Dimethyl Fumarate Reduces Oxidative Stress and Pronociceptive Immune Responses in a Murine Model of Complex Regional Pain Syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works through the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional, and immunological changes measured in a model of CRPS. METHODS: Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25 mg/kg/d orally, per os for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments and hindlimb weight-bearing data were collected. The markers of oxidative stress malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1ß and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wildtype fractured mice to B cell-deficient fractured muMT mice (mice lacking B cells and immunoglobulin) helped to assess the pronociceptive activity of humoral factors. RESULTS: We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy, and the accumulation of IgM in fractured limbs. The sera of fractured vehicle-treated but not DMF-treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice. CONCLUSIONS: Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation.

Commonalities and differences in abnormal peripheral metabolites between patients with fibromyalgia and complex regional pain syndrome.

Background: Fibromyalgia (FM) and complex regional pain syndrome (CRPS) share many pathological mechanisms related to chronic pain that could contribute to multifactorial pathological mechanisms.Methods: We investigated peripheral metabolites in FM and CRPS patients compared to healthy controls based on cross-sectional study.Results: Mean corpuscular hemoglobin (p < 0.001), mean corpuscular volume (p = 0.014), and total bilirubin levels (p = 0.017) were lower in FM patients than in healthy controls. On the other hand, CRPS patients showed lower levels of total bilirubin than healthy controls (p = 0.037). Creatinine level was lower in FM patients (p = 0.057) compared to healthy controls, particularly when comparing the low-hemoglobin subgroup among FM patients (p = 0.035) with the low-hemoglobin subgroup among healthy controls. Red blood cell count (r = -0.620, p = 0.031), hematocrit (r = -0.593, p = 0.042), and creatinine level (r = -0.598, p = 0.040) showed negative correlations with McGill Pain Questionnaire-Affective (MPQ-A) scores in FM patients. A negative correlation was observed between MCV and McGill Pain Questionnaire-Sensory scores (r = -0.680, p = 0.015) in CRPS patients.Conclusion: We found specific peripheral metabolites that may exhibit different tendency between FM and CRPS patients as well as some common metabolites, which may be associated with peripheral pathology in the patients. Considering this study had a few limitations such as a small sample sizes and using a liberal threshold of significance in the correlation analysis, future studies with larger sample sizes may be needed to generalize these findings.

Unilateral L4-dorsal root ganglion stimulation evokes pain relief in chronic neuropathic postsurgical knee pain and changes of inflammatory markers: part II whole transcriptome profiling.

BACKGROUND: In our recent clinical trial, increased peripheral concentrations of pro-inflammatory molecular mediators were determined in complex regional pain syndrome (CRPS) patients. After 3 months adjunctive unilateral, selective L4 dorsal root ganglion stimulation (L4-DRGSTIM), significantly decreased serum IL-10 and increased saliva oxytocin levels were assessed along with an improved pain and functional state. The current study extended molecular profiling towards gene expression analysis of genes known to be involved in the gonadotropin releasing hormone receptor and neuroinflammatory (cytokines/chemokines) signaling pathways. METHODS: Blood samples were collected from 12 CRPS patients for whole-transcriptome profiling in order to assay 18,845 inflammation-associated genes from frozen blood at baseline and after 3 months L4-DRGSTIM using PANTHER™ pathway enrichment analysis tool. RESULTS: Pathway enrichment analyses tools (GOrilla™ and PANTHER™) showed predominant involvement of inflammation mediated by chemokines/cytokines and gonadotropin releasing hormone receptor pathways. Further, screening of differentially regulated genes showed changes in innate immune response related genes. Transcriptomic analysis showed that 21 genes (predominantly immunoinflammatory) were significantly changed after L4-DRGSTIM. Seven genes including TLR1, FFAR2, IL1RAP, ILRN, C5, PKB and IL18 were down regulated and fourteen genes including CXCL2, CCL11, IL36G, CRP, SCGB1A1, IL-17F, TNFRSF4, PLA2G2A, CREB3L3, ADAMTS12, IL1F10, NOX1, CHIA and BDKRB1 were upregulated. CONCLUSIONS: In our sub-group analysis of L4-DRGSTIM treated CRPS patients, we found either upregulated or downregulated genes involved in immunoinflammatory circuits relevant for the pathophysiology of CRPS indicating a possible relation. However, large biobank-based approaches are recommended to establish genetic phenotyping as a quantitative outcome measure in CRPS patients. Trial registration The study protocol was registered at the 15.11.2016 on German Register for Clinical Trials (DRKS ID 00011267). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00011267.

Disruption of Homeostasis Based on the Right and Left Hemisphere in Patients with Complex Regional Pain Syndrome.

OBJECTIVE: Although the clinical features and pathophysiology of complex regional pain syndrome (CRPS) have been studied in the peripheral and central nervous systems, few plausible pathological interactions are known among the metabolites in these systems. Thus, the purpose of this study was to investigate abnormal relationships and interactions between peripheral metabolites and central neurometabolites in patients with CRPS. METHODS: Various metabolites and molecules were measured in the peripheral blood, and central neurometabolites in the right and left thalamus using proton magnetic resonance spectroscopy in 12 patients with CRPS and 11 healthy controls. Interactions between peripheral metabolites in blood and central neurometabolites in the right and left thalamus were also investigated. RESULTS: The interactions between peripheral and central metabolites were different in the right and left hemispheres of healthy subjects, suggesting the presence of right hemisphere-dependent energy homeostasis and left hemisphere-dependent acid-base homeostasis that enables effective functioning. The interactions between central and peripheral metabolites in CRPS patients were distinct from those in healthy individuals, supporting the possibility of abnormal interactions and disrupted homeostasis between peripheral and central metabolites, which may result from neuroinflammation and immune system dysfunction. CONCLUSION: To the authors' knowledge, this is the first report describing abnormal metabolic dysfunction and disrupted homeostasis in interactions between metabolites of the peripheral and central nervous systems in CRPS. The approach used to uncover hidden pathophysiologies will improve understanding of how chronic pain can disrupt homeostasis in interactions between two systems and how alternative metabolites can be activated to recover and compensate for pathological dysfunctions in patients with CRPS.

Brain Metabolites and Peripheral Biomarkers Associated with Neuroinflammation in Complex Regional Pain Syndrome Using [11C]-(R)-PK11195 Positron Emission Tomography and Magnetic Resonance Spectroscopy: A Pilot Study.

Ojjective: The aim of this study was to find peripheral biomarkers and central metabolites affecting neuroinflammation in complex regional pain syndrome (CRPS) patients using [11C]-(R)-PK11195 positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). Methods: Using MRS and PET, we measured associations between neurometabolites and neuroinflammation in 12 CRPS patients and 11 healthy controls. Also, we investigated various peripheral parameters that may affect neuroinflammation in CRPS. Results: We found positive correlations of Lipid (Lip)13a/total creatine (tCr) and Lip09/tCr with neuroinflammation, the distribution volume ratio (DVR) of [11C]-(R)-PK11195 in the right and left insula in CRPS patients. However, these correlations were not found in controls. High hemoglobin levels correlated with decreased neuroinflammation (the DVR of [11C]-(R)-PK11195) in the right thalamus and left insula in healthy controls. We found that high levels of glucose and pH correlated with increased neuroinflammation, but high levels of CO2, basophil, and creatinine were associated with decreased neuroinflammation in the left thalamus and the right and left insula in CRPS patients. Conclusions: This is the first report indicating that elevated neuroinflammation levels are associated primarily with lipids in the brain and pH, glucose, CO2, basophil, and creatinine in the peripheral parameters in CRPS patients. Our results suggest that characterizing the peripheral biomarkers and central metabolites affecting neuroinflammation is essential to understanding the pathophysiology of CRPS.

Electroacupuncture induces antihyperalgesic effect through endothelin-B receptor in the chronic phase of a mouse model of complex regional pain syndrome type I.

Complex regional pain syndrome (CRPS) is a disorder that involves abnormal inflammation and nerve dysfunction frequently resistant to a broad range of treatments. Peripheral nerve stimulation with electroacupuncture (EA) has been widely used in different clinical conditions to control pain and inflammation; however, the use of EA in the treatment of CRPS is under investigation. In this study, we explore the effects of EA on hyperalgesia and edema induced in an animal model of chronic post-ischemia pain (CPIP model) and the possible involvement of endothelin receptor type B (ETB) in this effect. Female Swiss mice were subjected to 3 h hind paw ischemia/reperfusion CPIP model. EA treatment produced time-dependent inhibition of mechanical and cold hyperalgesia, as well as edema in CPIP mice. Peripheral administration (i.pl.) of BQ-788 (10 nmol), an ETB antagonist, prevented EA-induced antihyperalgesia while intrathecal administration prolonged EA's effect. Additionally, peripheral pre-treatment with sarafotoxin (SRTX S6c, 30 pmol, ETB agonist) increased EA anti-hyperalgesic effect. Furthermore, the expression of peripheral ETB receptors was increased after EA treatments, as measured by western blot. These results may suggest that EA's analgesic effect is synergic with ETB receptor activation in the periphery, as well as central (spinal cord) ETB receptor blockade. These data support the use of EA as a nonpharmacological approach for the management of CRPS-I, in an adjuvant manner to ETB receptor targeting drugs.

Aberrant interactions of peripheral measures and neurometabolites with lipids in complex regional pain syndrome using magnetic resonance spectroscopy: A pilot study.

Background The aim of this study was to assess peripheral measures and central metabolites associated with lipids using magnetic resonance spectroscopy. Results Twelve patients with complex regional pain syndrome (CRPS) and 11 healthy controls participated. Using magnetic resonance spectroscopy, we measured the levels of lipid 13a (Lip13a) and lipid 09 (Lip09) relative to total creatine (tCr) levels in the right and left thalamus. We found negative correlations of Lip13a/tCr in the right thalamus with red blood cells or neutrophils, but a positive correlation between Lip13a/tCr and lymphocytes in the controls. We found negative correlations between Lip09/tCr and peripheral pH or platelets in the controls. There were positive correlations between Lip09a/tCr and myo-inositol/tCr, between Lip13a/tCr and N-acetylaspartate (NAA)/tCr, and between Lip09/tCr and NAA/tCr in healthy controls. On the other hand, there were positive correlations between Lip13a/tCr and Lip09/tCr and urine pH in CRPS patients. There were significant correlations between Lip13a/tCr or Lip09/tCr and different peripheral measures depending on the side of the thalamus (right or left) in CRPS patients. Conclusion This is the first report indicating that abnormal interactions of Lip13a and Lip09 in the thalamus with peripheral measures and central metabolites may mediate the complex pathophysiological mechanisms underlying CRPS.

Neuropeptide regulation of adaptive immunity in the tibia fracture model of complex regional pain syndrome.

BACKGROUND: Both dysfunctional neuropeptide signaling and immune system activation are characteristic of complex regional pain syndrome (CRPS). Unknown is whether substance P (SP) or calcitonin gene-related peptide (CGRP) support autoantibody production and, consequently, nociceptive sensitization. METHODS: These experiments involved the use of a well-characterized tibia fracture model of CRPS. Mice deficient in SP expression (Tac1-/-) and CGRP signaling (RAMP1-/-) were used to probe the neuropeptide dependence of post-fracture sensitization and antibody production. The deposition of IgM in the spinal cord, sciatic nerves, and skin was followed using Western blotting, as was expression of the CRPS-related autoantigen cytokeratin 16 (Krt16). Passive serum transfer to B-cell-deficient muMT mice was used to assess the production of functional autoantibodies in CRPS model mice. The use of immunohistochemistry allowed us to assess neuropeptide-containing fiber distribution and Langerhans cell abundance in mouse and human CRPS patient skin, while Langerhans cell-deficient mice were used to assess the functional contributions of these cells. RESULTS: Functional SP and CGRP signaling were required both for the full development of nociceptive sensitization after fracture and the deposition of IgM in skin and neural tissues. Furthermore, the passive transfer of serum from wildtype but not neuropeptide-deficient mice to fractured muMT mice caused enhanced allodynia and postural unweighting. Langerhans cells were increased in number in the skin of fracture mice and CRPS patients, and those increases in mice were reduced in neuropeptide signaling-deficient animals. Unexpectedly, Langerhans cell-deficient mice showed normal nociceptive sensitization after fracture. However, the increased expression of Krt16 after tibia fracture was not seen in neuropeptide-deficient mice. CONCLUSIONS: Collectively, these data support the hypothesis that neuropeptide signaling in the fracture limb of mice is required for autoantigenic IgM production and nociceptive sensitization. The mechanism may be related to neuropeptide-supported autoantigen expression.

Molecular signature of complex regional pain syndrome (CRPS) and its analysis.

INTRODUCTION: Complex Regional Pain Syndrome (CRPS) is a rare, but often disabling pain disease. Biomarkers are lacking, but several inflammatory substances have been associated with the pathophysiology. This review outlines the current knowledge with respect to target biomolecules and the analytical tools available to measure them. Areas covered: Targets include cytokines, neuropeptides and resolvins; analysis strategies are thus needed for different classes of substances such as proteins, peptides, lipids and small molecules. Traditional methods like immunoassays are of importance next to state-of-the art high-resolution mass spectrometry techniques and 'omics' approaches. Expert commentary: Future biomarker studies need larger cohorts, which improve subgrouping of patients due to their presumed pathophysiology, and highly standardized workflows from sampling to analysis.

Acute versus chronic phase mechanisms in a rat model of CRPS.

BACKGROUND: Tibia fracture followed by cast immobilization in rats evokes nociceptive, vascular, epidermal, and bone changes resembling complex regional pain syndrome (CRPS). In most cases, CRPS has three stages. Over time, this acute picture, allodynia, warmth, and edema observed at 4 weeks, gives way to a cold, dystrophic but still painful limb. In the acute phase (at 4 weeks post fracture), cutaneous immunological and NK1-receptor signaling mechanisms underlying CRPS have been discovered; however, the mechanisms responsible for the chronic phase are still unknown. The purpose of this study is to understand the mechanisms responsible for the chronic phases of CRPS (at 16 weeks post fracture) at both the peripheral and central levels. METHODS: We used rat tibial fracture/cast immobilization model of CRPS to study molecular, vascular, and nociceptive changes at 4 and 16 weeks post fracture. Immunoassays and Western blotting were carried out to monitor changes in inflammatory response and NK1-receptor signaling in the skin and spinal cord. Skin temperature and thickness were measured to elucidate vascular changes, whereas von Frey testing and unweighting were carried out to study nociceptive changes. All data were analyzed by one-way analysis of variance (ANOVA) followed by Neuman-Keuls multiple comparison test to compare among all cohorts. RESULTS: In the acute phase (at 4 weeks post fracture), hindpaw allodynia, unweighting, warmth, edema, and/or epidermal thickening were observed among 90 % fracture rats, though by 16 weeks (chronic phase), only the nociceptive changes persisted. The expression of the neuropeptide signaling molecule substance P (SP), NK1 receptor, inflammatory mediators TNFα, IL-1ß, and IL-6 and nerve growth factor (NGF) were elevated at 4 weeks in sciatic nerve and/or skin, returning to normal levels by 16 weeks post fracture. The systemic administration of a peripherally restricted IL-1 receptor antagonist (anakinra) or of anti-NGF inhibited nociceptive behaviors at 4 weeks but not 16 weeks. However, spinal levels of NK1 receptor, TNFα, IL-1ß, and NGF were elevated at 4 and 16 weeks, and intrathecal injection of an NK1-receptor antagonist (LY303870), anakinra, or anti-NGF each reduced nociceptive behaviors at both 4 and 16 weeks. CONCLUSIONS: These results demonstrate that tibia fracture and immobilization cause peripheral changes in neuropeptide signaling and inflammatory mediator production acutely, but central spinal changes may be more important for the persistent nociceptive changes in this CRPS model.

Bisphosphonates Inhibit Pain, Bone Loss, and Inflammation in a Rat Tibia Fracture Model of Complex Regional Pain Syndrome.

BACKGROUND: Bisphosphonates are used to prevent the bone loss and fractures associated with osteoporosis, bone metastases, multiple myeloma, and osteogenesis deformans. Distal limb fractures cause regional bone loss with cutaneous inflammation and pain in the injured limb that can develop into complex regional pain syndrome (CRPS). Clinical trials have reported that antiresorptive bisphosphonates can prevent fracture-induced bone loss, inhibit serum inflammatory cytokine levels, and alleviate CRPS pain. Previously, we observed that the inhibition of inflammatory cytokines or adaptive immune responses attenuated the development of pain behavior in a rat fracture model of CRPS, and we hypothesized that bisphosphonates could prevent pain behavior, trabecular bone loss, postfracture cutaneous cytokine upregulation, and adaptive immune responses in this CRPS model. METHODS: Rats underwent tibia fracture and cast immobilization for 4 weeks and were chronically administered either subcutaneously perfused alendronate or oral zoledronate. Behavioral measurements included hindpaw von Frey allodynia, unweighting, warmth, and edema. Bone microarchitecture was measured by microcomputed tomography, and bone cellular activity was evaluated by static and dynamic histomorphometry. Spinal cord Fos immunostaining was performed, and skin cytokine (tumor necrosis factor, interleukin [IL]-1, IL-6) and nerve growth factor (NGF) levels were determined by enzyme immunoassay. Skin and sciatic nerve immunoglobulin levels were determined by enzyme immunoassay. RESULTS: Rats with tibia fractures developed hindpaw allodynia, unweighting, warmth, and edema, increased spinal Fos expression and trabecular bone loss in the lumbar vertebra and bilateral distal femurs as measured by microcomputed tomography, increased trabecular bone resorption and osteoclast surface with decreased bone formation rates, increased cutaneous inflammatory cytokine and NGF expression, and elevated immunocomplex deposition in skin and nerve. Alendronate (60 µg/kg/d subcutaneously [s.c.]) or zoledronate (3 mg/kg/d orally) treatment for 28 days, started at the time of fracture, completely inhibited the development of hindpaw allodynia and reduced hindpaw unweighting by 44% ± 13% and 58% ± 5%, respectively. Orally administered zoledronate (3 mg/kg/d for 21 days) treatment also completely reversed established allodynia and unweighting when started at 4 weeks postfracture. Histomorphometric and microcomputed tomography analysis demonstrated that both the 3 and 60 µg/kg/d alendronate treatments reversed trabecular bone loss (an 88% ± 25% and 188% ± 39% increase in the ipsilateral distal femur BV/TV, respectively) and blocked the increase in osteoclast numbers and erosion surface observed in bilateral distal femurs and in L5 vertebra of the fracture rats. Alendronate treatment inhibited fracture-induced increases in hindpaw inflammatory mediators, reducing postfracture levels of tumor necrosis factor by 43% ± 9%, IL-1 by 60% ± 9%, IL-6 by 56% ± 14%, and NGF by 37% ± 14%, but had no effect on increased spinal cord Fos expression, or skin and sciatic nerve immunocomplex deposition. CONCLUSIONS: Collectively, these results indicate that bisphosphonate therapy inhibits pain, osteoclast activation, trabecular bone loss, and cutaneous inflammation in the rat fracture model of CRPS, data supporting the hypothesis that bisphosphonate therapy can provide effective multimodal treatment for CRPS.

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

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

Remission of hypersensitivity by simple weight load stimuli in a complex regional pain syndrome mouse model.

Painful sensitivity of the hand or foot are the most common and debilitating symptoms of complex regional pain syndrome (CRPS). Physical therapy is standard treatment for CRPS, but evidence supporting its efficacy is minimal and it can be essentially impossible for CRPS patients to actively exercise the painful limb. Using the well-characterized distal tibial fracture CRPS mouse model, we compared the therapeutic effects of several weeks of daily hindlimb loading versus rotarod walking exercise. The effects of loading and exercise were evaluated by weekly testing of hind-paw withdrawal thresholds to von Frey fibers and radiant heat, as well as measurements of paw and ankle edema. At 6 weeks after fracture, the mice were killed and the ipsilateral femur, spinal cord and L4/5 dorsal root ganglia, and hind-paw skin collected for PCR assays and paw skin Immunohistochemistry evaluation. Hindlimb loading reduced hind-paw von Frey allodynia and heat hyperalgesia and edema within a week and these effects persisted for at least a week after discontinuing treatment. These therapeutic effects of loading exceeded the beneficial effects observed with rotarod walking exercise in fracture mice. Levels of nerve growth factor and transient receptor potential vanilloid 1 (TRPV1) immunostaining in the hind-paw skin were increased at 6 weeks after fracture, and both loading and exercise treatment reduced increases. Collectively, these results suggest that loading may be an effective and possibly curative treatment in CRPS patients with sensitivity in the affected limb.

Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord.

BACKGROUND: CRPS is a painful, debilitating, and often-chronic condition characterized by various sensory, motor, and vascular disturbances. Despite many years of study, current treatments are limited by our understanding of the underlying mechanisms. Little is known on the molecular level concerning changes in gene expression supporting the nociceptive sensitization commonly observed in CRPS limbs, or how those changes might evolve over time. RESULTS: We used a well-characterized mouse tibial fracture/cast immobilization model of CRPS to study molecular, vascular and nociceptive changes. We observed that the acute (3 weeks after fracture) and chronic (7 weeks after fracture) phases of CRPS-like changes in our model were accompanied by unique alterations in spinal gene expression corresponding to distinct canonical pathways. For the acute phase, top regulated pathways were: chemokine signaling, glycogen degradation, and cAMP-mediated signaling; while for the chronic phase, the associated pathways were: coagulation system, granzyme A signaling, and aryl hydrocarbon receptor signaling. We then focused on the role of CcL2, a chemokine that we showed to be upregulated at the mRNA and protein levels in spinal cord tissue in our model. We confirmed its association with the nociceptive sensitization displayed in this model by demonstrating that the spinal but not peripheral administration of a CCR2 antagonist (RS504393) in CRPS animals could decrease mechanical allodynia. The spinal administration of CcL2 itself resulted in mechanical allodynia in control mice. CONCLUSIONS: Our data provide a global look at the transcriptional changes in the spinal cord that accompany the acute and chronic phases of CRPS as modeled in mice. Furthermore, it follows up on one of the top-regulated genes coding for CcL2 and validates its role in regulating nociception in the fracture/cast model of CRPS.

Inflammatory processes in complex regional pain syndromes.

Chronic Regional Pain Syndrome, previously known as reflex sympathetic dystrophy, has garnered increased attention in recent years. Many recent studies have attempted to better describe the etiology, pathogenesis, and treatment of symptoms. Despite recent advances to the fund of medical knowledge, the underlying pathophysiology and mechanisms involved in disease progression remain unclear. This review will synthesize and present current theories and data regarding the role of inflammatory cytokines and other mediators in both the central and peripheral nervous systems that contribute to the development and progression of CRPS.

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.

Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome.

BACKGROUND: Distal limb fracture in man can induce a complex regional pain syndrome (CRPS) with pain, warmth, edema, and cutaneous inflammation. In the present study substance P (SP, Tac1(-/-)) and CGRP receptor (RAMP1(-/-)) deficient mice were used to investigate the contribution of neuropeptide signaling to CRPS-like changes in a tibia fracture mouse model. Wildtype, Tac1(-/-), and RAMP1(-/-) mice underwent tibia fracture and casting for 3 weeks, then the cast was removed and hindpaw mechanical allodynia, unweighting, warmth, and edema were tested over time. Hindpaw skin was collected at 3 weeks post-fracture for immunoassay and femurs were collected for micro-CT analysis. RESULTS: Wildtype mice developed hindpaw allodynia, unweighting, warmth, and edema at 3 weeks post-fracture, but in the Tac1(-/-) fracture mice allodynia and unweighting were attenuated and there was no warmth and edema. RAMP1(-/-) fracture mice had a similar presentation, except there was no reduction in hindpaw edema. Hindpaw skin TNFα, IL-1ß, IL-6 and NGF levels were up-regulated in wildtype fracture mice at 3 weeks post-fracture, but in the Tac1(-/-) and RAMP1(-/-) fracture mice only IL-6 was increased. The epidermal keratinocytes were the cellular source for these inflammatory mediators. An IL-6 receptor antagonist partially reversed post-fracture pain behaviors in wildtype mice. CONCLUSIONS: In conclusion, both SP and CGRP are critical neuropeptide mediators for the pain behaviors, vascular abnormalities, and up-regulated innate immune responses observed in the fracture hindlimb. We postulate that the residual pain behaviors observed in the Tac1(-/-) and RAMP1(-/-) fracture mice are attributable to the increased IL-6 levels observed in the hindpaw skin after fracture.

Substance P signaling controls mast cell activation, degranulation, and nociceptive sensitization in a rat fracture model of complex regional pain syndrome.

BACKGROUND: Patients with complex regional pain syndrome have increased tryptase in the skin of the affected extremity indicating mast cell (MC) accumulation and degranulation, processes known to be mediated by substance P (SP). The dysregulation of SP release from primary afferent neurons is characteristic of complex regional pain syndrome. The authors hypothesized that SP acting through the neurokinin-1 receptor results in mast cell accumulation, degranulation, and nociceptive sensitization in a rat model of complex regional pain syndrome. METHODS: Groups of 6-10 rats underwent tibia fracture and hind limb casting for 4 weeks, and the hind paw skin was harvested for histologic and immunohistochemical analysis. The effects of a selective neurokinin-1 receptor antagonist (LY303870) and of direct SP intraplantar injection were measured. Dermal MC degranulation induced by sciatic nerve stimulation and the effects of LY303870 on this process were investigated. Finally, the antinociceptive effects of acute and chronic treatment with a MC degranulator (48/80) were tested. RESULTS: The authors observed that fracture caused MC accumulation, activation, and degranulation, which were inhibited by LY303870; the percentage of MCs in close proximity to peptidergic nerve fibers increased after fracture; electrical stimulation caused MC activation and degranulation, which was blocked by LY303870; intraplantar SP-induced MC degranulation and acute administration of 48/80 caused MC degranulation and enhanced postfracture nociception, but MC-depleted animals showed less sensitization. CONCLUSIONS: These results indicate that facilitated peptidergic neuron-MC signaling after fracture can cause MC accumulation, activation, and degranulation in the injured limb, resulting in nociceptive sensitization.

Microvascular Barrier Protection by microRNA-183 via FoxO1 Repression: A Pathway Disturbed in Neuropathy and Complex Regional Pain Syndrome.

Blood nerve barrier disruption and edema are common in neuropathic pain as well as in complex regional pain syndrome (CRPS). MicroRNAs (miRNA) are epigenetic multitarget switches controlling neuronal and non-neuronal cells in pain. The miR-183 complex attenuates hyperexcitability in nociceptors, but additional non-neuronal effects via transcription factors could contribute as well. This study explored exosomal miR-183 in CRPS and murine neuropathy, its effect on the microvascular barrier via transcription factor FoxO1 and tight junction protein claudin-5, and its antihyperalgesic potential. Sciatic miR-183 decreased after CCI. Substitution with perineural miR-183 mimic attenuated mechanical hypersensitivity and restored blood nerve barrier function. In vitro, serum from CCI mice und CRPS patients weakened the microvascular barrier of murine cerebellar endothelial cells, increased active FoxO1 and reduced claudin-5, concomitant with a lack of exosomal miR-183 in CRPS patients. Cellular stress also compromised the microvascular barrier which was rescued either by miR-183 mimic via FoxO1 repression or by prior silencing of Foxo1. PERSPECTIVE: Low miR-183 leading to barrier impairment via FoxO1 and subsequent claudin-5 suppression is a new aspect in the pathophysiology of CRPS and neuropathic pain. This pathway might help untangle the wide symptomatic range of CRPS and nurture further research into miRNA mimics or FoxO1 inhibitors.