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Methods

Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)‐hydroxy‐docosahexaenoic acid (17[R]‐HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)‐HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry.

Results

There was a positive correlation between resolvin receptor and interleukin‐6 (IL‐6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL‐1β, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)‐HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate–induced OA rat model.

Animals and model induction

Animal experiments were approved by the Nottingham University ethics committee, and all procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and conform to the guidelines of the International Association for the Study of Pain. Adult male Sprague‐Dawley rats (n = 166) were used (Charles River). All procedures and testing were performed in a blinded manner. The model of monosodium iodoacetate (MIA)–induced OA pain was generated as previously described . The medial meniscal transection (MNX) induction model of OA pain was based on previously described methods (see also Supplementary Methods, available on the Arthritis & Rheumatology web site at ).

Pharmacologic interventions and assessment of pain behavior

The 17(R)‐HDoHE (stock solution 100 μg/ml in ethanol) was diluted in normal sterile saline to provide a concentration of 1 ng/μl. The vehicle solution consisted of 1% ethanol solution in 99% saline. A series of different pharmacologic studies were performed using 300 ng of 17(R)‐HDoHE in 300 μl saline administered intraperitoneally. Study 1 determined the effects of a single injection of 17(R)‐HDoHE on pain behavior on day 14 after induction of the model. Study 2 determined the effects of repeated administration of 17(R)‐HDoHE (300 ng in 300 μl saline, every other day from day 14 until day 28 after induction of the model) on pain behavior in the MIA‐induced OA pain model and the MNX induction model of OA pain. Study 3 determined the effects of discontinuous administration of 17(R)‐HDoHE (300 ng in 300 μl saline, from day 14 to day 22 after induction of the model) on pain behavior, quantified until day 35 after induction of the model. All of the drug intervention studies were conducted in a blinded manner.

Reversal of MIA‐ and MNX‐induced OA pain by the D series precursor 17(R)‐HDoHE

To further investigate the potential mechanisms underlying the effects of17(R)‐HDoHE on OA‐induced pain behavior, the effects of repeated treatment with 17(R)‐HDoHE on joint pathology were determined (Figures A–C). Intraarticular injection of MIA was associated with a significant increase in chondropathy, synovitis, and chondrocyte appearance and increased numbers of subchondral osteoclasts (Figures D–G). Following repeated administration of 17(R)‐HDoHE (300 ng in 300 μl every other day from day 14 to day 28) there were no significant changes in any of these features of OA joint pathology (Figures D–G). Similarly, repeated treatment with 17(R)‐HDoHE did not alter MNX‐induced joint pathology (data not shown).

Spinal effects of 17(R)‐HDoHE correlated with behavioral analgesia

We previously reported a significant increase in GFAP immunofluorescence, a marker for astrogliosis, in the spinal cord at later time points in the MIA‐induced model of OA . Consistent with previous findings, GFAP immunofluorescence was significantly increased in the ipsilateral dorsal horn (Figures E and F) but not the contralateral dorsal horn (data not shown) of MIA‐treated rats that received vehicle, compared with saline‐treated controls. Repeated systemic administration of 17(R)‐HDoHE (300 ng in 300 μl every other day from day 14 to day 28) significantly inhibited GFAP immunofluorescence in the ipsilateral dorsal horn of the spinal cord in MIA‐treated rats, compared with vehicle‐treated rats with MIA‐induced OA pain (Figures E and F). Correlation analysis revealed that spinal GFAP expression was positively correlated with weight‐bearing asymmetry (Figure G) and negatively correlated with ipsilateral paw withdrawal thresholds (Figure H).

Methods

Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)‐hydroxy‐docosahexaenoic acid (17[R]‐HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)‐HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry.

Results

There was a positive correlation between resolvin receptor and interleukin‐6 (IL‐6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL‐1β, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)‐HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate–induced OA rat model.

Animals and model induction

Animal experiments were approved by the Nottingham University ethics committee, and all procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and conform to the guidelines of the International Association for the Study of Pain. Adult male Sprague‐Dawley rats (n = 166) were used (Charles River). All procedures and testing were performed in a blinded manner. The model of monosodium iodoacetate (MIA)–induced OA pain was generated as previously described . The medial meniscal transection (MNX) induction model of OA pain was based on previously described methods (see also Supplementary Methods, available on the Arthritis & Rheumatology web site at ).