Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses
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Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of “safe” values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium.
Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium.
Ninety percent of the endothelial cells survived when using 16.5 μJ pulses no closer than 200 μm to the endothelium, or pulses not exceeding 2 μJ at a distance of 50 μm.
The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 108 Pa/m.
Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium.
KeywordsCornea Keratoplasty Cell viability Ultrashort pulse Laser surgery Endothelium
The authors thank Gilles Thuret, Zhiguo He, and Philippe Gain from the research unit Biologie, Ingénierie et Imagerie de la Greffe de Cornée, Jean Monnet University, Saint-Étienne, France, for help with the staining protocol and for fruitful discussions. We also thank Ralf Brinkmann from the Institute of Biomedical Optics, Medizinische Universität Lübeck, Germany, for helpful discussions.
Compliance with ethical standards
The RTRA (Advanced Research Cluster) Triangle de la physique provided financial support in the form of a post-doctoral position for Z.E. and a complementary budget for material expenses and consumables.
A.C. and C.M. acknowledge support from the French DGA. The sponsors had no role in the design or conduct of this research.
Conflict of interest
All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements) or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
This article does not contain any studies with human participants or animals performed by any of the authors.
Porcine corneas were used under permission number 91 477 102, issued by the Direction départementale de la protection des populations, Essonne department, France.
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