Induction of polytrauma

The polytrauma was induced as previously described.[34] In brief, antibiotic prophylaxis (Ceftriaxon® 2 g) was administered before surgery and after every 24 h until sacrifice. Prior to initial trauma induction, the fraction of inspired O₂ (FiO₂) was set at 0.21 and the fluid administration was reduced to 10 ml/h. At this phase, the animals were allowed to descend into a hypothermic state following hemorrhagic shock period mimicking the pre-clinical scenario. The animal was positioned on the right side and a femur fracture was induced with a bolt shot on the right hind leg (Blitz-Kerner, turbocut JOBB GmbH, Germany, 9x17, Dynamit Nobel AG, Troisdorf, Germany). After being placed back in the dorsal position, blunt thoracic trauma with a bolt shot on the right dorsal lower thorax was induced. Finally, a midline-laparotomy and uncontrolled bleeding for 30 seconds after crosswise incision of the caudal liver lobe (4.5 x 4.5 cm) was induced. Using sterile gauze-compresses the liver was packed. Pressure-controlled haemorrhagic shock using exsanguination from right femoral artery was performed until a mean arterial blood pressure (MAP) of 40 ± 5 mm Hg was reached and maintained for 90 minutes.

Resuscitation started immediately after hemorrhagic shock by adjusting FiO₂ to baseline values, and re-infusing with previously withdrawn blood and additional fluids (Sterofundin ISO^®; 2 ml kg/BW/h). Rewarming was performed using forced-air warming systems until normothermia was reached (38.7–39.8°C).

After experimentation, clinical treatment of the open femur fracture was performed according to established trauma guidelines. The intensive care and complications management followed the standardized clinical protocols according to the latest recommendations of the European Resuscitation Council and Advanced Trauma Life Support (ATLS).[35, 36] After the observational period the animals were euthanized by using potassium chloride until cardiac arrest.