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Cell viability in the endothelium of porcine cornea exposed to ultrashort laser pulses at 1030nm

Poster Details

First Author: S.Hussain FRANCE

Co Author(s):    L. Kowalczuk   Z. Essaïdi   F. Alahyane   M. Schanne-Klein   M. Guedj   K. Plamann

Abstract Details



Purpose:

The corneal endothelium is a monolayer of cells at the posterior side of the cornea. The primary function of it is to keep the corneal hydration at a constant level and to thereby maintaining corneal transparency. Endothelial dysfunction can compromise visual acuity or even lead to blindness. In many cases, total or partial corneal grafting (keratoplasty) is indicated. Grafts can be prepared either mechanically by means of a microkeratome or by using a femtosecond laser. In the work we present here we have studied the viability of porcine corneal endothelial cells after keroplasty assisted by ultrashort laser pulses. Two sets of experiments were performed: the first one at a constant energy (variable depth) and the second at variable energy (constant depth). For a very high value of energy (~17µJ) and close to endothelial cells, we observed a noticeable decrease in the viable cell area. The exact mechanism behind the cell death has not been fully elucidated, however a plausible explanation for the observed cell viability behaviour would be the exposure to the shockwave that forms due to the interaction of femtosecond laser pulses with the tissue.

Setting:

Laboratoire d'Optique Appliquée ENSTA ParisTech - École Polytechnique - CNRS UMR 7639.

Methods:

After extraction from the eye the cornea was placed on an artificial chamber filled with balanced salt solution which was mounted onto an XYZ step motor unit. The laser beam was focused into the cornea from the anterior side by using a microscope objective with a numerical aperture of 0.45. The step motor module was controlled by a personal computer using the LabView programming language. Pre-programmed routines were used to produce planar ('lamellar') cuts inside the volume of cornea. Two sets of data were obtained, one was obtained with pulses of constant energy while the other at constant depth while varying the laser pulse energy. To evaluate cell viability we have used a live/dead cells staining kit and Nucleus staining (A. Pipparelli et.al. IOVS, 2011). The stained cornea was examined by using a fluorescence microscope. A typical image in RGB provides information about live cells (green), dead cells (red) and the nuclei of all the cells which are alive, dead, and dying (blue). By analyzing the images the available viable area was represented as a black area. The percent of the viable area is calculated through the available black area over the whole region of interest.

Results:

The first set of results was obtained by selecting a fixed value of energy and increasing the distance between the focal spot and the endothelial cells. For a distance of 50µm the percentage of the surface occupied by live cells is very low. We also performed experiments for 100, 200, 300 and 400µm; with increasing distance the percentage of the surface occupied by live cells approached 100%. We then performed another set of experiments by inducing a lamellar incision at a fixed distance of 50µm from the endothelium while subsequently decreasing the pulse energy from 17 to 2.15µJ. At 17µJ the viability of endothelial cells was zero and it starts to approach 100% of viable area beyond 5µJ. With the help of literature data we estimated the amplitude of shockwave corresponding to the experimental parameters. The estimated values show that cell death occurred when amplitudes of several tens of mega Pascal were reached; the cells remained alive at shockwave amplitudes below several mega Pascal.

Conclusions:

In this paper we have investigated the effect of ultrashort laser pulses on endothelial cells under experimental conditions comparable to those permitting to prepare endothelial grafts by ultrashort pulse lasers. The obtained results show a clear dependency of cell viability on pulse energy and laser focus distance to the endothelium; in particular our data permits the definition of 'safe' conditions with respect to pulse energy and distance to the endothelium. Although the exact mechanism behind cell death has not been identified. However, a plausible explanation for cell death during the laser procedure is the exposure of the cells to the shock wave generated by the laser-tissue interaction. FINANCIAL INTEREST: NONE

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