BACKGROUND Trauma and hypovolemic shock are associated with mitochondrial dysfunction and septic complications. We hypothesize that hypovolemic shock and resuscitation results in peripheral blood mononuclear cell (PBMC) mitochondrial dysfunction that is linked to immunosuppression. METHODS Using a decompensated shock model, Long-Evans rats were bled to a MAP of 40 mmHg until the blood pressure could no longer be maintained without fluid infusion. Shock was sustained by incremental infusion of Lactated Ringer’s solution (LR) until 40% of the shed volume had been returned (severe shock). Animals were resuscitated with 4X the shed volume in LR over 60 minutes (resuscitation). Control animals underwent line placement, but were not hemorrhaged. Animals were randomized to control (n=5), severe shock (n=5), or resuscitation (n=6) groups. At each time point, PBMC were isolated for mitochondrial function analysis using flow cytometry and high resolution respirometry. Immune function was evaluated by quantifying serum IL-6 and TNF-α after PBMC stimulation with lipopolysaccharide (LPS). The impact of plasma on mitochondrial function was evaluated by incubating PBMC’s harvested following severe shock with control plasma. PBMC’s from control animals were likewise mixed with plasma collected following resuscitation. Student’s t-test and Pearson correlations were performed (significance: p <0.05). RESULTS Following resuscitation, PBMCs demonstrated significant bioenergetic failure with a marked decrease in basal, maximal, and ATP-linked respiration. Mitochondrial membrane potential also decreased significantly by 50% following resuscitation. Serum IL6 increased, while LPS stimulated TNF-α production decreased dramatically following shock and resuscitation. Observed mitochondrial dysfunction correlated significantly with IL6 and TNF-α levels. PBMCs demonstrated significant mitochondrial recovery when incubated in control serum, whereas control PBMCs developed depressed function when incubated with serum collected following severe shock. CONCLUSION Mitochondrial dysfunction following hemorrhagic shock and resuscitation was associated with the inhibition of PBMC response to endotoxin that may lead to an immunosuppressed state.

INTRODUCTION Although mitochondrial dysfunction is thought to contribute to the development of post-traumatic organ failure, current techniques to assess mitochondrial function in tissues are invasive and clinically impractical. We hypothesized that mitochondrial function in peripheral blood mononuclear cells (PBMCs) would reflect cellular respiration in other organs during hemorrhagic shock and resuscitation (HS&R). METHODS Using a fixed pressure HS model, Long Evan’s rats were bled to a mean arterial pressure (MAP) of 40 mmHg. When blood pressure could no longer be sustained without intermittent fluid infusion (Decompensated HS), Lactated Ringer’s (LR) was incrementally infused to maintain the MAP at 40 mmHg until 40% of the shed blood volume was returned (Severe HS). Animals were then resuscitated with 4X total shed volume in LR over 60 minutes (Resuscitation). Control animals underwent the same surgical procedures, but were not hemorrhaged. Animals were randomized to Control (n=6), Decompensated HS (n=6), Severe HS (n=6) or Resuscitation (n=6) groups. Kidney, liver, and heart tissues as well as PBMC’s were harvested from animals in each group to measure mitochondrial oxygen consumption using high resolution respirometry. Flow cytometry was used to assess mitochondrial membrane potential (Ψm) in PBMCs. One-way ANOVA and Pearson correlations were performed. RESULTS Mitochondrial oxygen consumption decreased in all tissues, including PBMC’s, following Decompensated HS, Severe HS, and Resuscitation. However, the degree of impairment varied significantly across tissues during HS&R. Of the tissues investigated, PBMC mitochondrial oxygen consumption and Ψm provided the closest correlation to kidney mitochondrial function during HS (complex I: r =0.65; complex II: r=0.65; complex IV: r=0.52; p<0.05). This association, however, disappeared with resuscitation. A weaker association between PBMC and heart mitochondrial function was observed but no association was noted between PBMC and liver mitochondrial function. CONCLUSION All tissues including PBMC’s demonstrated significant mitochondrial dysfunction following HS&R. Although PBMC and kidney mitochondrial function correlated well during hemorrhagic shock, the variability in mitochondrial response across tissues over the spectrum of hemorrhagic shock and resuscitation limits the usefulness of using PBMC’s as a proxy for tissue-specific cellular respiration.