Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient.

The binding of oxygen to heme irons in hemoglobin promotes the binding of nitric oxide (NO) to cysteinebeta93, forming S-nitrosohemoglobin. Deoxygenation is accompanied by an allosteric transition in S-nitrosohemoglobin [from the R (oxygenated) to the T (deoxygenated) structure] that releases the NO group. S-nitrosohemoglobin contracts blood vessels and decreases cerebral perfusion in the R structure and relaxes vessels to improve blood flow in the T structure. By thus sensing the physiological oxygen gradient in tissues, hemoglobin exploits conformation-associated changes in the position of cysteinebeta93 SNO to bring local blood flow into line with oxygen requirements.

Oxygen-induced mitochondrial biogenesis in the rat hippocampus.

The hypothesis that damage to mitochondrial DNA by reactive oxygen species increases the activity of nuclear and mitochondrial transcription factors for mitochondrial DNA replication was tested in the in vivo rat brain. Mitochondrial reactive oxygen species generation was stimulated using pre-convulsive doses of hyperbaric oxygen and hippocampal mitochondrial DNA content and neuronal and mitochondrial morphology and cell proliferation were evaluated at 1, 5 and 10 days. Gene expression was subsequently evaluated to assess nuclear and mitochondrial-encoded respiratory genes, mitochondrial transcription factor A, and nuclear respiratory transcription factors-1 and -2. After 1 day, a mitochondrial DNA deletion emerged involving Complex I and IV subunit-encoding regions that was independent of overt neurological or cytological O(2) toxicity, and resolved before the onset of cell proliferation. This damage was attenuated by blockade of neuronal nitric oxide synthase. Compensatory responses were found in nuclear gene expression for manganese superoxide dismutase, mitochondrial transcription factor A, and nuclear respiratory transcription factor-2. Enhanced nuclear respiratory transcription factor-2 binding activity in hippocampus was accompanied by a nearly three-fold boost in mitochondrial DNA content over 5 days. The finding that O(2) activates regional mitochondrial DNA transcription, replication, and mitochondrial biogenesis in the hippocampus may have important implications for maintaining neuronal viability after brain injury.

Nitric oxide amplifies the excitatory to inhibitory neurotransmitter imbalance accelerating oxygen seizures.

CNS O2 toxicity is manifested most profoundly by generalized motor convulsions. The hypothesis was tested that HBO2 triggers seizures by an excitatory to inhibitory neurotransmitter imbalance produced by neuronal nitric oxide (NO) activity. Anesthetized rats were exposed to 5 ATA HBO2 for 75 min with or without prior inhibition of nNOS. Interstitial NO and amino acids: aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were determined in the striatum by microdialysis coupled with HPLC. Blood flow and EEG in the same striatal region were measured simultaneously. Rats treated with 7-NI showed no EEG spikes of O2 toxicity, while seizure latency for untreated rats was 63 +/- 7 min. Significant increases in NO metabolites and blood flow were observed in control rats before seizures. HBO2 did not change Glu significantly and increased Asp slightly whereas GABA decreased progressively by 37 +/- 7%. Pretreatment with 7-NI led to a significantly smaller decline in GABA. Overall, the simplified excitotoxicity index Glu/GABA increased significantly after 60 min of HBO2 in control but fell in rats treated with 7-NI. We conclude that HBO2-stimulated neuronal NO production promotes an imbalance between glutamatergic and GABAergic synaptic function implicated in the genesis of oxygen-induced seizures.

The roles of nitric oxide and carbon dioxide gas in the neurotoxic actions of oxygen under pressure.

The hypothesis that in conditions of hyperbaric oxygenation, nitric oxide (NO) modulates the vasodilatory effect of CO2 in the brain and thus accelerates the neurotoxic action of oxygen was verified experimentally. Conscious rats breathed atmospheric air or oxygen at 5 atm and blood flow in the striatum was measured before and after inhibition of carbonic anhydrase with acetazolamide, which causes retention of CO2 in the brain. Acetazolamide (35 mg/kg) increased blood flow in the animals when breathing air by 38 +/- 7.4% (p < 0.01), while preliminary inhibition of NO synthase with N(omega)-nitro-L-arginine-methyl ester (L-NAME, 30 mg/kg) significantly weakened its vasodilatory action. Inhibition of carbonic anhydrase in animals breathing hyperbaric oxygen at 5 atm prevented cerebral vasoconstriction, increased brain blood flow, and accelerated the development of oxygen convulsions. The vasodilatory effect of acetazolamide in hyperbaric oxygenation was significantly reduced in animals pretreated with the NO synthase inhibitor, such that the latent period of convulsions increased. The results obtained here provide evidence that in conditions of extreme hyperoxia, NO modulates the cerebral hyperemia developing in conditions of CO2 retention in the brain and accelerates the development of the neurotoxic actions of hyperbaric oxygen.

[ELECTRIC STIMULATION OF VAGUS NERVE MODULATES A PROPAGATION OF OXYGEN EPILEPSY IN RABBITS].

The activation of autonomic afferents (achieved through the vagus nerve (VN) electrical stimulation) on CNS O2 toxicity and cardiovascular function was investigated. In conscious rabbits at 5 ATA 02, prodromal signs of CNS O2 toxicity and convulsion latency were determined with and without vagus nerve (VN) stimulation. EEG, ECG and respiration were also recorded. In rabbits at 5 ATA, sympathetic overdrive and specific patterns on the EEG (synchronization of slow-waves), ECG (tachycardia) and respiration (respiratory minute volume increase) preceded motor convulsions. Vagus nerve stimulation increased parasympathetic component of autonomic drive and significantly delayed prodromal signs of oxygen toxicity and convulsion latency. Autonomic afferent input to the brain is a novel target for preventing CNS toxicity in HBO2.

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Production of hydroxyl radical in the hippocampus after CO hypoxia or hypoxic hypoxia in the rat.

Carbon monoxide poisoning produces both immediate and delayed neuronal injury in selective regions of the brain that is not readily explained on the basis of tissue hypoxia. One possibility is that cellular injury during and after CO poisoning is related to the production of reactive oxygen species (ROS) by the brain. In this study, we hypothesized that the extent of ROS generation in the brain would be greater after CO than after hypoxic hypoxia due to intracellular uptake of CO. We assessed hydroxyl radical (OH.) production by comparing the nonenzymatic hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid (2,3-DHBA) in the hippocampus of the rat by microdialysis during either CO hypoxia or an exposure to hypoxic hypoxia that produced similar PO2 and cerebral blood flow (CBF) values in the region of microdialysis. We found neither control animals nor animals exposed to 30 min of hypoxic hypoxia at a mean tissue PO2 of 15 mmHg demonstrated significant increases in 2,3-DHBA production in the hippocampus over the 2-h the exposure. In contrast, CO exposed rats which also developed brain PO2 values in the range of 15 mmHg showed highly significant increases in 2,3-DHBA production. We conclude that cerebral oxidative stress in the hippocampus of the rat during CO hypoxia in vivo is not a direct effect of decreased tissue oxygen concentration.

Nitric oxide and cerebral blood flow responses to hyperbaric oxygen.

We have tested the hypothesis that cerebral nitric oxide (NO) production is involved in hyperbaric O(2) (HBO(2)) neurotoxicity. Regional cerebral blood flow (rCBF) and electroencephalogram (EEG) were measured in anesthetized rats during O(2) exposure to 1, 3, 4, and 5 ATA with or without administration of the NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester), L-arginine, NO donors, or the N-methyl-D-aspartate receptor inhibitor MK-801. After 30 min of O(2) exposure at 3 and 4 ATA, rCBF decreased by 26-39% and by 37-43%, respectively, and was sustained for 75 min. At 5 ATA, rCBF decreased over 30 min in the substantia nigra by one-third but, thereafter, gradually returned to preexposure levels, preceding the onset of EEG spiking activity. Rats pretreated with N(omega)-nitro-L-arginine methyl ester and exposed to HBO(2) at 5 ATA maintained a low rCBF. MK-801 did not alter the cerebrovascular responses to HBO(2) at 5 ATA but prevented the EEG spikes. NO donors increased rCBF in control rats but were ineffective during HBO(2) exposures. The data provide evidence that relative lack of NO activity contributes to decreased rCBF under HBO(2), but, as exposure time is prolonged, NO production increases and augments rCBF in anticipation of neuronal excitation.

Nitric oxide production is enhanced in rat brain before oxygen-induced convulsions.

Central nervous system oxygen toxicity (CNS O2 toxicity) is preceded by release of hyperoxic vasoconstriction, which increases regional cerebral blood flow (rCBF). These increases in rCBF precede the onset of O2-induced convulsions. We have tested the hypothesis that hyperbaric oxygen (HBO2) stimulates NO* production in the brain that leads to hyperemia and anticipates electrical signs of neurotoxicity. We measured rCBF and EEG responses in rats exposed at 4 to 6 atmospheres (ATA) of HBO2 and correlated them with brain interstitial NO* metabolites (NO(x)) as an index of NO* production. During exposures to hyperbaric oxygen rCBF decreased at 4 ATA, decreased for the initial 30 min at 5 ATA then gradually increased, and increased within 30 min at 6 ATA. Changes in rCBF correlated positively with NO(x) production; increases in rCBF during HBO2 exposure were associated with large increases in NO(x) at 5 and 6 ATA and always preceded EEG discharges as a sign of CNS O2 toxicity. In rats pretreated with L-NAME, rCBF remained maximally decreased throughout 75 min of HBO2 at 4, 5 and 6 ATA. These data provide the first direct evidence that increased NO* production during prolonged HBO2 exposure is responsible for escape from hyperoxic vasoconstriction. The finding suggests that NO* overproduction initiates CNS O2 toxicity by increasing rCBF, which allows excessive O2 to be delivered to the brain.

Neutrophil sequestration and the effect of hyperbaric oxygen in a rat model of temporary middle cerebral artery occlusion.

A rat model of reversible occlusion of the middle cerebral artery was developed to assess the role of neutrophils and prophylactic hyperbaric oxygen (HBO2) on cerebral injury. Blood flow to the ipsilateral caudate putamen nucleus was reduced by approximately 50% during 2 h of arterial occlusion, but unaffected on the contralateral side. Neutrophil accumulation in brain was documented as myeloperoxidase concentration, which was elevated in both ipsilateral and contralateral cerebral hemispheres at 1 and 46 h after occlusion/reperfusion. HBO2 administered before ischemia at 2.8 atm abs for 45 min, as well as antibody-induced neutropenia, reduced neutrophil accumulation, functional neurologic deficits, and cerebral infarct volume. These data demonstrate that one mechanism for benefit of HBO2 is related to its ability to ameliorate post-ischemic injury by inhibiting neutrophil sequestration. This mechanism should be taken into consideration when choosing partial pressures of oxygen for investigational clinical protocols.

Measurement of cerebral blood flow in rats and mice by hydrogen clearance during hyperbaric oxygen exposure.

The hydrogen (H2) clearance method was adapted for the measurement of regional cerebral blood flow (rCBF) in anesthetized rats and mice during hyperbaric oxygen (HBO2) exposure. Polarographic platinum electrodes 0.1 mm in diameter were used to record H2 clearance curves from the parietal cortex (PC), substantia nigra (SN), and caudate putamen nucleus (CPN) after inhalation of 2.5% H2 in air. The system for H2 breathing under hyperbaric conditions was designed for remote operation from outside the chamber. The rCBF values (measured every 10 min) were calculated from the H2 clearance curves using the initial slope method. During air breathing control, rCBF values were similar to values reported using other methods. Considering all control rats together, blood flow (ml.100 g-1.min-1) was 89 +/- 3.6 in the SN, 78 +/- 4.7 in the CPN, and 76 +/- 6.7 in the PC. Blood flow (ml.100 g-1.min-1) for air-breathing mice was 108 +/- 11.4 in the SN and 74 +/- 8.8 in the CPN. During HBO2 exposure to 3 atm abs, rCBF in rats fell within 30 min by 26-39% (P < 0.05) and by 27-29% in mice (P < 0.05). HBO2 exposure to 4 atm abs induced maximal rCBF decreases in rats within 60 min by 37% (P < 0.01) in the SN and by 47% (P < 0.01) in the CPN. Breathing CO2 during HBO2 exposure to 4 atm abs reversed the vasoconstriction and led to a rCBF increase of 80-96% in rats. The H2 clearance method seems to be an accurate and sensitive technique for the repeated measurement of local CBF under hyperbaric conditions.

Dynamics of ion currents in the membrane of the isolated mollusc neuron under high pressure.

The action of helium (up to 101 abs. atm.) under high pressure on ionic currents through the cellular membrane was studied in experiments on isolated neurons of a gastropod. A method of intracellular dialysis which is new for physiological investigations under hyperbaric conditions was used. A substantial decrease in the inward (sodium) current with increased pressure was found. Its amplitude was decreased by 10-15% at 25 abs. atm. and by 64% at 101 abs. atm. as compared with the control. At the same time, a shift in the currentvoltage characteristics along the abscissa was not observed. Significant changes in the outward (potassium) current were not appreciable. The inhibition of the sodium current observed in these experiments with increased pressure is associated with a possible change in the structure of the cell membrane under the influence of pressure; this leads in its turn to shifts in the functioning of the membrane components participating in the generation of the nerve impulse.

Brain blood flow modulates the neurotoxic action of hyperbaric oxygen via neuronal and endothelial nitric oxide.

Studies on conscious rats with inhibition of NO synthase were used to assess the dynamics of brain blood flow and EEG traces during hyperbaric oxygenation at 4 or 5 atm. Oxygen at a pressure of 4 atm induced cerebral vasoconstriction in intact animals and decreased blood flow by 11-18% (p < 0.05) during 60-min exposure to hyperbaric oxygenation. Paroxysmal EEG activity and oxygen convulsions did not occur in rats at 4 atm of O2. At 5 atm, convulsive activity appeared on the EEG at 41 +/- 1.9 min, and blood flow decreased significantly during the first 20 min; blood flow increased by 23 +/- 9%, as compared with controls, (p < 0.01) before the appearance of convulsions on the EEG. Prior inhibition of NO synthase I (NOS I) and NO synthase III (NOS III) with N(omega)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) or inhibition only of NOS I with 7-nitroindazole (7-NI, 50 mg/kg) prevented the development of hyperoxic hyperemia and paroxysmal spikes on the EEG during hyperbaric oxygenation at 5 atm. These results show that hyperbaric oxygen induces changes in cerebral blood flow which modulate its neurotoxic action via nitric oxide synthesized both in neurons and in cerebral vessels.

Hyperoxic vasoconstriction in the brain is mediated by inactivation of nitric oxide by superoxide anions.

The hypothesis that decreases in brain blood flow during respiration of hyperbaric oxygen result from inactivation of nitric oxide (NO) by superoxide anions (O2(-)) is proposed. Changes in brain blood flow were assessed in conscious rats during respiration of atmospheric air or oxygen at a pressure of 4 atm after dismutation of O2(-) with superoxide dismutase or suppression of NO synthesis with the NO synthase inhibitor L-NAME. I.v. administration of superoxide dismutase increased brain blood flow in rats breathing air but was ineffective after previous inhibition of NO synthase. Hyperbaric oxygenation at 4 atm induced decreases in brain blood flow, though prior superoxide dismutase prevented hyperoxic vasoconstriction and increased brain blood flow in rats breathing hyperbaric oxygen. The vasodilatory effect of superoxide dismutase in hyperbaric oxygenation was not seen in animals given prior doses of the NO synthase inhibitor. These results provide evidence that one mechanism for hyperoxic vasoconstriction in the brain consists of inactivation of NO by superoxide anions, decreasing its basal vasorelaxing action.

[Intensity of the blood supply to various organs in alert cats]. / Intensivnost' krovosnabzheniia razlichnykh organov bodrstvuiushchikh koshek.

The blood flow was simultaneously measured in brain, kidney, intestine, spleen and hindlimbs of alert cats by means of hydrogen clearance method. The clearance curves were recorded with the aid of concentric electrodes set around intact veins of the organs. The following values of organ blood flow were obtained in alert cats at resting: brain--0.73 +/- 0.025 ml/g/min; kidney --1.83 +/- 0.16 ml/g/min; intestine--0.87 +/- 0.03 ml/g/min; spleen--0.61 +/- 0.026 ml/g/min; hind-limbs--0.28 +/- 0.01 ml/g/min. Methods of hydrogen clearance as well as the precision of organ blood flow measurements are discussed.

[The superslow bioelectrical activity of the brain in monkeys during the development of the high-pressure nervous syndrome]. / Sverkhmedlennaia bioélektricheskaia aktivnost' mozga obez'ian pri razvitii nervnogo sindroma vyskogo davleniia.

In immersions over 20 ata, symptoms occur known as the high pressure neural syndrome (HPNS). Regular changes of the infraslow electrical activity were revealed in development of the HPNS: the waves of the 20-sec period start developing, the average oscillation amplitudes becoming considerably higher. The inter-lead correlation becomes more obvious.

[A study of the intercapillary distribution of PO2 in the brain using microelectrodes]. / Issledovanie mezhkapilliarnogo raspredeleniia PO2 v golovnom mozge s pomoshch'iu mikroélektrodov

A simple method was suggested which enabled to estimate the intercapillary PO2 under different conditions. The intercapillary distribution of the cortex tissue oxygen pressure in normal conditions and in hypoxia was studied in unanesthetized and immobilized cats with the aid of microelectrodes. The absolute values obtained varied from 1 to 95 mm Hg. Initially high PO2 underwent maximum changes in hypoxia whereas lower PO2 values did not change significantly.

[The local blood supply of the brain in guinea pigs during the development of the high-pressure nervous syndrome]. / Lokal'noe krovosnabzhenie golovnogo mozga morskikh svinok pri razvitii nervnogo sindroma vysokogo davleniia.

Under continuous compression with normoxic helium-oxygen mixture up to 100 Ata with the velocity 1 Ata/min, guinea pigs developed successively tremor, myoclonias, seizures of clonic and tonic types. Blood supply of cerebral structures (cortex, black substance, caudate nucleus) during motor disorders increased depending on the stage of development of the high pressure neural syndrome. The role of cerebral circulation in the latter's pathogenesis is discussed.

[The structuro-functional modulus of the microcirculatory network of the cerebral cortex in rats]. / Strukturno-funktsional'nyi modul' mikrotsirkuliatornoi seti kory golovnogo mozga krys.

Complex investigation carried out in rats with the aid of polarographic microelectrodes revealed a mosaic distribution of blood supply within a small volume of nerve tissue in resting. The activation of cortical neurons was accompanied by local functional hyperemia and increased level of oxygen available. The hyperemia spread over a 200-300 mu area corresponding to the sides of neuronal columns. The data obtained have shown that the system of the cortex blood circulation and oxygen transport to neural tissues is based on a special microcirculatory module responsible for circulatory-metabolic supply of separate groups of neurons and composed as vertically oriented columns.

[Changes in the blood supply and oxygen tension of the brain following afferent somatic stimulation]. / Izmeneniia krovotoka i napriazheniia kisloroda v golovnom mozge pri afferentnoi somaticheskoi stimuliatsii

Effect of somatic afferent C fibres stimulation on rCBF (H2 - clearance method) and cerebral tissue pO2 was studied in 20 dogs. MABP and partial pressure of the arterial blood gases were stabilized before and during 3 min stimulation of the sciatic nerves. The blood flow was changed by 32% in the thalamus and by 17% in the hypothalamus during ipsilateral stimulation. Contralateral sciatic nerve stimulation caused smaller but still a significant fall of the rCBF and increase of the CVR in both regions. Parallel to these changes, tissue pO2 decreased by 19% in the hypothalamic and by 25% in the thalamic areas as compared with the prestimulation level.