Acute Anoxia Research Articles

Comparison of different methods of heart rate entropy analysis during acute anoxia superimposed on a chronic rat model of pulmonary hypertension

Acute life-threatening situations are particularly critical when superimposed on chronic diseases. The objective of this study was the assessment of heart rate (HR) dynamics during episodes of acute anoxia superimposed on a rat model of chronic pulmonary hypertension. In 10 adult Wistar rats, five weeks after pulmonary hypertension induction with Monocrotaline, we analysed eight 1-min HR segments, during episodes of baseline, mechanical ventilation and acute anoxia, using linear indices, approximate entropy (ApEn), sample entropy (SampEn) and multiscale entropy (MSE). The transition from baseline or mechanical ventilation to early anoxia was identified through almost all indices, but SampEn(2,0.6) was the index that better identified all the transitions. MSE presented limited performance, possibly due to the non-stationary nature and short duration of the acute anoxia episodes. A systematic evaluation of all computed HR indices may help to identify which indices or combination of indices more adequately discriminates and monitors critical acute events superimposed on chronic clinical conditions.

Synaptic Released Zinc Promotes Tau Hyperphosphorylation by Inhibition of Protein Phosphatase 2A (PP2A)

Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer disease (AD), and the tangle distribution largely overlaps with zinc-containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pretreatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser-396, Ser-404, Thr-231, and Thr-205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus were detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pretreatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.

ATM-dependent phosphorylation of 53BP1 in response to genomic stress in oxic and hypoxic cells

The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1Ser25in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1Ser25 is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1Ser25 activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1Ser25 phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1Ser25 in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1Ser25, and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.

The response of isolated cardiac muscle to acute anoxia: protective effect of adenosine triphosphate and creatine phosphate.

Abstract A standardized and reproduceable preparation is described that enables the effect of drugs to be examined on the response of isolated cardiac muscle to acute anoxia. There was a linear relation (r = 0·963) between tension developed in isolated, electrically-driven guinea-pig atria and the oxygen tension of the fluid surrounding the muscle. For any one atrial preparation the time taken, from the beginning of the anoxic period, for tension to be reduced by 50 % was constant for consecutive anoxic periods. ATP and creatine phosphate significantly increased this time and protected cardiac muscle against the consequences of anoxia; in concentrations that were without direct cardiac effects, a greater degree of protection was possible with creatine phosphate.

Oxygen-Sensing of L-type Calcium Channels in Rat Cardiomyocytes: The Possible Role of Hemoxygenase

Hemoxygenases (HO)-1 and −2 are enzymes that metabolize the heme group, generating CO, biliverdin and Fe+2. HO-2 is believed to be an O2 sensor in the carotid body controlling the release of dopamine as it needs O2 for its function. HO-2 is necessary for the anoxic activation of nociceptive neuron and binds to calmodulin in neurons, and the complex is modulated by Ca2+. Previously, we have found that L-type Ca2+ channel is suppressed by 22-43% with acute anoxia prior to metabolic impairment or run down of Ica . This property appeared to require the calmodulin-binding IQ motif on the cytoplasmic carboxy-tail of the channel. Here we probe whether HO mediates this rapid Ica-suppressant effect of anoxia thus serving as the O2 sensor of the heart. Freshly isolated rat cardiomyocites were maintained in Tyrode containing (in mM): 135NaCl, 5.4KCl, 10Hepes, 10glucose, 1MgCl2 and 2CaCl2). Internal solution used was (in mM): 15NaCl, 118CsCl, 5.5Glucose, 14EGTA, 3.92CaCl2, 10Hepes, 3MgATP, 0.5MgCl2. Solutions bubbled with 100% O2 or 100% N2 were rapidly (50ms) applied. IBa was measured replacing CaCl2 for BaCl2. HO inhibitors ZnPP-IX 100nM or SnPP-IX 100nM were added to either 100% O2 or 100% N2 solutions. Both the O2 removal and the inhibition of HO blocked about 30% of the ICa and IBa. Administration of anoxic solution did not further inhibit ICa in cells previously exposed to ZnPP-IX or SnPP-IX. We conclude that both anoxia and HO suppress ICa and IBa with the same intensity and kinetics and O2 sensing effect becomes negligible in cells exposed to HO inhibitors.

The anti-necrosis role of hypoxic preconditioning after acute anoxia is mediated by aldose reductase and sorbitol pathway in PC12 cells

It has been demonstrated that hypoxic preconditioning (HP) enhances the survival ability of the organism against the subsequent acute anoxia (AA). However, it is not yet clear whether necrosis induced by AA can be prevented by HP, and what are the underlying mechanisms. In this study, we examined the effect of HP (10% O(2), 48 h) on necrosis induced by AA (0% O(2), 24 h) in PC12 cells. We found that HP delayed the regulatory volume decrease and reduced cell swelling after 24 h of exposure to AA. Since aldose reductase (AR) is involved in cell volume regulation, we detected AR mRNA expression with reverse transcription-polymerase chain reaction (RT-PCR) techniques. The AR mRNA level was dramatically elevated by HP. Furthermore, an HP-induced decrease in cell injury was reversed by berberine chloride (BB), the inhibitor of AR. In addition, sorbitol synthesized from glucose catalyzed by AR is directly related to cell volume regulation. Subsequently, we tested sorbitol content in the cytoplasm. HP clearly elevated sorbitol content, while BB inhibited the elevation induced by HP. Further study showed that a strong inhibitor of sorbitol permease, quinidine, completely reversed the protection induced by HP after AA. These data provide evidence that HP prevents necrosis induced by AA and is mediated by AR and sorbitol pathway.

The Puzzling Death of Reinhard Heydrich

Summary Reinhard Heydrich, SS Police chief and“Protector” of Bohemia-Moravia waswounded by a grenade fragment during anassassination attempt in Prague on May27, 1942. Eight days after undergoing emer-gency surgery, he suddenly collapsed,slipped into coma and died the next morn-ing, on June 4, 1942. The cause of his deathhas remained obscure.The present article reviews the knowndetails of Heydrich’s medical history afterthe attack. The death of his physicians,the loss of his medical records, and an in-adequate post-mortem make an accuratediagnosis impossible. Massive pulmonaryembolus with acute cor pulmonale and ce-rebral anoxia is a reasonable assumption.That Heydrich died from botulism is mostunlikely. Short Biographies of Heydrich’sPhysicians 1. Dick, Walter (1899-1990). Sudeten Ger-man born in Bohemia. MD degree fromCharles V University (Prague) in 1925.Chief of Surgery at Bulovka Hospital(Prague) from 1940 to 1945. From 1945to his retirement in 1967, was professorof surgery at the Universities ofKlagenfurt, Bonn and Tubingen.2. Hohlbaum, Josef (1884-1945). Born inGerman Silesia. From 1924 to 1940taught surgery in Leipzig. From 1941to 1945 was chairman of the SurgeryDepartment at Charles V University(Prague). Remained in Prague in May,1945, was sentenced to forced labor bythe Czech authorities and was criticallywounded by a mine while clearing abuilding in Prague. Denied treatmentby his Czech colleagues, he escaped toLeipzig where he died from his legwounds.3. Sauerbruch, Ferdinand (1875-1951).

Heat shock protein (HSP 70) induced by mild heat shock inhibits sharp plasma osmolarity increases upon seawater transfer in rainbow trout ( Oncorhynchus mykiss)

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that “channel arrest” in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca2+-channel, underwent a 40–50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L− 1), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O2) provided indirect evidence that a reduction in Na+/K+-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na+/K+-ATPase activity of 30–40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na+/K+-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid “channel arrest” of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na+/K+-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Proteomic analysis of anoxia tolerance in the developing zebrafish embryo

While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of > 24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.

Linear and nonlinear heart-rate analysis in a rat model of acute anoxia

The objective of this study was the assessment of heart-rate (HR) dynamics with linear and nonlinear methods during episodes of mechanical ventilation and acute anoxia in rats. Namely, to assess whether linear and nonlinear HR analysis was able to discriminate acute anoxia from baseline in rats and if this was consistent with human foetal and adult studies. Five HR segments of 1 min duration, during baseline recording, mechanical ventilation and first, second and third minutes of induced acute anoxia, were analysed in ten adult Wistar rats. Linear time and frequency domain and nonlinear methods were used, namely mean HR (mHR), long-term irregularity (LTI), interval index (II), low frequency (LF) and high frequency (HF), approximate entropy (ApEn) and sample entropy (SampEn). New parameters for the entropy indices are proposed for the analysis of rats' HR. Bootstrap percentile confidence intervals and nonparametric statistical tests were used in the evaluation of the differences between segments. During mechanical ventilation a clear spectral band was detectable at the ventilation rate, but mHR, II and the ‘new’ entropy indices were the only significantly changed indices. In the transition from baseline–mechanical-ventilation to mechanical-ventilation–induced anoxia, a statistically significant decrease of mHR, II and entropy indices was observed, clearly discriminating these two instances, whereas most linear indices increased. With continued anoxia, most linear indices decreased significantly, whereas entropy remained stably low. These results are consistent with other foetal human and non-human studies and evidence that the rat model may be used for further research on linear and nonlinear analysis of heart-rate dynamics. The transition from baseline to acute anoxia was encompassed by signs of increased activation of the autonomic nervous system sympathetic branch, and decreased or blunted activity of the HR complexity regulatory centres.

Nerve growth factor inhibits Gd3+-sensitive calcium influx and reduces chemical anoxic neuronal death

To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca(2+)](i) was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 micromol/L MK-801 (glutamate receptor antagonist), 40 micromol/L CNQX (AMPA receptor antagonist) and 5 micromol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca(2+)](i) and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd(3+) (10 micromol/L) almost completely blocked KCN-mediated [Ca(2+)](i) elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca(2+)](i) increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 micromol/L) abolished NGF effects. It is concluded that Gd(3+)-sensitive calcium influx, which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd(3+)-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

Evidence of anoxia-induced channel arrest in the brain of the goldfish ( Carassius auratus)

The common goldfish ( Carassius auratus) is extremely anoxia tolerant and here we provide evidence that “channel arrest” in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl- d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca 2+-channel, underwent a 40–50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L − 1 ), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O 2) provided indirect evidence that a reduction in Na +/K +-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na +/K +-ATPase activity of 30–40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na +/K +-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid “channel arrest” of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na +/K +-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Spontaneous Umbilical Cord Hematoma

Spontaneous hematoma of the umbilical cord represents a rare cause of fetal morbidity and mortality and the outcome is poor in half of the cases. There are many risk factors, such as infections, morphologic anomalies, alterations of the vessel walls, prolapses, twisting and traction of the cord, but in many cases the causes remain unknown. We present 3 cases of umbilical cord hematoma which took place at the end of the pregnancy and were followed by perinatal death of the fetus. In the 3 cases, which were autopsied including macroscopical and histopathologic examination of the placenta and the umbilical cord, a cordonal pathology was present; in all cases, there were alterations of the vascular wall, and in the third case inflammatory vasculopathy was found. In all 3 cases, the cause of death was attributed to acute anoxia due to the cordonal hematoma.

Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-κB and ATF4

Under anoxia a coordinated, cytoprotective program is induced, called the unfolded protein response (UPR). Activating transcription factor 4 (ATF4) is a mediator of the UPR and activates a gene expression program, promoting tumour growth and survival under anoxia. A key gene induced by ATF4 under normoxic conditions is SKIP3. We characterized the induction of SKIP3 during anoxic exposure to determine whether UPR alone was sufficient or there was a more complex regulatory response to anoxia. There was temporal separation of acute hypoxia-inducible factor (HIF)-1alpha- and chronic ATF4-dependent gene expression programs. SKIP3 was regulated by chronic (48 h) rather than acute anoxia (<24 h) by a complex set of pathways and mechanisms, besides ATF4 induced by the classical UPR, there was transcriptional regulation by nuclear factor-kappa B (NF-kappaB) and RNA stabilization by HuR. Temporal activation of the NF-kappaB pathway under anoxia protected cells from negative consequences of the oxygen stress and involved the canonical signalling pathways that promote IkappaBA phosphorylation and degradation, and reduced mRNA level of the inhibitory protein IkappaBA followed by the translational repression of IkappaBA. We also show that SKIP3 acts as an inhibitor of NF-kappaB and ATF4-dependent transcription under anoxia and provides a regulatory feedback loop. Repression of the survival pathway NF-kappaB by SKIP3 sensitized cells to metabolic consequences of the anoxic stress. Thus, the response to anoxia is mediated by three pathways independently of HIF, suggesting that combined therapeutic approaches would be needed to maximize effects against this pathway.

Acute anoxia stimulates proliferation in adult neural stem cells from the rat brain

Hypoxic-ischemic damage is a major challenge for neuronal tissue. In the present study, we investigated the effects of anoxia and glucose deprivation on adult neural stem cells (NSCs) in vitro. We assessed glucose deprivation, anoxia and the combination of the latter separately. After 24 h of anoxia, cell numbers increased up to 60% compared to normoxic controls. Whereas nearly all normoxic cells incorporated the mitotic marker BrdU (99%), only 85% of the anoxic cells were BrdU-positive. Counting of interphase chromosomes showed 8-fold higher cell division activity after anoxia. The number of necrotic cells doubled after anoxia (14% compared to 7% after normoxia). Apoptosis was measured by two distinct caspases assays. Whereas the total caspase activity was reduced after anoxia, caspase 3/7 showed no alterations. Glucose deprivation and oxygen glucose deprivation both reduced cell viability by 56 and 53%, respectively. Under these conditions, total caspases activity doubled, but caspase 3/7 activity remained unchanged. Erythropoietin, which was reported as neuroprotective, did not increase cell viability in normoxia, but moderately under oxygen glucose deprivation by up to 6%. Erythropoietin reduced total caspase activity by nearly 30% under all the conditions, whereas caspase 3/7 activity was not affected. Our results show that anoxia increases proliferation and viability of adult NSCs, although a fraction of NSCs does not divide during anoxia. In conclusion, anoxia increased cell viability, cell number and proliferation in NSCs from the rat brain. Anoxia turned out to be a highly stimulating environmental for NSCs and NSCs died only when deprived of glucose. We conclude that the availability of glucose but not of oxygen is a crucial factor for NSC survival, regulating apoptotic pathways via caspases activity other than the caspases 3/7 pathway. Therefore, we conclude that NSCs are dying from glucose deprivation, not from hypoxic-ischemic damage.

Rapid effects of acute anoxia on spindle kinetochore interactions activate the mitotic spindle checkpoint

The dramatic chromosome instability in certain tumors might reflect a synergy of spindle checkpoint defects with hypoxic conditions. In Caenorhabditis elegans and Drosophila melanogaster, spindle checkpoint activation has been implicated in the response to acute anoxia. The activation mechanism is unknown. Our analyses in D. melanogaster demonstrate that oxygen deprivation affects microtubule organization within minutes. The rapid effects of anoxia are identical in wild-type and spindle checkpoint-deficient Mps1 mutant embryos. Therefore, the anoxia effects on the mitotic spindle are not a secondary consequence of spindle checkpoint activation. Some motor, centrosome and kinetochore proteins (dynein, Kin-8, Cnn, TACC, Cenp-C, Nuf2) are rapidly relocalized after oxygen deprivation. Kinetochores congress inefficiently into the metaphase plate and do not experience normal pulling forces. Spindle checkpoint proteins accumulate mainly within the spindle midzone and inhibit anaphase onset. In checkpoint-deficient embryos, mitosis is still completed after oxygen deprivation, although accompanied by massive chromosome missegregation. Inhibitors of oxidative phosphorylation mimic anoxia effects. We conclude that oxygen deprivation impairs the chromosome segregation machinery more rapidly than spindle checkpoint function. Although involving adenosine triphosphate (ATP)-consuming kinases, the spindle checkpoint can therefore be activated by spindle damage in response to acute anoxia and protect against aneuploidies.

Drosophila dMRP4regulates responsiveness to O2deprivation and development under hypoxia

For most vertebrates, oxygen is a prerequisite for survival. Although we have previously shown that Drosophila melanogaster is hypoxia tolerant, how this species senses O(2) deprivation and how it survives oxygen-limiting conditions are as yet poorly understood. We began to address this question by testing for anoxic responsiveness in Drosophila adult flies following overexpression of existing EP lines. In this screen, we identified Drosophila CG14709 gene as a homolog of the human multidrug resistance protein 4 (MRP4/ABCC4) that is tightly regulated to oxygen. Ubiquitous expression of dMRP4 in adult flies resulted in increased sensitivity to anoxia as they had longer recovery time from anoxic stupor. When exposed to 4% oxygen chronically (throughout its lifespan), constitutive expression of dMRP4 in larvae caused larval lethality due to growth arrest. Mutations of dMRP4 led to a hypersensitive response to acute anoxia in adult flies but had less impact on larval survival under chronic hypoxia compared with dMRP4 overexpression. Selective expression of this gene in neurons, but not in glia or muscles, mirrored the same phenotype as the ubiquitous one. Thus, our data suggest novel roles for MRP in vivo: 1) dMRP4 regulates the sensitivity to acute or chronic O(2) deprivation, and 2) dMRP expression in neurons is sufficient to induce the sensitivity to O(2) in the whole organism.

Acute anoxia attenuates sodium‐hydrogen exchange activity in isolated mammalian ventricular myocytes

H+ extrusion through sodium-hydrogen exchange (NHE) was studied in isolated rat ventricular myocytes by monitoring pHi recovery (SNARF fluorescence) from an acid load (20 mM NH4Cl prepulse; Hepes- buffered superfusates). Acute anoxia (1mM Na dithionite, 100% N2, 5-8 minutes) reduced resting pHi by ~ 0.2 units, and slowed recovery rate by ~ 60 %. The slowing could not be explained by increased endogenous H+i-generation, or by increased intrinsic intracellular buffering (which did not change). Reoxygenation induced a rapid, apparent rise of pHi (~ 0.2 units), which occurred in the presence of 30μM cariporide (NHE inhibitor), indicating it was independent of NHE. Subsequent removal of cariporide, then permitted additional pHi recovery, demonstrating that anoxic NHE-inhibition was reversed by reoxygenation. In conclusion, anoxia rapidly and reversibly attenuates NHE activity. Acute anoxic sensitivity is likely to contribute to the attenuation of NHE activity reported during myocardial ischaemia. The apparent reoxygenation-induced rise of pHi observed in the absence of NHE activity is the subject of further study.

Acute anoxia induces tau dephosphorylation in rat brain slices and its possible underlying mechanisms

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.

Heat-shock protein (Hsp70) protects glutamatergic synaptic transmission in cells of the rat olfactory cortex against acute anoxia in vitro.

Heat-shock protein (Hsp70) protects glutamatergic synaptic transmission in cells of the rat olfactory cortex against acute anoxia in vitro.