Hartmann 2017 Abstract MITOEAGLE Barcelona
Mitochondrial respiration of isolated peripheral blood mononuclear cells in an experimental porcine brain injury model.
Hartmann C (2017)
Event: MitoEAGLE Barcelona 2017
Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in patients with trauma . The rapid onset of mitochondrial dysfunction is a key participant in the pathophysiological development of TBI and is associated with a poor prognosis [2,3]. Structural and functional impairment of mitochondrial function, increased oxidative stress and inflammation are considered as crucial contributors to the development of a primary brain damage [4,5,6]. Secondary damage, to either the brain or the systemic circulation, is triggered by the primary injury, and can continue for days after the initial trauma and eventually lead to multi-organ failure . Immune cells are key mediators of the post-injury inflammatory response. Previous studies have assessed changes in mitochondrial function, reactive oxygen species (ROS) production and glycolytic metabolism in isolated human blood cells following TBI .
In order to characterize mitochondrial (dys-)function in peripheral blood mononuclear cells (PBMCs) of traumatized patients in a clinical setting, we are currently establishing different methods in healthy human volunteers. In addition, we are applying the protocols to a porcine trauma brain injury model, to analyze mitochondrial (dys-)function in PBMCs during a defined TBI trauma model. Understanding the role of PBMCs may help to develop disease-specific diagnostic procedures, such as biomarkers, and potential therapeutical approaches in the future.
Venous blood from healthy volunteers was collected in heparinized tubes. Arterial blood was collected from pig in heparinized tubes at defined time-points prior and up to 48h after TBI induction. Briefly, PBMCs were separated from whole blood by the density gradient centrifugation method using Histopaque solution. High-resolution respirometry (HRR) of intact PBMCs was measured in terms of routine respiration, LEAK-, and ET-capacity by using an Oxygraph-2k (Oroboros Instruments, Austria). ROS production was measured by fluorometry in a simultaneous approach using Amplex Red (AmR). Glucose metabolism was investigated by lactate production and glucose oxidation in order to characterize the pentose phosphate pathway by using a stable isotope approach.
The results presented here are obtained from healthy human volunteers. Further, we are applying our protocols to a porcine TBI model, which we are currently establishing in our laboratory. Our preliminary data suggests that the techniques, established in human PBMCs, can be used in a porcine TBI-model.
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Human, Pig Tissue;cell: Blood cells Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET
HRR: Oxygraph-2k, O2k-Fluorometer Event: A4 PMBCs, AmR
- Ulm Univ, Fac Medicine, Germany
- Di Saverio S, Gambale G, Coccolini F, Catena F, Giorgini E, Ansaloni L, Amadori N, Coniglio C, Giugni A, Biscardi A, Magnone S, Filicori F, Cavallo P, Villani S, Cinquantini F, Annicchiarico M, Gordini G, Tugnoli G (2014) Changes in the outcomes of severe trauma patients from 15-year experience in a Western European trauma ICU of Emilia Romagna region (1996-2010). A population cross-sectional survey study. Langenbecks Arch Surg 399:109-26.
- Gilmer LK, Roberts KN, Joy K, Sullivan PG, Scheff SW (2009) Early mitochondrial dysfunction after cortical contusion injury. J Neurotrauma 26:1271-80.
- Gross T, Schüepp M, Attenberger C, Pargger H, Amsler F (2012) Outcome in polytraumatized patients with and without brain injury. 56:1163-74.
- Larson BE, Stockwell DW, Boas S, Andrews T, Wellman GC, Lockette W, Freeman K (2012) Cardiac reactive oxygen species after traumatic brain injury. (2012) J Surg Res 173:e73-81.
- Kavakli et al. (2010) Scientific Research and Essays 5:2832-6.
- Liao Y, Liu P, Guo F, Zhang ZY, Zhang Z (2013) Oxidative burst of circulating neutrophils following traumatic brain injury in human. PLOS ONE 8:e68963