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

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

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

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

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

Nelfinavir mimics the effect of RuR on mitochondrial response to OGD.

<p><b>A–C,</b> - Confocal microscopy and semi-quantitative analysis of cytosolic cellular Ca²⁺ fluorescence (Fluo4) with simultaneous Ψm fluorescence (TMRE) in cells treated with vehicle (n = 7) or NLF (n = 7), or RuR (n = 7) at 1 hrs following OGD (12 hrs). * p<0.0001 compared to values compared to the naïve cells (100%), and ** p<0.0001 compared to the vehicles. <b>D–F</b> -Confocal microscopy and semi-quantitative analysis for mitochondria-specific Ca²⁺ (Rhod 2) fluorescence alone with Ψm (R123) fluorescence in naïve cells and cells pre-incubated (20 hrs) with vehicle or NLF (4.4 µM), RuR (10 µM). * p<0.0001 compared to naives, ** p<0.0001 compared to the NLF cells. n = 7 in each group. The area outlined with dashed line demonstrates cells with Ca²⁺overloaded mitochondria which lost their Ψm. Scale bar = 40 (merged images) and 20 µm.</p

Nelfinavir limits post-ischemic mitochondrial cytochrome C release, brain infarct volume and cellular mortality.

<p><b>A, B</b> - Western blot analysis for the presence of Cytochrome C (Cyt C) in cytosolic (Cytosol) and mitochondrial (Mitos) fractions obtained from the ipsilateral hemisphere at five hrs after HI insult. β-actin was used as a loading control and cytochrome C oxidase (COX IV) was used as a purity control of cytosolic fraction and loading control for mitochondrial fraction. Quantitative data presented in (B) are expressed in arbitrary OD units normalized to β-actin. Vehicle –treated mice, n = 3, NLF-treated mice, n = 4. <b>C, D</b> - Cerebral infarct volume and representative TTC-stained brain slices obtained at 24 hrs of reperfusion after HI in the vehicle and NLF-treated mice. <b>E, F</b> – Cell mortality at 6 hours of reperfusion following 12 hours of OGD in HT-22 cells treated with vehicle (n = 7), NLF (n = 7) or RuR (n = 7) compared to naives (n = 4). <b>E</b> - Representative images of HT-22 cells in different experimental conditions stained with Propidium Iodide (red) and Hoechst (blue). Note that amount of red cells predominates in OGD-vehicle group. Confocal microscopy. Scale bar = 50 µm. <b>F</b> - Quantitative evaluation of cell mortality. One-way Anova, only significant difference is shown.</p

Nelfinavir does not inhibit mitochondrial Ca²⁺ uniporter.

<p><b>A</b> – The experiment controlling mitochondrial specificity for TMRE and Rhodamine 123 (R123) fluoroprobes. Note a drastic decrease in Ψm fluorescence following FCCP (0.5 µM) supplementation. Scale bar = 10 µm. <b>B</b> – One of three highly reproducible tracings of mitochondrial Ca²⁺ buffering capacity in organelles pre-treated with RuR (1 µM), NLF (4.4 µM) or vehicle. Note, that only RuR completely inhibited Ca²⁺ up-take by mitochondria, while NLF, virtually, had no effect. <b>C</b> – One of the four highly reproducible tracings of changes in the safranin (Ψm) fluorescence in response to mitochondrial supplementation (indicated) and addition of 10 nmoles of Ca²⁺ pulses to mitochondria pre-incubated with RuR, NLF or vehicle. Note, only the RuR prevented the collapse of Ψm in response to Ca²⁺ challenge. Brain mitochondria were isolated from naïve p10 mice, substrate: succinate-glutamate (see also methods).</p

Nelfinavir improves mitochondrial function.

<p><b>A, B</b> – Intramitochondrial Ca²⁺ content (<b>A</b>) and representative tracings of Ca²⁺ release from mitochondria (<b>B</b>) in naïve (n = 4) and at the end of HI-insult in vehicle (n = 7) or NLF-treated (n = 7) mice. One-way ANOVA. *- p<0.0001 compared to naives. Mito (downward arrow) indicates addition of mitochondria (0.1 mg/ml). Digitonin (downward arrow) shows addition of Digitonin (10 mg/mg of the mitochondrial protein). Digitonin-induced nonspecific fluorescence curve (without mitochondria addition) is indicated as Digitonin. <b>C, D</b> – Mitochondrial Ca²⁺ buffering capacity at five hours of reperfusion (<b>C</b>), with representative tracings (<b>D</b>) in vehicle (n = 11) and NLF-treated (n = 14) HI-mice compared to naïve littermates (n = 7). Mitochondrial Ca²⁺ buffering capacity was defined by the amount of Ca²⁺ needed to open mPTP (spontaneous increase in Ca²⁺ fluorescence). <b>E, F</b> - Mitochondrial ADP-phosphorylating (state 3) and resting (state 4) respiration rates (E), with representative tracing (F) examined in naïve (n = 11) and at five hours of reperfusion in vehicle (n = 16) or NLF-treated mice (n = 15). * p<0.02 compared to Naïve and NLF treated mice.</p