Disinfection of Ocular Cells and Tissues by Atmospheric-Pressure Cold Plasma

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 and 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

Tailored approach to the treatment of vernal keratoconjunctivitis

To develop a standardized clinical grading system for the management of patients with vernal keratoconjunctivitis (VKC) and to identify the risk factors associated with a worsened outcome of the disease, including decrease of visual acuity and ocular complications development

Atmospheric-pressure cold plasma induces transcriptional changes in ex vivo human corneas

BACKGROUND: Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. OBJECTIVES: In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. RESULTS: Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than \ub1 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. CONCLUSIONS: These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant

qPCR analysis of selected genes at different time points.

<p>Two human corneas were used to evaluate the expression of specific genes by qPCR at selected time points from the exposure to APCP. To minimize the variability of response, both corneas, one used as negative control and the other exposed to 2 min APCP, were divided into three pieces, then collected at 3, 6 and 24 h post-treatment. Expression levels of the target genes, detected in the treated corneal sample relative to the untreated sample were normalized to GAPDH levels.</p

Differentially expressed corneal genes (DEGs) at 6 h after exposure to APCP.

<p>(a) over- and under-expressed DEGs are shown as common (overlapping area) or exclusive to the APCP (left) or APCP+NAC (right) treatments; (b) number of total (Baggerly’s test FDR p-value <0.01) and DEG, over- and under-expressed genes detected in HC1-HC6 samples, paired per condition.</p

Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.

<p>GAPDH was used as housekeeping gene, other genes were selected for RNA-seq validation (underlined) or time-related expression analysis (*)</p><p>Gene ID, Ensemble ID and related Forward and Reverse primers used in qPCR.</p

Detection of OGG1 in human corneas treated ex-vivo with APCP.

<p>Corneal tissues exposed for 2 min to APCP were analyzed by immunohistochemistry (a-d) and Western Blot (e-f). Frozen sections (5 μm) of corneas treated in the absence (a) or presence (b) of 10 mM NAC were incubated with polyclonal rabbit anti-OGG1 at 6 h post-treatment. Protein immunostaining (in red) was compared to that of untreated controls (c). Negative controls were prepared by omitting the primary antibody (d). For the Western Blot analysis, proteins were extracted at 6 and 24 h post-treatment: the OGG1 protein signal increased at 6 h, and was reduced in the presence of NAC, and returned to values comparable to that of controls within 24 h. Densitometric values of OGG1 autoradiographic bands were normalized to corresponding β-actin and expressed as percentage ± SE of the mean control value.</p

Hierarchical clustering performed on the whole normalized dataset.

<p>52,447 genes, Manhattan correlation, complete linkage.</p