Background In recent years, argon has been shown to exert neuroprotective effects in an array of models. However, the mechanisms by which argon exerts its neuroprotective characteristics remain unclear. Accumulating evidence imply that argon may exert neuroprotective effects via modulating the activation and polarization of microglia/macrophages after ischemic stroke. In the present study, we analyzed the underlying neuroprotective effects of delayed argon application until 7 days after reperfusion and explored the potential mechanisms. Methods Twenty-one male Wistar rats underwent transient middle cerebral artery occlusion or sham surgery randomly for 2 h using the endoluminal thread model. Three hours after transient middle cerebral artery occlusion induction and 1 h after reperfusion, animals received either 50% vol Argon/50% vol O 2 or 50% vol N 2 /50% vol O 2 for 1 h. The primary outcome was the 6-point neuroscore from 24 h to d7 after reperfusion. Histological analyses including infarct volume, survival of neurons (NeuN) at the ischemic boundary zone, white matter integrity (Luxol Fast Blue), microglia/macrophage activation (Iba1), and polarization (Iba1/Arginase1 double staining) on d7 were conducted as well. Sample size calculation was performed using nQuery Advisor + nTerim 4.0. Independent t test, one-way ANOVA and repeated measures ANOVA were performed, respectively, for statistical analysis (SPSS 23.0). Results The 6-point neuroscore from 24 h to d7 after reperfusion showed that tMCAO Ar group displayed significantly improved neurological performance compared to tMCAO N 2 group ( p = 0.026). The relative numbers of NeuN-positive cells in the ROIs of tMCAO Ar group significantly increased compared to tMCAO N 2 group ( p = 0.010 for cortex and p = 0.011 for subcortex). Argon significantly suppressed the microglia/macrophage activation as revealed by Iba1 staining ( p = 0.0076) and promoted the M2 microglia/macrophage polarization as revealed by Iba1/Arginase 1 double staining ( p = 0.000095). Conclusions Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion significantly alleviated neurological deficit within the first week and preserved the neurons at the ischemic boundary zone 7 days after stroke. Moreover, argon reduced the excessive microglia/macrophage activation and promoted the switch of microglia/macrophage polarization towards the anti-inflammatory M2 phenotype. Studies making efforts to further elucidate the protective mechanisms and to benefit the translational application are of great value.

In a previous study from our group, argon has shown to significantly attenuate brain injury, reduce brain inflammation and enhance M2 microglia/macrophage polarization until 7 days after ischemic stroke. However, the long-term effects of argon have not been reported thus far. In the present study, we analyzed the underlying neuroprotective effects and potential mechanisms of argon, up to 30 days after ischemic stroke. Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion demonstrated long-term neuroprotective effect by preserving the neurons at the ischemic boundary zone 30 days after stroke. Furthermore, the excessive microglia/macrophage activation in rat brain was reduced by argon treatment 30 days after ischemic insult. However, long-lasting neurological improvement was not detectable. More sensorimotor functional measures, age- and disease-related models, as well as further histological and molecular biological analyses will be needed to extend the understanding of argon’s neuroprotective effects and mechanism of action after ischemic stroke.

The poor translational success rate of preclinical stroke research may partly be due to inaccurate modelling of the disease. We provide data on transient middle cerebral artery occlusion (tMCAO) experiments including detailed intraoperative monitoring to elaborate predictors indicating experimental success (ischemia without occurrence of confounding pathologies).The tMCAO monitoring data (bilateral cerebral blood flow - CBF, heart rate - HR, mean arterial pressure - MAP) of 16 animals with an “ideal” outcome (MCA-ischemia) and 48 animals with additional or other pathologies (subdural hematoma or subarachnoid hemorrhage) were checked for their prognostic performance (receiver operating characteristic curve and area under the curve - AUC).Animals showing a decrease in the contralateral CBF at the time of MCA occlusion suffered from unintended pathologies. Implementation of baseline MAP in addition to baseline HR (AUC 0.83, 95% CI 0.68 to 0.97) increased prognostic relevance (AUC 0.89, 95% CI 0.79 to 0.98). Prediction performance improved when two additional predictors referring differences in left and right CBF were considered (AUC 1.00, 95% CI 1.0 to 1.0).Our data underline the importance of periinterventional monitoring to verify a successful experimental performance in order to ensure a disease model as homogeneous as possible.

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