This study is the first to systematically explore the neuroprotective effect of iAr under different therapeutic schedules in tMCAO-induced moderate and severe ischemic stroke models. Our results suggest that compared to the tMCAO group, the groups administered 39% and 79% argon administration at 1 h after stroke onset for 1 h and 3 h showed a significant reduction in infarct volume in the severe cerebral ischemia model, and neurological outcome was obviously improved in a dose-dependent manner with 3 h of treatment. The neuroprotective efficacy of 79% argon administration at 1 h after stroke onset for 3 h was further validated in a moderate stroke model. Moreover, we found that the optimal schedule of argon treatment reduced edema in both models.
The neuroprotective effects of inhaled argon on the infarct volume after ischemic stroke
We observed that argon administration improved the infarct volume in severe ischemic stroke at 1 h after stroke onset (during ischemia) but not at 3 h after stroke onset (during reperfusion) in exp 1. Consistent with our observations during ischemia, Ryang et al observed improved infarct volume when argon was administered for 1 h during ischemia after tMCAO induction. During reperfusion, David et al found that argon decreased the subcortical volume but increased the subcortical volumes of damaged brain regions when given 1 h after reperfusion (during reperfusion), which is partly consistent with our results. Coburn et al reported that the infarct volume was not affected by argon treatment with a 3 h delay after stroke onset and 1 h after reperfusion (during reperfusion). Similarly, Ma et al. suggested that the infarct size was not decreased when argon was administered for 24 h during reperfusion. Notably, our results showed that argon administered for 1 h, 3 h, or 24 h during ischemia decreased the infarct volume, especially at 1–3 h. We observed that during the trial by Ma et al, the initial application of argon for 24 h occurred during the reperfusion phase and, in our study, during the ischemic phase in exp 2. Overall, current evidence seems to show that iAr effectively improves the infarct volume when administered during ischemia, not reperfusion. The reasons for these results are unclear. One possible reason is that application of iAr immediately after reperfusion (3 h after ischemia) may miss the therapeutic window for neuroprotective intervention in the ischemic penumbra. Many animal and human experimental studies suggest that the therapeutic window for neuroprotective intervention in the ischemic penumbra is very brief, often less than 2 or 3 h[36, 37]. Another possible reason is that with the increase in the duration of ischemia, the ischemic core area is further enlarged, and ischemia-reperfusion injury may simply counteract or even exceed the protective effect of argon, as first proposed by Ma et al. Necroptosis and apoptosis are of great importance in cerebral ischemia-reperfusion injury, and the activation of necroptosis depends on reperfusion and is activated immediately after ischemic insult on reperfusion. Reperfusion itself followed by MCAO can lead to neuronal death due to overperfusion and hemorrhagic transformation, which aggravates ischemic brain injury[39, 40]. Zhuang et al. showed the effect of argon on the antiapoptotic signaling pathway in a neonatal rat asphyxia model. Therefore, we speculate that the antiapoptotic activity of argon may be offset or even concealed by this reperfusion injury to some extent. Moreover, recent literature has reported that treatment with argon promotes the switch of microglia/macrophage polarization toward the anti-inflammatory M2 phenotype. In addition, cerebral ischemia and ischemia coupled with reperfusion result in differing pathologic mechanisms and microglial morphological responses, and a spatiotemporal relationship exists between microglial morphology and evolving brain injury after ischemic stroke and reperfusion. We speculate that iAr can impact the balance between proinflammatory and anti-inflammatory cytokines to anti-inflammation after AIS and affects the infarct size under the appropriate combination of treatment timing and duration of treatment (for example, short-term intervention during ischemia). However, this is just our speculation, and additional experiments are needed to validate this hypothesis and further dissect the possible mechanisms involved.
Recovery of neurological function with inhaled argon after ischemic stroke
Functional recovery is one of the main end outcomes of stroke patients. Clinically, it is customary to divide the neurological impairment of patients with stroke into general functional injury and focal functional injury. The former reflects the patient's life state and global neurological function, while the latter reflects localized functional defects caused by local injury. Therefore, in addition to infarct volume, neurological deficits were assessed in this study according to Clark’s scoring system, reflecting the degree of neurological injury after ischemia, from the point of view of general functional injury and focal functional injury. Our in vivo results suggested that argon administered at 39 vol% and 79 vol% could improve neurologic outcome in exp 3 in a dose-dependent manner. In accordance with our results, previous studies suggest that argon exerts neuroprotective effects in a dose-dependent manner in different injury models (hypoxia vs. ischemia), such as models of traumatic brain injury (TBI), retinal ischemia/reperfusion injury (IRI) and cardiac arrest (CA). Similarly, Coburn et al. demonstrated significantly improved neurological performance using a 6-point neuroscore daily from 24 h to 7 d after reperfusion. In addition, Ma et al. found that neurologic outcome was significantly improved after 24 h of treatment with argon instituted after reperfusion. However, in contrast with its beneficial effect on the neurological outcome, David et al. found that 50 vol% argon inhaled during reperfusion for 3 h failed to improve MCAO-induced neurologic deficits. The results seem to indicate that even if argon is administered at similar time points (such as after reperfusion), it will lead to significant differences in neurological impairment due to different treatment protocols, such as durations and concentrations. Notably, however, in addition to the initial time point of iAr, the difference in neurologic assessment systems is one factor to be considered.
An interesting aspect of our research is that argon administration for 1 h or 24 h during ischemia reduced infarct volume, but neurologic outcomes were not improved. Another unanticipated result was that iAr caused dose-dependent improvement of neurological prognosis in our study, which was also inconsistent with observations of infarct volume. These results reflect those of Coburn et al., who found that with a 3 h delay after stroke onset and 1 h after reperfusion, argon significantly alleviated neurological deficits during the first week after stroke but failed to reduce the infarct volume. Additionally, these differences corroborate the ideas of Ma et al., who found that neurologic outcome was improved but that infarct size was not reduced when argon administration was delayed 2 h after permanent stroke onset or instituted after reperfusion. One possible explanation of this difference may be that although there is a correlation between the focal functional injury score and cerebral infarction volume of the Clark score, evidence from the recent literature indicates that histologic lesion size was less correlated with improved neurologic outcome, as Ma and colleagues clearly pointed out.
The influences of argon on edema after ischemic stroke
Few studies have addressed the effect of iAr on the complications of AIS. However, the complications of AIS have significant effects on the short-term and long-term prognosis of patients, and brain edema is a common complication of AIS and one of the main causes of death. Acute brain injury is linked to cellular edema, characterized by abnormal intracellular accumulation of water in brain cells, resulting in cellular swelling. Delayed reperfusion, whether by thrombolysis or endovascular therapy, can result in more severe brain edema and poorer clinical outcomes and increase the risk of mortality, hemorrhagic transformation, headache, and seizures[45, 46]. Brain edema is also an important prognostic indicator in the assessment of argon efficacy in severe ischemic stroke induced by delayed reperfusion. As previously reported[10, 11, 15, 47, 48], argon has been shown to exert a neuroprotective effect in in vivo tMCAO models induced by ischemia for 1–2 h, consistent with our findings. Our study showed that neuroprotection still existed in severe ischemic stroke induced by an increasing ischemic duration beyond those commonly used at clinically relevant time windows. The results showed that argon treatment in moderate and severe AIS models led to significantly reduced brain water content, infarct volume, neurologic function, and weight recovery at the same treatment time point, duration and concentration. Importantly, delayed reperfusion affects the ability of iAr to improve brain edema, infarction volume and neurologic function following tMCAO. The design of the present study allowed conclusions to be drawn regarding whether a higher concentration of argon inhaled for 3 h during ischemia would improve the complications characterized by brain edema in tMCAO model. Further studies are needed to clarify the underlying mechanism of this improvement and should pay more attention to argon-induced improvements of other complications of AIS, such as hemorrhagic transformation.
Limitations and prospective
There are several design limitations in our experiment that deserve special attention. First, an important limitation is the lack of data to measure potential medium- and long-term effects, which are clinically important for the prognosis of patients with acute ischemic stroke. Animals survived for only 24 h after reperfusion, which may contribute to some results that are not wholly consistent with the conclusions of our study at a later sampling time. Longer recovery times should be included in future studies, and functional imaging, such as MRI, can be applied to monitor the evolution of brain injury and treatment. Second, animal models other than rodents should be adopted, including large animal models[49, 50], such as pigs, dogs and monkeys. Further research should test the same strategies in larger species with basic diseases such as hypertension or diabetes and explore the impact of other biological variables such as sex and age. These factors were not explored in this study because of budgetary limitations. Finally, this study is limited because it considered only the timing, duration and concentration; however, the specific mechanism involved in this improvement was not investigated, and this is part of our future research contents and direction. Thus, further animal and clinical studies addressing these issues at length are needed. Despite these limitations, this study is the first to demonstrate the effects of argon on cellular edema after MCAO-induced severe cerebral ischemic stroke in mice. Moreover, our study adopted the synthesis of two kinds of neurobehavioral scores that are suitable for clinical practice, and the behavioral experiments were double blinded to limit bias.