A new multiple trauma model of the mouse

Abstract

Background: Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability.

Methods: Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d.

Results: A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1β (Interleukin-1β), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture.

Conclusions: The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions.

Fig. 1

a, b Apparatus used for the multiple trauma mouse model. A cylindrical weight of 500 g was dropped through a tube onto a plunger in contact with the chest of an anesthetized mouse. Mice were placed on a cross on a platform of acrylic glass immediately below the plunger. The plunger allowed induction of a bilateral pulmonary contusion. c Induction of femur fracture. Before a closed femur fracture was induced, the femur was stabilized using a cannula (Sterican 0.55 × 25 LILA lI) from the knee joint to the femoral neck. The method used to induce femur fracture was first described by Bonnarens. After primary wound closure, a standardized femur fracture was induced using a blunt guillotine device with a weight of 500 g (0.784 J)

Fig. 2

Weight of the mice after multiple trauma. The mouse numbers are equal to the order of the data. The weight of mice was determined before multiple trauma and before each mouse was sacrificed. A decrease in weight was observed during the first 24 h following trauma (shown for 6 h after trauma)

Fig. 3

Body temperature of the mice after multiple trauma. The mouse mice are equal to the order of the data. The body temperature (°C) of mice was measured before multiple trauma was induced and before each mouse was sacrificed. A drop in temperature was observed during the first 24 h after trauma (shown for 6 h after trauma)

Fig. 4

Hemoglobin measurements in mice after multiple trauma. The hemoglobin level in mice without trauma was compared to hemoglobin level in mice after multiple trauma. Five mice without trauma underwent retrobulbar puncture to measure hemoglobin as a control. Mice that underwent trauma were sacrificed by heart puncture at 6 h, 12 h, 24 h, 3 d, 7 d, 14 d, and 28 d after trauma. Data are shown for 5 mice each at 6, 12, and 24 h after trauma. Blood samples were analyzed immediately after puncture using a Radiometer ABL 700. Hemoglobin levels decreased up to 24 h

Fig. 5

CT scan of chest trauma. Computerized tomography was performed for 5 mice that died immediately after chest trauma. The CT scans revealed intrathoracic bleeding and hemopneumothorax. Scans were performed in the Molecular Imaging North Competence Center, CAU Kiel

Fig. 6

Microscopic evaluation of histological changes in the lung of mice after pulmonary contusion. Mice subjected to chest injury showed extensive intra-alveolar and intrabronchial hemorrhaging with consecutive atelectasis, while sham mice did not show such pulmonary changes

Fig. 7

a, b, c, d, e Cytokine analysis assessed by the Luminex assay. Serum samples of mice with an isolated femur fracture (Fx) and mice with chest trauma and femur fracture (TTFx) were analysed by multiplex immunoassay. All Data are shown as box-and-whisker plot with median and interquartile range. Concentration of different cytokines (IL-1β, IL-6, IL-10, IL-12p70 and TNFα) was measured in different groups of mice 0 h, 6 h, 12 h, 3 d and 7 d (n = 8) after trauma. Figures show regulation of IL-1α, IL-6, IL-10, IL-12p70 and TNFα after an isolated femur fracture from 0 h to 7 d after trauma and cytokine regulation after induction of chest trauma and femur fracture from 0 h to 7 d after trauma. Cytokine expression was compared respectively to Sham group