Marc Belhomme

Influence of Decompression Sickness on Vasomotion of Isolated Rat Vessels

Several studies have demonstrated that endothelial function is impaired following a dive even without decompression sickness. During this study we determined the effect of decompression sickness on endothelium-dependent and independent vasoreactivity. For this purpose twenty-seven male Sprague-Dawley rats were submitted to a simulated dive up to 1,000 kPa absolute pressure and divided into 3 groups: safe diving without decompression sickness or dives provoking mild or severe sickness. A fourth control group remained at atmospheric pressure. Endothelium-dependent and independent vasomotion was assessed ex vivo by measuring isometric tension in rings of abdominal aorta and mesenteric arteries. Dose-response curves were obtained with phenylephrine, acetylcholine and sodium nitroprusside. Acetylcholine-induced relaxation was measured in the presence of L-NAME, indometacin or both of them at once.Contraction was significantly decreased after each protocol compared with the control rats. Additionally, the response in animals from the severe group was significantly different from that of the safe and mild groups. Dose response curves for acetylcholine alone and in the presence of inhibitors remained unchanged. We did not observe differences in endothelium-dependent vasodilation after diving or in the presence of decompression sickness. Contractile response to phenylephrine was progressively impaired with increased decompression stress. These results may indicate smooth muscle injury.

Different effect of L-NAME treatment on susceptibility to decompression sickness in male and female rats

Vascular bubble formation results from supersaturation during inadequate decompression contributes to endothelial injuries, which form the basis for the development of decompression sickness (DCS). Risk factors for DCS include increased age, weight-fat mass, decreased maximal oxygen uptake, chronic diseases, dehydration, and nitric oxide (NO) bioavailability. Production of NO is often affected by diving and its expression-activity varies between the genders. Little is known about the influence of sex on the risk of DCS. To study this relationship we used an animal model of Nω-nitro-l-arginine methyl ester (l-NAME) to induce decreased NO production. Male and female rats with diverse ages and weights were divided into 2 groups: treated with l-NAME (in tap water; 0.05 mg·mL(-1) for 7 days) and a control group. To control the distribution of nitrogen among tissues, 2 different compression-decompression protocols were used. Results showed that l-NAME was significantly associated with increased DCS in female rats (p = 0.039) only. Weight was significant for both sexes (p = 0.01). The protocol with the highest estimated tissue pressures in the slower compartments was 2.6 times more likely to produce DCS than the protocol with the highest estimated tissue pressures in faster compartments. The outcome of this study had significantly different susceptibility to DCS after l-NAME treatment between the sexes, while l-NAME per se had no effect on the likelihood of DCS. The analysis also showed that for the appearance of DCS, the most significant factors were type of protocol and weight.

Antioxidants, endothelial dysfunction, and DCS: in vitro and in vivo study

Reactive oxygen species (ROS) production is a well-known effect in individuals after an undersea dive. This study aimed to delineate the links between ROS, endothelial dysfunction, and decompression sickness (DCS) through the use of antioxidants in vitro and in vivo. The effect of N-acetylcysteine (NAC) on superoxide and peroxynitrite, nitric oxide (NO) generation, and cell viability during in vitro diving simulation were analyzed. Also analyzed was the effect of vitamin C and NAC on plasma glutathione thiol and thiobarbituric acid reactive substances (TBARS), plasma angiotensin-converting enzyme (ACE) activity, and angiotensin-II and DCS morbidity during in vivo diving simulation. During an in vitro diving simulation, vascular endothelial cells showed overproduction of superoxide and peroxynitrite, obvious attenuation of NO generation, and promotion of cell death, all of which were reversed by NAC treatment. After in vivo diving simulation, plasma ACE activity and angiotensin-II level were not affected. The plasma level of glutathione thiol was downregulated after the dive, which was attenuated partially by NAC treatment. Plasma TBARS level was upregulated; however, either NAC or vitamin C treatment failed to prevent DCS morbidity. During in vitro simulation, endothelial superoxide and peroxynitrite-mediated oxidative stress were involved in the attenuation of NO availability and cell death. This study is the first attempt to link oxidative stress and DCS occurrence, and the link could not be confirmed in vivo. Even in the presence of antioxidants, ROS and bubbles generated during diving and/or decompression might lead to embolic or biochemical stress and DCS. Diving-induced oxidative stress might not be the only trigger of DCS morbidity.

Mechanism of action of antiplatelet drugs on decompression sickness in rats: a protective effect of anti-GPIIbIIIa therapy

Literature highlights the involvement of disseminated thrombosis in the pathophysiology of decompression sickness (DCS). We examined the effect of several antithrombotic treatments targeting various pathways on DCS outcome: acetyl salicylate, prasugrel, abciximab, and enoxaparin. Rats were randomly assigned to six groups. Groups 1 and 2 were a control nondiving group (C; n = 10) and a control diving group (CD; n = 30). Animals in Groups 3 to 6 were treated before hyperbaric exposure (HBE) with either prasugrel (n = 10), acetyl salicylate (n = 10), enoxaparin (n = 10), or abciximab (n = 10). Blood samples were taken for platelet factor 4 (PF4), thiobarbituric acid reactive substances (TBARS), and von Willebrand factor analysis. Onset of DCS symptoms and death were recorded during a 60-min observation period after HBE. Although we observed fewer outcomes of DCS in all treated groups compared with the CD, statistical significance was reached in abciximab only (20% vs. 73%, respectively, P = 0.007). We also observed significantly higher levels of plasmatic PF4 in abciximab (8.14 ± 1.40 ng/ml; P = 0.004) and enoxaparin groups (8.01 ± 0.80 ng/ml; P = 0.021) compared with the C group (6.45 ± 1.90 ng/ml) but not CD group (8.14 ± 1.40 ng/ml). Plasmatic levels of TBARS were significantly higher in the CD group than the C group (49.04 ± 11.20 μM vs. 34.44 ± 5.70 μM, P = 0.002). This effect was prevented by all treatments. Our results suggest that abciximab pretreatment, a powerful glycoprotein IIb/IIIa receptor antagonist, has a strong protective effect on decompression risk by significantly improving DCS outcome. Besides its powerful inhibitory action on platelet aggregation, we suggest that abciximab could also act through its effects on vascular function, oxidative stress, and/or inflammation.

Influence of decompression sickness on vasocontraction of isolated rat vessels.

Studies conducted in divers indicate that endothelium function is impaired following a dive even without decompression sickness (DCS). Our previous experiment conducted on rat isolated vessels showed no differences in endothelium-dependent vasodilation after a simulated dive even in the presence of DCS, while contractile response to phenylephrine was progressively impaired with increased decompression stress. This study aimed to further investigate the effect of DCS on vascular smooth muscle. Thirty-two male Sprague-Dawley rats were submitted to the same hyperbaric protocol and classified according to the severity of DCS: no-DCS (without clinical symptoms), mild-DCS, or severe-DCS (dead within 1 h). A control group remained at atmospheric pressure. Isometric tension was measured in rings of abdominal aorta and mesenteric arteries. Single dose contraction was assessed with KCl solution. Dose-response curves were obtained with phenylephrine and endothelin-1. Phenylephrine-induced contraction was observed in the presence of antioxidant tempol. Additionally, plasma concentrations of angiotensin II, angiotensin-converting enzyme, and thiobarbituric acid reactive substances (TBARS) were assessed. Response to phenylephrine was impaired only among mild-DCS in both vessels. Dose-response curves to endothelin-1 were impaired after mild-DCS in mesenteric and severe-DCS in aorta. KCl-induced contraction was affected after hyperbaric exposure regardless of DCS status in aorta only. These results confirm postdive vascular dysfunction is dependent on the type of vessel. It further evidenced that vascular dysfunction is triggered by DCS rather than by diving itself and suggest the influence of circulating factor/s. Diving-induced impairment of the L-type voltage-dependent Ca(2+) channels and/or influence of renin-angiotensin system is proposed.

Diving under a Microscope—A New Simple and Versatile In Vitro Diving Device for Fluorescence and Confocal Microscopy Allowing the Controls of Hydrostatic Pressure, Gas Pressures, and Kinetics of Gas Saturation

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Angiotensin Converting Enzyme Inhibitor Has a Protective Effect on Decompression Sickness in Rats

Introduction: Commercial divers, high altitude pilots, and astronauts are exposed to some inherent risk of decompression sickness (DCS), though the mechanisms that trigger are still unclear. It has been previously showed that diving may induce increased levels of serum angiotensin converting enzyme. The renin angiotensin aldosterone system (RAAS) is one of the most important regulators of blood pressure and fluid volume. The purpose of the present study was to control the influence of angiotensin II on the appearance of DCS. Methods: Sprague Dawley rats have been pre-treated with inhibitor of angiotensin II receptor type 1 (losartan; 10 mg/kg), angiotensin-converting enzyme (ACE) inhibitor (enalapril; 10 mg/kg), and calcium-entry blocker (nifedipine; 20 mg/kg). The experimental groups were treated for 4 weeks before exposure to hyperbaric pressure while controls were not treated. Seventy-five rats were subjected to a simulated dive at 1000 kPa absolute pressure for 45 min before starting decompression. Clinical assessment took place over a period of 60 min after surfacing. Blood samples were collected for measurements of TBARS, interleukin 6 (IL-6), angiotensin II (ANG II) and ACE. Results: The diving protocol induced 60% DCS in non-treated animals. This ratio was significantly decreased after treatment with enalapril, but not other vasoactive drugs. Enalapril did not change ANG II or ACE concentration, while losartant decreased post dive level of ACE but not ANG II. None of the treatment modified the effect of diving on TBARS and IL-6 values. Conclusion: Results suggests that the rennin angiotensin system is involved in a process of triggering DCS but this has to be further investigated. However, a vasorelaxation mediated process, which potentially could increase the load of inert gas during hyperbaric exposure, and antioxidant properties were excluded by our results.

Evidence of Heritable Determinants of Decompression Sickness in Rats.

Introduction Decompression sickness (DCS) is a complex and poorly understood systemic disease caused by inadequate desaturation after a decrease of ambient pressure. Strong variability between individuals is observed for DCS occurrence. This raises questions concerning factors that may be involved in the interindividual variability of DCS occurrence. This study aimed to experimentally assess the existence of heritable factors involved in DCS occurrence by selectively breeding individuals resistant to DCS from a population stock of Wistar rats. Methods Fifty-two male and 52 female Wistar rats were submitted to a simulated air dive known to reliably induce about 63% DCS: compression was performed at 100 kPa·min−1 up to 1000 kPa absolute pressure before a 45-min long stay. Decompression was performed at 100 kPa·min−1 with three decompression stops: 5 min at 200 kPa, 5 min at 160 kPa, and 10 min at 130 kPa. Animals were observed for 1 h to detect DCS symptoms. Individuals without DCS were selected and bred to create a new generation, subsequently subjected to the same hyperbaric protocol. This procedure was repeated up to the third generation of rats. Results As reported previously, this diving profile induced 67% of DCS, and 33% asymptomatic animals in the founding population. DCS/asymptomatic ratio was not initially different between sexes, although males were heavier than females. In three generations, the outcome of the dive significantly changed from 33% to 67% asymptomatic rats, for both sexes. Interestingly, survival in females increased sooner than in males. Conclusions This study offers evidence suggesting the inheritance of DCS resistance. Future research will focus on genetic and physiological comparisons between the initial strain and the new resistant population.