Musculoskeletal Polytrauma In The Setting of Ischemia/reperfusion Results In Increased Intramuscular Inflammation and Recruitment of Circulatory Macrophages
Michael Sorkin, MD, Charles Hwang, BS, John Li, MD, Nicole Patel, BS, Shuli Li, MD, PhD, Kaetlin Vasquez, MS, Shailesh Agarwal, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Severe musculoskeletal trauma as observed in crush injuries and prolonged ischemia as seen in flap reconstruction and composite tissue allotransplantation is associated with ischemia-reperfusion (IR) injury leading to an aberrant inflammatory response. While smaller muscle injuries heal by complete muscle regeneration, complex injuries result in aberrant healing with muscle fibrosis. However, the inflammatory cascade that is governing this process remains incompletely understood. Recently, neutrophil extracellular traps (NETs), extracellular DNA structures released by neutrophils, have been implicated in the initial phases of ischemic inflammation. In this study, we develop a mouse extremity polytrauma model of combined muscle trauma and IR injury to assess the initial inflammatory response.
METHODS Male C57BL/6 mice were randomized into three treatment groups: 1. Fibrosis model: Cardiotoxin (CTX) injection into the tibialis anterior (known fibrosis model); 2. IR model: hindlimb ischemia by occluding the femoral artery using a microvascular clamp for 3.5 hours followed by reperfusion for up to 5 days 3. Polytrauma model: IR plus CTX injection (n=4 mice/group). Bioluminescent imaging for myeloperoxidase as a readout of inflammation was analyzed as well as neutrophil elastase. Additionally, lower extremity muscle was harvested for flow cytometry to assess inflammatory cell subpopulations and histology.
RESULTS Macroscopic evaluation of muscle specimens revealed an evident area of necrosis and apparent muscle changes while the CTX alone and IR alone muscles appeared normal. In-vivo imaging revealed significantly increased inflammation as measured by myeloperoxidase and neutrophil elastase activity in mice undergoing IR+CTX than either of the other groups. This was further corroborated by flow cytometry where we found differences in inflammatory cell subpopulations with more inflammatory monocytes/macrophages and neutrophils recruited to the muscle injury site in the IR+CTX group (Figure, p(Monocytes: IR+CTX vs. CTX only)=.000002 and p(Neutrophils: IR+CTX vs. CTX only)=.037).
CONCLUSIONS Our findings demonstrate that combined IR+muscle injury (CTX) results in significantly increased muscle injury than either ischemia or muscle injury alone. This was characterized by an elevated inflammatory response with amplified recruitment of pro-inflammatory macrophages and neutrophils. This model can be utilized to examine the effects of musculoskeletal polytrauma on muscle regeneration and fibrosis. A better understanding and description of the inflammatory cascade after extremity IR injury will enable early intervention and prevention of post-traumatic muscle fibrosis.
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