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Andrea Leonardi, Elena Tarricone, Umberto Rosani, Matteo Zuin, Emilio Martines, Paola Venier, Paola Brun; Cornea transcriptome changes induced by Atmospheric-Pressure Cold Plasma for the treatment of ocular surface infections. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6264.
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© ARVO (1962-2015); The Authors (2016-present)
A prototype of an atmospheric pressure cold plasma (APCP) source has been developed for the treatment of corneal infections and for ocular surface sterilization. Two minutes of APCP treatment was previously found effective against various microorganisms but caused, in corneal fibroblast cultures, transitory ROS formation, expression of the ogg1 marker of oxidative damage and increase of apoptosis. In the present study we investigated the transcriptome changes of whole human cornea (HC) ex vivo exposed to the disinfectant dose of APCP.
The effects of exposure to APCP for 2 min on HC, in the absence or presence of the antioxidant N-acetyl L-cysteine (NAC) and related to unexposed controls were assessed at 6 h post-treatment by histological and immunohistochemical analysis, Western blotting and Illlumina RNA sequencing (RNA-seq). Additional HC were subsequently exposed to APCP in the same conditions to measure the expression of selected genes by quantitative Real-Time PCR (qRT-PCR) at 3, 6 and 24 h post-treatment.
Nearly 358 million of high quality reads were obtained, with about 50x106 per sample of uniquely aligned reads to the human genome (hg19 release). After data normalization and statistical analysis, 1114 differentially expressed genes (DEGs) (511 over- and 603 under-expressed) were identified in APCP-treated HCs and 1304 DEGs (773 over- and 531 under-expressed) in APCP-treated HC+NAC, with about one fourth of the total DEGs being modulated over 2 fold in the APCP-treated corneas. Gene expression signals consistent with APCP-induced oxidative stress are represented by the over-expression of inositol-trisphosphate 3-kinase C (ITPKC), many elements of the MAP kinase pathways, glutathione S-transferase M1 (GSTM1), cytochromes P450 (CYP4F11, CYP1A1) and glutathione peroxidase (GPX2). RNA-seq and qRT-PCR analysis indicated faint or no increase of ogg1 transcript at 6 h post-treatment.
Six hours after the exposure to APCP, the transcriptome of treated corneas revealed cells actively responding to oxidative stress with sparse evidence of genes involved in DNA damage sensing and DNA repair pathways. These results support the hypothesis of a transient and stimulatory cell response to the APCP pulse, and confirm the potential and safe use of APCP as a new strategy to prevent and treat ocular infections.
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