Extremity injuries with hemorrhage have been a significant cause of death in civilian medicine and on the battlefield. The use of a tourniquet as an intervention is necessary for treatment to an injured limb; however, the tourniquet and subsequent release results in serious acute ischemia-reperfusion (IR) injury in the skeletal muscle and neuromuscular junction (NMJ). Much evidence demonstrates that inflammation is an important factor to cause acute IR injury. To find effective therapeutic interventions for tourniquet-induced acute IR injuries, our current study investigated effect of dexamethasone, an anti-inflammatory drug, on tourniquet-induced acute IR injury in mouse hindlimb. In C57/BL6 mice, a tourniquet was placed on unilateral hindlimb (left hindlimb) at the hip joint for 3 h, and then released for 24 h to induce IR. Three hours of tourniquet and 24 h of release (24-h IR) caused gastrocnemius muscle injuries including rupture of the muscle sarcolemma and necrosis (42.8 ± 2.3% for infarct size of the gastrocnemius muscle). In the NMJ, motor nerve terminals disappeared, and endplate potentials were undetectable in 24-h IR mice. There was no gastrocnemius muscle contraction in 24-h IR mice. Western blot data showed that inflammatory cytokines (TNFα and IL-1β) were increased in the gastrocnemius muscle after 24-h IR. Treatment with dexamethasone at the beginning of reperfusion (1 mg/kg, i.p.) significantly inhibited expression of TNFα and IL-1β, reduced rupture of the muscle sarcolemma and infarct size (24.8 ± 2.0%), and improved direct muscle stimulation-induced gastrocnemius muscle contraction in 24-h IR mice. However, this anti-inflammatory drug did not improve NMJ morphology and function, and sciatic nerve-stimulated skeletal muscle contraction in 24-h IR mice. The data suggest that one-time treatment with dexamethasone at the beginning of reperfusion only reduced structural and functional impairments of the skeletal muscle but not the NMJ through inhibiting inflammatory cytokines.

Tourniquet application and its subsequent release cause serious injuries to the skeletal muscle, nerve, and neuromuscular junction (NMJ) due to mechanical compression and ischemia-reperfusion (IR). Monitoring structural and functional repair of the NMJ, nerve, and skeletal muscle after tourniquet-induced injuries is beneficial in exploring potential cellular and molecular mechanisms responsible for tourniquet-induced injuries, and for establishing effective therapeutic interventions. Here, we observed long-term morphological and functional changes of the NMJ in a murine model of tourniquet-induced hindlimb injuries. Unilateral hindlimbs of C57/BL6 mice were subjected to 3 h of tourniquet by placing an orthodontic rubber band, followed by varied periods of tourniquet release (1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks). NMJ morphology in the gastrocnemius muscle was imaged, and the endplate potential (EPP) was recorded to evaluate NMJ function. In NMJs, nicotinic acetylcholine receptor (nAChR) clusters normally displayed an intact, pretzel-like shape, and all nAChR clusters were innervated (100%) by motor nerve terminals. During 3 h of tourniquet application and varied periods of tourniquet release, NMJs in the gastrocnemius muscle were characterized by morphological and functional changes. At 1 day and 3 days of tourniquet release, nAChR clusters retained normal, pretzel-like shapes, whereas motor nerve terminals were completely destroyed and no EPPs recorded. From 1 to 6 weeks of tourniquet release, motor nerve terminals gradually regenerated, even reaching that seen in sham mice, whereas nAChR clusters were gradually fragmented with prolongation of tourniquet release. Additionally, the amplitude of EPPs gradually increased with prolongation of tourniquet release. However, even at 6 weeks after tourniquet release, the amplitude of EPPs did not restore to the level seen in sham mice (13.9 ± 1.1 mV, p < 0.05 vs. sham mice, 29.8 ± 1.0 mV). The data suggest that tourniquet application and subsequent release impair the structure and function of NMJs. Morphological change in motor nerve terminals is faster than in nAChR clusters in NMJs. Slow restoration of fragmented nAChR clusters possibly dampens neuromuscular transmission during the long phase following tourniquet release.

Severe injury and infection are often followed by accelerated protein catabolism and acute insulin resistance. This results in several effects that complicate and prolong recovery, including weakness, immobility, impaired wound healing, and organ dysfunction. Recent studies have demonstrated the development of GH resistance during severe inflammation, providing a potential mechanism for the protein loss that follows injury and infection. To understand this GH resistance, we recently developed a murine model of acute injury. Mice were subjected to soft-tissue injury, alone or combined with hemorrhage, and injected iv with GH 30, 60, or 90 minutes later. Hepatic GH signaling was measured via Western analysis. GH-induced signal transducer and activator of transcription 5 phosphorylation was decreased immediately after completion of the trauma procedure, and at 30 and 60 minutes, but further decreased by 90 minutes after trauma. Combined trauma and hemorrhage resulted in severely decreased GH-induced signal transducer and activator of transcription 5 phosphorylation compared with trauma alone, and this was true at all time points studied. Western analysis revealed an apparent decrease in the molecular weight of the hepatic GH receptor (GHR) after trauma and hemorrhage, but not trauma alone. Additional studies determined that the hemorrhage-induced decrease in receptor size was not due to changes in GHR N-linked glycosylation. These results suggest that GH sensitivity is rapidly impaired after acute injury and that trauma combined with hemorrhage results in a more severe form of GH resistance resulting from alteration or inactivation of hepatic GHR.