Characterization of pain-related behaviors in a rat model of acute-to-chronic low back pain: single vs. multi-level disc injury.

Introduction: Low back pain is the most common type of chronic pain. We examined pain-related behaviors across 18 weeks in rats that received injury to one or two lumbar intervertebral discs (IVD) to determine if multi-level disc injuries enhance/prolong pain. Methods: Twenty-three Sprague-Dawley adult female rats were used: 8 received disc puncture (DP) of one lumbar IVD (L5/6, DP-1); 8 received DP of two lumbar IVDs (L4/5 & L5/6, DP-2); 8 underwent sham surgery. Results: DP-2 rats showed local (low back) sensitivity to pressure at 6- and 12-weeks post-injury, and remote sensitivity to pressure (upper thighs) at 12- and 18-weeks and touch (hind paws) at 6, 12 and 18-weeks. DP-1 rats showed local and remote pressure sensitivity at 12-weeks only (and no tactile sensitivity), relative to Sham DP rats. Both DP groups showed reduced distance traveled during gait testing over multiple weeks, compared to pre-injury; only DP-2 rats showed reduced distance relative to Sham DP rats at 12-weeks. DP-2 rats displayed reduced positive interactions with a novel adult female rat at 3-weeks and hesitation and freezing during gait assays from 6-weeks onwards. At study end (18-weeks), radiological and histological analyses revealed reduced disc height and degeneration of punctured IVDs. Serum BDNF and TNFα levels were higher at 18-weeks in DP-2 rats, relative to Sham DP rats, and levels correlated positively with remote sensitivity in hind paws (tactile) and thighs (pressure). Discussion: Thus, multi-level disc injuries resulted in earlier, prolonged and greater discomfort locally and remotely, than single-level disc injury. BDNF and TNFα may have contributing roles.

Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats.

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing human nucleus pulposus cells (NPCs), generated by certain conditions in degenerating discs, that was associated with the onset of discogenic back pain. Single-cell transcriptomic analysis of human tissues showed a strong correlation between a specific cell subtype and the pain condition associated with the human degenerated disc, suggesting that they are pain-triggering. The application of IVD degeneration-associated exogenous stimuli to healthy NPCs in vitro recreated a pain-associated phenotype. These stimulated NPCs activated functional human iPSC-derived sensory neuron responses in an in vitro organ-chip model. Injection of stimulated NPCs into the healthy rat IVD induced local inflammatory responses and increased cold sensitivity and mechanical hypersensitivity. Our findings reveal a previously uncharacterized pain-inducing mechanism mediated by NPCs in degenerating IVDs. These findings could aid in the development of NPC-targeted therapeutic strategies for the clinically unmet need to attenuate discogenic LBP.

Adverse childhood experience is associated with an increased risk of reporting chronic pain in adulthood: a stystematic review and meta-analysis.

Background: Adverse childhood experiences (ACEs) have been shown to negatively affect health in adulthood. Estimates of associations between ACEs and chronic painful conditions are lacking.Objectives: This systematic review and meta-analysis aimed to evaluate associations between exposure to ACEs and chronic pain and pain-related disability in adults.Methods: We searched 10 electronic databases from inception to February 2023. We included observational studies assessing associations between direct ACEs (childhood sexual, physical, emotional abuse, or neglect) alone or in combination with indirect ACEs (witnessing domestic violence, household mental illness), and adult chronic pain (≥3 months duration) and pain-related disability (daily activities limited by chronic pain). Pairs of reviewers independently extracted data and assessed study risks of bias. Random-effect models were used to calculate pooled adjusted odds ratios [aOR]. Tau square [T2], 95% prediction intervals [95%PI] and I2 expressed the amount of heterogeneity, and meta-regressions and subgroup meta-analyses investigated sources of heterogeneity (PROSPERO: CRD42020150230).Results: We identified 85 studies including 826,452 adults of which 57 studies were included in meta-analyses. Study quality was generally good or fair (n = 70). The odds of reporting chronic pain in adulthood were significantly higher among individuals exposed to a direct ACE (aOR, 1.45, 95%CI, 1.38-1.53). Individuals reporting childhood physical abuse were significantly more likely to report both chronic pain (aOR, 1.50, 95CI, 1.39-1.64) and pain-related disability (1.46, 95CI, 1.03-2.08) during adulthood. Exposure to any ACEs alone or combined with indirect ACEs significantly increase the odds of adult chronic painful conditions (aOR, 1.53, 95%CI, 1.42-1.65) and pain-related disability (aOR, 1.29; 95%CI, 1.01-1.66). The risk of chronic pain in adulthood significantly increased from one ACE (aOR, 1.29, 95%CI, 1.22-1.37) to four or more ACEs (1.95, 95%CI, 1.73-2.19).Conclusions: Single and cumulative ACEs are significantly associated with reporting of chronic pain and pain-related disability as an adult.

Previous meta-analyses highlighted the negative impact of adverse childhood experiences on physical, psychological, and behavioural health across the lifespan.We found exposure to any direct adverse childhood experience, i.e. childhood sexual, physical, emotional abuse, or neglect alone or combined, increased the risk of reporting chronic pain and pain-related disability in adulthood.The risk of reporting chronic painful disorders increased with increasing numbers of adverse childhood experiences.

Sex Differences in the Serum Proteomic Profile During Acute Low Back Pain-A Preliminary Study of the Relationship to Future Low Back Pain.

The molecular processes driving the transition from acute to chronic low back pain (LBP) remain poorly understood and are likely to be sexually dimorphic. This study aimed to explore sex differences in the serum proteomic profile of people experiencing an acute LBP episode and determine if serum protein concentrations were associated with three-month outcome. Serum samples were collected through venepuncture from 30 female and 29 male participants experiencing an acute LBP episode. Serum samples underwent trypsin digestion and fractionation using hydrophobic interaction chromatography and were then analysed using mass-spectrometry. Mass-spectrometry spectra were searched in the Swissprot database for protein identification. Sex differences in protein abundance changes were evident upon inspection of fold changes. Multivariable data analysis identified 21 serum proteins during the acute episode that correctly classified 93% of males and 23 serum proteins that correctly classified 90% of females with ongoing LBP at 3 months. Pathway analysis suggested the differentially expressed proteins during acute LBP were frequently involved in immune, inflammatory, complement, or coagulation responses. This data provides preliminary evidence that biological processes during an acute LBP episode may contribute to the resolution, or persistence, of LBP symptoms at 3 months, however, these processes differ between males and females. PERSPECTIVE: Differential expression of serum proteins was observed between male and female participants during an acute LBP episode. This preliminary work provides a foundation for future research targeting distinct immune system processes in males and females that may interfere with the transition from acute to chronic LBP.

Genome wide association joint analysis reveals 99 risk loci for pain susceptibility and pleiotropic relationships with psychiatric, metabolic, and immunological traits.

Chronic pain is at epidemic proportions in the United States, represents a significant burden on our public health system, and is coincident with a growing opioid crisis. While numerous genome-wide association studies have been reported for specific pain-related traits, many of these studies were underpowered, and the genetic relationship among these traits remains poorly understood. Here, we conducted a joint analysis of genome-wide association study summary statistics from seventeen pain susceptibility traits in the UK Biobank. This analysis revealed 99 genome-wide significant risk loci, 65 of which overlap loci identified in earlier studies. The remaining 34 loci are novel. We applied leave-one-trait-out meta-analyses to evaluate the influence of each trait on the joint analysis, which suggested that loci fall into four categories: loci associated with nearly all pain-related traits; loci primarily associated with a single trait; loci associated with multiple forms of skeletomuscular pain; and loci associated with headache-related pain. Overall, 664 genes were mapped to the 99 loci by genomic proximity, eQTLs, and chromatin interaction and ~15% of these genes showed differential expression in individuals with acute or chronic pain compared to healthy controls. Risk loci were enriched for genes involved in neurological and inflammatory pathways. Genetic correlation and two-sample Mendelian randomization indicated that psychiatric, metabolic, and immunological traits mediate some of these effects.

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.

The Canadian version of the National Institutes of Health minimum dataset for chronic low back pain research: reference values from the Quebec Low Back Pain Study.

ABSTRACT: The National Institutes of Health (NIH) minimum dataset for chronic low back pain (CLBP) was developed in response to the challenge of standardizing measurements across studies. Although reference values are critical in research on CLBP to identify individuals and communities at risk of poor outcomes such as disability, no reference values have been published for the Quebec (Canada) context. This study was aimed to (1) provide reference values for the Canadian version of the NIH minimum dataset among individuals with CLBP in Quebec, both overall and stratified by gender, age, and pain impact stratification (PIS) subgroups, and (2) assess the internal consistency of the minimum data set domains (pain interference, physical function, emotional distress or depression, sleep disturbance, and PIS score). We included 2847 individuals living with CLBP who completed the baseline web survey of the Quebec Low Back Pain Study (age: 44.0 ± 11.2 years, 48.1% women) and were recruited through social media and healthcare settings. The mean score was 6.1 ± 1.8 for pain intensity. Pain interference, physical function, emotional distress or depression, sleep disturbance, and PIS scores were 12.9 ± 4.1, 14.4 ± 3.9, 9.8 ± 4.4, 13.0 ± 3.6, and 26.4 ± 6.6, respectively. Emotional distress or depression showed floor effects. Good-to-excellent internal consistency was found overall and by language, gender, and age subgroups for all domains (alpha: 0.81-0.93) and poor-to-excellent internal consistency for PIS subgroups (alpha: 0.59-0.91). This study presents reference values and recommendations for using the Canadian version of the NIH minimum dataset for CLBP that can be useful for researchers and clinicians.

Macrophages and Intervertebral Disc Degeneration.

The intervertebral disc (IVD) aids in motion and acts to absorb energy transmitted to the spine. With little inherent regenerative capacity, degeneration of the intervertebral disc results in intervertebral disc disease, which contributes to low back pain and significant disability in many individuals. Increasing evidence suggests that IVD degeneration is a disease of the whole joint that is associated with significant inflammation. Moreover, studies show elevated macrophage accumulation within the IVD with increasing levels of disease severity; however, we still need to understand the roles, be they causative or consequential, of macrophages during the degenerative process. In this narrative review, we discuss hallmarks of IVD degeneration, showcase evidence of macrophage involvement during disc degeneration, and explore burgeoning research aimed at understanding the molecular pathways regulating macrophage functions during intervertebral disc degeneration.

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.

Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms.

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.

The Role of Surface Chemistry in the Osseointegration of PEEK Implants.

Poly(etheretherketone) (PEEK) implants suffer from poor osseointegration because of chronic inflammation. In this study, we hypothesized that adding NH2 and COOH groups to the surface of PEEK could modulate macrophage responses by altering protein adsorption and improve its osseointegration. NH2 and COOH-functionalized PEEK surfaces induced pro- and anti-inflammatory macrophage responses, respectively, and differences in protein adsorption patterns on these surfaces were related to the varied inflammatory responses. The macrophage responses to NH2 surfaces significantly reduced the osteogenic differentiation of mesenchymal stem cells (MSCs). MSCs cultured on NH2 surfaces differentiated less than those on COOH surfaces even though NH2 surfaces promoted the most mineralization in simulated body fluid solutions. After 14 days in rat tibia unicortical defects, the bone around NH2 surfaces had thinner trabeculae and higher specific bone surface than the bone around unmodified implants; surprisingly, the NH2 implants significantly increased bone-binding over the unmodified implants, while COOH implants only showed a trend for increasing bone-binding. Taken together, these results suggest that both mineral-binding and immune responses play a role in osseointegration, and PEEK implant integration may be improved with mixtures of these two functional groups to harness the ability to reduce inflammation and bind bone strongly.

Low back pain definitions: effect on patient inclusion and clinical profiles.

Introduction: Numerous definitions of acute low back pain (aLBP) exist. The use of different definitions results in variability in reported prevalence or incidence, conflicting data regarding factors associated with the transition to chronic LBP (cLBP), and hampers comparability among studies. Objective: Here, we compare the impact of 3 aLBP definitions on the number of aLBP cases and participants' characteristics and explore the distribution of participants across definitions. Methods: A sample of 1264 participants from the Quebec Low Back Pain Study was included. Three definitions of aLBP were used: (1) not meeting the National Institutes of Health (NIH) cLBP definition ("nonchronic"), (2) pain beginning <3 months ago ("acute"), and (3) pain beginning <3 months with a preceding LBP-free period ("new episode"). Results: There were 847, 842, and 489 aLBP cases meeting the criteria for the 3 definitions, respectively. Participants included in the "nonchronic" had lower pain interference, greater physical function scores, and fewer participants reporting >5 years of pain than in the other definitions. Half the participants meeting the "acute" definition and one-third of participants meeting the "new episode" definition were also classified as cLBP based on the NIH definition. Conclusions: Our results highlight the importance of the definition used for aLBP. Different definitions influence the sample size and clinical profiles (group's characteristics). We recommended that cohort studies examining the transition from aLBP to cLBP ensure that the definitions selected are mutually exclusive (ie, participants included [aLBP] differ from the expected outcome [cLBP]).

Epigenetic signature of chronic low back pain in human T cells.

OBJECTIVE: Determine if chronic low back pain (LBP) is associated with DNA methylation signatures in human T cells that will reveal novel mechanisms and potential therapeutic targets and explore the feasibility of epigenetic diagnostic markers for pain-related pathophysiology. METHODS: Genome-wide DNA methylation analysis of 850,000 CpG sites in women and men with chronic LBP and pain-free controls was performed. 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. RESULTS: Analysis with the discovery cohort revealed a total of 2,496 and 419 differentially methylated CpGs in women and men, respectively. In women, most of these sites were hypomethylated and enriched in genes with functions in the extracellular matrix, in the immune system (ie, cytokines), or in epigenetic processes. In men, a unique chronic LBP DNA methylation signature was identified characterized by significant enrichment for genes from the major histocompatibility complex. Sex-specific polygenic DNA methylation scores were generated to estimate the pain status of each individual and confirmed in the validation cohort using pyrosequencing. CONCLUSION: This study reveals sex-specific DNA methylation signatures in human T cells that discriminates chronic LBP participants from healthy controls.

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.

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.

Protein Adsorption on Surfaces Functionalized with COOH Groups Promotes Anti-inflammatory Macrophage Responses.

Implants can induce a foreign body reaction that leads to chronic inflammation and fibrosis in the surrounding tissue. Macrophages help detect the foreign material, play a role in the inflammatory response, and may promote fibrosis instead of the desired tissue regeneration around implants. Implant surface properties impact macrophage responses by changing the nature of the adsorbed protein layer, but conflicting studies highlight the complexity of this relationship. In this study, the effect of surface chemistry on macrophage behavior was investigated with poly(styrene) surfaces containing common functional groups at similar surface densities. The protein layer was characterized to identify the proteins that adsorbed on the surfaces from the medium and the proteins secreted onto the surfaces by adherent macrophages. Of the surface chemistries studied, carboxylic acid (COOH) groups promoted anti-inflammatory responses from unstimulated macrophages and did not exacerbate inflammation upon stimulation. These surfaces also enhanced the adsorption of proteins involved in integrin signaling and promoted the secretion of proteins related to angiogenesis, integrin signaling, and cytokine signaling, which have been previously associated with improved biomaterial integration. Therefore, this study suggests that surface modification with COOH groups may help improve the integration of implants in the body by enhancing anti-inflammatory macrophage responses through altered protein adsorption.