Bubble formation in crustaceans following decompression from hyperbaric gas exposures

In vivo bubble formation was studied in various crustaceans equilibrated with high gas pressures and rapidly decompressed to atmospheric pressure. The species varied widely in susceptibility to bubble formation, and adults were generally more susceptible than larval stages. Bubbles did not form in early brine shrimp larvae unless equilibration pressures of at least 175 atm argon or 350 atm helium were used; for adult brine shrimp, copepods, and the larvae of crabs and shrimps, 100-125 atm argon or 175-225 atm helium were required. In contrast, bubbles formed in the leg joints of megalopa and adult crabs following decompression from only 3-10 atm argon; stimulation of limb movements increased this bubble formation, whereas inhibition of movements decreased it. High hydrostatic compressions applied before gas equilibration or slow compressions did not affect bubble formation. We concluded that circulatory systems, musculature, and storage lipids do not necessarily render organisms susceptible to bubble formation and that bubbles do not generally originate as preformed nuclei. In some cases, tribonucleation appears to be the cause of the bubbles.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

CARBON DIOXIDE AS A FACILITATING AGENT IN THE INITIATION AND GROWTH OF BUBBLES IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. Rats killed in a variety of ways (broken neck, nembutal, anoxia, electrocution) may undergo extensive bubble formation when subsequently decompressed from atmospheric pressure to simulated altitudes of 50,000 feet. On autopsy at sea level, large numbers of bubbles are found throughout the vascular system in the majority of animals. These bubbles appear to originate in small vessels deep within muscular regions, later spreading widely in arterial and venous systems. Dead rabbits and frogs also bubble profusely on decompression. 2. Bubble formation in dead animals is attributed primarily to the accumulation of CO₂, derived from residual cellular respiration after death, and from anaerobic glycolysis with attendant decomposition of bicarbonates in blood and tissue fluids. If anaerobic glycolysis is inhibited by using sodium iodoacetate as a lethal agent, bubble formation is greatly reduced or lacking on subsequent decompression. 3. Experiments in vitro suggest that high concentrations of CO₂ favor bubble formation by reducing the degree of mechanical disturbance necessary. 4. Administration of CO₂ in high concentrations to living frogs lowers the minimum altitude (pressure equivalent) at which bubble formation occurs, with exercise, in untreated animals. Pre-treatment with CO₂ also reduces the degree of muscular activity necessary for bubbles to form in frogs at higher altitudes. 5. Analyses have been made of the gas content of bubbles taken directly from the large veins of decompressed frogs and rats. In living animals the figures obtained indicate rapid equilibration with gas tensions in the blood. Bubbles taken from decompressed dead rats may contain 60–80 per cent CO₂. 6. The bearing of these experiments on the mechanisms of bubble initiation and growth in normal living animals is discussed. Reasons are given for suggesting that CO₂, due largely to its high dissolved concentration in localized active regions, may be an outstanding factor in the initiation and early growth of bubbles which in later stages are expanded and maintained principally by nitrogen.     

Intracellular gas supersaturation tolerances of erythrocytes and resealed ghosts.

Intact mammalian, avian, and amphibian erythrocytes were saturated with up to 300 atm nitrogen or argon gas and rapidly decompressed. Despite the profuse nucleation of gas bubbles in the suspending fluid, no evidence of intracellular gas bubble nucleation was found; all or most of the cells remained intact and little or no hemoglobin escaped. Internal bubbles were similarly absent from resealed ghosts of human erythrocytes as shown by lack of disintegration and by retention of an entrapped fluorescent compound. The absence of bubbles may indicate that much of the internal water does not have the same nucleation properties as external water.     

Gas supersaturation tolerances in amoeboid cells before and after ingestion of bubble-promoting particles.

Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macrophages and macrophage-like tumor cells, and for cells of the slime mold Dictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogen-supersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 x 10(7) Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.     

MUSCULAR ACTIVITY AND BUBBLE FORMATION IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. Muscular activity during decompression causes bubble formation in the blood of intact bullfrogs. The amount of gas liberated depends on the degrees of muscular activity and supersaturation (as influenced by altitude). In decompressed dissected bullfrogs, bubbles appear in veins leading from active but not from inactive muscles. 2. Muscular activity during decompression similarly causes bubble formation in rats. Bubbles appear in veins coming from muscles, and often in the lymphatic system. Quiescent rats do not form bubbles. 3. Violent muscular activity before decompression favors bubble formation in bullfrogs during ensuing decompression, but it is less effective than exercise during decompression. The effect persists in large frogs for about an hour. 4. Pre-oxygenation for 2 to 4 hours before decompression reduces the incidence of bubble formation in decompressed bullfrogs. It thus has the same effect on bubble formation in bullfrogs as it does on the "bends" in man. The effect is presumably due to removal of nitrogen. 5. Possible mechanisms by which muscular activity causes bubble formation are discussed. The effects of mechanical agitation and of metabolic CO₂ are considered to be the dominant factors.     

Gas supersaturation tolerances in amoeboid cells before and after ingestion of bubble-promoting particles

Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macro-phages and macrophage-like tumor cells, and for cells of the slime moldDictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogensupersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 × 10⁷ Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.     

THE RELATION OF EXERCISE TO BUBBLE FORMATION IN ANIMALS DECOMPRESSED TO SEA LEVEL FROM HIGH BAROMETRIC PRESSURES

1. Bullfrogs (Rana catesbiana) and rats have been subjected to high barometric pressures and studied for bubble formation on subsequent decompression to sea level. Pressures varying from 3 to 60 pounds per square inch, in excess of atmospheric pressure, were used. 2. Muscular activity after decompression is necessary for bubble formation in bullfrogs after pressure treatment throughout the above range. Anesthetized frogs remained bubble-free following decompression. Rats compressed at 15 to 45 pounds per square inch likewise did not contain bubbles unless exercised on return to sea level. 3. Bubbles form without voluntary muscular activity in anesthetized rats previously subjected to pressure of 60 pounds per square inch. Small movements involved in breathing and other vital activities are believed sufficient to initiate bubbles in the presence of very high supersaturations of N₂. 4. Bubbles appear (with exercise) in rats previously compressed at 15 pounds per square inch, and in bullfrogs subjected to pressure at levels as low as 3 pounds per square inch above atmospheric pressure. The percentage drop in pressure necessary for bubble formation is less in compressed animals than in those decompressed from sea level to simulated altitudes. 5. The action of exercise on bubble formation in compressed frogs and rats is attributed to mechanical factors associated with muscular activity, combined with the high supersaturation of N₂. CO₂ probably is not greatly involved, since its concentration does not reach supersatuation, as it does at high altitude. 6. Anoxia following decompression from high barometric pressures has no observable facilitating effect on bubble formation.     

Promotion of gas bubble formation by ingested nuclei in the ciliate, Tetrahymena pyriformis

Cells of the ciliate Tetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1-0.5 X 10(7) Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

ADDITIONAL MECHANISMS FOR THE ORIGIN OF BUBBLES IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. A heavy ingestion of frothy emulsified fat by rats and bullfrogs does not increase susceptibility to bubble formation when the animals are decompressed 2 to 72 hours later. This indicates that gaseous films (bubble nuclei) initially present do not pass across the intestinal wall with the digested fat, and also that high fat content per se in the lymph and blood does not increase susceptibility to bubble formation. 2. Liquid caprylic acid injected into veins of bullfrogs crystallizes when the frogs are cooled. The crystallization causes bubbles to form without muscular activity on subsequent decompression. Cooling normal bullfrogs to 1–2°C. fails, however, to crystallize any substances occurring naturally in the animals that might act in a similar manner. 3. When bullfrogs are cooled (e.g. to –5° to –10°C.) until ice forms in the blood vessels, and are then warmed and decompressed, bubbles form in the absence of exercise. Crystallization of water in the body thus forms nuclei or even small bubbles that persist. If only one foot is frozen, bubbles originate in the frozen foot. In some cases visible bubbles were observed in thawed feet at sea level (i.e. without decompression). When frog''s blood is partly frozen in test tubes or in tied off sections of veins, bubbles will appear on decompression in the absence of mechanical agitation. The practical relation of this phenomenon to flight at high altitude should not be overlooked. 4. Fracturing a leg bone (tibia or femur) in a frog induces bubble formation on subsequent decompression. Bubble nuclei, which persist for ½ to 1 hour, are probably formed as a result of the intense mechanical disturbance when the bone snaps. Fracturing of bone is considerably more effective than crushing muscles for producing bubbles in frogs.     

Promotion of gas bubble formation by ingested nuclei in the ciliate,Tetrahymena pyriformis

Cells of the ciliateTetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1−0.5×10⁷ Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Optimal oxygen pressure and time for reduced bubble formation in the N2-saturated decompressed prawn.

Bubbles that grow during decompression are believed to originate from preexisting gas micronuclei. We showed that pretreatment of prawns with 203 kPa oxygen before nitrogen loading reduced the number of bubbles that evolved on decompression, presumably owing to the alteration or elimination of gas micronuclei (Arieli Y, Arieli R, and Marx A. J Appl Physiol 92: 2596-2599, 2002). The present study examines the optimal pretreatment for this assumed crushing of gas micronuclei. Transparent prawns were subjected to various exposure times (0, 5, 10, 15, and 20 min) at an oxygen pressure of 203 kPa and to 5 min at different oxygen pressures (PO2 values of 101, 151, 203, 405, 608, and 810 kPa), before nitrogen loading at 203 kPa followed by explosive decompression. After the decompression, bubble density and total gas volume were measured with a light microscope equipped with a video camera. Five minutes at a PO2 of 405 kPa yielded maximal reduction of bubble density and total gas volume by 52 and 71%, respectively. It has been reported that 2-3 h of hyperbaric oxygen at bottom pressure was required to protect saturation divers decompressed on oxygen against decompression sickness. If there is a shorter pretreatment that is applicable to humans, this will be of great advantage in diving and escape from submarines.     

Effect of oxygen breathing on micro oxygen bubbles in nitrogen-depleted rat adipose tissue at sea level and 25 kPa altitude exposures

The standard treatment of altitude decompression sickness (aDCS) caused by nitrogen bubble formation is oxygen breathing and recompression. However, micro air bubbles (containing 79% nitrogen), injected into adipose tissue, grow and stabilize at 25 kPa regardless of continued oxygen breathing and the tissue nitrogen pressure. To quantify the contribution of oxygen to bubble growth at altitude, micro oxygen bubbles (containing 0% nitrogen) were injected into the adipose tissue of rats depleted from nitrogen by means of preoxygenation (fraction of inspired oxygen = 1.0; 100%) and the bubbles studied at 101.3 kPa (sea level) or at 25 kPa altitude exposures during continued oxygen breathing. In keeping with previous observations and bubble kinetic models, we hypothesize that oxygen breathing may contribute to oxygen bubble growth at altitude. Anesthetized rats were exposed to 3 h of oxygen prebreathing at 101.3 kPa (sea level). Micro oxygen bubbles of 500-800 nl were then injected into the exposed abdominal adipose tissue. The oxygen bubbles were studied for up to 3.5 h during continued oxygen breathing at either 101.3 or 25 kPa ambient pressures. At 101.3 kPa, all bubbles shrank consistently until they disappeared from view at a net disappearance rate (0.02 mm(2) × min(-1)) significantly faster than for similar bubbles at 25 kPa altitude (0.01 mm(2) × min(-1)). At 25 kPa, most bubbles initially grew for 2-40 min, after which they shrank and disappeared. Four bubbles did not disappear while at 25 kPa. The results support bubble kinetic models based on Fick's first law of diffusion, Boyles law, and the oxygen window effect, predicting that oxygen contributes more to bubble volume and growth during hypobaric conditions. As the effect of oxygen increases, the lower the ambient pressure. The results indicate that recompression is instrumental in the treatment of aDCS.     

Mathematical model of gas bubble evolution in a straight tube.

Deep sea divers suffer from decompression sickness (DCS) when their rate of ascent to the surface is too rapid. When the ambient pressure drops, inert gas bubbles may form in blood vessels and tissues. The evolution of a gas bubble in a rigid tube filled with slowly moving fluid, intended to simulate a bubble in a blood vessel, is studied by solving a coupled system of fluid-flow and gas transport equations. The governing equations for the fluid motion are solved using two techniques: an analytical method appropriate for small nondeformable spherical bubbles, and the boundary element method for deformable bubbles of arbitrary size, given an applied steady flow rate. A steady convection-diffusion equation is then solved numerically to determine the concentration of gas. The bubble volume, or equivalently the gas mass inside the bubble for a constant bubble pressure, is adjusted over time according to the mass flux at the bubble surface. Using a quasi-steady approximation, the evolution of a gas bubble in a tube is obtained. Results show that convection increases the gas pressure gradient at the bubble surface, hence increasing the rate of bubble evolution. Comparing with the result for a single gas bubble in an infinite tissue, the rate of evolution in a tube is approximately twice as fast. Surface tension is also shown to have a significant effect. These findings may have important implications for our understanding of the mechanisms of inert gas bubbles in the circulation underlying decompression sickness.     

A new gas lesion syndrome in man, induced by "isobaric gas counterdiffusion".

Normal men have been found to develop pruritis and gas bubble lesions in the skin, and disruption of vestibular function, when breathing nitrogen or neon with oxygen while surrounded by helium at increased ambient pressure. This phenomenon, which occurs at stable ambient pressures, at 1 or many ATA, has been designated the "isobaric gas counterdiffusion syndrome." In a series of analyses and experiments in vivo and in vitro the cause of the syndrome has been established as due to gas accumulation and development of gas bubbles in tissues as a result of differences in selective diffusivities, for various respired and ambient gases, in the tissue substances between capillary blood and the surrounding atmosphere. The phenomenon here described in man is an initial stage of a process shown later in animals to progress to continuous, massive, lethal, intravascular gas embolization.     

Effect of a short-acting NO donor on bubble formation from a saturation dive in pigs.

It has previously been reported that a nitric oxide (NO) donor reduces bubble formation from an air dive and that blocking NO production increases bubble formation. The present study was initiated to see whether a short-acting NO donor (glycerol trinitrate, 5 mg/ml; Nycomed Pharma) given immediately before start of decompression would affect the amount of vascular bubbles during and after decompression from a saturation dive in pigs. A total of 14 pigs (Sus scrofa domestica of the strain Norsk landsvin) were randomly divided into an experimental (n = 7) and a control group (n = 7). The pigs were anesthetized with ketamine and alpha-chloralose and compressed in a hyperbaric chamber to 500 kPa (40 m of seawater) in 2 min, and they had 3-h bottom time while breathing nitrox (35 kPa O(2)). The pigs were all decompressed to the surface (100 kPa) at a rate of 200 kPa/h. During decompression, the inspired Po(2) of the breathing gas was kept at 100 kPa. Thirty minutes before decompression, the experimental group received a short-acting NO donor intravenously, while the control group were given equal amounts of saline. The average number of bubbles seen during the observation period decreased from 0.2 to 0.02 bubbles/cm(2) (P < 0.0001) in the experimental group compared with the controls. The present study gives further support to the role of NO in preventing vascular bubble formation after decompression.     

Tolerance of bacteria to extreme gas supersaturations.

Bacteria without (Escherichia coli and Corynebacterium xerosis) and with gas vacuoles (Microcyclus aquaticus) were saturated with Ar or N₂ gas at pressures up to 300 atm and then rapidly decompressed. The resulting intracellular gas supersaturations had no effect on the viability of the bacteria except when the gas vesicles were purposely kept intact by slow pressurization rates. Thus no gas bubbles form within the cells even at these extreme supersaturations. This contradicts earlier interpretations of the cause of the disruptive effect on various cells by gas pressurization and decompression.     

Observation and quantification of gas bubble formation on a mechanical heart valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.     

A model for influence of exercise on formation and growth of tissue bubbles during altitude decompression

In response to exercise performed before or after altitude decompression, physiological changes are suspected to affect the formation and growth of decompression bubbles. We hypothesized that the work to change the size of a bubble is done by gas pressure gradients in a macro- and microsystem of thermodynamic forces and that the number of bubbles formed through time follows a Poisson process. We modeled the influence of tissue O(2) consumption on bubble dynamics in the O(2) transport system in series against resistances, from the alveolus to the microsystem containing the bubble and its surrounding tissue shell. Realistic simulations of experimental decompression procedures typical of actual extravehicular activities were obtained. Results suggest that exercise-induced elevation of O(2) consumption at altitude leads to bubble persistence in tissues. At the same time, exercise-enhanced perfusion leads to an overall suppression of bubble growth. The total volume of bubbles would be reduced unless increased tissue motion simultaneously raises the rate of bubble formation through cavitation processes, thus maintaining or increasing total bubble volume, despite the exercise.