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Background: Neurocytokines may upregulate or downregulate neuropathic pain. We hypothesized that dextrose (D-glucose) injections for therapeutic purposes (dextrose prolotherapy: DPT) in painful knee osteoarthritis (KOA) would favorably affect synovial-fluid neurocytokine concentrations. Methods: Twenty participants with grade IV symptomatic KOA received synovial-fluid aspiration followed by dextrose or simulated dextrose injections, followed by the reverse after one week. All participants then received open-label dextrose injections monthly for 6 months, with serial assessments of walking pain at 20 min for 9 months, as well as synovial-neurocytokine-concentration measurements (calcitonin gene-related peptide, substance P (SP), and neuropeptide Y (NPY)) at one week and three months. Results: Clinically important analgesia was observed at 20 min and for 9 months post dextrose injection. One -week synovial-fluid SP concentration rose by 111% (p = 0.028 within groups and p = 0.07 between groups) in the dextrose-injected knees compared to synovial-fluid aspiration only. Three-month synovial-fluid NPY concentration dropped substantially (65%; p < 0.001) after open-label dextrose injection in all knees. Conclusions: Prompt and medium-term analgesia after intra-articular dextrose injection in KOA was accompanied by potentially favorable changes in synovial-fluid neurocytokines SP and NPY, respectively, although these changes were isolated. Including neurocytokines in future assessments of DPT to elucidate mechanisms of action is recommended. Full article