Clinical-Like Cryotherapy in Acute Knee Arthritis Protects Neuromuscular Junctions of Quadriceps and Reduces Joint Inflammation in Mice

Rheumatoid arthritis is an autoimmune and inflammatory disease that affects synovial joint tissues and skeletal muscle. Clinical-like cryotherapy benefits signs of joint inflammation in knee osteoarthritis after 60 days of anterior cruciate ligament transection surgery. However, it is unknown whether it also benefits acute knee arthritis (e.g., reduces inflammatory process and protects neuromuscular junction [NMJ] and muscle fibers). We aimed to analyze the effects of clinical-like cryotherapy on NMJ and quadriceps muscle fibers in a model of acute knee arthritis. Twenty-four male C57BL/6 mice (20 to 25 g) were randomly allocated into three groups: control (mice with no intervention), antigen-induced arthritis (AIA; mice sensitized and immunized with intra-articular [i.a.] injection of methylated bovine serum albumin [mBSA]), and AIA+cryotherapy (mice sensitized, immunized with i.a. injection of mBSA, and submitted to a clinical-like cryotherapy protocol). Twenty-one days after sensitization, arthritis was induced in immunized mice via i.a. injection of mBSA (100 mu g/joint). Two clinical-like cryotherapy sessions (crushed ice pack for 20 min) were applied two hours apart. The first session was applied immediately after i.a. injection of mBSA. The quadriceps was removed two hours after the second clinical-like cryotherapy session for morphological analysis of muscle fibers (cross-sectional area), frequency distribution of muscle fiber area (%), and NMJ (area, perimeter, and maximum diameter). Gene expressions of mRNA involved in NMJ signaling (gamma-nAChR, alpha 1-nAChR, epsilon-nAChR, Agrin-MusK-Rapsyn, alpha-dystrobrevin, and utrophin) and atrophy (muscle RING-finger protein-1 and Atrogin-1) pathways were analyzed. Inflammatory signs were assessed in knee joint (swelling, articular surface temperature, and neutrophil migration in synovial fluid). Regarding morphological analysis of muscle fibers, 180 to 270 and >270 mu m(2) classes were higher in the AIA+cryotherapy than the AIA group. Area, perimeter, and maximum diameter of NMJ also increased in the AIA+cryotherapy compared with the control group. Agrin mRNA expression increased in the AIA+cryotherapy compared with the control and AIA groups. In the atrophy pathway, Atrogin-1 increased compared with the control and AIA groups. The AIA+cryotherapy group reduced knee swelling and neutrophil migration compared with the AIA group. In conclusion, clinical-like cryotherapy increased Agrin expression, contributing to NMJ maintenance and increased Atrogin-1 expression, thus protecting NMJ and muscle fiber. Furthermore, clinical-like cryotherapy reduced inflammatory signs (swelling and neutrophil migration) of acute knee arthritis.