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

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.

Figure 1. Time-dependent effects of APCP on microorganisms.

Microorganisms (1×10⁴ CFU) were treated with APCP for different lengths of time, plated out and grown under optimal conditions. Microorganism survival was evaluated by the colony count assay and reported as a percentage of CFU/ml calculated in untreated samples. Data are reported as mean ± SE (error bars) of four independent experiments.

Figure 2. Time-dependent effects of APCP on fibroblast and keratocyte viability.

(A) 4×10⁴ human conjunctival fibroblasts and (B) 5×10⁴ human keratocytes in 24-well culture dishes covered with 200 µl of medium were treated with APCP for different exposure times. The MTT test was performed 1 and 24 hours after treatment. At 1 hour, the viability of cells treated for up to 2 minutes was not reduced whereas a significant reduction of viability was found after 5 minutes of exposure (•P<0.05 compared to control values). The viability of both cells types significantly increased after 24 hours of culture (*P<0.05 compared to 1 hour of treatment). Data are reported as mean ± SE (error bars) of three independent experiments.

Figure 3. Reactive oxygen species (ROS) generated by APCP treatment generated in human cells.

Primary cultures of human keratocytes were pre-treated with 5 mM N-acetylcystein (NAC) and exposed for 2 minutes to APCP. Cells were cultured under optimal conditions and then evaluated at different times for intracellular ROS production using the FACS-activated 2′,7′-dichlorofluorescein diacetate probe, collecting at least 10,000 events. ROS formation is expressed as percentage of fluorescent intensity. Data are reported as mean ± SE (error bars) of the percentage of fluorescence observed at least two independent experiments. (*P<0.05 compared to respective control; •P<0.05 compared to 2-minute exposure to plasma).

Figure 4. Effects of APCP treatment on human cell cycle progression.

Cultured human keratocytes (panel A) and conjunctival fibroblasts (panel B) were exposed for 2 minutes to APCP with or without a 5 mM-pretreatment with NAC and subsequently cultured under optimal conditions. Cells were stained with propidium iodide and the cell cycle progression was analyzed by flow cytometry, collecting at least 10,000 events. Data are from at least two independent experiments and are expressed as mean ± SE (error bars) of percentage of fluorescence intensity in cells within each cycling phase. At 12 and 24 hours post-APCP, dead cells (sub G0) were no longer evident in samples pre-treated with NAC (*P<0.05 compared to cells exposed to APCP without NAC pretreatment). Percentages (y axes) indicate the fraction of cells within the drawn intervals of time (x axes).

Figure 5. Effects of APCP treatment on externalization of phosphatidylserine.

Cultured human keratocytes (panel A) and conjunctival fibroblasts (panel B) were exposed for 2 minutes to APCP with or without a 5 mM-pretreatment with NAC and subsequently cultured under optimal conditions. The externalization of phosphatidylserine was analyzed by flow cytometry after double staining of the cells with Annexin-V-FITC and propidium iodide, collecting at least 10,000 events. Single or double positive cells are expressed as a percentage of fluorescent intensity. In cells pre-treated with NAC, the increased percentage of AnnexinV/PI positive cells at 2 and 6 hours post-treatment returned to control values after 12 hours. Data are expressed as mean ± SE (error bars) of at least two independent experiments.

Figure 6. 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.

Figure 7. Effects of APCP treatment on ex-vivo human corneas.

The tissue morphology of corneas exposed for 5 minutes to APCP was evaluated. Samples were embedded in paraffin, cut into 5 µm sections, and stained with hematoxylin and eosin. Light microscopic staining was compared to that of unexposed control tissues. A and B: control sections at 100 (A) and 200 (B) magnification; A1 and B1: treated sections at 100 (A1) and 200 (B1) magnification.

Figure 8. Tunel test on APCP treated corneal tissue.

Apoptotic cells in 5 µm paraffin-embedded corneal tissues exposed for 2 minutes to APCP were identified by labelling DNA strand breaks with biotin-labeled deoxynucleotides and peroxidase. The immunoreaction product was visualized using 3,3′-diaminobenzidine and light microscopy. No significant apoptotic effects were evident in corneal tissues treated with APCP or only with helium (He). Data are expressed as the mean ± SE (error bars) of at least two independent experiments.

Figure 9. APCP-induced oxidative burst elicited short-term adaptive responses in mammalian cells.

Human keratocytes were exposed for 2 minutes to APCP and cultured under optimal conditions. The mRNA transcript levels specific for human nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, panel A), DNA glycosylase OGG1 (panel B) and glutathione peroxidase 1 (GPX1, panel C) were evaluated by quantitative real-time PCR. Expression of the target genes was normalized to the endogenous levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± SE (error bars) of gene copies in 5 µg of loaded total RNA, obtained from two independent experiments. ^* P<0.05 vs untreated cells. Protein expression of GPX and OGG1 in keratocytes was confirmed by Western blot analysis of samples taken at different time points after APCP treatment (panel D).

Figure 10. Antiseptic APCP effects on human corneas ex vivo.

Human corneal explants were kept wet in sterile PBS. Corneas were scarred using a needle and infected with 1×10² CFU bacteria and incubated for 16 hours at 37°C to allow for bacterial growth and colonization. Corneas were then exposed for 2 minutes to APCP and the wounded areas were immediately stroked using a sterile swab. Inocula were serially diluted, plated out and incubated under optimal conditions. Surviving bacteria were assessed by colony count assay and reported as CFU/ml. Data are reported as mean ± SE (error bars) of at least three independent experiments.

Figure 11. APCP treatment elicited ROS-dependent adaptive responses in ex-vivo corneas.

Human corneal tissues exposed for 2 minutes to APCP were kept wet at 37°C. At different time periods after exposure, tissues were dissected and snap frozen for RNA and protein extraction. The mRNA transcript levels specific for human glutathione peroxidase 1 (GPX1, panel A) and glycosylase OGG1 (panel B) were evaluated by quantitative real-time PCR. Expression of the target genes was normalized to the endogenous levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± SE of gene copy number in 5 µg of loaded total RNA obtained from two experiments. ^* P<0.05 vs untreated cells. Protein expression of GPX and OGG1 was evaluated by Western blot analysis (panel C).

Figure 12. Analysis of thymine dimers (TD) in the nuclei of APCP corneal tissue.

Corneal specimens exposed to APCP for 5 minutes were treated with anti-TD and counterstained with Hoechst. TD signals were not observed. Only the Hoechst staining was evident and localized in the nuclei of APCP-treated tissues;. Positive controls were prepared by exposing the tissue sections to UV rays at 254 nm for 10 minutes; negative controls were slides not incubated with the anti-TD antibody.