Genetic evidence of the function of Phox2a-expressing anterolateral system neurons in the transmission of chronic pain.

The development of the chronic neuropathic pain state often originates at the level of peripheral sensory neurons, whose abnormal function elicits central sensitization and maladaptive plasticity in the nociceptive circuits of the spinal dorsal horn. These changes eventually reach supraspinal areas bringing about cognitive and affective co-morbidities of chronic pain such as anxiety and depression. This transmission presumably relies on the function of spinal projection neurons at the origin of the anterolateral system (AS). However, the identity of these neurons and the extent of their functional contribution remain unknown. Here, we asked these questions in the context of the mouse AS neurons that require the transcription factor Phox2a for their normal target connectivity and function in transmitting acute nociceptive information to the brain. To this end, we examined the effects of a spinal cord-specific loss of Phox2a (Phox2acKO) on the development of central sensitization evoked by the spared nerve injury (SNI) model of chronic pain. We found that SNI-treated Phox2acKO mice developed normal reflexive spinal responses such as mechanical allodynia evidenced by a decreased withdrawal threshold to von Frey filament stimulation and dynamic brush. On the other hand, Phox2acKO attenuated the development of cold but not mechanical hyperalgesia, in behavioral paradigms that require the relay of nociceptive information to the brain. Furthermore, Phox2acKO attenuated anxio-depressive-like behaviors evoked by SNI, measured by performance in the open field test and tail suspension test. Thus, Phox2a AS neurons play a critical role in the generation and maintenance of chronic neuropathic pain.

Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis.

BACKGROUND: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 (ENT1-/-), a preclinical model of DISH, and behavioral indicators of pain. METHODS: A longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1-/- mice at 2, 4, and 6 months. At endpoint, spinal cords were isolated for immunohistochemical analysis of astrocytes (GFAP), microglia (IBA1), and nociceptive innervation (CGRP). RESULTS: Increased spine calcification in ENT1-/- mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1-/- mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1-/- mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1-/- mice compared to wild-type, suggesting an increase in nociceptive innervation. CONCLUSION: These data suggest that ENT1-/- mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.

Sex-specific effects of neuropathic pain on long-term pain behavior and mortality in mice.

ABSTRACT: Human epidemiological studies suggest that chronic pain can increase mortality risk. We investigated whether this was true in mice so that underlying mechanisms might be identified. At 10 weeks of age, C57BL/6 mice of both sexes received sham or spared nerve injury (SNI) surgery producing neuropathic pain. Mice were weighed monthly, tested behaviorally for mechanical and cold sensitivity and guarding behavior every 3 months postsurgery, and otherwise left undisturbed in their cages until death by natural causes. Evidence of pain over the lifespan displayed a strikingly sex-specific pattern. Male mice displayed largely stable mechanical and cold hypersensitivity and guarding at 6 to 30 months post-SNI. By contrast, female mice displayed a biphasic temporal pattern of mechanical hypersensitivity and guarding behavior, with a complete resolution of SNI-induced pain behavior at 6 to 9 months post-SNI followed by the return of pain thereafter. Mouse lifespan was not significantly altered by SNI in either sex nor was frailty as assessed by cage inspection in the last 6 months of life. However, in male mice with SNI, we observe a significant correlation between average lifetime mechanical hypersensitivity and lifespan, such that death occurred sooner in male mice exhibiting more evidence of chronic pain. This relationship was not observed in female SNI mice nor in sham-operated mice of either sex. This experiment is the first to investigate pain behavior over an entire adult lifetime and suggests that biology of relevance to human chronic pain is being ignored by the very short timespans of most extant preclinical pain research.

TAK-242 treatment and its effect on mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice.

PURPOSE: Intervertebral disc (IVD) degeneration is accompanied by mechanical and gene expression changes to IVDs. SPARC-null mice display accelerated IVD degeneration, and treatment with (toll-like receptor 4 (TLR4) inhibitor) TAK-242 decreases proinflammatory cytokines and pain. This study examined if chronic TAK-242 treatment impacts mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice. METHODS: Male and female SPARC-null and WT mice aged 7-9 months were given intraperitoneal injections with TAK-242 or an equivalent saline vehicle for 8 weeks (3x/per week, M-W-F). L2-L5 spinal segments were tested in cyclic axial tension and compression. Gene expression analysis (RT-qPCR) was performed on male IVD tissues using Qiagen RT2 PCR arrays. RESULTS: SPARC-null mice had decreased NZ length (p = 0.001) and increased NZ stiffness (p < 0.001) compared to WT mice. NZ length was not impacted by TAK-242 treatment (p = 0.967) despite increased hysteresis energy (p = 0.024). Tensile stiffness was greater in SPARC-null mice (p = 0.018), and compressive (p < 0.001) stiffness was reduced from TAK-242 treatment in WT but not SPARC-null mice (p = 0.391). Gene expression analysis found upregulation of 13 ECM and 5 inflammatory genes in SPARC-null mice, and downregulation of 2 inflammatory genes after TAK-242 treatment. CONCLUSIONS: TAK-242 had limited impacts on SPARC-null mechanical properties and did not attenuate NZ mechanical changes associated with IVD degeneration. Expression analysis revealed an increase in ECM and inflammatory gene expression in SPARCnull mice with a reduction in inflammatory expression due to TAK-242 treatment. This study provides insight into the role of TLR4 in SPARC-null mediated IVD degeneration.

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.

The methyl donor S-adenosyl methionine reverses the DNA methylation signature of chronic neuropathic pain in mouse frontal cortex.

Chronic pain is associated with persistent but reversible structural and functional changes in the prefrontal cortex (PFC). This stable yet malleable plasticity implicates epigenetic mechanisms, including DNA methylation, as a potential mediator of chronic pain-induced cortical pathology. We previously demonstrated that chronic oral administration of the methyl donor S-adenosyl methionine (SAM) attenuates long-term peripheral neuropathic pain and alters global frontal cortical DNA methylation. However, the specific genes and pathways associated with the resolution of chronic pain by SAM remain unexplored. OBJECTIVE: To determine the effect of long-term therapeutic exposure to SAM on the DNA methylation of individual genes and pathways in a mouse neuropathic pain model. METHODS: Male CD-1 mice received spared nerve injury or sham surgery. Three months after injury, animals received SAM (20 mg/kg, oral, 3× a week) or vehicle for 16 weeks followed by epigenome-wide analysis of frontal cortex. RESULTS: Peripheral neuropathic pain was associated with 4000 differentially methylated genomic regions that were enriched in intracellular signaling, cell motility and migration, cytoskeletal structure, and cell adhesion pathways. A third of these differentially methylated regions were reversed by SAM treatment (1415 regions representing 1013 genes). More than 100 genes with known pain-related function were differentially methylated after nerve injury; 29 of these were reversed by SAM treatment including Scn10a, Trpa1, Ntrk1, and Gfap. CONCLUSION: These results suggest a role for the epigenome in the maintenance of chronic pain and advance epigenetic modulators such as SAM as a novel approach to treat chronic pain.

Disc degeneration spreads: long-term behavioural, histologic and radiologic consequences of a single-level disc injury in active and sedentary mice.

STUDY DESIGN: A multi-cohort, case-control rodent study. PURPOSE: Investigate the long-term behavioural, histologic and radiologic consequences on the complete lumbar spine of L4/5 intervertebral disc (IVD) injury in mice and determine if increased physical activity mitigates the observed changes. METHODS: Cohorts of 2-month-old CD1 female mice underwent a single ventral puncture of the L4/5 IVD. 0.5-, 3- or 12-months after injury, general health (body weight and locomotor capacity), behavioural signs of axial discomfort (tail suspension, grip strength and FlexMaze assays) and radiating pain (von Frey and acetone tests) were assessed. Experimental groups with free access to an activity wheel in their home cages were including in the 12-month cohort. Lumbar disc status was determined using colorimetric staining and radiologic (X-ray and T2-MRI) analysis. Innervation was measured by immunoreactivity for PGP9.5 and calcitonin gene-related peptide. RESULTS: No changes in general health or persistent signs of axial discomfort were observed up to one year post-injury. In contrast, signs of radiating pain developed in injured mice at 3 months post-injury, persisted up to 12 months and were reversed by long-term physical activity. At 12-months post-injury, degeneration was observed in non-injured lumbar discs. Secondary degenerating IVDs were similar to the injured discs by X-ray (narrowing) and T2-MRI (internal disc disruption) but did not show abnormal innervation. Increased physical activity had no impact on mechanically injured IVDs, but attenuated disc narrowing at other lumbar levels. CONCLUSIONS: Mechanical injury of L4/5-IVDs induces delayed radiating pain and degeneration of adjacent discs; increased physical activity positively mitigated both.

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.

Multifidus Muscle Fiber Type Distribution is Changed in Mouse Models of Chronic Intervertebral Disc Degeneration, but is not Attenuated by Whole Body Physical Activity.

STUDY DESIGN: Case-controlled animal study. OBJECTIVE: The aim of this study was to investigate whether multifidus muscle fiber type distribution changes in models of interverbal disc (IVD) degeneration and whether this is resolved by physical activity (PA). SUMMARY OF BACKGROUND DATA: The loss of slow type I muscle fibers in the multifidus muscle in people with low back pain is contentious. Data from animal models of IVD degeneration suggest some discrepancies in human studies might be explained by the dependence of slow muscle fiber changes and their underlying mechanisms, on the time since injury and progression of IVD degeneration. It is not yet resolved what changes are apparent once the chronic phase is established. It is also not known whether muscle fiber changes can be resolved by whole body PA. This study aimed to examine slow fiber distribution in the multifidus muscle in models of IVD injury or spontaneous degeneration in animals with or without exposure to PA. METHODS: Two models of IVD degeneration were used. The first model used a genetically modified mouse (SPARC-null) that spontaneously develops IVD degeneration. The second model involved a surgically induced IVD lesion to induce degeneration. Mice in each study were allocated to housing with or without a running wheel for PA. At 12 months of age, the multifidus muscle was harvested. Slow muscle fiber distribution and the mRNA expression of genes associated with muscle fiber type transformation were examined. RESULTS: The proportion and cross-sectional area of slow muscle fibers were reduced in both models of IVD degeneration compared to controls, without evidence of ongoing fiber transformation. Whole-body PA did not attenuate these alterations. CONCLUSION: Results confirmed slow muscle fiber loss in the multifidus in the chronic phase of IVD degeneration induced spontaneously and by injury. Whole-body PA did not attenuate changes to muscle fiber distribution. More specific approaches to muscle activation might be required to achieve more complete reversal of muscle fiber changes, with potential implications for therapy in humans.Level of Evidence: N/A.

Diet-induced obesity leads to behavioral indicators of pain preceding structural joint damage in wild-type mice.

INTRODUCTION: Obesity is one of the largest modifiable risk factors for the development of musculoskeletal diseases, including intervertebral disc (IVD) degeneration and back pain. Despite the clinical association, no studies have directly assessed whether diet-induced obesity accelerates IVD degeneration, back pain, or investigated the biological mediators underlying this association. In this study, we examine the effects of chronic consumption of a high-fat or high-fat/high-sugar (western) diet on the IVD, knee joint, and pain-associated outcomes. METHODS: Male C57BL/6N mice were randomized into one of three diet groups (chow control; high-fat; high-fat, high-sugar western diet) at 10 weeks of age and remained on the diet for 12, 24, or 40 weeks. At endpoint, animals were assessed for behavioral indicators of pain, joint tissues were collected for histological and molecular analysis, serum was collected to assess for markers of systemic inflammation, and IBA-1, GFAP, and CGRP were measured in spinal cords by immunohistochemistry. RESULTS: Animals fed obesogenic (high-fat or western) diets showed behavioral indicators of pain beginning at 12 weeks and persisting up to 40 weeks of diet consumption. Histological indicators of moderate joint degeneration were detected in the IVD and knee following 40 weeks on the experimental diets. Mice fed the obesogenic diets showed synovitis, increased intradiscal expression of inflammatory cytokines and circulating levels of MCP-1 compared to control. Linear regression modeling demonstrated that age and diet were both significant predictors of most pain-related behavioral outcomes, but not histopathological joint degeneration. Synovitis was associated with alterations in spontaneous activity. CONCLUSION: Diet-induced obesity accelerates IVD degeneration and knee OA in mice; however, pain-related behaviors precede and are independent of histopathological structural damage. These findings contribute to understanding the source of obesity-related back pain and the contribution of structural IVD degeneration.

Mechanical Consequence of Induced Intervertebral Disc Degeneration in the SPARC-Null Mouse.

Intervertebral disc (IVD) degeneration is associated with low back pain (LBP) and accompanied by mechanical changes to the spine. Secreted protein acidic and rich in cysteine (SPARC) is a protein that contributes to the functioning and maintenance of the extracellular matrix. SPARC-null mice display accelerated IVD degeneration and pain-associated behaviors. This study examined if SPARC-null mice also display altered spine mechanics as compared to wild-type (WT) mice. Lumbar spines from SPARC-null (n = 36) and WT (n = 18) mice aged 14-25 months were subjected to cyclic axial tension and compression to determine neutral zone (NZ) length and stiffness. Three separate mechanical tests were completed for each spine to determine the effect of the number of IVDs tested in series (one versus two versus three IVDs). SPARC-null spine NZs were both stiffer (p < 0.001) and smaller in length (p < 0.001) than WT spines. There was an effect of the number of IVDs tested in series for NZ length but not NZ stiffness when collapsed across condition (SPARC-null and WT). Correlation analysis revealed a weak negative correlation (r = -0.24) between age and NZ length in SPARC-null mice and a weak positive correlation (r = 0.30) between age and NZ stiffness in WT mice. In conclusion, SPARC-null mice had stiffer and smaller NZs than WT mice, regardless of the number of IVDs in series being tested. The increased stiffness of these IVDs likely influences mobility at these spinal joints thereby potentially contributing to low back pain.

The transition from acute to chronic pain: dynamic epigenetic reprogramming of the mouse prefrontal cortex up to 1 year after nerve injury.

Chronic pain is associated with persistent structural and functional changes throughout the neuroaxis, including in the prefrontal cortex (PFC). The PFC is important in the integration of sensory, cognitive, and emotional information and in conditioned pain modulation. We previously reported widespread epigenetic reprogramming in the PFC many months after nerve injury in rodents. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation at the onset of acute pain or how it progresses as pain transitions from acute to chronic. We hypothesize that acute pain after injury results in rapid and persistent epigenetic remodelling in the PFC that evolves as pain becomes chronic. We further propose that understanding epigenetic remodelling will provide insights into the mechanisms driving pain-related changes in the brain. Epigenome-wide analysis was performed in the mouse PFC 1 day, 2 weeks, 6 months, and 1 year after peripheral injury using the spared nerve injury in mice. Spared nerve injury resulted in rapid and persistent changes in DNA methylation, with robust differential methylation observed between spared nerve injury and sham-operated control mice at all time points. Hundreds of differentially methylated genes were identified, including many with known function in pain. Pathway analysis revealed enrichment in genes related to stimulus response at early time points, immune function at later time points, and actin and cytoskeletal regulation throughout the time course. These results emphasize the importance of considering pain chronicity in both pain research and in treatment optimization.

The geriatric pain experience in mice: intact cutaneous thresholds but altered responses to tonic and chronic pain.

Older individuals have an elevated risk for chronic pain as half of all individuals over 65 years old have at least one chronic pain condition. Unfortunately, relevant assessment tools and recommendations for chronic pain management targeting older adults are lacking. This study explores changes in response to pain between young (2-3 months old) and geriatric (20-24 months old) ages using mice. Although cutaneous thresholds to brisk stimuli (von Frey and radiant heat assays) were not affected, behavioral responses to tonic stimuli (acetone and capsaicin assays) were more pronounced in geriatric animals. After nerve injury, geriatric mice present an altered neuropathic pain profile with hypersensitivity to mechanical stimuli but not acetone and an impairment in conditioned noxious stimuli avoidance. This altered behavioral response pattern was associated with an abnormal monoaminergic signature in the medial prefrontal cortex, suggesting decreased COMT function. We conclude that young and geriatric mice exhibit different behavioral and physiological responses to the experience of pain, suggesting that knowledge and practices must be adjusted for geriatric populations.

Long-term histological analysis of innervation and macrophage infiltration in a mouse model of intervertebral disc injury-induced low back pain.

Low back pain (LBP) is a leading cause of global disability. Multiple anatomical, cellular, and molecular factors are implicated in LBP, including the degeneration of lumbar intervertebral discs (IVDs). We previously described a mouse model that displays behavioral symptoms of chronic LBP. Here, we investigated the development of pathological innervation and macrophage infiltration into injured IVDs following a puncture injury in mice over 12 months. 2-month old CD1 female mice underwent a single puncture of the ventral L4/5 IVD using a 30G needle, and were sacrificed 4 days and 0.5-, 3-, 6- and 12-months post-injury. Severity of disc degeneration was assessed using colorimetric staining. IVD innervation was measured by PGP9.5-immunoreactivity (-ir) and calcitonin gene-related peptide-ir (CGRP-ir). Macrophage accumulation into IVDs was detected by F4/80-ir. Mechanical IVD injury resulted in severe degeneration and increased PGP9.5-ir nerve fiber density starting at 4 days that persisted for up to 12 months and dorsal herniations began to occur at 3 months. CGRP-ir was also upregulated in injured IVDs, with the largest increase at 12 months after injury. Infiltration of F4/80-ir macrophages was observed in injured IVDs by day 4 both dorsally and ventrally, with the latter diminishing in the later stage. Persistent LBP is a complex disease with multiple underlying pathologies. By highlighting pathological changes in IVD innervation and inflammation, our study suggests that strategies targeting these mechanisms might be useful therapeutically.

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.

Dysregulation of the Inflammatory Mediators in the Multifidus Muscle After Spontaneous Intervertebral Disc Degeneration SPARC-null Mice is Ameliorated by Physical Activity.

STUDY DESIGN: A longitudinal case-control animal model. OBJECTIVE: The aim of this study was to investigate the inflammatory pathways active in the multifidus muscle after spontaneous intervertebral disc degeneration (IDD), and whether these IDD-related muscle changes can be ameliorated by exercise. SUMMARY OF BACKGROUND DATA: A pro-inflammatory response is present in the multifidus muscle after an intervertebral disc lesion and has been proposed to drive the structural alterations present during low back pain. However, it is not known whether spontaneous IDD produces an inflammatory response. Furthermore, exercise/physical activity produces a strong anti-inflammatory response, but its effectiveness in ameliorating inflammation in the multifidus is unknown. We assessed the inflammatory profile of the multifidus and the effectiveness of physical activity as a treatment using an animal model of spontaneous model of IDD. METHODS: Wild-type and SPARC null mice that were sedentary or housed with a running wheel were used in this study. Multifidus muscle segments were harvested from L2-L6 from the mice at 9 months of age after they had undergone a magnetic resonance imaging (MRI) scan to determine levels with IDD. The inflammatory profile of the multifidus was examined using quantitative polymerase chain reaction (PCR) assays. RESULTS: Spontaneous IDD in the SPARC-null mice caused a dysregulation of interleukin (IL)-1ß, IL6, transforming growth factor-beta (TGFß1), and adiponectin expression. More specifically, the proximity and degree of IDD was related to levels of IL-1ß expression. Physical activity reduced the pro-inflammatory response to IDD in the multifidus. IL-1ß, tumor necrosis factor (TNF), IL-10, adiponectin, and leptin levels were lower in the physically active group. CONCLUSION: These results reveal that spontaneous IDD causes dysregulation of the inflammatory pathways active in the multifidus muscle. These alterations were related to the severity of IDD and were prevented by physical activity. LEVEL OF EVIDENCE: N/A.

Anti-nerve growth factor therapy attenuates cutaneous hypersensitivity and musculoskeletal discomfort in mice with osteoporosis.

INTRODUCTION: The prevalence of osteoporosis is increasing with the aging population and is associated with increased risk of fracture and chronic pain. Osteoporosis is currently treated with bisphosphonate therapy to attenuate bone loss. We previously reported that improvement in bone mineral density is not sufficient to reduce osteoporosis-related pain in an ovariectomy (OVX)-induced mouse model of osteoporosis, highlighting the need for new treatments. Targeting of nerve growth factor (NGF) with sequestering antibodies is a promising new direction for the treatment of musculoskeletal pain including back pain and arthritis. Its efficacy is currently unknown for osteoporotic pain. OBJECTIVE: To investigate the efficacy of anti-NGF antibody therapy on osteoporotic pain in an OVX-induced mouse model. METHODS: Ovariectomy- and sham-operated mice were injected with an anti-NGF antibody (10 mg/kg, intraperitoneally, administered 2×, 14 days apart), and the effect on behavioural indices of osteoporosis-related pain and on sensory neuron plasticity was evaluated. RESULTS: Treatment with anti-NGF antibodies attenuated OVX-induced hypersensitivity to mechanical, cold, and heat stimuli on the plantar surface of the hind paw. The OVX-induced impairment in grip force strength, used here as a measure of axial discomfort, was partially reversed by anti-NGF therapy. No changes were observed in the rotarod or open-field tests for overall motor function and activity. Finally, anti-NGF treatment attenuated the increase in calcitonin gene-related peptide-immunoreactive dorsal root ganglia neurons observed in OVX mice. CONCLUSION: Taken together, these data suggest that anti-NGF antibodies may be useful in the treatment of prefracture hypersensitivity that is reported in 10% of patients with osteoporosis.

Low back pain and disc degeneration are decreased following chronic toll-like receptor 4 inhibition in a mouse model.

OBJECTIVE: Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP) but current treatment is limited. Toll-like receptors (TLRs) on disc cells are activated by endogenous extracellular matrix (ECM) fragments and modulate degeneration in vitro. This study investigated whether inhibiting TLR4 slows disc degeneration and reduces behavioral signs of LBP in vivo. DESIGN: 7-9-month old wild-type and secreted protein acidic and rich in cysteine (SPARC)-null (a model of disc degeneration and LBP) male mice were treated with TAK-242 (TLR4 inhibitor) once, and following a 10-day washout, mice were treated 3 times/week for 8 weeks. Behavioral signs of axial discomfort and radiating leg pain were assessed weekly with the grip force assay and acetone test, respectively. Following treatment, pain-related spinal cord changes were evaluated and lumbar discs were excised and cultured. Cytokine secretion from discs was evaluated with protein arrays. RESULTS: SPARC-null mice displayed elevated signs of axial and radiating pain at baseline compared to wild-type. Chronic, but not acute, TLR4 inhibition reduced behavioral signs of pain compared to vehicle. SPARC-null mice have increased calcitonin gene-related peptide (CGRP)- and glial fibrillary acidic protein (GFAP)-immunoreactivity (astrocyte marker) in the dorsal horn compared to wild-type, which is reduced by chronic TLR4 inhibition. Ex vivo degenerating discs from SPARC-null mice secrete increased levels of many pro-inflammatory cytokines, which chronic TLR4 inhibition reduced. CONCLUSION: Chronic TLR4 inhibition decreased behavioral signs of LBP, pain-related neuroplasticity and disc inflammation in SPARC-null mice. TAK-242 inhibits TLR4 activation within discs, as evidenced by decreases in cytokine release. Therefore, TLRs are potential therapeutic targets to slow disc degeneration and reduce pain.

Delayed onset of persistent discogenic axial and radiating pain after a single-level lumbar intervertebral disc injury in mice.

Low back pain (LBP) is associated with both axial discomfort and radiating leg pain. Although intervertebral discs are suspected as the source of pain in some individuals, the relationship between disc degeneration and back pain remains controversial. The goals of this study were to investigate the long-term impact of L4/L5 disc puncture on disc degeneration and the subsequent emergence, persistence, and underlying mechanisms of axial and radiating LBP in mice. L4 to L5 discs were punctured on the ventral aspect with a 30 gauge needle in 3-month old female CD1 mice, and the development of behavioral signs of axial discomfort (tail suspension and grip force), radiating hypersensitivity (von Frey and acetone), and motor impairment (rotarod) were monitored. Disc degeneration was assessed using X-ray, T2-magnetic resonance imaging, and histology, and persisted for up to 1 year. Innervation was quantified by immunohistochemistry using the pan-neuronal marker PGP9.5. Behavioural signs of axial discomfort peaked 3 to 9 months after injury. During the peak, local nerve density was increased. A transient increase in hypersensitivity to cold, suggestive of radiating pain, was observed 2 weeks after injury. Radiating pain then reemerged 9 to 12 months after injury in half the animals and correlated with increased dorsal innervation and reduced disc height at these late time points. In summary, a single-level disc injury is sufficient to induce prolonged disc degeneration and delayed axial and radiating pain. This model will be useful to investigate underlying mechanisms and potential therapeutic strategies for discogenic LBP.

Chronic Osteoporotic Pain in Mice: Cutaneous and Deep Musculoskeletal Pain Are Partially Independent of Bone Resorption and Differentially Sensitive to Pharmacological Interventions.

Although the pathological changes in osteoporotic bones are well established, the characterization of the osteoporotic pain and its appropriate treatment are not fully elucidated. We investigated the behavioral signs of cutaneous and deep musculoskeletal pain and physical function; time-dependent changes in bone mineral density (BMD) and the emergence of the behavioral phenotype; and the effects of pharmacological interventions having different mechanisms of action (chronic intraperitoneal administration of pamidronate [0.25 mg/kg, 5x/week for 5 weeks] versus acute treatment with intraperitoneal morphine [10 mg/kg] and pregabalin [100 mg/kg]) in a mouse model of ovariectomized or sham-operated mice 6 months following surgery. We observed reduced BMD associated with weight gain, referred cutaneous hypersensitivity, and deep musculoskeletal pain that persisted for 6 months. Chronic bisphosphonate treatment, 6 months after ovariectomy, reversed bone loss and hypersensitivity to cold, but other behavioral indices of osteoporotic pain were unchanged. While the efficacy of acute morphine on cutaneous pain was weak, pregabalin was highly effective; deep musculoskeletal pain was intractable. In conclusion, the reversal of bone loss alone is insufficient to manage pain in chronic osteoporosis. Additional treatments, both pharmacological and nonpharmacological, should be implemented to improve quality of life for osteoporosis patients.