Argon treatment 3 h after stroke onset and 1 h after reperfusion improved neurological performance during the first week after ischemic stroke. The morphological analysis showed that argon administration preserved the neurons in cortex and subcortex of the ischemic boundary zone whereas the infarct volume and white matter integrity were not affected. In addition, argon alleviated the excessive microglia/macrophage activation in rat CNS. More interestingly, treatment with argon promoted the switch of microglia/macrophage polarization towards the anti-inflammatory M2 phenotype. The findings of the influences of argon on microglia/macrophage activation and polarization may lead to new understandings of the mechanisms involved in the neuroprotective effects of argon.

The neuroprotective effects of argon after ischemic stroke

The first description of the neuroprotective properties of argon could be dated back to 1998 [38]. Thereafter, argon was proven to be an effective neuroprotective agent in a series of models, ranging from in vitro models, such as oxygen and glucose deprivation (OGD) in cortical neuronal cell cultures and brain slices, focal mechanical trauma in organotypic hippocampal slice cultures and apoptosis in human neuroblastoma cells, to in vivo models including retinal ischemia-reperfusion injury, neonatal hypoxia-ischemia brain injury, cardiac arrest, cerebral ischemia injury, and subarachnoid hemorrhage [18,19,20,21,22,23,24,25,26,27]. Within the context of cerebral ischemia injury, argon was shown to reduce the infarct volume in cortex and basal ganglia and relieve the composite adverse outcome 24 h after reperfusion when administered for 1 h until reperfusion with an 1 h delay after tMCAO induction [18]. In the present study, we further verified that, with a 3 h delay after stroke onset and 1 h after reperfusion, argon significantly alleviated neurological deficit during the first week after stroke and promoted neuronal survival in the cortex and subcortex of the ischemic boundary zone 7 days after reperfusion. However, the infarct volume and white matter integrity were not affected by argon treatment.

Although statistical analysis showed that the course of left CBF did not differ between tMCAO Ar and tMCAO N 2 groups in the present study, the degree of CBF reduction within the infarct period and CBF recovery after reperfusion was not identical. Animals in tMCAO N 2 group had relatively larger blood flow reduction and delayed interval to complete reperfusion. These variations between tMCAO Ar and tMCAO N 2 groups may influence the histological and behavioral outcomes and might contribute to the inconsistency of the infarct volume and neuroscore results in the present study. Nevertheless, the calculated sample size was based on the primary outcome, namely the 6-point neuroscore from 24 h to d7 after reperfusion. Larger sample size might be needed to detect the difference in infarct volume. In humans, the size of the lesion does not always correlate with functional impairments [39]. In animal studies, the inconsistency of infarct size and functional outcomes has also been reported [40,41,42,43]. Except for the influence of the infarct core, the functional and/or structural reorganization of the remaining brain may also play important roles after ischemic stroke. In particular, the peri-infarct tissue is an important target for neurorepair and neuroprotective therapies [44]. The present findings, namely the increased number of surviving neurons at the IBZ along with the improved functional outcome generated by argon treatment, do support this point of view. Other functional and/or structural alterations including cerebral vasoconstriction, blood-brain barrier permeability, synaptic plasticity, axonal remodeling, neurogenesis, microglia/macrophage activation, astrocyte reactivity, and angiogenesis at the IBZ, even within the non-affected hemisphere, are of importance to uncover the panorama of argon’s neuroprotective effects after ischemic stroke [8, 37, 42, 45]. Meanwhile, a battery of functional assessments with respect to different aspects of the sensorimotor function as well as the cognitive function would also be beneficial to further comprehend the protective capacity of the treatment.

The influences of argon on microglia/macrophage activation and polarization after ischemic stroke

Previous studies found that both the general microglia/macrophages and the M2 phenotype peaked at d7 after ischemic stroke insult [36, 46]. Thus, this observational time point was used in the present study to achieve the optimal exploration into the changes of microglia/macrophage activation and polarization after argon treatment.

In 2012, Fahlenkamp and colleagues first discussed the influence of argon on microglia activation using an in vitro model of LPS-induced inflammation in microglia cell cultures [29]. The authors demonstrated that administration of argon suppressed the expression of the pro-inflammatory cytokine IL-1β. Later on, using cortical neuronal cell cultures subjected to OGD, Zhang and colleagues showed that argon exposure attenuated neuronal cell death and reduced the expression of the pro-inflammatory cytokines TNF-α and IL-6 [24]. In the present study, using an in vivo cerebral ischemia injury model, we demonstrated that argon significantly attenuated the excessive microglia/macrophage activation in rat CNS caused by the insult.

In recent years, researchers have put great effort to explore the mechanisms of argon’s neuroprotective effects. So far, they are mainly related to the anti-apoptotic and the anti-oxidative stress abilities. However, the anti-neuroinflammatory potential of argon has been emerging with time [24, 28, 47]. Several receptors, such as toll-like receptors, purinergic receptors, chemokine receptor CCR2, Fc receptors, receptor for advanced glycation end products (RAGE), cysteinyl leukotriene receptor 2, galectin-3, and CD36, were shown to be involved in mediating microglia activation and its functions including inflammation, motility, migration, phagocytosis, and survival during ischemic stroke [8]. Among these receptors, the TLRs have been widely studied and were confirmed to be essential for microglia-mediated inflammation [8, 10]. In a clinical trial, Brea and colleagues found that TLR2 and TLR4 were associated with poor outcome and correlated with higher serum levels of IL-1β, TNF-α, and IL-6 in patients [48]. In animal studies, stimulation or inhibition of TLR2 and TLR4 resulted in corresponding changes in pro-inflammatory cytokines, brain infarct volume, and functional outcomes [8]. Notably, in models of apoptosis in human neuroblastoma cells and ischemia-reperfusion injury in retina, argon was proven to mediate neuroprotection via inhibiting TLR2 and TLR4 [25, 49]. Whether argon affects the microglia/macrophage activation triggered by ischemic stroke via modulating TLRs and other above-mentioned receptors and how this relates to the neuroprotective effects of the agent require further elucidation.

Over the past few years, increasing evidence manifested that M1-M2 microglia/macrophage polarization is involved in ischemic stroke insult [17, 36, 50]. Generally, the microglia/macrophages could be divided into two phenotypes, the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. The M1 phenotype is characterized by a high production of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, CC-chemokine ligand (CCL) 2, and C-X-C motif chemokine (CXCL) 10, reactive oxygen species (ROS), nitric oxide (NO), and inducible nitric oxide synthase (iNOS) as well as proteolytic enzymes matrix metalloproteinase (MMP)-9 and MMP-3 [8, 10]. In contrast, the M2 phenotype is characterized by enhanced expression of Arg1, Ym1, insulin-like growth factor (IGF)-1, CD206, chitinase 3-like 3, and Fizz1 and is capable of producing anti-inflammatory cytokines, such as IL-10, transforming growth factor (TGF)-β, IL-4, and IL-13 [8, 10]. Hu and colleagues reported that local microglia and newly recruited macrophages assumed the M2 phenotype at early stages of ischemic stroke but gradually transformed into the M1 phenotype in the peri-infarct region [50]. In the present study, we first revealed that administration of argon significantly promoted the switch of microglia/macrophage polarization towards M2 phenotype after ischemic stroke. In addition, except for serving as a prominent M2 marker, Arg1 is generally considered as a protective and regeneration promoting enzyme, since it counteracts the excessive production of cytotoxic NO and is responsible for the generation of polyamines and collagen [37, 51].

A series of intracellular molecules including signal transducer and activator of transcription (STAT) family members, peroxisome proliferator-activated receptor γ (PPARγ), interferon regulatory factors (IRFs), and microRNAs were identified as key modulators of microglia/macrophage polarization [17]. Interestingly, using a model of apoptosis in human neuroblastoma cells, Ulbrich and colleagues observed that argon affected the phosphorylation and binding activity of STAT3, and that inhibition of STAT3 attenuated argon’s anti-apoptotic effect [25]. It is reasonable to assume that argon influences the activation and function of STAT3, which in turn produces an effect on microglia/macrophage polarization, and ultimately leads to behavioral and histological improvements after ischemic stroke. However, further studies will be needed to confirm this hypothesis. Moreover, some signaling pathways, such as mammalian target of rapamycin complex 1 (mTORC1) pathway and 5′ AMP-activated protein kinase (AMPK) pathway, were also identified recently to be essential for microglia/macrophage polarization after stroke [52, 53]. The roles of these molecules and pathways in the regulation of microglia/macrophage polarization and the neuroprotection induced by argon treatment should be further investigated.

Limitations and prospective

The design of a 3 h interval of argon administration after stroke onset and the prolonged observational period would provide valuable data to guide the development of new therapeutic strategies. Furthermore, the novel findings with regard to the microglia/macrophage activation and polarization would give rise to more comprehensive understandings of the mechanisms of argon’s neuroprotective effects. However, several limitations exist in the present study. Further studies are advisable to confirm and extend the findings here.

The present study observed the effects of argon treatment up to 7 days after stroke event. According to the Stroke Therapy Academic Industry Roundtable recommendations, studies conducted at least 2 to 3 weeks or longer after stroke onset would be needed to demonstrate a sustained benefit by candidate neuroprotective agents [54]. Meanwhile, behavioral assessments with respect to different aspects of the sensorimotor function and the cognitive function, as well as evaluations of multi-faceted histological changes after treatment are of importance.

In most of the animal studies, cerebral ischemia was induced in young healthy animals. In contrast, stroke occurs in humans as a result of the natural progression of underlying diseases or risk factors, such as aging, hypertension, and diabetes [54]. Thus, studies performed in age-related or disease-related models may be of great interest and would extend the understanding of argon’s neuroprotective effect after stroke.

As mentioned above, the degree of CBF reduction within the infarct period and CBF recovery after reperfusion was not strictly the same in tMCAO Ar and tMCAO N 2 groups. The variations may have impact on the research findings. Therefore, further studies should strive to maintain the stability and consistency of the tMCAO procedure. A larger sample size may also be beneficial.

In the present study, due to limited resources, Luxol Fast Blue myelin staining was used to assess the white matter damage after stroke. Although Luxol Fast Blue staining was proven to be an effective method to detect the demyelination in CNS [33], more specific and sensitive method such as immunohistochemical staining of myelin basic protein (MBP) may provide more valuable information.

Despite the comparability of blood gas analysis parameters between tMCAO Ar and tMCAO N 2 groups, mild hypercapnia was detected in both groups in the present study. Hypercapnia may lead to cerebral vasodilation, increased intracranial pressure as well as cerebral edema. These effects could be even more severe when an intracranial disease, such as stroke, exists. Studies in the future should try to avoid hypercapnia so that its’ potential interference with cerebral pathophysiologic changes in the context of ischemic stroke could be excluded.

The generation of a dose-response curve is critical for neuroprotective drug candidates. 50% vol argon was used in the present study based on previous reports of in vitro and in vivo investigations [18,19,20]. However, there is a need to further assess the ideal concentration, timing, and duration of argon application in in vivo tMCAO models.