Search

Chronic back pain poses a significant health concern. Though back pain is often attributed to disc degeneration (DD), intervertebral disc (IVD) degeneration is frequently experienced without pain. Why does DD lead to discogenic LBP in some cases and remain asymptomatic in others? A growing body of evidence suggests epigenetic modifications, such as DNA methylation, contribute to pathological DD. Understanding DNA methylation in discogenic LBP is crucial due to its reversibility, offering a promising therapeutic target. The goal of this study was to identify DNA methylation patterns associated with painful vs. pain-free DD. In a case involving a subject with LBP and DD at multiple IVD levels, a lumbar discogram was performed before surgery to pinpoint the discs causing pain. Despite severe degeneration, one spinal level (L5/S1) scored 0/10 on the discogram while L3/L4 and L4/L5 produced pain reports of 10/10 and 8/10, respectively. Next, DNA was isolated from the nucleus pulposus of discs collected during surgery. Methylation analysis was conducted using Illumina Infinium MethylationEPIC v2.0 BeadChip. SeSAMe pipeline in R determined methylation levels (beta values). After filtering for greater than 20% effect size, 315 gene-associated promoter sites were identified. Pathway enrichment analysis revealed terms related to nerve growth (e.g., TRK receptor binding, substrate adhesion-dependent cell spreading, netrin-1 signaling), as well as immune processes (e.g., neutrophil extravasation, T-cell polarity, CD28 co-stimulation). These findings suggest a potential epigenetic mechanism behind the disconnect between DD and pain; thus, raising the possibility of diagnostic or therapeutic strategies for discerning or treating painful disc degeneration.

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.

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.

ABSTRACTBackground: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 I2expressed 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.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) and is associated with widespread changes in gene expression, extracellular matrix (ECM) degradation, inflammation, and innervation. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation in degenerated IVDs. The objective of this study was to investigate changes in DNA methylation in degenerating human IVDs from chronic LBP patients. Human lumbar IVD segments at L4/5 level including control (n=4) and degenerating (n=6) were homogenized in liquid nitrogen with a mortar and pestle and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. Epigenome-wide 5mC mapping was performed following bisulfite sequencing using the Infinium MethylationEPIC BeadChip 850k Arrays. All analysis was performed in the R package RnBeads 2.0. Principal component analysis and hierarchical clustering analysis revealed group differences between healthy and degenerating IVDs. Pathway analysis revealed enrichment in genes related to ECM organization, signaling by receptor tyrosine kinases and signaling by interleukins. Further analysis at the single gene promoter level showed hypermethylation in ECM-related genes linked to IVD degeneration including sparc and col2a1 and hypomethylation of pro-inflammatory cytokines associated with IVD degeneration including IL-1b and IL-8. These changes in methylation would be expected to result in loss of ECM and increased inflammation. Persistent reprogramming of gene expression by DNA methylation could drive IVD degeneration and inflammation. Understanding the role of DNA methylation in IVD pathology may provide a scientific framework for the development of new therapeutic approaches.

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 (MS). MS 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 three 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 resolution, or persistence, of LBP symptoms at three months, however, these processes differ between males and females.

Systemically or centrally administered agmatine (decarboxylated arginine) prevents, moderates, or reverses opioid-induced tolerance and self-administration, inflammatory and neuropathic pain, and sequelae associated with ischemia and spinal cord injury in rodents. These behavioral models invoke the N-methyl-D-aspartate (NMDA) receptor/nitric-oxide synthase cascade. Agmatine (AG) antagonizes the NMDA receptor and inhibits nitric-oxide synthase in vitro and in vivo, which may explain its effect in models of neural plasticity. Agmatine has been detected biochemically and immunohistochemically in the central nervous system. Consequently, it is conceivable that agmatine operates in an anti-glutamatergic manner in vivo; the role of endogenous agmatine in the central nervous system remains minimally defined. The current study used an immunoneutralization strategy to evaluate the effect of sequestration of endogenous agmatine in acute opioid analgesic tolerance in mice. First, intrathecal pretreatment with an anti-AG IgG (but not normal IgG) reversed an established pharmacological effect of intrathecal agmatine: antagonism of NMDA-evoked behavior. This result justified the use of anti-AG IgG to sequester endogenous agmatine in vivo. Second, intrathecal pretreatment with the anti-AG IgG sensitized mice to induction of acute spinal tolerance of two micro-opioid receptor-selective agonists, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin and endomorphin-2. A lower dose of either agonist that, under normal conditions, produces moderate or no tolerance was tolerance-inducing after intrathecal pretreatment of anti-AG IgG (but not normal IgG). The effect of the anti-AG IgG lasted for at least 24 h in both NMDA-evoked behavior and the acute opioid tolerance. These results suggest that endogenous spinal agmatine may moderate glutamate-dependent neuroplasticity.

Poly(etheretherketone) (PEEK) implants suffer from poor osseointegration because of chronic inflammation. In this study, we hypothesized that adding NH2and COOH groups to the surface of PEEK could modulate macrophage responses by altering protein adsorption and improve its osseointegration. NH2and 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 NH2surfaces significantly reduced the osteogenic differentiation of mesenchymal stem cells (MSCs). MSCs cultured on NH2surfaces differentiated less than those on COOH surfaces even though NH2surfaces promoted the most mineralization in simulated body fluid solutions. After 14 days in rat tibia unicortical defects, the bone around NH2surfaces had thinner trabeculae and higher specific bone surface than the bone around unmodified implants; surprisingly, the NH2implants 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.

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.

Agonists acting at alpha(2)-adrenergic receptors (alpha(2)ARs) produce antinociception and synergize with opioids. The alpha(2)ARs are divided into three subtypes, alpha(2A)AR, alpha(2B)AR, and alpha(2C)AR. Most alpha(2)AR agonists require alpha(2A)AR activation to produce antinociception and opioid synergy. The same subtype also mediates the side effect of sedation, which limits the clinical utility of these compounds. Identification of a non-alpha(2A)AR-mediated antinociceptive agent would enhance the therapeutic utility of alpha(2)AR agonists in pain management. Previous studies have suggested that the alpha(2)AR agonist ST91 [2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride] has a nonsedating, non-alpha(2A)AR mechanism of action. We examined the pharmacology of spinal ST91 and its interaction with the delta-opioid agonist deltorphin II (Tyr-d-Ala-Phe-Glu-Val-Val-Gly amide) in mice lacking either functional alpha(2A)ARs or alpha(2C)ARs. All drugs were administered by direct lumbar puncture, and drug interactions were evaluated using isobolographic analysis. In contrast to the majority of alpha(2)AR agonists, ST91 potency was only moderately reduced (3-fold) in the absence of the alpha(2A)AR. Studies with the alpha(2)AR subtype-preferring antagonists BRL-44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate) and prazosin [[4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone] and the pan-alpha(2)AR antagonist SKF-86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine) suggest a shift from alpha(2A)AR to the other alpha(2)AR subtype(s) in the absence of alpha(2A)AR. Antinociceptive synergy with deltorphin II was preserved in the absence of either alpha(2A)AR or alpha(2C)AR. In conclusion, ST91 activates both alpha(2A)AR and non-alpha(2A)AR subtypes to produce spinal antinociception and opioid synergy. This study confirms that the spinal pharmacology of ST91 differs from that of other alpha(2)AR agonists and extends those data to include spinal synergy with opioid agonists. The unique profile of ST91 may be advantageous in pain management.

Intrathecal agmatine (decarboxylated arginine) moderates induction of neuropathic pain, spinal cord injury, and opioid tolerance in rodents. An endogenous central nervous system molecule and N-methyl-D-aspartate receptor antagonistnitric oxide synthase inhibitor, agmatine may be a neuromodulator. We evaluated depolarization-induced release of agmatine from purified spinal nerve terminals (synaptosomes). Agmatine immunoreactivity was observed colocalized or closely apposed to some synaptophysin- andor synaptotagmin-labeled structures. A temperature- and concentration-dependent uptake of 3H-agmatine into synaptosomes was observed, consistent with an uptake mechanism. Potassium-induced depolarization resulted in release of 3H- agmatine from the synaptosomes in a Ca2-dependent manner, consistent with a neuromodulatory function. These results agree with previous reports of agmatine uptake into synaptosomes of the brain and extend those results to include stimulated release and a spinal site of activity.

Moxonidine is a mixed a2-adrenergic and imidazoline receptor agonist with an improved side effect profile compared to clonidine. Intrathecal (i.t.) moxonidine has been found to possess analgesic activity that, in contrast to the majority of a2-adrenoceptor (a2AR) agonists, does not require activation of the a2AAR subtype, which mediates many of the side effects associated with a2AR use. In addition, moxonidine (i.t.) interacts in a synergistic manner with opioid agonists and this synergy is retained in neuropathic pain states. Moxonidine may therefore be clinically useful in the treatment of chronic neuropathic pain, either alone or as a coadjuvant with opioids.

To determine the test characteristics of a self-report questionnaire, the Beck Depression Inventory, when used as a screening test for depression in a population of ambulatory pregnant women.

TWO highly-selective μ-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous μ-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.

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.

Introduction Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) levels increase during disc degeneration and evidence suggests NGF promotes disc innervation, neuronal sensitization and low back pain, making NGF a possible therapeutic target. However, current anti-NGF therapeutics have limited efficacy and safety concerns. Mechanisms leading to increased NGF in the disc are poorly understood and the molecular signaling mechanisms regulating NGF in inflammatory mediator rich environments, such as a degenerating disc, are unknown. During degeneration, proteoglycans and extra-cellular matrix (ECM) proteins are degraded and fragmented. ECM fragments can act as endogenous danger signal ligands for toll-like receptors (TLR). TLR activation induces cytokine and chemokine expression, and could thus regulate expression of inflammatory mediators, such as IL-1β or TNFα, during disc degeneration. NGF is often increased in environments with elevated levels of inflammatory mediators. Therefore, we hypothesized that TLR activation in the disc induces NGF expression.Materials and Methods Non-degenerate human intervertebral discs from organ donors without a history of low back pain were collected for cell isolation. NP and AF tissues were separated and enzymatically digested. Cells were treated with IL-1β (control), peptidoglycan (PGN, TLR2 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Neutralizing antibodies against TLR2 were used to prevent TLR2 activation. Activated cell-signaling pathways following IL-1β and PGN treatment were evaluated by western blot and immunofluorescence. Small molecule inhibitors blocking p38 MAPK (SB203580) and NF-κB (BMS-345541) activity were added to cell cultures in combination with IL-1β and PGN. NGF gene expression was evaluated by qRT-PCR after 6, 12 and 24 hours, and NGF protein levels were examined by western blot and ELISA after 48 hours.Results TLR 2 activation significantly increased NGF gene expression in NP and AF cells following 6, 12 and 24 hours of treatment, while TLR 4 activation had little effect on NGF expression. TLR 2 activation significantly increased NGF protein secretion after 48 hours in NP and AF cells while TLR 4 activation did not increase NGF protein levels. TLR 2 neutralization with antibodies showed that TLR 2 is required for PGN induced NGF expression. TLR 2 activation of the p38 MAPK, ERK1/2, JNK and NF-κB signaling pathways was analyzed using phosphorylation specific antibodies. TLR activation increased p38 MAPK, ERK1/2 and p65 (NF- κB pathway) phosphorylation in disc cells compared with untreated cells. Furthermore, immunofluorescence found that TLR activation induces p65 translation to the nucleus, indicative of NF-κB signaling. Inhibiting NF-κB signaling decreased TLR 2 induced NGF expression.Conclusion This study found that TLR 2 activation directly regulates NGF expression via NF-κB signaling in human intervertebral disc cells. These findings represent a novel regulatory mechanism of NGF in the IVD. In the central nervous system NF-κB distinct signaling pathways regulate NGF. Therefore, these finding may provide therapeutic strategies to target NGF and low back pain without affecting the central nervous system.