L. R. Klingbeil1,2, G. Piraino1, P. W. Hake1, J. R. Ledford1, B. Zingarelli1 1Cincinnati Children’s Hospital Medical Center,Department Of Critical Care,Cincinnati, OH, USA 2University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA
Introduction: Despite advances in the management of trauma victims, mortality from hemorrhagic shock and the ensuing multiple organ dysfunction syndrome (MODS), remains high. Pediatric patients have a lower incidence of MODS, including acute lung injury, than adult patients. The molecular mechanisms underlying this age-dependent susceptibility to MODS are not fully understood. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy and metabolic recovery through mitochondrial biogenesis. Following activation by phosphorylation, AMPK regulates mitochondrial biogenesis via peroxisome proliferator-activated receptor γ co-activator α (PGC1-α), which can also be regulated by sirtuin 1(Sirt-1). We hypothesize that these metabolic signaling pathways are altered in the lung during hemorrhagic shock and are age-dependent.
Methods: Hemorrhagic shock was induced in anesthetized young male (2-4 months old) and mature mice (9-10 months old) by withdrawing blood from the femoral artery to a mean arterial pressure (MAP) of 30 mmHg for 90 minutes. The mice were then resuscitated with the shed blood plus two times that amount in Lactated Ringer’s solution. Mice were sacrificed 3 hours after resuscitation and lungs were harvested for biochemical assays. Sham mice underwent a similar surgical preparation but were not bled.
Results: After hemorrhagic shock, there was marked neutrophil infiltration, as evaluated by myeloperoxidase (MPO) assay, in the lung of young mice when compared to the sham mice (201.3±19.4 versus 132.9±17.7 U/100mg tissue, p<0.05) indicating increased inflammation after hemorrhage. Mature mice also exhibited higher neutrophil infiltration in the lung after hemorrhagic shock when compared to mature sham mice (222.1±30.1 versus 167.6±12.9 U/100mg tissue, p<0.05). At western blot analysis, there was a significant increase of nuclear pAMPK (1.7±0.06 relative intensity) in lung of young mice after hemorrhagic shock when compared to age-matched sham mice (1.0±0.04 relative intensity, p<0.05) and was associated with maintenance of normal levels of PGC1-α and Sirt-1, thus suggesting the capability to mount a metabolic response. Interestingly, nuclear levels of pAMPK were significantly increased in sham mature mice (2.2±0.01 relative intensity) when compared with young sham animals (p<0.05). However, after hemorrhagic shock in lung of mature mice, there was a significant decrease in pAMPK expression (1.2±0.21 relative intensity), which was associated with a marked down-regulation of PGC1-α and Sirt-1 when compared to sham age-matched mice, thus suggesting the impairment to promote a metabolic recovery after stress.
Conclusion: Our data suggests that during hemorrhagic shock, metabolic repair mechanisms are activated in the lung and involve AMPK-dependent pathways. However, this restorative process diminishes in old age.