Tourniquet-induced ischemia creates increased risk of organ dysfunction and mortality following delayed limb amputation.

Tourniquets are critical for the control of traumatic extremity hemorrhage. In this study, we sought to determine, in a rodent blast-related extremity amputation model, the impact of prolonged tourniquet application and delayed limb amputation on survival, systemic inflammation, and remote end organ injury. Adult male Sprague Dawley rats were subjected to blast overpressure (120±7 kPa) and orthopedic extremity injury consisting femur fracture, one-minute soft tissue crush injury (20 psi), ± 180 min of tourniquet-induced hindlimb ischemia followed by delayed (60 min of reperfusion) hindlimb amputation (dHLA). All animals in the non-tourniquet group survived whereas 7/21 (33%) of the animals in the tourniquet group died within the first 72 h with no deaths observed between 72 and 168 h post-injury. Tourniquet induced ischemia-reperfusion injury (tIRI) likewise resulted in a more robust systemic inflammation (cytokines and chemokines) and concomitant remote pulmonary, renal, and hepatic dysfunction (BUN, CR, ALT. AST, IRI/inflammation-mediated genes). These results indicate prolonged tourniquet application and dHLA increases risk of complications from tIRI, leading to greater risk of local and systemic complications including organ dysfunction or death. We thus need enhanced strategies to mitigate the systemic effects of tIRI, particularly in the military prolonged field care (PFC) setting. Furthermore, future work is needed to extend the window within which tourniquet deflation to assess limb viability remains feasible, as well as new, limb-specific or systemic point of care tests to better assess the risks of tourniquet deflation with limb preservation in order to optimize patient care and save both limb and life.

Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury.

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.