Low-temperature Plasma Device For The Treatment Of Corneal Infections

Abstract

Purpose: : Low temperature plasma is a partially ionized gas that has been considered for material processing and sterilization. A prototype of a plasma source has been developed. Preliminary studies demonstrated that cold plasma significantly reduced bacterial viability. The objective of the present study was to evaluate the effects of plasma on human cells and tissues and to explore the use of plasma as an antimicrobial agent for the treatment of corneal infections.

Methods: : Effects of the plasma on conjunctival fibroblasts and corneal keratocytes were assessed at different exposure times. The MTT assay was used to analyze the viability of cells after plasma exposure. ROS production in microorganisms and cell cultures was evaluated using the 2’,7’-dichlorodihydrofluorescein test. The micronucleus test (MNT) was used to ascertain the induction of DNA damage in human cells. Human corneas were infected ex-vivo with P. aerugionosa or S. aureus and treated with plasma for 5 minutes. Bacterial colony forming units (CFU) were then counted and histological observations were performed in parallel. Apoptosis and UV damage of corneal tissue were also analyzed with the Tunel test and immunohistochemical analysis.

Results: : Treatment of human cells did not cause any significant reduction of viability. High levels of ROS were observed in microorganisms and human cell cultures treated with plasma. Plasma application to infected corneas caused a significant reduction of microbial viability compared to untreated specimens, with no evidence of morphological damage observed by histological analysis. No induction of thymine dimer formation or apoptosis was observed in treated corneal tissues. Conversely, a significant increase in the frequency of micronucleated cells was found. However this effect disappeared 13 days after the treatment.

Conclusions: : Cold plasma reduced the viability of microorganisms but not that of human cells. The measurable effects consisted in high ROS production, probably associated with its antimicrobial activity. Despite the studies on human corneas in toto have shown that the treatment does not alter corneal tissues further studies are necessary to confirm the early clastogenicity of plasma application and to investigate the evolution of DNA damage in treated cells.

Keywords: cornea: clinical science • keratitis • cell survival