Treatment with inhaled Argon: a systematic review of pre-clinical and clinical studies with meta-analysis on neuroprotective effect.

BACKGROUND: Argon (Ar) has been proposed as a potential therapeutic agent in multiple clinical conditions, specifically in organ protection. However, conflicting data on pre-clinical models, together with a great variability in Ar administration protocols and outcome assessments, have been reported. The aim of this study was to review evidence on treatment with Ar, with an extensive investigation on its neuroprotective effect, and to summarise all tested administration protocols. METHODS: Using the PubMed database, all existing pre-clinical and clinical studies on the treatment with Ar were systematically reviewed (registration: https://doi.org/10.17605/OSF.IO/7983D). Study titles and abstracts were screened, extracting data from relevant studies post full-text review. Exclusion criteria included absence of full text and non-English language. Furthermore, meta-analysis was also performed to assess Ar potential as neuroprotectant agent in different clinical conditions: cardiac arrest, traumatic brain injury, ischemic stroke, perinatal hypoxic-ischemic encephalopathy, subarachnoid haemorrhage. Standardised mean differences for neurological, cognitive and locomotor, histological, and physiological measures were evaluated, through appropriate tests, clinical, and laboratory variables. In vivo studies were evaluated for risk of bias using the Systematic Review Center for Laboratory Animal Experimentation tool, while in vitro studies underwent assessment with a tool developed by the Office of Health Assessment and Translation. FINDINGS: The systematic review detected 60 experimental studies (16 in vitro, 7 ex vivo, 31 in vivo, 6 with both in vitro and in vivo) investigating the role of Ar. Only one clinical study was found. Data from six in vitro and nineteen in vivo studies were included in the meta-analyses. In pre-clinical models, Ar administration resulted in improved neurological, cognitive and locomotor, and histological outcomes without any change in physiological parameters (i.e., absence of adverse events). INTERPRETATION: This systematic review and meta-analysis based on experimental studies supports the neuroprotective effect of Ar, thus providing a rationale for potential translation of Ar treatment in humans. Despite adherence to established guidelines and methodologies, limitations in data availability prevented further analyses to investigate potential sources of heterogeneity due to study design. FUNDING: This study was funded in part by Italian Ministry of Health-Current researchIRCCS and by Ministero della Salute Italiano, Ricerca Finalizzata, project no. RF 2019-12371416.

Comparative assessment of direct and indirect cold atmospheric plasma effects, based on helium and argon, on human glioblastoma: an in vitro and in vivo study.

Recent research has highlighted the promising potential of cold atmospheric plasma (CAP) in cancer therapy. However, variations in study outcomes are attributed to differences in CAP devices and plasma parameters, which lead to diverse compositions of plasma products, including electrons, charged particles, reactive species, UV light, and heat. This study aimed to evaluate and compare the optimal exposure time, duration, and direction-dependent cellular effects of two CAPs, based on argon and helium gases, on glioblastoma U-87 MG cancer cells and an animal model of GBM. Two plasma jets were used as low-temperature plasma sources in which helium or argon gas was ionized by high voltage (4.5 kV) and frequency (20 kHz). In vitro assessments on human GBM and normal astrocyte cell lines, using MTT assays, flow cytometry analysis, wound healing assays, and immunocytochemistry for Caspase3 and P53 proteins, demonstrated that all studied plasma jets, especially indirect argon CAP, selectively induced apoptosis, hindered tumor cell growth, and inhibited migration. These effects occurred concurrently with increased intracellular levels of reactive oxygen species and decreased total antioxidant capacity in the cells. In vivo results further supported these findings, indicating that single indirect argon and direct helium CAP therapy, equal to high dose Temozolomide treatment, induced tumor cell death in a rat model of GBM. This was concurrent with a reduction in tumor size observed through PET-CT scan imaging and a significant increase in the survival rate. Additionally, there was a decrease in GFAP protein levels, a significant GBM tumor marker, and an increase in P53 protein expression based on immunohistochemical analyses. Furthermore, Ledge beam test analysis revealed general motor function improvement after indirect argon CAP therapy, similar to Temozolomide treatment. Taken together, these results suggest that CAP therapy, using indirect argon and direct helium jets, holds great promise for clinical applications in GBM treatment.

Chemiluminescent Analysis of Oxidative Metabolism in Rat Blood under the Influence of Argon and Helium.

We studied the nature of the action of course treatment with argon and helium (1 min, 3 procedures) on the oxidative metabolism in rat blood plasma. The study was performed on 30 Wistar rats divided into 3 groups (n=10 in each group): intact and 2 experimental (treatment of the skin of the back with a stream of argon and helium, respectively). After completion of the treatment course, the intensity of free radical processes, the total antioxidant activity, and malondialdehyde concentration were evaluated in the blood plasma. It was found that argon and helium gas flows provide stimulation of antioxidant systems, but the mechanisms of their effect were different. Treatment with helium did not affect the intensity of free radical processes, but significantly increased the overall antioxidant activity of blood plasma and reduced malondialdehyde concentration in comparison with the effect of argon flow.

Antimicrobial activity of cold atmospheric-pressure argon plasma combined with chicory (Cichorium intybus L.) extract against P. aeruginosa and E. coli biofilms.

The present study reports a significant combined antibacterial activity of Cichorium intybus L. (known as Chicory) natural extract with cold atmospheric-pressure argon plasma treatment against multi-drug resistant (MDR) Gram-negative bacteria. To detect reactive species that are generated in the argon plasma, optical emission spectra were recorded. The molecular bands were allocated to the hydroxyl radicals (OH) and neutral nitrogen molecules (N2). Moreover, the atomic lines form the emitted spectra were determined to argon atoms (Ar) and the oxygen atoms (O), respectively. The results revealed that Chicory extract treatment at a concentration of 0.043 g/ml reduced the metabolic activity of P. aeruginosa cells by 42%, while, a reduced metabolic activity of 50.6% was found for E. coli biofilms. Moreover, the combination of Chicory extract with 3 min Ar-plasma introduced a synergistic effect, so that it exhibited a significantly reduced metabolic activity of P. aeruginosa to 84.1%, and E. coli ones to 86.7%, respectively. The relationship between cell viability and membrane integrity of P. aeruginosa and E. coli biofilms treated with Chicory extract and argon plasma jet were also analyzed by CLSM. It was found that after the combined treatment, a noticeable membrane disruption was formed. Besides, it was concluded that E. coli biofilms showed a higher sensitivity to Ar-plasma than P. aeruginosa biofilm at longer plasma exposure times. This study suggests that the anti-biofilm therapy based on a combined effect of Chicory extract and cold argon plasma treatment can serve as a considerable green method for treatment of antimicrobial MDR bacteria.

Argon-stimulated nitric oxide production and its function in alfalfa cadmium tolerance.

Recent results showed that argon may have great potential in both medicines (especially) and agriculture. However, how argon positively influences crop physiology remains elusive. Here, we observed that the stimulation of nitric oxide (NO) production upon cadmium (Cd) stress in hydroponic alfalfa root tissues was strengthened by argon-rich water and/or a NO-releasing compound. The pharmacological results further indicated that above potential source of NO stimulation achieved by argon might be attributed to NO synthase (NOS) and nitrate reductase (NR). Under hydroponic and pot conditions, the improvement of Cd tolerance elicited by argon, confirmed by the alleviation in the plant growth inhibition, oxidative damage, and Cd accumulation, was sensitive to the scavenger of NO. These results suggested a crucial role of argon-induced NO synthesis in response to Cd stress. Subsequent evidence showed that the improved iron homeostasis and increased S-nitrosylation were also dependent on argon-stimulated NO. Above results were matched with the transcriptional profiles of representative target genes involved in heavy metal detoxification, antioxidant defence, and iron homeostasis. Taken together, our results clearly indicated that argon-stimulated NO production contributes to Cd tolerance by favoring important defense strategies against heavy metal exposure.

The effect of argon cold atmospheric plasma on the metabolism and demineralization of oral plaque biofilms.

Objective: The aim of this study was to design and optimize a cold atmospheric plasma (CAP) device that could be applied in an oral environment and to study its effects on plaque biofilm metabolism and regrowth, as well as microbial flora composition and enamel demineralization. Method: CAP was obtained through a dielectric barrier discharge device; the optical properties were analyzed using emission spectroscopy. The electrochemical analysis of plasma devices includes voltametric characteristic curves and Lissajous. The Streptococcus mutans (UA159) and saliva biofilms were treated in vitro, and the effects of CAP on biofilm metabolism were investigated using MTT and lactate dehydrogenase assays. The duration of antibacterial activity on biofilms was examined, scanning electron microscopy was used to observe the morphology of biofilms, and 16S rRNA sequencing was used to explore the influence of CAP on the microbial flora composition of saliva biofilms. An in vitro model of biofilm-enamel demineralization was designed, and the effect of CAP on enamel demineralization was evaluated by micro surface hardness and micro-CT analysis. Results: CAP had antibacterial proliferative ability toward Streptococcus mutans biofilms and saliva biofilms and was stronger than ultraviolet under the same tested conditions. After 24 h, the antibacterial effect disappeared, which proved the short-term timeliness of its bactericidal ability. CAP can inhibit the acid production of biofilms, and its inhibitory effect on saliva biofilms can be extended to 24 h. CAP had a strong ability to regulate the composition of plaque biofilms, especially for Lactococcus proliferation, a major acid-producing bacterium in microcosm biofilms. The CAP-treated enamels were more acid-tolerant than non-treated controls. Conclusion: CAP had an explicit bactericidal effect on caries-related biofilms, which is a short-term antibacterial effect. It can inhibit the acid production of biofilms and has a downregulation effect on Lactococcus in saliva biofilms. CAP can help reduce demineralization of enamel.

Argon mitigates post-stroke neuroinflammation by regulating M1/M2 polarization and inhibiting NF-κB/NLRP3 inflammasome signaling.

Neuroinflammation plays a vital role in cerebral ischemic stroke (IS). In the acute phase of IS, microglia are activated toward the pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Argon, an inert gas, can reduce neuroinflammation and alleviate ischemia/reperfusion (I/R) injury. However, whether argon regulates M1/M2 polarization to protect against I/R injury as well as the underlying mechanism has not been reported. In this study, we analyzed the activation and polarization of microglia after I/R injury with or without argon administration and explored the effects of argon on NLRP3 inflammasome-mediated inflammation in microglia in vitro and in vivo. The results showed that argon application inhibited the activation of M1 microglia/macrophage in the ischemic penumbra and the expression of proteins related to NLRP3 inflammasome and pyroptosis in microglia. Argon administration also inhibited the expression and processing of IL-1ß, a primary pro-inflammatory cytokine. Thus, argon alleviates I/R injury by inhibiting pro-inflammatory reactions via suppressing microglial polarization toward M1 phenotype and inhibiting the NF-κB/NLRP3 inflammasome signaling pathway. More importantly, we showed that argon worked better than the specific NLRP3 inflammasome inhibitor MCC950 in suppressing neuroinflammation and protecting against cerebral I/R injury, suggesting the therapeutic potential of argon in neuroinflammation-related neurodegeneration diseases as a potent gas inhibitor of the NLRP3 inflammasome signaling pathway.

Positive Neuroprotective Effect of Argon Inhalation after Photochemically Induced Ischemic Stroke Model in Rats.

We studied the effect of 2-h inhalation of argon-oxygen mixture (Ar 70%/O2 30%) after photochemically induced stroke and on days 2 and 3 after stroke modeling on the severity of neurological deficit and brain damage (by MRI data) in Wistar rats. Neurological deficit was assessed within 14 days using the limb placement test. MRI and histological study of the brain with an assessment of the size of damage were performed on day 14 after ischemia. Significant differences were obtained in limb placement scores on days 3, 7, and 14, as well as in the volume of ischemic focus by MRI in comparison with the control (ischemia+N2 70%/O2 30%). Inhalation of argon-oxygen mixture for 2 h a day over 3 days after photoinduced stroke decreased the volume of brain damage by 2 times and reduced the severity of neurological deficit.

Effects of Argon Gas Plasma Treatment on Biocompatibility of Nanostructured Titanium.

In this study, we applied argon plasma treatment to titanium surfaces with nanostructures deposited by concentrated alkali treatment and investigated the effects on the surface of the material and the tissue surrounding an implant site. The results showed that the treatment with argon plasma removed carbon contaminants and increased the surface energy of the material while the nanoscale network structure deposited on the titanium surface remained in place. Reactive oxygen species reduced the oxidative stress of bone marrow cells on the treated titanium surface, creating a favorable environment for cell proliferation. Good results were observed in vitro evaluations using rat bone marrow cells. The group treated with argon plasma exhibited the highest apatite formation in experiments using simulated body fluids. The results of in vivo evaluation using rat femurs revealed that the treatment improved the amount of new bone formation around an implant. Thus, the results demonstrate that argon plasma treatment enhances the ability of nanostructured titanium surfaces to induce hard tissue differentiation and supports new bone formation around an implant site.

Some Beneficial Effects of Inert Gases on Blood Oxidative Metabolism: In Vivo Study.

The aim of the study was to assess the effect of external use of inert gases (helium and argon) on the state of free radical processes in vivo. The experiment was performed on 30 male Wistar stock rats (age-3 months, weight-200-220 g.), randomly distributed into 3 equal groups. The first group of animals was intact (n = 10). The animals of the second and third groups were treated with argon and helium streams, respectively. Our research has allowed us to establish that the studied inert gases have a modulating effect on the state of oxidative metabolism of rat blood, and the nature of this effect is directly determined by the type of gas. The results of this study allowed us to establish the potential antioxidant effect of the helium stream, mainly realized due to the activation of the catalytic properties of the enzymatic link of the antioxidant system of rat blood plasma. At the same time, the revealed features of shifts in oxidative metabolism during treatment with argon flow include not only stimulation of the antioxidant system but also the pronounced induction of free radical oxidation. Thus, the conducted studies made it possible to verify the specificity of the response of the oxidative metabolism of blood plasma to the use of inert gases, depending on their type.

Plasma treatment effects on destruction and recovery of Porphyromonas gingivalis biofilms.

The objective of this study was to investigate the treatment effects of non-thermal atmospheric gas plasmas (NTAP) on destruction and the recovery (or re-colonization) of Porphyromonas gingivalis (P. gingivalis) in biofilms. P. gingivalis is a well-known keystone periodontal pathogen strongly associated with periodontal diseases, especially periodontitis. P. gingivalis biofilms were formed on stainless steel coupons and treated for 1, 2, and 5 minutes by NTAP of pure argon gas and argon+oxygen gas mixture. MTT assay, colony forming unit (CFU) counting assay and confocal laser scanning microscopy (CLSM) were used to assess the destruction efficiency. In addition, the plasma treated biofilms were re-cultured in the medium supplemented with antibiotics and oxidative stress sources to determine the synergy of the NTAP with other antimicrobial agents. The results showed the plasma treatment could result in 2.7 log unit reduction in bacterial load. The recovered biofilm CFU with NTAP treatment combined with sub minimal inhibition concentration of amoxicillin was 0.33 log units less than the biofilm treated with amoxicillin alone. The recovered biofilm CFU in NTAP groups was about 2.0 log units less than that in the untreated controls under H2O2 treatment. There was approximately 1.0 log unit reduction of biofilm CFU in plasma treated biofilm compared with untreated control under paraquat treatment. The plasma treated biofilms exhibited less resistance to amoxicillin and greater susceptibility to hydrogen peroxide (H2O2) and paraquat, suggesting that NTAP may enhance biofilm susceptibility to host defense. These in vitro findings suggested that NTAP could be a novel and effective treatment method of oral biofilms that cause periodontal diseases.

Plasma of Argon Treatment of the Implant Surface, Systematic Review of In Vitro Studies.

This paper aims to review the evidence of the cellular activity on titanium samples exposed to Plasma of Argon (PoA) treatment. A systematic review was carried out based on the PRISMA statement by searching the Cochrane Library, PubMed, Web of Science, EMBASE and Scopus, up to October 2020. Papers were selected according to PICOS format that is: Population (P): osteoblasts, fibroblasts, gingival cells; Intervention (I): PoA disinfection treatment; Comparison (C): untreated controls; Outcome (O): cell culture; Setting (S): in vitro assays. The quality assessment was performed according to the CRIS Guidelines (Checklist for Reporting In vitro Studies). A total of 661 articles were found, of which 16 were included. The quality assessment revealed an overall poor quality of the studies analyzed. In vitro studies on the potential of PoA showed a potential effect in promoting higher cell adhesion and protein adsorption in the earliest times (hours). This outcome was not so evident when later stages of cell growth on the surfaces were tested and compared to the control groups. Only one study was conducted in vivo on a human sample regarding abutment cleaning. No meta-analysis was conducted because of the variety of experimental settings, mixed methods and different cell lines studied. PoA seems to be effective in promoting cell adhesion and protein adsorption. The duration of this effect remains unclear. Further evidence is required to demonstrate the long-term efficacy of the treatment and to support the use of PoA treatment in clinical practice.

An unexpected discovery toward argon-rich water amelioration of cadmium toxicity in Medicago sativa L.

Argon has organ-protective effects on animals. However, whether or how argon influences plant responses remains elusive. In this study, we discovered that the growth inhibition of hydroponically cultured alfalfa seedlings under 100 µM CdCl2 condition was significantly ameliorated by 100 % saturated argon-rich water (ARW). Less Cd uptake and accumulation were also observed in both root and shoot parts, which could be explained by the modified root cell walls, including the increased cell wall thickness, lignin content, and demethylation degree of covalently bound and ion-bound pectin, as well as the down-regulated expression of natural-resistance-associated-macrophage protein1 (Nramp1) encoding a heavy metal ion transporter in root tissue. The hindered Cd translocation from root to shoot achieved by ARW addition was validated by the decreased expression of heavy metal ATPase 2/4 (HMA2/4) in roots and decreased Cd content in xylem saps. The reestablished glutathione (GSH) homeostasis and redox balance, two important indicators of plant defense against Cd poisoning, were also observed. Further greenhouse experiments demonstrated that the phenotypic and physiological performances of alfalfa plants cultured in Cd-contaminated soil were significantly improved by irrigating with ARW. Above results implied that ARW confers plants tolerance against cadmium toxicity by impairing Cd uptake and accumulation and restoring GSH and redox homeostasis. These findings might open a new window for understanding argon biology in higher plants.

Inactivation of Escherichia coli by atmospheric pressure plasma jet in water.

The main aim of this work is inactivation of Escherichia coli in water using a laboratory-scale radio-frequency atmospheric pressure Argon plasma jet. This bacterium is widely present in the environment, especially in drinking water, and its pathogenic effects are very harmful. For this purpose, an Argon flow rate of 3.5 slm, maximum plasma power of 200 W, and discharge frequency of 13.56 MHz was conducted to generate a uniform plasma plume for water treatment. 150 ml of drinking water contaminated by E. coli was exposed to the radiation of plasma placed about 3 cm within the water, the treatment time varied from 2 to 6 minutes at 100, 150, and 200 W of plasma input power. The temperature of the plume, discharge current and voltage, and electron density were all measured to characterize the plasma. Active species such as excited molecules, ions, and radicals produced in the plasma in water were detected using the optical emission spectroscopy method. The decreasing behavior of live bacteria versus exposure time and plasma jet input power was observed, and finally, at the discharge power of 200 W and 6 min, an effective inactivation was achieved and the number of bacteria reduced from 92×104 to less than 1.7 MPN/100 ml.

Neuroprotection by the noble gases argon and xenon as treatments for acquired brain injury: a preclinical systematic review and meta-analysis.

BACKGROUND: The noble gases argon and xenon are potential novel neuroprotective treatments for acquired brain injuries. Xenon has already undergone early-stage clinical trials in the treatment of ischaemic brain injuries, with mixed results. Argon has yet to progress to clinical trials as a treatment for brain injury. Here, we aim to synthesise the results of preclinical studies evaluating argon and xenon as neuroprotective therapies for brain injuries. METHODS: After a systematic review of the MEDLINE and Embase databases, we carried out a pairwise and stratified meta-analysis. Heterogeneity was examined by subgroup analysis, funnel plot asymmetry, and Egger's regression. RESULTS: A total of 32 studies were identified, 14 for argon and 18 for xenon, involving measurements from 1384 animals, including murine, rat, and porcine models. Brain injury models included ischaemic brain injury after cardiac arrest (CA), neurological injury after cardiopulmonary bypass (CPB), traumatic brain injury (TBI), and ischaemic stroke. Both argon and xenon had significant (P<0.001), positive neuroprotective effect sizes. The overall effect size for argon (CA, TBI, stroke) was 18.1% (95% confidence interval [CI], 8.1-28.1%), and for xenon (CA, TBI, stroke) was 34.1% (95% CI, 24.7-43.6%). Including the CPB model, only present for xenon, the xenon effect size (CPB, CA, TBI, stroke) was 27.4% (95% CI, 11.5-43.3%). Xenon, both with and without the CPB model, was significantly (P<0.001) more protective than argon. CONCLUSIONS: These findings provide evidence to support the use of xenon and argon as neuroprotective treatments for acquired brain injuries. Current evidence suggests that xenon is more efficacious than argon overall.

High-pressure crystallography shows noble gas intervention into protein-lipid interaction and suggests a model for anaesthetic action.

In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs - a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia.

Experimental Evaluation of the Effect of Argon Cold Plasma on Oxidative Metabolism of the Blood.

We studied the effect of course exposure to argon cold plasma (ten 1- and 2-min procedures) on some parameters of the oxidative metabolism of rat blood plasma. The intensity of free radical processes, the total antioxidant activity, and malondialdehyde concentration in rat plasma were evaluated. It was found that 2-min exposure to argon cold plasma and nonionized argon stream produce a prooxidant effect, while 1-min exposure argon plasma led to stimulation of the antioxidant reserves of the blood.

Timely and Appropriate Administration of Inhaled Argon Provides Better Outcomes for tMCAO Mice: A Controlled, Randomized, and Double-Blind Animal Study.

BACKGROUND: Inhaled argon (iAr) has shown promising therapeutic efficacy for acute ischemic stroke and has exhibited impressive advantages over other inert gases as a neuroprotective agent. However, the optimal dose, duration, and time point of iAr for acute ischemic stroke are unknown. Here, we explored variable iAr schedules and evaluated the neuroprotective effects of acute iAr administration on lesion volume, brain edema, and neurological function in a mouse model of cerebral ischemic/reperfusion injury. METHODS: Adult ICR (Institute of Cancer Research) mice were randomly subjected to sham, moderate (1.5 h), or severe (3 h) transient middle cerebral artery occlusion (tMCAO). One hour after tMCAO, the mice were randomized to variable iAr protocols or air. General and focal deficit scores were assessed during double-blind treatment. Infarct volume, overall recovery, and brain edema were analyzed 24 h after cerebral ischemic/reperfusion injury. RESULTS: Compared with those in the tMCAO-only group, lesion volume (p < 0.0001) and neurologic outcome (general, p < 0.0001; focal, p < 0.0001) were significantly improved in the group administered iAr 1 h after stroke onset (during ischemia). Short-term argon treatment (1 or 3 h) significantly improved the infarct volume (1 vs. 24 h, p < 0.0001; 3 vs. 24 h, p < 0.0001) compared with argon inhalation for 24 h. The concentration of iAr was confirmed to be a key factor in improving focal neurological outcomes relative to that in the tMCAO group, with higher concentrations of iAr showing better effects. Additionally, even though ischemia research has shown an increase in cerebral damage proportional to the ischemia time, argon administration showed significant neuroprotective effects on infarct volume (p < 0.0001), neurological deficits (general, p < 0.0001; focal, p < 0.0001), weight recovery (p < 0.0001), and edema (p < 0.0001) in general, particularly in moderate stroke. CONCLUSIONS: Timely iAr administration during ischemia showed optimal neurological outcomes and minimal infarct volumes. Moreover, an appropriate duration of argon administration was important for better neuroprotective efficacy. These findings may provide vital guidance for using argon as a neuroprotective agent and moving to clinical trials in acute ischemic stroke.

Post-stroke treatment with argon preserved neurons and attenuated microglia/macrophage activation long-termly in a rat model of transient middle cerebral artery occlusion (tMCAO).

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.