Immuno-spin trapping after Intraarticular Procedures Shows Coordination between Radical Production, Iron Metabolism, and Mitochondria in Articular Chondrocytes

Prior examinations of pathological effects of surgery have focused on inflammation. We sought to capture the effect of surgical injury on joint redox status. Endpoints were assessed in C57B6J mice 24 h after destabilization of the medial meniscus (DMM) using markers of protein oxidation alongside a non-biased measure of oxidative stress independent of individual reactive oxygen species. DMPO mean intensity, a marker of radical production, was analyzed using generalized linear mixed models with FDR-adjusted pairwise comparisons of marginal means between DMM and paired CC limbs. Potential differences between paired DMM and CC limbs in percentage DMPO-positive cells were analyzed with Wilcoxon signed-rank tests. The remaining redox and mitochondrial markers were compared between independent samples using Kruskal-Wallis tests with FDR-adjusted post hoc Wilcoxon rank sum tests. The highest basal radical production (DMPO mean intensity) in CC limbs was seen in synovium (mean (95% confidence interval) 35.8(26.8-48.0)AU, followed by cartilage (57.2(48.8-67)AU, p=0.008) then meniscus (6.5(3.9-10.9)AU, p<0.001) tissue. Paired DMM limbs had similar values for both synovium (38.8(31.1-48.4)AU), p=0.655) and meniscus (8.3(6.0-11.7)AU, p=0.493) but lower values for cartilage tissue (43.4(36.4-41.7), p=0.037) compared with CC limbs. DMM decreased chondrocyte radicals with minimal impact elsewhere. Intraarticular injections of prospective treatment vehicles constructed with xanthan gum (XG+DMM) or hyaluronic acid (HA+DMM) were examined. Compared to DMM, XG+DMM had greater protein nitration (median[IQR] XG+DMM 53.6[33.2-69.7] vs DMM=32.0[25.2-38.1], p=0.064) and oxidative stress (XG+DMM 37.0[33.5-51.8] vs DMM 26.0[25.1-27.3], p=0.009), but decreased synovial integrity (XG+DMM 41.7[32.2-46.4] vs DMM 67.6[54.1-75.6], p<0.001). In contrast, HA pretreatment was protective of cartilage oxidative function, preserving radical production (CC 58.4[55.4-63.9]; DMM 40.9[33.3-48.3]; HA+DMM 62.4[34.6-64.1], p=0.113 for DMM vs HADMM), protein nitration (CC 38[37-43]; DMM 8.0[7.0-12.0]; HA+DMM 44.0[7.0-65.0], p=0.071 for DMM vs HADMM), iron metabolism (CC 35[29.8-39.5]; DMM 4.5[1.0-29.0]; HA+DMM 73.5[34.0-85.5], p=0.024 for DMM vs HA+DMM), and mitochondrial staining (CC 20.5[17.8-23.9]; DMM 6.9 [3.2-11.5]; HA+DMM 32.4[17.9-35.0], p=0.024) for DMM vs HA+DMM. Redox effects from surgery in mice are more nuanced than might be expected, with decreases in chondrocyte oxidative metabolism the most apparent outcome. We propose perioperative HA treatment might blunt the negative effects intraarticular surgery.