Attenuation of lipopolysaccharide-induced oxidative stress and apoptosis in fetal pulmonary artery endothelial cells by hypoxia

Pulmonary vascular endothelial injury resulting from lipopolysaccharide (LPS) and oxygen toxicity contributes to vascular simplification seen in the lungs of premature infants with bronchopulmonary dysplasia. Whether the severity of endotoxin-induced endothelial injury is modulated by ambient oxygen tension (hypoxic intrauterine environment vs. hyperoxic postnatal environment) remains unknown. We posited that ovine fetal pulmonary artery endothelial cells (FPAEC) will be more resistant to LPS toxicity under hypoxic conditions (20–25 Torr) mimicking the fetal milieu. LPS (10 μg/ml) inhibited FPAEC proliferation and induced apoptosis under normoxic conditions (21% O₂) in vitro. LPS-induced FPAEC apoptosis was attenuated in hypoxia (5% O₂) and exacerbated by hyperoxia (55% O₂). LPS increased intracellular superoxide formation, as measured by 2-hydroxyethidium (2-HE) formation, in FPAEC in normoxia and hypoxia. 2-HE formation in LPS-treated FPAEC increased in parallel with the severity of LPS-induced apoptosis in FPAEC, increasing from hypoxia to normoxia to hyperoxia. Differences in LPS-induced apoptosis between hypoxia and normoxia were abolished when LPS-treated FPAEC incubated in hypoxia were pretreated with menadione to increase superoxide production. Apocynin decreased 2-HE formation, and attenuated LPS-induced FPAEC apoptosis under normoxic conditions. We conclude that ambient oxygen concentration modulates the severity of LPS-mediated injury in FPAEC by regulating superoxide levels produced in response to LPS.     

Heart rate responses to altered ambient oxygen in early (days 3–9) chick embryos in the intact egg

Normal heart rate (HR), and the HR responses to hypoxia and hyperoxia during early heart development in chick embyros have not been studied in detail, particularly in undisturbed embryos within the intact egg. HR was measured in day 3–9 chick embryos at 38 °C using relatively noninvasive impedance cardiography. Embryos were exposed to air (control) and to hypoxic (10% O₂) or hyperoxic (100% O₂) gas for a 2-h or 4-h period, during which HR was continually monitored. Control (normoxic) HR increased from about 150 beats per min (bpm) on day 3 to about 240 bpm on days 7–9. HR in very early embryos showed a variety of moderate responses to hypoxia (all survived), but as development progressed beyond day 6, hypoxic exposure induced a profound bradycardia that frequently terminated in death before the end of the measurement period. In contrast to the marked developmental changes in hypoxic sensitivity, HR showed little response to hyperoxia throughout development, suggesting no “hypoxic drive” to HR. We speculate that hypoxia has little effect early in development because of the embryo's small absolute O₂ demand, but as the embryo grows, hypoxia represents a progressively more severe perturbation. Although general trends were identified, there was considerable variation in both HR and HR responses to ambient O₂ changes between individuals of the same developmental stage. Accepted: 16 December 1998     

Cardiorespiratory response to hypoxia in persons with different levels of creativity

The correlation between the parameters of creativity and tolerance to experimentally induced normobaric hypoxia has been studied in young healthy subjects. The subjects inhaled a low-oxygen gas mixture (10% O₂) until the arterial hemoglobin oxygen saturation (SaO₂) decreased to 80%. In the recovery period, the subjects breathed normoxic air until the SaO₂ returned to its initial value. The parameters of creativity, including the originality and fluency of producing images in Torrance’s subtests of Circles and Incomplete figures, as well as the originality and fluency of sentences composed by using nouns from distant semantic categories, were evaluated before conducting hypoxic tests. Positive relationships were found between the values of figurative originality and tolerance to hypoxia, i.e., by the time of decrease in SaO₂ and the coefficient that reflects the rate of recovery of the respiratory function. Analysis of the reactivity of the cardiovascular system showed negative correlations between the parameters of creativity, namely, the figurative originality and verbal fluency, and the heart rate under the conditions of hypoxia, as well as after the recovery of the functions of the cardiorespiratory system. An increase in the capacity for original figurative thinking also corresponded to smaller differences in the values of the heart rate during the recovery and at rest. These correlations between the characteristics of creative thinking and reactions of the cardiovascular and pulmonary systems to experimentally induced hypoxia indicate that there is a common mechanism of nervous regulation underlying adaptive behavior and maintaining homeostasis.     

Effect of acute hypoxia on respiratory muscle fatigue in healthy humans

Background

Greater diaphragm fatigue has been reported after hypoxic versus normoxic exercise, but whether this is due to increased ventilation and therefore work of breathing or reduced blood oxygenation per se remains unclear. Hence, we assessed the effect of different blood oxygenation level on isolated hyperpnoea-induced inspiratory and expiratory muscle fatigue.

Methods

Twelve healthy males performed three 15-min isocapnic hyperpnoea tests (85% of maximum voluntary ventilation with controlled breathing pattern) in normoxic, hypoxic (SpO₂ = 80%) and hyperoxic (FiO₂ = 0.60) conditions, in a random order. Before, immediately after and 30 min after hyperpnoea, transdiaphragmatic pressure (P_di,tw ) was measured during cervical magnetic stimulation to assess diaphragm contractility, and gastric pressure (P_ga,tw ) was measured during thoracic magnetic stimulation to assess abdominal muscle contractility. Two-way analysis of variance (time x condition) was used to compare hyperpnoea-induced respiratory muscle fatigue between conditions.

Results

Hypoxia enhanced hyperpnoea-induced P_di,tw and P_ga,tw reductions both immediately after hyperpnoea (P_di,tw : normoxia -22 ± 7% vs hypoxia -34 ± 8% vs hyperoxia -21 ± 8%; P_ga,tw : normoxia -17 ± 7% vs hypoxia -26 ± 10% vs hyperoxia -16 ± 11%; all P < 0.05) and after 30 min of recovery (P_di,tw : normoxia -10 ± 7% vs hypoxia -16 ± 8% vs hyperoxia -8 ± 7%; P_ga,tw : normoxia -13 ± 6% vs hypoxia -21 ± 9% vs hyperoxia -12 ± 12%; all P < 0.05). No significant difference in P_di,tw or P_ga,tw reductions was observed between normoxic and hyperoxic conditions. Also, heart rate and blood lactate concentration during hyperpnoea were higher in hypoxia compared to normoxia and hyperoxia.

Conclusions

These results demonstrate that hypoxia exacerbates both diaphragm and abdominal muscle fatigability. These results emphasize the potential role of respiratory muscle fatigue in exercise performance limitation under conditions coupling increased work of breathing and reduced O₂ transport as during exercise in altitude or in hypoxemic patients.     

Influence of Hypoxic Interval Training and Hyperoxic Recovery on Muscle Activation and Oxygenation in Connection with Double-Poling Exercise

Here, we evaluated the influence of breathing oxygen at different partial pressures during recovery from exercise on performance at sea-level and a simulated altitude of 1800 m, as reflected in activation of different upper body muscles, and oxygenation of the m. triceps brachii. Ten well-trained, male endurance athletes (25.3±4.1 yrs; 179.2±4.5 cm; 74.2±3.4 kg) performed four test trials, each involving three 3-min sessions on a double-poling ergometer with 3-min intervals of recovery. One trial was conducted entirely under normoxic (No) and another under hypoxic conditions (Ho; F_iO₂ = 0.165). In the third and fourth trials, the exercise was performed in normoxia and hypoxia, respectively, with hyperoxic recovery (HOX; F_iO₂ = 1.00) in both cases. Arterial hemoglobin saturation was higher under the two HOX conditions than without HOX (p<0.05). Integrated muscle electrical activity was not influenced by the oxygen content (best d = 0.51). Furthermore, the only difference in tissue saturation index measured via near-infrared spectroscopy observed was between the recovery periods during the NoNo and HoHOX interventions (P<0.05, d = 0.93). In the case of HoHo the athletes’ P_mean declined from the first to the third interval (P < 0.05), whereas P_mean was unaltered under the HoHOX, NoHOX and NoNo conditions. We conclude that the less pronounced decline in P_mean during 3 x 3-min double-poling sprints in normoxia and hypoxia with hyperoxic recovery is not related to changes in muscle activity or oxygenation. Moreover, we conclude that hyperoxia (F_iO₂ = 1.00) used in conjunction with hypoxic or normoxic work intervals may serve as an effective aid when inhaled during the subsequent recovery intervals.     

Effects of oxygen fraction in inspired air on force production and electromyogram activity during ergometer rowing

Six male rowers rowed maximally for 2500 m in ergometer tests during normoxia (fractional concentration of oxygen in inspired air, F _IO₂ 0.209), in hyperoxia (F _IO₂ 0.622) and in hypoxia (F _IO₂ 0.158) in a randomized single-blind fashion. Oxygen consumption (V˙O₂), force production of strokes as well as integrated electromyographs (iEMG) and mean power frequency (MPF) from seven muscles were measured in 500-m intervals. The iEMG signals from individual muscles were summed to represent overall electrical activity of these muscles (sum-iEMG). Maximal force of a stroke (F _max) decreased from the 100% pre-exercise maximal value to 67 (SD 12)%, 63 (SD 15)% and 76 (SD 13)% (P<0.05 to normoxia, ANOVA) and impulse to 78 (SD 4)%, 75 (SD 14)% and 84 (SD 7)% (P<0.05) in normoxia, hypoxia and hyperoxia, respectively. A strong correlation between F _max and V˙O₂ was found in normoxia but not in hypoxia and hyperoxia. The mean sum-iEMG tended to be lower (P<0.05) in hypoxia than in normoxia but hyperoxia had no significant effect on it. In general, F _IO₂ did not affect MPF of individual muscles. In conclusion, it was found that force output during ergometer rowing was impaired during hypoxia and improved during hyperoxia when compared with normoxia. Moreover, the changes in force output were only partly accompanied by changes in muscle electrical activity as sum-iEMG was affected by hypoxic but not by hyperoxic gas. The lack of a significant correlation between F _max and V˙O₂ during hypoxia and hyperoxia may suggest a partial uncoupling of these processes and the existence of other limiting factors in addition to V˙O₂. Accepted: 2 June 1997     

Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos

During the first day of hatching, the developing chicken embryo internally pips the air cell and relies on both the lungs and chorioallantoic membrane (CAM) for gas exchange. Our objective in this study was to examine respiratory and cardiovascular responses to acute changes in oxygen at the air cell or the rest of the egg during internal pipping. We measured lung (V·_O2lung) and CAM (V·_O2CAM) oxygen consumption independently before and after 60 min exposure to combinations of hypoxia, hyperoxia, and normoxia to the air cell and the remaining egg. Significant changes in V·_O2total were only observed with combined egg and air cell hypoxia (decreased V·_O2total) or egg hyperoxia and air cell hypoxia (increased V·_O2total). In response to the different O₂ treatments, a change in V·_O2lung was compensated by an inverse change in V·_O2CAM of similar magnitude. To test for the underlying mechanism, we focused on ventilation and cardiovascular responses during hypoxic and hyperoxic air cell exposure. Ventilation frequency and minute ventilation (V_E) were unaffected by changes in air cell O₂, but tidal volume (V_T) increased during hypoxia. Both V_T and V_E decreased significantly in response to decreased P_CO2. The right-to-left shunt of blood away from the lungs increased significantly during hypoxic air cell exposure and decreased significantly during hyperoxic exposure. These results demonstrate the internally pipped embryo's ability to control the site of gas exchange by means of altering blood flow between the lungs and CAM.     

Responses of Drosophila melanogaster to atypical oxygen atmospheres

We examined physiological phenotypes of Drosophila melanogaster in hypoxic to hyperoxic atmospheres. We performed measurements on life span or behavioural function in 5, 21, 40, 60, and 80% O₂, and combined this with literature data for 2% and 100% O₂. O₂ incubation resulted in a concentration-dependent reduction of life span in both hypoxia and hyperoxia, though different measures of life span were affected differently. We also examined how behavioural and metabolic functions were affected by exposure to hyperoxia (up to 60% O₂). Climbing behaviour was measured as a fast (4 s) and slow (55 s) response in a negative geotaxis assay. In normoxia, both measures of climbing response declined exponentially until disappearing completely. Interestingly, survivorship was very high until the loss of climbing ability, after which it dropped rapidly. This pattern appeared accelerated in 40% O₂. However, while flies in 60% O₂ also apparently lost their fast climbing ability immediately prior to the drop in survivorship, they maintained considerable climbing ability over the longer trial. Metabolism, measured by CO₂ release, did not change with age in normoxic flies, but was significantly lower in flies exposed to hyperoxia, particularly as the flies aged. There was, however, a slight increase in water loss rate with age in normoxia, while in hyperoxia, water loss was reduced. Uniquely, the water loss rates of flies in 60% O₂ doubled immediately prior to the end of their life span. Because ageing results in generally irreversible functional declines, we examined if functional declines in hyperoxia (60% O₂) were also irreversible, or whether some functioning could recover after a return to normoxia. After 7 days of recovery, water loss rates decreased, CO₂ exhalation slightly increased, and climbing ability was partially recovered. Therefore, the effect of O₂ on D. melanogaster function is non-linear, may be reversible, and may include unique phenotypes that arise at some O₂ concentrations, and not others.     

Noninvasive estimation of human tissue respiration with wavelet-analysis of oxygen saturation and blood flow oscillations in skin microvessels

Laser Doppler flowmetry, laser spectrophotometry of oxygen saturation, and the fluorescence determination of the NADH/FAD ratio were carried out in 30 subjects in the upper limb skin zones with and without arteriolovenular anastomoses (AVAs). It was demonstrated that the wavelet-analysis of oxygen saturation and blood flow oscillations in microvessels was an efficient approach to noninvasive estimation of the skin oxygen extraction (OE) and oxygen consumption (OC) rates. OE = (SaO₂ ? SvO₂)/SaO₂, where SaO₂ (%) and SvO₂ (%) are the oxygen saturations of arterial and venular blood, respectively. If the cardiac (A_c, perfusion units, p.u.) to respiratory rhythm amplitude (A_r, p.u.) ratio A_c/A_r ?? 1, SvO₂ = SO₂. If A_c/A_r > 1, SvO₂ = SO₂/(A_c/A_r). OC = M _nutr (SaO₂ ?? SvO₂) in p.u. · %O₂, where M _nutr is the nutritive blood flow value in p.u. M _nutr = M/SI, where SI is the shunting index of blood flow in microvessels. The perfusion, OE, and OC values were higher in the skin with AVAs than in the skin without AVAs. The perfusion and oxygen saturation values were more variable in the skin with AVAs. The oxygen diffusing from the tiniest arterioles and capillaries is the most important for tissue metabolism. The contribution of the total perfusion and the oxygen diffusion from arterioles to tissue metabolism increased under the tissue ischemia conditions.     

Blunted pulmonary pressor responses to hypoxia in blood perfused, ventilated lungs isolated from oxygen toxic rats: Possible role of prostaglandins

To determine the effects of high oxygen (O₂) tension on pulmonary vascular reactivity, we exposed rats either to 100% O₂ for 48 hrs or 40% O₂ for 3 to 5 weeks. Lungs from all rats were isolated, blood perfused and ventilated, and pressor responses to airway hypoxia and to infused angiotensin II were measured. We found that chronic subtoxic hyperoxia did not augment subsequent hypoxic vasoconstriction, and that 48 hrs of 100% O₂ markedly blunted hypoxic vasoconstriction. Meclofenamate restored hypoxic vasoconstriction to control levels in the lungs with blunted responses. Evidence for O₂ toxicity in the lungs exposed to 100% O₂ included interstitial swelling with alveolar exudates seen by light microscopy, and lung edema by water content calculations. We conclude that 1) chronic subtoxic hyperoxia does not influence subsequent hypoxic vasoconstriction, and 2) a dilator prostaglandin produced in the lung is a potent inhibitor of hypoxic vasoconstriction in O₂ toxic lungs.     

The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy

Blood flow and pO₂ changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F₂BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F₂BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO₂ and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO₂, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24 h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO₂ compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.     

Endothelial NOS expression and ischemia-reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions

Induction of endothelial nitric oxide synthase (eNOS) contributes to the mechanism of heart protection against ischemia-reperfusion damage. We analyzed the effects of hypoxia and hyperoxia on eNOS expression in isolated working rat hearts after ischemia-reperfusion damage. Adult male Wistar rats were submitted to chronic hypoxia (2 weeks) and hyperoxia (72 h). The hearts were submitted to 15 min of ischemia and reperfused for 60 min, then we evaluated hemodynamic parameters and creatine phosphokinase (CPK) release. eNOS expression was estimated by RT-PCR; enzyme localization was evaluated by immunohistochemistry and the eNOS protein levels were detected by Western blot. All hemodynamic parameters in hypoxic conditions were better with respect to other groups. The CPK release was lower in hypoxic (P<0.01) than in normoxic and hyperoxic conditions. The eNOS deposition was significantly higher in the hypoxic group versus the normoxic or hyperoxic groups. The eNOS protein and mRNA levels were increased by hypoxia versus both other groups. Chronic hypoxic exposure may decrease injury and increase eNOS protein and mRNA levels in heart subjected to ischemia-reperfusion.     

Augmentation of hypoxic respiration after brief hyperoxia in the anesthetized cat: Putative function of GABA<Subscript>A</Subscript> neurotransmission

In this study, we attempted to determine the role of GABA neurotransmission in augmentation of hypoxic respiration by antecedent hyperoxic breathing. The experiments were performed in anesthetized, paralyzed and vagotomized cats divided into control and bicuculline (a GABA_A receptor blocker)-injected groups. The experimental protocol consisted of exposing the animals to successive hypoxic-hyperoxic-hypoxic conditions. Respiration was assessed using phrenic electroneurograms, from which the peak phrenic height, a surrogate of the tidal volume component, and respiratory rate were obtained, and their product, the respiratory minute output, was calculated. We found that prior hyperoxic ventilation increased the subsequent respiratory response to hypoxia by an average of 23.5%, compared with the preoxygen response. This increase was driven by volume respiration. The biphasic character of the hypoxic respiratory response, consisting of stimulatory and depressant phases, was sustained. Bicuculline abolished the augmentative effect on hypoxic respiration of prior hyperoxia, which suggests that oxygenation induces GABA_A-mediated hyperexcitability of respiratory neurons, possibly by the liberation of reactive oxygen species. We concluded that GABA neurotransmission is pertinent to the effect of hyperoxia on hypoxic respiratory reactivity.     

Hypoxic avoidance behaviour in cod (Gadus morhua L.): The effect of temperature and haemoglobin genotype

Hypoxia can influence fish growth, survival and on larger scales, population structure. These effects may be influenced by water temperature, and may vary intra-specifically with genotype. In Atlantic cod (Gadus morhua L.), the two haemoglobin homozygotes (Hb-I?11 and Hb-I?22) vary in oxygen affinity at different temperatures, which is thought to correspond to variation in hypoxia tolerance. We therefore tested if hypoxic avoidance behaviour in cod 1) depends on ambient temperature and 2) is modified by haemoglobin genotype. In a laminar flow choice box, we subjected juvenile cod to an initial phase of non-escapable hypoxia, and a subsequent recovery phase, where one habitat was kept at 20% O₂ saturation while the other was raised in steps to full saturation. The experiment was performed at 5 and 15 °C with Hb-I?11 and Hb-I?22 cod. Cod responded to inescapable hypoxia by reducing their overall swimming speed and then, at the initial levels of the recovery phase, avoiding the most hypoxic habitat, irrespective of temperature or genotype. Fish recovered quickly as O₂ levels increased, as evidenced by increased swimming speed and time spent in the most hypoxic habitat. The avoidance response depended strongly on temperature: the relative reduction in speed and avoidance of the most hypoxic habitat was more pronounced at 15 than at 5 °C. During the recovery phase, stressed fish initially maintained a higher swimming speed in the most hypoxic habitat. However, as O₂ increased, swimming speed in both habitats converged. This point of convergence occurred at a lower O₂ saturation at 5 °C. Fish ventilation rate in inescapable hypoxia was also higher at 15 °C. Haemoglobin genotype did not influence either ventilation rates or the nature of the hypoxic avoidance response at either temperature, but Hb-I?11 cod swam faster than Hb-I?22 cod in normoxia at 15 °C. Our results indicate that increased temperature limits the ability of cod of both haemoglobin genotypes to exploit hypoxic habitats. This may have negative future consequences for coastal cod stocks in light of increasing global temperatures and eutrophication in coastal waters.     

Hyperoxia increases oxygen radical production in rat lung homogenates

Lung damage during hyperoxia has been postulated to be due to increased rates of local organ oxygen radical production. Lung homogenate respiration was inhibited with cyanide, and residual respiration was used as an indicator of electron diversion to O₂^? and H₂O₂. Cyanide-resistant respiration in lung homogenates, supplemented with 1 mm NADH, increased linearly with oxygen tension, and accounted for 7% of total respiration in air and for 17% of total respiration when homogenates were incubated in 80% oxygen. Exposure of rats to 85% oxygen for 7 days induces tolerance to the lethal effects of 100% oxygen. Rats which previously breathed 85% oxygen for 7 days had a greater CN^?-resistant respiration than control rats. This implies that adaptation to hyperoxia does not include decreased lung tissue oxygen radical production as indicated by CN^?-resistant respiration. One possible explanation for the increased CN^?-resistant respiration in oxygen tolerant rat lungs is that they contain increased cell mass. Lung homogenates of rats exposed to 85% oxygen for 7 days also had 2.5 times greater thiobarbituric acid positive material than controls, indicating that increased lung lipid peroxidation occurs as a consequence of hyperoxia. Incubation of normal rat lung homogenates under hyperoxic conditions also acutely increased lipid peroxidation, which could be inhibited by both superoxide dismutase and catalase. This confirms that hyperoxia enhances cellular production of O₂^? and H₂O₂ and implies an essential role for both O₂^? and H₂O₂ in hyperoxic lung damage.     

NF-κB involvement in hyperoxia-induced myocardial damage in newborn rat hearts

Premature newborns are frequently exposed to hyperoxia ventilation and some literature data indicate the possibility of hyperoxia-induced myocardial damage. Since nuclear factor κB (NF-κB) is a crucial signaling molecule involved in physiological response to hyperoxia in different cell types as well as in various tissues, our attention has been focused on the role played by NF-κB pathway in response to moderate and severe hyperoxia exposure in rat neonatal heart tissue. Akt and IκBα levels, involved in NF-κB activation, along with the balance between apoptotic and survival pathways have also been investigated. Experimental design of the study has involved exposure of newborn rats to room air (controls), 60 % O₂ (moderate hyperoxia), or 95 % O₂ (severe hyperoxia) for the first two postnatal weeks. Morphological analysis shows a less compact tissue in rat heart exposed to moderate hyperoxia and a decreased number of nuclei in samples exposed to severe hyperoxia. A significant increase of NF-κB positive nuclei percentage and p-IκBα expression in samples exposed to 95 % hyperoxia compared to control and to 60 % hyperoxia is evidenced; in parallel, an increase of pAkt/Akt ratio in both samples exposed to 95 and 60 % hyperoxia is shown. Furthermore, a more evident cytochrome c/Apaf-1 immunocomplex and a decreased Bcl2 expression in 95 % hyperoxia-exposed sample compared to 60 % exposed one is evidenced. In conclusion, our findings suggest the involvement of the NF-κB pathway and Akt signaling in the mechanisms of myocardial hyperoxic damage in the newborns, with particular reference to the induction of oxidative stress-related apoptosis.     

Postnatal Hyperoxia Exposure Differentially Affects Hepatocytes and Liver Haemopoietic Cells in Newborn Rats

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O₂ (moderate hyperoxia), or 95% O₂ (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.     

Severe Blunt Muscle Trauma in Rats: Only Marginal Hypoxia in the Injured Area

Background

After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O₂ supply were combined with immunological detection of hypoxic cells to estimate O₂ conditions within the injured muscle area.

Materials and Methods

Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized “weight-drop” device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O₂ saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α.

Results

Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O₂ saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied.

Conclusions

In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O₂ supply is clearly increased. This increased O₂ supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.