A total of 12 animals (n = 3 in each group) underwent myocardial and neuronal sampling for histopathology analysis because only 3 animals survived to 96 h in both Ctrl and TH groups. Representative micrographs of the HE-stained left ventricular are shown in Figure 4. Animals in the Ctrl group exhibited distinctive contraction bands and infiltration of a large number of immune cells. Compared with control, minor irregularities and few immune cells were observed in the three experimental groups. Representative images of the hippocampal CA1 region are shown in Figure 5. Many degenerated neurons with hypereosinophilic cytoplasm and dark shrunken nuclei were detectable in the Ctrl and TH groups. On the contrary, only few degenerated neurons and a lot of remaining viable neurons were observed in H2 and H2+TH groups. The overall myocardial damage score was significantly lower in TH, H2 and H2+TH groups compared to Ctrl (Fig. 6A). In addition, the H2+TH group had significantly reduced myocardial damage score compared to TH group. The neuronal damage score was also markedly reduced in H2 and H2+TH groups (Fig. 6B), compared either with Ctrl, or with TH.
As hydrogen was reported to reduce infarct size and to improve neurological function in a rat model of middle cerebral artery occlusion, the effects of hydrogen have been studied on other animal models cardiac arrest and CPR (9). Hayashida et al. (10) first reported that inhalation of mixed gas comprising 2% hydrogen and 98% oxygen for 2 h at the beginning of CPR improved brain and cardiac function to an extent comparable to mild hypothermia in VF model of rats. The authors subsequently observed that inhalation of mixed gas comprising 1.3% hydrogen and 26% oxygen after ROSC for 2 h also improved neurological recovery and survival (11). Consistent with these studies, we observed that 96-h survival was greatly improved by inhaling 2% hydrogen for 2 h immediately after ROSC in animals with intrinsic cardiac disease, either under normothermia or hypothermia. The improved outcome can be attributed to both ameliorated postresuscitation myocardial dysfunction and brain injury. Furthermore, our results suggested that the beneficial effects of hydrogen outperform mild hypothermia in rats with spontaneous hypertension. This result was in agreement with previous animal studies. In isolated perfused hearts, Hayashida et al. (29) demonstrated that inhalation of hydrogen gas reduced infarct size without altering hemodynamic parameters, preventing deleterious left ventricular remodeling. Moreover, inhalation of hydrogen also prevented PCAS-associated increases in left ventricular end-diastolic pressure and serum IL-6 (10), which was not observed in animals treated with hypothermia (30). In a rat model of asphyxial cardiac arrest, hydrogen inhalation was also demonstrated to be superior to mild hypothermia in improving cardiac function and neurological outcome in our previous study (12).
The observation that hydrogen was superior to hypothermia on reducing postresuscitation cerebral injury can be explained by the difference in potential mechanisms of action. On one side, the protective role of hydrogen is primarily through selective reactive oxygen species attenuation (27, 31), whereas the physiological effects of hypothermia are multiple mechanisms of action blocking the cascade of ischemia on many levels (23). Although both therapies protect the organs by suppressing free radicals, inflammatory and apoptotic processes associated with reperfusion injury, inhaled hydrogen acts more rapidly because it is permeable to cell membranes and diffuses into ischemic tissues and cells in a timely fashion (32, 32). In addition, hydrogen specifically quenches detrimental reactive oxygen species while maintaining the metabolic oxidation-reduction reaction (31, 32). In contrast, hypothermia reduces the production of radical oxygen species through diminishing tissue metabolism (23, 33). On the other side, hydrogen has low toxicity with little drug–drug interaction and there is no reported potential side effect up to now (31–35). On the contrary, a number of adverse effects have been reported in the majority of patients treated with hypothermia, including pneumonia, sepsis, arrhythmia, electrolyte imbalance, bleeding, shivering, and coagulopathy (25, 36, 37). These advantages together with its effectiveness, suggest that hydrogen may be a good candidate in the management of PCAS as an effective therapy.
We recognize several limitations in our study. First, even though SHR is widely used for experimental studies because of its reliable spontaneous development of hypertension and hypertensive complications, the essential hypertension and destructive process may be different in rats compared with human. Second, the animals did not receive active antihypertensive treatment before induction of cardiac arrest. The results therefore should be more carefully interpreted when applied into clinical practice. Third, although we showed that hydrogen inhalation was superior to mild hypothermia for neuroprotection, the exact mechanism is still undetermined.
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