François Guerrero

Bubble Formation and Endothelial Function Before and After 3 Months of Dive Training

INTRODUCTION It has been suggested that repeated compression-decompression cycles reduce diver susceptibility to decompression sickness (DCS). This study examined whether intensive scuba dive training would reduce bubble formation and modulate endothelial function as shown by skin circulation. METHODS There were 22 military divers who were studied before and after a 90-d program of physical training and open-sea air diving (mean 67 dives total). Skin blood flow in the forearm was measured at rest (baseline), during post-occlusive hyperemia (endothelium-dependent vasodilatation), and with local heating to 42 degrees C (maximal vasodilatation). Subjects were also examined by pulsed Doppler for venous bubbles 30, 60, and 90 min after surfacing from a hyperbaric exposure to 400 kPa (30 msw) for 30 min in a dry chamber. RESULTS None of the divers experienced DCS during the training period. There was no change in weight, body mass index, maximal oxygen uptake, or endothelial function. Bubble grades by the Kisman Integrated Severity Score were significantly decreased immediately after the diving training period (3.6 +/- 9.2 vs. 16.4 +/- 14.3) and increased 3 mo after this period (10.3 +/- 13.9 vs. 3.6 +/- 9.2). DISCUSSION The results highlight that repeated scuba dives and regular physical exercise activity reduce bubble formation and probably have a protective effect against DCS risk. Although this phenomenon has been observed for decades, the mechanism remains complex and the results cannot elucidate the effects of physical exercise and NO production. Bubble formation could activate the stress response which could be the basis for diving acclimatization.

Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives.

PURPOSE Excessive reactive oxygen species (ROS) is considered a consequence of hyperoxia and a major contributor to diving-derived vascular endothelial damage and decompression sickness. The aims of this work were: 1) to directly observe endothelial ROS production during simulated air dives as well as its relation with both mitochondrial activity and cell survival; and 2) to determine which ambient factor during air diving (hydrostatic pressure or oxygen and/or nitrogen partial pressure) is responsible for the observed modifications. METHODS In vitro diving simulation was performed with bovine arterial endothelial cells under real-time observation. The effects of air diving, hydrostatic, oxygen and nitrogen pressures, and N-acetylcysteine (NAC) treatment on mitochondrial ROS generation, mitochondrial membrane potential and cellular survival during simulation were investigated. RESULTS Vascular endothelial cells performing air diving simulation suffered excessive mitochondrial ROS, mitochondrial depolarization, and cell death. These effects were prevented by NAC: after NAC treatment, the cells presented no difference in damage from nondiving cells. Oxygen diving showed a higher effect on ROS generation but lower impacts on mitochondrial depolarization and cell death than hydrostatic or nitrogen diving. Nitrogen diving had no effect on the inductions of ROS, mito-depolarization, or cell death. CONCLUSION This study is the first direct observation of mitochondrial ROS production, mitochondrial membrane potential and cell survival during diving. Simulated air SCUBA diving induces excessive ROS production, which leads to mitochondrial depolarization and endothelial cell death. Oxygen partial pressure plays a crucial role in the production of ROS. Deleterious effects of hyperoxia-induced ROS are potentiated by hydrostatic pressure. These findings hold new implications for the pathogenesis of diving-derived endothelial dysfunction.

Progressive Induction of Type 2 Diabetes: Effects of a Reality–Like Fructose Enriched Diet in Young Wistar Rats

Purpose The aim of this study was to characterize short and medium-lasting effects of fructose supplementation on young Wistar rats. The diet was similar to actual human consumption. Methods Three week old male rats were randomly divided into 2 groups: control (C; n = 16), fructose fed (FF; n = 16) with a fructose enriched drink for 6 or 12 weeks. Bodyweight, fasting glycemia and systolic blood pressure were monitored. Glucose tolerance was evaluated using an oral glucose tolerance test. Insulinemia was measured concomitantly and enable us to calculate insulin resistance markers (HOMA-IR, Insulin Sensitivity Index for glycemia: ISI-gly). Blood chemistry analyses were performed. Results After six weeks of fructose supplementation, rats were not overweight but presented increased fasting glycemia, reduced glucose tolerance, and lower insulin sensitivity compared to control group. Systolic blood pressure and heart weight were also increased without any change in renal function (theoretical creatinine clearance). After twelve weeks of fructose supplementation, FF rats had increased bodyweight and presented insulin resistance (higher HOMA-IR, lower ISI-gly). Rats also presented higher heart volume and lower ASAT/ALAT ratio (presumed liver lesion). Surprisingly, the Total Cholesterol/Triglycerides ratio was increased only after six weeks of fructose supplementation, predicting a higher LDL presence and thus a higher risk of developing cardiovascular disease. This risk was no longer present after twelve weeks of a fructose enriched diet. Conclusion On young Wistar rats, six weeks of fructose supplementation is sufficient to induce signs of metabolic syndrome. After twelve weeks of fructose enriched diet, rats are insulin resistant. This model enabled us to study longitudinally the early development of type 2 diabetes.

Effect of training frequency on endothelium-dependent vasorelaxation in rats

Background Moderate physical activity enhances endothelium-dependent vasorelaxation. Whether the frequency of exercise affects endothelial function is unclear. The purpose of this study was to investigate the effects of various frequencies of training on endothelium-dependent vasorelaxation. Design Male Wistar rats were trained for 8 weeks on a treadmill at various frequencies [1 (Ex1), 3 (Ex3) or 5 days/week (Ex5)] and compared with age-matched sedentary animals (SED). A control group allowed us to assess endothelial function before the exercise protocol. Rings of thoracic aorta were precontracted with phenylephrine. Results Endothelium-independent relaxation elicited by sodium nitroprusside was similar in all groups. The maximal response elicited by acetylcholine (ACh) was not different between groups, whereas pD2 values (−logEC50, EC50 being the concentration of ACh that elicited 50% of the maximal response) significantly correlated with frequency of training, nitro-L-arginine methyl ester (L-NAME) reduced the relaxation elicited by 10−7mol/l ACh or higher in control and all trained groups, and by 10−6mol/l ACh or higher in SED group. Indomethacin inhibited the vasodilating response to 10−7mol/l ACh or higher in control, SED and Ex1 groups, and to 10−8mol/l or more in Ex3 and Ex5 animals. Tetraethylammonium attenuated the response to 10−6mol/l ACh or higher in control and SED groups and to 10−7mol/l or more in all trained animals. Conclusion This data suggest that decreased ACh-induced vasorelaxation after physical inactivity may result from impairment of endothelial nitric oxide synthase, prostacyclin and endothelium-derived hyperpolarizing factor pathways. This effect is prevented by training in a frequency-dependent manner.

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.

Effect of decompression-induced bubble formation on highly trained divers microvascular function

We previously showed microvascular alteration of both endothelium‐dependent and ‐independent reactivity after a single SCUBA dive. We aimed to study mechanisms involved in this postdive vascular dysfunction. Ten divers each completed three protocols: (1) a SCUBA dive at 400 kPa for 30 min; (2) a 41‐min duration of seawater surface head immersed finning exercise to determine the effect of immersion and moderate physical activity; and (3) a simulated 41‐min dive breathing 100% oxygen (hyperbaric oxygen [HBO]) at 170 kPa in order to analyze the effect of diving‐induced hyperoxia. Bubble grades were monitored with Doppler. Cutaneous microvascular function was assessed by laser Doppler. Endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) reactivity was tested by iontophoresis. Endothelial cell activation was quantified by plasma Von Willebrand factor and nitric oxide (NO). Inactivation of NO by oxidative stress was assessed by plasma nitrotyrosine. Platelet factor 4 (PF4) was assessed in order to determine platelet aggregation. Blood was also analyzed for measurement of platelet count. Cutaneous vascular conductance (CVC) response to ACh delivery was not significantly decreased by the SCUBA protocol (23 ± 9% before vs. 17 ± 7% after; P = 0.122), whereas CVC response to SNP stimulation decreased significantly (23 ± 6% before vs. 10 ± 1% after; P = 0.039). The HBO and immersion protocols did not affect either endothelial‐dependent or ‐independent function. The immersion protocol induced a significant increase in NO (0.07 ± 0.01 vs. 0.12 ± 0.02 μg/mL; P = 0.035). This study highlighted change in microvascular endothelial‐independent but not ‐dependent function in highly trained divers after a single air dive. The results suggest that the effects of decompression on microvascular function may be modified by diving acclimatization.

The Effects of Ionotropic Agonists of Excitatory Amino Acids on the Release of Arginine Vasopressin in Rat Hypothalamic Slices

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose‐dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 × 10−8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 ± 7.6% of controls when the slices were obtained from chronically (7 days) salt‐loaded rats. Addition of 1 × 10−7 M NMDA or 1 × 10−6 M kainate to the incubation medium antagonized the salt loading‐induced increase in vasopressin release. Incubation with 1 × 10−4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 × 10−7 M NMDA or 1 × 10−6 M kainate or 1 × 10−6 M AMPA. Incubation with 1 × 10−5 M phaclophen (a GABAB antagonist) and 1 × 10−5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 × 10−7 M NMDA. Incubation with 1 × 10−6 M GABA alone decreased vasopressin secretion to 64.6 ± 6.9% of control values. The inhibitory effect of GABA did not change when 1 × 10−7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.

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.

Effect of simulated air dive and decompression sickness on the plasma proteome of rats

Decompression sickness (DCS) is a poorly understood systemic disease caused by inadequate desaturation following a reduction in ambient pressure. Although recent studies highlight the importance of circulating factors, the available data are still puzzling. In this study, we aimed to identify proteins and biological pathways involved in the development of DCS in rats.

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.