Isoflurane Anesthesia Initiated at the Onset of Reperfusion Attenuates Oxidative and Hypoxic-Ischemic Brain Injury

This study demonstrates that in mice subjected to hypoxia-ischemia (HI) brain injury isoflurane anesthesia initiated upon reperfusion limits a release of mitochondrial oxidative radicals by inhibiting a recovery of complex-I dependent mitochondrial respiration. This significantly attenuates an oxidative stress and reduces the extent of HI brain injury. Neonatal mice were subjected to HI, and at the initiation of reperfusion were exposed to isoflurane with or without mechanical ventilation. At the end of HI and isoflurane exposure cerebral mitochondrial respiration, H₂O₂ emission rates were measured followed by an assessment of cerebral oxidative damage and infarct volumes. At 8 weeks after HI navigational memory and brain atrophy were assessed. In vitro, direct effect of isoflurane on mitochondrial H₂O₂ emission was compared to that of complex-I inhibitor, rotenone. Compared to controls, 15 minutes of isoflurane anesthesia inhibited recovery of the compex I-dependent mitochondrial respiration and decreased H₂O₂ production in mitochondria supported with succinate. This was associated with reduced oxidative brain injury, superior navigational memory and decreased cerebral atrophy compared to the vehicle-treated HI-mice. Extended isoflurane anesthesia was associated with sluggish recovery of cerebral blood flow (CBF) and the neuroprotection was lost. However, when isoflurane anesthesia was supported with mechanical ventilation the CBF recovery improved, the event associated with further reduction of infarct volume compared to HI-mice exposed to isoflurane without respiratory support. Thus, in neonatal mice brief isoflurane anesthesia initiated at the onset of reperfusion limits mitochondrial release of oxidative radicals and attenuates an oxidative stress. This novel mechanism contributes to neuroprotective action of isoflurane. The use of mechanical ventilation during isoflurane anesthesia counterbalances negative effect of isoflurane anesthesia on recovery of cerebral circulation which potentiates protection against reperfusion injury

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies

A Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted--ischemia--and then restored--reperfusion--leading to a burst of reactive oxygen species (ROS) from mitochondria. It has been tacitly assumed that ROS production during IR is a non-specific consequence of oxygen interacting with dysfunctional mitochondria upon reperfusion. Recently, this view has changed, suggesting that ROS production during IR occurs by a defined mechanism. Here we survey the metabolic factors underlying IR injury and propose a unifying mechanism for its causes that makes sense of the huge amount of disparate data in this area and provides testable hypotheses and new directions for therapies.Work in our laboratories is supported by the Medical Research Council (UK) and the British Heart Foundation. E.T.C. is supported by a Human Frontiers Science Program fellowship.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.cmet.2015.12.00

DHA but Not EPA Emulsions Preserve Neurological and Mitochondrial Function after Brain Hypoxia-Ischemia in Neonatal Mice

Background and Purpose Treatment with triglyceride emulsions of docosahexaenoic acid (tri-DHA) protected neonatal mice against hypoxia-ischemia (HI) brain injury. The mechanism of this neuroprotection remains unclear. We hypothesized that administration of tri-DHA enriches HI-brains with DHA/DHA metabolites. This reduces Ca2+-induced mitochondrial membrane permeabilization and attenuates brain injury. Methods: 10-day-old C57BL/6J mice following HI-brain injury received tri-DHA, tri-EPA or vehicle. At 4–5 hours of reperfusion, mitochondrial fatty acid composition and Ca2+ buffering capacity were analyzed. At 24 hours and at 8–9 weeks of recovery, oxidative injury, neurofunctional and neuropathological outcomes were evaluated. In vitro, hyperoxia-induced mitochondrial generation of reactive oxygen species (ROS) and Ca2+ buffering capacity were measured in the presence or absence of DHA or EPA. Results: Only post-treatment with tri-DHA reduced oxidative damage and improved short- and long-term neurological outcomes. This was associated with increased content of DHA in brain mitochondria and DHA-derived bioactive metabolites in cerebral tissue. After tri-DHA administration HI mitochondria were resistant to Ca2+-induced membrane permeabilization. In vitro, hyperoxia increased mitochondrial ROS production and reduced Ca2+ buffering capacity; DHA, but not EPA, significantly attenuated these effects of hyperoxia. Conclusions: Post-treatment with tri-DHA resulted in significant accumulation of DHA and DHA derived bioactive metabolites in the HI-brain. This was associated with improved mitochondrial tolerance to Ca2+-induced permeabilization, reduced oxidative brain injury and permanent neuroprotection. Interaction of DHA with mitochondria alters ROS release and improves Ca2+ buffering capacity. This may account for neuroprotective action of post-HI administration of tri-DHA

Mitochondrial Dysfunction and Permeability Transition in Neonatal Brain and Lung Injuries

This review discusses the potential mechanistic role of abnormally elevated mitochondrial proton leak and mitochondrial bioenergetic dysfunction in the pathogenesis of neonatal brain and lung injuries associated with premature birth. Providing supporting evidence, we hypothesized that mitochondrial dysfunction contributes to postnatal alveolar developmental arrest in bronchopulmonary dysplasia (BPD) and cerebral myelination failure in diffuse white matter injury (WMI). This review also analyzes data on mitochondrial dysfunction triggered by activation of mitochondrial permeability transition pore(s) (mPTP) during the evolution of perinatal hypoxic-ischemic encephalopathy. While the still cryptic molecular identity of mPTP continues to be a subject for extensive basic science research efforts, the translational significance of mitochondrial proton leak received less scientific attention, especially in diseases of the developing organs. This review is focused on the potential mechanistic relevance of mPTP and mitochondrial dysfunction to neonatal diseases driven by developmental failure of organ maturation or by acute ischemia-reperfusion insult during development

DHA but Not EPA Emulsions Preserve Neurological and Mitochondrial Function after Brain Hypoxia-Ischemia in Neonatal Mice.

Treatment with triglyceride emulsions of docosahexaenoic acid (tri-DHA) protected neonatal mice against hypoxia-ischemia (HI) brain injury. The mechanism of this neuroprotection remains unclear. We hypothesized that administration of tri-DHA enriches HI-brains with DHA/DHA metabolites. This reduces Ca2+-induced mitochondrial membrane permeabilization and attenuates brain injury.10-day-old C57BL/6J mice following HI-brain injury received tri-DHA, tri-EPA or vehicle. At 4-5 hours of reperfusion, mitochondrial fatty acid composition and Ca2+ buffering capacity were analyzed. At 24 hours and at 8-9 weeks of recovery, oxidative injury, neurofunctional and neuropathological outcomes were evaluated. In vitro, hyperoxia-induced mitochondrial generation of reactive oxygen species (ROS) and Ca2+ buffering capacity were measured in the presence or absence of DHA or EPA.Only post-treatment with tri-DHA reduced oxidative damage and improved short- and long-term neurological outcomes. This was associated with increased content of DHA in brain mitochondria and DHA-derived bioactive metabolites in cerebral tissue. After tri-DHA administration HI mitochondria were resistant to Ca2+-induced membrane permeabilization. In vitro, hyperoxia increased mitochondrial ROS production and reduced Ca2+ buffering capacity; DHA, but not EPA, significantly attenuated these effects of hyperoxia.Post-treatment with tri-DHA resulted in significant accumulation of DHA and DHA derived bioactive metabolites in the HI-brain. This was associated with improved mitochondrial tolerance to Ca2+-induced permeabilization, reduced oxidative brain injury and permanent neuroprotection. Interaction of DHA with mitochondria alters ROS release and improves Ca2+ buffering capacity. This may account for neuroprotective action of post-HI administration of tri-DHA

Nelfinavir inhibits intra-mitochondrial calcium influx and protects brain against hypoxic-ischemic injury in neonatal mice.

Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca(2+)-induced membrane permeabilization, greater ADP-phosphorylating activity and reduced cytochrome C release during reperfusion. Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca(2+) content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca(2+) release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca(2+) content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca(2+) uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca(2+) influx, and did not prevent Ca(2+) induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca(2+) uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca(2+) flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca(2+) overload → mitochondrial membrane permeabilization → secondary energy failure

Attenuation of oxidative damage by targeting mitochondrial complex I in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Establishing sustained reoxygenation/reperfusion ensures not only the recovery, but may initiate a reperfusion injury in which oxidative stress plays a major role. This study offers the mechanism and this mechanism-specific therapeutic strategy against excessive release of reactive oxygen species (ROS) associated with reperfusion-driven recovery of mitochondrial metabolism. AIMS AND METHODS: In neonatal mice subjected to cerebral hypoxia-ischaemia (HI) and reperfusion, we examined conformational changes and activity of mitochondrial complex I with and without post-HI administration of S-nitrosating agent, MitoSNO. Assessment of mitochondrial ROS production, oxidative brain damage, neuropathological and neurofunctional outcomes were used to define neuroprotective strength of MitoSNO. A specificity of reperfusion-driven mitochondrial ROS production to conformational changes in complex I was examined in-vitro. RESULTS: HI deactivated complex I, changing its conformation from active form (A) into the catalytically dormant, de-active form (D). Reperfusion rapidly converted the D-form into the A-form and increased ROS generation. Administration of MitoSNO at the onset of reperfusion, decelerated D→A transition of complex I, attenuated oxidative stress, and significantly improved neurological recovery. In cultured neurons, after simulated ischaemia-reperfusion injury, MitoSNO significantly reduced ROS generation and neuronal mortality. In isolated mitochondria subjected to anoxia-reoxygenation, MitoSNO restricted ROS release during D→A transitions. CONCLUSION: Rapid D→A conformation in response to reperfusion reactivates complex I. This is essential not only for metabolic recovery, but also contributes to excessive release of mitochondrial ROS and reperfusion injury. We propose that the initiation of reperfusion should be followed by pharmacologically-controlled gradual reactivation of complex I.This study was supported by NIH grant, NS100850 (V.T.), by MRC grant MR/L007339/1 (A.G.) and MC_U105663142 and by a Wellcome Trust Investigator award110159/Z/15/Z (M.P.M)

Metabolism of the Cysteine S-Conjugate of Busulfan Involves a Beta-Lyase Reaction

The present work documents the first example of an enzyme-catalyzed beta-elimination of a thioether from a sulfonium cysteine S-conjugate. beta-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic beta-elimination reaction at pH 7.4 and 37 degrees C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5\u27-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway