In vivo study on the repair of rat Achilles tendon injury treated with non-thermal atmospheric-pressure helium microplasma jet.

Non-thermal atmospheric-pressure plasma (NTAPP) has been widely studied for clinical applications, e.g., disinfection, wound healing, cancer therapy, hemostasis, and bone regeneration. It is being revealed that the physical and chemical actions of plasma have enabled these clinical applications. Based on our previous report regarding plasma-stimulated bone regeneration, this study focused on Achilles tendon repair by NTAPP. This is the first study to reveal that exposure to NTAPP can accelerate Achilles tendon repair using a well-established Achilles tendon injury rat model. Histological evaluation using the Stoll's and histological scores showed a significant improvement at 2 and 4 weeks, with type I collagen content being substantial at the early time point of 2 weeks post-surgery. Notably, the replacement of type III collagen with type I collagen occurred more frequently in the plasma-treated groups at the early stage of repair. Tensile strength test results showed that the maximum breaking strength in the plasma-treated group at two weeks was significantly higher than that in the untreated group. Overall, our results indicate that a single event of NTAPP treatment during the surgery can contribute to an early recovery of an injured tendon.

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.

Enhanced Fat Graft Viability and Remodeling Using a Helium-based Radiofrequency Device to Prepare the Recipient Site.

BACKGROUND: Improvements to autologous fat grafting for soft tissue augmentation are needed to overcome the unpredictable volume retention. Approaches such as fat harvesting and processing, injection technique, preparation of the recipient site, and supplemental biologics are topics of ongoing research. Here, an energy-based device was investigated as a stimulatory tool for recipient site preparation for improving fat graft retention. OBJECTIVE: The objective was to measure the stimulatory responses in fat grafts after 4 weeks when using a helium-based radiofrequency device to pretreat the recipient tissue. METHODS: Using an autologous fat grafting mouse model, the inguinal fat pad was grafted in a small cranial pocket after either a saline injection alone (control) or a saline injection followed by pretreatment (treated). The fat pad was resected after 4 weeks, sectioned and stained with immunofluorescence markers to investigate tissue remodeling. RESULTS: Pretreatment resulted in higher viability of adipocytes, a higher concentration of viable ASCs in areas of adipose tissue regeneration, and localized macrophages in the areas of regeneration when compared to the control. There was no observable difference in vascularity or angiogenesis. The staining for ASCs was higher in the pretreated group in comparison with the control group (5.0% vs. 3.3%, p=0.36) when using a pixel classifier in QuPath in the viable adipose tissue regions. CONCLUSIONS: The use of a helium-based radiofrequency device as a pretreatment tool appears to increase the viability of the adipose tissue likely due to higher concentration of ASCs. The apparent increase in viable ASCs may be due to enhanced proliferation or paracrine recruitment of these cells in response to the helium-based radiofrequency treatment. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 . Bullet List of Important Points: Pretreatment of the fat graft recipient site increases the viability of the adipose tissue after 4 weeks in comparison with the control grafts. The increased viability is likely due to the observed increase in adipose-derived stem cells in the pretreated group. Pretreatment enhanced the adipose tissue remodeling as colocalization of adipose-derived stem cells and macrophages showed an active remodeling, whereas the control group exhibited more necrotic and fibrotic tissue.

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.

Comparative analysis of helium and air surface micro-discharge plasma treatment on the microbial reduction and quality attributes of beef slices.

This work aimed to compare the effects of helium and air surface micro-discharge (SMD) plasma on the microbial safety and quality of beef tissues. For the beef tissue model, the concentration and diffusion depth of hydroxyl radical and ozone have different change patterns over plasma treatment time and distance in helium and air SMD plasma. The inactivation efficiency of helium plasma depended on the plasma treatment time and distance, while the inactivation efficiency of air plasma only depended on the treatment time. For the fresh beef slices, air SMD plasma treatment exhibited a higher antimicrobial activity against S. aureus and E. coli than helium SMD plasma treatment (1.5 versus 0.9; 0.9 versus 0.28 log CFU/g at 10 min). However, air SMD plasma treatment caused more adverse effects on beef quality, leading to a smooth surface, extensive lipid oxidation, protein structure damage, low pH and discoloration compared to helium SMD plasma treatment. This work provides valuable guidelines for the working gas choice in the practical application of plasma to meat decontamination.

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.

NiTi Alloys Exposure Alters miR-124 Expression in Physiological and Osteoarthritic Osteoblasts.

PURPOSE OF THE STUDY Nitinol (NiTi) is a biomaterial widely used in medicine based on super-elastic and shape memory properties. miR-124 has a key role in inflammatory process, osteoblasts differentiation, and mineralization. The aim of study was evaluating the differences in gene expression of miR-124 of human physiological osteoblasts (HOB) and human osteoarthritic osteoblasts (OSBA) as a response to NiTi alloy in different heat treatments. MATERIAL AND METHODS The cells were cultivated with NiTi discs with/without addition of bacterial lipopolysaccharide (LPS) for 72 hours. MicroRNAs were isolated, underwent reverse transcription and were analyzed by RT-PCR. RESULTS As a response to LPS, HOB overexpressed miR-124, while in OSBA expression change did not occur. Overexpression was also observed in both cell lines as a response to hydrogen and helium treated NiTi discs. HOB expressed significantly higher amount of miR-124 than OSBA as a response to hydrogen treatment of NiTi discs. In addition, hydrogen treatment caused significantly higher expression in HOB than LPS. The combination of NiTi disc and LPS treatment in HOB didn't cause any expression changes. Comparing to LPS-only treatment, the expression in HOB with combination of LPS and alloy was significantly lower. In OSBA, the expression was increased by the combination of LPS and hydrogen disc, in case of helium disc, the expression was decreased. CONCLUSIONS In conclusion, human physiological and osteoarthritic osteoblasts respond to NiTi alloy with both surface (hydrogen and helium atmosphere) treatment by overexpression of miR-124. The effect of LPS as inflammatory modulator suggests the presence of an "anti-inflammatory preconditioning" in osteoarthritic osteoblasts, as physiological osteoblasts overexpression was significantly higher. Key words: nitinol, osteoblast, miR-124, lipopolysaccharide.

Cold Atmospheric Plasma Jet Treatment Improves Human Keratinocyte Migration and Wound Closure Capacity without Causing Cellular Oxidative Stress.

Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10−120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.

Anti-Melanoma Capability of Contactless Cold Atmospheric Plasma Treatment.

In this study, we demonstrated that the widely used cold atmospheric plasma (CAP) jet could significantly inhibit the growth of melanoma cells using a contactless treatment method, The flow rate of helium gas was a key operational parameter to modulate electromagnetic (EM) effect on melanoma cells. Metal sheets with different sizes could be used as a strategy to control the strength of EM effect. More attractive, the EM effect from CAP could penetrate glass/polystyrene barriers as thick as 7 mm. All these discoveries presented the profound non-invasive nature of a physically based CAP treatment, which provided a solid foundation for CAP-based cutaneous/subcutaneous tumor therapy.

Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles.

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Cold atmospheric pressure He-plasma jet and plasma ball interactions with the Venus flytrap: Electrophysiology and side effects.

Cold atmospheric pressure radio frequency plasma (CAPP) can play an important role in agriculture, medicine, biophysical and bioelectrochemical applications, disinfection and sterilization, synthesis of different compounds, nitrogen fixation, and treatment of surfaces. Here we found that reactive oxygen and nitrogen species, UV-Vis photons, and high-frequency strong electromagnetic fields with an amplitude of a few kV produced by a cold plasma jet can interact with bio-tissue and damage it if the plasma treatment is long enough. The electrophysiological effects of CAPP treatment of bio-tissue and electrical signals transmission were measured in the Venus flytrap. The plasma ball does not produce any visible side effects on the Venus flytrap, but induces electrical signals in bio-tissue with very high amplitude. Plasma (Kirlian) photography shows the existence of a blue aura around the plasma ball due to a corona discharge. Understanding the mechanisms of interactions between CAPP and bio-tissue and preventing side effects can contribute to the application of plasma technology in medicine and agriculture. The use of cold plasma in medicine and agriculture should be monitored for side effects from strong high-frequency electro-magnetic fields, UV photons, and reactive oxygen and nitrogen species to protect against undesirable consequences.

The effects of heliox non-saturation diving on the cardiovascular system and cognitive functions.

The purpose of this study was to investigate the effects of a single bout of heliox non-saturation diving on the cardiovascular system and cognitive function. Ten recreational scuba divers (10 males, ∼35 years old) participated in this study. These subjects made two pool dives within a one-week interval, alternating gases with compressed air (21% O2, 79% N2) and with heliox (21% O2 and 79% He). The depth was to 26 meters over a 20-minute duration. The results showed that heliox diving significantly increased blood O2 saturation by 1.15% and significantly decreased blood lactate levels by ∼57% when compared with air diving (P ≺ 0.05). However, there were no significant differences in resting heart rate, systolic or diastolic pressure, core body blood pressure, and pulse wave velocity between the heliox and air dives. The Stroop test showed that the heliox dive significantly increased cognitive function compared with the air dive in both the simple test (Offtime) and interference test (Ontime) (P ≺ 0.05). It was concluded that the heliox dive increases blood O2 saturation and decreases blood lactate concentration when compared with air dives. These conditions are likely to help divers reduce hypoxia in the water, reduce the risk of loss of consciousness, reduce fatigue and allow them to dive for longer. Heliox diving may also help judgment and risk coping skills in the water due to the improvement of cognitive ability as compared to air breathing dives.

Neuroprotection of dopamine neurons by xenon against low-level excitotoxic insults is not reproduced by other noble gases.

Using midbrain cultures, we previously demonstrated that the noble gas xenon is robustly protective for dopamine (DA) neurons exposed to L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate uptake used to generate sustained, low-level excitotoxic insults. DA cell rescue was observed in conditions where the control atmosphere for cell culture was substituted with a gas mix, comprising the same amount of oxygen (20%) and carbon dioxide (5%) but 75% of xenon instead of nitrogen. In the present study, we first aimed to determine whether DA cell rescue against PDC remains detectable when concentrations of xenon are progressively reduced in the cell culture atmosphere. Besides, we also sought to compare the effect of xenon to that of other noble gases, including helium, neon and krypton. Our results show that the protective effect of xenon for DA neurons was concentration-dependent with an IC50 estimated at about 44%. We also established that none of the other noble gases tested in this study protected DA neurons from PDC-mediated insults. Xenon's effectiveness was most probably due to its unique capacity to block NMDA glutamate receptors. Besides, mathematical modeling of gas diffusion in the culture medium revealed that the concentration reached by xenon at the cell layer level is the highest of all noble gases when neurodegeneration is underway. Altogether, our data suggest that xenon may be of potential therapeutic value in Parkinson disease, a chronic neurodegenerative condition where DA neurons appear vulnerable to slow excitotoxicity.

Helium-Induced Changes in Circulating Caveolin in Mice Suggest a Novel Mechanism of Cardiac Protection.

The noble gas helium (He) induces cardioprotection in vivo through unknown molecular mechanisms. He can interact with and modify cellular membranes. Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma-membrane-containing caveolin (Cav) proteins that are critical in protection of the heart. Mice (C57BL/6J) inhaled either He gas or adjusted room air. Functional measurements were performed in the isolated Langendorff perfused heart at 24 h post He inhalation. Electron paramagnetic resonance spectrometry (EPR) of samples was carried out at 24 h post He inhalation. Immunoblotting was used to detect Cav-1/3 expression in whole-heart tissue, exosomes isolated from platelet free plasma (PFP) and membrane fractions. Additionally, transmission electron microscopy analysis of cardiac tissue and serum function and metabolomic analysis were performed. In contrast to cardioprotection observed in in vivo models, the isolated Langendorff perfused heart revealed no protection after He inhalation. However, levels of Cav-1/3 were reduced 24 h after He inhalation in whole-heart tissue, and Cav-3 was increased in exosomes from PFP. Addition of serum to muscle cells in culture or naïve ventricular tissue increased mitochondrial metabolism without increasing reactive oxygen species generation. Primary and lipid metabolites determined potential changes in ceramide by He exposure. In addition to direct effects on myocardium, He likely induces the release of secreted membrane factors enriched in caveolae. Our results suggest a critical role for such circulating factors in He-induced organ protection.

The radiobiological effects of He, C and Ne ions as a function of LET on various glioblastoma cell lines.

The effects of the charged ion species 4He, 12C and 20Ne on glioblastoma multiforme (GBM) T98G, U87 and LN18 cell lines were compared with the effects of 200 kVp X-rays (1.7 keV/µm). These cell lines have different genetic profiles. Individual GBM relative biological effectiveness (RBE) was estimated in two ways: the RBE10 at 10% survival fraction and the RBE2Gy after 2 Gy doses. The linear quadratic model radiosensitivity parameters α and ß and the α/ß ratio of each ion type were determined as a function of LET. Mono-energetic 4He, 12C and 20Ne ions were generated by the Heavy Ion Medical Accelerator at the National Institute of Radiological Sciences in Chiba, Japan. Colony-formation assays were used to evaluate the survival fractions. The LET of the various ions used ranged from 2.3 to 100 keV/µm (covering the depth-dose plateau region to clinically relevant LET at the Bragg peak). For U87 and LN18, the RBE10 increased with LET and peaked at 85 keV/µm, whereas T98G peaked at 100 keV/µm. All three GBM α parameters peaked at 100 keV/µm. There is a statistically significant difference between the three GBM RBE10 values, except at 100 keV/µm (P < 0.01), and a statistically significant difference between the α values of the GBM cell lines, except at 85 and 100 keV/µm. The biological response varied depending on the GBM cell lines and on the ions used.

Effect of helium preconditioning on neurological decompression sickness in rats.

Decompression sickness (DCS) occurs because of an excessively rapid and extensive reduction of the ambient pressure. Bubble-induced spinal cord ischemia is generally considered as a part of neurological DCS pathogenesis. Because helium preconditioning (HPC) recently demonstrated beneficial properties against ischemic damage, we hypothesized that HPC may decrease the neurological deficits of DCS in rats. Seventy-five male Sprague-Dawley rats were divided into a non-HPC group ( n = 25) and a HPC group ( n = 25) and 25 naive animals that were euthanized for histological examination ( n = 5) or anesthetized for baseline somatosensory evoked potential (SSEP) recordings ( n = 20). To induce DCS, rats were compressed with air to a pressure of 709 kPa for 60 min and decompressed at a rate of 203 kPa/min. HPC was administered as three episodes of 79% helium-21% oxygen mixture inhalation for 5 min interspersed with 5 min of air breathing. We found that HPC resulted in significantly decreased DCS incidence and delay of DCS onset. HPC also improved animal performance on the grip test after decompression and significantly ameliorated decompression-induced decrease of platelet number. Furthermore, the incidence of abnormal SSEP waves and histological spinal lesions was significantly reduced by HPC. We conclude that HPC can decrease the occurrence of DCS and ameliorate decompression-induced neurological deficits. NEW & NOTEWORTHY Helium preconditioning ameliorates decompression-induced neurological deficits in rats. Helium breathing before air dives may prevent neurological deficit and attenuate symptoms after decompression.

Determination of proper treatment time for in vivo blood coagulation and wound healing application by non-thermal helium plasma jet.

The aim of this research was to determine the time of blood coagulation of in vivo cuts that treated by the non-thermal atmospheric pressure plasma jet. Also, the effect of different treatment times on wound healing has been studied. The non-thermal atmospheric pressure plasma jet working in helium gas has been used. The averaged treatment time of 8.6 s for in vivo cuts on the Balb/c mouse liver showed the complete blood coagulation. Also, the effect of tretament time on wound healing has been studied by applying plasma on the wounds for different times (10, 20, 30, 40 and 50 s). It was obtained from morphological analysis that the treatment groups of 30 s, 40 s and 50 s cause the wounds to be healed faster than the groups 10 s and 20 s.The histological analysis showed that in 30 s and 40 s treatment time groups, the repair process of treated wounds has been accelerated, while for 50 s group, it has not been completed, yet. The 30 s treatment time has been chosen because of imposing lower dose to living tissue. The treated wound area reduction ratios against the control wound reduction ratios for 30 s group were obtained 78%, 77% and 63% at the 3rd, 5th and 8th days, respectively.

Amplitude-modulated cold atmospheric pressure plasma jet for treatment of oral candidiasis: In vivo study.

The aim of this study was to establish an effective and safe protocol for in vivo oral candidiasis treatment with atmospheric plasma jets. A novel amplitude-modulated cold atmospheric pressure plasma jet (AM-CAPPJ) device, operating with Helium, was tested. In vitro assays with Candida albicans biofilms and Vero cells were performed in order to determine the effective parameters with low cytotoxicity. After the determination of such parameters, the protocol was evaluated in experimentally induced oral candidiasis in mice. AM-CAPPJ could significantly reduce the viability of C. albicans biofilms after 5 minutes of plasma exposure when compared to the non-exposed group (p = 0.0033). After this period of exposure, high viability of Vero cells was maintained (86.33 ± 10.45%). Also, no late effects on these cells were observed after 24 and 48 hours (83.24±15.23% and 88.96±18.65%, respectively). Histological analyses revealed significantly lower occurrence of inflammatory alterations in the AM-CAPPJ group when compared to non-treated and nystatin-treated groups (p < 0.0001). Although no significant differences among the values of CFU/tongue were observed among the non-treated group and the groups treated with AM-CAPPJ or nystatin (p = 0.3201), histological analyses revealed marked reduction in candidal tissue invasion. In conclusion, these results point out to a clinical applicability of this protocol, due to the simultaneous anti-inflammatory and inhibitory effects of AM-CAPPJ with low cytotoxicity.