Ran Arieli

Hyperbaric oxygen therapy for reduction of secondary brain damage in head injury: an animal model of brain contusion.

Cerebral contusions are one the most frequent traumatic lesions and the most common indication for secondary surgical decompression. The purpose of this study was to investigate the physiology of perilesional secondary brain damage and evaluate the value of hyperbaric oxygen therapy (HBOT) in the treatment of these lesions. Five groups of five Sprague-Dawley rats each were submitted to dynamic cortical deformation (DCD) induced by negative pressure applied to the cortex. Cerebral lesions produced by DCD at the vacuum site proved to be reproducible. The study protocol entailed the following: (1) DCD alone, (2) DCD and HBOT, (3) DCD and post-operative hypoxia and HBOT, (4) DCD, post-operative hypoxia and HBOT, and (5) DCD and normobaric hyperoxia. Animals were sacrificed after 4 days. Histological sections showed localized gross tissue loss in the cortex at injury site, along with hemorrhage. In all cases, the severity of secondary brain damage was assessed by counting the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3-positive cells in successive perilesional layers, each 0.5 mm thick. Perilesional TUNEL positive cells suggested the involvement of apoptosis in group 1 (12.24% of positive cells in layer 1). These findings were significantly enhanced by post-operative hypoxia (31.75%, p < 0.001). HBOT significantly reduced the severity and extent of secondary brain damage expressed by the number of TUNEL positive cells in each layer and the volume of the lesion (4.7% and 9% of TUNEL positive cells in layer 1 in groups 2 and 4 respectively, p < 0.0001 and p < 0.003). Normobaric hyperoxia also proved to be beneficial although in a lesser extent. This study demonstrates that the vacuum model of brain injury is a reproducible model of cerebral contusion. The current findings also suggest that HBOT may limit the growth of cerebral contusions and justify further experimental studies.

Heat acclimation prolongs the time to central nervous system oxygen toxicity in the rat: Possible involvement of HSP72

Oxygen toxicity of the central nervous system (CNS-OT) can occur during diving with oxygen-enriched gas mixtures, or during hyperbaric medical treatment. CNS-OT is characterised by convulsions and sudden loss of consciousness, which may be fatal in diving. Heat acclimation is known to provide cross-tolerance to various forms of stress in different organs, including the brain. We hypothesised that heat acclimation may delay the onset of CNS-OT in the rat. Male Sprague-Dawley rats were acclimated to an ambient temperature of 32 degrees C for 4 weeks. Rats in the control group were kept at 24 degrees C. Both groups were exposed to oxygen at 608 kPa. EEG was recorded continuously until the appearance of the first electrical discharge preceding clinical convulsions. CO(2) production was measured simultaneously with the EEG. Latency to CNS-OT was measured and brain samples were taken for evaluation of heat shock protein 72 (HSP72) levels by Western blot analysis at the end of the acclimation period and during 4 weeks of deacclimation. Latency to CNS-OT was twice as long in the heat-acclimated rat, with insignificant changes in CO(2) production. This prolongation continued for 2 weeks during deacclimation. There was a significant increase in the level of HSP72 following heat acclimation, with a subsequent decrease during deacclimation. We conclude that heat acclimation prolongs latency to CNS-OT in a way that does not involve changes in metabolic rate. During deacclimation there was a linear relationship between latency to CNS oxygen toxicity and the level of HSP72. A possible beneficial effect of HSP72 is discussed.

Combined effect of denucleation and denitrogenation on the risk of decompression sickness in rats

We previously hypothesized that the number of bubbles emerging on decompression from a dive, and the resultant risk of decompression sickness (DCS), may be reduced by a process whereby effective gas micronuclei that might otherwise have formed bubbles on decompression are shrunk and eliminated. In a procedure defined by us as denucleation, exposure to hyperbaric oxygen (HBO) would result in oxygen replacing the resident gas in the micronuclei, to be subsequently consumed by the mitochondria when the oxygen pressure is reduced. Support for the validity of our hypothesis may be found in our previous studies on the transparent prawn and the reduction of DCS in the rat. In all of these studies, HBO pretreatment was given before supersaturation with inert gas at high pressure. The purpose of the present study was to compare DCS outcome in rats that underwent nitrogen washout (denitrogenation) alone (9 min O(2) at 507 kPa) after exposure to air at high pressure (33 min at 1,266 kPa), and rats treated by both procedures (denitrogenation + denucleation; 8 min of O(2) breathing followed by 5 min air breathing, both at 507 kPa) after high-pressure air exposure. This was done with the same nitrogen load in both groups before the final decompression (a nitrogen pressure of 467 kPa in fatty and 488 kPa in aqueous tissue). Six of 20 rats in the denitrogenation + denucleation group died, compared with 13 in the denitrogenation group (P < 0.03). Three rats in the denitrogenation + denucleation group suffered mild DCS, recovering completely within 2 h of decompression. The present study indicates an advantage in considering both denitrogenation and denucleation before decompression. This may have practical application before escape from a disabled submarine, when aborting a technical dive, or in the preparation of aviators for high altitude.

Latency to CNS oxygen toxicity in rats as a function of PCO2 and PO2

Abstract Central nervous system (CNS) oxygen toxicity can occur as convulsions and loss of consciousness, without any premonitory symptoms. We have made a quantitative study of the effect of inspired carbon dioxide on sensitivity to oxygen toxicity in the rat. Rats were exposed to four oxygen pressures (PO2; 456, 507, 608 and 709 kPa) and an inspired partial pressure of carbon dioxide (PCO2) in the range 0–12 kPa until the appearance of the electroencephalograph first electrical discharge (FED) that preceds the clinical convulsions. Exposures were conducted at a thermoneutral temperature of 27°C. Latency to the FED decreased linearly with the increase in PCO2 at all four PO2 values studied. This decrease, which is probably related to the cerebral vasodilatory effect of carbon dioxide, reached a minimal value that remained constant on further elevation of PCO2. The slopes (absolute value) and intercepts of latency to the FED as a function of carbon dioxide decreased with the increase in PO2. This log-linear relationship made possible the derivation of equations that describe latency to the FED as a function of both PO2 and PCO2 in the PCO2– dependent range: Latency (min) = e(5.19−0.0040PO2)−e(2.77−0.0034PO2) × PCO2 (kPa), and in the PCO2-independent range: Latency(min) = e(2.44−0.0009PO2). A PCO2 as low as 1 kPa significantly reduced the latency to the FED. It is suggested that in closed-circuit oxygen diving, any accumulation of carbon dioxide should be avoided in order to minimize the risk of CNS oxygen toxicity.

Decompression sickness bubbles: Are gas micronuclei formed on a flat hydrophobic surface?

It is a long-standing hypothesis that the bubbles which evolve as a result of decompression have their origin in stable gas micronuclei lodged in hydrophobic crevices, micelles of surface-active molecules, or tribonucleation. Recent findings supported by atomic force microscopy have indicated that tiny, flat nanobubbles form spontaneously on smooth, hydrophobic surfaces submerged in water. We propose that these nanobubbles may be the gas micronuclei responsible for the bubbles that evolve to cause decompression sickness. To support our hypothesis, we used hydrophilic and monolayer-covered hydrophobic smooth silicon wafers. The experiment was conducted in three main stages. Double distilled water was degassed at the low pressure of 5.60 kPa; hydrophobic and hydrophilic silicon wafers were placed in a bowl of degassed water and left overnight at normobaric pressure. The bowl was then placed in the hyperbaric chamber for 15 h at a pressure of 1013 kPa (=90 m sea water). After decompression, bubbles were observed and photographed. The results showed that bubbles only evolved on the hydrophobic surfaces following decompression. There are numerous hydrophobic surfaces within the living body (e.g., in the large blood vessels), which may thus be the sites where nanobubbles that serve as gas micronuclei for bubble evolution following decompression are formed.

Recovery time constant in central nervous system O2 toxicity in the rat

Abstract The development of oxygen toxicity can be delayed by intermittent periods of normoxia. However, there is no accepted procedure for quantifing the recovery during normoxia. A cumulative oxygen toxicity index - K, when K reaches a critical value (Kc) and the toxic effect is manifested, can be calculated using the equation K = te2 × PO2c where te is hyperoxic exposure time and PO2 is oxygen pressure and c is a power parameter. Recovery during normoxia (reducing K) is calculated by the equation K2 = K1 × e−rtr where tr is recovery time, r being the recovery time constant. A combination of accumulation of oxygen toxicity and its recovery can be used to calculate central nervous system oxygen toxicity. In protocol A (n = 25), r was calculated for rats exposed either continuously to 608 kPa oxygen or to PO2 = 608 kPa followed by a period of normoxia (3.5% O2), with a subsequent return to PO2 = 608 kPa until appearance of the first electrical discharge (FED) in the electroencephalogram which precedes clinical convulsions. In protocol B (n = 22), predicted latency to the FED was compared to measured latency for seven different exposures to hyperbaric oxygen (HBO), followed by a period of normoxia and further HBO exposure. Recovery followed an exponential path, with r = 0.31 (SD 0.12) min−1. The predicted latency to FED in protocol B correlated with the measured latencies. Calculation of the recovery of the CNS oxygen toxicity agreed with the previously suggested exponential recovery of the hypoxic ventilatory response and was probably a general recovery process. We concluded that recovery can be applied to the design of various hyperoxic exposures.

Dynamics of gas micronuclei formed on a flat hydrophobic surface, the predecessors of decompression bubbles.

It is a long-standing hypothesis that the bubbles which evolve as a result of decompression have their origin in stable gas micronuclei. In a previous study (Arieli and Marmur, 2011), we used hydrophilic and monolayer-covered hydrophobic smooth silicon wafers to show that nanobubbles formed on a flat hydrophobic surface may be the gas micronuclei responsible for the bubbles that evolve to cause decompression sickness. On decompression, bubbles appeared only on the hydrophobic wafers. The purpose of the present study was to examine the dynamics of bubble evolution. The numbers of bubbles after decompression were greater with increasing hydrophobicity. Bubbles appeared after decompression from 150 kPa, and their density increased with elevation of the exposure pressure (and supersaturation), up to 400 kPa. The normal force of attraction between the hydrophobic surface and the bubble, as determined from the volume of bubbles leaving the surface of the wafer, was 38×10(-5) N and the tangential force was 20×10(-5) N. We discuss the correlation of these results with previous reports of experimental decompression and bubble formation, and suggest to consider appropriate modification of decompression models.

Evolution of bubbles from gas micronuclei formed on the luminal aspect of ovine large blood vessels.

It has been shown that tiny gas nanobubbles form spontaneously on a smooth hydrophobic surface submerged in water. These nanobubbles were shown to be the source of gas micronuclei from which bubbles evolved during decompression of silicon wafers. We suggest that the hydrophobic inner surface of blood vessels may be a site of nanobubble production. Sections from the right and left atria, pulmonary artery and vein, aorta, and superior vena cava of sheep (n=6) were gently stretched on microscope slides and exposed to 1013 kPa for 18 h. Hydrophobicity was checked in the six blood vessels by advancing contact angle with a drop of saline of 71±19°, with a maximum of about 110±7° (mean±SD). Tiny bubbles ~30 μm in diameter rose vertically from the blood vessels and grew on the surface of the saline, where they were photographed. All of the blood vessels produced bubbles over a period of 80 min. The number of bubbles produced from a square cm was: in the aorta, 20.5; left atrium, 27.3; pulmonary artery, 17.9; pulmonary vein, 24.3; right atrium, 29.5; superior vena cava, 36.4. More than half of the bubbles were present for less than 2 min, but some remained on the saline-air interface for as long as 18 min. Nucleation was evident in both the venous (superior vena cava, pulmonary artery, right atrium) and arterial (aorta, pulmonary vein, left atrium) blood vessels. This newly suggested mechanism of nucleation may be the main mechanism underlying bubble formation on decompression.

Thermal status of wet-suited divers using closed circuit O2 apparatus in sea water of 17–18.5°C

Abstract A wet suit may not provide adequate thermal protection when diving in moderately cold water (17–18°C), and any resultant mild hypothermia may impair performance during prolonged diving. We studied heat exchange during a dive to a depth of 5 m in sea water (17–18.5°C) in divers wearing a full wet suit and using closed-circuit oxygen breathing apparatus. Eight fin swimmers dived for 3.1 h and six underwater scooter (UWS) divers propelled themselves through the water for 3.7 h. The measurements taken throughout the dive were the oxygen pressure in the cylinder and skin and rectal temperatures (Tre). Each subject also completed a cold score questionnaire. The Tre decreased continuously in all subjects. Oxygen consumption in the fin divers (1.40 l · min−1) was higher than that of the UWS divers (1.05 l · min−1). The mean total insulation was 0.087°C · m2 · W−1 in both groups. Mean body insulation was 37% of the total insulation (suit insulation was 63%). The reduction in Tre over the 1st hour was related to subcutaneous fat thickness. There was a correlation between cold score and Tre at the end of 1 h, but not after that. A full wet suit does not appear to provide adequate thermal protection when diving in moderately cold water.

Ex vivo bubble production from ovine large blood vessels: Size on detachment and evidence of “active spots”

Nanobubbles formed on the hydrophobic silicon wafer were shown to be the source of gas micronuclei from which bubbles evolved during decompression. Bubbles were also formed after decompression on the luminal surface of ovine blood vessels. Four ovine blood vessels: aorta, pulmonary vein, pulmonary artery, and superior vena cava, were compressed to 1013 kPa for 21 h. They were then decompressed, photographed at 1-s intervals, and bubble size was measured on detachment. There were certain spots at which bubbles appeared, either singly or in a cluster. Mean detachment diameter was between 0.7 and 1.0 mm. The finding of active spots at which bubbles nucleate is a new, hitherto unreported observation. It is possible that these are the hydrophobic spots at which bubbles nucleate, stabilise, and later transform into the gas micronuclei that grow into bubbles. The possible neurological effects of these large arterial bubbles should be further explored.