Decompressive Pathology in Cetaceans Based on an Experimental Pathological Model.

Decompression sickness (DCS) is a widely known clinical syndrome in human medicine, mainly in divers, related to the formation of intravascular and extravascular gas bubbles. Gas embolism and decompression-like sickness have also been described in wild animals, such as cetaceans. It was hypothesized that adaptations to the marine environment protected them from DCS, but in 2003, decompression-like sickness was described for the first time in beaked whales, challenging this dogma. Since then, several episodes of mass strandings of beaked whales coincidental in time and space with naval maneuvers have been recorded and diagnosed with DCS. The diagnosis of human DCS is based on the presence of clinical symptoms and the detection of gas embolism by ultrasound, but in cetaceans, the diagnosis is limited to forensic investigations. For this reason, it is necessary to resort to experimental animal models to support the pathological diagnosis of DCS in cetaceans. The objective of this study is to validate the pathological results of cetaceans through an experimental rabbit model wherein a complete and detailed histopathological analysis was performed. Gross and histopathological results were very similar in the experimental animal model compared to stranded cetaceans with DCS, with the presence of gas embolism systemically distributed as well as emphysema and hemorrhages as primary lesions in different organs. The experimental data reinforces the pathological findings found in cetaceans with DCS as well as the hypothesis that individuality plays an essential role in DCS, as it has previously been proposed in animal models and human diving medicine.

Blood Gene Expression and Vascular Function Biomarkers in Professional Saturation Diving.

Saturation diving is an established way to conduct subsea operations with human intervention. While working, the divers must acclimatize to the hyperbaric environments. In this study, genome-wide gene expression and selected plasma biomarkers for vascular function were investigated. We also examined whether antioxidant vitamin supplements affected the outcome. The study included 20 male professional divers, 13 of whom took vitamin C and E supplements in doses of 1,000 and 30 mg daily during saturation periods that lasted 7-14 days. The dives were done in a heliox atmosphere with 40 kPa oxygen partial pressure (ppO2) to a depth of 100-115 m of sea-water (msw), from which the divers performed in-water work excursions to a maximum depth of 125 msw with 60 kPa ppO2. Venous blood was collected immediately before and after saturation. Following gene expression profiling, post-saturation gene activity changes were analyzed. Protein biomarkers for inflammation, endothelial function, and fibrinolysis: Il-6, CRP, ICAM-1, fibrinogen, and PAI-1, were measured in plasma. Post-saturation gene expression changes indicated acclimatization to elevated ppO2 by extensive downregulation of factors involved in oxygen transport, including heme, hemoglobin, and erythrocytes. Primary endogenous antioxidants; superoxide dismutase 1, catalase, and glutathione synthetase, were upregulated, and there was increased expression of genes involved in immune activity and inflammatory signaling pathways. The antioxidant vitamin supplements had no effect on post-saturation gene expression profiles or vascular function biomarkers, implying that the divers preserved their homeostasis through endogenous antioxidant defenses.

The future of diving research in Norway.

Norway has a long tradition of quality research within the field of baromedicine. With the discovery of oil in the North Sea, it became important to establish scientific research facilities to overcome immediate challenges, but also to work towards long-term goals. For the diving community, an understanding of the pathophysiology of decompression sickness (DCS) has been one of the major forces to maintain focus on the importance of scientific research in this field. In addition to oil, the aquaculture and fish farming industries are increasing in size and are Norway's second biggest export industry today. It also requires underwater workers for the inspection and repair of underwater structures and fishnets. The importance of health and safety for the underwater worker was identified early on by the offshore industry. The Norwegian Petroleum Safety Authority publishes a yearly report that identifies all offshore diving activity. The last reported incident of DCS was in 2002, whilst the last fatal saturation diving accident was in 1987. In-shore diving operations in Norway are regulated through the Norwegian Labour Inspection Authority and here the track record is different; since 1979 there have been 28 fatalities, and they continue to occur. At the Norwegian University of Science and Technology (NTNU), there has been a research group investigating barophysiology since the early 1980s. Led by Professor Alf O Brubakk, this research has been recognized internationally and has provided ground-breaking insights into the pathophysiology of DCS. This has included the identification of vascular gas bubbles through the use of ultrasound and identifying the importance of both protecting the vascular endothelium to maintain fitness to dive and also regular physical activity to reduce the risk of the adverse effects of diving. The group has educated many students, physiologists, engineers, medical doctors and researchers, all in the spirit of Professor Brubakk who considered that education was at least as important as the research itself. In 2008, Professor Brubakk was concerned about the future, as he was soon to retire. Great effort was put into perpetuating his position but this process ended when the University axed the only professorial position in environmental physiology in Norway. Today, there is only one non-permanent barophysiology research position at NTNU. This position and all research activity is dependent on external funding, so the education and research environment has changed drastically. Whilst there are clinicians in Norway working at different hyperbaric centres who participate in research related to barophysiology, this is not their primary task. With the lack of funding to include education and students in research, the rich history of barophysiological research at NTNU will be at an end. In Norway, the majority of grant-funded scientific programmes last only three years, so it has not been easy to recruit or to keep expertise between grants. So, who is planning for long-term research efforts in Norway? Whilst there are obvious challenges left to study in barophysiology, there is a lack of understanding amongst those responsible for decision-making and funding of the importance of having an academic-based research centre for diving research. NTNU, one of the world's most advanced hyperbaric laboratories, built up at considerable capital expense to investigate the pathophysiology of diving and decompression, is about to be closed and dismantled. At a time when the off-shore industry is putting greater focus on finding better solutions for safer underwater work environments, and in-shore diving is facing huge challenges due to a worrying level of serious accidents and increasing activity, there is no political drive in Norway to acknowledge the importance of maintaining the research facilities that support this industry. If the door does close on the NTNU facility, it will take many years and substantial funding to re-establish a modern research centre. Most importantly, it will be impossible to bring new students into the field of barophysiology in the foreseeable future. Whilst the off-shore oil industry has a finite future, aquaculture and other in-shore activities requiring diving support continue to expand. Good barophysiological research in established centres will be essential to support these industries into the future. Alf Brubakk often quoted an old Chinese proverb: "When planning for a month, sow rice, when planning for a year, plant trees, when planning for a decade, train and educate men". In Norway, we are only planting trees.

Observed decompression sickness and venous bubbles following 18-msw dive profiles using RN Table 11.

The venous bubble load in the body after diving may be used to infer risk of decompression sickness (DCS). Retrospective analysis of post-dive bubbling and DCS was made on seven studies. Each of these investigated interventions, using an 18 meters of sea water (msw) air dive profile from Royal Navy Table 11 (Mod Air Table), equivalent to the Norwegian Air tables. A recent neurological DCS case suggested this table was not safe as thought. Two-hundred and twenty (220) man-dives were completed on this profile. Bubble measurements were made following 219 man-dives, using Doppler or 2D ultrasound measurements made on the Kisman-Masurel and Eftedal-Brubakk scales, respectively. The overall median grade was KM/EB 0.5 and the overall median maximum grade was KM/EB 2. Two cases of transient shoulder discomfort ("niggles") were observed (0.9% (95% CL 0.1% - 3.3%)) and were treated with surface oxygen. One dive, for which no bubble measurements were made, resulted in a neurological DCS treated with hyperbaric oxygen. The DCS risk of this profile is below that predicted by models, and comparison of the cumulative incidence of DCS of these data to the large dataset compiled by DCIEM [1, 2] show that the incidence is lower than might be expected.

Bubbles Quantified In vivo by Ultrasound Relates to Amount of Gas Detected Post-mortem in Rabbits Decompressed from High Pressure.

The pathophysiological mechanism of decompression sickness is not fully understood but there is evidence that it can be caused by intravascular and autochthonous bubbles. Doppler ultrasound at a given circulatory location is used to detect and quantify the presence of intravascular gas bubbles as an indicator of decompression stress. In this manuscript we studied the relationship between presence and quantity of gas bubbles by echosonography of the pulmonary artery of anesthetized, air-breathing New Zealand White rabbits that were compressed and decompressed. Mortality rate, presence, quantity, and distribution of gas bubbles elsewhere in the body was examined postmortem. We found a strong positive relationship between high ultrasound bubble grades in the pulmonary artery, sudden death, and high amount of intra and extra vascular gas bubbles widespread throughout the entire organism. In contrast, animals with lower bubble grades survived for 1 h after decompression until sacrificed, and showed no gas bubbles during dissection.

Differentiation at necropsy between in vivo gas embolism and putrefaction using a gas score.

Gas bubble lesions consistent with decompression sickness in marine mammals were described for the first time in beaked whales stranded in temporal and spatial association with military exercises. Putrefaction gas is a post-mortem artifact, which hinders the interpretation of gas found at necropsy. Gas analyses have been proven to help differentiating putrefaction gases from gases formed after hyperbaric exposures. Unfortunately, chemical analysis cannot always be performed. Post-mortem computed tomography is used to study gas collections, but many different logistical obstacles and obvious challenges, like the size of the animal or the transport of the animal from the stranding location to the scanner, limit its use in stranded marine mammals. In this study, we tested the diagnostic value of an index-based method for characterizing the amount and topography of gas found grossly during necropsies. For this purpose, putrefaction gases, intravenously infused atmospheric air, and gases produced by decompression were evaluated at necropsy with increased post-mortem time in New Zealand White Rabbits using a gas score index. Statistical differences (P<0.001) were found between the three experimental models immediately after death. Differences in gas score between in vivo gas embolism and putrefaction gases were found significant (P<0.05) throughout the 67h post-mortem. The gas score-index is a new and simple method that can be used by all stranding networks, which has been shown through this study to be a valid diagnostic tool to distinguish between fatal decompression, iatrogenic air embolism and putrefaction gases at autopsies.

Consensus guidelines for the use of ultrasound for diving research.

The International Meeting on Ultrasound for Diving Research produced expert consensus recommendations for ultrasound detection of vascular gas bubbles and the analysis, interpretation and reporting of such data. Recommendations for standardization of techniques to allow comparison between studies included bubble monitoring site selection, frequency and duration of monitoring, and use of the Spencer, Kisman-Masurel or Eftedal-Brubakk scales. Recommendations for reporting of results included description of subject posture and provocation manoeuvres during monitoring, reporting of untransformed data and the appropriate use of statistics. These guidelines are available from www.dhmjournal.com.

Could some aviation deep vein thrombosis be a form of decompression sickness?

Aviation deep vein thrombosis is a challenge poorly understood in modern aviation. The aim of the present project was to determine if cabin decompression might favor formation of vascular bubbles in commercial air travelers. Thirty commercial flights were taken. Cabin pressure was noted at take-off and at every minute following, until the pressure stabilized. These time-pressure profiles were imported into the statistics program R and analyzed using the package SCUBA. Greatest pressure differentials between tissues and cabin pressures were estimated for 20, 40, 60, 80 and 120 min half-time compartments. Time to decompress ranged from 11 to 47 min. The greatest drop in cabin pressure was from 1022 to 776 mBar, equivalent to a saturated diver ascending from 2.46 msw depth. Mean pressure drop in flights >2 h duration was 193 mBar, while mean pressure drop in flights <2 h was 165 mBar. The greatest drop in pressure over 1 min was 28 mBar. Over 30 commercial flights it was found that the drop in cabin pressure was commensurate with that found to cause bubbles in man. Both the US Navy and the Royal Navy mandate far slower decompression from states of saturation, being 1.7 and 1.9 mBar/min respectively. The median overall rate of decompression found in this study was 8.5 mBar/min, five times the rate prescribed for USN saturation divers. The tissues associated with hypobaric bubble formation are likely slower than those associated with bounce diving, with 60 min a potentially useful index.

S100B and NSE serum concentrations after simulated diving in rats.

The purpose of this study was to assess whether one could detect S100 calcium-binding protein B (S100B) and neuron-specific enolase (NSE) in serum of rats after a simulated dive breathing air, with the main hypothesis that the serum concentrations of S100B and NSE in rats will increase above pre-exposure levels following severe decompression stress measured as venous gas emboli (VGE). The dive group was exposed to a simulated air dive to 700 kPa for 45 min. Pulmonary artery was monitored for vascular gas bubbles by ultrasound. Pre- and postdive blood samples were analyzed for S100B and NSE using commercially available Elisa kits. There was no increase in serum S100B or NSE after simulated diving and few of the animals were showing high bubble grades after the dives. The present study examined whether the protein biomarkers S100B and NSE could be found in serum from rats after exposure to a simulated dive to 700 kPa for 45 min breathing air. There were no differences in serum concentrations before versus after the dive exposure. This may be explained by the lack of vascular gas bubbles after the dives.

Evaluating PAI-1 as a biomarker for stress in diving: human serum total PAI-1 is unaltered after 2 h dry exposures to 280 kPa hyperbaric air.

Plasminogen activator inhibitor (PAI-1) is induced in the vasculature and secreted into the vascular lumen in response to inflammation and oxidative stress. We have previously reported a fivefold increase in plasma PAI-1 from rats exposed to 708 kPa hyperbaric air. In the current study we assess the potential of human serum total PAI-1 as a biomarker for stress in compressed air diving. Eleven recreational divers, nine males and two females, completed four 2 h hyperbaric air exposures to 280 kPa in a pressure chamber over a period of 2 weeks. The air pressure corresponds to a diving depth of 18 m in water. Serum was collected before the study and again 3 h 30 min after completion of each hyperbaric exposure. All samples were taken in the afternoon to minimize the contribution of circadian variation. The analysis revealed no change in serum total PAI-1 after hyperbaric exposures within the group of divers (P = 0.064), but significant interindividual differences persisted throughout the study (P < 0.0005). A case of decompression sickness after the third round of hyperbaric exposure did not affect PAI-1. In conclusion, compressed air exposure to 280 kPa does not affect serum total PAI-1, and significant interindividual variation in PAI-1 levels may limit its usefulness as a biomarker. This does, however, not give a complete answer regarding PAI-1 in physiologically stressful dives. Further studies with different exposures and timing are needed for that.

Ultrasound detection of vascular decompression bubbles: the influence of new technology and considerations on bubble load.

INTRODUCTION: Diving often causes the formation of 'silent' bubbles upon decompression. If the bubble load is high, then the risk of decompression sickness (DCS) and the number of bubbles that could cross to the arterial circulation via a pulmonary shunt or patent foramen ovale increase. Bubbles can be monitored aurally, with Doppler ultrasound, or visually, with two dimensional (2D) ultrasound imaging. Doppler grades and imaging grades can be compared with good agreement. Early 2D imaging units did not provide such comprehensive observations as Doppler, but advances in technology have allowed development of improved, portable, relatively inexpensive units. Most now employ harmonic technology; it was suggested that this could allow previously undetectable bubbles to be observed. METHODS: This paper provides a review of current methods of bubble measurement and how new technology may be changing our perceptions of the potential relationship of these measurements to decompression illness. Secondly, 69 paired ultrasound images were made using conventional 2D ultrasound imaging and harmonic imaging. Images were graded on the Eftedal-Brubakk (EB) scale and the percentage agreement of the images calculated. The distribution of mismatched grades was analysed. RESULTS: Fifty-four of the 69 paired images had matching grades. There was no significant difference in the distribution of high or low EB grades for the mismatched pairs. CONCLUSIONS: Given the good level of agreement between pairs observed, it seems unlikely that harmonic technology is responsible for any perceived increase in observed bubble loads, but it is probable that our increasing use of 2D ultrasound to assess dive profiles is changing our perception of 'normal' venous and arterial bubble loads. Methods to accurately investigate the load and size of bubbles developed will be helpful in the future in determining DCS risk.

Differentiation at autopsy between in vivo gas embolism and putrefaction using gas composition analysis.

Gas embolism can arise from different causes (iatrogenic accidents, criminal interventions, or diving related accidents). Gas analyses have been shown to be a valid technique to differentiate between putrefaction gases and gas embolism. In this study, we performed systematic necropsies at different postmortem times in three experimental New Zealand White Rabbits models: control or putrefaction, infused air embolism, and compression/decompression. The purpose of this study was to look for qualitative and quantitative differences among groups and to observe how putrefaction gases mask in vivo gas embolism. We found that the infused air embolism and compression/decompression models had a similar gas composition prior to 27-h postmortem, being typically composed of around 70-80 % of N(2) and 20-30 % of CO(2), although unexpected higher CO(2) concentrations were found in some decompressed animals, putting in question the role of CO(2) in decompression. All these samples were statistically and significantly different from more decomposed samples. Gas composition of samples from more decomposed animals and from the putrefaction model presented hydrogen, which was therefore considered as a putrefaction marker.

Simulated dive in rats lead to acute changes in cerebral blood flow on MRI, but no cerebral injuries to grey or white matter.

In this study, the effect of a simulated dive on rat brain was investigated using several magnetic resonance imaging (MRI)-methods and immunohistochemistry. Rats were randomly assigned to a dive- or a control group. The dive group was exposed to a simulated air dive to 600 kPa for 45 min. Pulmonary artery was monitored for vascular gas bubbles by ultrasound. MRI was performed 1 h after decompression and at one and 2 weeks after the dive with a different combination of MRI sequences at each time point. Two weeks after decompression, rats were sacrificed and brains were prepared for histology. Dived rats had a different time-curve for the dynamic contrast-enhanced MRI signal than controls with higher relative signal intensity, a tendency towards longer time to peak and a larger area under the curve for the whole brain on the acute MRI scan. On MRI, 1 and 2 weeks after dive, T2-maps showed no signal abnormalities or morphological changes. However, region of interest based measurements of T2 showed higher T2 in the brain stem among decompressed animals than controls after one and 2 weeks. Microscopical examination including immunohistochemistry did not reveal apparent structural or cellular injuries in any part of the rat brains. These observations indicate that severe decompression does not seem to cause any structural or cellular injury to the brain tissue of the rat, but may cause circulatory changes in the brain perfusion in the acute phase.

Observation of increased venous gas emboli after wet dives compared to dry dives.

INTRODUCTION: Testing of decompression procedures has been performed both in the dry and during immersion, assuming that the results can be directly compared. To test this, the aim of the present paper was to compare the number of venous gas bubbles observed following a short, deep and a shallow, long air dive performed dry in a hyperbaric chamber and following actual dives in open water. METHODS: Fourteen experienced male divers participated in the study; seven performed dry and wet dives to 24 metres' sea water (msw) for 70 minutes; seven divers performed dry and wet dives to 54 msw for 20 minutes. Decompression followed a Bühlmann decompression procedure. Immediately following the dive, pulmonary artery bubble formation was monitored for two hours. The results were graded according to the method of Eftedal and Brubakk. RESULTS: All divers completed the dive protocol, none of them showed any signs of decompression sickness. During the observation period, following the shallow dives, the bubbles increased from 0.1 bubbles per cm ² after the dry dive to 1.4 bubbles per cm ² after the wet dive. Following the deep dives, the bubbles increased from 0.1 bubbles per cm ² in the dry dive to 2.4 bubbles per cm ² in the wet dive. Both results are highly significant (P = 0.0001 or less). CONCLUSIONS: The study has shown that diving in water produces significantly more gas bubble formation than dry diving. The number of venous gas bubbles observed after decompression in water according to a rather conservative procedure, indicates that accepted standard decompression procedures nevertheless induce considerable decompression stress. We suggest that decompression procedures should aim at keeping venous bubble formation as low as possible.

Venous and arterial bubbles at rest after no-decompression air dives.

PURPOSE: During SCUBA diving, breathing at increased pressure leads to a greater tissue gas uptake. During ascent, tissues may become supersaturated, and the gas is released in the form of bubbles that typically occur on the venous side of circulation. These venous gas emboli (VGE) are usually eliminated as they pass through the lungs, although their occasional presence in systemic circulation (arterialization) has been reported and it was assumed to be the main cause of the decompression sickness. The aims of the present study were to assess the appearance of VGE after air dives where no stops in coming to the surface are required and to assess their potential occurrence and frequency in the systemic circulation. METHODS: Twelve male divers performed six dives with 3 d of rest between them following standard no-decompression dive procedures: 18/60, 18/70, 24/30, 24/40, 33/15, and 33/20 (the first value indicates depth in meters of sea water and the second value indicates bottom time in minutes). VGE monitoring was performed ultrasonographically every 20 min for 120 min after surfacing. RESULTS: Diving profiles used in this study produced unexpectedly high amounts of gas bubbles, with most dives resulting in grade 4 (55/69 dives) on the bubble scale of 0-5 (no to maximal bubbles). Arterializations of gas bubbles were found in 5 (41.7%) of 12 divers and after 11 (16%) of 69 dives. These VGE crossovers were only observed when a large amount of bubbles was concomitantly present in the right valve of the heart. CONCLUSIONS: Our findings indicate high amounts of gas bubbles produced after no-decompression air dives based on standardized diving protocols. High bubble loads were frequently associated with the crossover of VGE to the systemic circulation. Despite these findings, no acute decompression-related pathology was detected.

Immersion before dry simulated dive reduces cardiomyocyte function and increases mortality after decompression.

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.

S100B and its relation to intravascular bubbles following decompression.

INTRODUCTION: When neurological damage occurs in divers, it is considered to be caused by gas bubbles. Entrapment of these bubbles may lead to cellular injury and cerebral oedema. S100B is a protein biomarker that is released in CNS injuries and the concentration is related to the amount of brain damage. METHODS: A total of 27 rats were randomly assigned to one of three groups. Group I served as controls (n = 9). Group II (n = 7) underwent a simulated dive to 400 kPa and Group III to 700 kPa (n = 11). In groups II and III, venous gas bubble scores were evaluated by ultrasound during the first hour after surfacing. The amount of S100B in serum after the dives was tested using a commercial ELISA kit. Bubble grades were compared to S100B protein concentrations. RESULTS: The average level of S100B was significantly higher in rats compressed to 700 kPa compared to the control rats, (P = 0.038) and the rats compressed to 400 kPa, (P = 0.003). There was no difference in S100B concentration between groups I and II. Following the dive to 700 kPa, there were significantly higher bubble grades observed than following the dive to 400 kPa (P = 0.001). CONCLUSION: The correlation between bubble grade and an increase in serum protein level of S100B indicates that this protein may be useful as a biomarker for neurological damage caused by decompression.

Aortic function in rats after decompression without ultrasonically detectable bubble formation.

BACKGROUND: Several studies have demonstrated an adverse effect of bubbles on endothelial function. The degree of dysfunction appears to be related to the number of bubbles present. The aim of the study was to determine whether decompression without bubble formation visible by ultrasound had any effect on arterial endothelial function. METHODS: We decompressed 21 Sprague-Dawley rats weighing 215-260 g from 700 kPa (approximately 6.9 ATA) in a dry hyperbaric chamber followed by a 1-h observation period and measured aortic endothelial-dependent relaxation to acetylcholine. Later, we determined the specific weight of the brain as a measure of edema formation and vascular bubbles in the arterial circulation. RESULTS: No bubbles were seen in the pulmonary arteries of seven rats. We found a significant lower vasodilatory response to acetylcholine in the decompressed rats (44% +/- 14%) compared to the control rats (58% +/- 12%) as a sign of endothelial dysfunction. There was no significant difference between the two groups in the specific gravity of the brain. CONCLUSION: We conclude that measurable arterial dysfunction in the aorta can occur even if no visible venous bubble formation is seen. There are no results in this study suggesting that these rats had damaged blood-brain barriers or brain edema.