Exercise attenuates low back pain and alters epigenetic regulation in intervertebral discs in a mouse model.

BACKGROUND CONTEXT: Chronic low back pain (LBP) is a multifactorial disorder with complex underlying mechanisms, including associations with intervertebral disc (IVD) degeneration in some individuals. It has been demonstrated that epigenetic processes are involved in the pathology of IVD degeneration. Epigenetics refers to several mechanisms, including DNA methylation, that have the ability to change gene expression without inducing any change in the underlying DNA sequence. DNA methylation can alter the entire state of a tissue for an extended period of time and thus could potentially be harnessed for long-term pain relief. Lifestyle factors, such as physical activity, have a strong influence on epigenetic regulation. Exercise is a commonly prescribed treatment for chronic LBP, and sex-specific epigenetic adaptations in response to endurance exercise have been reported. However, whether exercise interventions that attenuate LBP are associated with epigenetic alterations in degenerating IVDs has not been evaluated. PURPOSE: We hypothesize that the therapeutic efficacy of physical activity is mediated, at least in part, at the epigenetic level. The purpose of this study was to use the SPARC-null mouse model of LBP associated with IVD degeneration to clarify (1) if IVD degeneration is associated with altered expression of epigenetic regulatory genes in the IVDs, (2) if epigenetic regulatory machinery is sensitive to therapeutic environmental intervention, and (3) if there are sex-specific differences in (1) and/or (2). STUDY DESIGN: Eight-month-old male and female SPARC-null and age-matched control (WT) mice (n=108) were assigned to exercise (n=56) or sedentary (n=52) groups. Deletion of SPARC is associated with progressive IVD degeneration and behavioral signs of LBP. The exercise group received a circular plastic home cage running wheel on which they could run freely. The sedentary group received an identical wheel secured in place to prevent rotation. After 6 months, the results obtained in each group were compared. METHODS: After 6 months of exercise, LBP-related behavioral indices were determined, and global DNA methylation (5-methylcytosine) and epigenetic regulatory gene mRNA expression in IVDs were assessed. This project was supported by the Canadian Institutes for Health Research. The authors have no conflicts of interest. RESULTS: Lumbar IVDs from WT sedentary and SPARC-null sedentary mice had similar levels of global DNA methylation (%5-mC) and comparable mRNA expression of epigenetic regulatory genes (Dnmt1,3a,b, Mecp2, Mbd2a,b, Tet1-3) in both sexes. Exercise attenuated LBP-related behaviors, decreased global DNA methylation in both WT (p<.05) and SPARC-null mice (p<.01) and reduced mRNA expression of Mecp2 in SPARC-null mice (p<.05). Sex-specific effects of exercise on expression of mRNA were also observed. CONCLUSIONS: Exercise alleviates LBP in a mouse model. This may be mediated, in part, by changes in the epigenetic regulatory machinery in degenerating IVDs. Epigenetic alterations due to a lifestyle change could have a long-lasting therapeutic impact by changing tissue homeostasis in IVDs. CLINICAL SIGNIFICANCE: This study confirmed the therapeutic benefits of exercise on LBP and suggests that exercise results in sex-specific alterations in epigenetic regulation in IVDs. Elucidating the effects of exercise on epigenetic regulation may enable the discovery of novel gene targets or new strategies to improve the treatment of chronic LBP.

Effect of voluntary running activity on mRNA expression of extracellular matrix genes in a mouse model of intervertebral disc degeneration.

INTRODUCTION: Low back pain (LBP), a leading cause of global disability, is often associated with intervertebral disc degeneration (IDD). Exercise therapy is recommended for chronic LBP management and affects many tissues and organ systems. However, the ability of exercise to repair the extracellular matrix (ECM) in degenerating discs is unclear. The aims of the study were to examine mRNA expression of ECM structural components (collagen I, II, X, aggrecan) and regulators of matrix turnover (matrix metalloproteinases (MMP)-3, - 9, - 13, ADAMTS-4, - 5, TIMP1-4, CCN2) between age-matched (a) wild-type and secreted protein acidic and rich in cysteine (SPARC)-null, (b) sedentary and active, and (c) male and female mice. METHODS: At 8 months of age, male and female SPARC-null and wild-type control mice received a home cage running wheel or a control, fixed wheel for 6 months. Deletion of the SPARC gene results in progressive IDD beginning at 2 to 4 months of age. Increased activity was confirmed, and qPCR was performed on excised lumbar discs. RESULTS: Male SPARC-null mice expressed less aggrecan mRNA than wild-type controls. After 6 months of running, collagen, MMP3, and MMP13 expression was increased in male and MMP3 was increased in female SPARC-null mice. Sex differences were observed in wild-type mice and in response to IDD and long-term running. CONCLUSIONS: Voluntary running results in changes in mRNA consistent with increased ECM turnover and disc regeneration. Improved disc ECM might contribute to the beneficial effects of exercise on LBP and may create an intradiscal environment hospitable to regenerative therapies. Sex-specific differences should be considered in the development of disc-targeting therapies.

Prenatal maternal stress is associated with increased sensitivity to neuropathic pain and sex-specific changes in supraspinal mRNA expression of epigenetic- and stress-related genes in adulthood.

Exposure to prenatal maternal stress impacts adult behavioral outcomes and has been suggested as a risk factor for chronic pain. However, the neurobiological mechanisms implicated are not well-characterized. In this study, we analyzed the effect of a prenatal maternal stress on the development of neuropathic pain-related behaviours and gene expression in the frontal cortex and hippocampus in adult offspring following chronic constriction injury of the sciatic nerve in male and female CD1 mice. Nerve injury-induced mechanical hypersensitivity was amplified in both male and female prenatally-stressed offspring, suggesting that prenatal stress exacerbates pain after injury. Analysis of mRNA expression of genes related to epigenetic regulation and stress responses in the frontal cortex and hippocampus, brain structures implicated in chronic pain, showed distinct sex and region-specific patterns of dysregulation. In general, mRNA expression was most frequently altered in the male hippocampus and effects of prenatal stress were more prevalent than effects of nerve injury in both supraspinal areas. These findings demonstrate the impact of prenatal stress on behavioral sensitivity to a painful injury. Changes in the expression of epigenetic- and stress-related genes suggest a possible mechanism by which the early life stress becomes embedded in the central nervous system. Increased understanding of the interactions among early-life stress, sex, and pain may lead to the identification of novel therapeutic targets and epigenetic drugs for the treatment of chronic pain disorders.

The immobilization of fibronectin- and fibroblast growth factor 2-derived peptides on a culture plate supports the attachment and proliferation of human pluripotent stem cells.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.

Interleukin-8 as a therapeutic target for chronic low back pain: Upregulation in human cerebrospinal fluid and pre-clinical validation with chronic reparixin in the SPARC-null mouse model.

BACKGROUND: Low back pain (LBP) is the leading global cause of disability and is associated with intervertebral disc degeneration (DD) in some individuals. However, many adults have DD without LBP. Understanding why DD is painful in some and not others may unmask novel therapies for chronic LBP. The objectives of this study were to a) identify factors in human cerebrospinal fluid (CSF) associated with chronic LBP and b) examine their therapeutic utility in a proof-of-concept pre-clinical study. METHODS: Pain-free human subjects without DD, pain-free human subjects with DD, and patients with chronic LBP linked to DD were recruited and lumbar MRIs, pain and disability levels were obtained. CSF was collected and analyzed by multiplex cytokine assay. Interleukin-8 (IL-8) expression was confirmed by ELISA in CSF and in intervertebral discs. The SPARC-null mouse model of progressive, age-dependent DD and chronic LBP was used for pre-clinical validation. Male SPARC-null and control mice received systemic Reparixin, a CXCR1/2 (receptors for IL-8 and murine analogues) inhibitor, for 8 weeks. Behavioral signs of axial discomfort and radiating pain were assessed. Following completion of the study, discs were excised and cultured, and conditioned media was evaluated with a protein array. FINDINGS: IL-8 was elevated in CSF of chronic LBP patients with DD compared to pain-free subjects with or without DD. Chronic inhibition with reparixin alleviated low back pain behaviors and attenuated disc inflammation in SPARC-null mice. INTERPRETATION: These studies suggest that the IL-8 signaling pathway is a viable therapy for chronic LBP. FUND: Supported by NIH, MMF, CIHR and FRQS.