Pro-oxidant effects of normobaric hyperoxia in rat tissues

Rats were exposed to 100% O₂ atmosphere for 12, 36 or 48 h, and their lungs, brain, liver and kidneys were studied for signs of oxidative damage. Oxidative damage at molecular level was estimated by: (1) the appearance of conjugated diene double bonds and (2) the amount of fluorescent chromolipids in lipids extracted from tissues. As important intracellular regulators of oxidative stress, the response of enzymes detoxifying reactive oxygen species was also studied. Macroscopically, the brain and the lungs were most susceptible to oxygen-induced effects. As an indication of oxidative tissue damage, hyperoxia caused accumulation of fluorescent chromolipids in brain and lung tissues, whereas diene conjugation did not reveal any signs of lipid peroxidation. Accumulation of fluorescent chromolipids was most prominent in the brain, where 99 and 138% increases over the control were detected after 36 and 48 h hyperoxia, respectively. Fluorescent chromolipids appeared in urine already before their concentrations were elevated in tissues. The activity of superoxide dismutase in the brain was initially decreased, followed then by a slight induction of activity at the later time-points. Pulmonary and hepatic catalase activities were markedly decreased after prolonged (36 and 48 h) hyperoxia. In conclusion, fluorescent chromolipid formation seems to be a sensitive indicator of hyperoxia-induced oxidative damage in rat tissues. The lipid peroxidation-derived fluorescent chromolipids are eliminated from the body via urinary excretion. Moreover, impaired detoxication of reactive oxygen may be implicated in tissue damage due to hyperoxia.     

Stimulation of ventilation by normobaric hyperoxia in exercising dogs

In order to describe the factors which, during hyperoxic exercise, can counteract the chemoreceptor-mediated inhibition of ventilation by O2, minute ventilation (VE) and the pulmonary gas exchange were studied breath-by-breath in four dogs running on a treadmill (5 km x h(-1)) for 10 min during and following exposure to O2 of different durations. We found that a brief inhalation of O2 applied during the steady state of the VE response provoked a reduction in VE by 6.5 +/- 0.9 l x min(-1) whereas hyperoxia applied 2 min before the onset of exercise and maintained for 2.5 min during the running tests had a significantly weaker effect on VE (-1.8 +/- 0.2 l x min(-1), P < 0.05). The rise in pulmonary CO2 output (VCO2) during the prolonged O2 exposure was less than in normoxic exercise leading to a deficit of CO2 eliminated by the lungs of 181 ml. The return to air breathing provoked a rise in VE, which reached within 73 s a much higher level than the control tests (22.9 +/- 3.6 vs. 19.5 +/- 2.2 l x min(-1), P < 0.05); VE then subsided to control levels with a long exponential decline. The CO2 deficit during O2 breathing, was fully compensated after recovery in air within 6 min. No stimulatory effect on ventilation was observed at rest at the cessation of a similar exposure to O2 despite a higher end-tidal PCO2 (+4 +/- 1 mmHg) than in exercise. In conclusion, the stimulatory effect of O2 during exercise can be clearly revealed after recovery in air and seems to operate through a more complex mechanism than that thought to be involved at rest. We propose that the changes in CO2 stores in the exercising muscles could contribute to O2-induced stimulation during exercise, possibly through stimulation of muscle afferents responding to local circulatory changes. Finally, the observation that during continuous dopamine (DA) infusion (5 microg x kg(-1) x min(-1)) the VE response to recovery in air was only a slow decrease, suggests that the arterial chemoreceptors potentiate O2-induced hyperventilation, or that the vascular actions of DA counteract part of the effects provoked by CO2 accumulation in the exercising muscles.     

Pro-oxidant effects of normobaric hyperoxia in rat tissues.

Rats were exposed to 100% O2 atmosphere for 12, 36 or 48 h, and their lungs, brain, liver and kidneys were studied for signs of oxidative damage. Oxidative damage at molecular level was estimated by: (1) the appearance of conjugated diene double bonds and (2) the amount of fluorescent chromolipids in lipids extracted from tissues. As important intracellular regulators of oxidative stress, the response of enzymes detoxifying reactive oxygen species was also studied. Macroscopically, the brain and the lungs were most susceptible to oxygen-induced effects. As an indication of oxidative tissue damage, hyperoxia caused accumulation of fluorescent chromolipids in brain and lung tissues, whereas diene conjugation did not reveal any signs of lipid peroxidation. Accumulation of fluorescent chromolipids was most prominent in the brain, where 99 and 138% increases over the control were detected after 36 and 48 h hyperoxia, respectively. Fluorescent chromolipids appeared in urine already before their concentrations were elevated in tissues. The activity of superoxide dismutase in the brain was initially decreased, followed then by a slight induction of activity at the later time-points. Pulmonary and hepatic catalase activities were markedly decreased after prolonged (36 and 48 h) hyperoxia. In conclusion, fluorescent chromolipid formation seems to be a sensitive indicator of hyperoxia-induced oxidative damage in rat tissues. The lipid peroxidation-derived fluorescent chromolipids are eliminated from the body via urinary excretion. Moreover, impaired detoxication of reactive oxygen may be implicated in tissue damage due to hyperoxia.     

Influence of normobaric hypoxia on learning capacity of different aged rats

In this study it was tested to what extent subchronic normobaric hypoxia (10% oxygen by volume) influences the learning performance of rats of different ages (4, 20, 30 months). The animals were presented with two successive conditioning patterns (FR 10/DRL). After acquisition of the FR 10 under normoxic conditions in a Skinner box the rats needed to reestablish the learned behavior under normoxia and further under hypoxia. Following this, the rats had to learn the DRL in a test chamber under the conditions of reduced oxygen. Their performance was compared with that of control animals which had to accomplish the tasks under normoxia. No age differences were observed under normoxia in learning the FR 10. However, the reestablishment of FR 10 under hypoxic conditions was less well performed by old rats than younger ones. The young rats (4 months) had a high efficiency level on the DRL-schedule under normoxia which was not impaired by hypoxia. The old rats (30 months) had considerably lower performance level under normoxia which was not further reduced in individuals by hypoxia. The performance of the middle group (20 months) was, under normoxia, at an intermediate level, while oxygen reduction led to a pronounced reduction in efficiency.     

The changes in neuromuscular excitability with normobaric hyperoxia in humans

Based on previous observations in hyperbaric hyperoxia, we hypothesized that normobaric hyperoxia, often used during general anaesthesia and resuscitation, might also induce a neuromuscular excitability. In heathy volunteers, we studied the consequences of a 50 min period of pure oxygen breathing on the neuromuscular conduction time (CT), the amplitude of the compound evoked muscle potential (M-wave), the latency and amplitude of the Hoffman reflex (H reflex) and the electromyographic tonic vibratory response (TVR) of the flexor digitorum superficialis muscle to explore the proprioceptive reflex loop. Hyperoxia-induced oxidative stress was measured by the changes in blood markers of lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and antioxidant response (reduced ascorbic acid, RAA). During hyperoxia, the M-wave amplitude increased, both CT and H reflex latency were shortened, and the H reflex amplitude increased. By contrast, TVR significantly decreased. Concomitantly, an oxidative stress was assessed by increased TBARS and decreased RAA levels. This study shows the existence of dual effects of hyperoxia, which facilitates the muscle membrane excitability, nerve conduction and spinal reflexes, but reduces the gain of the proprioceptive reflex loop. The activation of the group IV muscle afferents by hyperoxia and the resulting oxidative stress might explain the TVR depression.     

Oxygen transport in the rat brain cortex at normobaric hyperoxia

The distribution of oxygen tension (PO₂) in microvessels and in the tissues of the rat brain cortex on inhaling air (normoxia) and pure oxygen at atmospheric pressure (normobaric hyperoxia) was studied with the aid of oxygen microelectrodes (diameter?= 3–6?μm), under visual control using a contact optic system. At normoxia, the PO₂ of arterial blood was shown to decrease from [mean (SE)] 84.1?(1.3)?mmHg in the aorta to about 60.9?(3.3)?mmHg in the smallest arterioles, due to the permeability of the arteriole walls to oxygen. At normobaric hyperoxia, the PO₂ of the arterial blood decreased from 345 (6)?mmHg in the aorta to 154?(11)?mmHg in the smallest arterioles. In the blood of the smallest venules at normoxia and at normobaric hyperoxia, the differences between PO₂ values were smoothed out. Considerable differences between PO₂ values at normoxia and at normobaric hyperoxia were found in tissues at a distance of 10–50?μm from the arteriole walls (diameter?= 10–30?μm). At hyperbaric hyperoxia these values were greater than at normoxia, by 100–150?mmHg. In the long-run, thorough measurements of PO₂ in the blood of the brain microvessels and in the tissues near to the microvessels allowed the elucidation of quantitative changes in the process of oxygen transport from the blood to the tissues after changing over from the inhalation of air to inhaling oxygen. The physiological, and possibly pathological significance of these changes requires further analysis.     

Heart rate variability in healthy volunteers during normobaric and hyperbaric hyperoxia

Inhaled supranormal partial pressure of oxygen induces bradycardia and peripheral vasoconstriction. The exact mechanism of the decreasing heart rate is not clear, but the autonomic nervous system is partly involved. In the present study the role of the autonomic nervous system in hyperoxic bradycardia was evaluated by using the power spectral analysis of heart rate variability. Ten healthy volunteers participated in four experiments: (i) hyperbaric oxygen treatment (100% oxygen at 2.5 ATA), (ii) hyperbaric air treatment (O₂ 21% at 2.5 ATA), (iii) oxygen treatment at normal pressure (100% O₂, 1 ATA) and (iv) air breathing at normal pressure (21% O₂, 1 ATA). During the experiments, ECG was registered and subjected to power spectral analysis. The volunteers rated their perception of temperature, ear discomfort, sweating and excitement on a visual analogue scale. Statistical comparison of the results of the four trials was conducted with a two-way ANOVA for repeated measurements. Heart rate decreased during all interventions, but there were no statistically significant differences between the sessions. High frequency variability of heart rate variability and Hayano’s index of HF power increased and LF/HF ratio decreased with increasing partial pressure of oxygen. Our results suggest, that normobaric and hyperbaric hyperoxia increase parasympathetic influence in the regulation of the heart.     

Heart rate variability in healthy volunteers during normobaric and hyperbaric hyperoxia.

Inhaled supranormal partial pressure of oxygen induces bradycardia and peripheral vasoconstriction. The exact mechanism of the decreasing heart rate is not clear, but the autonomic nervous system is partly involved. In the present study the role of the autonomic nervous system in hyperoxic bradycardia was evaluated by using the power spectral analysis of heart rate variability. Ten healthy volunteers participated in four experiments: (i) hyperbaric oxygen treatment (100% oxygen at 2.5 ATA), (ii) hyperbaric air treatment (O2 21% at 2.5 ATA), (iii) oxygen treatment at normal pressure (100% O2, 1 ATA) and (iv) air breathing at normal pressure (21% O2, 1 ATA). During the experiments, ECG was registered and subjected to power spectral analysis. The volunteers rated their perception of temperature, ear discomfort, sweating and excitement on a visual analogue scale. Statistical comparison of the results of the four trials was conducted with a two-way ANOVA for repeated measurements. Heart rate decreased during all interventions, but there were no statistically significant differences between the sessions. High frequency variability of heart rate variability and Hayano's index of HF power increased and LF/HF ratio decreased with increasing partial pressure of oxygen. Our results suggest, that normobaric and hyperbaric hyperoxia increase parasympathetic influence in the regulation of the heart.     

Combination effects of normobaric hyperoxia and edaravone on focal cerebral ischemia-induced neuronal damage in mice

We evaluated the potential neuroprotective effects of combination treatment with normobaric hyperoxia (NBO) and edaravone, a potent scavenger of hydroxyl radicals, on acute brain injuries after stroke. Mice subjected to 2-h filamental middle cerebral artery occlusion were treated with NBO (95% O2, during the ischemia) alone, with edaravone (1.5 mg/kg, intravenously after the ischemia) alone, with both of these treatments (combination), or with vehicle. The histological and neurological score were assessed at 22-h after reperfusion. Infarct volume was significantly reduced in the combination group [36.3+/-6.7 mm3 (n=10) vs. vehicle: 65.5+/-5.9 mm3 (n=14) P<0.05], but not in the two monotherapy-groups [NBO: 50.5+/-5.8 mm3 (n=14) and edaravone: 56.7+/-5.8 mm3 (n=10)]. The combination therapy reduced TUNEL-positive cells in the ischemic boundary zone both in cortex [6.0+/-1.4 x 10(2)/mm2 (n=5) vs. vehicle: 18.9+/-2.4 x 10(2)/mm2 (n=5), P<0.01] and subcortex [11.6+/-1.5 x 10(2)/mm2 (n=5) vs. vehicle: 22.5+/-2.1 x 10(2)/mm2 (n=5), P<0.01]. NBO and combination groups exhibited significantly reduced neurological deficit scores at 22-h after reperfusion (vs. vehicle, P<0.05). Combination therapy with NBO plus edaravone prevented the neuronal damage after focal cerebral ischemia and reperfusion in mice, compared with monotherapy of NBO or edaravone.     

Effects of hyperoxia on bronchial wall dimensions and lung mechanics in rats

The effects of exposure to hyperoxic conditions (> 95 kPa at normobaric pressure) on bronchial wall dimensions and lung mechanics were examined in adult rats. Measurements of baseline pulmonary resistance and changes in pulmonary resistance following acetylcholine aerosol inhalation were made in rats exposed to hyperoxia for 48 and 60 h and in control rats exposed to air. Exposures for 48 h were carried out in humid (80% relative humidity) or dry (35–40% relative humidity) conditions. Morphometric measurements of airway wall thickness in lobar bronchi were made in separate groups of similarly exposed rats. Exposure to hyperoxia was associated with an increase in baseline pulmonary resistance (control rats 0.043 (0.016) cmH₂O ml^-1 s^-1, 60 h exposed rats 0.125 (0.042) cmH₂O ml^-1 s^-1) but hyper-responsiveness to acetylcholine inhalation did not occur. Thickness of the airway wall and its subdivisions, epithelium, lamina propria and muscularis, was not altered by hyperoxic exposure in humid conditions. However, epithelial thickening in the lobar bronchi was observed in rats exposed for 48 h to hyperoxia in dry conditions compared to rats exposed in humid conditions (mean (SD) thickness 13.2 (3.3) μm for controls, 14.5 (1.5) μm for humid exposed rats and 16.5 (3.3) μm for dry exposed rats). The increase in pulmonary resistance caused by hyperoxic exposure is unlikely to be due to airway damage as airway hyper-responsiveness did not occur, and is more likely to be associated with the development of alveolar oedema. Environmental humidity may modulate lung damage induced by hyperoxia, as exposure in dry conditions was associated with significant epithelial thickening.     

Hypoxic ventilatory responses in rats after hypercapnic hyperoxia and intermittent hyperoxia

Perinatal hyperoxia attenuates the adult hypoxic ventilatory response in rats. Hyperoxia might elicit this plasticity by inhibiting chemoreceptor activity during early life. Thus, we hypothesized that stimulating chemoreceptors with CO(2) during hyperoxia or interrupting hyperoxia with periods of normoxia would reduce the effects of hyperoxia on the hypoxic ventilatory response. Rats were born and raised in 60% O(2) for the first two postnatal weeks. Two groups were simultaneously exposed to either sustained hypercapnia (5% CO(2)) or intermittent hypercapnia (alternating 1-h exposures to 0 and 7.5% CO(2)) while another group was exposed to only intermittent hyperoxia (alternating 1-h exposures to 21 and 60% O(2)). Hypoxic ventilatory responses were assessed at 6-10 weeks of age by whole-body plethysmography. Rats exposed to intermittent hypercapnia during hyperoxia or to intermittent hyperoxia exhibited greater increases in ventilation-to-metabolism ratio ( VE/VO2 ) in response to 12.5% O(2) than rats exposed to hyperoxia alone (both P<0.05), although responses were generally less than those of normoxia-reared controls; a similar trend was observed for rats exposed to sustained hypercapnia during hyperoxia (P=0.053). These data suggest that activity-dependent mechanisms contribute to hyperoxia-induced developmental plasticity, although contributions from additional mechanisms cannot be excluded.     

Biochemical and morphological changes in rat brain synaptosomes after exposure to normobaric hyperoxia in vivo.

Adult rats were submitted to normobaric hyperoxia for 1 to 24 h, then the brain synaptosomes were isolated and their metabolic and morphologic properties were studied. Hyperoxia lasting 1-2 h significantly increased the content of thiobarbituric acid-reactive material (TBAR) and decreased the level of protein thid groups. During the next 5-8 h of hyperoxia SH groups as well as TBAR content became almost normal, reflecting adaptation of the animals to an elevated oxygen tension. After 24 h of hyperoxia a maximal increase in the TBAR content and parallel fall in protein thiol groups were noted. Simultaneously, significant morphological differences between control synaptosomes and synaptosomes isolated from rats exposed to 24 h oxygenation were observed in electron microscopy. The high-affinity dopamine uptake in hyperoxic synaptosomes was significantly increased in all experimental groups. A specific high sensitivity of the dopamine uptake system in synaptoplasmatic membranes to the free radical modification of the membrane structure is suggested.     

Delayed hyperbaric oxygenation is more effective than early prolonged normobaric hyperoxia in experimental focal cerebral ischemia

Hyperbaric (HBO) and normobaric (NBO) oxygen therapy have been shown to be neuroprotective in focal cerebral ischemia. In previous comparative studies, NBO appeared to be less effective than HBO. However, the experimental protocols did not account for important advantages of NBO in the clinical setting such as earlier initiation and prolonged administration. Therefore, we compared the effects of early prolonged NBO to delayed HBO on infarct size and functional outcome. We also examined whether combining NBO and HBO is of additional benefit. Wistar rats underwent filament-induced middle cerebral artery occlusion (MCAO) for 150 min. Animals breathed either air, 100% O(2) at ambient pressure (NBO; initiated 30 min after MCAO) 100% O(2) at 3 atm absolute (HBO; initiated 90 min after MCAO), or a sequence of NBO and HBO. Infarct volumes and neurological outcome (Garcia score) were examined 7d after MCAO. HBO (174+/-65 mm(3)) significantly reduced mean infarct volume by 31% compared to air (251+/-59 mm(3)) and by 23% compared to NBO treated animals (225+/-63 mm(3)). In contrast, NBO failed to decrease infarct volume significantly. Treatment with NBO+HBO (185+/-101 mm(3)) added no additional benefit to HBO alone. Neurological deficit was significantly smaller in HBO treated animals (Garcia score: 13.3+/-1.2) than in animals treated with air (12.1+/-1.4), but did not differ significantly from NBO (12.4+/-0.9) and NBO+HBO (12.8+/-1.1). In conclusion, HBO is a more effective therapy than NBO in transient experimental ischemia even when accounting for delayed treatment-onset of HBO. The combination of NBO and HBO results in no additional benefit.