Serum levels of hydroxylated metabolites of arachidonic acid cross-sectionally and longitudinally predict knee pain progression: an observational cohort study.

OBJECTIVE: To examine associations between serum oxylipins, which regulate tissue repair and pain signalling, and knee pain/radiographic osteoarthritis (OA) at baseline and knee pain at 3 year follow-up. METHOD: Baseline, and 3 year follow-up, knee pain phenotypes were assessed from 154 participants in the Knee Pain in the Community (KPIC) cohort study. Serum and radiographic Kellgren and Lawrence (KL) and Nottingham line drawing atlas OA scores were collected at baseline. Oxylipin levels were quantified using liquid chromatography coupled with mass spectrometry. Associations were measured by linear regression and receiver operating characteristics (ROC). RESULTS: Serum levels of 8,9-epoxyeicosatrienoic acid (EET) (ß(95% confidence intervals (CI)) = 1.809 (-0.71 to 2.91)), 14,15-dihydroxyeicosatrienoic acid (DHET) (ß(95%CI) = 0.827 (0.34-1.31)), and 12-hydroxyeicosatetraenoic acid (HETE) (ß(95%CI) = 4.090 (1.92-6.26)) and anandamide (ß(95%CI) = 3.060 (1.35-4.77)) were cross-sectionally associated with current self-reported knee pain scores (numerical rating scale (NRS) item 3, average pain). Serum levels of 9- (ß(95%CI) = 0.467 (0.18-0.75)) and 15-HETE (ß(95%CI) = 0.759 (0.29-1.22)), 14-hydroxydocosahexaenoic acid (ß(95%CI) = 0.483(0.24-0.73)), and the ratio of 8,9-EET:DHET (ß(95%CI) = 0.510(0.19-0.82)) were cross-sectionally associated with KL scores. Baseline serum concentrations of 8,9-EET (ß(95%CI) = 2.166 (0.89-3.44)), 5,6-DHET (ß(95%CI) = 152.179 (69.39-234.97)), and 5-HETE (ß(95%CI) = 1.724 (0.677-2.77) showed positive longitudinal associations with follow-up knee pain scores (NRS item 3, average pain). Combined serum 8,9-EET and 5-HETE concentration showed the strongest longitudinal association (ß(95%CI) = 1.156 (0.54-1.77) with pain scores at 3 years, and ROC curves distinguished between participants with no pain and high pain scores at follow-up (area under curve (95%CI) = 0.71 (0.61-0.82)). CONCLUSIONS: Serum levels of a combination of hydroxylated metabolites of arachidonic acid may have prognostic utility for knee pain, providing a potential novel approach to identify people who are more likely to have debilitating pain in the future.

The challenges of treating osteoarthritis pain and opportunities for novel peripherally directed therapeutic strategies.

Osteoarthritis (OA) is a chronic joint disease that represents an increasingly substantial global burden. Joint pain is the most significant symptom of OA. Unfortunately, current pharmacological treatments for OA pain are often not wholly efficacious, or are associated with serious adverse effects. This lack of effective pain relief has seen the prescription of opioids for OA pain increase over the past decades. The long-term adverse effects of prescribed opioids alongside the increasing prevalence of OA pain highlights the need for alternative analgesics. Understanding the mechanisms that drive this chronic joint pain is crucial for the development of novel analgesics. OA is a heterogeneous disease, and this is reflected by the diversity of pain phenotypes in people with the disease. Herein, we review current understanding of the biological changes at the joint and within the central nervous system that drive this chronic pain. We particularly focus on the most recent advances in our understanding of the peripheral nociceptive mechanisms that underlie chronic OA pain and highlight how targeting peripheral OA inflammation may open up opportunities for novel analgesics.

Interplay between cellular changes in the knee joint, circulating lipids and pain behaviours in a slowly progressing murine model of osteoarthritis.

BACKGROUND: Synovial inflammation has known contributions to chronic osteoarthritis (OA) pain, but the potential role in transitions from early to late stages of OA pain is unclear. METHODS: The slowly progressing surgical destabilization of the medial meniscus (DMM) murine OA model and sham control, was used in male C57BL/6J mice to investigate the interplay between knee inflammation, plasma pro- and anti-inflammatory oxylipins and pain responses during OA progression. Changes in joint histology, macrophage infiltration, chemokine receptor CX3CR1 expression, weight bearing asymmetry, and paw withdrawal thresholds were quantified 4, 8 and 16 weeks after surgery. Plasma levels of multiple bioactive lipid mediators were quantified using liquid chromatography with tandem mass-spectrometry (LC-MS/MS). RESULTS: Structural joint damage was evident at 8 weeks post-DMM surgery onwards. At 16 weeks post-DMM surgery, synovial scores, numbers of CD68 and CD206 positive macrophages and pain responses were significantly increased. Plasma levels of oxylipins were negatively correlated with joint damage and synovitis scores at 4 and 8 weeks post-DMM surgery. Higher circulating levels of the pro-resolving oxylipin pre-cursor 17-HDHA were associated with lower weight bearing asymmetry at week 16. CONCLUSIONS: The transition to chronic OA pathology and pain is likely influenced by both joint inflammation and plasma oxylipin mediators of inflammation and levels of pro-resolution molecules. SIGNIFICANCE: Using a slow progressing surgical model of osteoarthritis we show how the changing balance between local and systemic inflammation may be of importance in the progression of pain behaviours during the transition to chronic osteoarthritis pain.

Clinical and Preclinical Evidence for Roles of Soluble Epoxide Hydrolase in Osteoarthritis Knee Pain.

OBJECTIVE: Chronic pain due to osteoarthritis (OA) is a major clinical problem, and existing analgesics often have limited beneficial effects and/or adverse effects, necessitating the development of novel therapies. Epoxyeicosatrienoic acids (EETs) are endogenous antiinflammatory mediators, rapidly metabolized by soluble epoxide hydrolase (EH) to dihydroxyeicosatrienoic acids (DHETs). We undertook this study to assess whether soluble EH-driven metabolism of EETs to DHETs plays a critical role in chronic joint pain associated with OA and provides a new target for treatment. METHODS: Potential associations of chronic knee pain with single-nucleotide polymorphisms (SNPs) in the gene-encoding soluble EH and with circulating levels of EETs and DHETs were investigated in human subjects. A surgically induced murine model of OA was used to determine the effects of both acute and chronic selective inhibition of soluble EH by N-[1-(1-oxopropy)-4-piperidinyl]-N'-(trifluoromethoxy)phenyl]-urea (TPPU) on weight-bearing asymmetry, hind paw withdrawal thresholds, joint histology, and circulating concentrations of EETs and DHETs. RESULTS: In human subjects with chronic knee pain, 3 pain measures were associated with SNPs of the soluble EH gene EPHX2, and in 2 separate cohorts of subjects, circulating levels of EETs and DHETs were also associated with 3 pain measures. In the murine OA model, systemic administration of TPPU both acutely and chronically reversed established pain behaviors and decreased circulating levels of 8,9-DHET and 14,15-DHET. EET levels were unchanged by TPPU administration. CONCLUSION: Our novel findings support a role of soluble EH in OA pain and suggest that inhibition of soluble EH and protection of endogenous EETs from catabolism represents a potential new therapeutic target for OA pain.

Anxiety enhances pain in a model of osteoarthritis and is associated with altered endogenous opioid function and reduced opioid analgesia.

INTRODUCTION: Negative affect, including anxiety and depression, is prevalent in chronic pain states such as osteoarthritis (OA) and associated with greater use of opioid analgesics, potentially contributing to present and future opioid crises. OBJECTIVES: We tested the hypothesis that the interaction between anxiety, chronic pain, and opioid use results from altered endogenous opioid function. METHODS: A genetic model of negative affect, the Wistar-Kyoto (WKY) rat, was combined with intra-articular injection of monosodium iodoacetate (MIA; 1 mg) to mimic clinical presentation. Effects of systemic morphine (0.5-3.5 mg·kg-1) on pain behaviour and spinal nociceptive neuronal activity were compared in WKY and normo-anxiety Wistar rats 3 weeks after MIA injection. Endogenous opioid function was probed by the blockade of opioid receptors (0.1-1 mg·kg-1 systemic naloxone), quantification of plasma ß-endorphin, and expression and phosphorylation of spinal mu-opioid receptor (MOR). RESULTS: Monosodium iodoacetate-treated WKY rats had enhanced OA-like pain, blunted morphine-induced analgesia, and greater mechanical hypersensitivity following systemic naloxone, compared with Wistar rats, and elevated plasma ß-endorphin levels compared with saline-treated WKY controls. Increased MOR phosphorylation at the master site (serine residue 375) in the spinal cord dorsal horn of WKY rats with OA-like pain (P = 0.0312) indicated greater MOR desensitization. CONCLUSIONS: Reduced clinical analgesic efficacy of morphine was recapitulated in a model of high anxiety and OA-like pain, in which endogenous opioid tone was altered, and MOR function attenuated, in the absence of previous exogenous opioid ligand exposure. These findings shed new light on the mechanisms underlying the increased opioid analgesic use in high anxiety patients with chronic pain.

Refining surgical models of osteoarthritis in mice and rats alters pain phenotype but not joint pathology.

The relationship between osteoarthritis (OA) structural change and pain is complex. Surgical models of OA in rodents are often rapid in onset, limiting mechanistic utility and translational validity. We aimed to investigate the effect of refining surgical small rodent models of OA on both joint pathology and pain behaviour. Adult male C57BL/6 mice (n = 76, 10-11 weeks of age at time of surgery) underwent either traditional (transection of the medial meniscotibial ligament [MMTL]) or modified (MMTL left intact, transection of the coronary ligaments) DMM surgery, or sham surgery. Adult male Sprague Dawley rats (n = 76, weight 175-199g) underwent either modified meniscal transection (MMNX) surgery (transection of the medial meniscus whilst the medial collateral ligament is left intact) or sham surgery. Pain behaviours (weight bearing asymmetry [in mice and rats] and paw withdrawal thresholds [in rats]) were measured pre-surgery and weekly up to 16 weeks post-surgery. Post-mortem knee joints were scored for cartilage damage, synovitis, and osteophyte size. There was a significant increase in weight bearing asymmetry from 13 weeks following traditional, but not modified, DMM surgery when compared to sham operated mice. Both traditional and modified DMM surgery led to similar joint pathology. There was significant pain behaviour from 6 weeks following MMNX model compared to sham operated control rats. Synovitis was significant 4 weeks after MMNX surgery, whereas significant chondropathy was first evident 8 weeks post-surgery, compared to sham controls. Pain behaviour is not always present despite significant changes in medial tibial plateau cartilage damage and synovitis, reflecting the heterogeneity seen in human OA. The development of a slowly progressing surgical model of OA pain in the rat suggests that synovitis precedes pain behaviour and that chondropathy is evident later, providing the foundations for future mechanistic studies into the disease.

Peripheral brain-derived neurotrophic factor contributes to chronic osteoarthritis joint pain.

Brain-derived neurotrophic factor (BDNF) and the high-affinity receptor tropomyosin receptor kinase B (TrkB) have important roles in neuronal survival and in spinal sensitization mechanisms associated with chronic pain. Recent clinical evidence also supports a peripheral role of BDNF in osteoarthritis (OA), with synovial expression of TrkB associated with higher OA pain. The aim of this study was to use clinical samples and animal models to explore the potential contribution of knee joint BDNF/TrkB signalling to chronic OA pain. Brain-derived neurotrophic factor and TrkB mRNA and protein were present in knee synovia from OA patients (16 women, 14 men, median age 67 years [interquartile range: 61-73]). There was a significant positive correlation between mRNA expression of NTRK2 (TrkB) and the proinflammatory chemokine fractalkine in the OA synovia. Using the surgical medial meniscal transection (MNX) model and the chemical monosodium iodoacetate (MIA) model of OA pain in male rats, the effects of peripheral BDNF injection, vs sequestering endogenous BDNF with TrkB-Fc chimera, on established pain behaviour were determined. Intra-articular injection of BDNF augmented established OA pain behaviour in MIA rats, but had no effect in controls. Intra-articular injection of the TrkB-Fc chimera acutely reversed pain behaviour to a similar extent in both models of OA pain (weight-bearing asymmetry MIA: -11 ± 4%, MNX: -12 ± 4%), compared to vehicle treatment. Our data suggesting a contribution of peripheral knee joint BDNF/TrkB signalling in the maintenance of chronic OA joint pain support further investigation of the therapeutic potential of this target.

Cell type-specific super-resolution imaging reveals an increase in calcium-permeable AMPA receptors at spinal peptidergic terminals as an anatomical correlate of inflammatory pain.

Spinal hyperexcitability is a key event in the development of persistent pain, and arises partly from alterations in the number and localization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors. However, determining precisely where these changes occur is challenging due to the requirement for multiplex labelling and nanoscale resolution. The recent development of super-resolution light microscopy provides new tools to address these challenges. Here, we apply combined confocal/direct STochastic Optical Reconstruction Microscopy (dSTORM) to reveal changes in calcium-permeable subunits of AMPA-type glutamate receptors (GluA1) at identified spinal cord dorsal horn (SCDH) peptidergic axon terminals in a model of inflammatory pain. L4/5 lumbar spinal cord was collected from adult male C57BL/6J mice 24 hours after unilateral hind paw injection of saline or 1% carrageenan (n = 6/group). Tissue was immunolabelled for markers of peptidergic axon terminals (substance P; SP), presynaptic active zones (Bassoon), and GluA1. Direct stochastic optical reconstruction microscopy revealed a 59% increase in total GluA1 immunolabelling in the SCDH in the carrageenan group, which was not detected by confocal microscopy. Cell type-specific analyses identified a 10-fold increase in GluA1 localized to SP structures, and identified GluA1 nanodomains that scaled with behavioural hypersensitivity, and were associated with synaptic release sites. These findings demonstrate that dSTORM has the sensitivity and power to detect nanoscale anatomical changes in the SCDH, and provides new evidence for synaptic insertion of GluA1-AMPA-Rs at spinal peptidergic nociceptive terminals in a model of inflammatory pain.

The impact of anxiety on chronic musculoskeletal pain and the role of astrocyte activation.

Anxiety and depression are associated with increased pain responses in chronic pain states. The extent to which anxiety drives chronic pain, or vice versa, remains an important question that has implications for analgesic treatment strategies. Here, the effect of existing anxiety on future osteoarthritis (OA) pain was investigated, and potential mechanisms were studied in an animal model. Pressure pain detection thresholds, anxiety, and depression were assessed in people with (n = 130) or without (n = 100) painful knee OA. Separately, knee pain and anxiety scores were also measured twice over 12 months in 4730 individuals recruited from the general population. A preclinical investigation of a model of OA pain in normo-anxiety Sprague-Dawley (SD) and high-anxiety Wistar Kyoto (WKY) rats assessed underlying neurobiological mechanisms. Higher anxiety, independently from depression, was associated with significantly lower pressure pain detection thresholds at sites local to (P < 0.01) and distant from (P < 0.05) the painful knee in patients with OA. Separately, high anxiety scores predicted increased risk of knee pain onset in 3274 originally pain-free people over the 1-year period (odds ratio = 1.71; 95% confidence interval = 1.25-2.34, P < 0.00083). Similarly, WKY rats developed significantly lower ipsilateral and contralateral hind paw withdrawal thresholds in the monosodium iodoacetate model of OA pain, compared with SD rats (P = 0.0005). Linear regressions revealed that baseline anxiety-like behaviour was predictive of lowered paw withdrawal thresholds in WKY rats, mirroring the human data. This augmented pain phenotype was significantly associated with increased glial fibrillary acidic protein immunofluorescence in pain-associated brain regions, identifying supraspinal astrocyte activation as a significant mechanism underlying anxiety-augmented pain behaviour.

Analgesic effects of the cathepsin K inhibitor L-006235 in the monosodium iodoacetate model of osteoarthritis pain.

INTRODUCTION: The mounting evidence that osteoclasts play an important role in osteoarthritis (OA) pain lead us to investigate the effects of L-006235, a potent and selective inhibitor of cathepsin K, on pain behaviour and joint pathology in a model of OA pain. METHODS: Effects of preventative (30 and 100 mg/kg) and therapeutic (100 mg/kg) oral dosing with L-006235 on weight-bearing asymmetry, hind paw withdrawal thresholds, cartilage and bone pathology, synovial inflammation, and drug exposure were studied in the monosodium iodoacetate rat model of OA pain. RESULTS: Preventative L-006235 inhibited weight-bearing asymmetry from day 14, with this measure nearly abolished by the higher dose. In the same treatment setting, L-006235 prevented lowering of hind paw withdrawal thresholds from day 7. Exposure to L-006235 in plasma was higher for the 100 mg/kg dose, compared with 30 mg/kg. Therapeutic dosing with L-006235 from day 14 significantly inhibited weight-bearing asymmetry, compared with monosodium iodoacetate vehicle rats. Regression analysis revealed a significant interaction coefficient of the effects of L-006235 on weight-bearing asymmetry and synovitis score, but not for cartilage damage nor osteophyte scores. CONCLUSION: Our novel finding that cathepsin K inhibition is analgesic in a clinically relevant model of OA pain provides new evidence for the therapeutic potential of this target.