Tourniquet use following blast-associated complex lower limb injury and traumatic amputation promotes end organ dysfunction and amplified heterotopic ossification formation

Abstract

Abstract Background Traumatic heterotopic ossification (tHO) is characterized by ectopic bone formation in extra-skeletal sites leading to impaired wound healing, entrapment of neurovascular structures, pain, and reduced range of motion. HO has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan and can compound recovery by causing difficulty with prosthesis limb wearing. Tourniquet use to control catastrophic limb hemorrhage prior to surgery has become almost ubiquitous during this time, with the recognition the prolonged use may risk an ischemia reperfusion injury and associated complications. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care has not been explored. Methods Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of tourniquet use on tHO formation. Adult male rats were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. Pneumatic tourniquet (250–300 mmHg) applied proximal to the injured limb for 150-min was compared to a control group without tourniquet, before a trans-femoral amputation was performed. Outcome measures were volume to tHO formation at 12 weeks and changes in proteomic and genomic markers of early tHO formation between groups. Results At 12 weeks, volumetric analysis with microCT imaging revealed a 70% increase in total bone formation (p = 0.007) near the site of injury compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to tourniquet usage had increased expression of danger-associated molecular patterns (DAMPs) and end organ damage as early as 6 h and as late as 7 days post injury. The expressions of pro-inflammatory cytokines and chemokines and osteochondrogenic genes using quantitative RT-PCR similarly revealed increased expression as early as 6 h post injury, and these genes along with hypoxia associated genes remained elevated for 7 days compared to no tourniquet use. Conclusion These findings suggest that tourniquet induced ischemia leads to significant increases in key transcription factors associated with early endochondral bone formation, systemic inflammatory and hypoxia, resulting in increased HO formation.