Associations between preoperative clinical data and radiological osteoarthritis with glutamate, interleukin-6, receptor activator of nuclear factor kappa-b ligand, and osteoprotegerin in dogs with naturally occurring cranial cruciate ligament disease

Abstract Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. Objectives 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Methods Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA]. Results canine population (n=14) was of various breeds, aged between 2–10 years and weighing 17.1–45.5Kg; 42.86% male; 57.14% female; 83.33% males and 62.5% females were neutered. Lameness scores varied from 1 and 4 (average 2.07±1.12) and radiographic OA scores from 18 and 36 (average 27.86±5.11). Individual correlations in concentrations with respect to age, weight, lameness score (1–4) and OA scores (15–60) were tested. SF [glu] and lameness score were inversely correlated with higher levels of lameness corresponding to lower SF [glu] (P=0.0141). SF [RANKL] inversely correlated with weight (P=0.0045) and lameness score (P=0.0135), and serum [RANKL] inversely correlated with weight (P=0.0437). There was also a negative correlation between SF and serum [OPG] and weight (P=0.0165 and P=0.0208, respectively). No other significant correlations were detected. Overall, [glu] and [IL-6] are increased in SF compared to serum/plasma, by 12.84 and 1.28, respectively, whereas all the remaining biomarkers are higher (2–3 times) in the serum/plasma compared to SF. Principal component analysis (PCA) and Pearson correlation coefficient matrix [IL-6/glu/RANKL/OPG] (n=7) showed SF [IL-6] correlates with SF [glu] (rs=0.64) and strong positive correlations between SF/serum [RANKL] and SF/serum [OPG] (rs 0.68–0.96). Conclusions Dogs with CrCL disease show an association between the bone remodelling markers RANKL and OPG, and the inflammatory cytokine IL-6, and to a lesser extent SF [glu]. Therapeutics targeting bone remodelling, IL-6 or GluR/[glu] may be of interest for the management of OA in dogs. Declaration of Interest (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.