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Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma.

Brun P, Brun P, Vono M, Venier P, Tarricone E, Deligianni V, Martines E, Zuin M, Spagnolo S, Cavazzana R, Cardin R, Castagliuolo I, Valerio AL, Leonardi A - PLoS ONE (2012)

Bottom Line: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas.Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units.Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe.

View Article: PubMed Central - PubMed

Affiliation: Histology Unit, Department of Biomedical Sciences, University of Padua, Padua, Italy. paola.brun@unipd.it

ABSTRACT

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage.

Methodology/principal findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2), was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis.

Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses.

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Related in: MedlinePlus

Tunel test on atmospheric pressure cold plasma (APCP) exposed keratocytes.Subconfluent keratocytes seeded onto coverslips in multiwell culture dishes were treated for 2 minutes with APCP. After 24 hours, fluorescein-12-dUTP-labeled DNA apoptotic cells were identified by fluorescence microscopy. No significant apoptotic effects were evident 24 hours after treatment. Data are expressed as mean ± SE (error bars) of at least two independent experiments.
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pone-0033245-g006: Tunel test on atmospheric pressure cold plasma (APCP) exposed keratocytes.Subconfluent keratocytes seeded onto coverslips in multiwell culture dishes were treated for 2 minutes with APCP. After 24 hours, fluorescein-12-dUTP-labeled DNA apoptotic cells were identified by fluorescence microscopy. No significant apoptotic effects were evident 24 hours after treatment. Data are expressed as mean ± SE (error bars) of at least two independent experiments.

Mentions: To characterize the form of cell death, we performed a biparametric cytofluorimetric analysis using propidium iodide (PI) and AnnexinV-FITC to stain DNA and phosphatidylserine (PS) residues, respectively. Flow cytometry data indicated that 2 minutes of APCP exposure increased AnnexinV/PI positive cells at 2 and 6 hours post-treatment. However, the percentage of AnnexinV and PI-positive cells returned to control values at 12 hours post-treatment (Figure 5). This trend was confirmed by the TUNEL test on keratocyte cultures treated with APCP for 2 minutes and monitored from 24 hours to 6 days post-treatment (Figure 6). Cells pre-treated with NAC were more resistant to APCP-induced cell apoptosis (Fig. 5).


Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma.

Brun P, Brun P, Vono M, Venier P, Tarricone E, Deligianni V, Martines E, Zuin M, Spagnolo S, Cavazzana R, Cardin R, Castagliuolo I, Valerio AL, Leonardi A - PLoS ONE (2012)

Tunel test on atmospheric pressure cold plasma (APCP) exposed keratocytes.Subconfluent keratocytes seeded onto coverslips in multiwell culture dishes were treated for 2 minutes with APCP. After 24 hours, fluorescein-12-dUTP-labeled DNA apoptotic cells were identified by fluorescence microscopy. No significant apoptotic effects were evident 24 hours after treatment. Data are expressed as mean ± SE (error bars) of at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303808&req=5

pone-0033245-g006: Tunel test on atmospheric pressure cold plasma (APCP) exposed keratocytes.Subconfluent keratocytes seeded onto coverslips in multiwell culture dishes were treated for 2 minutes with APCP. After 24 hours, fluorescein-12-dUTP-labeled DNA apoptotic cells were identified by fluorescence microscopy. No significant apoptotic effects were evident 24 hours after treatment. Data are expressed as mean ± SE (error bars) of at least two independent experiments.
Mentions: To characterize the form of cell death, we performed a biparametric cytofluorimetric analysis using propidium iodide (PI) and AnnexinV-FITC to stain DNA and phosphatidylserine (PS) residues, respectively. Flow cytometry data indicated that 2 minutes of APCP exposure increased AnnexinV/PI positive cells at 2 and 6 hours post-treatment. However, the percentage of AnnexinV and PI-positive cells returned to control values at 12 hours post-treatment (Figure 5). This trend was confirmed by the TUNEL test on keratocyte cultures treated with APCP for 2 minutes and monitored from 24 hours to 6 days post-treatment (Figure 6). Cells pre-treated with NAC were more resistant to APCP-induced cell apoptosis (Fig. 5).

Bottom Line: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas.Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units.Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe.

View Article: PubMed Central - PubMed

Affiliation: Histology Unit, Department of Biomedical Sciences, University of Padua, Padua, Italy. paola.brun@unipd.it

ABSTRACT

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage.

Methodology/principal findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2), was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis.

Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses.

Show MeSH
Related in: MedlinePlus