Aleksandra Mazur

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.

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.

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.

Intravital investigation of rat mesenteric small artery tone and blood flow

Substantial information on rat mesenteric small artery physiology and pharmacology based on in vitro experiments is available. Little is known about the relevance of this for artery function in vivo. We here present an intravital model where rat mesenteric small artery diameters are studied under isolated and controlled conditions in situ with simultaneous measurement of blood flow. The responses of the isolated arteries vary with the anaesthetic used, and they are quantitatively but not qualitatively different from the responses seen in vitro.

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.

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.

A ternary model of decompression sickness in rats

BACKGROUND Decompression sickness (DCS) in rats is commonly modelled as a binary outcome. The present study aimed to develop a ternary model of predicting probability of DCS in rats, (as no-DCS, survivable-DCS or death), based upon the compression/decompression profile and physiological characteristics of each rat. METHODS A literature search identified dive profiles with outcomes no-DCS, survivable-DCS or death by DCS. Inclusion criteria were that at least one rat was represented in each DCS status, not treated with drugs or simulated ascent to altitude, that strain, sex, breathing gases and compression/decompression profile were described and that weight was reported. A dataset was compiled (n=1602 rats) from 15 studies using 22 dive profiles and two strains of both sexes. Inert gas pressures in five compartments were estimated. Using ordinal logistic regression, model-fit of the calibration dataset was optimised by maximum log likelihood. Two validation datasets assessed model robustness. RESULTS In the interpolation dataset the model predicted 10/15 cases of nDCS, 3/3 sDCS and 2/2 dDCS, totalling 15/20 (75% accuracy) and 18.5/20 (92.5%) were within 95% confidence intervals. Mean weight in the extrapolation dataset was more than 2SD outside of the calibration dataset and the probability of each outcome was not predictable. DISCUSSION This model is reliable for the prediction of DCS status providing the dive profile and rat characteristics are within the range of parameters used to optimise the model. The addition of data with a wider range of parameters should improve the applicability of the model.