Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses

Abstract : Purpose : Clinical surgical laser systems based on ultrashort pulse lasers have become increasingly widespread in corneal surgery. Some forms of keratoplasty require performing laser cuts close to the endothelium, using “safe” values concerning incision depth and pulse energy in order to avoid damaging the endothelium. We present a study on the viability of endothelial cells of porcine corneas exposed to ultrashort laser pulses. The thresholds for cell death in terms of laser pulse energy and closeness of the impacts to the endothelium were compared to numerical simulations of the maximum shock wave amplitude at the corneal endothelium. Methods : Experiments were performed with a laboratory set-up based on a laser emitting 730 fs pulses at a wavelength of 1030 nm in corneas from eyes of pigs aged of about 6 months. Lamellar cuts were induced, first, using pulse energies of 16.5 µJ at distances from the endothelium from 50 µm to 400 µm, and then, at a fixed distance of 50 µm from the endothelium with pulse energies from 2 µJ to 14 µJ. The triple Hoechst 33342-Ethidium-Calcein staining protocol was used to determine the viability of endothelial cells after the laser cut. Numerical simulations of the Euler equations for compressible fluids provided pressure level estimates at the corneal endothelium. The broadening of the laser spot in the volume of the cornea was taken into account for the calculations. Results : Ninety percent of the endothelial cells survived when using 16.5 µJ pulses not closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed cell viability percentage with the calculated estimates of the shock wave amplitudes generated by the laser pulses yielded cell death thresholds at amplitudes in the Megapascal range. Conclusions : Our results permit to define limits in terms of pulse energy and distance of the incision to the endothelium within which the viability of the endothelial cells is preserved. The parameters used for current forms of corneal surgery assisted by ultrashort pulse lasers are compatible with these limits. However, these limits will need to be taken into account for the development of future laser routines working closely to the endothelium as cell death threshold may be reached under those conditions.