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Purpose: Recently, cerebral autoregulation indices based on moving correlation indices between mean arterial pressure (MAP) and cerebral oximetry (NIRS, ORx) or transcranial Doppler (TCD)-derived middle cerebral artery flow velocity (Mx) have been introduced to clinical practice. In a pilot study, we aimed to evaluate the validity of these indices using incremental lower body negative pressure (LBNP) until presyncope representing beginning cerebral hypoperfusion as well as lower body positive pressure (LBPP) with added mild hypoxia to induce cerebral hyperperfusion in healthy subjects. Methods: Five male subjects received continuous hemodynamic, TCD and NIRS monitoring. Decreasing levels of LBNP were applied in 5-minute steps until subjects reached presyncope. Increasing levels of LBPP were applied stepwise up to 20 or 25 mmHg. Normobaric hypoxia was added until an oxygen saturation of 84% was reached. This was continued for 10 minutes. ORx and Mx indices were calculated using previously described methods. Results: Both Indices showed an increase > 0.3 indicating impaired cerebral autoregulation during presyncope. However, there was no significant difference in Mx at presyncope compared to baseline (p = 0.168). Mean arterial pressure and cardiac output decreased only in presyncope, while stroke volume was decreased at the last pressure level. Neither Mx nor ORx showed significant changes during LBPP or hypoxia. Agreement between Mx and ORx was poor during the LBNP and LBPP experiments (R2 = 0.001, p = 0.3339). Conclusion: Mx and ORx represent impaired cerebral autoregulation, but in Mx this may not be distinguished sufficiently from baseline. LBPP and hypoxia are insufficient to reach the upper limit of cerebral autoregulation as indicated by Mx and ORx. [ABSTRACT FROM AUTHOR]

Background: The SafeBoosC II, randomised clinical trial, showed that the burden of cerebral hypoxia was reduced with the combination of near infrared spectroscopy and a treatment guideline in extremely preterm infants during the first 72 hours after birth. We have previously reported that a high burden of cerebral hypoxia was associated with cerebral haemorrhage and EEG suppression towards the end of the 72-hour intervention period, regardless of allocation. In this study we describe the associations between the burden of cerebral hypoxia and the 2-year outcome. Methods: Cerebral oxygenation was continuously monitored from 3 to 72 hours after birth in 166 extremely preterm infants. At 2 years of age 114 of 133 surviving children participated in the follow-up program: medical examination, Bayley II or III test and the parental Ages and Stages Questionnaire. The infants were classified according to the burden of hypoxia: within the first three quartiles (n = 86, low burden) or within in the 4th quartile (n = 28, high burden). All analyses were conducted post hoc. Results: There were no statistically significant differences between the quantitative assessments of neurodevelopment in the groups of infants with the low burden of cerebral hypoxia versus the group of infants with the high burden of cerebral hypoxia. The infants in the high hypoxia burden group had a higher–though again not statistically significant—rate of cerebral palsy (OR 2.14 (0.33–13.78)) and severe developmental impairment (OR 4.74 (0.74–30.49). Conclusions: The burden of cerebral hypoxia was not significantly associated with impaired 2-year neurodevelopmental outcome in this post-hoc analysis of a feasibility trial. [ABSTRACT FROM AUTHOR]

Objective: Infants with Congenital Heart Disease (CHD) are at risk for developmental delays, though the mechanisms of brain injury that impair development are unknown. Potential causes could include cerebral hypoxia and cerebrovascular instability. We hypothesized that we would detect significantly reduced cerebral oxygen saturation and greater cerebrovascular instability in CHD infants compared to the healthy controls. Methods: We performed a secondary analysis on a sample of 43 term infants (28 CHD, 15 healthy controls) that assessed prospectively in temporal cross-section before or at 12 days of age. CHD infants were assessed prior to open-heart surgery. Cerebral oxygen saturation levels were estimated using Near-Infrared Spectroscopy, and cerebrovascular stability was assessed with the response of cerebral oxygen saturation after a postural change (supine to sitting). Results: Cerebral oxygen saturation was 9 points lower in CHD than control infants in both postures (β = -9.3; 95%CI = -17.68, -1.00; p = 0.028), even after controlling for differences in peripheral oxygen saturation. Cerebrovascular stability was significantly impaired in CHD compared to healthy infants (β = -2.4; 95%CI = -4.12, -.61; p = 0.008), and in CHD infants with single ventricle compared with biventricular defects (β = -1.5; 95%CI = -2.95, -0.05; p = 0.04). Conclusion: CHD infants had cerebral hypoxia and decreased cerebral oxygen saturation values following a postural change, suggesting cerebrovascular instability. Future longitudinal studies should assess the associations of cerebral hypoxia and cerebrovascular instability with long-term neurodevelopmental outcomes in CHD infants. [ABSTRACT FROM AUTHOR]

The time-critical 'can't intubate, can't oxygenate' [CICO] emergency post-induction of anaesthesia is rare, but one which, should it occur, requires Anaesthetists to perform rapid emergency front of neck access [FONA] to the trachea, restoring oxygenation, and preventing death or brain hypoxia. The UK Difficult Airway Society [DAS] has directed all Anaesthetists to be trained with surgical cricothyroidotomy [SCT] as the primary emergency FONA method, sometimes referred to as 'Cric' as a shorthand. We present a longitudinal analysis using a classical approach to Grounded Theory methodology of ten Specialist Trainee Anaesthetists' data during a 6-month training programme delivered jointly by Anaesthetists and Surgeons. We identified with a critical realist ontology and an objectivist epistemology meaning data interpretation was driven by participants' narratives and accepted as true accounts of their experience. Our theory comprises three themes: 'Identity as an Anaesthetist'; 'The Role of a Temporary Surgeon'; and 'Training to Reconcile Identities', whereby training facilitated the psychological transition from a 'bloodless Doctor' (Anaesthetist) to becoming a 'temporary Surgeon'. The training programme enabled Specialist Trainees to move between the role of control and responsibility (Identity as an Anaesthetist), through self-described 'failure' and into a role of uncertainty about one's own confidence and competence (The Role of a Temporary Surgeon), and then return to the Anaesthetist's role once the airway had been established. Understanding the complexity of an intervention and providing a better insight into the training needs of Anaesthetic trainees, via a Grounded Theory approach, allows us to evaluate training programmes against the recognised technical and non-technical needs of those being trained. [ABSTRACT FROM AUTHOR]

Brain tissue oxygen tension (PbtO2)-guided care, a therapeutic strategy to treat or prevent cerebral hypoxia through modifying determinants of cerebral oxygen delivery, including arterial oxygen tension (PaO2), end-tidal carbon dioxide (ETCO2), and mean arterial pressure (MAP), has recently been introduced. Studies have reported that cerebral hypoxia occurs after cardiac arrest in the absence of hypoxemia or hypotension. To obtain preliminary information on the degree to which PbtO2 is responsive to changes in the common target variables for PbtO2-guided care in conditions without hypoxemia or hypotension, we investigated the relationships between the common target variables for PbtO2-guided care and PbtO2 using data from an experimental study in which the animals did not experience hypoxemia or hypotension after resuscitation. We retrospectively analyzed 170 sets of MAP, ETCO2, PaO2, PbtO2, and cerebral microcirculation parameters obtained during the 60-min post-resuscitation period in 10 pigs resuscitated from ventricular fibrillation cardiac arrest. PbtO2 and cerebral microcirculation parameters were measured on parietal cortices exposed through burr holes. Multiple linear mixed effect models were used to test the independent effects of each variable on PbtO2. Despite the absence of arterial hypoxemia or hypotension, seven (70%) animals experienced cerebral hypoxia (defined as PbtO2 <20 mmHg). Linear mixed effect models revealed that neither MAP nor ETCO2 were related to PbtO2. PaO2 had a significant linear relationship with PbtO2 after adjusting for significant covariates (P = 0.030), but it could explain only 17.5% of the total PbtO2 variance (semi-partial R2 = 0.175; 95% confidence interval, 0.086–0.282). In conclusion, MAP and ETCO2 were not significantly related to PbtO2 in animals without hypoxemia or hypotension during the early post-resuscitation period. PaO2 had a significant linear association with PbtO2, but its ability to explain PbtO2 variance was small. [ABSTRACT FROM AUTHOR]

Real-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constant independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. Based on published RG validation studies involving hypoxia the present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex–in order to identify RGs that are stably expressed under these experimental conditions–using several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Using the Geometric mean rank, Pgk1 was identified as the most stable gene. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. Altogether, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each individual experimental paradigm, considering the limitations of the statistical methods used for this aim. [ABSTRACT FROM AUTHOR]

Background: High altitude associated hypobaric hypoxia is one of the cellular and environmental perturbation that alters proteostasis network and push the healthy cell towards loss of muscle mass. The present study has elucidated the robust proteostasis network and signaling mechanism for skeletal muscle atrophy under chronic hypobaric hypoxia (CHH). Methods: Male Sprague Dawley rats were exposed to simulated hypoxia equivalent to a pressure of 282 torr for different durations (1, 3, 7 and 14 days). After CHH exposure, skeletal muscle tissue was excised from the hind limb of rats for biochemical analysis. Results: Chronic hypobaric hypoxia caused a substantial increase in protein oxidation and exhibited a greater activation of ER chaperones, glucose-regulated protein-78 (GRP-78) and protein disulphide isomerase (PDI) till 14d of CHH. Presence of oxidized proteins triggered the proteolytic systems, 20S proteasome and calpain pathway which were accompanied by a marked increase in [Ca2+]. Upregulated Akt pathway was observed upto 07d of CHH which was also linked with enhanced glycogen synthase kinase-3β (GSk-3β) expression, a negative regulator of Akt. Muscle-derived cytokines, tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-©) and interleukin-1β (IL-1β) levels significantly increased from 07d onwards. CHH exposure also upregulated the expression of nuclear factor kappa-B (NF-κB) and E3 ligase, muscle atrophy F-box-1 (Mafbx-1/Atrogin-1) and MuRF-1 (muscle ring finger-1) on 07d and 14d. Further, severe hypoxia also lead to increase expression of ER-associated degradation (ERAD) CHOP/ GADD153, Ub-proteasome and apoptosis pathway. Conclusions: The disrupted proteostasis network was tightly coupled to degradative pathways, altered anabolic signaling, inflammation, and apoptosis under chronic hypoxia. Severe and prolonged hypoxia exposure affected the protein homeostasis which overwhelms the muscular system and tends towards skeletal muscle atrophy. [ABSTRACT FROM AUTHOR]

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection. [ABSTRACT FROM AUTHOR]

Background: Determination of the blood oxyhemoglobin saturation in the retinal vessels of the eye can be achieved through spectrophotometric retinal oximetry which provides access to the state of oxyhemoglobin saturation in the central nervous system circulation. The purpose of this study was to test the capability of the Oxymap T1 oximeter to detect systemic hypoxemia and the effect of supplemental oxygen on retinal vessel oxyhemoglobin saturation. Methods: Oxygen saturation of hemoglobin in retinal arterioles and venules was measured in 11 subjects with severe chronic obstructive pulmonary disease (COPD) on long term oxygen therapy. Measurements were made with and without their daily supplemental oxygen. Eleven healthy age and gender matched subjects were measured during ambient air breathing for comparison of oxyhemoglobin saturation in retinal arterioles and venules. Retinal arteriolar oxyhemoglobin saturation in COPD subjects inspiring ambient air was compared with finger pulse oximetry and blood samples from radial artery. Results: COPD subjects had significantly lower oxyhemoglobin saturation during ambient air breathing than healthy controls in both retinal arterioles (87.2%±4.9% vs. 93.4%±4.3%, p = 0.02; n = 11) and venules (45.0%±10.3% vs. 55.2%±5.5%, p = 0.01). Administration of their prescribed supplemental oxygen increased oxyhemoglobin saturation in retinal arterioles (87.2%±4.9% to 89.5%±6.0%, p = 0.02) but not in venules (45.0%±10.3% to 46.7%±12.8%, p = 0.3). Retinal oximetry values were slightly lower than radial artery blood values (mean percentage points difference = -5.0±5.4, 95% CI: -15.68 to 5.67) and finger pulse oximetry values (-3.1±5.5, 95% CI: -14.05 to 7.84). Conclusions: The noninvasive Oxymap T1 retinal oximetry detects hypoxemia in central nervous system vessels in patients with severe COPD compared with healthy controls. The instrument is sensitive to changes in oxygen breathing but displays slightly lower measures than finger pulse oximetry or radial artery measures. With further technological improvement, retinal oximetry may offer noninvasive “on-line” measurement of oxygen levels in central circulation in general anesthesia and critically ill patients. [ABSTRACT FROM AUTHOR]

Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. We detected that IP3R1 accumulates specially in CA1, and this spatial pattern did not change after neonatal anoxia. Interestingly, we observed that anoxia triggers translocation of IP3R1 to nucleus in hippocampal cells. We were able to observe that anoxia changes distribution of IP3R1 immunofluorescence signals, as revealed by cluster size analysis. We next examined the role of IP3R1 in the neuronal cell loss triggered by neonatal anoxia. Intrahippocampal injection of non-specific IP3R1 blocker 2-APB clearly reduced the number of Fluoro-Jade C and Tunel positive cells, revealing that activation of IP3R1 increases cell death after neonatal anoxia. Finally, we aimed to disclose mechanistics of IP3R1 in cell death. We were able to determine that blockade of IP3R1 did not reduced the distribution and pixel density of activated caspase 3-positive cells, indicating that the participation of IP3R1 in neuronal cell loss is not related to classical caspase-mediated apoptosis. In summary, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ventilatory acclimatization to hypoxia is a time-dependent increase in ventilation and the hypoxic ventilatory response (HVR) that involves neural plasticity in both carotid body chemoreceptors and brainstem respiratory centers. The mechanisms of such plasticity are not completely understood but recent animal studies show it can be blocked by administering ibuprofen, a nonsteroidal anti-inflammatory drug, during chronic hypoxia. We tested the hypothesis that ibuprofen would also block the increase in HVR with chronic hypoxia in humans in 15 healthy men and women using a double-blind, placebo controlled, cross-over trial. The isocapnic HVR was measured with standard methods in subjects treated with ibuprofen (400mg every 8 hrs) or placebo for 48 hours at sea level and 48 hours at high altitude (3,800 m). Subjects returned to sea level for at least 30 days prior to repeating the protocol with the opposite treatment. Ibuprofen significantly decreased the HVR after acclimatization to high altitude compared to placebo but it did not affect ventilation or arterial O2 saturation breathing ambient air at high altitude. Hence, compensatory responses prevent hypoventilation with decreased isocapnic ventilatory O2-sensitivity from ibuprofen at this altitude. The effect of ibuprofen to decrease the HVR in humans provides the first experimental evidence that a signaling mechanism described for ventilatory acclimatization to hypoxia in animal models also occurs in people. This establishes a foundation for the future experiments to test the potential role of different mechanisms for neural plasticity and ventilatory acclimatization in humans with chronic hypoxemia from lung disease. [ABSTRACT FROM AUTHOR]

Aging is associated with an increased risk of seizures/epilepsy. Stroke (ischemic or hemorrhagic) and cardiac arrest related brain injury are two major causative factors for seizure development in this patient population. With either etiology, seizures are a poor prognostic factor. In spite of this, the underlying pathophysiology of seizure development is not well understood. In addition, a standardized treatment regimen with anticonvulsants and outcome assessments following treatment has yet to be established for these post-ischemic seizures. Previous studies have modeled post-ischemic seizures in adult rodents, but similar studies in aging/aged animals, a group that mirrors a higher risk elderly population, remain sparse. Our study therefore aimed to investigate early-onset seizures in aging animals using a hypoxia-ischemia (HI) model. Male C57 black mice 18-20-month-old underwent a unilateral occlusion of the common carotid artery followed by a systemic hypoxic episode (8% O2 for 30 min). Early-onset seizures were detected using combined behavioral and electroencephalographic (EEG) monitoring. Brain injury was assessed histologically at different times post HI. Convulsive seizures were observed in 65% of aging mice post-HI but not in control aging mice following either sham surgery or hypoxia alone. These seizures typically occurred within hours of HI and behaviorally consisted of jumping, fast running, barrel-rolling, and/or falling (loss of the righting reflex) with limb spasms. No evident discharges during any convulsive seizures were seen on cortical-hippocampal EEG recordings. Seizure development was closely associated with acute mortality and severe brain injury on brain histological analysis. Intra-peritoneal injections of lorazepam and fosphenytoin suppressed seizures and improved survival but only when applied prior to seizure onset and not after. These findings together suggest that seizures are a major contributing factor to acute mortality in aging mice following severe brain ischemia and that early anticonvulsive treatment may prevent seizure genesis and improve overall outcomes. [ABSTRACT FROM AUTHOR]

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury. [ABSTRACT FROM AUTHOR]

The relative importance of environmental hypoxia due to global climate change on organismal ability to adapt to chemical insult and/or mechanisms of these responses is not well understood. Therefore, we have studied the effects of combined exposure to perfluorooctane sulfonamide (PFOSA) and chemically induced hypoxia on membrane lipid profile and homeostasis. Primary salmon hepatocytes were exposed to PFOSA at 0, 25 and 50 µM singly or in combination with either cobalt chloride (CoCl2: 0 and 150 µM) or deferroxamine (DFO: 0 and 100 µM) for 24 and 48 h. CoCl2 and DFO were used to induce cellular hypoxia because these two chemicals have been commonly used in animal experiments for this purpose and have been shown to increase hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) levels. Fatty acid (FA) profiles were determined by GC-MS, while gene expression patterns were determined by quantitative PCR. Hypoxic condition was confirmed with time-related increases of HIF-1α mRNA levels in CoCl2 and DFO exposed cells. In general, significant alterations of genes involved in lipid homeostasis were predominantly observed after 48 h exposure. Gene expression analysis showed that biological responses related to peroxisome proliferation (peroxisome proliferator-activated receptors (PPARs) and acyl coenzyme A (ACOX)) and FA desaturation (Δ5- and Δ6-desaturases: FAD5 and FAD6, respectively) and elongation (FAE) were elevated slightly by single exposure (i.e. either PFOSA, CoCl2 or DFO exposure alone), and these responses were potentiated in combined exposure conditions. Principal component analysis (PCA) showed a clustering of peroxisome proliferation responses at transcript levels and FA desaturation against membrane FAs levels whose changes were explained by PFOSA and chemically induced hypoxia exposures. Overall, our data show that most of the observed responses were stronger in combined stressor exposure conditions, compared to individual stressor exposure. In general, our data show that hypoxia may, singly or in combination with PFOSA produce deleterious health, physiological and developmental consequences through the alteration of membrane lipid profile in organisms. [ABSTRACT FROM AUTHOR]

Edaravone, a potent antioxidant, may improve thrombolytic therapy because it benefits ischemic stroke patients on its own and mitigates adverse effects of tissue plasminogen activator (tPA) in preclinical models. However, whether the combined tPA-edaravone therapy is more effective in reducing infarct size than singular treatment is uncertain. Here we investigated this issue using a transient hypoxia-ischemia (tHI)-induced thrombotic stroke model, in which adult C57BL/6 mice were subjected to reversible ligation of the unilateral common carotid artery plus inhalation of 7.5% oxygen for 30 min. While unilateral occlusion of the common carotid artery suppressed cerebral blood flow transiently, the addition of hypoxia triggered reperfusion deficits, endogenous thrombosis, and attenuated tPA activity, leading up to infarction. We compared the outcomes of vehicle-controls, edaravone treatment, tPA treatment at 0.5, 1, or 4 h post-tHI, and combined tPA-edaravone therapies with mortality rate and infarct size as the primary end-points. The best treatment was further compared with vehicle-controls in behavioral, biochemical, and diffusion tensor imaging (DTI) analyses. We found that application of tPA at 0.5 or 1 h – but not at 4 h post-tHI – significantly decreased infarct size and showed synergistic (p<0.05) or additive benefits with the adjuvant edaravone treatment, respectively. The acute tPA-edaravone treatment conferred >50% reduction of mortality, ∼80% decline in infarct size, and strong white-matter protection. It also improved vascular reperfusion and decreased oxidative stress, inflammatory cytokines, and matrix metalloproteinase activities. In conclusion, edaravone synergizes with acute tPA treatment in experimental thrombotic stroke, suggesting that clinical application of the combined tPA-edaravone therapy merits investigation. [ABSTRACT FROM AUTHOR]

Background: For oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall. Objective: In our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids. Methods: BSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O2) and hypoxic (1% and 5% O2) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay. Results: Proliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O2, whereas 5% O2 reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium. Conclusion: Hypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling. [ABSTRACT FROM AUTHOR]

Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD), a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL) loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1β was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R1 and IL-1R1 in APC+ OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM) and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1β in primary cultured astrocytes as well as their corresponding receptors in primary culture APC+ oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1β, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats. [ABSTRACT FROM AUTHOR]

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that leads to progressive weakness of the respiratory and limb muscles. Consequently, most patients with ALS exhibit progressive hypoventilation, which worsens during sleep. The aim of this study was to evaluate the relationship between nocturnal hypoxia and cognitive dysfunction and to assess the pattern of nocturnal hypoxia in patients with ALS. Method: Twenty-five patients with definite or probable ALS underwent neuropsychologic testing, nocturnal pulse oximetry, and capnography. Patients were grouped according to the presence of nocturnal hypoxia (SpO2<95% for ≥10% of the night) and their clinical characteristics and cognitive function were compared. Results: Compared to patients without nocturnal hypoxia, those with nocturnal hypoxia (n = 10, 40%) had poor memory retention (p = 0.039) and retrieval efficiency (p = 0.045). A cluster-of-desaturation pattern was identified in 7 patients (70%) in the Hypoxia Group. Conclusions: These results suggest that nocturnal hypoxia can be related to cognitive dysfunction in ALS. In addition, a considerable number of patients with ALS may be exposed to repeated episodes of deoxygenation–reoxygenation (a cluster-of-desaturation pattern) during sleep, which could be associated with the generation of reactive oxygen species. Further studies are required to define the exact causal relationships between these phenomena, the exact manifestations of nocturnal cluster-of-desaturation patterns, and the effect of clusters of desaturation on ALS progression. [ABSTRACT FROM AUTHOR]

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment. [ABSTRACT FROM AUTHOR]

Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders. [ABSTRACT FROM AUTHOR]

Background: Intra-uterine growth restriction (IUGR) has been associated with increased predisposition to age-related complications. We tested the hypothesis that rat offspring models of IUGR would exhibit exacerbated, age-related retinal dysfunction. Methods: Female Sprague-Dawley rats (maintained at 11.5% O2 from gestational day 15 to 21 to induce IUGR) and control offspring (maintained at 21% O2 throughout pregnancy) had retinal function assessed at 2 months (young) and 14 months of age (aged) with electroretinogram (ERG) recordings. Retinal anatomy was assessed by immunofluorescence. Results: Deficits in rod-driven retina function were observed in aged IUGR offspring, as evidenced by reduced amplitudes of dark-adapted mixed a-wave Vmax (by 49.3%, P<0.01), b-wave Vmax (by 42.1%, P<0.001) and dark-adapted peak oscillatory potentials (by 42.3%, P<0.01). In contrast to the rod-driven defects specific to aged IUGR offspring, light adapted ERG recordings revealed cone defects in young animals, that were stationary until old age. At 2 months, IUGR offspring had amplitude reductions for both b-wave (Vmax by 46%, P<0.01) and peak oscillatory potential (Vmax by 38%, P<0.05). Finally, defects in cone-driven responses were further confirmed by reduced maximal photopic flicker amplitudes at 2 (by 42%, P<0.001) and 14 months (by 34%, P = 0.06) and critical flicker fusion frequencies at 14 months (Control: 42±1 Hz, IUGR: 35±2 Hz, P<0.05). These functional changes were not paralleled by anatomical losses in IUGR offspring retinas. Conclusions: These data support that the developing retina is sensitive to stressors, and that pathways governing cone- and rod-driven function differ in their susceptibilities. In the case of prenatal hypoxia, cone- and rod-driven dysfunction manifest at young and old ages, respectively. We must, therefore, take into account the specific impact that fetal programming might exert on age-related retinal dystrophies when considering related diagnoses and therapeutic applications. [ABSTRACT FROM AUTHOR]

It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult. [ABSTRACT FROM AUTHOR]

Background: Monitoring cerebral saturation is increasingly seen as an aid to management of patients in the operating room and in neurocritical care. How best to manipulate cerebral saturation is not fully known. We examined cerebral saturation with graded changes in carbon dioxide tension while isoxic and with graded changes in oxygen tension while isocapnic. Methodology/Principal Findings: The study was approved by the Research Ethics Board of the University Health Network at the University of Toronto. Thirteen studies were undertaken in healthy adults with cerebral oximetry by near infrared spectroscopy. End-tidal gas concentrations were manipulated using a model-based prospective end-tidal targeting device. End-tidal carbon dioxide was altered ±15 mmHg from baseline in 5 mmHg increments with isoxia (clamped at 110±4 mmHg). End-tidal oxygen was changed to 300, 400, 500, 80, 60 and 50 mmHg under isocapnia (37±2 mmHg). Twelve studies were completed. The end-tidal carbon dioxide versus cerebral saturation fit a linear relationship (R2 = 0.92±0.06). The end-tidal oxygen versus cerebral saturation followed log-linear behaviour and best fit a hyperbolic relationship (R2 = 0.85±0.10). Cerebral saturation was maximized in isoxia at end-tidal carbon dioxide of baseline +15 mmHg (77±3 percent). Cerebral saturation was minimal in isocapnia at an end-tidal oxygen tension of 50 mmHg (61±3 percent). The cerebral saturation during normoxic hypocapnia was equivalent to normocapnic hypoxia of 60 mmHg. Conclusions/Significance: Hypocapnia reduces cerebral saturation to an extent equivalent to moderate hypoxia. [ABSTRACT FROM AUTHOR]

Hypoxic ischemic encephalopathy (HIE) affects 2-3 per 1000 full-term neonates. Up to 75% of newborns with severe HIE die or have severe neurological handicaps. Stem cell therapy offers the potential to replace HIE-damaged cells and enhances the autoregeneration process. Our laboratory implanted Multipotent Astrocytic Stem Cells (MASCs) into a neonatal rat model of hypoxia-ischemia (HI) and demonstrated that MASCs move to areas of injury in the cortex and hippocampus. However, only a small proportion of the implanted MASCs differentiated into neurons. MASCs injected into control pups did not move into the cortex or differentiate into neurons. We do not know the mechanism by which the MASCs moved from the site of injection to the injured cortex. We found neurotrophins present after the hypoxic-ischemic milieu and hypothesized that neurotrophins could enhance the migration and differentiation of MASCs. Using a Boyden chamber device, we demonstrated that neurotrophins potentiate the in vitro migration of stem cells. NGF, GDNF, BDNF and NT-3 increased stem cell migration when compared to a chemokinesis control. Also, MASCs had increased differentiation toward neuronal phenotypes when these neurotrophins were added to MASC culture tissue. Due to this finding, we believed neurotrophins could guide migration and differentiation of stem cell transplants after brain injury. [ABSTRACT FROM AUTHOR]

Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles) that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors. [ABSTRACT FROM AUTHOR]

Background: Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied. Methods and Findings: Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate. Conclusions: Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells. [ABSTRACT FROM AUTHOR]

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O2). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca2+-activated K+ channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 μM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons. [ABSTRACT FROM AUTHOR]

Establishment of marine protected areas, including fully protected marine reserves, is one of the few management tools available for local communities to combat the deleterious effect of large scale environmental impacts, including global climate change, on ocean ecosystems. Despite the common hope that reserves play this role, empirical evidence of the effectiveness of local protection against global problems is lacking. Here we show that marine reserves increase the resilience of marine populations to a mass mortality event possibly caused by climate-driven hypoxia. Despite high and widespread adult mortality of benthic invertebrates in Baja California, Mexico, that affected populations both within and outside marine reserves, juvenile replenishment of the species that supports local economies, the pink abalone Haliotis corrugata, remained stable within reserves because of large body size and high egg production of the protected adults. Thus, local protection provided resilience through greater resistance and faster recovery of protected populations. Moreover, this benefit extended to adjacent unprotected areas through larval spillover across the edges of the reserves. While climate change mitigation is being debated, coastal communities have few tools to slow down negative impacts of global environmental shifts. These results show that marine protected areas can provide such protection. [ABSTRACT FROM AUTHOR]

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and nonphysiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O2 tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency. [ABSTRACT FROM AUTHOR]

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model. [ABSTRACT FROM AUTHOR]

Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans. [ABSTRACT FROM AUTHOR]

Background: Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress. Methodology and Principal Findings: Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M77, thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the proinflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and proapoptosis genes. Conclusion: A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure. [ABSTRACT FROM AUTHOR]

For more than a century oxygen has been known to be one of the most powerful radiosensitizers. However, despite decades of preclinical and clinical research aimed at overcoming tumor hypoxia, little clinical progress has been made so far. Ionizing radiation damages DNA through generation of free radicals. In the presence of oxygen these lesions are chemically modified, and thus harder to repair while hypoxia protects cells from radiation (Oxygen enhancement ratio (OER)). Breast cancer stem cells (BSCSs) are protected from radiation by high levels of free radical scavengers even in the presence of oxygen. This led us to hypothesize that BCSCs exhibit an OER of 1. Using four established breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SUM159PT) and primary breast cancer samples, we determined the number of BCSCs using cancer stem cell markers (ALDH1, low proteasome activity), compared radiation clonogenic survival and mammosphere formation under normoxic and hypoxic conditions, and correlated these results to the expression levels of key members of the free radical scavenging systems. The number of BCSCs increased with increased aggressiveness of the cancer. This correlated with increased radioresistance (SF8Gy), and decreasing OERs. When cultured as mammospheres, breast cancer cell lines and primary samples were highly radioresistant and not further protected by hypoxia (OER~1). We conclude that because BCSCs are protected from radiation through high expression levels of free radical scavengers, hypoxia does not lead to additional radioprotection of BCSCs. [ABSTRACT FROM AUTHOR]

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli. [ABSTRACT FROM AUTHOR]

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities. [ABSTRACT FROM AUTHOR]

Maternal obesity results in a number of obstetrical and fetal complications with both immediate and long-term consequences. The increased prevalence of obesity has resulted in increasing numbers of women of reproductive age in this high-risk group. Since many of these obese women have been subjected to hypercaloric diets from early childhood we have developed a rodent model of life-long maternal obesity to more clearly understand the mechanisms that contribute to adverse pregnancy outcomes in obese women. Female Sprague Dawley rats were fed a control diet (CON - 16% of calories from fat) or high fat diet (HF - 45% of calories from fat) from 3 to 19 weeks of age. Prior to pregnancy HF-fed dams exhibited significant increases in body fat, serum leptin and triglycerides. A subset of dams was sacrificed at gestational day 15 to evaluate fetal and placental development. The remaining animals were allowed to deliver normally. HF-fed dams exhibited a more than 3-fold increase in fetal death and decreased neonatal survival. These outcomes were associated with altered vascular development in the placenta, as well as increased hypoxia in the labyrinth. We propose that the altered placental vasculature may result in reduced oxygenation of the fetal tissues contributing to premature demise and poor neonatal survival. [ABSTRACT FROM AUTHOR]

Background: Recent studies have emphasized causative links between microRNAs (miRNAs) deregulation and tumor development. In hepatocellular carcinoma (HCC), more and more miRNAs were identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools. This study aimed to investigate the functional significance and regulatory mechanism of the miR-199a2/214 cluster in HCC progression. Methods and Findings: In this study, we showed that miR-214, as well as miR-199a-3p and miR-199a-5p levels were significantly reduced in the majority of examined 23 HCC tissues and HepG2 and SMMC-7721 cell lines, compared with their nontumor counterparts. To further explore the role of miR-214 in hepatocarcinogenesis, we disclosed that the ER stressinduced pro-survival factor XBP-1 is a target of miR-214 by using western blot assay and luciferase reporter assay. Reexpression of miR-214 in HCC cell lines (HepG2 and SMMC-7721) inhibited proliferation and induced apoptosis. Furthermore, ectopic expression of miR-214 dramatically suppressed the ability of HCC cells to form colonies in vitro and to develop tumors in a subcutaneous xenotransplantation model of the BALB/c athymic nude mice. Moreover, reintroduction of XBP-1s attenuated miR-214-mediated suppression of HCC cells proliferation, colony and tumor formation. To further understand the mechanism of the miR-199a/214 cluster down-expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia-induced unfolded protein response (UPR) suppresses the expression of the miR-199a/214 cluster in HCC cells. By promoter analysis of the miR-199a2/214 gene, we conjectured NFkB as a potential negative regulator. We further found that UPR and LPS-induced NFkB activation suppressed miR-199a2/214 transcription, and this suppression was reversed by NFkB inhibition in HCC cells. [ABSTRACT FROM AUTHOR]

Background: Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. Methods and Findings: Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acidinduced differentiation of neuroblastoma cells. Conclusions: Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells. [ABSTRACT FROM AUTHOR]

Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxicregulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS. [ABSTRACT FROM AUTHOR]

Background: Inflammatory cytokines are detected in the plasma of patients with renal cell carcinoma (RCC) and are associated with poor prognosis. However, the primary cell type involved in producing inflammatory cytokines and the biological significance in RCC remain unknown. Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and production of pro-inflammatory gene products. Solid tumors are often heterogeneous in oxygen tension together suggesting that hypoxia may play a role in inflammatory processes in RCC. Epithelial cells have been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status and a role for AMPK in the regulation of cell inflammatory processes has recently been demonstrated. Methods and Principal Findings: We have identified for the first time that interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted solely from RCC cells exposed to hypoxia. Furthermore, we demonstrate that the NADPH oxidase isoform, Nox4, play a key role in hypoxia-induced IL-6 and IL-8 production in RCC. Finally, we have characterized that enhanced levels of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion. Conclusions/Significance: Together, our data identify novel mechanisms by which AMPK and Nox4 may be linked to inflammation-induced RCC metastasis and that pharmacological activation of AMPK and/or antioxidants targeting Nox4 may represent a relevant therapeutic intervention to reduce IL-6- and IL-8-induced inflammation and cell invasion in RCC. [ABSTRACT FROM AUTHOR]

Background: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. Methodology/Finding: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 mM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NOmediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. Conclusion: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia. [ABSTRACT FROM AUTHOR]

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing. [ABSTRACT FROM AUTHOR]

Background: Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic betacells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxiadependent manner. Methodology/Principal Findings: Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Leprdb/db but not non-diabetic Leprdb/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. Conclusions/Significance: Glucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes. [ABSTRACT FROM AUTHOR]

Background Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. Results Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by NG-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O2 consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. Conclusion Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background: Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes. Methodology/Principal Findings: BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10-1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2a protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2a protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2a protein and EPO mRNA. Conclusions/Significance: Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes. [ABSTRACT FROM AUTHOR]

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors. [ABSTRACT FROM AUTHOR]

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse. [ABSTRACT FROM AUTHOR]

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 59 proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a doseand time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl 2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases. [ABSTRACT FROM AUTHOR]

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged fromapproximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD. [ABSTRACT FROM AUTHOR]