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Endothelium and Diving
Abstract
Diving, as a method for exploring and exploiting the underwater world, requires equipment and methods that have been available for only about 200 years. The first practical procedures for decompression from dives were developed by Boycott and colleagues (1) at the beginning of the 1900s. With the development of the self-contained underwater breathing apparatus (SCUBA) in the 1940s, divers were able to swim freely, and saturation diving methods developed in the 1950s allowed divers to stay under pressure for weeks. Divers may reach a depth of 50 m using air as their breathing gas. Beyond 50 m, it is necessary to employ helium mixtures, owing to depth-related toxic effects of nitrogen and excessive oxygen tension. For a review of the general effects of diving on the organism, the reader is referred to Bennett and Elliott (2). The major risk of injury associated with diving relates to decompression upon return to the surface. Gas will be taken up in solution by the tissues of the body during the dive proportional to the depth, and the uptake is exponentially related to the time spent under pressure. The gas content at the bottom is given by Henry's law: where P is partial pressure of the gas and L is the solubility coefficient. Upon returning to the surface, this excess gasmust be eliminated. Gas elimination follows an exponential curve, with time constants determined by blood flow to the different tissues. If pressure is reduced faster than gas can be eliminated, the partial pressure of gas in the tissue will be higher than the environmental pressure.
... It is speculated to be a combination of its vasodilating effect and its ability to reduce the hydrophobicity and hence the adhesiveness of bubble precursors to the endothelium surface [2]. The endothelium is the innermost lining of blood vessels and is the first tissue to come in contact with the intravascular bubbles formed during decompression [6]. Endothelial cells, which make up the endothelium, are therefore a suitable in vitro model for investigating the effects of diving and hyperoxia on NO generation. ...
Nitric oxide (NO) may protect against gas bubble formation and risk of decompression sickness. We have previously shown that the crucial co-factor tetrahydrobiopterin (BH4) is oxidized in a dose-dependent manner when exposed to hyperoxia similar to diving conditions but with minor effects on the NO production by nitric oxide synthase. By manipulating the intracellular redox state, we further investigated the relationship between BH4 levels and production of NO in human endothelial cells (HUVECs). HUVECs were cultured with and without ascorbic acid (AA) and the glutathione (GSH) synthesis inhibitor buthionine sulfoximine, prior to hyperoxic exposure. The levels of biopterins and GSH were determined in cell lysates while the production of NO was determined in intact cells. Omitting AA resulted in a 91% decrease in BH4 levels (0.49 ± 0.08 to 0.04 ± 0.01 pmol/10⁶ cells, p⟨0.001) at 20 kPa oxygen (O2), and 88% decrease (0.24 ± 0.03 to 0.03 ± 0.01 pmol/10⁶ cells, p=0.01) after exposure to 60 kPa O2. The NO generation was decreased by 23% (74.5 ± 2.2 to 57.3 ± 5.6 pmol/min/mg protein, p⟨0.001) at 20 kPa O2, but no significant change was observed at 60 kPa O2. GSH depletion had no effects on the NO generation. No correlation was found between NO generation and the corresponding intracellular BH4 concentration (p=0.675, r=-0.055) or the BH4 to BH2 ratio (p=0.983, r=0.003), determined across 18 in vitro experiments. Decreased BH4 in HUVECs, due to hyperoxia or lack of ascorbic acid, does not imply corresponding decreases in NO generation.
... Endothelial cells form the endothelium -the innermost lining of blood vessels. It is the endothelium that first comes into contact with bubbles formed during decompression [11]. Endothelial NO is produced within endothelial cells, mainly by the isoform eNOS [7]. ...
Purpose:
Nitric oxide (NO) has been shown to protect against bubble formation and the risk of decompression sickness. We hypothesize that oxidation of tetrahydrobiopterin (BH4) leads to a decreased production of NO during simulated diving.
Methods:
Human umbilical vein endothelial cells (HUVEC) were exposed to hyperoxia or simulated diving for 24 hours. The levels of biopterins (BH4, BH2 and B) were determined by LC-MS/MS, and the production of NO by monitoring the conversion of L-arginine to L-citrulline.
Results:
Exposure to hyperoxia decreased BH4 in a dose-dependent manner; by 48 ± 15% following exposure to 40 kPa O2 (P⟨0.001 vs. control at 20 kPa O2), and 70 ± 16% following exposure to 60 kPa O2. Exposure to 40 kPa O2 decreased NO production by 25 ± 9%, but there was no further decrease when increasing oxygen exposure to 60 kPa (25 ± 10%). No additional effects of simulated diving were observed, indicating no additive or synergistic effects of hyperbaria and hyperoxia on the BH4 level or NO generation.
Conclusion:
NO generation in intact human endothelial cells was decreased by simulated diving, as well as by hyperoxic exposure, while BH4 levels seem to be affected only by hyperoxia. Hence, the results suggest that BH4 is not the sole determinant of NO generation in HUVEC.
... Heat shock proteins are involved in folding and unfolding of other proteins (13) and are expressed in response to various stressors such as hyperoxia, hypoxia, heat, cold, exercise, some heavy metals and drugs, and many of these factors are involved in diving (14). HSP60, a member of this family, is highly expressed in vitro in endothelial cells. ...
Skin and ear infections, primarily caused by Pseudomonas aeruginosa (P. aeruginosa), are recurrent problems for saturation divers, whereas infections caused by P. aeruginosa are seldom observed in healthy people outside saturation chambers. Cystic fibrosis (CF) patients suffer from pulmonary infections by P. aeruginosa, and it has been demonstrated that CF patients have high levels of autoantibodies against Heat shock protein 60 (HSP60) compared to controls, probably due to cross-reacting antibodies induced by P. aeruginosa. The present study investigated whether rats immunised with P. aeruginosa produced autoantibodies against their own HSP60 and whether diving influenced the level of circulating anti-HSP60 antibodies.
A total of 24 rats were randomly assigned to one of three groups ('immunised', 'dived' and 'immunised and dived'). The rats in group 1 and 3 were immunised with the bacteria P. aeruginosa, every other week. Groups 2 and 3 were exposed to simulated air dives to 400 kPa (4 ata) with 45 min bottom time, every week for 7 weeks. Immediately after surfacing, the rats were anaesthetised and blood was collected from the saphenous vein. The amount of anti-HSP60 rat antibodies in the serum was analysed by enzyme linked immunosorbent assay.
The immunised rats (group 1) showed a significant increase in the level of autoantibodies against HSP60, whereas no autoantibodies were detected in the dived rats (group 2). The rats both immunised and dived (group 3) show no significant increase in circulating autoantibodies against HSP60. A possible explanation may be that HSP60 is expressed during diving and that cross-reacting antibodies are bound.
... Th e fi rst part of our study will be a pilot involving a relatively small number of animals. We will extract RNA from the endothelial lining of rat aortas following dives using well FROM BUBBLES TO ALTERED GENE EXPRESSION It is well documented that gas bubbles occur both on the arterial and venous side of the circulation following decompression from a dive, and we have increasing knowledge of the damaging eff ects these bubbles have on the endothelium (Brubakk et al., 2007). Changes in the physical or chemical surroundings of any organism, such as those experienced on ascent from diving, are likely to trigger adaptive changes in gene expression patterns. ...
Changes in the physical or chemical surroundings of any organism, such as the pressure
changes experienced on ascent from diving, are likely to trigger adaptive changes in gene expression
patterns. However, the molecular responses are not necessarily uniform. Considerable variation
in both constitutive and inducible gene expression is seen between and even within individuals at
diff erent times. Th e expression of a specifi c gene is regulated both by the DNA-sequence of the gene
itself and by epigenetic modifi cations that alter a gene’s availability for transcription without changing
the sequence of DNA. A diver’s capacity for adaptation to decompression may vary according
to his or her individual genetic and epigenetic make up. Some of the genes involved in nitric oxide
homeostasis in the endothelium display considerable individual diff erences in activity, which is
known to trigger susceptibility to a number of diseases. Th is could indicate a link between endothelial
dysfunction and decompression sickness (DCS) making the endothelium a highly interesting
organ to study. We plan to examine alterations in genetic expression profi les of RNA in vascular
endothelium in situ following decompression using rats as experimental models. We further plan to
look for epigenomic variations between individual animals by examining the methylation pattern
of cytosine in genomic DNA. By doing so, we hope to gain knowledge of genetic responses to the
physical and biochemical changes experienced in diving. Ultimately the goal is to be able to better
predict individual risk of developing DCS, and possibly also to prevent or relieve disease by means
of preconditioning or pharmacological intervention.
... Bubbles are believed mainly to form on the venous side of the circulation (9) but may become arterial bubbles through shunts in the lungs or the heart. Vascular bubbles may lead to detrimental effects on the organism, both by endothelial dysfunction and ischemic effects in, for instance, brain tissue (3). ...
Diving and decompression performed under immersed conditions have been shown to reduce cardiac function. The mechanisms for these changes are not known. The effect of immersion before a simulated hyperbaric dive on cardiomyocyte function was studied. Twenty-three rats were assigned to four groups: control, 1 h thermoneutral immersion, dry dive, and 1 h thermoneutral immersion before a dive (preimmersion dive). Rats exposed to a dive were compressed to 700 kPa, maintained for 45 min breathing air, and decompressed linearly to the surface at a rate of 50 kPa/min. Postdive, the animals were anesthetized and the right ventricle insonated for bubble detection using ultrasound. Isolation of cardiomyocytes from the left ventricle was performed and studied using an inverted fluorescence microscope with video-based sarcomere spacing. Compared with a dry dive, preimmersion dive significantly increased bubble production and decreased the survival time (bubble grade 1 vs. 5, and survival time 60 vs. 17 min, respectively). Preimmersion dive lead to 18% decreased cardiomyocyte shortening, 20% slower diastolic relengthening, and 22% higher calcium amplitudes compared with controls. The protein levels of the sarco-endoplasmic reticulum calcium ATPase (SERCA2a), Na+/Ca2+ exchanger (NCX), and phospholamban phosphorylation in the left ventricular tissue were significantly reduced after both dry and preimmersion dive compared with control and immersed animals. The data suggest that immersion before a dive results in impaired cardiomyocyte and Ca2+ handling and may be a cellular explanation to reduced cardiac function observed in humans after a dive.
... Vascular gas bubbles are thought to evolve in nearly all decompressions and may cause damage to or reduce the function of the endothelium resulting in decompression sickness (DCS) (Brubakk et al. 2007 ). A study in experienced divers showed that arterial endothelial function, evaluated by flow-mediated dilatation (FMD), was reduced after a single air dive where very few venous gas bubbles could be detected in the pulmonary artery (Brubakk et al. 2005). ...
Nitric oxide (NO) seems to be related to bubble formation and endothelial dysfunction resulting in decompression sickness. Bubble formation can be affected by aerobic exercise or manipulating NO. A prior heat stress (HS) has been shown to confer protection against decompression sickness in rats. An important question was if the oxidative environment experienced during diving limits the availability of the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4). Human endothelial cells were used to investigate how HS and simulated diving affected NO synthesis and defense systems such as heat shock protein 70 (HSP70) and glutathione (GSH). BH4 was measured using a novel LC-MS/MS method and NOS by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Increased pO(2) reduced BH4 levels in cells in a dose-dependent manner independently of high pressure. This effect may result in decreased generation of NO by NOS. The BH4 decrease seemed to be abolished when cells were exposed to HS prior to hyperoxia. NOS enzyme was unaffected by increased pO(2) but substantially reduced after HS. The BH4 level seemed to a minor extent to be dependent upon GSH and probably to a higher degree dependent on other antioxidants such as ascorbic acid. A simulated dive at 60 kPa O(2) had a potentiating effect on the heat-induced HSP70 expression, whereas GSH levels were unaffected by hyperoxic exposure. HS, hyperoxia, and dive affected several biochemical parameters that may play important roles in the mechanisms protecting against the adverse effects of saturation diving.
Background and Purpose: Leukocyte cell surface adhesion molecule CD11b, decorated with CD15s, plays a critical role in the regulation of P 2 integrin function during neutrophile endothelial transmigration. Hyperbaric oxygenation reduces neutrophil-endothelial cell adhesion, which is mediated by Mac-1 (CD11b/CD18) beta(2)-integrin. Materials and Methods: This study investigated the expression of CD15 and CD15s, on leukocytes following repeated trimix dives in two series: in the first series 7 divers performed 6 consecutive dives from 55-80 m, while in the second series 7 divers performed 3 consecutive dives from 63-65 m. A more intense dive profile was used in the second series, as can be seen from the longer total dive time. Five divers took part in each of the two series. CD15 and CD15s were determined before and after the 1st and the last dive. Results and Conclusions: Leukocyte subpopulations were not elevated after either the first or last dives in series I Only CD15+CD15s+ granulocytes were significantly decreased after the 1st dive. In the second series the monocyte proportion was increased and lymphocytes decreased within the total leukocyte population, while CD15s+ monocytes and CD14+CD15s+ granulocytes were elevated after the 1st dive. CD15+CD14+ granulocytes were decreased after the 1st and the last dive in the second series, while CD15s+ granulocytes were decreased only after the last dive in the second series. The current findings of decreased endothelial selectin ligand CD15s expression on CD15+ granulocytes after certain dives point to the role of this subpopulation in the endothelial damage prevention.
A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for the quantification of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and biopterin (B) in human umbilical vein endothelial cells (HUVECs). Freshly prepared cell samples were treated with a mixture consisting of 0.2M trichloroacetic acid (TCA) and a cocktail of various antioxidants in order to precipitate proteins and other cellular components and to stabilize red/ox conditions in the lysates. Chromatography of the cell lysates was performed on a Poroshell 120 SB-C18 column (2.7μm, 150×2.1mm) using a stepwise gradient elution made from two mobile phases. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) at positive ion mode. Total chromatographic run time was 23min. The method was validated for analysis in HUVECs, and the limits of quantification were 1nM for BH4 and BH2 and 2.5nM for B. Standard curves were linear in the concentration ranges of 1 to 100nM for BH4 and BH2 and 2.5 to 100nM for B. The current study reports a novel method for the simultaneous and direct quantification of BH4, BH2, and B in a single injection.
Although decompression procedures have been improved over the years, decompression still remains a significant problem in diving. While there is universal agreement that the basic problem of decompression is gas coming out of solution, forming bubbles when pressure is reduced, the exact mechanism of decompression injury is not known. Furthermore, the wide variety of clinical symptoms and the significant difference in individual susceptibility makes identification of the mechanisms involved difficult. Using ultrasound, vascular gas bubbles have been detected in most decompressions, and these bubbles can act on the endothelial lining of blood vessels resulting in impaired endothelial function. Normal endothelial function is a major indicator of cardiovascular health and thus a reduction in vascular bubble formation and hence the risk of endothelial injury is an important goal in decompression. Even if vascular gas bubbles may not be the only adverse effect of decompression, vascular gas bubbles and their adverse effects on the endothelium may be a useful model for decompression injury. This review claims that endothelial dysfunction may be a possible main mechanism for neurological decompression injuries and describes some of the effects of vascular gas bubbles on the endothelium. Furthermore, as the formation of vascular gas bubbles can be significantly influenced by physical exercise and the use of nitric oxide, a novel approach to reducing the risk of decompression injury is suggested.
Heat stress prior to diving has been shown to confer protection against endothelial damage due to decompression sickness.
Several lines of evidence indicate a relation between such protection and the heat shock protein (HSP)70 and HSP90 and the
major cellular red-ox determinant, glutathione (GSH). The present study has used human endothelial cells as a model system
to investigate how heat stress and simulated diving affect these central cellular defense molecules. The results demonstrated
for the first time that a simulated dive at 2.6MPa (26bar) had a potentiating effect on the heat-induced expression of HSP70,
increasing the HSP70 concentration on average 54 times above control level. In contrast, a simulated dive had no significant
potentiating effect on the HSP90 level, which might be due to the higher baseline level of HSP90. Both 2 and 24-h dive had
similar effects on the HSP70 and HSP90, suggesting that the observed effects were independent of duration of the dive. The
rapid HSP response following a 2-h dive with a decompression time of 5min might suggest that the effects were due to compression
or pressure per se rather than decompression and may involve posttranslational processing of HSP. The exposure order seemed
to be critical for the HSP70 response supporting the suggestion that the potentiating effect of dive was not due to de novo
synthesis of HSP70. Neither heat shock nor a simulated dive had any significant effect on the intracellular GSH level while
a heat shock and a subsequent dive increased the total GSH level approximately 62%. Neither of these conditions seemed to
have any effect on the GSH red-ox status.
KeywordsEndothelial cells-Diving-Decompression-Heat shock protein-Glutathione
Decompression sickness (DCS) may result from damage to the endothelium caused by the gas bubbles formed during decompression and may be related to nitric oxide (NO) production by nitric oxide synthase (NOS). Heat stress prior to diving has been shown to protect animals from DCS, and by simulating this treatment in human endothelial cells (HUVEC) we have shown that a simulated dive performed subsequent to a heat stress potentiated the heat-induced expression of HSP70 and increased the level of the antioxidant glutathione (GSH). Since operational saturation diving is performed at an increased oxygen level, HUVEC have been exposed to heat stress and simulated diving at 40 kPa O(2), comparing the response on HSP70, HSP90 and GSH level to the effects previously observed at 20 kPa O(2). In addition, we wanted to investigate the effect on both endothelial NOS (eNOS) protein and enzymatic activity. The present results showed that a heat stress (45°C, 1 h) decreased the NOS activity and the protein markedly. Hyperoxia (40 kPa) alone or a dive either at 20 or 40 kPa O(2),had no effects on NOS activity or protein. At 40 kPa O(2) a simulated dive after heat stress potentiated the HS-induced HSP70 response, whereas the heat-induced HSP90 response decreased. GSH levels were found to be inversely related to NOS activity and protein expression, and might be explained by a possible post-translational regulation by glutathionylation of eNOS protein. The results add to the limited knowledge of these critical factors in cellular defence mechanisms that can prevent injury during decompression.
A key process in the pathophysiological steps leading to decompression sickness (DCS) is the formation of inert gas bubbles. The adverse effects of decompression are still not fully understood, but it seems reasonable to suggest that the formation of venous gas emboli (VGE) and their effects on the endothelium may be the central mechanism leading to central nervous system (CNS) damage. Hence, VGE might also have impact on the long-term health effects of diving. In the present review, we highlight the findings from our laboratory related to the hypothesis that VGE formation is the main mechanism behind serious decompression injuries. In recent studies, we have determined the impact of VGE on endothelial function in both laboratory animals and in humans. We observed that the damage to the endothelium due to VGE was dose dependent, and that the amount of VGE can be affected both by aerobic exercise and exogenous nitric oxide (NO) intervention prior to a dive. We observed that NO reduced VGE during decompression, and pharmacological blocking of NO production increased VGE formation following a dive. The importance of micro-nuclei for the formation of VGE and how it can be possible to manipulate the formation of VGE are discussed together with the effects of VGE on the organism. In the last part of the review we introduce our thoughts for the future, and how the enigma of DCS should be approached.
Abstract: Following decompression, there is a considerable difference in detected venous gas emboli (VGE) between individuals; this study is an exploration of the role surface tension may play in the differences. We measured serum surface tension in 26 anesthetized pigs before (predive) and after a dive (postdive) to 300 kPa for 3 hours. Gas bubbles in the pulmonary artery were monitored continuously from the beginning of decompression and continued throughout 120 minutes after the dive. Maximum bubble levels were reached about 30 minutes after surfacing. Predive surface tension was significantly higher than postdive values (66.8 +/- 1.0 dynes/cm, n=26) vs. (66.4 +/- 1.0 dynes/cm, n=26). We found a significant negative correlation between predive surface tension and the number of bubbles that were generated as a result of the dive. A significant negative correlation was also observed between the generated vascular bubbles and postdive surface tension. We conclude that small surface tension differences between individuals may influence vascular bubble formation, and that formation of VGE may itself lower surface tension.
Using our recently described model of acute lung injury in rats after systemic activation of complement by cobra venom factor (CVF), we demonstrated that pretreatment of animals with human milk apolactoferrin (in its native or derivatized form), but not iron-saturated lactoferrin, provides significant protection against complement- and neutrophil-mediated lung injury. The synthetic iron chelator deferoxamine mesylate also affords protection from lung injury. The protective effects of apolactoferrin are not related to a blocking of CVF-induced complement activation. We also demonstrated that infusion of ionic iron, especially Fe3+, greatly potentiates lung vascular injury after systemic complement activation. Finally, protection from lung injury occurs in animals pretreated with the potent scavenger of hydroxyl radicals (OH.), dimethyl sulfoxide. Based on transmission electron microscopy, CVF-treated rats show leukoaggregates and endothelial cell destruction in interstitial pulmonary capillaries, along with intraalveolar hemorrhage and fibrin deposition. In animals protected with apolactoferrin, deferoxamine mesylate, or dimethyl sulfoxide, the morphological studies reveal leukoaggregates but no endothelial cell damage, hemorrhage, or fibrin deposition. These data support the concept that tissue injury that is complement and neutrophil dependent may be related to generation of OH. derived from H2O2 after leukocytic activation.
Plasma levels of the anaphylatoxin C5a were measured in 19 divers performing repeated air dives. Blood samples were collected immediately before the first dive and 2 h after the first and the second or third dive. Serum obtained at the same times was subjected to complement activation in vitro by air bubbles. Six divers developed symptoms of decompression sickness (DCS). Most intravascular bubbles were observed in divers with the lowest plasma levels of C5a. Postdive plasma levels of C5a did not increase compared with predive levels, nor were postdive levels significantly different after two or three dives compared with the first dive. Repeated dives did not influence the amounts of C5a generated in vitro. Neither plasma levels of C5a nor C5a generated in vitro were significantly different in divers who experienced symptoms of DCS vs. divers without symptoms of DCS. We conclude that plasma level of C5a and measurement of C5a generation in vitro cannot be used to predict DCS.
Complement activation induced by air bubbles in rabbit and human sera was studied by measuring the generation of anaphylatoxin des-Arg-C5a. des-Arg-C5a was quantified by sandwich enzyme-linked immunosorbent assays based on neoepitope-specific anti-des-Arg-C5a monoclonal antibodies. Air bubbles were continuously introduced to serum via a calibrated microflowmeter, and the serum was incubated at 37 degrees C for 30 min. Air bubbles clearly generated increased amounts of des-Arg-C5a compared with corresponding levels in control serum, and a dose-dependent effect was also noted. Strong positive correlations between des-Arg-C5a concentrations in control sera and sera incubated with air bubbles at a flow of 0.5 ml/min were found. To study variation over time, serum was obtained at regular intervals from six rabbits and from six healthy humans during 66- and 196-day periods, respectively. A pronounced intraindividual variability over time was thus observed. The reason for the large variability is at present unknown. We conclude that the sensitivity of complement to activation by air bubbles is not an inherent, static feature of the complement system of an individual. Therefore single-point analysis of complement activation by air bubbles appears to be an inappropriate parameter by which to differentiate a "sensitive" or "insensitive" complement system between individuals.
Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe.
During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver.
The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.
Previous studies have shown that gas bubbles activate the complement system in vitro, generating C5a. The effect of anti-C5a monoclonal antibody 4B1C11 in preventing endothelial damage caused by decompression in the pulmonary artery of the rabbit was examined. The endothelial response was measured using tension measurements in the blood vessel wall. The mean bubble count for all rabbits (n = 24) was 4.2+/-3.1 bubbles x cm(-2), and ranged from 0 to 15 bubbles x cm(-2). Animals with many bubbles showed significantly more vascular damage than those with fewer bubbles. Anti-C5a monoclonal antibody could not prevent endothelial damage than that occurred after exposure to this level of gas bubbles. The maximum number of gas bubbles present is important for the endothelial damage. We speculate that the endothelial damage observed was mainly mechanical. A possible beneficial effect of anti-C5a antibody can thus be masked at a high degree of bubble generation. This study, together with a previous paper, demonstrates that gas bubbles cause endothelial damage from decompression both in the pig and in the rabbit.
Pulmonary function was measured in 152 professional saturation divers and in a matched control group of 106 subjects. Static lung volumes, dynamic lung volumes and flows, transfer factor for carbon monoxide (T1CO), transfer volume per unit alveolar volume (KCO), delta-N2, and closing volume (CV) were measured and compared with reference values from recent Scandinavian studies, British submariners, and the European Community for Coal and Steel (ECCS) recommended reference values. Diving exposure was assessed as years of diving experience, total number of days in saturation and depth, and as the product of days in saturation and mean depth. Divers had significantly lower values for forced expired volume in one second (FEV1), FEV1/forced vital capacity (FVC) ratio, FEF25-75%, FEF75-85%, FEF50%, FEF75%, T1CO, and KCO compared with the controls and a significantly higher CV. There was a positive correlation between diving exposure and CV, whereas the other variables had negative correlations with diving exposure. Values for the control group were not different from the predictive values of Scandinavian reference studies or British submariners, although the ECCS standard predicted significantly lower values for the lung function variables both in divers and the control group. The pattern of the differences in lung function variables between the divers and controls is consistent with small airways dysfunction and with the transient changes in lung function found immediately after a single saturation dive. The association between reduced pulmonary function and previous diving exposure further indicates the presence of cumulative long term effects of diving on pulmonary function.
A detailed investigation using light and electron microscopes is reported. The objects identified as nuclei are found in both distilled water and gelatin, and they resemble ordinary gas bubbles. Radii are on the order of 1 mu m or less and can be three orders of magnitude smaller. The number density decreases exponentially with increasing radius. A gas filling is implied by the observation that nuclei expand when the pressure decreases and contract when it rises. The occurrence of nuclear clusters and of binary or osculating nuclei suggests that stabilization is achieved via surfactant films. The monolayer thickness of these films, estimated from the thicknesses of bilayer septa, is (20 plus or minus 7) A. Many nuclei are embedded in reservoirs of surface-active material made visible by osmium-tetroxide straining. Electron microscope sections are hardened by infiltrating gelatin with epoxy. Reservoirs, encased in epoxy, form microbubble chambers in which the coalescence and bursting of nuclei can be studied during extended exposures to the electron beam.
Recently a new cavitation model was proposed in which bubble formation in aqueous media is initiated by spherical gas nuclei stabilized by surface-active membranes of varying gas permeability. By tracking the changes in nuclear radius that are caused by increases or decreases in ambient pressure, the varying-permeability model has provided precise quantitative descriptions of several bubble counting experiments carried out with supersaturated gelatin. The model has also been used to calculate diving tables and to predict levels of incidence for decompression sickness in a variety of animal species. The model equations, in their present form, are essentially static and can be derived by requiring mechanical or chemical equilibrium at each setting in a rudimentary pressure schedule. The time dependence of the evolution of an individual nucleus from one equilibrium state to another is examined, and a statistical process by which the equilibrium size distribution of an entire population of nuclei may be generated or regenerated is then investigated.
The solubility of air bubbles is observed in a variety of experiments. Freely rising bubbles exhibit a solution rate more than twice that of bubbles which are stationary: i.e., trapped on the walls of the container. The theory of solution of stationary bubbles is given but the theory of a free bubble is difficult and has not been solved. Stationary bubble observations lead to a determination of the coefficient of diffusivity: 2.9×10-5 cm2/sec at 27°C; the temperature dependence is considerable, obeying Einstein's equation relating diffusion and viscosity. The effect of surface contamination on diffusion is analyzed; bubble solution in the presence of contaminants is not greatly altered. Experiments are described showing that air bubbles of dimension less than 1μ lodged on hydrophobic particles are not soluble and can exist indefinitely. It is shown that hydrophobic particle residues frequently remain after bubble solution, even in relatively pure water. These residues function as bubble nuclei for boiling or for cavitation.
Eucaryotic unicellular (a yeast, a cellular slime mold, and various protozoans) and two multicellular (aschelminths) microorganisms were saturated with gas at high pressures and rapidly decompressed. No effect was observed with pressures of argon up to 125 atm, nitrogen up to 175 atm, and helium up to 350 atm, showing that the induced gas supersaturations did not cause intracellular bubbles to form. With 25--50 atm higher gas pressures, the decompression usually produced killing and cell rupture, although differences in tolerances existed among the various organisms. Substantial fractions of the populations survived gas supersaturations well above the threshold values for massive spontaneous nucleation of bubbles in the water. When killing occurred, external rather than internal bubbles appeared to be the cause. Even with the 300 atm argon or nitrogen pressures, yeast cells were unaffected, apparently because of the external protection provided by their cell wall. It is concluded that the gas supersaturations required for intracellular formation of bubbles generally are at least equal to and probably higher than the bubble nucleation thresholds for water or aqueous solutions.
C5a is an 11,000-Da complement-derived inflammatory glycoprotein that has been shown to mediate inflammatory reactions in vitro as well as in vivo in human skin. The C5a degradation product, C5a des Arg, is rapidly formed after exposure of C5a to serum carboxypeptidase N and may represent the relevant C5-derived inflammatory peptide in vivo. To examine the biologic activity of human C5a des Arg in vivo and to compare it with that seen with human C5a, we purified and characterized homogeneous preparations of human C5a and C5a des Arg and injected them intradermally into seven normal volunteers. C5a des Arg exhibited biochemical and biologic properties in vitro that were different from those of C5a. When injected into human skin, C5a des Arg was less potent than C5a, in respect to both minimal dose eliciting wheal and flare reactions and maximal wheal and flare elicited at a given dose, but C5a des Arg still elicited cutaneous wheal and flare reactions at physiologically relevant concentrations. Histologically, C5a des Arg skin test sites showed dense polymorphonuclear neutrophil-rich infiltrates associated with leukocytoclasis, dermal mast cell degranulation, and endothelial cell swelling. These were virtually indistinguishable from reactions elicited by C5a and occurred with concentrations attainable in vivo. Cutaneous wheal and flare reactions elicited by either C5a or C5a des Arg were partially inhibited by H1 antihistamines but were unaffected by selected nonsteroidal anti-inflammatory agents.
The consequences of complement activation and the symptoms of decompression sickness are similar. Consequently, the relation between the sensitivity of individuals to complement activation by air bubbles and their susceptibility to decompression sickness has been examined. Plasma samples from 34 individuals were incubated with air bubbles, and the concentration of the fluid phase metabolites of complement activation C3a, C4a, and C5a were measured with radioimmunoassays. It was found that both the anaphylatoxins C3a and C5a were produced by the presence of air bubbles but that the anaphylatoxin C4a was not. This finding indicates that air bubbles activate the complement system by the alternate pathway. One group of individuals was found to be particularly sensitive to complement activation by this pathway. They produced 3.3 times more C3a and 5.3 times more C5a in their plasma samples incubated with air bubbles as did the other group. Sixteen individuals were subjected to a series of pressure profiles that were severe enough to produce bubbles in their circulatory system that could be detected by Doppler ultrasonic monitoring. The group of individuals that had been identified as being more sensitive to complement activation by the alternate pathway was also found to be more susceptible to decompression sickness.
This review focuses upon the endothelium of the cerebral microcirculation. We will discuss the endothelium in three contexts: the interaction of endothelium with platelets; the role of endothelium as a source of mediators controlling vascular tone; and the role of endothelium in maintaining permeability barriers residing in cerebral blood vessels. With respect to the interaction of endothelium with platelets, and the role of endothelium in modulating microvascular tone, we will stress that endothelial denudation in cerebral microvessels is not required in order to initiate other platelet aggregation or the loss of endothelial mediators of tone. Minimal morphologic alterations of endothelial cells may be the only structural indication of profound loss of normal properties. It is apparent that microvascular endothelium in general, and brain microvascular endothelium in particular, possesses properties which distinguish it from endothelium of large blood vessels. The consequence of even minor damage to endothelium of brain microvessels has profound consequences leading to platelet aggregation, loss of normal control of vascular diamater, and alterations in permeability.
Substances that stimulate smooth muscle have been previously implicated in the pathogenesis of decompression sickness. This concept was strongly supported by the demonstration that compounds that combine activities against histamine, bradykinin, and serotonin prevent or ameliorate decompression sickness. This communication deals with the prophylactic effect of cyproheptadine (Periactin), a drug exhibiting such pharmacologic properties. More than 500 obese mice were used. Experimental groups, subcutaneously injected with cyproheptadine (0.5-40 mg/kg) prior to compression, and corresponding control animals were simultaneously subjected to 75 psig air pressure for 6 h and then rapidly decompressed. Most control animals exhibited signs of decompression sickness (chokes, scratching, twitching, convulsions, paralysis) and died. Gross and histologic examination revealed gas bubbles in vessels and tissues, perivascular edema, and other changes. In cyproheptadine-treated animals the incidence and severity of clinical manifestations and pathologic alterations were reduced, and mortality was marked decreased. Statistically significant results were obtained with doses of 2.5-10.0 mg/kg. The 5-mg/kg does lowered mortality by 45.9%. These results support the proposed pathogenetic concept and suggest a potential preventive treatment for human subjects.
During development of a pig model of neurologic decompression illness (DCI) we noted that treadmill-trained pigs seemed less likely to develop DCI than sedentary pigs. The phenomenon was formally investigated. Twenty-four immature, male, castrated, pure-bred Yorkshire swine were conditioned by treadmill running, while 34 control pigs remained sedentary. All pigs (weight 18.75-21.90 kg) were dived on air to 200 feet of seawater (fsw) in a dry chamber. Bottom time was 24 min. Decompression rate was 60 fsw/min. Pigs that developed neurologic DCI were treated by recompression. Pigs without neurologic signs were considered neurologically normal if they ran on the treadmill without gait disturbance at 1 and 24 h postdive. Of the 24 exercise-conditioned pigs, only 10 (41.7%) developed neurologic DCI, compared to 25 of 34 (73.5%) sedentary pigs (X2 = 5.97; P = < 0.015). Neither mean carcass density (adiposity) nor mean age were significantly different between groups. No patent foramen ovale was detected at necropsy. An additional control group of 24 pigs was dived to clarify the influence of weight. The results suggest that the risk of neurologic DCI is reduced by physical conditioning, and the effect is independent of differences in age, adiposity, and weight.
To clarify the influence of diving activity on the central nervous system, we studied 10 amateur and 10 professional deceased divers with emphasis on the presence of subacute or chronic pathologic changes in the spinal cord. Of the 10 professional divers (median age 38 yr; range 29-52; median experience in excess of 13 yr), 7 were experienced saturation divers. Five had dived to a maximum depth of 150 meters of seawater, the 2 others to 300 and 500 msw, respectively. Five of the professional divers had experienced decompression sickness. The experience of the amateur divers (median age 29 yr; range 17-51) varied from a few dives to many years of recreational diving. The spinal cords were formalin-fixated and routinely processed for neuropathologic examination, which included light microscopy after immunostaining for glial fibrillary acidic protein and monocyte-macrophage-microglial markers. The microscopic examination did not reveal previous spinal cord damage. Thus, diving activity, saturation diving to extreme depths included, does not in itself seem to lead to necrosis, degeneration, or scar formation in the human spinal cord.
Interference with the dynamics of cerebrospinal fluid may lead to loss of ependymal lining in the ventricles of the brain. The ependymal loss of the lateral ventricles under the corpus callosum at the level of the commisura anterior was measured in 21 diver brains and in a control material of 15 neurologic and non-neurologic brains. The divers were sport divers and professional divers with and without saturation exposure. The different groups were compared with respect to the mean loss of ependymal cells (in percentage). A statistically significant higher loss of ependyma was found in the total number of divers than in the controls. There was no significant difference between the group of sport divers and the control group. The largest loss of ependymal cells was found in the professional divers without saturation experience. Statistically, this loss was significantly larger than the loss of ependymal cells in the controls.
In this study, the levels of activated complement fragments C3a and C5a were measured on 11 U.S. Navy divers as they performed a 28-day saturation dive to a pressure equivalent of 1,000 feet of seawater (fsw, 31.3 atm abs). Two subjects developed symptoms consistent with the high pressure nervous syndrome (HPNS) and three were treated for type I DCS (joint pain only). These events allowed us to test two hypotheses: a) alterations in C3a or C5a levels during compression are related to the occurrence of HPNS and b) increases in complement fragments are an indicator of decompression stress associated with type I DCS. There was no correlation between changes in C3a and C5a levels during compression and the diagnosis of HPNS. Our results suggest that an increase in C3a and C5a levels during saturation diving correlates with decompression stress and the clinical diagnosis of type I DCS.
The aim of this study was to determine whether venous gas embolism after a single air dive, evaluated using precordial Doppler monitoring, was associated with alterations in spirometry, lung volumes, arterial blood gases, or pulmonary diffusing capacity for carbon monoxide (DLCO). Postdive time course monitoring of pulmonary function was undertaken in 10 professional divers exposed to absolute air pressure of 5.5 bar for 25 min in a dry walk-in chamber. The US Navy decompression table was followed. Venous bubbles were detected by precordial Doppler monitoring. Two types of decompression were used: air and 100% O2 applied for 21 min during decompression stops. Spirometry, flow-volume, and body plethysmography parameters were unchanged after the dive with air decompression (AD) as well as with O2 decompression (OD). A significant reduction in arterial PO2, on average 20 Torr, was found after the dive with AD. DLCO was decreased in all divers 20, 40, 60, and 80 min after diving with AD (P < 0.001), whereas it was not significantly decreased after diving with OD. Maximal DLCO decrease of approximately 15% occurred 20 min postdive. In AD diving, maximum bubble grade for each individual vs. maximum DLCO reduction correlated significantly (r = 0.85, P = 0.002), as well as DLCO vs. arterial PO2 (r = 0.64, P = 0.017). In conclusion, a reduction in pulmonary diffusing capacity is observed in parallel with the appearance of venous bubbles detected by precordial Doppler. We suggest that bubbles cause pulmonary microembolization, triggering a complex sequence of events that remains to be resolved. Measuring DLCO complements Doppler bubble detection in postdiving assessment of pulmonary function.
The correlation is low between the occurrence of gas bubbles in the pulmonary artery, called venous gas emboli (VGE), and subsequent decompression illness (DCI). The correlation improves when a "grade" of VGE is considered; a zero to four categorical classification based on the intensity and duration of the VGE signal from a Doppler bubble detector. Additional insight about DCI might come from an analysis of the time course of the occurrence of VGE. Using the NASA Hypobaric Decompression Sickness Databank, we compared the time course of the VGE outcome between 322 subjects who exercised and 133 Doppler technicians who did not exercise to evaluate the role of physical activity on the VGE outcome and incidence of DCI. We also compared 61 subjects with VGE and DCI with 110 subjects with VGE but without DCI to identify unique characteristics about the time course of the VGE outcome to try to discriminate between DCI and no-DCI cases. The VGE outcome as a function of time showed a characteristic short lag, rapid response, and gradual recovery phase that was related to physical activity at altitude and the presence or absence of DCI. The average time for DCI symptoms in a limb occurred just before the time of the highest fraction of VGE in the pulmonary artery. It is likely, but not certain, that an individual will report a DCI symptom if VGE are detected early in the altitude exposure, the intensity or grade of VGE rapidly increases from a limb region, and the intensity or grade of VGE remains high.
Leukocyte infiltration plays a major role in ischemia-associated organ dysfunction and damage. A crucial step for extravasation of white blood cells is binding of leukocyte beta-integrins to endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). To test for direct effects of oxygen on this process we studied ICAM-1 and VCAM-1 expression in human dermal microvascular and umbilical vein endothelial cells (EC) exposed to different oxygen tensions in the absence or presence of tumor necrosis factor-alpha (TNF-alpha). Hypoxia (95% N2-5% CO2) resulted in a downregulation of basal but not TNF-alpha-induced expression of ICAM-1 and VCAM-1. Subsequent rises in oxygen (21, 40, or 95% O2) led to marked increase of ICAM-1 and VCAM-1 cell surface and mRNA expression in both EC types, which after 16 h amounted to about one-third to one-half of maximal TNF-alpha-induced expression. This increase was greatest after 0.5-h hypoxia and was blunted with prolonged hypoxic preincubation. Exposure of cells preincubated under "normoxic" (21% O2) conditions to hyperoxia (40 or 95% O2) also enhanced expression of both adhesion molecules, but the increase was lower than in cells preexposed to hypoxia. The nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) enhanced ICAM-1 and VCAM-1 expression under basal and hypoxic conditions, but in the presence of L-NAME, levels in reoxygenated cells were not higher than basal levels. Moreover, the oxygen-induced rise could be mimicked by addition of H2O2 to normoxic cells, and the oxygen-induced expression of VCAM-1 but not of ICAM-1 was inhibited by addition of the free radical scavengers superoxide dismutase, N-acetyl-L-cysteine, and pyrrolidinedithiocarbamate. These data indicate that an increase in oxygen availability stimulates ICAM-1 and VCAM-1 expression on micro- and macrovascular EC, which may contribute to adhesion and transmigration of different leukocyte populations in ischemia-reperfusion injuries.
Recent experimental and clinical data have shown that physical training is an important therapeutic intervention in the management of patients with chronic heart failure (CHF), improving central hemodynamics and attenuating peripheral abnormalities (endothelial dysfunction and skeletal myopathy) characterizing the progression of the syndrome. Additionally, physical training seems to beneficially modulate peripheral immune responses of CHF expressed by elevated circulating proinflammatory cytokines, soluble cellular adhesion molecules and soluble apoptosis signaling molecules, resulting in improvement in exercise capacity of CHF patients. This article summarizes current knowledge about the beneficial role of physical training in CHF, as well as about traditional and novel mechanisms contributing to the physical training-induced improvement in clinical performance of CHF patients.
There is considerable variability in individual susceptibility to altitude decompression sickness (DCS). The Air Force Research Laboratory Altitude DCS Research Database consists of extensive information on 2980 altitude exposures conducted with consistent procedures and endpoint criteria. We used this database to quantify the variation in susceptibility and determine if anthropometric and/or physiologic variables could be used to predict DCS risk.
There were 240 subjects who participated in at least 4 of 70 exposure profiles in which between 5 and 95% of all subjects tested developed DCS symptoms. A subject/study ratio (SSR) was calculated by dividing the DCS experienced by a subject during all their exposures by the DCS incidence for all subjects who participated in the identical exposures. The SSR was used to identify the relative susceptibility of subjects for use in analyzing possible relationships between DCS susceptibility and the variables of height, weight, body mass index, age, percent body fat, and aerobic capacity.
The DCS incidence was 46.5% during 1879 subject-exposures by subjects exposed at least 4 times. A significant relationship existed between higher DCS susceptibility and the combination of lower aerobic capacity and greater weight (p < 0.05).
Despite a correlation, less than 13% of the variation in DCS susceptibility was accounted for by the best combination of variables, weight and VO2max.
Differences in DCS susceptibility cover a wide range and appear to be related to some anthropometric and physiologic variables. However, there was insufficient correlation to allow prediction of an individual's susceptibility.
Moving bubbles have been observed in the blood during or after decompression using ultrasonic techniques. It has been proposed that these may grow from nuclei housed on the blood vessel wall. One candidate for bubble nucleation is hydrophobic crevices. This work explores the growth of gas pockets that might exist in conical crevices and the release of bubbles from these crevices under decompression. An existing dynamic mathematical model for the stability of gas pockets in crevices [Chappell, M.A., Payne, S.J., in press. A physiological model of gas pockets in crevices and their behavior under compression. Respir. Physiol. Neurobiol.] is extended to include the behavior as the gas pocket reaches the crevice mouth and bubbles seed into the bloodstream. The behavior of the crevice bubble is explored for a single inert gas, both alone and with metabolic gases included. It was found that the presence of metabolic gases has a significant effect on the behavior under decompression and that this appears to be due to the high diffusivity of these gases.
Intravital microscopic techniques were used to examine the mechanisms underlying bradykinin-induced leukocyte/endothelial cell adhesive interactions (LECA) and venular protein leakage (VPL) in single postcapillary venules of the rat mesentery. The effects of bradykinin superfusion to increase LECA and VPL were prevented by coincident topical application of either a bradykinin-B(2) receptor antagonist, a cell-permeant superoxide dismutase (SOD) mimetic or antioxidant, or inhibitors of cytochrome P-450 epoxygenase (CYPE) or protein kinase C (PKC) but not by concomitant treatment with either SOD, a mast cell stabilizer, or inhibitors of nitric oxide synthase, cyclooxygenase, xanthine oxidase, NADPH oxidase, or platelet-activating factor. Immunoneutralizing P-selectin or intercellular adhesion molecule-1 (ICAM-1) completely prevented bradykinin-induced leukocyte adhesion and emigration but did not affect VPL. On the other hand, stabilization of F-actin with phalloidin prevented bradykinin-induced leukocyte emigration and VPL but did not alter leukocyte adhesion. These data indicate that bradykinin induces LECA in rat mesenteric venules via a B(2)-receptor-initiated, CYPE-, oxidant- and PKC-mediated, P-selectin- and ICAM-1-dependent mechanism. Bradykinin also produced VPL, an effect that was initiated by stimulation of B(2) receptors and involved CYPE and PKC activation, oxidant generation, and cytoskeletal reorganization but was independent of leukocyte adherence and emigration.