Plasma Damage Control: From Biomolecules to Cells and Skin.

The antibacterial and cell-proliferative character of atmospheric pressure plasma jets (APPJs) helps in the healing process of chronic wounds. However, control of the plasma-biological target interface remains an open issue. High vacuum ultraviolet/ultraviolet (VUV/UV) radiation and RONS flux from plasma may cause damage of a treated tissue; therefore, controlled interaction is essential. VUV/UV emission from argon APPJs and radiation control with aerosol injection in plasma effluent is the focus of this research. The aerosol effect on radiation is studied by a fluorescent target capable of resolving the plasma oxidation footprint. In addition, DNA damage is evaluated by plasmid DNA radiation assay and cell proliferation assay to assess safety aspects of the plasma jet, the effect of VUV/UV radiation, and its control with aerosol injection. Inevitable emission of VUV/UV radiation from plasmas during treatment is demonstrated in this work. Plasma has no antiproliferative effect on fibroblasts in short treatments (t < 60 s), while long exposure has a cytotoxic effect, resulting in decreased cell survival. Radiation has no effect on cell survival in the medium due to absorption. However, a strong cytotoxic effect on the attached fibroblasts without the medium is apparent. VUV/UV radiation contributes 70% of the integral plasma effect in induction of single- and double-strand DNA breaks and cytotoxicity of the attached cells without the medium. Survival of the attached cells increases by 10% when aerosol is introduced between plasma and the cells. Injection of aerosol in the plasma effluent can help to control the plasma-cell/tissue interaction. Aerosol droplets in the effluent partially absorb UV emission from the plasma, limiting photon flux in the direction of the biological target. Herein, cold and safe plasma-aerosol treatment and a safe operational mode of treatment are demonstrated in a murine model.

Investigation of toxicity and mutagenicity of cold atmospheric argon plasma.

Cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. So far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. Thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the MicroPlaSter ß® , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the XTT assay in keratinocytes and fibroblasts and the HGPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 Chinese hamster cells. The tested clinical parameter of a 2 min cold atmospheric argon plasma treatment revealed no relevant toxicity on keratinocytes (viability: 76% ± 0.17%) and on fibroblasts (viability: 81.8 ± 0.10) after 72 hr as compared to the untreated controls. No mutagenicity was detected in the HGPRT assay with V79 cells even after repetitive CAP treatments of 2-10 min every 24 hr for up to 5 days. In contrast, UV-C irradiation of V79 cells, used as a positive control in the HGPRT test, led to DNA damage and mutagenic effects. Our findings indicate that cold atmospheric plasma using the MicroPlaSter ß® shows negligible effects on keratinocytes and fibroblasts but no mutagenic potential in the HGPRT assay, indicating a new contact free safe technology. Environ. Mol. Mutagen. 58:172-177, 2017. © 2017 Wiley Periodicals, Inc.

Risk assessment of a cold argon plasma jet in respect to its mutagenicity.

Cold atmospheric pressure plasmas represent a favorable option for the treatment of heat sensitive materials and human or animal tissue. Beneficial effects have been documented in a variety of medical conditions, e.g., in the treatment of chronic wounds. It is assumed that the main mechanism of the plasma's efficacy is mediated by a stimulating dissipation of energy via radiation and/or chemical energy. Although no evidence on undesired side effects of a plasma treatment has yet been presented, skepticism toward the safety of the exposure to plasma is present. However, only little data regarding the mutagenic potential of this new treatment option is available. Accordingly, we investigated the mutagenic potential of an argon plasma jet (kinpen) using different testing systems in accordance with ISO norms and multiple cell lines: a HPRT1 mutation assay, a micronucleus formation assay, and a colony formation assay. Moderate plasma treatment up to 180 s did not increase genotoxicity in any assay or cell type investigated. We conclude that treatment with the argon plasma jet kinpen did not display a mutagenic potential under the test conditions applied and may from this perspective be regarded as safe for the use in biomedical applications.

Is argon plasma coagulation an effective and safe treatment option for patients with chronic radiation proctitis after high doses of radiotherapy? / ¿Es la coagulación con plasma argón una opción terapéutica eficaz y segura en pacientes con proctitis crónica actínica tras dosis altas de radioterapia?

Introduction: In severe cases refractory to medical treatment, APC appears to be the preferred alternative to control persistent rectal bleeding of patients with chronic radiation proctitis. Although successful outcomes have been demonstrated in patients previously treated with moderate doses of radiotherapy, there is reluctance towards its indication due to the concern of severe adverse events in patients treated with high doses of radiation. Objectives: The aim of this study was to assess the efficacy and toxicity of APC in the management of bleeding radiationinduced proctitis in patients treated with high doses of radiation for prostate cancer. Methods and materials: Data from 30 patients were treated with APC due to chronic radiation proctitis, were reviewed retrospectively. All cases had prostate cancer and 9 of them (30 %) underwent previous radical prostatectomy. The median dose of conformal 3D External Beam Radiotherapy (EBRT) delivered was 74 Gy (range 46-76). Median rectal D1cc and D2cc was 72.5 and 72.4 Gy respectively. Median rectal V70, V60 and V40 was 12, 39.5 and 80 %. Cardiovascular and digestive disease, diabetes, smoking behaviour, lowest haemoglobin and transfusion requirements were recorded. Indications for treatment with APC were anemia and persistent bleeding despite medical treatment. Argon gas flow was set at 1.8 l/min with an electrical power setting of 50 W. Results: Median age of all patients was 69.6 years. The median lowest haemoglobin level was 9.6 g/dL. Median time between completion of radiotherapy and first session of APC as 13 months. Ninety-four therapeutic sessions were performed (median 3 sessions). Median time follow-up was 14.5 months (range 2-61). Complete response with resolved rectal bleeding was achieved in 23 patients (77 %), partial response in 5 (16 %) and no control in 2 (6 %). No patients required transfusion following therapy. Two patients developed long-term (> 6 weeks) grade 2 rectal ulceration and grade 2 rectal incontinence, respectively. Conclusions: The argon plasma coagulation is an effective and safe management option in patients with medically refractory rectal bleeding after high doses of radiation for prostate cancer (AU)

No disponible

Unusual hand frostbite caused by refrigerant liquids and gases.

BACKGROUND: The refrigerant liquids and gases used widely in industry, farming and medicine for their cooling properties may cause severe frostbite. Despite their widespread use, only a few reports on frostbite of the hand involving these liquids and gases have been published. In this study, the circumstances accompanying these injuries, several adjunctive therapies and preventive measures are discussed. METHODS: A retrospective analysis of hand frostbite injuries was conducted between June 2005 to June 2009 in a burn care center in Istanbul, Turkey. Seventeen patients (13 men, 4 women) were treated for hand frostbite injuries due to contact with refrigerant liquids and gases. RESULTS: There was a preponderance of male patients (76.5%). Ages ranged from 22 to 52 years (mean age, 30.82 years). Eleven patients were treated conservatively. The hospital stay for treatment of their burns ranged from 16 to 52 days, with a mean stay of 30 days. CONCLUSION: Frostbite injuries of the hand are uncommon and their etiologies vary. Thus, the low incidence of these injuries and limited experience in handling rare cases of this nature may lead to misjudgments in treatment that can have grave consequences. Decreasing the exposure time is an important first step in the treatment approach. After exposure to gas, quick delivery of the patient to a burn center is essential.

Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy.

PURPOSE: Retinal microglia have been implicated in the pathogenesis of various retinal diseases, but their basic function and cellular phenotype remain incompletely understood. Here, the authors used a novel ex vivo retinal imaging preparation to examine the behavioral phenotype of living retinal microglia in intact tissue and in response to injury. METHODS: Fluorescence-labeled microglia in retinal explants from CX3CR1(+/GFP) transgenic mice were observed using time-lapse confocal imaging. High spatial and temporal resolution imaging parameters were used to follow dynamic microglial behavior in real time. RESULTS: Under normal conditions, resting retinal microglia are not static in structure but instead exhibit extensive structural dynamism in their cellular processes. Process movements are highly random in direction but are balanced to maintain overall cellular symmetry and arbor size. At rest, however, these exuberant process movements do not result in overt cellular migration. After focal laser injury, microglial processes increase significantly in their motility and direct themselves toward the injury site. Microglia rapidly transition their morphologies from symmetric to polarized toward the laser lesion. Microglia also transition from a fixed to a migratory phenotype, translocating through tissue while retaining their ramified morphology. CONCLUSIONS: Retinal microglia normally occupying uninjured tissue display a continuous, dynamic behavior that suggests functions of tissue surveillance and intercellular communication. Microglial behavior is highly regulated by, and immediately responsive to, focal tissue injury and may constitute a therapeutic cellular response to focal laser photocoagulation. Ex vivo live imaging in the retina is an experimental approach well suited to the study of dynamic aspects of microglial physiology.

Behaviour of pigs exposed to mixtures of gases and the time required to stun and kill them: welfare implications.

Pigs were exposed individually to either 90 per cent argon in air (anoxia), a mixture of 30 per cent carbon dioxide and 60 per cent argon in air (hypercapnic anoxia) or 80 to 90 per cent carbon dioxide in air (hypercapnic hypoxia) and the times to loss of posture, the onset and duration of convulsions, vocalisation and cessation of gagging (respiratory arrest) were determined. The duration of convulsions and the time to onset of respiratory arrest were longer when the pigs were exposed to argon than when they were exposed to the mixture of carbon dioxide and argon or to the high concentration of carbon dioxide in air. A second experiment was carried out under commercial conditions to determine the duration of unconsciousness and insensibility based on the response to a nose prick, and the incidence of death induced by exposing pigs to either 90 per cent argon in air or a mixture of 30 per cent carbon dioxide and 60 per cent argon in air for different periods. The results showed that when pigs were exposed for three minutes to either argon or the mixture of carbon dioxide and argon they should be bled within 25 seconds from the end of exposure to the gas to prevent them regaining consciousness during bleeding. When the pigs were exposed to either argon or the mixture of carbon dioxide and argon for five minutes and bleeding out began within 45 seconds they did not regain consciousness or suffer convulsions while being bled. The majority of the pigs died when they were exposed to argon for seven minutes, and all of them died when they were exposed to the mixture of carbon dioxide and argon for seven minutes.

Controlled atmosphere stunning of poultry.

For more than 20 yr Stork PMT has carried out a lot of research into stunning and killing of poultry with electrical and mechanical methods and by applying gas mixtures. For the past 3 yr, research was carried out under the European umbrellas (AIR and EUREKA) together with BOC, The Spelderholt, CIVO, and Bristol University. This research program involved tests on broiler stunning or killing with various gas mixtures. The effect on meat quality, bleeding, and plucking was studied, as well as the use of gas as a humane way of killing broilers. The study of meat quality was based on tenderness tests conducted on the shear force principle, meat color, and drip and cooking losses. The percentage of blood loss in bleeding, blood spots, and the amount of blood in the veins in relation to the gas mixtures and time of hanging were measured. The effect of gas killing on the plucking characteristics was studied by determining the feather release force and product handling. The behavior of the birds, reflexes, the onset of convulsions, electroencephalograms, and evoked responses were studied in relation to the humanness of gas killing. Anoxia generated through argon or a mixture of argon and carbon dioxide or hypercapnic hypoxia seems to be very promising. The tests revealed that meat tenderness and drip losses will improve. The blood spots, especially those on the thighs and breasts caused by stunning and hanging, disappear altogether. It also appeared that animal welfare will be drastically improved. From a technological point of view, broiler killing in a controlled gas atmosphere is considered to be the optimal process. However, successful introduction of the process requires legislative changes and poultry processors must be made aware of its economic benefits.

Inactivation of food microorganisms by high-pressure carbon dioxide treatment with or without explosive decompression.

In order to elucidate the sterilization mechanism underlying the explosive decompression system, baker's yeast was pressurized with CO2, N2O, N2, or Ar gas at 40 atm and 40 degrees C for 4h, and then explosively discharged. The survival ratio was markedly decreased only by the treatments with CO2 and N2O, which are relatively soluble gases in water, suggesting that the microorganisms' death may be highly correlated with gas absorption by the cells. Lower decompression rates to atmospheric pressure, however, led to neither any lower reduction of remaining cells nor any smaller release of total cellular proteins. Furthermore, operating with a longer treatment time and smaller number of repetitions was usually more lethal than with a shorter time and more frequent repetition. From these results, most of the yeast cells appear to have been sterilized during the pressurization process. The spore cells of B. megaterium are considered to have been killed in a somewhat different manner, because of their distinct sensitivity to the applied gases.

Effect of trophic factors on delayed neuronal death induced by in vitro ischemia in cultivated hippocampal and cortical neurons.

The effect of trophic factors on neuronal survival after 30 min oxygen and glucose deprivation (in vitro ischemia) was studied in primary hippocampal and cortical neuronal cultures of rat. In vitro ischemia was produced at 37 degrees C by placing cultures in glucose-free medium, the oxygen content of which was removed by gassing with pure argon. After in vitro ischemia neurons were allowed to recover either in serum-free minimal essential medium (MEM) or in MEM containing 5% native horse serum, 100 ng/ml basic fibroblast growth factor (bFGF) or 10 ng/ml transforming growth factor-beta 1 (TGF-beta 1), respectively. Cultures that recovered in serum-free medium suffered a progressive type of neuronal injury: survival of either cortical or hippocampal neurons declined from about 60% after 1 h to 50% after 3 h, 40% after 6 h and less than 20% after 24 h. Addition of serum proteins to the incubation medium did not influence early survival (up to 3-6 h) but significantly improved survival after 24 h (more than 40% in both hippocampal and cortical cultures). Addition of TGF-beta 1 and bFGF had only minor effects. These data show that serum reduces delayed ischemic cell death by a mechanism which is different from that of TGF-beta 1 or bFGF protection.

Carcase and meat quality in broilers either killed with a gas mixture or stunned with an electric current under commercial processing conditions.

1. Carcase and meat quality were evaluated under commercial conditions in 400 broilers either killed with a mixture of 30% carbon dioxide and 60% argon in air or stunned with a 50 Hz AC with clipped sine wave. 2. Compared with electrical stunning, killing broilers with the gas mixture eliminated or substantially reduced the prevalence of carcase and meat quality defects. 3. The results also showed that killing broilers with a mixture of 30% carbon dioxide and 60% argon would enable filleting (deboning) to be performed at 4 h post mortem without adversely affecting the cook loss or texture of breast meat.

Effect of the method of killing, interval between killing and neck cutting and blood vessels cut on blood loss in broilers.

1. Broiler chickens were killed using 90% argon in air, or 30% carbon dioxide and 60% argon in air or 120 mA per bird in a waterbath with a 50 Hz alternating electric current. Ventral or unilateral neck cutting was performed at 1, 3 or 5 min after killing. In addition, a group of broilers was stunned with 120 mA per bird in a waterbath using 1500 Hz alternating current and were bled out-with a ventral neck cut within 20 s from stunning. 2. Blood leaving the neck wound was collected in a bin placed on an electronic balance and a computer program calculated the cumulative blood loss up to 100 s after neck cutting. 3. Bleed-out was significantly affected by killing method and time of neck cutting. Broilers killed with the carbon dioxide-argon mixture bled-out less than those killed with argon or 50 Hz electric current. When compared with the 1 min neck cutting interval, a delay of 3-or 5 min resulted in a lower bleed-out. High frequency electrical stunning and ventral neck cutting within 20 s resulted in a slightly higher bleed-out than those recorded for the killing methods. However, within argon killing, a delay of 3 or 5 min in ventral or unilateral neck cutting had no significant effect on the bleed-out. In broilers killed with the carbon dioxide-argon mixture a 3 min delay in ventral neck cutting or a 5 min delay in unilateral neck cutting resulted in lower bleed-out. 4. Neck cutting of broilers within 5 min after argon killing or 3 min after killing with the carbon dioxide-argon mixture would result in a satisfactory bleed-out.

Susceptibility of hippocampal and cortical neurons to argon-mediated in vitro ischemia.

Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon-induced in vitro ischemia. Neuronal cultures were exposed at 37 degrees C for 10-30 min to argon-equilibrated glucose-free medium. During argon equilibration, Po2 declined to < 2.5 torr within 1 min and stabilized shortly later at approximately 1.3 torr. After 30 min of in vitro ischemia, total adenylate was < 45% and ATP content < 15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 nM. Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to approximately 90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to < 35%. Cytosolic calcium activity continued to rise after ischemia and reached values of approximately 500 nM after 3 h. The new argon-induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.

Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam.

Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E = 7.1 MeV/nucleon, LET = 1590 keV/micron) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colony-forming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colony-forming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 microns2; it was 39 microns2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN.(ABSTRACT TRUNCATED AT 250 WORDS)

Time to loss of somatosensory evoked potentials and onset of changes in the spontaneous electroencephalogram of turkeys during gas stunning.

The times to the loss of somatosensory evoked potentials (SEPs) and the onset of suppressed and isoelectric electroencephalogram (EEG) were investigated in turkeys as they were stunned with gas mixtures consisting of one of three mixtures: (A) 30 per cent carbon dioxide and 60 per cent argon in air (2 per cent residual oxygen and 8 per cent residual nitrogen); (B) 90 per cent argon in air (2 per cent residual oxygen and 8 per cent residual nitrogen); (C) 65 per cent carbon dioxide in air (7 per cent residual oxygen and 28 per cent residual nitrogen). The time to the loss of SEPs, EEG suppression and the onset of an isoelectric EEG, respectively, were 22, 16 and 35 seconds in mixture A, 44, 41 and 101 seconds in mixture B, and 15, 15 and 67 seconds in mixture C. Stunning turkeys with mixture A or B would be suitable under commercial conditions. Mixture C, containing 65 per cent carbon dioxide in air, is considered on humanitarian grounds to be unacceptable for stunning turkeys owing to the pungency of the carbon dioxide at this concentration.

Changes in rat respiratory behavior under varying PCO2 levels and argon content in hyperbaric Ar-O2-CO2 atmospheres.

An anesthetized rat preparation has been used for breathing studies in hyperbaric Ar-O2-CO2 atmospheres in which total saturation pressure was varied in the range 1-19.1 ATA and PCO2 was set at levels of 0.0, 0.05 or 0.10 ATA. The variables monitored were respiration rate and an isotonic measure of diaphragm-twitch amplitude during contracture/relaxation (twitch index). The results indicate that: (1) hyperbaric Ar-O2 atmospheres are able to maintain the animal in stable condition for several hours; (2) elevation in PCO2 level produces enhancements in respiration rate and twitch index that are markedly dependent on the PAr level; (3) successive increases in PAr are able to lower progressively the effects on both breathing parameters produced by either 0.05 or 0.10 ATA of CO2, with full abolition of both effects of inhaled CO2 in the PAr range 14-19 ATA; and (4) the intrinsic ability of hyperbaric argon to abolish respiratory enhancement by CO2 is greater than that of hyperbaric helium. These results have been discussed in terms of possible CNS loci at which hyperbaric argon (or helium) is able to antagonize centrally induced effects from dissolved molecular species derived from inspired CO2, and in relation to the broader task of control of excitatory/inhibitory centers of the CNS during diving by manipulation of the partial pressures of diluent components of breathing-gas mixtures.

Narcotic potency of N2, A, and N2O evaluated by the physical performance of mouse colonies at simulated depths.

The physical performance of colonies of deer mice was studied in various inert gas environments at pressures up to 31 ATA. The mice were housed in habitats wherein their diurnal running activity and social interactions could be monitored. By transferring the portable habitats and mouse colonies to a high pressure chamber, the effects of elevated inert gas pressures were studied in socially and ecologically intact surroundings. Analysis of wheel-running performance showed that either 1.1 atm nitrous oxide, 7.2 atm argon, or 20.5 atm nitrogen reduced running activity to 50% of its control value. Behavioral observations revealed a deterioration of physical performance and social interaction with increasing inert gas pressures. A comparison was made between ED50 (the dose that will depress a particular response by 50%) values obtained by studying wheel-running activity and those published for single-reflex responses.