Persistent inflammatory pain is linked with anxiety-like behaviors, increased blood corticosterone, and reduced global DNA methylation in the rat amygdala.

Chronic pain increases the risk of developing anxiety, with limbic areas being likely neurological substrates. Despite high clinical relevance, little is known about the precise behavioral, hormonal, and brain neuroplastic correlates of anxiety in the context of persistent pain. Previous studies have shown that decreased nociceptive thresholds in chronic pain models are paralleled by anxiety-like behavior in rats, but there are conflicting ideas regarding its effects on the stress response and circulating corticosterone levels. Even less is known about the molecular mechanisms through which the brain encodes pain-related anxiety. This study examines how persistent inflammatory pain in a rat model would impact anxiety-like behaviors and corticosterone release, and whether these changes would be reflected in levels of global DNA methylation in brain areas involved in stress regulation. Complete Freund's adjuvant (CFA) or saline was administered in the right hindpaw of adult male Wistar rats. Behavioral testing included the measurement of nociceptive thresholds (digital anesthesiometer), motor function (open field test), and anxiety-like behaviors (elevated plus maze and the dark-light box test). Corticosterone was measured via radioimmunoassay. Global DNA methylation (enzyme immunoassay) as well as DNMT3a levels (western blotting) were quantified in the amygdala, prefrontal cortex, and ventral hippocampus. CFA administration resulted in persistent reduction in nociceptive threshold in the absence of locomotor abnormalities. Increased anxiety-like behaviors were observed in the elevated plus maze and were accompanied by increased blood corticosterone levels 10 days after pain induction. Global DNA methylation was decreased in the amygdala, with no changes in DNMT3a abundance in any of the regions examined. Persistent inflammatory pain promotes anxiety -like behaviors, HPA axis activation, and epigenetic regulation through DNA methylation in the amygdala. These findings describe a molecular mechanism that links pain and stress in a well-characterized rodent model.

The hippocampal extracellular matrix regulates pain and memory after injury.

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

Autoinflammatory and autoimmune contributions to complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a highly enigmatic syndrome typically developing after injury or surgery to a limb. Severe pain and disability are common among those with chronic forms of this condition. Accumulating evidence suggests that CRPS may involve both autoinflammatory and autoimmune components. In this review article, evidence for dysfunction of both the innate and adaptive immune systems in CRPS is presented. Findings from human studies in which cytokines and other inflammatory mediators were measured in the skin of affected limbs are discussed. Additional results from studies of mediator levels in animal models are evaluated in this context. Similarly, the evidence from human, animal, and translational studies of the production of autoantibodies and the potential targets of those antibodies is reviewed. Compelling evidence of autoinflammation in skin and muscle of the affected limb has been collected from CRPS patients and laboratory animals. Cytokines including IL-1ß, IL-6, TNFα, and others are reliably identified during the acute phases of the syndrome. More recently, autoimmune contributions have been suggested by the discovery of self-directed pain-promoting IgG and IgM antibodies in CRPS patients and model animals. Both the autoimmune and the autoinflammatory components of CRPS appear to be regulated by neuropeptide-containing peripheral nerve fibers and the sympathetic nervous system. While CRPS displays a complex neuroimmunological pathogenesis, therapeutic interventions could be designed targeting autoinflammation, autoimmunity, or the neural support for these phenomena.

Nonpharmacological Interventions in Targeting Pain-Related Brain Plasticity.

Chronic pain is a highly prevalent and debilitating condition that is frequently associated with multiple comorbid psychiatric conditions and functional, biochemical, and anatomical alterations in various brain centers. Due to its widespread and diverse manifestations, chronic pain is often resistant to classical pharmacological treatment paradigms, prompting the search for alternative treatment approaches that are safe and efficacious. The current review will focus on the following themes: attentional and cognitive interventions, the role of global environmental factors, and the effects of exercise and physical rehabilitation in both chronic pain patients and preclinical pain models. The manuscript will discuss not only the analgesic efficacy of these therapies, but also their ability to reverse pain-related brain neuroplasticity. Finally, we will discuss the potential mechanisms of action for each of the interventions.

Novel cytogenic and neurovascular niches due to blood-brain barrier compromise in the chronic pain brain.

BACKGROUND: The mechanisms by which painful injuries are linked to the multitude of pain-related comorbidities and neuroplastic changes in the brain remain poorly understood. Here we propose a model that relies on epi-neuronal communication through the vascular system to effect various brain structures. Specifically, we hypothesize that the differential vulnerability of the blood-brain barrier (BBB) in different brain regions is associated with region-specific neuroplastic and neurovascular changes that are in turn associated with particular pain-related comorbidities. PRESENTATION OF THE HYPOTHESIS: We will present our hypothesis by focusing on two main points: (A) chronic pain (CP) is associated with differential BBB compromise. (B) Circulating mediators leaking through the BBB create cytogenic and neovascular niches associated with pain-related co-morbidities. TESTING THE HYPOTHESIS: Pre-clinically, our hypothesis can be tested by observing, in parallel, BBB compromise, (neo)vascularization, neurogenesis, and their co-localization in animal pain models using imaging, microscopy, biochemical and other tools. Furthermore, the BBB can be experimentally damaged in specific brain regions, and the consequences of those lesions studied on nociception and associated comorbidities. Recently developed imaging techniques allow the analysis of blood brain barrier integrity in patients providing a route for translation of the laboratory findings. Though perhaps more limited, post-mortem examination of brains with available pain histories constitutes a second approach to addressing this hypothesis. IMPLICATIONS OF THE HYPOTHESIS: Understanding changes in BBB permeability in chronic pain conditions has clear implications both for understanding the pathogenesis of chronic pain and for the design of novel treatments to prevent chronic pain and its consequences. More broadly, this hypothesis may help us to understand how peripheral injuries impact the brain via mechanisms other than commonly studied efferent sensory pathways.

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.

Differential Efficacy of Ketamine in the Acute versus Chronic Stages of Complex Regional Pain Syndrome in Mice.

BACKGROUND: Complex regional pain syndrome (CRPS) is a painful, disabling, and often chronic condition, where many patients transition from an acute phase with prominent peripheral neurogenic inflammation to a chronic phase with evident central nervous system changes. Ketamine is a centrally acting agent believed to work through blockade of N-methyl-D- aspartate receptors and is being increasingly used for the treatment of refractory CRPS, although the basis for the drug's effects and efficacy at different stages of the syndrome remains unclear. METHODS: The authors used a mouse model of CRPS (n = 8 to 12/group) involving tibia fracture/cast immobilization to test the efficacy of ketamine (2 mg kg day; 7 days) or vehicle infusion during acute (3 weeks after fracture) and chronic (7 weeks after fracture) stages. RESULTS: Acute-phase fracture mice displayed increased limb temperature, edema, and nociceptive sensitization that were not reduced by ketamine. Fracture mice treated with ketamine during the chronic phase showed reduced nociceptive sensitization that persisted beyond completion of the infusion. During this chronic phase, ketamine also reduced latent nociceptive sensitization and improved motor function at 18 weeks after fracture. No side effects of the infusions were identified. These behavioral changes were associated with altered spinal astrocyte activation and expression of pain-related proteins including N-methyl-D-aspartate receptor 2b, Ca/calmodulin-dependent protein kinase II, and brain-derived neurotrophic factor. CONCLUSIONS: Collectively, these results demonstrate that ketamine is efficacious in the chronic, but not acute, stage of CRPS, suggesting that the centrally acting drug is relatively ineffective in early CRPS when peripheral mechanisms are more critical for supporting nociceptive sensitization.

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

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

Acute restraint stress regulates brain DNMT3a and promotes defensive behaviors in male rats.

Depending on its duration and severity, stress may contribute to neuropsychiatric diseases such as depression and anxiety. Studies have shown that stress impacts the hypothalamic-pituitary-adrenal (HPA) axis, but its downstream molecular, behavioral, and nociceptive effects remain unclear. We hypothesized that a 2-hour single exposure to acute restraint stress (ARS) activates the HPA axis and changes DNA methylation, a molecular mechanism involved in the machinery of stress regulation. We further hypothesized that ARS induces anxiety-like and risk assessment behavior and alters nociceptive responses in the rat. We employed biochemical (radioimmunoassay for corticosterone; global DNA methylation by enzyme immunoassay and western blot for DNMT3a expression in the amygdala, ventral hippocampus, and prefrontal cortex) and behavioral (elevated plus maze and dark-light box for anxiety and hot plate test for nociception) tests in adult male Wistar rats exposed to ARS or handling (control). All analyses were performed 24 h after ARS or handling. We found that ARS increased corticosterone levels in the blood, increased the expression of DNMT3a in the prefrontal cortex, promoted anxiety-like and risk assessment behaviors in the elevated plus maze, and increased the nociceptive threshold observed in the hot plate test. Our findings suggest that ARS might be a helpful rat model for studying acute stress and its effects on physiology, epigenetic machinery, and behavior.

Peripheral nerve injury is accompanied by chronic transcriptome-wide changes in the mouse prefrontal cortex.

BACKGROUND: Peripheral nerve injury can have long-term consequences including pain-related manifestations, such as hypersensitivity to cutaneous stimuli, as well as affective and cognitive disturbances, suggesting the involvement of supraspinal mechanisms. Changes in brain structure and cortical function associated with many chronic pain conditions have been reported in the prefrontal cortex (PFC). The PFC is implicated in pain-related co-morbidities such as depression, anxiety and impaired emotional decision-making ability. We recently reported that this region is subject to significant epigenetic reprogramming following peripheral nerve injury, and normalization of pain-related structural, functional and epigenetic abnormalities in the PFC are all associated with effective pain reduction. In this study, we used the Spared Nerve Injury (SNI) model of neuropathic pain to test the hypothesis that peripheral nerve injury triggers persistent long-lasting changes in gene expression in the PFC, which alter functional gene networks, thus providing a possible explanation for chronic pain associated behaviors. RESULTS: SNI or sham surgery where performed in male CD1 mice at three months of age. Six months after injury, we performed transcriptome-wide sequencing (RNAseq), which revealed 1147 differentially regulated transcripts in the PFC in nerve-injured vs. control mice. Changes in gene expression occurred across a number of functional gene clusters encoding cardinal biological processes as revealed by Ingenuity Pathway Analysis. Significantly altered biological processes included neurological disease, skeletal muscular disorders, behavior, and psychological disorders. Several of the changes detected by RNAseq were validated by RT-QPCR and included transcripts with known roles in chronic pain and/or neuronal plasticity including the NMDA receptor (glutamate receptor, ionotropic, NMDA; grin1), neurite outgrowth (roundabout 3; robo3), gliosis (glial fibrillary acidic protein; gfap), vesicular release (synaptotagmin 2; syt2), and neuronal excitability (voltage-gated sodium channel, type I; scn1a). CONCLUSIONS: This study used an unbiased approach to document long-term alterations in gene expression in the brain following peripheral nerve injury. We propose that these changes are maintained as a memory of an insult that is temporally and spatially distant from the initial injury.

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.

Rapamycin Augmentation of Chronic Ketamine as a Novel Treatment for Complex Regional Pain Syndrome.

This case report describes the dramatic clinical response of refractory chronic complex regional pain syndrome to combined immunomodulatory treatment. Ketamine and rapamycin markedly minimized pain historically associated with suicidal behavior, increased baseline activity, and allowed for a reduction in palliative polypharmacy. The piecewise mechanism of action is unclear given multiple postulated targets, such as microglia, astroglia, T-regulatory cells, B-regulatory cells, or neurons. Relevant laboratory and genetic information may allow the application of this treatment to other affected individuals.

Is there hemispheric specialization in the chronic pain brain?

Organismal bilateral symmetry is associated with near-identical halves of the central nervous system, with certain functions displaying specialization through one brain hemisphere. The processing of pain in the brain as well as brain plasticity in the context of painful injuries have garnered much attention in recent decades. Noninvasive brain imaging studies in pain-free human subjects have identified multiple brain regions that are linked to the sensory and affective components of pain. Longlasting adaptations in brains of chronic pain sufferers have likewise been described, suggesting a mechanism for pain chronification. Invasive molecular and biochemical studies in animal models have expanded on these findings, with added emphasis on the role of specific genes and molecules involved. To date, the extent of hemispheric asymmetry in the context of pain is not well-understood. This topical review evaluates the evidence of hemispheric specialization observed in humans and rodent models of pain and compares it to findings where such asymmetry is absent. Our review shows conflicting information regarding the existence of pain-related asymmetry, and if so, the side to which it can be localized. This could be due to the heterogeneity of pain processing pathways, heterogeneity in study parameters, as well as differences in data reporting. With the advent of progressively sophisticated non-invasive tools that can be used in human subjects, in addition to more precise methods to visualize and control specific brain regions or neuronal ensembles in animal models, we predict that the next few decades will witness a better understanding of the supraspinal control and processing of chronic pain, including the role of each of its hemispheres.

DNA methylation of SPARC and chronic low back pain.

BACKGROUND: The extracellular matrix protein SPARC (Secreted Protein, Acidic, Rich in Cysteine) has been linked to degeneration of the intervertebral discs and chronic low back pain (LBP). In humans, SPARC protein expression is decreased as a function of age and disc degeneration. In mice, inactivation of the SPARC gene results in the development of accelerated age-dependent disc degeneration concurrent with age-dependent behavioral signs of chronic LBP.DNA methylation is the covalent modification of DNA by addition of methyl moieties to cytosines in DNA. DNA methylation plays an important role in programming of gene expression, including in the dynamic regulation of changes in gene expression in response to aging and environmental signals. We tested the hypothesis that DNA methylation down-regulates SPARC expression in chronic LBP in pre-clinical models and in patients with chronic LBP. RESULTS: Our data shows that aging mice develop anatomical and behavioral signs of disc degeneration and back pain, decreased SPARC expression and increased methylation of the SPARC promoter. In parallel, we show that human subjects with back pain exhibit signs of disc degeneration and increased methylation of the SPARC promoter. Methylation of either the human or mouse SPARC promoter silences its activity in transient transfection assays. CONCLUSIONS: This study provides the first evidence that DNA methylation of a single gene plays a role in chronic pain in humans and animal models. This has important implications for understanding the mechanisms involved in chronic pain and for pain therapy.

Garments and Footwear for Chronic Pain.

In most human societies, wearing clothing and shoes, particularly in public settings, is commonplace and may even be legally required. Consequently, there is an abundance of clothing and footwear options for individuals of different ages, genders, body shapes and catering to different needs such as workwear and active-wear. However, many of the available options may not be viable for the millions and pain sufferers worldwide, indicating a need for adaptive apparel for the pain patient. In this perspective manuscript, we focus on the availability and efficacy of clothing designed to prevent pain in the general population as well as reduce or treat pain in pain patients. Furthermore, we put forth some considerations for the construction of adaptive garments. Such efforts and needed and could significantly improve well-being and quality of life in the pain patient.

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

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

Spinal matrix metalloproteinase 8 regulates pain after peripheral trauma.

It is well documented that pain chronification requires a host of plastic mechanisms at the spinal cord (SC) level, including alterations in neuronal and glial structure and function. Such cellular plasticity necessitates the existence of a plastic extracellular matrix (ECM). Here, we describe a key role for ECM remodeling in the regulation of chronic pain following peripheral injury. Three weeks following tibia fracture in mice, we show increased levels of MMP8 in the SC. Furthermore, we show that the pharmacological or genetic downregulation of MMP8 ameliorates the pain phenotype observed after injury. These results delineate an extracellular mechanism for pain chronification, thereby improving our mechanistic understanding of pain and providing novel therapeutic venues that go beyond targeting individual cell types.

Nociceptive and Cognitive Changes in a Murine Model of Polytrauma.

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

Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome.

OBJECTIVE: Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. METHODS: A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and CRPS human sera, to the recombinant protein. RESULTS: We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS patients showed increased autoantibody binding to recombinant kRT16 protein. CONCLUSIONS: Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.