Acute Hyperbaric Oxygenation, Contrary to Intermittent Hyperbaric Oxygenation, Adversely Affects Vasorelaxation in Healthy Sprague‐Dawley Rats due to Increased Oxidative Stress

Figures

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  Figure 1. ACh-induced relaxation (AChIR) of isolated rat aorta rings in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups. AChIR was significantly impaired in the A-HBO2 group when compared to the other groups of rats with a 10−9–10−5 M ACh concentration. A-HBO2 rats exhibited lower sensitivity to ACh compared to other groups of rats (table). Half maximal effective concentration (EC50) presents concentration of ACh (M) which induces a response halfway between the baseline and maximum. LogEC50 values (shown in the corresponding tables) were compared by a one-way ANOVA test.

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  Figure 2. Mechanisms of AChIR response of isolated rat aorta rings in the CTRL (a), A-HBO2 (b), 24H-HBO2 (c), and 4D-HBO2 (d) groups of rats. Used concentrations: ACh 10−9 to 10−5 M, L-NAME 3 × 10−4 M, and TEMPOL 100 μM. Half maximal effective concentration (EC50) presents the concentration of ACh (M) which induces a response halfway between the baseline and maximum. The presence of L-NAME significantly reduced AChIR of isolated rat aortic rings in all experimental groups. TEMPOL administration significantly increased the AChIR response in the A-HBO2 group of rats, while it did not induce any significant change in the AChIR of isolated rat aortic rings in other groups. Data were compared by two-way ANOVA and Bonferroni post hoc tests. Tables within figures present sensitivity of aortic rings to ACh in the CTRL (a), A-HBO2 (b), 24H-HBO2 (c), and 4D-HBO2 (d) groups of rats. Sensitivity to ACh in the presence of L-NAME was significantly decreased compared to the basic response or response to ACh in the presence of TEMPOL in all experimental groups of rats. LogEC50 values were compared by one-way ANOVA followed by a Holm-Sidak pairwise multiple comparison.

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  Figure 3. Relaxation to acetylcholine in the presence of eNOS inhibitor L-NAME (a) and superoxide scavenger TEMPOL (b) in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups of rats. The presence of L-NAME inhibited AChIR more prominently in CTRL compared to 24H-HBO2 and 4D-HBO2 groups, and in A-HBO2 that in the 4D-HBO2 group of rats. AChIR in the presence of TEMPOL was lower in 4D-HBO2 than in the 24H-HBO2 group for ACh concentrations 10−8 and 10−7 M. Data were compared by two-way ANOVA and Bonferroni post hoc tests. Tables within figures present sensitivity of aortic rings to ACh in the presence of L-NAME (a) and TEMPOL (b) in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups of rats. Sensitivity to ACh in the presence of L-NAME did not differ among experimental groups of rats. Sensitivity to ACh in the presence of TEMPOL was significantly decreased in the 4D-HBO2 group compared to the other groups of rats. LogEC50 values were compared by one-way ANOVA followed by a Holm-Sidak pairwise multiple comparison.

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  Figure 4. Hypoxia-induced relaxation (HIR) response of isolated rat aorta rings in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups. HIR was significantly lower in A-HBO2 compared to all other groups of rats. The 24-HBO2 and 4D-HBO2 groups exhibited significantly increased HIR compared to CTRL and A-HBO2 groups of rats.

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  Figure 5. Relaxation to hypoxia in the presence of superoxide scavenger TEMPOL in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups of rats. TEMPOL administration significantly increased HIR compared to baseline measurement in the A-HBO2 and 24H-HBO2 groups of rats.

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  Figure 6. SNP-induced relaxation (SNPIR) of isolated rat aorta rings in the CTRL, A-HBO2, 24H-HBO2, and 4D-HBO2 groups. There was no difference in SNPIR among experimental groups of rats. Data were compared by two-way ANOVA and Bonferroni post hoc tests. The CTRL group exhibited lower sensitivity to SNP compared to the A-HBO2 and 24H-HBO2 groups of rats (†p < 0.05); the 4D-HBO2 group exhibited lower sensitivity to SNP compared to the 24H-HBO2 group of rats (‡p < 0.05) (table). Half maximal effective concentration (EC50) presents concentration of SNP (M) which induces a response halfway between the baseline and maximum. LogEC50 values (shown in corresponding tables) were compared by one-way ANOVA test.

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  Figure 7. (a) DHE superoxide fluorescence intensity measurement in rat aorta. The increase in superoxide levels in the aortas of rats exposed to hyperbaric oxygen was prevented by incubation with TEMPOL. Data are shown as means ± SEM of DHE fluorescence intensity units. ∗Significant increase in superoxide levels compared with control value before any treatment (p < 0.05; n = 9 rats). (b) In situ fluorescence of superoxide after dying of aortic rings with DHE color in all tested groups before and after incubation with TEMPOL. The color intensity is proportional to the amount of superoxide. The images were made on the Zeiss Axioskop MOT2 microscope, using the Olympus DP70 camera with the Zeiss filter set 15 (546 nm wavelength for excitation and 590 nm for emission with a beam splitter at 580 nm).

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  Figure 8. (a) DHE superoxide fluorescence intensity measurement in the rat femoral artery. The increase in superoxide levels in the artery of rats exposed to a single session of hyperbaric oxygen was prevented by incubation with TEMPOL. Data are shown as means ± SEM of DHE fluorescence intensity units. ∗Significant increase in superoxide levels compared with control value before any treatment (p < 0.05; n = 4 rats). (b) In situ fluorescence of superoxide after dying of the femoral artery with DHE color in all tested groups before and after incubation with TEMPOL. The color intensity is proportional to the amount of superoxide. The images were made on the Zeiss Axioskop MOT2 microscope, using the Olympus DP70 camera with the Zeiss filter set 15 (546 nm wavelength for excitation and 590 nm for emission with a beam splitter at 580 nm).

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  Figure 9. DHE superoxide fluorescence intensity measurement in the rat aorta before and after incubation with L-NAME. The superoxide levels in the aortas of rats exposed to a single session of hyperbaric oxygen was decreased by incubation with L-NAME compared to baseline. Data are shown as means ± SEM of DHE fluorescence intensity units. ∗Significant decrease in superoxide levels after L-NAME incubation compared with baseline value (p < 0.05; n = 9 rats).