Quercetin supplementation attenuates airway hyperreactivity and restores airway relaxation in rat pups exposed to hyperoxia.

Hyperoxia exposure of immature lungs contributes to lung injury and airway hyperreactivity. Up to now, treatments of airway hyperreactivity induced by hyperoxia exposure have been ineffective. The aim of this study was to investigate the effects of quercetin on hyperoxia-induced airway hyperreactivity, impaired relaxation, and lung inflammation. Newborn rats were exposed to hyperoxia (FiO2 > 95%) or ambient air (AA) for seven days. Subgroups were injected with quercetin (10 mg·kg-1·day-1). After exposures, tracheal cylinders were prepared for in vitro wire myography. Contraction to methacholine was measured in the presence or absence of organ bath quercetin and/or Nω-nitro-L-arginine methyl ester (L-NAME). Relaxation responses were evoked in preconstricted tissues using electrical field stimulation (EFS). Lung tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) levels were measured by enzyme-linked immunosorbent assay (ELISA). A P < 0.05 was considered statistically significant. Contractile responses of tracheal smooth muscle (TSM) of hyperoxic animals were significantly increased compared with AA animals (P < 0.001). Treatment with quercetin significantly reduced contraction in hyperoxic groups compared with hyperoxic control (P < 0.01), but did not have any effect in AA groups. In hyperoxic animals, relaxation of TSM was significantly reduced compared with AA animals (P < 0.001), while supplementation of quercetin restored the lost relaxation in hyperoxic groups. Incubation of preparations in L-NAME significantly reduced the quercetin effects on both contraction and relaxation (P < 0.01). Treatment of hyperoxic animals with quercetin significantly decreased the expression of TNF-α and IL-1ß compared with hyperoxic controls (P < 0.001 and P < 0.01, respectively).The findings of this study demonstrate the protective effect of quercetin on airway hyperreactivity and suggest that quercetin might serve as a novel therapy to prevent and treat neonatal hyperoxia-induced airway hyperreactivity and inflammation.

Attenuation of hyperoxic acute lung injury by Lycium barbarum polysaccharide via inhibiting NLRP3 inflammasome.

Lycium barbarum polysaccharide (LBP), an active component from Goji berry which is a traditional Chinese medicine, has anti-inflammatory and antioxidant features. The aim of our study was to investigate whether LBP has any role in hyperoxia-induced acute lung injury (ALI). Using a murine model of hyperoxia-induced ALI, we investigate the effect of LBP on pulmonary pathological changes as well as Sirtuin 1 (SIRT1) and the nucleotide binding domain and leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. Exposure to 100% oxygen for 72 h in male C57BL/6 mice resulted in increased protein levels of tumor necrosis factor-α and interleukin-1ß in lung tissues, and aggravated lung histological alterations. These hyperoxia-induced changes and mortality were improved by LBP. LBP markedly suppressed the activation of NLRP3 inflammasome both in vivo and in vitro. Moreover, LBP upregulated SIRT1 expression compared with vehicle-treated group. Importantly, knockdown of SIRT1 reversed the inhibitory effect of LBP on NLRP3 inflammasome activation in vitro. LBP meliorated hyperoxia-induced ALI in mice by SIRT1-dependent inhibition of NLRP3 inflammasome activation.

Hyperbaric oxygen toxicity in brain: A case of hyperoxia induced hypoglycemic brain syndrome.

Hyperbaric oxygen exposure is a recent hazzard for higher animals that originated as humans began underwater construction, exploration, and sports. Exposure can lead to abnormal brain EEG, convulsions, and death, the time to onset of each stage of pathology decreasing with increase in oxygen pressure. We provide evidence that hyperoxia, through oxidative phosphorylation, increases the energy state ([ATP]/[ADP][Pi]) of cells critical to providing glucose to cells behind the blood brain barrier (BBB). Brain cells without an absolute dependence on glucose metabolism; i.e. those having sufficient ATP synthesis using lactate and glutamate as oxidizable substrates, are not themselves very adversely affected by hyperoxia. The increased energy state and decrease in free [AMP], however, suppress glucose transport through the blood brain barrier (BBB) and into cells behind the BBB. Glucose has to pass in sequence through three steps of transport by facilitated diffusion and transporter activity for each step is regulated in part by AMP dependent protein kinase. The physiological role of this regulation is to increase glucose transport in response to hypoxia and/or systemic hypoglycemia. Hyperoxia, however, through unphysiological decrease in free [AMP] suppresses 1) glucose transport through the BBB (endothelial GLUT1 transporters) into cerebrospinal fluid (CSF); 2) glucose transport from CSF into cells behind the BBB (GLUT3 transporters) and (GLUT4 transporters). Cumulative suppression of glucose transport results in local regions of hypoglycemia and induces hypoglycemic failure. It is suggested that failure is initiated at axons and synapses with insufficient mitochondria to meet their energy requirements.

Lycium barbarum polysaccharide reduces hyperoxic acute lung injury in mice through Nrf2 pathway.

INTRODUCTION: The disruption of the balance between antioxidants and oxidants plays a vital role in the pathogenesis of acute lung injury (ALI). Evidence has shown that Lycium barbarum polysaccharide (LBP) has antioxidant feature. We examined the efficacy and mechanisms of LBP on hyperoxia-induced acute lung injury (ALI) in the present study. MATERIALS AND METHODS: C57BL/6 wild-type (WT) mice and nuclear factor erythroid 2-related factor 2 (Nrf2)-deficient (Nrf2-/-) mice were used in the present study. LBP was fed by gavages once daily for 1 week. Then, the mice were exposed to hyperoxia or room air for 72 h. Additional dosage of LBP was given per 24 h. RESULTS: Reactive oxygen species production was increased in WT mice exposed to hyperoxia. Inflammatory cytokines including interleukin (IL)-1ß as well as IL-6, and inflammatory cells were increased infiltration in the lung after 3 days hyperoxia exposure. Hyperoxia exposure also induced pulmonary edema and histopathological changes. These hyperoxia-induced changes were improved in LBP treated group. Moreover, elevated activities of heme oxygenase-1 and glutathione peroxidase and enhanced activation of Nrf2 were observed in mice treated with LBP. However, the benefit of LBP on hyperoxic ALI was abolished in Nrf2-/- mice. Moreover, our cell study showed that the LBP-induced activation of Nrf2 was dampened in pulmonary microvascular endothelial cells when the AMPK signal was inhibited by siRNA. CONCLUSIONS: LBP improves hyperoxic ALI via Nrf2-dependent manner. The LBP-induced activation of Nrf2 is mediated, at least in part, by AMPK pathway.

Human umbilical cord-derived mesenchymal stem cells protect from hyperoxic lung injury by ameliorating aberrant elastin remodeling in the lung of O2-exposed newborn rat.

The incidence and mortality rates of bronchopulmonary dysplasia (BPD) remain very high. Therefore, novel therapies are imminently needed to improve the outcome of this disease. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) show promising therapeutic effects on oxygen-induced model of BPD. In our experiment, UC-MSCs were intratracheally delivered into the newborn rats exposed to hyperoxia, a well-established BPD model. This study demonstrated that UC-MSCs reduce elastin expression stimulated by 90% O2 in human lung fibroblasts-a (HLF-a), and inhibit HLF-a transdifferentiation into myofibroblasts. In addition, the therapeutic effects of UC-MSCs in neonatal rats with BPD, UC-MSCs could inhibit lung elastase activity and reduce aberrant elastin expression and deposition in the lung of BPD rats. Overall, this study suggested that UC-MSCs could ameliorate aberrant elastin expression in the lung of hyperoxia-induced BPD model which may be associated with suppressing increased TGFß1 activation.

Protective Effects of Fetal Zone Steroids Are Comparable to Estradiol in Hyperoxia-Induced Cell Death of Immature Glia.

Impaired neurodevelopment in preterm infants is caused by prematurity itself; however, hypoxia/ischemia, inflammation, and hyperoxia contribute to the extent of impairment. Because preterm birth is accompanied by a dramatic decrease in 17ß-estradiol (E2) and progesterone, preliminary clinical studies have been carried out to substitute these steroids in preterm infants; however, they failed to confirm significantly improved neurologic outcomes. We therefore hypothesized that the persistently high postnatal production of fetal zone steroids [mainly dehydroepiandrosterone (DHEA)] until term could interfere with E2-mediated protection. We investigated whether E2 could reduce hyperoxia-mediated apoptosis in three immature glial cell types and detected the involved receptors. Thereafter, we investigated protection by the fetal zone steroids DHEA, 16α-hydroxy-DHEA, and androstenediol. For DHEA, the involved receptors were evaluated. We examined aromatases, which convert fetal zone steroids into more estrogenic compounds. Finally, cotreatment was compared against single hormone treatment to investigate synergism. In all cell types, E2 and fetal zone steroids resulted in significant dose-dependent protection, whereas the mediating receptors differed. The neuroprotection by fetal zone steroids highly depended on the cell type-specific expression of aromatases, the receptor repertoire, and the potency of the fetal zone steroids toward these receptors. No synergism in fetal zone steroid and E2 cotreatment was detected in two of three cell types. Therefore, E2 supplementation may not be beneficial with respect to neuroprotection because fetal zone steroids circulate in persistently high concentrations until term in preterm infants. Hence, a refined experimental model for preterm infants is required to investigate potential treatments.

Normobaric hyperoxia markedly reduces brain damage and sensorimotor deficits following brief focal ischaemia.

'True' transient ischaemic attacks are characterized not only clinically, but also radiologically by a lack of corresponding changes on magnetic resonance imaging. During a transient ischaemic attack it is assumed that the affected tissue is penumbral but rescued by early spontaneous reperfusion. There is, however, evidence from rodent studies that even brief focal ischaemia not resulting in tissue infarction can cause extensive selective neuronal loss associated with long-lasting sensorimotor impairment but normal magnetic resonance imaging. Selective neuronal loss might therefore contribute to the increasingly recognized cognitive impairment occurring in patients with transient ischaemic attacks. It is therefore relevant to consider treatments to reduce brain damage occurring with transient ischaemic attacks. As penumbral neurons are threatened by markedly constrained oxygen delivery, improving the latter by increasing arterial O2 content would seem logical. Despite only small increases in arterial O2 content, normobaric oxygen therapy experimentally induces significant increases in penumbral O2 pressure and by such may maintain the penumbra alive until reperfusion. Nevertheless, the effects of normobaric oxygen therapy on infarct volume in rodent models have been conflicting, although duration of occlusion appeared an important factor. Likewise, in the single randomized trial published to date, early-administered normobaric oxygen therapy had no significant effect on clinical outcome despite reduced diffusion-weighted imaging lesion growth during therapy. Here we tested the hypothesis that normobaric oxygen therapy prevents both selective neuronal loss and sensorimotor deficits in a rodent model mimicking true transient ischaemic attack. Normobaric oxygen therapy was applied from the onset and until completion of 15 min distal middle cerebral artery occlusion in spontaneously hypertensive rats, a strain representative of the transient ischaemic attack-prone population. Whereas normoxic controls showed normal magnetic resonance imaging but extensive cortical selective neuronal loss associated with microglial activation (present both at Day 14 in vivo and at Day 28 post-mortem) and marked and long-lasting sensorimotor deficits, normobaric oxygen therapy completely prevented sensorimotor deficit (P < 0.02) and near-completely Day 28 selective neuronal loss (P < 0.005). Microglial activation was substantially reduced at Day 14 and completely prevented at Day 28 (P = 0.002). Our findings document that normobaric oxygen therapy administered during ischaemia nearly completely prevents the neuronal death, microglial inflammation and sensorimotor impairment that characterize this rodent true transient ischaemic attack model. Taken together with the available literature, normobaric oxygen therapy appears a promising therapy for short-lasting ischaemia, and is attractive clinically as it could be started at home in at-risk patients or in the ambulance in subjects suspected of transient ischaemic attack/early stroke. It may also be a straightforward adjunct to reperfusion therapies, and help prevent subtle brain damage potentially contributing to long-term cognitive and sensorimotor impairment in at-risk populations.

Neonatal hyperoxia induces alterations in neurotrophin gene expression.

Each year in the United States, nearly 500,000 infants a year are born prematurely. Babies born before 35 weeks gestation are often placed on ventilators and/or given supplemental oxygen. This increase in oxygen, while critical for survival, can cause long-term damage to lungs, retinas and brains. In particular, hyperoxia causes apoptosis in neurons and alters glial activity. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are members of the neurotrophin family of proteins that function to promote the growth, differentiation and development of the nervous system. We hypothesized that hyperoxia can alter the regulation of these genes and by doing so adversely affect the development of the brain. We predicted that mice exposed to hyperoxic conditions would have differences in BDNF and GDNF mRNA expression and relative level of methylated promoter regions coinciding with differences in the relative levels of DNMT1 and DNMT3a mRNA expression. To test this hypothesis, newborn C57Bl/6 mice and their littermates were placed in hyperoxic or normoxic conditions from postnatal day 7 to 12. There were significant decreases in BDNF mRNA expression in the prefrontal cortex following hyperoxia, but a significant increase in the isocortex. GDNF mRNA expression was significantly increased in both the isocortex and prefrontal cortex following hyperoxia. DNMT1 mRNA expression was significantly decreased in the isocortex but significantly increased in the prefrontal following hyperoxia. Together these data suggest that short-term exposure to hyperoxic conditions can affect the regulation and expression of BDNF and GDNF potentially leading to alterations in neural development.

DHA suppresses chronic apoptosis in the lung caused by perinatal inflammation.

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model.

Hyperbaric oxygen preconditioning protects lung against hyperoxic acute lung injury in rats via heme oxygenase-1 induction.

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. Previous studies have shown hyperbaric oxygen preconditioning (HBO-PC) had a protective effect on oxidative injury. In the present study, we investigated the effect of HBO-PC on HALI in rats. The results demonstrated that HBO-PC ameliorated the lung biochemical and histological alterations induced by hyperoxia, decreased oxidative products but increased antioxidant enzymes. Furthermore, HBO-PC up-regulated heme oxygenase-1 (HO-1) mRNA and activity in lung tissues. The administration of HO-1 inhibitor, Zinc protoporphyrin IX, abolished its protective effects. The data showed that HBO-PC could protect rats against HALI and the anti-oxidative effect may be related to the up-regulation of HO-1.

Thioredoxin-deficient mice, a novel phenotype sensitive to ambient air and hypersensitive to hyperoxia-induced lung injury.

Pulmonary oxygen toxicity is a major clinical problem for patients undergoing supplemental oxygen therapy. Thioredoxin (Trx) is an endogenous antioxidant protein that regenerates oxidatively inactivated proteins. We examined how Trx contributes to oxygen tolerance by creating transgenic mice with decreased levels of functional thioredoxin (dnTrx-Tg) using a dominant-negative approach. These mice showed decreased Trx activity in the lung although the expression of mutant protein is three times higher than the wild-type mice. Additionally, we found that these mice showed increased oxidation of endogenous Trx in room air. When exposed to hyperoxia (>90% O2) for 4 days, they failed to recover and showed significant mortality. Even in normal oxygen levels, these mice displayed a significant decrease in aconitase and NADH dehydrogenase activities, decreased mitochondrial energy metabolism, increased p53 and Gadd45α expression, and increased synthesis of proinflammatory cytokines. These effects were further increased by hyperoxia. We also generated mice overexpressing Trx (Trx-Tg) and found they maintained lung redox balance during exposure to high oxygen and thus were resistant to hyperoxia-induced lung injury. These mice had increased levels of reduced Trx in the lung in normoxia as well as hyperoxia. Furthermore, the levels of aconitase and NADH dehydrogenase activities were maintained in these mice concomitant with maintenance of mitochondrial energy metabolism. The genotoxic stress markers such as p53 or Gadd45α remained in significantly lower levels in hyperoxia compared with dnTrx-Tg or wild-type mice. These studies establish that mice deficient in functional Trx exhibit a phenotype of sensitivity to ambient air and hypersensitivity to hyperoxia.

Oxygen dose responsiveness of human fetal airway smooth muscle cells.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 µM acetylcholine (ACh) and 10 µM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

[Apoptosis of the hypothalamus neurosecretory cells in stress and ageing: the role of immune modulators].

There is assumption about active role of immune modulators in cell death process. The involvement of interferon-alpha and cycloferon in apoptosis regulation of hypothalamic neurons of mice during stress and aging was studied. We determined the expression of apoptosis markers (Bcl-2, Mcl-1, Bax) in comparison with apoptosis level. We have found that immune modulators suppress activity of nonapeptidergic neurons. Thus, interferon-alpha treatment reduces synthesis of Bcl-2; cycloferon treatment inhibits expression of Bax and Bcl-2. So the role of immune modulators in neuron apoptosis depends on the stage of ontogenesis and type of immune modulator. Cycloferon is able to reduce the level of age-dependent apoptosis of neurons in aging, but under stress condition both interferon-alpha and cycloferon act as protectors of cell death.

Environmental damage to the retina and preconditioning: contrasting effects of light and hyperoxic stress.

PURPOSE: Environmental stress (bright light, hypoxia) can "condition" retinal photoreceptors, increasing their resistance to subsequent stress. The present study tests whether another photoreceptor-lethal stress, hyperoxia, can induce similar resistance. METHODS: Vulnerability to hyperoxia was tested in young adult C57BL/6J mice exposed to 1000 lux cyclic light for 1 week or to 50% O2 for 1 week and then to 75% O2 for 2 weeks. Vulnerability to light was tested in Balb/cJ mice exposed to 300 lux cyclic light for 2 days or to 75% O2 for 2 weeks and then to 1000 lux cyclic light for 1 week. Retinas were analyzed for photoreceptor death, levels of stress-related proteins (GFAP, FGF-2, MnSOD, acrolein), and the regulation of candidate neuroprotective genes (HSP70.1, Ledgf, FGF-13, Timp2). RESULTS: Light preconditioning did not cause measurable death of photoreceptors but reduced photoreceptor death induced by subsequent hyperoxic or light stress, reduced levels of stress-related proteins, and maintained the length and organization of photoreceptor outer segments. Hyperoxic preconditioning caused measurable cell death but provided no protection against subsequent hyperoxic or light stress. Of the four candidate neuroprotective proteins examined, the regulation of only one (Timp2) seemed associated with the neuroprotection observed. CONCLUSIONS: Light preconditioning, causing only minimal damage to photoreceptors, induced protection against subsequent stress from both hyperoxia and light. By contrast, hyperoxic preconditioning caused measurable photoreceptor damage but induced no protection against light or hyperoxia. These data suggest a separation between stress-induced damage to photoreceptors and the upregulation of protective mechanisms, encouraging the search for ways to protect the retina without damaging it.

[Permeability of rat neurohypophysial blood vessels for neurophysins and ferritin in acute hyperoxia].

The time course of the release of vasopressin-binding (nicotine-stimulated) and oxytocin-binding (estrogen-stimulated) neurophysins (NPs) into the rat pituitary and blood serum has been studied during the convulsive phase of hyperbaric oxygenation (HBO) and the postconvulsive period (PCP). The ultrastructure of the posterior pituitary (neurohypophysis) and the state of the blood-pituitary barrier in the caudal region of the gland have been studied with the use of ferritin as an exogenous marker of vascular permeability.

Contributions of nitric oxide synthase isoforms to pulmonary oxygen toxicity, local vs. mediated effects.

Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% O2 at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase (nNOS(-/-)), endothelial nitric oxide synthase (eNOS(-/-)), inducible nitric oxide synthase (iNOS(-/-)), extracellular superoxide dismutase (SOD3(-/-)), or glutathione peroxidase 1 (GPx1(-/-)), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NBO2) and hyperbaric hyperoxia (HBO2) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NBO2, and SOD3 provides an important defense. But in HBO2, nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HBO2-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways.

Respiratory plasticity after perinatal hyperoxia is not prevented by antioxidant supplementation.

Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its chemoafferent neurons. In this study, we tested the hypothesis that hyperoxia elicits this plasticity through the increased production of reactive oxygen species (ROS). Rats were born and raised in 60% O(2) for the first two postnatal weeks while treated with one of two antioxidants: vitamin E (via milk from mothers whose diet was enriched with 1000 IU vitamin E kg(-1)) or a superoxide dismutase mimetic, manganese(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP; via daily intraperitoneal injection of 5-10 mg kg(-1)); rats were subsequently raised in room air until studied as adults. Peripheral chemoreflexes, assessed by carotid sinus nerve responses to cyanide, asphyxia, anoxia and isocapnic hypoxia (vitamin E experiments) or by hypoxic ventilatory responses (MnTMPyP experiments), were reduced after perinatal hyperoxia compared to those of normoxia-reared controls (all P<0.01); antioxidant treatment had no effect on these responses. Similarly, the carotid bodies of hyperoxia-reared rats were only one-third the volume of carotid bodies from normoxia-reared controls (P <0.001), regardless of antioxidant treatment. Protein carbonyl concentrations in the blood plasma, measured as an indicator of oxidative stress, were not increased in neonatal rats (2 and 8 days of age) exposed to 60% O(2) from birth. Collectively, these data do not support the hypothesis that perinatal hyperoxia impairs peripheral chemoreceptor development through ROS-mediated oxygen toxicity.

Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats.

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.

Glutamine protects mitochondrial structure and function in oxygen toxicity.

Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.

Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice.

Keratinocyte growth factor (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -2 and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs.