The optimization conditions of establishing an H9c2 cardiomyocyte hypoxia/reoxygenation injury model based on an AnaeroPack System

Ischemia–reperfusion (I/R) injury is a major cause of cardiomyocyte apoptosis after vascular recanalization, which was mimicked by a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes in vitro. In this study, we explored an optimal H/R duration procedure using the AnaeroPack System. To study the H/R procedure, cardiomyocytes were exposed to the AnaeroPack System with sugar and serum-free medium, followed by reoxygenation under normal conditions. Cell injury was detected through lactate dehydrogenase (LDH) and cardiac troponin (c-Tn) release, morphological changes, cell apoptosis, and expression of apoptosis-related proteins. The results showed that the damage to H9c2 cells increased with prolonged hypoxia time, as demonstrated by increased apoptosis rate, LDH and c-Tn release, HIF-1α expression, as well as decreased expression of Bcl-2. Furthermore, hypoxia for 10 h and reoxygenation for 6 h exhibited the highest apoptosis rate and damage and cytokine release; in addition, cells were deformed, small, and visibly round. After 12 h of hypoxia, the majority of the cells were dead. Taken together, this study showed that subjecting H9c2 cells to the AnaeroPack System for 10 h and reoxygenation for 6 h can achieve a practicable and repeatable H/R injury model.     

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.     

Pathological alterations of astrocytes in stroke-prone spontaneously hypertensive rats under ischemic conditions

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension, and more than 95% of them die of cerebral stroke. We showed the vulnerability of neuronal cells of SHRSP/Izm rats. Furthermore, we analyzed the characteristics of SHRSP/Izm astrocytes during a stroke. It is known that the proliferating ability of SHRSP/Izm astrocytes is significantly enhanced compared with those in the normotensive Wistar Kyoto rats (WKY/Izm) strain. Conversely, the ability of SHRSP/Izm astrocytes to form tight junctions (TJ) was attenuated compared with astrocytes from WKY/Izm rats. During the stress of hypoxia and reoxygenation (H/R), lactate production, an energy source for neuronal cells, decreased in SHRSP/Izm astrocytes in comparison with the WKY/Izm strain. Moreover, during H/R, SHRSP/Izm astrocytes decreased their production of glial cell line-derived neurotrophic factor (GDNF) in comparison with WKY/Izm astrocytes. Furthermore, SHRSP/Izm rats decreased production of l-serine, compared with WKY/Izm rats following nitric oxide (NO) stimulation. Additionally, in H/R, astrocytes of SHRSP/Izm rats expressed adhesion molecules such as VCAM-1 at higher levels.It is possible that all of these differences between SHRSP/Izm and WKY/Izm astrocytes are not associated with the neurological disorders in SHRSP/Izm. However, attenuated production of lactate and reduced GDNF production in astrocytes may reduce required energy levels and weaken the nutritional status of SHRSP/Ism neuronal cells. We suggest that the attenuation of astrocytes’ functions accelerates neuronal cell death during stroke, and may contribute to the development of strokes in SHRSP/Izm. In this review, we summarize the altered properties of SHRSP/Izm astrocytes during a stroke.     

Exposure of Astrocytes to Hypoxia/Reoxygenation Enhances Expression of Glucose-Regulated Protein 78 Facilitating Astrocyte Release of the Neuroprotective Cytokine Interleukin 6

Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78-kDa polypeptide that demonstrated sequence identity with glucose-regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high-molecular-weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti-sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R-induced astrocyte release of IL-6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL-6 under the stress of H/R.     

Endothelial NOS-derived nitric oxide prevents injury resulting from reoxygenation in the hypoxic lung

Abstract

To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.     

Upregulation of cardiac NO/NOS system during short-term hypoxia and the subsequent reoxygenation period

Hypoxia/reoxygenation (H/R) reportedly influences nitric oxide (NO) production and NO synthase (NOS) expression in the heart. Nonetheless, a number of works have shown controversial results regarding the changes that the cardiac NO/NOS system undergoes under such situations. Therefore, this study aims to clarify the behaviour of this system in the hypoxic heart by investigating seven different reoxygenation times. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0, 2, 12, 24, 48, 72 h, and 5 days) in a novel approach to address the events provoked by assaults under such circumstances. Endothelial and inducible NOS (eNOS and iNOS) mRNA and protein expression, as well as enzymatic activity and enzyme location were determined. NO levels were indirectly quantified as nitrate/nitrite, and other S-nitroso compounds (NOx), which would act as NO-storage molecules. The results showed a significant increase in eNOS mRNA, protein and activity, as well as in NOx levels immediately after hypoxia, while iNOS protein and activity were induced throughout the reoxygenation period. These findings indicate that, not only short-term hypoxia, but also the subsequent reoxygenation period upregulate cardiac NO/NOS system until at least 5 days after the hypoxic stimulus, implying major involvement of this system in the changes occurring in the heart in response to H/R.     

Oxygen tension regulates the maturation of the blood-brain barrier.

The oxygen tension during the development of vascular systems influences vascular vessel formation through regulating angiogenesis. We studied the effect of hypoxia/reoxygenation (H/R) to explain its role in concert with astrocytes involvement in the development of the blood-brain barrier (BBB). On the basis of the fact that the disappearance of hypoxic regions and the decreased expression of vascular endothelial growth factor (VEGF) were observed by immunohistochemistry in a development-dependent manner in rat cerebral cortex, we examined the effects of astrocytes on the BBB-like properties of ECV304 cells by exposing astrocytes to H/R. Conditioned medium of reoxygenated astrocytes inhibited [(3)H]thymidine incorporation and tube formation of ECV 304 cells. When astrocytes were exposed to reoxygenation, the expression of VEGF was reduced, whereas the expression of angiopoietin-1 and thrombospondin-1 was enhanced. Moreover, [(3)H]sucrose permeability assay revealed that astrocytes enhance the barrier function of ECV 304 cells in coculture model within 5 h of reoxygenation. Correspondingly, the occludin expression of ECV 304 cells was slightly increased by the conditioned medium of reoxygenated astrocytes. In conclusion, our study suggests that reoxygenation of astrocytes may act as a significant driving force for the maturation of the BBB during brain development through oxygen-regulated gene(s).     

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.     

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.     

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.     

Adiponectin up-regulates the expression of T-cadherin in cardiomyocytes injured by hypoxia/reoxygenation

The aim of the present study was to investigate the effects of adiponectin (APN) on the expression of T-cadherin in cultured Sprague-Dawley (SD) rat cardiomyocytes injured by hypoxia/reoxygenation (H/R). Primary myocardial cells from neonatal rats were obtained by enzymatic digestion. The cells were divided into control group, H/R group and H/R+APN (3, 10, 20 and 30 μg/mL) groups. The H/R group was incubated in anoxic environment (anoxic solution saturated with high concentration N2) for 3 h, and then in the reoxygenation environment (the reoxygenation solution saturated with pure oxygen) for 1 h. The H/R+APN group was pretreated with different concentrations of APN for 24 h prior to the initiation of H/R. The content of lactate dehydrogenase (LDH) was measured by chemistry chromatometry. Cellular apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of T-cadherin was detected by RT-PCR and Western blotting. The results showed that, compared with control group, the apoptotic rate and release of LDH were significantly increased in the H/R group, whereas the expressions of T-cad mRNA and protein were decreased. Pretreating with APN significantly and dose-dependently decreased apoptotic rate and LDH release, and up-regulated T-cad mRNA and protein level in rat neonatal cardiomyocytes under H/R conditions. These results suggest that APN may protect cardiomyocytes against H/R-induced injury by up-regulating H/R-decreased T-cad expression.     

Role of large-conductance Ca²+-activated K+ channels in adenosine A₁ receptor-mediated pharmacological postconditioning in H9c2 cells

Ischaemic postconditioning is a phenomenon whereby short periods of ischaemia applied during the start of reperfusion protect the myocardium from the damaging consequences of reperfusion. As such, pharmacological-induced postconditioning represents an attractive therapeutic strategy for reducing reperfusion injury during cardiac surgery and following myocardial infarction. The primary aim of this study was to determine the role of large-conductance Ca2(+)-activated potassium channels (BK(Ca) channels) in adenosine A? receptor-induced pharmacological postconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. H9c2 cells were exposed to 6 h hypoxia (0.5% O?) followed by 18 h reoxygenation (H/R) after which cell viability was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. The adenosine A? receptor agonist N?-cyclopentyladenosine (CPA; 100 nmol/L) or the BK(Ca) channel opener NS1619 (10 μmol/L) were added for 30 min at the start of reoxygenation following 6 h hypoxic exposure. Where appropriate, cells were treated (15 min) before pharmacological postconditioning with the BK(Ca) channel blockers paxilline (1 μmol/L) or iberiotoxin (100 nmol/L). Pharmacological postconditioning with CPA or NS1619 significantly reduced H/R-induced LDH release. Treatment with paxilline or iberiotoxin attenuated adenosine A? receptor and NS1619-induced pharmacological postconditioning. These results have shown for the first time that BK(Ca) channels are involved in adenosine A? receptor-induced pharmacological postconditioning in a cell model system.     

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

Differential regulation and function of Fas expression on glial cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.