Mitochondrial bioenergetic alterations after focal traumatic brain injury in the immature brain

Abstract

Traumatic brain injury (TBI) is one of the leading causes of death in children worldwide. Emerging evidence suggests that alterations in mitochondrial function are critical components of secondary injury cascade initiated by TBI that propogates neurodegeneration and limits neuroregeneration. Unfortunately, there is very little known about the cerebral mitochondrial bioenergetic response from the immature brain triggered by traumatic biomechanical forces. Therefore, the objective of this study was to perform a detailed evaluation of mitochondrial bioenergetics using high-resolution respirometry in a high-fidelity large animal model of focal controlled cortical impact injury (CCI) 24 h post-injury. This novel approach is directed at analyzing dysfunction in electron transport, ADP phosphorylation and leak respiration to provide insight into potential mechanisms and possible interventions for mitochondrial dysfunction in the immature brain in focal TBI by delineating targets within the electron transport system (ETS). Development and application of these methodologies have several advantages, and adds to the interpretation of previously reported techniques, by having the added benefit that any toxins or neurometabolites present in the ex-vivo samples are not removed during the mitochondrial is olation process, and simulates the in situ tricarboxylic acid (TCA) cycle by maximizing key substrates for convergent flow of electrons through both complexes I and II. To investigate alterations in mitochondrial function after CCI, ipsilateral tissue near the focal impact site and tissue from the corresponding contralateral side were examined. Respiration per mg of tissue was also related to citrate synthase activity (CS) and calculated flux control ratios (FCR), as an attempt to control for variability in mitochondrial content. Our biochemical analysis of complex interdependent pathways of electron flow through the electron transport system, by most measures, reveals a bilateral decrease in complex I-driven respiration and an increase in complex II-driven respiration 24 h after focal TBI. These alterations in convergent electron flow though both complex I and II-driven respiration resulted in significantly lower maximal coupled and uncoupled respiration in the ipsilateral tissue compared to the contralateral side, for all measures. Surprisingly, increases in complex II and complex IV activities were most pronounced in the contralateral side of the brain from the focal injury, and where oxidative phosphorylation was increased significantly compared to sham values. We conclude that 24 h after focal TBI in the immature brain, there are significant alterations in cerebral mitochondrial bioenergetics, with pronounced increases in complex II and complex IV respiration in the contralateral hemisphere. These alterations in mitochondrial bioenergetics present multiple targets for therapeutic intervention to limit secondary brain injury and support recovery.

Mitochondrial response in a toddler-aged swine model following diffuse non-impact traumatic brain injury

“…As a starting point for our initial large animal studies we have chosen 24 hours as our terminal time-point for two critical reasons: 1) We have documented significant injury with neuropathology, imaging and behavior at this time point in our model of diffuse TBI (Jaber et al, 2015; Kilbaugh et al, 2011; 2015; Weeks et al, 2014) 2) other investigators have documented bioenergetic and mitochondrial alterations in rodents following diffuse TBI at this particular time point. The hippocampus and cortex were studied as the areas of interest for two critical factors: 1) Previous studies form our laboratory have documented significant neuropathology and mitochondrial dysfunction within these regions following RNR (Coats and Margulies, 2006; Eucker et al, 2011; Kilbaugh et al, 2011; Weeks et al, 2014) 2) If our hypothesis was consistent with previous findings in adults and infants, and these areas of interest do exhibit alterations in mitochondrial functional pathways; then, these findings would be instrumental in the evaluation of neurobehavioral outcomes in future studies, linking mitochondrial bioenergetics and long-term outcomes (S.…”

“…The instrument was calibrated daily, as previously described, and respiration measurements were obtained at a constant 37°C with the addition of tissue homogenates to a final concentration of 1 mg per ml of Mir05 buffer (Kilbaugh et al, 2015). Oxygen consumption and oxygen flux recorded using Datlab software (5.1, Oroboros Instruments, Innsbruck, Austria)…”

“…A substrate, uncoupler, inhibitor titration (SUIT) protocol previously used was specifically designed for porcine brain tissue (Kilbaugh et al, 2015). Complex specific substrates and inhibitors allowed for the assessment of respiratory capacities of the integrated electron transport system (ETS) (Figure 1).…”

“…Complex I (CI), and complex II (CII) respiratory capacities in brain tissue were evaluated separately; as well as with pathways of convergent electron input through the Q-junction (CI + II) using succinate and nicotinamide adenine dinucleotide (NADH)-linked substrates (Gnaiger, 2009). An optimized dose of digitonin, 1 μl (50 mg/ml), necessary to achieve accurate and consistent oxidative phosphorylation capacity within synaptasomes using water-soluble substrates, and is necessary to achieve similar results in brain tissue homogenates and isolated brain mitochondria (Kilbaugh et al, 2015; 2011). Exogenously administered cytochrome c did not induce a significant effect on mitochondrial respiration at the optimal digitonin dose, indicating an intact outer mitochondrial membrane (data not shown) (Brustovetsky et al, 2002; Sims and Blass, 1986).…”

“…Oxygen flux traces were measured automatically with a customized Matlab (Mathworks, Natick, MA) (Kilbaugh et al, 2015). Statistical evaluation was performed using JMP Pro (SAS Institute Inc., version 10.0, Cary, NC, USA).…”