Analysis of Single- and Double-Stranded DNA Damage in Osteoblastic Cells after Hyperbaric Oxygen Exposure.

(1) Background: Hyperbaric oxygen (HBO) exposure induces oxidative stress that may lead to DNA damage, which has been observed in human peripheral blood lymphocytes or non-human cells. Here, we investigated the impact of hyperbaric conditions on two human osteoblastic cell lines: primary human osteoblasts, HOBs, and the osteogenic tumor cell line SAOS-2. (2) Methods: Cells were exposed to HBO in an experimental hyperbaric chamber (4 ATA, 100% oxygen, 37 °C, and 4 h) or sham-exposed (1 ATA, air, 37 °C, and 4 h). DNA damage was examined before, directly after, and 24 h after exposure with an alkaline comet assay and detection of γH2AX+53BP1 colocalizing double-strand break (DSB) foci and apoptosis. The gene expression of TGFß-1, HO-1, and NQO1, involved in antioxidative functions, was measured with qRT-PCR. (3) Results: The alkaline comet assay showed significantly elevated levels of DNA damage in both cell lines after 4 h of HBO, while the DSB foci were similar to sham. γH2AX analysis indicated a slight increase in apoptosis in both cell lines. The increased expression of HO-1 in HOB and SAOS-2 directly after exposure suggested the induction of an antioxidative response in these cells. Additionally, the expression of TGF-ß1 was negatively affected in HOB cells 4 h after exposure. (4) Conclusions: in summary, this study indicates that osteoblastic cells are sensitive to the DNA-damaging effects of hyperbaric hyperoxia, with the HBO-induced DNA damage consisting largely of single-strand DNA breaks that are rapidly repaired.

Low Concentrations of C5a Complement Receptor Antibodies Are Linked to Disease Activity and Relapse in Antineutrophil Cytoplasmic Autoantibody-Associated Vasculitis.

OBJECTIVE: To examine concentrations of circulating antibodies targeting C3a and C5a complement receptors in antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) and analyze their association with disease activity. METHODS: Concentrations of antibodies against C3a and C5a complement receptors (anti-C3aR and anti-C5aR) and plasma complement fragments C3a and C5a were determined in patients with AAV (n = 110; granulomatosis with polyangiitis [GPA; n = 82] or microscopic polyangiitis [MPA; n = 28]), systemic lupus erythematosus (SLE) patients as disease controls (n = 36), and healthy donors (n = 220). C3aR and C5aR expression by circulating neutrophils, monocytes, and T cells was analyzed using flow cytometry. Clinical data were assessed at time of serum sampling and during follow-up for 60 months. RESULTS: In AAV, anti-C3aR and anti-C5aR antibodies were decreased (P = 0.0026 and P ≤ 0.0001, respectively). In remission, anti-C3aR antibody concentrations rose to values comparable to healthy donors, whereas anti-C5aR antibody concentrations did not. In GPA, anti-C5a and anti-C5aR antibody concentrations inversely correlated with each other (r = -0.6831, P = 0.0127). In newly diagnosed GPA, decreased concentrations of anti-C5aR antibodies but not anti-C3aR antibodies were associated with disease activity (P = 0.0009). Moreover, low anti-C5aR antibodies were associated with relapse in GPA (hazard ratio 3.54, P = 0.0009) and MPA (hazard ratio 4.41, P = 0.0041). The frequency of C5aR-expressing cells within T cell populations was increased in GPA (P = 0.0021 for CD4+ T cells; P = 0.0118 for CD8+ T cells), but not in MPA. CONCLUSION: Low concentrations of anti-C5aR antibodies reflect disease activity and are associated with an increased risk for relapse in AAV.

Diving ergospirometry with suspended weights: breathing- and fin-swimming style matter.

PURPOSE: Scuba diving is a complex condition including elevated ambient pressure, limited air supply, increased breathing work, and unfamiliar fin-swimming. Earlier approaches to assess diving specific data did not comprehensively address these aspects. We first present an underwater ergospirometry system and then test the hypothesis that both breathing characteristics and fin-swimming style affect the air consumption. METHODS/PARTICIPANTS: A suspended-weights ergospirometry system was mounted inside a hyperbaric chamber. Ergo group: 25 divers (24.6 ± 4.1 years); three set-ups: dry normobaric cycling (75-225 W), dry cycling at 20 m simulated depth (75-225 W), fin-swimming at 20 m (5-8 kg suspended weights). Style group: 20 other divers (24.6 ± 4.1 years): fin-swimming at 20 m (5-8 kg) with regard to ventilation ([Formula: see text]E) and fin-swimming style. RESULTS: Ergo group: linear heart rate and oxygen uptake ([Formula: see text]O2) increases with both 50 W-bicycle steps and suspended-weights ergometry (r = 0.97). During hyperbaric conditions, [Formula: see text]E was less increased versus normobaric conditions. Style group: the more efficient hip/thigh-oriented style shifted towards the knee/calf-oriented style. [Formula: see text]E and [Formula: see text]O2 were higher in beginners (< 100 dives) versus advanced divers (≥ 100 dives). Significant differences on the 5 kg-step: [Formula: see text]E: 31.5 ± 7.1 l/min vs. 23.7 ± 5.9 l/min and [Formula: see text]O2: 1.6 ± 0.3 l/min vs. 1.2 ± 0.3 l/min. A comparison is presented, in addition to illustrate the impact of differences in breathing characteristics and fin-swimming style. CONCLUSIONS: Diving ergospirometry with suspended weights in a hyperbaric chamber allows for comprehensive studies. Little diving experience in terms of breathing characteristics and fin-swimming style significantly increases [Formula: see text]E thereby increasing the risk of running-out-of-air.

Assessment of a dive incident using heart rate variability.

INTRODUCTION: Scuba diving likely has an impact on the autonomic nervous system (ANS). In the course of conducting trials of underwater ECG recording for measurement of heart rate variability, there was an unexpected stressful event; one participant's regulator iced and began to free-flow. METHODS: A custom-made, water- and pressure-tight aluminum housing was used to protect a portable Holter monitor. ECGs were recorded in three experienced divers who witnessed an unplanned moderately stressful incident during diving. The ECG signals were analysed for measures of heart rate variability (HRV). RESULTS: Analysis for different short-term HRV measures provided consistent results if periods of interest were appropriately time-aligned. There was improvement in sympatho-vagal balance. One diver unexpectedly exhibited an increase in both sympathetic and vagal activity shortly after the incident. CONCLUSIONS: A conventional open-water dive affected the ANS of experienced recreational divers as measured by HRV which provides a global evaluation of the ANS and alterations in its two branches. The heart rate variability data gathered from several participating divers around the time of this event illustrate the potential utility of this variable in quantifying stress during diving. HRV data may be useful in addressing relevant diving related questions such as effects of cold, exercise or different breathing gases on ANS function.

Does oxygen-enriched air better than normal air improve sympathovagal balance in recreational divers?An open-water study.

Effects of the hyperbaric environment on the autonomic nervous system (ANS) in recreational divers are not firmly settled. Aim of this exploratory study was to (1) assess ANS changes during scuba diving via recordings of electrocardiograms (ECG) and to (2) study whether nitrox40 better improves sympathovagal balance over air. 13 experienced divers (~40yrs) performed two open-water dives each breathing either air or nitrox40 (25m/39min). 3-channel ECGs were recorded using a custom-made underwater Holter-monitor. The underwater Holter system proved to be safe. Air consumption exceeded nitrox40 consumption by 12% (n = 13; p < 0.05). Both air and nitrox40 dives reduced HR (10 vs 13%; p < 0.05). The overall HRV (pNN50: 82 vs 126%; p < 0.05) and its vagal proportion (RMSSD: 33 vs 50%; p < 0.05) increased during the dive. Moreover, low (LF: 61 vs 47%) and high (HF: 71 vs 140%) frequency power were increased (all p < 0.05), decreasing the ratio of LF to HF (22 vs 34%). : Conventional open-water dives distinctly affect the ANS in experienced recreational divers, with sympathetic activation less pronounced than vagal activation thereby improving the sympathovagal balance. Nitrox40 delivered two positive results: nitrox40 consumption was lower than air consumption, and nitrox40 better improved the sympathovagal balance over air.

Effect of hyperoxia on the immune status of oxygen divers and endurance athletes.

Physical activity, particularly that, exerted by endurance athletes, impacts the immune status of the human body. Prolonged duration and high-intensity endurance training lead to increased production of reactive oxygen species (ROS) and thereby to oxidative stress. Military combat swimmers (O2-divers) are regularly exposed to hyperbaric hyperoxia (HBO) in addition to intensive endurance training intervals. They are, therefore, exposed to extreme levels of oxidative stress. Several studies support that the intensity of oxidative stress essentially determines the effect on immune status. The aim of this study was to comparatively characterise peripheral blood mononuclear cells (PBMCs) of O2-divers (military combat swimmers), endurance athletes (amateur triathletes), and healthy control volunteers with respect to DNA fragmentation, immune status and signs of inflammation. Furthermore, it was investigated how PBMCs from these groups responded acutely to exposure to HBO. We showed that DNA fragmentation was comparable in PBMCs of all three groups under basal conditions directly after HBO exposure. However, significantly higher DNA fragmentation was observed in O2-divers 18 hours after HBO, possibly indicating a slower recovery. O2-divers also exhibited a proinflammatory immune status exemplified by an elevated number of CD4+CD25+ T cells, elevated expression of proinflammatory cytokine IL-12, and diminished expression of anti-inflammatory TGF-ß1 compared to controls. Supported by a decreased basal gene expression and prolonged upregulation of anti-oxidative HO-1, these data suggest that higher oxidative stress levels, as present under intermitted hyperbaric hyperoxia, e.g. through oxygen diving, promote a higher inflammatory immune status than oxidative stress through endurance training alone.

Decompression illness with hypovolemic shock and neurological failure symptoms after two risky dives: a case report.

Hypovolemia is known to be a predisposing factor of decompression illness (DCI) while diving. The typical clinically impressive neurological symptoms of DCI may distract from other symptoms such as an incipient hypovolemic shock. We report the case of a 61-year-old male Caucasian, who presented with an increasing central and peripheral neural failure syndrome and massive hypovolemia after two risky dives. Computed tomography (CT) scans of the chest and Magnetic resonance imaging scans of the head revealed multiple cerebral and pulmonary thromboembolisms. Transesophageal echocardiography showed a patent foramen ovale (PFO). Furthermore, the patient displayed hypotension as well as prerenal acute kidney injury with elevated levels of creatinine and reduced renal clearance, indicating a hypovolemic shock. Early hyperbaric oxygen (HBO) therapy reduced the neurological deficits. After volume expansion of 11 liters of electrolyte solution (1000 mL/h) the cardiopulmonary and renal function normalized. Hypovolemia increases the risk of DCI during diving and that of hypovolemic shock. Early HBO therapy and fluid replacement is crucial for a favorable outcome.

The measurement of Eustachian tube function in a hyperbaric chamber using an ear canal microphone.

OBJECTIVE: The purpose of this study was to further the understanding of the opening of the Eustachian tube in relation to changes in barometric pressure. DESIGN: An ear canal microphone was used to measure the specific sounds related to tube opening and possible eardrum movements. Five subjects with normal tube function were examined in a hyperbaric chamber (up to 304 kPa). All active and passive equalization events were recorded and correlated with the subjectively perceived pressure regulation in the measured ear. RESULTS: The signals recorded were clear and reproducible. The acoustic analysis distinguished between the different kinds of equalization. Subjective impressions were confirmed by the recorded frequency of acoustic phenomena (clicks). During compression, the sequence of active equalization manoeuvres was in a more regular and steady pattern than during decompression, when the click sounds varied. CONCLUSION: The study established a simple technical method for analyzing the function of the Eustachian tube and provided new information about barometric pressure regulation of the middle ear.

Mechanical ventilation and resuscitation under water: Exploring one of the last undiscovered environments--A pilot study.

INTRODUCTION: Airway management, mechanical ventilation and resuscitation can be performed almost everywhere--even in space--but not under water. The present study assessed the technical feasibility of resuscitation under water in a manikin model. METHODS: Tracheal intubation was assessed in a hyperbaric chamber filled with water at 20 m of depth using the Pentax AWS S100 video laryngoscope, the Fastrach™ intubating laryngeal mask and the Clarus optical stylet with guidance by a laryngeal mask airway (LMA) and without guidance. A closed suction system was used to remove water from the airways. A test lung was ventilated to a maximum depth of 50 m with a modified Oxylator(®) EMX resuscitator with its expiratory port connected either to a demand valve or a diving regulator. Automated chest compressions were performed to a maximum depth of 50 m using the air-driven LUCAS™ 1. RESULTS: The mean cumulative time span for airway management until the activation of the ventilator was 36 s for the Fastrach™, 57 s for the Pentax AWS S100, 53s for the LMA-guided stylet and 43 s for the stylet without LMA guidance. Complete suctioning of the water from the airways was not possible with the suction system used. The Oxylator(®) connected to the demand valve ventilated at 50 m depth with a mean ventilation rate of 6.5 min(-1) vs. 14.7 min(-1) and minute volume of 4.5 l min(-1) vs. 7.6 l min(-1) compared to the surface. The rate of chest compression at 50 m was 228 min(-1) vs. 106 min(-1) compared to surface. The depth of compressions decreased with increasing depth. CONCLUSION: Airway management under water appears to be feasible in this manikin model. The suction system requires further modification. Mechanical ventilation at depth is possible but modifications of the Oxylator(®) are required to stabilize ventilation rate and administered minute volumes. The LUCAS™ 1 cannot be recommended at major depth.

Efficacy of ventilation and ventilation adjuncts during in-water-resuscitation--a randomized cross-over trial.

INTRODUCTION: Drowning is a common cause of death in young adults. The 2010 guidelines of the European Resuscitation Council call for in-water-resuscitation (IWR). There has been controversy about IWR amongst emergency and diving physicians for decades. The aim of the present study was assessing the efficacy of IWR. METHODS: In this randomized cross-over trial, nineteen lifeguards performed a rescue manoeuvre over a 100 m distance in open water. All subjects performed the procedure four times in random order: with no ventilation (NV) and transportation only, mouth-to-mouth ventilation (MMV), bag-mask-ventilation (BMV) and laryngeal tube ventilation (LTV). Tidal volumes, ventilation rate and minute-volumes were recorded using a modified Laerdal Resusci Anne manikin. Furthermore, water aspiration and number of submersions of the test mannequin were assessed, as well as the physical effort of the lifeguard rescuers.One lifeguard subject did not complete MMV due to exhaustion and was excluded from analysis. RESULTS: NV was the fastest rescue manoeuvre (advantage ∼40s). MMV and LTV were evaluated as efficient and relatively easy to perform by the lifeguards. While MMV (mean 199 ml) and BMV (mean 481 ml) were associated with a large amount of aspirated water, aspiration was significantly lower in LTV (mean 118 ml). The efficacy of ventilation was consistently good in LTV (Vt=447 ml), continuously poor in BMV (Vt=197) and declined substantially during MMV (Vt=1,019 ml initially and Vt=786 ml at the end). The physical effort of the lifeguards was remarkably higher when performing IWR: 3.7 in NV, 6.7 in MMV, 6.4 in BMV and 4.8 in LTV as measured on the 0-10 visual analogue scale. CONCLUSION: IWR in open water is time consuming and physically demanding. The IWR training of lifeguards should put more emphasis on a reduction of aspiration. The use of ventilation adjuncts like the laryngeal tube might ease IWR, reduce aspiration of water and increase the efficacy of ventilation during IWR.

Benzoate hydroxylation: a measure of oxidative stress in divers.

Hyperoxia may facilitate the formation of reactive oxygen species. Recent experiments indicated signs of oxidative stress after 3.5 h hyperoxic diving. We analyzed in the urine of healthy, 100% O2-breathing male volunteers before and after 45 min seawater diving (170 kPa) or 30 min resting at 280 kPa in a pressure chamber (HBO) for sub-fractions of hydroxybenzoate (HB), monohydroxybenzoate (MHB), and of dihydroxybenzoate (DHB). Measurements were performed by HPLC and electrochemical or UV-detection. Additionally, urinary concentrations of thiobarbituric acid-reactive substances (TBARS) and of creatinine (CREA) were analyzed by standard colorimetric assays. During HBO treatment, TBARS, DHB, 2,4-DHB, and 3,4-DHB increased significantly. MHB and CREA did not change. 2,4- and 3,4-DHB-alterations correlated with changes in TBARS. Diving induced urine dilution and stimulated oxygen consumption. Urinary TBARS and HB rose significantly higher during diving at 170 kPa than during HBO at 280 kPa. A different pattern in urinary sub-fractions of DHB could be observed in divers: 2,6 > 2,3 > 2,5 > 3,4. Changes in 2,6- and 2,5-DHB correlated significantly with alterations in TBARS. 2,6-DHB probably indicated renal oxidant stress similar to previously described animal experiments. It is concluded that analyzing urinary HB may provide a sensitive measure to quantify and qualify oxidant stress in divers.