatic GERD patients who were being treated with a proton pump inhibitor (PPI), 50% had pathologic esophageal acid exposure.Aim: We considered the possibility that the high prevalence of pathologic esophageal reflux might simply have resulted from calculating acidity as time pH < 4.Methods: We calculated integrated acidity and time pH < 4 from the 49 recordings of 24-hour gastric and esophageal pH from the Stanford study as well as from another study of 57 GERD subjects, 26 of whom were treated for 8 days with 20 mg omeprazole or 20 mg rabeprazole in a 2-way crossover fashion.Results: The prevalence of pathologic 24-hour esophageal reflux in both studies was significantly higher when measured as time pH < 4 than when measured as integrated acidity. This difference was entirely attributable to a difference between the two measures during the nocturnal period. Nocturnal gastric acid breakthrough was not a useful predictor of pathologic nocturnal esophageal reflux.Conclusion: In GERD subjects treated with a PPI, measuring time esophageal pH < 4 will significantly overestimate the prevalence of pathologic esophageal acid exposure over 24 hours and during the nocturnal period. [ABSTRACT FROM AUTHOR]

Abstract: Previous preclinical and translational studies suggest that tissue trauma related to bony fracture and intervertebral disk disruption initiates the formation of pronociceptive antibodies that support chronic musculoskeletal pain conditions. This study tested this hypothesis in the monosodium iodoacetate (MIA) mouse model of osteoarthritis (OA) and extended the findings using OA patient samples. Monosodium iodoacetate was injected unilaterally into the knees of male and female wild-type (WT) and muMT mice (lacking B cells) to induce articular cartilage damage. Repeated nociceptive behavioral testing was performed, and serum was collected for antibody isolation and passive transfer experiments. Serum antibodies collected from patients with OA were tested in MIA-treated muMT mice. Biochemical analyses were performed on knee joint tissues. Monosodium iodoacetate-treated WT mice developed chronic ipsilateral hindlimb allodynia, hyperalgesia, and unweighting, but these pain behaviors were absent in MIA-treated muMT mice, indicating that cartilage injury-induced pain is B-cell dependent. IgM accumulation was observed in the knee tissues of MIA-treated mice, and intra-articular injection of IgM from MIA-treated mice into MIA-treated muMT mice caused nociceptive sensitization. Similarly, intra-articular injection of IgM from patients with OA was pronociceptive in muMT MIA mice and control subject IgM had no effect. Monosodium iodoacetate-injected joints demonstrate elevated levels of complement component 5a (C5a) and C5a receptor blockade using intra-articular PMX-53-reduced sensitization. These data suggest that MIA-treated mice and patients with OA generate pronociceptive antibodies, and further support the pronociceptive autoimmunity hypothesis for the transition from tissue injury to chronic musculoskeletal pain., (Copyright © 2024 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Introduction: Chronic pain is a common and problematic consequence of injuries with few proven methods for prevention or treatment. In addition to pain, functional limitations and neuropsychiatric changes such as cognitive impairment and anxiety worsen outcomes., Objectives: To determine whether inhibiting activation of the adaptive immune response after limb fracture would reduce pain, functional loss, memory changes, and anxiety., Methods: These experiments used a murine tibial fracture/cast immobilization model that develops these adverse outcomes. Adaptive immunity was blocked using the immunosuppressant FK506 beginning at the time of fracture., Results: The administration of FK506 reduced mechanical allodynia and hind limb unweighting for weeks after cast removal as well as nonevoked pain measures. Fracture was associated with working memory loss in the Y-maze assay in vehicle- but not FK506-treated mice. Object recognition memory was not improved with FK506 after fracture. Also, vehicle- but not FK506-treated mice developed an anxiety phenotype. Impaired running wheel performance after cast removal over the following 2 weeks was not improved with FK506 administration. In addition, FK506 treatment blocked Immunoglobulin M (IgM) accumulation in the skin of the fractured limbs, and hippocampal enhancement of matrix metalloproteinase-8 expression, a metalloproteinase associated with neuroplastic changes after injuries, was completely blocked., Conclusion: Taken together, our results show that blocking the adaptive immune response after limb trauma reduces the severity of nociceptive and biological changes. The same treatment may reduce the adverse consequences of anxiety and memory deficits using some measures, but other measures of memory are not affected, and activity is not enhanced., Competing Interests: The authors have no conflicts of interest to declare. This work was supported by Department of Defense award W81XWH2010911 (J.D.C.), R01NS094438 (W.S.K.), R01NS117340 and the VA Merit Review award I01RX001475 (J.D.C.). The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

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., Competing Interests: J.v.d.H. was employed at Delta Phenomics B.V., for which she was also involved as Study Director for the reported study. Currently she is employed at Danone Nutricia Research without any direct or indirect relation to the work reported here. The other authors have nothing to disclose., (Thieme. All rights reserved.)

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., (Copyright © 2022 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a “work of the United States Government” for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Published by Elsevier Inc.)

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., Competing Interests: The authors have no conflicts of interest to declare. This study was funded by NIH NINDS R01NS102836 to S.K. Ajit and NIH NINDS R01NS117340 and Department of Veterans Affairs I01RX001475 to J.D. Clark. The authors thank Dr. Bradley Nash for critical reading of the article.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.)

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., (Published by Elsevier Inc.)

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.

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 α 2 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 α 2 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., Competing Interests: Declaration of Competing Interest No competing financial interests exist., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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-HT 3 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-HT 3 receptor antagonists or other measures which rebalance descending pain modulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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.

Chronic pain is a common consequence of traumatic brain injury (TBI) that can increase the suffering of a patient and pose a significant challenge to rehabilitative efforts. Unfortunately, the mechanisms linking TBI to pain are poorly understood, and specific treatments for TBI-related pain are still lacking. Our laboratory has shown that TBI causes pain sensitization in areas distant to the site of primary injury, and that changes in spinal gene expression may underlie this sensitization. The aim of this study was to examine the roles that pain modulatory pathways descending from the brainstem play in pain after TBI. Deficiencies in one type of descending inhibition, diffuse noxious inhibitory control (DNIC), have been suggested to be responsible for the development of chronic pain by allowing excess and uncontrolled afferent nociceptive inputs. Here we expand our knowledge of pain after TBI in two ways: (1) by outlining the neuropathology in pain-related centers of the brain and spinal cord involved in DNIC using the rat lateral fluid percussion (LFP) model of TBI, and (2) by evaluating the effects of a potent histone acetyl transferase inhibitor, anacardic acid (AA), on LFP-induced pain behaviors and neuropathology when administered for several days after TBI. The results revealed that TBI induces transient mechanical allodynia and a chronic persistent loss of DNIC. Further, while short-term AA treatment can block acute nociceptive sensitization and some early neuropathological changes, this treatment neither prevented the loss of DNIC nor did it alter long-term neuropathological changes in the brain or spinal cord.

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.

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. In these studies we pursued the hypothesis that TBI pain sensitization is associated with histone acetylation in the rat lateral fluid percussion model. Some animals received hindpaw incisions in addition to TBI to mimic polytrauma. Neuropathological analysis of brain tissue from sham and TBI animals revealed evidence of bleeding, breakdown of the blood brain barrier, in the cortex, hippocampus, thalamus and other structures related to pain signal processing. Mechanical allodynia was measured in these animals for up to eight weeks post-injury. Inhibitors of histone acetyltransferase (HAT) and histone deacetylase (HDAC) were used to probe the role of histone acetylation in such pain processing. We followed serum markers including glial fibrillary acidic protein (GFAP), neuron-specific enolase 2 (NSE) myelin basic protein (MBP) and S100β to gauge TBI injury severity. Our results showed that TBI caused mechanical allodynia in the hindpaws of the rats lasting several weeks. Hindpaws contralateral to TBI showed more rapid and profound sensitization than ipsilateral hindpaws. The inhibition of HAT using curcumin 50 mg/kg s.c reduced mechanical sensitization while the HDAC inhibitor suberoylanilide hydroxamic acid 50 mg/kg i.p. prolonged sensitization in the TBI rats. Immunohistochemical analyses of spinal cord tissue localized changes in the level of acetylation of the H3K9 histone mark to dorsal horn neurons. Taken together, these findings demonstrate that TBI induces sustained nociceptive sensitization, and changes in spinal neuronal histone proteins may play an important role.