Prehypoxic Levels Research Articles

Basal and Hypoxic Respiratory Behaviors in Aged 6‐OHDA‐Induced SN Lesion PD Rat Model: Effects of the 5‐HT1A Receptor Agonist Buspirone

Parkinson’s disease (PD) is progressive neurodegenerative disease, with the diagnosis of idiopathic PD occurring in humans at ~60 years of age. Most studies, however, use young adult (≤3 month old) rats to investigate PD‐related abnormalities in various physiological systems, with the most commonly used experimental rat PD model being generated by unilateral injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA) into specific regions of the nigrostriatal pathway. While this model results in the loss of dopaminergic neurons and the subsequent changes in downstream neurotransmitter signaling that are characteristic of PD, it does not consider potential contributions of physical changes to the brain and neurotransmitter signaling that are part of normal aging on the progression of PD. To better understand age‐related influences and potential contributions of PD‐related abnormalities in serotonergic (5‐HT) neurotransmission on respiratory dysfunction (which is a common non‐motor symptom) in PD, we investigated basal and hypoxic (12% O2; 90s) respiratory behaviors before and after acute administration of the frequently prescribed anxiolytic 5‐HT1A receptor agonist buspirone (BUS; 0.5 mg/kg, iv) in an aged (~9 month old) female Sprague‐Dawley PD rat model produced by unilateral 6‐OHDA injection into the substantia nigra (SN). For these experiments, diaphragm EMG activity was recorded and quantified from spontaneously breathing urethane‐anesthetized rats at 2‐weeks after SN 6‐OHDA or vehicle (control) injections. We found that 6‐OHDA‐injected rats have a lower basal breathing frequency (P<0.05) due to shorter expiratory duration (TE; P<0.05) than vehicle‐injected rats, but both 6‐OHDA‐ and vehicle‐injected rats exhibit significant increases in breathing frequency (P≤0.001) and EMG amplitude (P≤0.023) during hypoxia; the magnitude of the peak frequency increase in 6‐OHDA‐injected rats was slightly greater (P=0.083). Both 6‐OHDA‐ and vehicle‐injected rats also showed similar post‐hypoxic frequency decline (P≤0.003), but only vehicle‐injected rats displayed a slight and sustained post‐hypoxic amplitude depression (P<0.1). In vehicle‐injected rats, acute BUS injection produced a progressive decrease in basal EMG amplitude (P=0.066) and a persistent increase in breathing frequency (P=0.064) by decreasing inspiratory duration (TI; P<0.001) while in 6‐OHDA‐injected rats, a transient initial increase in EMG amplitude (P=0.043) and a persistent decrease in TI (P=0.005) and TE (P=0.089) were noted. In both 6‐OHDA‐ and vehicle‐injected rats, BUS injection also slightly exaggerated the initial hypoxic frequency increase, but blunted the amplitude increase with amplitude progressively declining to near pre‐hypoxic levels before the end of the hypoxic exposure. BUS injection also shortened the duration of post‐hypoxic EMG amplitude depression in vehicle‐injected rats. These data demonstrate that some differences in basal and hypoxic respiratory behaviors exist between older 6‐OHDA‐ and vehicle‐injected rats, and that some respiratory behaviors of these rat models are differentially impacted by BUS‐induced effects on respiratory control. We suggest that this older PD rat model be further evaluated.

Activation of Astrocytes in the Persistence of Post-hypoxic Respiratory Augmentation.

Acute hypoxia increases ventilation. After cessation of hypoxia loading, ventilation decreases but remains above the pre-exposure baseline level for a time. However, the mechanism of this post-hypoxic persistent respiratory augmentation (PHRA), which is a short-term potentiation of breathing, has not been elucidated. We aimed to test the hypothesis that astrocytes are involved in PHRA. To this end, we investigated hypoxic ventilatory responses by whole-body plethysmography in unanesthetized adult mice. The animals breathed room air, hypoxic gas mixture (7% O2, 93% N2) for 2min, and again room air for 10min before and after i.p. administration of low (100mg/kg) and high (300mg/kg) doses of arundic acid (AA), an astrocyte inhibitor. AA suppressed PHRA, with the high dose decreasing ventilation below the pre-hypoxic level. Further, we investigated the role of the astrocytic TRPA1 channel, a putative ventilatory hypoxia sensor, in PHRA using astrocyte-specific Trpa1 knockout (asTrpa1−/−) and floxed Trpa1 (Trpa1f/f) mice. In both Trpa1f/f and asTrpa1−/− mice, PHRA was noticeable, indicating that the astrocyte TRPA1 channel was not directly involved in PHRA. Taken together, these results indicate that astrocytes mediate the PHRA by mechanisms other than TRPA1 channels that are engaged in hypoxia sensing.

Astrocytes mediate the post‐hypoxic persistent respiratory augmentation

Acute hypoxia increases ventilation. After brief hypoxic exposure, switchback of inspired gas to room air is accompanied by short-term potentiation of breathing, i.e., ventilation remains elevated above the baseline level. Short-term potentiation of breathing has been thought to play a stabilizing role in control of breathing, e.g., prevention of periodic breathing. However, the mechanism of this potentiation is unclear. We aimed to test the hypothesis that astrocytes mediate this potentiation, i.e., post-hypoxic persistent respiratory augmentation (PHRA), and performed a series of in vitro and in vivo experiments. In vitro, isolated brainstem-spinal cord preparations of neonatal rats were superfused with artificial cerebrospinal fluid (aCSF) without or with arundic acid (500 µM), an inhibitory modulator of astrocytic function. Both groups were sequentially superfused with oxygenated (95% O2, 5% CO2) and hypoxic (95% N2, 5% CO2) aCSF for 5 min, and again with oxygenated aCSF for 17 min. Hypoxia universally increased the respiratory burst rate. Without arundic acid, the burst rate transiently decreased with reoxygenation, and then recovered to and remained at the pre-hypoxic level until the end of the post-hypoxic observation. Contrarily, the burst rate steadily decreased throughout the post-hypoxic phase with arundic acid, declining below the pre-hypoxic level. In vivo, the hypoxic ventilatory response was investigated by whole-body plethysmography in unanesthetized adult mice. The animals breathed room air, hypoxic gas (7% O2, 93% N2) for 2 min, and again room air for 10 min before and after i.p. administration of low (100 mg/kg) and high (300 mg/kg) doses of arundic acid. Minute ventilation in the post-hypoxic recovery was maintained above the pre-hypoxic level without arundic acid. However, it was suppressed with arundic acid. Notably with high dose arundic acid, it decreased below the pre-hypoxic level. Finally, we investigated the significance of the astrocytic TRPA1 channel, which is a ventilatory hypoxic sensor, in the PHRA using astrocyte-specific Trpa1 knockout (asTrpa1-/-) and floxed Trpa1 (Trpa1f/f) mice. Although the PHRA was observed in both Trpa1f/f and as Trpa1-/- mice, it was weaker in asTrpa1-/- as compared to Trpa1f/f mice. This observation indicates that the astrocytic TRPA1 channel plays a role in the PHRA but this by itself is not sufficient, suggesting the existence of additional key factors. These in vitro and in vivo experimental findings collectively demonstrate that astrocytes mediate the PHRA partially through the TRPA1 channel.

Impact of aging on basal and hypoxic respiratory behaviors in short‐ and long‐term 6‐OHDA‐induced SN lesion PD rat models

Parkinson's Disease (PD) is a progressive neurodegenerative disease often associated with aging, as it typically occurs in individuals over the age of 50. As such, investigating features and mechanisms of PD becomes complicated since molecular changes to dopaminergic (DA) neurons, subsequent neuronal loss, and downstream alterations to non-DA neurotransmitter systems can progress over decades to cause the various motor and non-motor symptoms (NMS), which can be further compounded by changes caused by normal aging. Amongst the earliest symptoms to arise in PD patients, respiratory abnormalities are a common feature, and ongoing work in our laboratory has focused on characterizing respiratory dysfunction phenotypes in preclinical PD rat models produced by 6-hydroxydopamine (6-OHDA) neurotoxin-induced lesion of the nigrostriatal pathway. These studies are typically conducted in rats aged 3-4 months at 2-4 weeks following lesion. To begin to understand the impact of aging on PD-related respiratory abnormalities, the current study evaluated basal and hypoxic (12% O2; 90s) respiratory behaviors in ~38-week-old urethane-anesthetized spontaneously breathing female Sprague-Dawley rats that received a unilateral substantia nigra (SN) 6-OHDA injection at either 2-weeks (short-term lesion; STL) or 24-weeks (long-term lesion; LTL) before being studied; SN vehicle (STV; LTV) injections served as controls. We found that LTL rats had higher basal breathing frequencies and slightly reduced burst-to-burst frequency and amplitude variability than LTV rats (which is similar to our observations in the younger SN 6-OHDA vs vehicle injected rats) while STL rats had lower breathing frequencies with slightly increased burst-to-burst amplitude variability compared to STV rats, with STV rats exhibiting breathing frequencies similar to LTL rats. The overall burst-to-burst frequency variability was greater in LTL and LTV rats compared to STL and STV rats, but STL rats exhibited greater burst-to-burst inspiratory duration (TI) and expiratory duration (TE) variability compared to STV rats; LTL rats had reduced burst-to-burst variability in these behaviors compared to LTV rats. Evaluation of the hypoxic ventilatory response revealed that LTL rats exhibit a blunted increase in frequency and amplitude in response to hypoxia compared to LTV and STL rats, with STL rats exhibiting an exaggerated hypoxic frequency increase compared to STV rats. While the increases in frequency were primarily mediated by decreases in TE in all groups, LTV rats also had reduced TI and they exhibited a larger post-hypoxic frequency decline due to a substantial increase in TE. Regardless of lesion (or vehicle injection) time, all of the older rats had a substantial delay in recovery following hypoxia. In contrast, younger SN 6-OHDA (compared to vehicle) injected rats exhibited an exaggerated frequency but blunted amplitude response to hypoxia, which rapidly recovered to pre-hypoxic levels. These data demonstrate that in older rats, the timing of the lesion may differentially affect the components underlying PD-related abnormalities in respiratory behaviors. Additional studies are needed to identify the mechanisms responsible.

Time course of alterations in chemical control of breathing in conscious spontaneously breathing 6‐OHDA SN‐lesioned Parkinson's Disease rat model

Respiratory dysfunction in Parkinson's Disease (PD) patients manifests as a variety of altered breathing patterns and chemical control of breathing deficits that are suggested to result from impairment of central respiratory control. Ongoing work in our laboratory has identified time‐dependent alterations in both basal inspiratory motor output and chemical control of breathing in the 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced unilateral substantia nigra (SN) lesion rat model. With reference to alterations in chemical control of breathing, our studies have been limited to examination of breathing behaviors at specified time points in acute terminal experiments; thus, the time course underlying the progressive nature of the changes observed is not known. Here, we begin to address this issue by examining the hypoxic (HVR; 12% O2, 3 min) and hypercapnic (HCVR; 7% CO2, 5 min) ventilatory responses using whole body plethysmography before and weekly for 4‐weeks after administration of 6‐OHDA into the SN of adult female Sprague‐Dawley rats; vehicle injected rats served as a control. For the HVR, we found that before injection into the SN, minute ventilation (MV) was increased by ~50% increase, which was mediated predominantly by an increase in breathing frequency (fb) but increases in tidal volume (VT) were noted in some rats; similar observations were noted in vehicle injected rats at the time points examined although some variability in magnitude of the responses were noted at various time points (but not in any specific pattern). In contrast, in 6‐OHDA injected rats, a markedly blunted HVR (~0–10% MV increase), in which fb and VT were minimally altered from pre‐hypoxic levels, was noted; this HVR alteration in behavior was stable over the time course examined albeit in a subset of rats, an HVR began to re‐emerge at the 4‐week post‐lesion time point. For the HCVR, we found that before injection into the SN, MV more than doubled due to similar magnitude increases in both fb and VT. In vehicle injected rats, there was a progressive increase in the magnitude of the HCVR ventilatory behaviors (MV, fb, VT) that stabilized over the 3–4 week post injection time point. In contrast in 6‐OHDA injected rats, the peak of the HCVR MV increase was similar at 5‐min of CO2 exposure, but the response during the initial 4‐min of CO2 exposure was blunted, especially at the 2–3 week post‐lesion time point. This deficit appeared to be mediated by attenuated fb and VT responses. These observations suggest that in conscious 6‐OHDA SN‐lesioned rats, chemical control of breathing undergoes progressive alterations albeit ventilatory behaviors associated with the HVR and HCVR appear to be differentially affected. It remains to be determined whether the ventilatory impairments noted in this PD rat model persist beyond the 4‐week time point studied here.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia-driven changes in glycolytic and tricarboxylic acid cycle metabolites of two nodulated soybean genotypes

Oxygen deprivation triggers changes at different levels of carbon and nitrogen metabolism, which may differ between plant-genotypes. The aim of this study was to evaluate the hypoxia-induced alterations of carbon and nitrogen metabolites in relation to alanine aminotransferase (AlaAT; EC 2.6.1.2) activity in two genotypes of nodulated soybean (Glycine max). Nodulated soybean plants (Fundacep 53 RR and BRS Macota) were grown in vermiculite and transferred to a hydroponic system at the early reproductive stage. The root system was subjected to hypoxia by continuously flushing the solution with N2 gas for 24 or 72h. For recovery, after 72h in hypoxia, plants returned to normoxic conditions after transfer to vermiculite for 24 and 72h. Root and nodule organic acids and amino acids were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively. Relative expression of AlaAT and AlaAT activity were also verified in both genotypes. Plants of Fundacep and Macota genotypes responded distinctly to hypoxia. In root tissues, Fundacep presented higher pyruvate and lactate accumulation than Macota, indicating higher glycolytic and fermentation rates. Furthermore, Fundacep responded more effectively on recovery by restoring pre-hypoxic levels of the metabolites. Although the amino acid composition did not differ between the genotypes, there was a clear link between glycolysis and the Krebs-cycle via increased gene expression and activity of AlaAT allied to succinate accumulation in roots of Fundacep. This may represents a metabolic advantage for this genotype over Macota with regard to hypoxia tolerance.

Acyl Ghrelin Improves Synapse Recovery in an In Vitro Model of Postanoxic Encephalopathy.

Comatose patients after cardiac arrest have a poor prognosis. Approximately half never awakes as a result of severe diffuse postanoxic encephalopathy. Several neuroprotective agents have been tested, however without significant effect. In the present study, we used cultured neuronal networks as a model system to study the general synaptic damage caused by temporary severe hypoxia and the possibility to restrict it by ghrelin treatment. Briefly, we applied hypoxia (pO2 lowered from 150 to 20 mmHg) during 6 h in 55 cultures. Three hours after restoration of normoxia, half of the cultures were treated with ghrelin for 24 h, while the other, non-supplemented, were used as a control. All cultures were processed immunocytochemically for detection of the synaptic marker synaptophysin. We observed that hypoxia led to drastic decline of the number of synapses, followed by some recovery after return to normoxia, but still below the prehypoxic level. Additionally, synaptic vulnerability was selective: large- and small-sized neurons were more susceptible to synaptic damage than the medium-sized ones. Ghrelin treatment significantly increased the synapse density, as compared with the non-treated controls or with the prehypoxic period. The effect was detected in all neuronal subtypes. In conclusion, exogenous ghrelin has a robust impact on the recovery of cortical synapses after hypoxia. It raises the possibility that ghrelin or its analogs may have a therapeutic potential for treatment of postanoxic encephalopathy.

Waterlogging-induced changes in fermentative metabolism in roots and nodules of soybean genotypes

Waterlogging blocks the oxygen supply to the root system which inhibits respiration, and greatly reduces the energy status of cells that affect important metabolic processes. This study evaluated fermentative metabolism and carbohydrate contents in the root system of two soybean (Glycine max L. Merril) genotypes under hypoxic and post-hypoxic conditions. Nodulated plants (genotypes Fundacep 53 RR and BRS Macota) were grown in vermiculite and transferred to a hydroponic system at the reproductive stage. The root system was submitted to hypoxia by flowing N2 (nitrogen) gas in a solution for 24 and 72 h. For recovery, plants returned to normoxia condition by transfer to vermiculite for 24 and 72 h. Fermentative enzyme activity, levels of anaerobic metabolites and carbohydrate content were all quantified in roots and nodules. The activity of alcohol dehydrogenase, pyruvate decarboxylase and lactate dehydrogenase enzymes, as well as the content of ethanol and lactate, increased with hypoxia in roots and nodules, and subsequently returned to pre-hypoxic levels in the recovery phase in both genotypes. Pyruvate content increased in nodules and decreased in roots. Sugar and sucrose levels increased in roots and decreased in nodules under hypoxia in both genotypes. Fundacep RR 53 was more responsive to the metabolic effects caused by hypoxia and post-hypoxia than BRS Macota, and it is likely that these characteristics contribute positively to improving adaptation to oxygen deficiency.

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia–reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia–reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.

Bioluminescent imaging of hepatic caspase‐3 activity in mice

Apoptosis‐mediated damage of cells and organs is a serious concern in various pathological situations. Here, we demonstrate caspase‐3 activity by the luciferase‐based optical probe of live liver cells challenged by various stimuli, and also in vivo imaging of caspase‐3 activity of the post‐ischemic liver.METHODSWe developed a novel probe (pcFluc‐DEVD) of cyclic luciferase reflecting caspase‐3 activity. By transfecting an adenovirus cording for pcFluc‐DEVD (AdpcFluc‐DEVD), we investigated whether this probe monitors apoptosis in live AML12 liver cells by staurosporine (STS), Fas‐ligand (FasL) or hypoxia/reoxygenation (H/R), and in the post‐ischemic liver in mice.RESULTSSTS and FasL activate caspase‐3 in caspase‐8‐independent and dependent manner, respectively. First, we measured caspase‐3 activity in STS‐ or FasL‐treated AML12 cells transiently transfected with AdpcFluc‐DEVD. In live AML12 cells, the signals from the probe started to increase immediately and peaked at 2–4 hr after 1 μM STS and 2 μg/ml Jo2 administration, respectively. In both stimuli, caspase‐3 activity was paralleled with apoptotic cell death. Next, we measured caspase‐3 activity in post‐hypoxic AML12 cells by this probe. During hypoxia, caspase‐3 activity dropped to the bottom level. However, it rapidly recovered to the pre‐hypoxic level after reoxygenation and started to rise until 4 – 6 hr post‐H/R.Then, we tried to evaluate in vivo apoptosis by measuring caspase‐3 activity in mouse hepatic ischemia/reperfusion (I/R). 60min of hepatic ischemia caused great increase of capase‐3 activity at 6 – 9 hr post‐I/R, whereas 30 min of liver ischemia caused small increase at 2 hr post‐I/R (Figure).CONCLUSIONSThe caspase3 probe successfully illustrated apoptotic damage in vitro and in vivo. Visualizing apoptotic damage of cells/organs may provide important insights into various physio‐pathological conditions.

Molecular Basis of Neocytolysis.

Abstract 2093The principal function of red blood cells (RBCs) is to deliver oxygen via hemoglobin resulting in the formation of reactive oxygen species (ROS), which mandates the existence of an ROS protective mechanism. Hypoxia increases RBC mass by increasing hypoxia-inducible factors (HIFs) levels. This is mainly mediated by HIFs-regulated erythropoietin. Upon return to normoxia, RBC mass is overcorrected by the destruction of newly formed RBCs, a process termed neocytolysis. Neocytolysis was first described in astronauts and people descending from high-altitude, and likely accounts for the rapid transition from polycythemia to anemia in neonates upon leaving the fetal hypoxic environment. The molecular mechanism of neocytolysis has not yet been elucidated. Reticulocytes newly released from the marrow contain ribosomes and mitochondria which are removed by multiple mechanisms, including mitochondrial loss by autophagy regulated by HIFs-controlled Bnip3L gene. FOXO3a is a HIF-regulated transcription factor that controls ROS-scavenging enzymes. Mature RBCs cannot respond to rapid changes of ROS levels because of their inability to produce new proteins. We hypothesized that after hypoxic exposure and return to normoxia, there is an increase in reticulocytes' ROS (mediated by Bnip3L), causing preferential destruction of the hypoxia-born young RBCs having decreased activity of FOXO3a-controlled ROS-scavenging enzymes.We developed a mouse model of neocytolysis that replicates RBC changes observed in humans to test our hypothesis, with awareness that RBCs live ∼120 days in humans and ∼45 days in mice. C57/BL6 mice were maintained at 12% oxygen (equivalent to 4500 m altitude) for 10 days followed by return to ambient air. We measured the following parameters: hematocrit (hct), reticulocyte count, RBC survival, mitochondrial mass, mRNA and the activity of ROS-scavenging enzymes, Bnip3L and FOXO3a mRNA, and ROS content in peripheral blood cells before and after hypoxic exposure.Hypoxic exposure increased hct by 22%; upon return to normoxia, hct reached normal levels at post-hypoxia days (PHD) 4–7, then continued to decrease by 28% at PHD 7–10, while plasma volume did not significantly change until its decrease at PHD 10 (p=0.049). Reticulocyte count increased by 35% following hypoxia, then decreased by 33% at PHD 4 compared to pre-hypoxic levels. Reticulocytes recovered by 50% by PHD 7, then normalized at PHD 14. To measure RBCs survival, we labeled them with biotin prior to hypoxic exposure and found preferential destruction of newly formed RBCs in the initial phase of normoxic return. This was accompanied by a dramatic increase of ROS level at PHD 0 and PHD 4 confined only to RBCs (and preferentially to young RBCs, i.e. 2.6 fold higher at PHD 4 in CD71+/TER119+ compared to CD71−/TER119+ in older RBCs) but not to platelets, neutrophils, B and T cells, or monocytes. We demonstrated that the ROS changes were accompanied by a commensurate increase of mitochondrial mass that was limited to reticulocytes; i.e. mitochondrial mass increased in reticulocytes by 40%, with an inverse relation to reticulocytes’ Bnip3L transcript levels (reduced by 35%). Furthermore, we show the transcripts and activity of FOXO3a-regulated ROS scavenging enzyme catalase were reduced at PHD 0 and PHD 4 by 30% and recovered at PHD 10, accompanied by commensurate changes in FOXO3a mRNA. However, there were no similar changes in glutathione peroxidase and superoxide dismutase. These data demonstrate that the increase in ROS is due to both excessive generation in mitochondria and insufficient scavenging due to a decrease in catalase activity.We conclude that there are three different phases in the response of RBC mass to transient hypoxia: 1) Hypoxia increases erythropoiesis, resulting in polycythemia; 2) Upon return to normoxia, the increased RBC mass is overcorrected below the initial levels by neocytolysis; 3) After 10–14 days, RBC mass normalizes. We demonstrate that neocytolysis is mediated by ROS in reticulocytes, with increased mitochondrial mass due to downregulation of Bnip3L. These increased ROS cannot be scavenged by young RBCs with decreased catalase activity. These two processes lead in concert to neocytolysis. These studies reveal a novel role for mitochondria-derived ROS in the regulation of red cell mass. Disclosures:No relevant conflicts of interest to declare.

Neuronal hypoxia-reoxygenation and nitric oxide: A protective role of autophagy

In stroke, hypoxia and reoxygenation induces glutamate excitotoxicity, nitric oxide is overproduced and can damage mitochondria and be detrimental to cellular bioenergetics and survival. Macroautophagy of modified proteins and mitophagy of modified mitochondria may provide a mechanism for the neurons to deal with nitric oxide-induced cellular damage. The role of autophagy in hypoxia-reoxygenation induced mitochondrial damage is not clear. We hypothesized that autophagy plays a protective role in neuronal hypoxia-reoxygenation damage and tested this in cultured rat cortical primary neurons. Cellular bioenergetic responses to DetaNONO under hypoxia conditions and during reoxygenation using Seahorse bioscience XF24 extra flux analyzer were examined. We found that 250 μM DetaNONO did not decrease basal oxygen consumption rate (OCR) in the 30 min pre-hypoxia condition. In response to hypoxia untreated cells maintained the same OCR in hypoxia as normoxia, whereas NO exposed cells exhibited a gradual decrease in mitochondrial respiration. Upon reoxygenation, the respiration in DetaNONO-treated cells recovered to similar levels to pre-hypoxia levels. Inhibition of autophagy by 3-MA alone did not affect cellular bioenergetics in hypoxia-reoxgenated conditions. Interestingly, inhibition of autophagy by 3-MA in the DetaNONO-treated cells completely resulted in a severe bioenergetic dysfunction during reoxygenation. These studies provide new insights into understanding how autophagy and NO may play an interactive role in neuroinflammation-induced cellular damage which is pertinent to our understanding of the pathology in stroke.

Preinspiratory and inspiratory hypoglossal motor output during hypoxia-induced plasticity in the rat

Respiratory-related discharge in the hypoglossal (XII) nerve is composed of preinspiratory (pre-I) and inspiratory (I) activity. Our first purpose was to test the hypothesis that hypoxia-induced plasticity in XII motor output is differentially expressed in pre-I vs. I XII bursting. Short-term potentiation (STP) of XII motor output was induced in urethane-anesthetized, vagotomized, and ventilated rats by exposure to isocapnic hypoxia (PaO2 of approximately 35 Torr). Both pre-I and I XII discharge abruptly increased at beginning of hypoxia (i.e., acute hypoxic response), and the relative increase in amplitude was much greater for pre-I (507+/-46% baseline) vs. I bursting (257+/-16% baseline; P<0.01). In addition, STP was expressed in I but not pre-I bursting following hypoxia. Specifically, I activity remained elevated following termination of hypoxia but pre-I bursting abruptly returned to prehypoxia levels. Our second purpose was to test the hypothesis that STP of I XII activity results from recruitment of inactive or "silent" XII motoneurons (MNs) vs. rate coding of active MNs. Single fiber recordings were used to classify XII MNs as I, expiratory-inspiratory, or silent based on baseline discharge patterns. STP of I XII activity following hypoxia was associated with increased discharge frequency in active I and silent MNs but not expiratory-inspiratory MNs. We conclude that the expression of respiratory plasticity is differentially regulated between pre-I and I XII activity. In addition, both recruitment of silent MNs and rate coding of active I MNs contribute to increases in XII motor output following hypoxia.

Rhinoceros beetles modulate spiracular opening to regulate haemolymph pH

Insects are the most diverse and numerous terrestrial air-breathing animals, yet a comprehensive understanding of their respiratory physiology remains elusive. This is clearly seen in the debate surrounding the origins and function of discontinuous gas exchange cycles (DGCs) displayed by many insects at rest. While many hypotheses have been proposed to explain the function of DGCs, no general underlying mechanism driving patterns of insect ventilation has been universally accepted. In the present study, we simultaneously measured in vivo pH fluctuations and patterns of CO2 emission in rhinoceros beetles Xylotrupes ulysses. Beetles were restrained within a flow-through respirometry chamber with a fibre optic pH probe implanted in the haemolymph through the dorsal surface of their thorax. DGCs caused haemolymph pH to fluctuate by 0.1 units, with internal pH declining while the spiracles were closed and rapidly climbing when the spiracles opened. Exposure to hypoxia (5% oxygen in nitrogen) caused the spiracles to open widely and elicited a large burst of CO2, causing haemolymph pH to increase from 7.0 to 7.3. If the spiracles were held open by continued exposure to hypoxia, the pH remained high. Once normoxia was restored, the beetles held their spiracles shut, and only resumed normal opening behaviour once internal pH declined to pre-hypoxia levels. The protracted breath hold following hypoxic exposure indicates that the beetles preferentially retain CO2 in order to restore their haemolymph pH. We therefore propose that insects modulate gas exchange to regulate internal pH, and that DGCs represent an extreme example of this strategy.

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 alpha (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre-hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.

Complex level alterations of the 2f 1−f 2 distortion product due to hypoxia in the guinea pig

It is controversially discussed inasmuch acute hearing disorders might originate from impaired cochlear circulation. Hypoxia-specific alterations of inner ear parameters measurable in patients with acute sensorineural hearing loss would therefore be of great interest. Aim of this study was to characterize hypoxia-related alterations of the 2f (1)-f (2) distortion product. Nine guinea pigs were anaesthetized by i.m. administration of Midazolam, Medetomidin and Fentanyl. For introduction of hypoxia, the spontaneously breathing animals were offered a gas mixture of N(2)O and O(2) containing either 21 or 12-13% O(2). Distortion product otoacoustic emissions (DPOAEs) were continuously monitored at f (2) = 16 kHz; f (2)/f (1) = 1, 2; DP-definition = 2f (1)-f (2); L (1) = 65 dB and L (2) = 55 dB, while inhaled oxygen was switched from 21 to 12-13% and back. Oxygen saturation (SaO(2)) was continuously monitored. Data from an hypoxic interval were only used for further data processing if DPOAE levels were stable before and after hypoxia. Six hypoxic intervals in five animals fulfilled the stability criterion. During the hypoxic interval with the highest measured SaO(2) (75%), no alterations of DPOAE levels were observed. During the remaining five hypoxic intervals, when SaO(2) ranged between 57 and 70%, DPOAE levels were on average lower with an increased standard deviation compared to mean pre-hypoxic levels. Mean decrease correlated with the decrease of SaO(2 )(r = 0.90, P = 0.014). Alterations followed a characteristic time course-when hypoxia was started, DPOAE levels exhibited a short increase before they decreased and remarkably destabilized. After re-oxygenation DPOAE levels showed a pronounced level decrease, while SaO(2) already had recovered to pre-hypoxic values. After reaching a minimum, DPOAE levels slowly recovered to pre-hypoxic values. The decrease of DPOAE levels during hypoxia and the post-hypoxic level alterations have similarly been described by other authors before, while the distinct destabilization and transiently increased DPOAE levels have not been explicitly mentioned. A micromechanical mechanism that might explain a transient level increase and the post-hypoxic DPOAE level changes is discussed.

Short-term effects of normobaric hypoxia on the human spleen

Spleen contraction resulting in an increase in circulating erythrocytes has been shown to occur during apnea. This effect, however, has not previously been studied during normobaric hypoxia whilst breathing. After 20 min of horizontal rest and normoxic breathing, five subjects underwent 20-min of normobaric hypoxic breathing (12.8% oxygen) followed by 10 min of normoxic breathing. Ultrasound measurements of spleen volume and samples for venous hemoglobin concentration (Hb) and hematocrit (Hct) were taken simultaneously at short intervals from 20 min before until 10 min after the hypoxic period. Heart rate, arterial oxygen saturation (SaO(2)) and respiration rate were recorded continuously. During hypoxia, a reduction in SaO(2) by 34% (P < 0.01) was accompanied by an 18% reduction in spleen volume and a 2.1% increase in both Hb and Hct (P < 0.05). Heart rate increased 28% above baseline (P < 0.05). Within 3 min after hypoxia SaO(2) had returned to pre-hypoxic levels, and spleen volume, Hb and Hct had all returned to pre-hypoxic levels within 10 min. Respiratory rate remained stable throughout the protocol. This study of short-term exposure to eupneic normobaric hypoxia suggests that hypoxia plays a key role in triggering spleen contraction and subsequent release of stored erythrocytes in humans. This response could be beneficial during early altitude acclimatization.

Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid,Astronotus ocellatus

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67+/-0.01 mg l(-1) at 28+/-1 degrees C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

Gaseous metabolism of the chicken embryo and hatchling during post-hypoxic recovery

In neonatal mammals, the drop in oxygen consumption (VO2) during moderate degrees of hypoxia is a manifestation of metabolic depression, and occurs without anaerobic energy compensation. We examined the possibility that embryos also respond to hypoxia with a similar hypometabolic response, by measuring the extent of the O2 debt during post-hypoxic recovery. In chicken embryos at incubation days 11 (E11) and 16 (E16), and hatchlings on the day of hatching (H1), VO2 and carbon dioxide production (VCO2) were measured with an open flow system. The protocol consisted of 1h in air, followed by 40 min in hypoxia (either 17% or 11% O2) and additional 45 min of post-hypoxic recovery. VO2 dropped in hypoxia, more with 11% than 17% O2, and more the younger the animal. During post-hypoxic recovery VO2 returned to, but did not exceed, the pre-hypoxic level, indicating that no O2 debt was contracted during hypoxia. In H1, the changes of VCO2 during hypoxia and post-hypoxia matched those of VO2. Differently, in the embryos, the changes in VCO2 during hypoxia and post-hypoxic recovery were minimal. This phenomenon is explained by changes in the large CO2 stores of the eggs, which buffer the changes in CO2 output of aerobic origin. We conclude that in the chicken embryo and hatchling the energetic shortfall during the hypoxic decrease in VO2 is not compensated by anaerobic energy supply, and represents a phenomenon of metabolic depression.

Reoxygenation with 100% Oxygen Versus Room Air: Late Neuroanatomical and Neurofunctional Outcome in Neonatal Mice with Hypoxic-Ischemic Brain Injury

Study investigated neuroutcome in mice subjected at 7-8 d of life to hypoxic-ischemic brain injury (HI) followed by 30 min of reoxygenation with 100% O(2) (Re-O(2)) or room air (Re-Air). At 24 h of recovery, mouse reflexes were tested. At 7 wks after HI spatial orientation and memory were assessed in the same mice. Mortality rate was recorded at 24 h and at 7 wks of recovery. In separate cohort of mice, changes in cerebral blood flow (CBF) during HI-insult and reoxygenation were recorded. Re-O(2)versus Re-Air mice exhibited significantly delayed geotaxis reflex. Adult Re-O(2)versus Re-Air mice exhibited significantly better spatial learning and orientation with strong tendency toward better preserved memory. Histopathology revealed significantly less hippocampal atrophy in Re-O(2)versus Re-Air mice. Following a hypoxia-induced hypoperfusion, Re-O(2) re-established CBF in the ipsilateral side to the prehypoxic level significantly faster than Re-Air. The mortality was higher among Re-O2 versus Re-Air mice, although, it did not reach statistical significance. Re-O(2)versus Re-Air restores CBF significantly faster and results in better late neuroutcome. However, greater early motor deficit and higher mortality rate among Re-O(2)versus Re-Air mice suggest that Re-O(2) may be deleterious at the early stage of recovery.