Morphological and functional changes in rat thyroid gland after a year following chronic exposure to low and intermediate doses of γ-radiation.

INTRODUCTION: Thyroid function depends on iodine uptake by the body as well as on exposure to various harmful environmental hazards (stress, ionizing radiation). AIM: The aim of the work was to assess the effect of exposure to low and intermediate doses of external γ-radiation on the thyroid structure and function in young female rats at remote periods after radiation. MATERIALS AND METHODS: Forty female rats were used to study remote effects of external γ-radiation exposure during 20 d (at daily doses of 0.1, 0.25 and 0.5 Gy) on the functional activity (levels of thyroid hormones, iodine metabolism) and the morphological structure of the rat thyroid) after 12 months following the radiation exposure. RESULTS: An increase in thyroid mass and a decrease in total thyroid protein concentration along with a reduction of blood T3 and T4 was shown only in rat groups exposed to 0.25 and 0.5 Gy. Both the concentration of total iodine and its protein-bound fraction (1.2-1.4 fold, p < .01) and the protein-bound to total iodine ratio were decreased in the thyroids of all irradiated animals. The 0.1-Gy group showed elevated thyroperoxidase (TPO) activity along with increased catalase activity, which may indicate the activation of iodine oxidation by thyrocytes. Only the 0.5-Gy group demonstrated reduced urinary excretion of iodine (2.1 fold, p < .01).The reduction of thyroid function at radiation doses of 0.25 and 0.5 Gy was characterized by a microfollicular structure and the development of atrophic changes in the parenchyma, desquamation of thyroid epithelium and an increase in epithelium proliferation. The diameter of the thyrocyte nuclei was increased in rats exposed to 0.25 and 0.5 Gy, which indicates functional tension of thyrocytes. CONCLUSION: Our research shows that after a year, the exposure to external γ-radiation of 0.1, 0.25 and 0.5-Gy caused changes in the structure and function of the rat thyroid which are manifested by the development of hypothyroiditis (0.5 Gy), 'subclinical' hypothyroiditis (0.25 Gy) and functional tension of thyrocytes. The mechanisms of thyroid dysfunction - impaired- uptake of iodine and its organification against the background of activation of free radical processes - suggest disturbances in the function of the sodium/iodide symporter (NIS), TPO and thyroglobulin synthesis. In contrast to the intermediate doses, the effects of the 0.1-Gy dose were mostly found at the remote periods compared to the earlier periods (180 days).

Mitochondrial calcium buffering depends upon temperature and is associated with hypothermic neuroprotection against hypoxia-ischemia injury.

Hypothermia (HT) is a standard of care in the management of hypoxic-ischemic brain injury (HI). However, therapeutic mechanisms of HT are not well understood. We found that at the temperature of 32°C, isolated brain mitochondria exhibited significantly greater resistance to an opening of calcium-induced permeability transition pore (mPTP), compared to 37°C. Mitochondrial calcium buffering capacity (mCBC) was linearly and inversely dependent upon temperature (25°C-37°C). Importantly, at 37°C cyclosporine A did not increase mCBC, but significantly increased mCBC at lower temperature. Because mPTP contributes to reperfusion injury, we hypothesized that HT protects brain by improvement of mitochondrial tolerance to mPTP activation. Immediately after HI-insult, isolated brain mitochondria demonstrated very poor mCBC. At 30 minutes of reperfusion, in mice recovered under normothermia (NT) or HT, mCBC significantly improved. However, at four hours of reperfusion, only NT mice exhibited secondary decline of mCBC. HT-mice maintained their recovered mCBC and this was associated with significant neuroprotection. Direct inverted dependence of mCBC upon temperature in vitro and significantly increased mitochondrial resistance to mPTP activation after therapeutic HT ex vivo suggest that hypothermia-driven inhibition of calcium-induced mitochondrial mPTP activation mechanistically contributes to the neuroprotection associated with hypothermia.

How many molecules of mitochondrial complex I are in a cell?

Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD+ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis. Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 105 complex I molecules/cell. Our method can be used for accurate quantification of complex I content.

Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart.

Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.

Developmental window of vulnerability to white matter injury driven by sublethal intermittent hypoxemia.

BACKGROUND: In the developing brain, the death of immature oligodendrocytes (OLs) has been proposed to explain a developmental window for vulnerability to white matter injury (WMI). However, in neonatal mice, chronic sublethal intermittent hypoxia (IH) recapitulates the phenotype of diffuse WMI without affecting cellular viability. This work determines whether, in neonatal mice, a developmental window of WMI vulnerability exists in the absence of OLs lineage cellular death. METHODS: Neonatal mice were exposed to cell-nonlethal early or late IH stress. The presence or absence of WMI phenotype in their adulthood was defined by the extent of sensorimotor deficit and diffuse cerebral hypomyelination. A separate cohort of mice was examined for markers of cellular degeneration and OLs maturation. RESULTS: Compared to normoxic littermates, only mice exposed to early IH stress demonstrated arrested OLs maturation, diffuse cerebral hypomyelination, and sensorimotor deficit. No cellular death associated with IH was detected. CONCLUSIONS: Neonatal sublethal IH recapitulates the phenotype of diffuse WMI only when IH stress coincides with the developmental stage of primary white matter myelination. This signifies a contribution of cell-nonlethal mechanisms in defining the developmental window of vulnerability to diffuse WMI. IMPACT: The key message of our work is that the developmental window of vulnerability to the WMI driven by intermittent hypoxemia exists even in the absence of excessive OLs and other cells death. This is an important finding because the existence of the developmental window of vulnerability to WMI has been explained by a lethal-selective sensitivity of immature OLs to hypoxic and ischemic stress, which coincided with their differentiation. Thus, our study expands mechanistic explanation of a developmental window of sensitivity to WMI by showing the existence of cell-nonlethal pathways responsible for this biological phenomenon.

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

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.

NPD1 rapidly targets mitochondria-mediated apoptosis after acute injection protecting brain against ischemic injury.

Mitochondria-related cell death pathways play a major role in ischemic brain injury. Thus, mitochondrial "protective" molecules could be considered for new therapeutic regimens. We recently reported that acute administration of docosahexaenoic acid (DHA) triglyceride lipid emulsion, immediately after hypoxic-ischemic (HI) insult, markedly attenuated brain infarct size. This was associated with an early change of DHA-derived specialized pro-resolving mediator (SPM) profiles. Specifically, DHA treatment induced a 50% increase of neuroprotectin D1 (NPD1) levels in ischemic brain. Based on these findings, we questioned if direct administration of NPD1 after HI injury also affords neuroprotection, and if so, by what mechanisms. Using HI insult to mimic ischemic stroke in neonatal mice, we observed that acute intraperitoneal injection of NPD1 immediately after HI injury prevented the expansion of the ischemic core by ~40% and improved coordination and motor abilities compared to the control group. At 7 days after HI injury, NPD1 treatment decreased ipsilateral hemisphere atrophy and preserved motor functions in wire-holding and bridge-crossing tests compared to control littermates. Brain mitochondria, isolated at 4 h after reperfusion from mice treated with NPD1, showed an increase in the capacity to buffer calcium after HI injury, as result of the preservation of mitochondrial membranes. Further, NPD1 induced a reduction of mitochondrial BAX translocation and oligomerization, attenuated cytochrome C release and decreased AIF nuclear translocation. To confirm whether NPD1 acts as BAX inhibitor, we evaluated NPD1 action co-administrated with a pro-apoptotic agent, staurosporine, using mouse embryonic fibroblasts as in vitro model of apoptosis. NPD1 exposure markedly decreased mitochondria-mediated apoptosis, blocking BAX translocation from cytosol to mitochondria and subsequently reducing caspase-3 activation. Our findings provide novel evidence that the neuroprotective action of NPD1 is elicited rapidly in the first few hours after ischemic injury and is associated with both preserved mitochondrial membrane structure and reduced BAX mitochondrial translocation and activation.

Cyclophilin D-dependent oligodendrocyte mitochondrial ion leak contributes to neonatal white matter injury.

Postnatal failure of oligodendrocyte maturation has been proposed as a cellular mechanism of diffuse white matter injury (WMI) in premature infants. However, the molecular mechanisms for oligodendrocyte maturational failure remain unclear. In neonatal mice and cultured differentiating oligodendrocytes, sublethal intermittent hypoxic (IH) stress activated cyclophilin D-dependent mitochondrial proton leak and uncoupled mitochondrial respiration, leading to transient bioenergetic stress. This was associated with development of diffuse WMI: poor oligodendrocyte maturation, diffuse axonal hypomyelination, and permanent sensorimotor deficit. In normoxic mice and oligodendrocytes, exposure to a mitochondrial uncoupler recapitulated the phenotype of WMI, supporting the detrimental role of mitochondrial uncoupling in the pathogenesis of WMI. Compared with WT mice, cyclophilin D-knockout littermates did not develop bioenergetic stress in response to IH challenge and fully preserved oligodendrocyte maturation, axonal myelination, and neurofunction. Our study identified the cyclophilin D-dependent mitochondrial proton leak and uncoupling as a potentially novel subcellular mechanism for the maturational failure of oligodendrocytes and offers a potential therapeutic target for prevention of diffuse WMI in premature infants experiencing chronic IH stress.

Redox-Dependent Loss of Flavin by Mitochondrial Complex I in Brain Ischemia/Reperfusion Injury.

Aims: Brain ischemia/reperfusion (I/R) is associated with impairment of mitochondrial function. However, the mechanisms of mitochondrial failure are not fully understood. This work was undertaken to determine the mechanisms and time course of mitochondrial energy dysfunction after reperfusion following neonatal brain hypoxia-ischemia (HI) in mice. Results: HI/reperfusion decreased the activity of mitochondrial complex I, which was recovered after 30 min of reperfusion and then declined again after 1 h. Decreased complex I activity occurred in parallel with a loss in the content of noncovalently bound membrane flavin mononucleotide (FMN). FMN dissociation from the enzyme is caused by succinate-supported reverse electron transfer. Administration of FMN precursor riboflavin before HI/reperfusion was associated with decreased infarct volume, attenuation of neurological deficit, and preserved complex I activity compared with vehicle-treated mice. In vitro, the rate of FMN release during oxidation of succinate was not affected by the oxygen level and amount of endogenously produced reactive oxygen species. Innovation: Our data suggest that dissociation of FMN from mitochondrial complex I may represent a novel mechanism of enzyme inhibition defining respiratory chain failure in I/R. Strategies preventing FMN release during HI and reperfusion may limit the extent of energy failure and cerebral HI injury. The proposed mechanism of acute I/R-induced complex I impairment is distinct from the generally accepted mechanism of oxidative stress-mediated I/R injury. Conclusion: Our study is the first to highlight a critical role of mitochondrial complex I-FMN dissociation in the development of HI-reperfusion injury of the neonatal brain. Antioxid. Redox Signal. 31, 608-622.

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.

Krebs cycle metabolites and preferential succinate oxidation following neonatal hypoxic-ischemic brain injury in mice.

BackgroundReverse electron transport (RET) driven by the oxidation of succinate has been proposed as the mechanism of accelerated production of reactive oxygen species (ROS) in post-ischemic mitochondria. However, it remains unclear whether upon reperfusion, mitochondria preferentially oxidase succinate.MethodsNeonatal mice were subjected to Rice-Vannucci model of hypoxic-ischemic brain injury (HI) followed by assessment of Krebs cycle metabolites, mitochondrial substrate preference, and H2O2 generation rate in the ischemic brain.ResultsWhile brain mitochondria from control mice exhibited a rotenone-sensitive complex-I-dependent respiration, HI-brain mitochondria, at the initiation of reperfusion, demonstrated complex-II-dependent respiration, as rotenone minimally affected, but inhibition of complex-II ceased respiration. This was associated with a 30-fold increase of cerebral succinate concentration and significantly elevated H2O2 emission rate in HI-mice compared to controls. At 60 min of reperfusion, cerebral succinate content and the mitochondrial response to rotenone did not differ from that in controls.ConclusionThese data are the first ex vivo evidence, that at the initiation of reperfusion, brain mitochondria transiently shift their metabolism from complex-I-dependent oxidation of NADH toward complex II-linked oxidation of succinate. Our study provides a critical piece of support for existence of the RET-dependent mechanism of elevated ROS production in reperfusion.

PI3Kγ/δ and NOTCH1 Cross-Regulate Pathways That Define the T-cell Acute Lymphoblastic Leukemia Disease Signature.

PI3K/AKT and NOTCH1 signaling pathways are frequently dysregulated in T-cell acute lymphoblastic leukemias (T-ALL). Although we have shown that the combined activities of the class I PI3K isoforms p110γ and p110δ play a major role in the development and progression of PTEN-null T-ALL, it has yet to be determined whether their contribution to leukemogenic programing is unique from that associated with NOTCH1 activation. Using an Lmo2-driven mouse model of T-ALL in which both the PI3K/AKT and NOTCH1 pathways are aberrantly upregulated, we now demonstrate that the combined activities of PI3Kγ/δ have both overlapping and distinct roles from NOTCH1 in generating T-ALL disease signature and in promoting tumor cell growth. Treatment of diseased animals with either a dual PI3Kγ/δ or a γ-secretase inhibitor reduced tumor burden, prolonged survival, and induced proapoptotic pathways. Consistent with their similar biological effects, both inhibitors downregulated genes involved in cMYC-dependent metabolism in gene set enrichment analyses. Furthermore, overexpression of cMYC in mice or T-ALL cell lines conferred resistance to both inhibitors, suggesting a point of pathway convergence. Of note, interrogation of transcriptional regulators and analysis of mitochondrial function showed that PI3Kγ/δ activity played a greater role in supporting the disease signature and critical bioenergetic pathways. Results provide insight into the interrelationship between T-ALL oncogenic networks and the therapeutic efficacy of dual PI3Kγ/δ inhibition in the context of NOTCH1 and cMYC signaling. Mol Cancer Ther; 16(10); 2069-82. ©2017 AACR.

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.

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.

Mild intermittent hypoxemia in neonatal mice causes permanent neurofunctional deficit and white matter hypomyelination.

Very low birth weight (VLBW) premature infants experience numerous, often self-limited non-bradycardic episodes of intermittent hypoxemia (IH). We hypothesized that these episodes of IH affect postnatal white matter (WM) development causing hypomyelination and neurological handicap in the absence of cellular degeneration. Based on clinical data from ten VLBW neonates; a severity, daily duration and frequency of non-bradycardic IH episodes were reproduced in neonatal mice. Changes in heart rate and cerebral blood flow during IH were recorded. A short-term and long-term neurofunctional performance, cerebral content of myelin basic protein (MBP), 2'3' cyclic-nucleotide 3-phosphodiesterase (CNPase), electron microscopy of axonal myelination and the extent of cellular degeneration were examined. Neonatal mice exposed to IH exhibited no signs of cellular degeneration, yet demonstrated significantly poorer olfactory discrimination, wire holding, beam and bridge crossing, and walking-initiation tests performance compared to controls. In adulthood, IH-mice demonstrated no alteration in navigational memory. However, sensorimotor performance on rota-rod, wire-holding and beam tests was significantly worse compared to naive littermates. Both short- and long-term neurofunctional deficits were coupled with decreased MBP, CNPase content and poorer axonal myelination compared to controls. In neonatal mice mild, non-ischemic IH stress, mimicking that in VLBW preterm infants, replicates a key phenotype of non-cystic WM injury: permanent hypomyelination and sensorimotor deficits. Because this phenotype has developed in the absence of cellular degeneration, our data suggest that cellular mechanisms of WM injury induced by mild IH differ from that of cystic periventricular leukomalacia where the loss of myelin-producing cells and axons is the major mechanism of injury.

Mechanical ventilation causes pulmonary mitochondrial dysfunction and delayed alveolarization in neonatal mice.

Hyperoxia inhibits pulmonary bioenergetics, causing delayed alveolarization in mice. We hypothesized that mechanical ventilation (MV) also causes a failure of bioenergetics to support alveolarization. To test this hypothesis, neonatal mice were ventilated with room air for 8 hours (prolonged) or for 2 hours (brief) with 15 µl/g (aggressive) tidal volume (Tv), or for 8 hours with 8 µl/g (gentle) Tv. After 24 hours or 10 days of recovery, lung mitochondria were examined for adenosine diphosphate (ADP)-phosphorylating respiration, using complex I (C-I)-dependent, complex II (C-II)-dependent, or cytochrome C oxidase (C-IV)-dependent substrates, ATP production rate, and the activity of C-I and C-II. A separate cohort of mice was exposed to 2,4-dinitrophenol (DNP), a known uncoupler of oxidative phosphorylation. At 10 days of recovery, pulmonary alveolarization and the expression of vascular endothelial growth factor (VEGF) were assessed. Sham-operated littermates were used as control mice. At 24 hours after aggressive MV, mitochondrial ATP production rates and the activity of C-I and C-II were significantly decreased compared with control mice. However, at 10 days of recovery, only mice exposed to prolonged-aggressive MV continued to exhibit significantly depressed mitochondrial respiration. This was associated with significantly poorer alveolarization and VEGF expression. In contrast, mice exposed to brief-aggressive or prolonged-gentle MV exhibited restored mitochondrial ADP-phosphorylation, normal alveolarization and pulmonary VEGF content. Exposure to DNP fully replicated the phenotype consistent with alveolar developmental arrest. Our data suggest that the failure of bioenergetics to support normal lung development caused by aggressive and prolonged ventilation should be considered a fundamental mechanism for the development of bronchopulmonary dysplasia in premature neonates.

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.

The oxygen free radicals originating from mitochondrial complex I contribute to oxidative brain injury following hypoxia-ischemia in neonatal mice.

Oxidative stress and Ca(2+) toxicity are mechanisms of hypoxic-ischemic (HI) brain injury. This work investigates if partial inhibition of mitochondrial respiratory chain protects HI brain by limiting a generation of oxidative radicals during reperfusion. HI insult was produced in p10 mice treated with complex I (C-I) inhibitor, pyridaben, or vehicle. Administration of P significantly decreased the extent of HI injury. Mitochondria isolated from the ischemic hemisphere in pyridaben-treated animals showed reduced H(2)O(2) emission, less oxidative damage to the mitochondrial matrix, and increased tolerance to the Ca(2+)-triggered opening of the permeability transition pore. A protective effect of pyridaben administration was also observed when the reperfusion-driven oxidative stress was augmented by the exposure to 100% O(2) which exacerbated brain injury only in vehicle-treated mice. In vitro, intact brain mitochondria dramatically increased H(2)O(2) emission in response to hyperoxia, resulting in substantial loss of Ca(2+) buffering capacity. However, in the presence of the C-I inhibitor, rotenone, or the antioxidant, catalase, these effects of hyperoxia were abolished. Our data suggest that the reperfusion-driven recovery of C-I-dependent mitochondrial respiration contributes not only to the cellular survival, but also causes oxidative damage to the mitochondria, potentiating a loss of Ca(2+) buffering capacity. This highlights a novel neuroprotective strategy against HI brain injury where the major therapeutic principle is a pharmacological attenuation, rather than an enhancement of mitochondrial oxidative metabolism during early reperfusion.

Mild hypoxemia during initial reperfusion alleviates the severity of secondary energy failure and protects brain in neonatal mice with hypoxic-ischemic injury.

Reperfusion triggers an oxidative stress. We hypothesized that mild hypoxemia in reperfusion attenuates oxidative brain injury following hypoxia-ischemia (HI). In neonatal HI-mice, the reperfusion was initiated by reoxygenation with room air (RA) followed by the exposure to 100%, 21%, 18%, 15% oxygen for 60 minutes. Systemic oxygen saturation (SaO(2)), cerebral blood flow (CBF), brain mitochondrial respiration and permeability transition pore (mPTP) opening, markers of oxidative injury, and cerebral infarcts were assessed. Compared with RA-littermates, HI-mice exposed to 18% oxygen exhibited significantly decreased infarct volume, oxidative injury in the brain mitochondria and tissue. This was coupled with improved mitochondrial tolerance to mPTP opening. Oxygen saturation maintained during reperfusion at 85% to 95% was associated (r=0.57) with the best neurologic outcome. Exposure to 100% or 15% oxygen significantly exacerbated brain injury and oxidative stress. Compared with RA-mice, hyperoxia dramatically increased reperfusion CBF, but exposure to 15% oxygen significantly reduced CBF to values observed during the HI-insult. Mild hypoxemia during initial reperfusion alleviates the severity of HI-brain injury by limiting the reperfusion-driven oxidative stress to the mitochondria and mPTP opening. This suggests that at the initial stage of reperfusion, a slightly decreased systemic oxygenation (SaO(2) 85% to 95%) may be beneficial for infants with birth asphyxia.