Evidence of a genetic background predisposing to complex regional pain syndrome type 1.

BACKGROUND: Complex regional pain syndrome type 1 (CRPS-1) is a rare, disabling and sometimes chronic disorder usually arising after a trauma. This exploratory study examined whether patients with chronic CRPS-1 have a different genetic profile compared with those who do not have the condition. METHODS: Exome sequencing was performed to seek altered non-synonymous SNP allele frequencies in a discovery cohort of well-characterised patients with chronic CRPS-1 (n=34) compared with population databases. Identified SNP alleles were confirmed by Sanger sequencing and sought in a replication cohort (n=50). Gene expression of peripheral blood macrophages was assessed. RESULTS: In the discovery cohort, the rare allele frequencies of four non-synonymous SNPs were statistically increased. The replication cohort confirmed this finding. In a chronic pain cohort, these alleles were not overexpressed. In total, 25 out of 84 (29.8%) patients with CRPS-1 expressed a rare allele. The SNPs were rs41289586 in ANO10, rs28360457 in P2RX7, rs1126930 in PRKAG1 and rs80308281 in SLC12A9. Males were more likely than females to have a rare SNP allele, 8 out of 14 (57.1%) vs 17 out of 70 (24.3%) (Fisher's p=0.023). ANO10, P2RX7, PRKAG1 and SLC12A9 were all expressed in macrophages from healthy human controls. CONCLUSION: A single SNP in each of the genes ANO10, P2RX7, PRKAG1 and SLC12A9 was associated with developing chronic CRPS-1, with more males than females expressing these rare alleles. Our work suggests the possibility that a permissive genetic background is an important factor in the development of CRPS-1.

Identification of Potential Inflammation-Related Genes and Key Pathways Associated with Complex Regional Pain Syndrome.

Complex regional pain syndrome (CRPS) is a chronic pain that affects the extremities after a trauma or nerve injury with no definite established treatment. The mechanisms mediating CRPS are not completely elucidated. Thus, we conducted a bioinformatics analysis to identify hub genes and key pathways to determine strategies for more effective treatments of CRPS. Finally, there is only one expression profile of GSE47063 in terms of homo sapiens-based CRPS from the Gene Expression Omnibus (GEO) database, which included four patients and five controls. We explored the differentially expressed genes (DEGs) in the dataset and conducted Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the potential hub genes. A protein-protein interaction (PPI) network was also established; then, according to the score of each hub genes, we used R software to draw the nomogram model to predict the rate of CRPS. Furthermore, GSEA analysis was estimated and assessed by the normalized enrichment score (NES). From the GO and KEGG analysis, we identified the top five hub genes (MMP9, PTGS2, CXCL8, OSM, TLN1); all of the selected DEGs were mainly enriched in their inflammatory response. In addition, the GSEA analysis showed complement and coagulation cascades also play an important role in CRPS. This study, to our knowledge, is the first to conduct further PPI network and GSEA analyses. Thus, targeting excessive inflammation could offer new therapeutic methods for CRPS and related physical and psychiatric disorders.

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.

Hsa-miR-605 regulates the proinflammatory chemokine CXCL5 in complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.

Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENT The contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

Molecular Aspects of Regional Pain Syndrome.

The purpose of this review is to summarize the pathophysiology of complex regional pain syndrome (CRPS), the underlying molecular mechanisms, and potential treatment options for its management. CRPS is a multifactorial pain condition. CRPS is characterized by prolonged or excessive pain and changes in skin color and temperature, and/or swelling in the affected area, and is generally caused by stimuli that lead to tissue damage. An inflammatory response involving various cytokines and autoantibodies is generated in response to acute trauma/stress. Chronic phase pathophysiology is more complex, involving the central and peripheral nervous systems. Various genetic factors involved in the chronicity of pain have been identified in CRPS patients. As with other diseases of complex pathology, CRPS is difficult to treat and no single treatment regimen is the same for two patients. Stimulation of the vagus nerve is a promising technique being tested for different gastrointestinal and inflammatory diseases. CRPS is more frequent in individuals of 61-70 years of age with a female to male ratio of 3 : 1. Menopause, migraine, osteoporosis, and asthma all represent risk factors for CRPS and in smokers the prognosis appears to be more severe. The pathophysiological mechanisms underlying CRPS involve both inflammatory and neurological pathways. Understanding the molecular basis of CRPS is important for its diagnosis, management, and treatment. For instance, vagal nerve stimulation might have the potential for treating CRPS through the cholinergic anti-inflammatory pathway.

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.

What is normal trauma healing and what is complex regional pain syndrome I? An analysis of clinical and experimental biomarkers.

Complex regional pain syndrome (CRPS) typically develops after fracture or trauma. Many of the studies so far have analyzed clinical and molecular markers of CRPS in comparison with healthy or pain controls. This approach, however, neglects mechanisms occurring during physiological trauma recovery. Therefore, we compared the clinical phenotype, sensory profiles, patient-reported outcomes, and exosomal immunobarrier microRNAs (miRs) regulating barrier function and immune response between CRPS and fracture controls (FCs) not fulfilling the CRPS diagnostic criteria. We included upper-extremity FCs, acute CRPS I patients within 1 year after trauma, a second disease control group (painful diabetic polyneuropathy), and healthy controls. Fracture controls were not symptoms-free, but reported some pain, disability, anxiety, and cold pain hyperalgesia in quantitative sensory testing. Patients with CRPS had higher scores for pain, disability, and all patient-reported outcomes. In quantitative sensory testing, ipsilateral and contralateral sides differed significantly. However, on the affected side, patients with CRPS were more sensitive in only 3 parameters (pinprick pain and blunt pressure) when compared to FCs. Two principal components were identified in the cohort: pain and psychological parameters distinguishing FC and CPRS. Furthermore, the immunobarrier-protective hsa-miR-223-5p was increased in plasma exosomes in FCs with normal healing, but not in CRPS and healthy controls. Low hsa-miR-223-5p was particularly observed in subjects with edema pointing towards barrier breakdown. In summary, normal trauma healing includes some CRPS signs and symptoms. It is the combination of different factors that distinguish CRPS and FC. Fracture control as a control group can assist to discover resolution factors after trauma.

DNA methylation profiles are associated with complex regional pain syndrome after traumatic injury.

Factors contributing to development of complex regional pain syndrome (CRPS) are not fully understood. This study examined possible epigenetic mechanisms that may contribute to CRPS after traumatic injury. DNA methylation profiles were compared between individuals developing CRPS (n = 9) and those developing non-CRPS neuropathic pain (n = 38) after undergoing amputation following military trauma. Linear Models for Microarray (LIMMA) analyses revealed 48 differentially methylated cytosine-phosphate-guanine dinucleotide (CpG) sites between groups (unadjusted P's < 0.005), with the top gene COL11A1 meeting Bonferroni-adjusted P < 0.05. The second largest differential methylation was observed for the HLA-DRB6 gene, an immune-related gene linked previously to CRPS in a small gene expression study. For all but 7 of the significant CpG sites, the CRPS group was hypomethylated. Numerous functional Gene Ontology-Biological Process categories were significantly enriched (false discovery rate-adjusted q value <0.15), including multiple immune-related categories (eg, activation of immune response, immune system development, regulation of immune system processes, and antigen processing and presentation). Differentially methylated genes were more highly connected in human protein-protein networks than expected by chance (P < 0.05), supporting the biological relevance of the findings. Results were validated in an independent sample linking a DNA biobank with electronic health records (n = 126 CRPS phenotype, n = 19,768 non-CRPS chronic pain phenotype). Analyses using PrediXcan methodology indicated differences in the genetically determined component of gene expression in 7 of 48 genes identified in methylation analyses (P's < 0.02). Results suggest that immune- and inflammatory-related factors might confer risk of developing CRPS after traumatic injury. Validation findings demonstrate the potential of using electronic health records linked to DNA for genomic studies of CRPS.

Exosome microRNA signatures in patients with complex regional pain syndrome undergoing plasma exchange.

BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE.

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.

Analysis of gene expression profile microarray data in complex regional pain syndrome.

The aim of the present study was to predict key genes and proteins associated with complex regional pain syndrome (CRPS) using bioinformatics analysis. The gene expression profiling microarray data, GSE47603, which included peripheral blood samples from 4 patients with CRPS and 5 healthy controls, was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) in CRPS patients compared with healthy controls were identified using the GEO2R online tool. Functional enrichment analysis was then performed using The Database for Annotation Visualization and Integrated Discovery online tool. Protein­protein interaction (PPI) network analysis was subsequently performed using Search Tool for the Retrieval of Interaction Genes database and analyzed with Cytoscape software. A total of 257 DEGs were identified, including 243 upregulated genes and 14 downregulated ones. Genes in the human leukocyte antigen (HLA) family were most significantly differentially expressed. Enrichment analysis demonstrated that signaling pathways, including immune response, cell motion, adhesion and angiogenesis were associated with CRPS. PPI network analysis revealed that key genes, including early region 1A binding protein p300 (EP300), CREB­binding protein (CREBBP), signal transducer and activator of transcription (STAT)3, STAT5A and integrin α M were associated with CRPS. The results suggest that the immune response may therefore serve an important role in CRPS development. In addition, genes in the HLA family, such as HLA­DQB1 and HLA­DRB1, may present potential biomarkers for the diagnosis of CRPS. Furthermore, EP300, its paralog CREBBP, and the STAT family genes, STAT3 and STAT5 may be important in the development of CRPS.

Analysis of Common Single Nucleotide Polymorphisms in Complex Regional Pain Syndrome: Genome Wide Association Study Approach and Pooled DNA Strategy.

OBJECTIVE: The objective of this study was to use a genome-wide association (GWAS) approach and pooled DNA strategy to search for new genomic loci associated with complex regional pain syndrome (CRPS). DESIGN: The study cohort consisted of 230 patients with established diagnosis of CRPS. The control group consisted of 230 age- and gender-matched subjects without chronic pain. We tested the association of common single nucleotide polymorphisms (SNPs), genotyped using a high-density microarray platform, with CRPS phenotype. This was followed by individual genotyping of the most significant SNPs identified in the microarray genomic scan, in both original discovery (N = 115) and independent verification (N = 115) groups of patients with CRPS, as well as in the appropriate matched control subjects. RESULTS: The results of our study provide no support for the initial hypothesis of the existence of an association between any investigated genomic targets (including GWAS for all genomic loci available on the microarray, and focused scan of the HLA locus on chromosome 6) and CRPS phenotype. CONCLUSIONS: Despite the fact that we interrogated about 83% of all of common SNPs in the human genome, we did not find evidence that any of the investigated common SNPs may be associated with CRPS phenotype.

Hsa-miR-34a mediated repression of corticotrophin releasing hormone receptor 1 regulates pro-opiomelanocortin expression in patients with complex regional pain syndrome.

BACKGROUND: Ketamine provides relief for a subset of patients with complex regional pain syndrome (CRPS). The poor responders had a lower body mass index (BMI) relative to responders. Regulation of proopiomelanocortin (POMC) expression is crucial in normal body weight homeostasis. The main objectives of this study were to investigate the mechanisms underlying lower BMI characterizing CRPS patients responding poorly to intravenous ketamine therapy and identify potential biomarkers for predicting response. METHODS: We investigated POMC transcript levels in blood from CRPS patients grouped as responders and poor responders to ketamine therapy. Plasma levels of ß-endorphin, ACTH and α-MSH were measured by ELISA. We previously identified differential expression of small noncoding microRNA hsa-miR-34a in blood between responders and poor responders. We investigated whether a 11-fold downregulation of hsa-miR-34a in poor responders relative to responders is contributing to the differences in POMC levels by targeting POMC regulator CRHR1. Binding of miR-34a to CRHR1 was assessed using reporter assay; changes in mRNA and protein levels of CRHR1 were used to determine the regulation of CRHR1 by miR-34a. RNA from blood of CRPS and control subjects were used for quantitative PCR for CRHR1. RESULTS: Though ketamine treatment did not alter POMC expression, poor responders had higher levels of POMC mRNA than responders, both before and after treatment. Corticotropin-releasing hormone (CRH) is a key regulator of POMC expression and the biological effects are mediated through its receptor corticotrophin releasing hormone receptor 1 (CRHR1). We show that hsa-miR-34a is a negative regulator of CRHR1; overexpression of hsa-miR-34a in Jurkat cells resulted in reduction of CRH-mediated POMC expression. Poor responders had higher expression of CRHR1 transcripts than responders, indicating a regulatory role for miR-34a. In addition, we found positive correlations between the pretreatment levels of miR-34a to BMI and response to ketamine therapy. CONCLUSIONS: Our findings indicate a mechanism by which hsa-miR-34a can regulate the CRH/CRHR1/POMC axis and may influence BMI. Studies in larger patient cohorts are required to confirm the biomarker utility of circulating hsa-miR-34a levels in predicting treatment response to ketamine therapy.

Increased prevalence of two mitochondrial DNA polymorphisms in functional disease: Are we describing different parts of an energy-depleted elephant?

About 20% of the population suffers from "functional syndromes". Since these syndromes overlap greatly in terms of co-morbidity, pathophysiology (including aberrant autonomic activity) and treatment responses, common predisposing genetic factors have been postulated. We had previously showed that two common mitochondrial DNA (mtDNA) polymorphisms at positions 16519 and 3010 are statistically associated with the functional syndromes of migraine, cyclic vomiting syndrome and non-specific abdominal pain. Herein, among individuals with mtDNA haplogroup H (HgH), the presence of these two mtDNA polymorphisms were ascertained in additional functional syndromes: chronic fatigue syndrome, complex regional pain syndrome, sudden infant death syndrome, and major depressive disorder. Polymorphic prevalence rates were compared between disease and control groups, and within each disease group in participants with and without specific clinical findings. In all four conditions, one or both of the polymorphisms was significantly associated with the respective condition and/or co-morbid functional symptomatology. Thus, we conclude that these two mtDNA polymorphisms likely modify risk for the development of multiple functional syndromes, likely constituting a proportion of the postulated common genetic factor, at least among individuals with HgH. Pathophysiology likely involves broad effects on the autonomic nervous system.

Epigenetic modification of DRG neuronal gene expression subsequent to nerve injury: etiological contribution to complex regional pain syndromes (Part I).

DRG is of importance in relaying painful stimulation to the higher pain centers and therefore could be a crucial target for early intervention aimed at suppressing primary afferent stimulation. Complex regional pain syndrome (CRPS) is a common pain condition with an unknown etiology. Recently added new information enriches our understanding of CRPS pathophysiology. Researches on genetics, biogenic amines, neurotransmitters, and mechanisms of pain modulation, central sensitization, and autonomic functions in CRPS revealed various abnormalities indicating that multiple factors and mechanisms are involved in the pathogenesis of CRPS. Epigenetics refers to mitotically and meiotically heritable changes in gene expression that do not affect the DNA sequence. As epigenetic modifications potentially play an important role in inflammatory cytokine metabolism, neurotransmitter responsiveness, and analgesic sensitivity, they are likely key factors in the development of chronic pain. In this dyad review series, we systematically examine the nerve injury-related changes in the neurological system and their contribution to CRPS. In this part, we first reviewed and summarized the role of neural sensitization in DRG neurons in performing function in the context of pain processing. Particular emphasis is placed on the cellular and molecular changes after nerve injury as well as different models of inflammatory and neuropathic pain. These were considered as the potential molecular bases that underlie nerve injury-associated pathogenesis of CRPS.

Genome-wide expression profiling of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a chronic, progressive, and devastating pain syndrome characterized by spontaneous pain, hyperalgesia, allodynia, altered skin temperature, and motor dysfunction. Although previous gene expression profiling studies have been conducted in animal pain models, there genome-wide expression profiling in the whole blood of CRPS patients has not been reported yet. Here, we successfully identified certain pain-related genes through genome-wide expression profiling in the blood from CRPS patients. We found that 80 genes were differentially expressed between 4 CRPS patients (2 CRPS I and 2 CRPS II) and 5 controls (cut-off value: 1.5-fold change and p<0.05). Most of those genes were associated with signal transduction, developmental processes, cell structure and motility, and immunity and defense. The expression levels of major histocompatibility complex class I A subtype (HLA-A29.1), matrix metalloproteinase 9 (MMP9), alanine aminopeptidase N (ANPEP), l-histidine decarboxylase (HDC), granulocyte colony-stimulating factor 3 receptor (G-CSF3R), and signal transducer and activator of transcription 3 (STAT3) genes selected from the microarray were confirmed in 24 CRPS patients and 18 controls by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We focused on the MMP9 gene that, by qRT-PCR, showed a statistically significant difference in expression in CRPS patients compared to controls with the highest relative fold change (4.0±1.23 times and p = 1.4×10(-4)). The up-regulation of MMP9 gene in the blood may be related to the pain progression in CRPS patients. Our findings, which offer a valuable contribution to the understanding of the differential gene expression in CRPS may help in the understanding of the pathophysiology of CRPS pain progression.

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.

Genetic HLA associations in complex regional pain syndrome with and without dystonia.

UNLABELLED: We previously showed evidence for a genetic association of the human leukocyte antigen (HLA) system and complex regional pain syndrome (CRPS) with dystonia. Involvement of the HLA system suggests that CRPS has a genetic component with perturbed regulation of inflammation and neuroplasticity as possible disease mechanisms. However, it is at present unclear whether the observed association with HLA-B62 and HLA-DQ8 in CRPS patients with dystonia also holds true for patients without dystonia. Therefore, we tested the possible association with HLA-B62 and HLA-DQ8 in a clinically homogeneous group of 131 CRPS patients without dystonia. In addition, we investigated the possible association with other alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ loci. We showed an increased prevalence of HLA-DQ8 (molecularly typed as HLA-DQB1*03:02; OR = 1.65 [95% CI 1.12-2.42], P = .014) in CRPS without dystonia, whereas no association was observed for HLA-B62 (molecularly typed as HLA-B*15:01; OR = 1.22 [95% CI .78-1.92], P = .458). Our data suggest that CRPS with and CRPS without dystonia may be genetically different, but overlapping, disease entities because only HLA-DQ8 is associated with both. The findings also indicate that distinct biological pathways may play a role in both CRPS subtypes. PERSPECTIVE: This study is the first to replicate a specific HLA region conferring genetic risk for the development of CRPS. Moreover, associations of HLA-DQ8 with both CRPS with and CRPS without dystonia, and HLA-B62 only with CRPS with dystonia, suggest that these disease entities may be genetically different, but overlapping.