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

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Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord
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  This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formattedPDF and full text (HTML) versions will be made available soon. Acute and chronic phases of complex regional pain syndrome in mice areaccompanied by distinct transcriptional changes in the spinal cord Molecular Pain   2013,  9 :40 doi:10.1186/1744-8069-9-40Joseph J Gallagher ( jjg1@stanford.edu)Maral Tajerian (maral@stanford.edu)Tianzhi Guo (wxtguo@yahoo.com)Xiaoyou Shi (xyshi@stanford.edu)Wenwu Li (wenwuli@stanford.edu)Ming Zheng (mzheng@stanford.edu)Gary Peltz (gpeltz@stanford.edu)Wade Kingery (wkingery@stanford.edu)J David Clark (djclark@stanford.edu) ISSN  1744-8069 Article type  Research Submission date  8 May 2013 Acceptance date  6 August 2013 Publication date  8 August 2013 Article URL  http://www.molecularpain.com/content/9/1/40This peer-reviewed article can be downloaded, printed and distributed freely for any purposes (seecopyright notice below).Articles in  Molecular Pain   are listed in PubMed and archived at PubMed Central.For information about publishing your research in  Molecular Pain   or any BioMed Central journal, gotohttp://www.molecularpain.com/authors/instructions/ For information about other BioMed Central publications go tohttp://www.biomedcentral.com/  Molecular Pain  © 2013 Gallagher  et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord Joseph J Gallagher 1,2  Email: jjg1@stanford.edu Maral Tajerian 1,2  Email: maral@stanford.edu Tianzhi Guo 3  Email: wxtguo@yahoo.com Xiaoyou Shi 1,2  Email: xyshi@stanford.edu Wenwu Li 1,2,3  Email: wenwuli@stanford.edu Ming Zheng 2  Email: mzheng@stanford.edu Gary Peltz 2  Email: gpeltz@stanford.edu Wade Kingery 3  Email: wkingery@stanford.edu J David Clark  1,2,*  Email: djclark@stanford.edu 1  Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304, USA 2  Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA, USA 3  Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA *  Corresponding author. Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA, USA  Abstract 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. Keywords Complex regional pain syndrome, CcL2, Chemokine, Chronic Pain, Spinal Cord, Microarray Analysis, Transcriptome, Pathway Analysis Background Complex regional pain syndrome (CRPS) is a painful, debilitating, and often-chronic condition with an estimated incidence rate of 26.2 per 100,000 person years [1]. While acute CRPS sometimes improves with early and aggressive physical therapy, CRPS present for a period of one year or more seldom spontaneously resolves. The syndrome encompasses a disparate collection of signs and symptoms involving the sensory, motor and autonomic nervous systems, cognitive deficits, bone demineralization, skin growth changes and vascular dysfunction [2]. Current therapies for CRPS including physical, interventional, pharmacological, rehabilitative and alternative are limited in their effectiveness, and none are  routinely curative of the chronic condition [3,4]. The acute phase of the condition is often characterized by edema and warmth, and is thought to be supported by neurogenic inflammation [5-7]. Alterations in CNS structure and function may be more important to the sustained pain and neurocognitive features of the chronic phase of the CRPS [8] The molecular analysis of peripheral mechanisms supporting CRPS has been extensive. Human studies have focused on dysfunctional signaling through autonomic and peptidergic neurons [9-11]. Other work has shown abnormal levels of cytokines in the skin of CRPS limbs [12]. More recently changes in the adaptive system of immunity have been demonstrated [13]. To better understand these changes, a tibial fracture/cast immobilization model of CRPS has been developed in rodents displaying nociceptive sensitization, bone demineralization, edema and warmth [14,15]. This model also recapitulates the human observations of abnormal peripheral neural signaling and cytokine generation, particularly in the first several days after removal of cast immobilization. The fracture/cast model has not to this point been utilized for the purpose of understanding the more chronic features of CRPS, though nociceptive changes are persistent for months in these animals. The development of pain involves a complicated sequence of events ranging from changes in neuronal properties [16] to alterations in gene transcription and protein levels [17,18]. While many successes have been achieved through the selection of individual molecules for study as they participate in pain, a complementary approach has been to study changes in the expression levels of large numbers of genes in hypothesis-free fashion using expression arrays and similar molecular tools. A recent meta-analysis of pain related gene expression studies on spinal cord and dorsal root ganglion tissue revealed both similarities and differences across a range of pain models [19]. These authors discovered that two genes, Reg3b (regenerating islet-derived 3 beta; pancreatitis-associated protein) and CcL2 (chemokine [C-C motif] ligand 2), were up-regulated in almost every dataset, included in their analysis, suggesting possible core roles in supporting persistent pain. To this point no array-based studies have been provided using a model of CRPS, a condition which has features separate from most acute, inflammatory and neuropathic etiologies of pain [10]. Furthermore, the striking transition of CRPS from an acute to a more chronic state suggests that analyses need to be conducted at more than one timepoint. Very few array-based analyses have commented on changes in gene expression over time. We hypothesized that array-based spinal cord gene expression studies using the fracture-cast model of CRPS would reveal timepoint-dependent genes and pathways relatively unique to the CRPS model, that a set of core genes might be shared with other models of persistent pain, and that at least one significantly changed gene could be shown to be functionally related to nociceptive sensitization in this model. Results CRPS mice exhibit transient increases in temperature and edema in addition to long-lasting nociceptive sensitization in the injured limb Ipsilateral and contralateral measurements of hindpaw temperature and edema, two commonly observed symptoms in CRPS patients, were performed at 3, 5 and 7 weeks post-fracture. Both temperature and edema demonstrated similar profiles, exhibiting transients increases identified at the 3-week, but not the 5- or 7-week timepoints. An increased  temperature in the contralateral hindpaw, as compared to baseline, was observed at the 3-week timepoint only (Figure 1A,B). Mechanical hypersensitivity, assessed using Von Frey filaments, identified a significant and persistent reduction – up to 7 weeks post-fracture - in the injured hindpaw compared to measurements of the contralateral hindpaw or control mice. Inspection of the mechanical hypersensitivity of the contralateral hindpaw suggested a modest decrease that did not reach significance at the 3 and 5 weeks (Figure 1C). Reduced weight bearing was present in the injured hindpaw and persisted at both timepoints assessed, extending from 3 to 7 weeks after fracture (Figure 1D). Figure 1   Physiological and behavioral changes in CRPS mice.  CRPS mice display increased temperature (A)  and edema (B)  on the affected hindpaw at 3 weeks post-fracture. In addition, they show signs of mechanical allodynia (C)  and decreased weight bearing (D)  for up to 7 weeks after fracture. **p<0.01, *** p<0.001. n=8/group. Errors bars=S.E.M. Microarray expression analysis from ipsilateral spinal cord identifies distinct profiles at 3 and 7 weeks post-fracture. We identified the genes significantly regulated at the 3- and 7-week timepoint, with respect to the control group (absolute fold change > 1.5, adjusted p-value < 0.05). 199 genes were identified as significantly regulated at the 3-week timepoint with 98 of these genes also identified as significantly regulated at the 7-week timepoint. 62 genes were unique to the 7-week timepoint. Half of the 122 genes with increased expression at the 3 week timepoint maintained increased expression at the 7-week timepoint (61 genes), while 38 of the 97 genes with decreased expression at the 3-week timepoint maintained decreased expression at the 7-week timepoint with the remaining genes showing no differential expression at the 7-week timepoint. No genes were identified as reversing their expression from over expressed to under expressed or vice versa between timepoints.154 genes are significantly regulated at the 7-week timepoint with 103 genes increased and 51 genes decreased (Additional file 1: Table S1; Additional file 2: Table S2). CRPS results in altered transcriptional programs in the spinal cord Ingenuity Pathway Analysis (IPA) identified specific networks that were dysregulated in the spinal cord 3 and 7 weeks following fracture. Canonical pathways with a cutoff p-value < 0.05 were considered statistically significant. Canonical pathways, indicating wide changes in currently known pathways, were shown to differ between the two timepoints. At 3 weeks, the main regulated pathways were chemokine signaling, glycogen degradation II, cAMP-mediated signaling, glycogen degradation III, role of IL-17A in arthritis, agranulocyte adhesion and diapedesis, IL-22 signaling, agrin interactions at neuromuscular junction, IL-17A signaling in gastric cells, and role of JAK family kinases in IL-6-type cytokine signaling. At the 7-week timepoint, the effected pathways were: coagulation system, granzyme A Signaling, aryl hydrocarbon receptor signaling, acute phase response signaling, granulocyte adhesion and diapedesis, agranulocyte adhesion and diapedesis, MSP-RON signaling pathway, thiosulfate disproportionation III (Rhodanese), GM-CSF signaling, and agrin interactions at neuromuscular junction (Figure 2). In addition to these canonical pathways, we identified the following top biological functions associated, to different degrees, with both timepoints: Cellular movement, cancer, cardiovascular system development and function, organismal development, nutritional disease, cell death and survival, nucleic acid metabolism, small molecule biochemistry, cell-to-cell signaling and interaction, molecular transport, hematological system development and function, immune cell trafficking,
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