Сравнительный анализ механического и фемтолазер-ассистированного методов выкраивания лимбальных мини-трансплантатов в эксперименте

Abstract

Original article Experimental study of manual and femtosecond laser-assisted methods for cutting limbal mini-transplants B.E. Malyugin, S.A. Borzenok, O.N. Nefedova, D.S. Ostrovskii, M.Yu. Gerasymov, A.V. Shatskikh S. Fyodorov Eye Microsurger y Federal State Institution, Moscow, Russian Federation A. Yevdokimov Moscow State University of Medicine and Dentistr y, Moscow, Russian Federation Purpose. To define optimal parameters of femtosecond laser (FSL) (Femto LDV Z8, Ziemer, Switzerland) for cutting limbal mini-transplants to be further used for limbal stem cells (LSC) transplantation and compare the results with manual cutting technique using microsurgical blade. Material and methods. At first step, performed on 3 porcine and 3 cadaveric eyes, the optimal energy parameters of FSL were selected, which ensured the excision of limbal mini-transplants. At the second step, 8 eyes from 4 postmortem donors were used. In the lower and upper parts of the limbus, limbal fragments were cut out into 8 mini-transplants with a FSL (experimental group). Mini-grafts were also formed manually in symmetrical areas of each eye using dosed diamond blade (control group). The structure of the limbal bed after removing mini-transplants was studied by light and scanning electron microscopy. The effect of FSL energy on the viability of LSC was determined by intravital staining using Live & Dead dye. Finally, on the third step, the assessment of reversible and irreversible apoptosis was performed by culturing corneoscleral discs containing mini-transplants not separated from the limbal bed. The obtained samples were cultivated in a medium DMEM/F12 for 7 days followed by immunohistochemical examination. Results. The optimal parameters of FSL operation are determined by energy levels- 100, 110 and 120%. Histological analysis of the limbal residual bed zone showed that the smoothest surface was obtained at energies 110 and 120%. This fact was also confirmed by scanning electron microscopy. When stained with the Live & Dead dye, damage on the lateral and inner sides of limbal mini-transplants was revealed, mainly in the experimental group. After 7 days of cultivation in the cut area in both groups, no expression of apoptosis markers was observed. Conclusion. Cutting out the limbal zone containing LSCs using FSL is safe and allows obtaining limbal mini-grafts. The most optimal parameters of laser operation in the limbal zone were selected – a planar cut at depth of 250 µm using 110% laser energy. Key words: femtosecond laser, limbal stem cells, apoptosis, limbal stem cell deficiency syndrome, scanning electron microscopy, light microscopy, cultivation, histology