The Systemic Effect of Burn Injury and Trauma on Muscle and Bone Mass and Composition

Abstract

Background: By understanding the global inflammatory effects on distant myopathies, surgeons can better guide the rehabilitative process for burn patients. The authors tested the systemic effect of burn injury on distant injured muscle and native bone using immunohistochemistry and validated a new morphometric analytic modality to reproducibly quantify muscle atrophy using computed tomographic imaging.

Methods: In vivo studies were performed on C57/BL6 mice using an Achilles tenotomy with concurrent burn injury model. Total muscle and bone (tibia and fibula) volume/density were quantified near the site of Achilles tenotomy using micro-computed tomography at 5, 7, and 9 to 12 weeks after surgery. The impact of burn injury on the inflammatory cascade [nuclear factor (NF)-κB, p-NF-κB] and the interconnected protein catabolism signaling pathway (Atrogin-1) was assessed by immunohistochemistry.

Results: Muscle volume and density at the site of Achilles tenotomy in burned mice were significantly diminished compared with nonburned mice at 5 weeks and 9 to 12 weeks. Similar decreases in muscle volume and density were observed when comparing tenotomy to no tenotomy. Cortical bone health remained stable in burn/tenotomy mice compared with tenotomy. Muscle atrophy was associated with up-regulation of p-NF-κB, NF-κB, and Atrogin-1 assessed by immunohistochemistry.

Conclusions: Burn injury significantly decreases muscle volume and density. Increased muscle atrophy using our computed tomographic morphometric analysis correlated with a significant increase in intramuscular inflammatory markers and proteolysis enzymes. This study demonstrates a unique characterization of how burn injuries may worsen local myopathy. Moreover, it provides a novel approach for quantifying muscle atrophy over an expanded period.

Fig. 1

Burn injury promotes muscular atrophy. Micro–computed tomographic imaging was used to quantify muscle volume (above) and muscle density (below) at 5-, 7-, and 9- to 12-week time points (*p < 0.05; **p < 0.01).

Fig. 2

The defined three-dimensional region of interest overlaid in yellow dictates the boundaries of muscle used in micro-computed tomographic analysis for muscle volume and density.

Fig. 3

Bone response remains stable despite tenotomy and/or burn insults. Micro–computed tomography was used to quantify bone volume (above), bone mineral content (center), and bone mineral density (below) at 5-, 7-, and 9- to 12-week time points (*p < 0.05; **p < 0.01).

Fig. 4

The yellow overlay in the left pane (left) delineates the region of interest that defined the borders of the section of analyzed bone. A representative section of analyzed bone is shown (right).

Fig. 5

NF-κB and p-NF-κB indicate an elevated expression of p-NF-κB associated with burn status. Micrographs of NF-κB staining shown for burn and sham mice across 5-day and 3-week time points at 2× and 40× magnification levels. Red hashed line indicates the plane of cross-section used in tissue samples (above). Scale bar = 200 µm. Micrographs of p-NF-κB staining shown for burn and sham mice across 5-day and 3-week time points at 2× and 40× magnification (center). Representative stained nuclei are indicated by black arrow. Representative nonstained nuclei are indicated by red arrow. Scale bar = 200 µm. Quantification of nuclei of the gastrocnemius muscle from p-NF-κB slides at 40× are shown graphically for both burn and sham mice (below).

Fig. 6

Atrogin-1 staining shows stark increases in cytoplasmic expression in early burn. Micrographs of Atrogin-1 staining shown for burn and sham mice across 5-day and 3-week time points at 2× and 40× magnification. Red hashed line indicates the plane of cross-section used in tissue samples. Scale bar = 200 µm.