Hydrogen gas with extracorporeal cardiopulmonary resuscitation improves survival after prolonged cardiac arrest in rats

Abstract

Background: Despite the benefits of extracorporeal cardiopulmonary resuscitation (ECPR) in cohorts of selected patients with cardiac arrest (CA), extracorporeal membrane oxygenation (ECMO) includes an artificial oxygenation membrane and circuits that contact the circulating blood and induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. There is currently no pharmacological treatment that has been proven to improve outcomes after CA/ECPR. We aimed to test the hypothesis that administration of hydrogen gas (H₂) combined with ECPR could improve outcomes after CA/ECPR in rats.

Methods: Rats were subjected to 20 min of asphyxial CA and were resuscitated by ECPR. Mechanical ventilation (MV) was initiated at the beginning of ECPR. Animals were randomly assigned to the placebo or H₂ gas treatment groups. The supplement gas was administered with O₂ through the ECMO membrane and MV. Survival time, electroencephalography (EEG), brain functional status, and brain tissue oxygenation were measured. Changes in the plasma levels of syndecan-1 (a marker of endothelial damage), multiple cytokines, chemokines, and metabolites were also evaluated.

Results: The survival rate at 4 h was 77.8% (7 out of 9) in the H₂ group and 22.2% (2 out of 9) in the placebo group. The Kaplan-Meier analysis showed that H₂ significantly improved the 4 h-survival endpoint (log-rank P = 0.025 vs. placebo). All animals treated with H₂ regained EEG activity, whereas no recovery was observed in animals treated with placebo. H₂ therapy markedly improved intra-resuscitation brain tissue oxygenation and prevented an increase in central venous pressure after ECPR. H₂ attenuated an increase in syndecan-1 levels and enhanced an increase in interleukin-10, vascular endothelial growth factor, and leptin levels after ECPR. Metabolomics analysis identified significant changes at 2 h after CA/ECPR between the two groups, particularly in D-glutamine and D-glutamate metabolism.

Conclusions: H₂ therapy improved mortality in highly lethal CA rats rescued by ECPR and helped recover brain electrical activity. The underlying mechanism might be linked to protective effects against endothelial damage. Further studies are warranted to elucidate the mechanisms responsible for the beneficial effects of H₂ on ischemia-reperfusion injury in critically ill patients who require ECMO support.

Keywords: Extracorporeal cardiopulmonary resuscitation; Extracorporeal membrane oxygenation; Heart arrest; Hydrogen; Ischemia reperfusion injury.

Fig. 1

Experimental protocol for extracorporeal cardiopulmonary resuscitation (ECPR) in a rat model of prolonged asphyxia cardiac arrest (CA). V-A ECMO: veno-arterial extracorporeal membrane oxygenation; MV: mechanical ventilation; ROSC: return of spontaneous circulation; ECG: electrocardiogram; EtCO₂: end-tidal carbon dioxide

Fig. 2

H₂ improved survival and brain recovery after CA/ECPR. A Survival rates during the first 4 h after CA. *log-rank P = 0.025 vs. placebo group. B Percentage of animals exhibiting responses to either or both the stimuli. n = 9 per group

Fig. 3

Representative electroencephalogram (EEG) wave forms showing brain electrical recovery after CA/ECPR. The bar indicates 100 s. Arrows indicate the time point of EEG disappearance

Fig. 4

Changes in brain tissue oxygenation (PtO₂, % change from baseline) during CA and ECPR with and without H₂. n = 7 per group (two-way repeated-measures ANOVA followed by Sidak’s correction, F = 2.51). **P = 0.009 vs. the placebo, ^##P = 0.007, ^###P = 0.0002, ^####P < 0.0001 vs. the baseline in the H₂ group

Fig. 5

Changes in the A esophageal temperature (Teso), B mean arterial pressure (MAP), C heart rate (HR), D dP/dt_max, E dP/dt_min, and F central venous pressure (CVP) during CA and ECPR. Two-way repeated-measures ANOVA followed by Sidak’s correction for post-hoc comparisons were used. F values for Teso, MAP, HR, dP/dt_max, dP/dt_min, and CVP were 1.89, 0.77, 0.58, 0.56, 1.18, and 1.15, respectively. Data are presented as the mean ± SEM. BL baseline, CA cardiac arrest; ECMO extracorporeal membrane oxygenation. ^#P < 0.05 vs. the baseline in the placebo group; *P < 0.05 vs. the baseline in the H₂ group; ^aP < 0.05 between groups

Fig. 6

Effects of H₂ on plasma mediators after CA/ECPR. Plasma levels of A syndecan-1, B interleukin (IL)-10, C vascular endothelial growth factor (VEGF), and D leptin at the baseline and at 2 h post-ECPR in animals treated with the placebo and H₂. n = 8 per group. Two-way repeated-measures ANOVA followed by Sidak’s correction for post-hoc comparisons were used. F values for Syndecan-1, IL-10, VEGF, and leptin were 1.47, 2.94, 3.97, and 6.76, respectively. Data are presented as the mean ± SEM

Fig. 7

Metabolomic analysis. A Heat map comparisons of differential metabolite alterations between groups. The heatmap was generated with MetaboAnalyst 5.0 using normalized data (log transformation, Auto scaling) and Euclidean distance. B Enrichment analysis. Metabolite set enrichment analysis (MSEA) was conducted to evaluate the impact of individual metabolite alterations between the placebo and H₂ groups. MSEA identified d-glutamine and d-glutamate metabolism (FDR = 1.62 × 10⁻²) as significantly discriminated among the groups. C Changes in plasma l-glutamic acid after CA/ECPR with and without H₂ (one-way repeated-measures ANOVA followed by Sidak’s correction, F = 8.33)