Models of lower extremity damage in mice: time course of organ damage and immune response

Abstract

Background: Post-traumatic inflammatory changes have been identified as major causes of altered organ function and failure. Both hemorrhage and soft tissue damage induce these inflammatory changes. Exposure to heterologous bone in animal models has recently been shown to mimic this inflammatory response in a stable and reproducible fashion. This follow-up study tests the hypothesis that inflammatory responses are comparable between a novel trauma model ("pseudofracture", PFx) and a bilateral femur fracture (BFF) model.

Materials and methods: In C57BL/6 mice, markers for remote organ dysfunction and inflammatory responses were compared in four groups (control/sham/BFF/PFx) at the time points 2, 4, and 6 h.

Results: Hepatocellular damage in BFF and PFx was highly comparable in extent and evolution, as shown by similar levels of NFkappaB activation and plasma ALT. Pulmonary inflammatory responses were also comparably elevated in both trauma models as early as 2 h after trauma as measured by myeloperoxidase activity (MPO). Muscle damage was provoked in both BFF and PFx mice over the time course, although BFF induced significantly higher AST and CK levels. IL-6 levels were also similar with early and sustained increases over time in both trauma models.

Conclusions: Both BFF and PFx create similar reproducible inflammatory and remote organ responses. PFx will be a useful model to study longer term inflammatory effects that cannot be studied using BFF.

Figure 1

Hepatocellular damage in mice at 2, 4, and 6h time points in control (n=5), sham (S, n=6), bilateral femur fracture (BFF, n=8) and pseudofracture (PFx, n=8) groups. A: Plasma ALT levels. Results show mean ± SEM; *=p<0.05 vs control; #=p<0.05 vs sham). B: EMSA showing NFκB activation in liver tissue (representative result of three assays).

Figure 2

Pulmonary myeloperoxidase (MPO) activity at 2, 4, and 6h time points in control (n=5), sham (S, n=6), bilateral femur fracture (BFF, n=8) and pseudofracture (PFx, n=8) groups. MPO activity was standardized to protein concentration of lung tissue lysates. Results show mean ± SEM; * = p<0.05 vs control, # = p<0.05 vs sham.

Figure 3

Muscular damage in mice at 2, 4, and 6h time points as determined by plasma levels of A: CK and B: AST in control (n=5), sham (S, n=6), bilateral femur fracture (BFF, n=8) and pseudofracture (PFx, n=8) groups. Results show mean ± SEM; + = p<0.05 vs PFx, * = p<0.05 vs control, # = p<0.05 vs sham.

Figure 4. Histological changes in skeletal muscle 6h after BFF and PFx

A: Control; B: PFx; C: BFF; D: PFx through hematoma; E and F: BFF through hematoma.

Figure 5

Inflammatory response as measured by IL-6 levels at 2, 4, and 6h time points in control (n=5), sham (S, n=6), BFF and PFx mice (n=8 per group). Levels for Control and Sham at 2h and 6h time points are not detectable (ND; level of detection: 5.2 pg/mL). Results show mean ± SEM; * = p<0.05 vs control, # = p<0.05 vs sham.

Figure 6. Immunosuppression after 48h of PFx

Splenocyte proliferation in response to Concanavalin A treatment in control (n=8) and PFx (n=4) mice at 48h after injury. Results show mean ± SEM; * = p<0.05 vs control.