Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification

Abstract

Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation.

Fig 1. Schematic of the microplasma jet setup with plasma zones and a sketch of the biomedical treatments.

A) On the top of the liquid and B) inside the liquid medium.

Fig 2. Thermal imaging of microplasma and environment in respect to treatment time.

Typical images with marked temperature positions are presented for 3 cases; a)-c) free standing jet spreading into open air, d)-f) microplasma jet on the top of the liquid, and g)-i) microplasma jet inserted into liquid medium.

Fig 3. Real-time monitoring of morphological changes of aLC-LEC during indirect plasma treatment.

A doublet of adherent LECs were selected and treated by the microplasma on top of the medium for 30 s. The monitored targeted cells in time after the treatment are labeled by the red dotted line.

Fig 4. Real-time monitoring of morphological changes of aLC-LEC during direct plasma treatment.

A doublet of adherent LECs were selected and treated by the microplasma directly inside the medium for 30 s. The monitored targeted cells in time after the treatment are labeled by the red dotted line.

Fig 5. Comparison of the morphological changes evoked in a LEC.

Indirectly and directly treated with plasma are being shown for the same plasma treatment time of 30 s.

Fig 6. Cell death analysis and migration potential of the treated cells.

3 representative cases are being shown: a) immunostaining againt expression of Caspase 8 (an apoptosis marker), and b) CXCR4 (cell migration marker); c) DAPI stained cells (marker for DNA fragmentation) for assessment of cell death. The red-dotted line presents boundary of a single cell in a). while in c), it presents the treated area of impact with apoptotic bodies being formed.

Fig 7. The initiated liquid chemistry with microplasma treatment on top or inside the liquid medium.

a) Hydrogen peroxide and b) nitrite concentrations are being shown in respect to different treatment times.

Fig 8. Effect of He gas flow on the cell morphological responses.

Indirectly and directly treated with plasma are being shown.

Fig 9. The morphological changes connected to the apoptosis induction in the cells in respect to microplasma treatment over a given time and consequent incubation of cells for a) indirect and b) direct treatments.

The legend shows multiple potential apoptosis stages, which are highly likely for certain treatment after 24h, which are medium value of optical and fluorescent observation taking under consideration Gaussian distribution of results.