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) 24h 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 isolation 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 24h 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 24h 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.

Keywords: Axonal injury; Bioenergetics; Contusion; Mitochondria; Pediatric traumatic brain injury; Swine.

Fig. 1

Graphical representation of the substrate, uncoupler, inhibitor titration (SUIT) protocol used to study the integrated function of individual components of the electron transport system (ETS). Induced respiratory states and respiratory complexes activated are outlined by boxes: ROX, residual oxygen consumption due to non-mitochondrial respiration induced by the inhibition of the complex III by antimycin A; ETS (ETS_{CI + CII}), respiration that is uncoupled capacity from ATP synthase induced by the optimum titration of the protonophore FCCP; LEAK (LEAK_{CI + CII}), State 4_o, is the resting non-phosphorylating electron transfer across the mitochondrial inner membrane due to uncoupling of ATP synthase by Olgiomycin; OXPHOS (OXPHOS_{CI + CII}) coupled capacity of oxidative phosphorylation measure convergent respiration of both complex I (Malate, Pyruvate, Glutamate) and II (Succinate) substrates. The SUIT protocol employed in these experiments also utilized the complex I inhibitor, Rotenone, to measure individual complex II-driven respiration (ETS_CII) separately from complex I. In addition, TMPD/ascorbate and azide were administered to measure complex IV respiration.

Fig. 2

Citrate synthase activity. Citrate synthase (CS) activity per mg of tissue 24 h after controlled cortical impact (CCI). There were no differences between sides of the brain in the sham animals, (p = 0.99). Following focal CCI injury, there was a nearly 50% reduction in CS activity in the ipsilateral peri-contusional side (9.9 ± 0.7 μmol/mL/min), compared to both the ipsilateral CS activity in sham animals, 18.2 ± 0.8 μmol/mL/min, (p < 0.001) and contralateral tissue in injured animals, 16.4 ± 0.6 μmol/mL/min, (p < 0.01). All values are mean ± SEM. Contra: contralateral side of brain, Ipsi: ipsilateral side of brain.

Fig. 3

Mitochondrial flux control ratios from CCI injured and shams normalized by electron transport system (ETS) capacity (ETS_{CI + CII}) measured 24 h post TBI. Sham contralateral (Contra) and ipsilateral (Ipsi) sides were not significantly different for any measure. A) Controlled cortical impact (CCI) injury resulted in a significant decrease in the contribution of complex I respiration (OXHPHOS_CI) to maximal electron transport (ETS_{CI + CII}), in injured tissue bilaterally, compared to corresponding sham regions. Complex II-driven respiration (ETS_CII) was significantly increased in injured tissue bilaterally, compared to corresponding sham regions. Maximal coupled, phosphorylating respiration (OXPHOS_{CI + CII}), stimulated by both complex I and complex II substrates, was significantly decreased post-CCI in the injured ipsilateral tissue compared to injured contralateral tissue and compared to ipsilateral sham. B) There was no significant alteration in CIV driven respiration post-CCI compared to corresponding sham in either side. State 4_o (LEAK_{CI + CII}) was significantly reduced in the ipsilateral and contralateral brain post-CCI compared to corresponding sham. Presented as mean ± SEM. For definitions of respiratory states and substrates utilized please see Fig. 1. Bars, p < 0.05. CCI: controlled cortical impact.