A murine multiple-injury model for the study of thromboinflammation

ABSTRACT INTRODUCTION Neutrophil Extracellular Traps (NETs) contribute to trauma-induced coagulopathy. We aimed to develop a murine polytrauma model that induces thrombo-inflammatory response i.e., NETosis and accelerated thrombin generation. METHODS Wild-type male mice (n = 10, age 8 – 12 weeks) underwent polytrauma (gastrocnemius crush, femur fracture, and laparotomy) and were compared to an uninjured control group (n = 10). Mice were euthanized by cardiac puncture performed 3 hours after injury. Whole blood samples were immediately processed to platelet poor plasma for thrombin generation kinetics (Calibrated Automated Thrombogram), myeloperoxidase (MPO), and von Willebrand Factor (vWF) quantification. Immunohistochemistry of lung tissue was performed to assess for Citruillinated H3 (Cit H3) and MPO. A NETosis cluster was defined as 3+ neutrophils staining for Cit H3 at 400 X magnification (CitH3 cluster). Data presented either as mean (SD) or median [IQR] with p < 0.05 significant. Sham and trauma treated animals were compared by the 2-sample Wilcoxon ranksum test. RESULTS Animals subjected to polytrauma had accelerated thrombin generation compared to controls with greater peak height (nM) (61.3 [41.2, 73.2] vs. 28.4 [19.5, 37.5], p = 0.035) and shorter time to peak (min) (3.37 [2.81, 3.81] vs. 4.5 [4.08, 4.75], p = 0.046). Markers of neutrophil activation were greater following polytrauma than in controls (MPO (ng/ml) 961.1 [858.1, 1116.8] vs. 481.3 [438.0, 648.9], p = 0.004). NETosis, as evidenced by the above defined number of CitH3 clusters in the lung, was greater in polytrauma than in controls (mean 3 [2.9] vs. 0.2 [0.7], p = 0.009). CONCLUSIONS This is the first study to demonstrate that NETosis and accelerated thrombin generation can be induced using a murine polytrauma model, as early as three hours following injury. STUDY TYPE Animal study LEVEL OF EVIDENCE NA