Project description:We performed genome-wide DNA methylation analysis of 850,000 CpG sites in women and men with chronic Low Back Pain (LBP) and pain free-controls. T cells were isolated (Discovery Cohort, n=32) and used to identify differentially methylated CpG sites, and gene ontologies and molecular pathways were identified.T cells were isolated (Discovery Cohort, n=32) and used to identify differentially methylated CpG sites, and gene ontologies and molecular pathways were identified. A polygenic DNA methylation score for LBP was generated in both women and men. Validation was performed in an independent cohort (Validation Cohort, n=63) of chronic LBP and healthy controls. Analysis with the Discovery Cohort revealed a total of 2,496 and 419 differentially methylated CpGs in women and men, respectively. The majority of these sites were hypo-methylated in women and enriched in genes with functions in the extracellular matrix, the immune system (i.e. cytokines) or in epigenetic processes. In men, we identified a unique chronic LBP DNA methylation signature characterized by significant enrichment for genes from the major histocompatibility complex. A sex-specific polygenic DNA methylation score was generated to evaluate the pain status of each individual and confirmed in The Validation Cohort using pyrosequencing.
Project description:Background. Inter- and intra-individual fluctuations in pain intensity pose a major challenge to treatment efficacy, with a majority perceiving their pain relief as inadequate. Recent preclinical studies have identified circadian rhythmicity as a potential contributor to these fluctuations and therapeutic target. Methods. We therefore sought to determine the impact of these rhythms in people with chronic low back pain (CLBP) through a detailed characterization, including questionnaires to evaluate biopsychosocial characteristics, ecological momentary assessment (7-day e-diaries at 8:00/14:00/20:00) to assess pain fluctuations, and intra-day blood transcriptomics (8:00/20:00) to identify genes/pathways of interest. Results. While most individuals displayed constant or variable/mixed pain phenotypes, a distinct subset had daily fluctuations of increasing pain scores (>30% change in intensity over 12-hours in ≥4/7 days). This population had no opioid users, better biopsychosocial profiles, and differentially expressed transcripts relative to other pain phenotypes. The circadian-governed neutrophil degranulation pathway was particularly enriched among arhythmic individuals; the link between neutrophil degranulation and opioid use was further confirmed in a separate CLBP cohort. Conclusion. Our findings identify pain rhythmicity and the circadian expression of neutrophil degranulation pathways as indicators of CLBP outcomes, which may help provide a personalized approach to phenotyping biopsychosocial characteristics and medication use. This highlights the need to better understand the impact of circadian rhythmicity across chronic pain conditions.
Project description:Chronic low back pain (cLBP) lacks clear physiological explanations, and the treatment options are of limited effect. Here, we elucidate the underlying biology of cLBP in a subgroup of patients with Modic changes type I (suggestive of inflammatory vertebral bone marrow lesions) by correlating gene expression in blood with patient-reported outcomes on disability and pain intensity and explore sex-differences. Patients were included from the placebo group of a clinical study on patients with cLBP and Modic changes. Blood was collected at the time of inclusion, after three months, and after one year, and gene expression was measured at all time points by high-throughput RNA sequencing. The patients reported disability using the Roland Morris Disability Questionnaire, and pain intensity was assessed as a mean of three scores on a 0-10 numeric rating scale: current LBP, worst LBP within the last two weeks, and mean LBP within the last two weeks. The gene expression profiles were then correlated to the reported outcomes. Changes in gene expression over time correlated significantly with changes in both disability and pain. The findings showed distinct patterns in men and women, with negligible overlap in correlated genes between the sexes. The genes involved were enriched in immunological pathways, particularly T cell receptor complex and immune responses related to neutrophils. Several of the genes harbour polymorphisms that previously have been found to be associated with chronic pain. Men and women had distinct sets of correlating genes, suggesting gender differences in the underlying biology of disability and pain in these patients.
Project description:This study presents the first application of comprehensive single-nuclei RNA sequencing (snRNA-seq) of dorsal root ganglion (DRG) neurons in a rat model using Parse technology. Our dataset includes two experimental models: (1) control rats representing both sexes and (2) rats with disc-associated chronic lower back pain, alongside sham-treated animals. By leveraging high-resolution transcriptomic profiling, this study provides novel insights into the molecular landscape of DRG neurons, offering a valuable resource for understanding the cellular mechanisms underlying chronic pain.
Project description:We used transcriptome-wide data to investigate the molecular pathophysiological mechanisms in peripheral blood immune cells at the transcriptome-wide level that underlie the transition of acute to chronic low back pain.
Project description:Gene expression correlates with disability and pain intensity in patients with chronic low back pain and Modic changes in a sex-specific manner
Project description:Not all patients with nerve injury develop neuropathic pain. The extent of nerve damage and age at the time of injury are two of the few risk factors identified to date. In addition, preclinical studies show that neuropathic pain variance is heritable. To define such factors further, we performed a large-scale gene profiling experiment which plotted global expression changes in the rat dorsal root ganglion in three peripheral neuropathic pain models. This resulted in the discovery that the potassium channel alpha subunit KCNS1, involved in neuronal excitability, is constitutively expressed in sensory neurons and markedly downregulated following nerve injury. KCNS1 was then characterized by an unbiased network analysis as a putative pain gene, a result confirmed by single nucleotide polymorphism association studies in humans. A common amino acid changing allele, the 'valine risk allele', was significantly associated with higher pain scores in five of six independent patient cohorts assayed (total of 1359 subjects). Risk allele prevalence is high, with 18-22% of the population homozygous, and an additional 50% heterozygous. At lower levels of nerve damage (lumbar back pain with disc herniation) association with greater pain outcome in homozygote patients is P = 0.003, increasing to P = 0.0001 for higher levels of nerve injury (limb amputation). The combined P-value for pain association in all six cohorts tested is 1.14 E-08. The risk profile of this marker is additive: two copies confer the most, one intermediate and none the least risk. Relative degrees of enhanced risk vary between cohorts, but for patients with lumbar back pain, they range between 2- and 3-fold. Although work still remains to define the potential role of this protein in the pathogenic process, here we present the KCNS1 allele rs734784 as one of the first prognostic indicators of chronic pain risk. Screening for this allele could help define those individuals prone to a transition to persistent pain, and thus requiring therapeutic strategies or lifestyle changes that minimize nerve injury. Microarrays were run on mRNA extracted from adult rat L4 and L5 DRGs cells after 3,7,21,40 hours after three different sciatic nerve lesions [Spared Nerve Injury (SNI); Chronic Constriction Injury (CCI); Spinal Nerve Ligation (Ch) with Sham controls (SH)].
Project description:Single-nuclei RNA Sequencing Reveals Distinct Transcriptomic Signatures of Rat Dorsal Root Ganglia in a Disc-associated Chronic Low Back Pain Model
Project description:Background: Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) in humans and canines. IVD degeneration affects the structure and function of both the disc and the innervating dorsal root ganglion (DRG) neurons. Pre-clinical animal models are necessary for elucidating the mechanisms of IVD degeneration (IVDD) and the pain signaling pathways involved in discogenic back pain. The chondrodystrophic (CD) dog exhibits similar characteristics to the clinical population affected by IVDD-associated LBP. However, further investigation of the translational tools to study these conditions and the efficacy of novel treatments is needed in this canine model. The objectives of the present study are to: (1) assess the changes in the structure and function of the IVD and DRG, including pain behaviors, in response to injury using a comprehensive set of outcome measures and (2) evaluate the efficacy of potential therapeutics in mitigating these pathologic changes due to injury in the CD canine model. Methods: Retired female research beagles underwent spinal surgery where T11/T12, T12/T13, and T13/L1 IVDs were identified and punctured with a needle containing either a protease-activated receptor 2 antagonist (PAR2A) and cromolyn sodium (CS) solution (N=3) or phosphate-buffered saline (PBS) (N=3). Pain phenotyping and related outcomes were assessed longitudinally or at the 12-week endpoint via RNA-seq on the DRG, von Frey analysis, FitBark activity, and C-reactive protein (CRP) plasma levels. Changes in the structure/function of the IVD were assessed via MRI, mechanics, Dimethylmethylene Blue Assay (DMMB), histological staining using Picrosirius red/ Alcian Blue (PR/AB) and fluoroscopy, and electrophysiology on the DRG neurons. Results: We evaluated a comprehensive series of outcome measures to determine the effects of IVD injury on the structure/function of the canine IVD and DRG, and pain in the in vivo CD dog model of IVDD and back pain. Specifically, we established methods to obtain high-quality messenger RNA from canine DRGs to perform bulk RNA-seq. We demonstrated that injury to the disc resulted in significant upregulation of inflammatory and pain-signaling genes, and downregulation of developmental genes in the adjacent innervating DRG neurons. Additionally, we isolated and cultured viable neurons from canine DRGs and found through whole-cell patch-clamp that that DRGs innervating the injured disc demonstrated altered voltage-gated sodium channel (VGSCs) activity compared to controls. Using T2 weighted MRI, we demonstrated that relaxation time in punctured discs was reduced in four out of the six dogs compared to internal control discs, indicating potential compositional changes in these injured IVDs. No significant effect of PAR2A and CS treatment was observed in this small cohort.Conclusion: This study evaluated a rigorous series of outcome measures to determine the effects of IVD injury on the disc joint and pain in a CD canine in vivo model of back pain. This was the first study to investigate the effects of disc injury on canine DRG transcriptome and whole-cell patch clamping on canine DRG neurons. Results support the CD dog as a clinically relevant translational model for studying IVDD and LBP, and for evaluating the potential efficacy of novel therapeutics in mitigating the changes associated with these conditions.