Deep layers morphology study in human corneas by the use of confocal microscopy

Title: Deep layers morphology study in Human corneas by the use of confocal microscopy Authors: Luca Menabuoni, Alex Malandrini, Annalisa Canovetti, Ivo Lenzetti, Luca Buzzonetti, Paola Valente, Francesca Tatini, Giada Magni, Francesca Rossi Purpose: to investigate the morphology of predescemetic collagen layers in human corneas, in order to find correlation with age, pathology and to find out new indications to surgical manoeuvres. Setting: Nuovo Opsedale S. Stefano, Prato, Italy; Ospedale Pediatrico Bambino Gesù, Rome, Italy Methods: corneas from patients udergoing trasnplantation and healthy corneas not suitable for transplantation were collected. The samples were grouped into two: one group is from 20 adult subjects aged 59 ± 18 y.o., and the other group is from 15 young subjects, aged 12±5 y.o. The analysis of the deep stroma was performed by confocal microscopy: the corneas were sectioned and incubated with Collagen VI primary antibody, Alexa Fluor-conjugated secondary antibody, and with Phalloidin-Atto 550. Slices were mounted with fluoroshield containing DAPI and visualized using a confocal Leica SP8 scanning microscope. Images were acquired in z-series scans at 0.3 µm intervals. Results: Confocal microscopy analysis of posterior cornea has been performed in order to investigate relevant parameters such as cellular distribution and morphology, and collagen VI characteristics. A predescemetic layer with a higher concentration of collagen I and some samples with a higher concentration of collagen VI was found. High variability due to age and pathology was found. Conclusions: It seems that there is a different distribution of collagen type I and collagen type VI in the deep layers of the cornea, depending on age and pathology. A larger study is needed to improve statistics. Financial Disclosure: all the authors have no financial interests