In vivo confocal microscopy confirms the presence of new predescemetic cellular layer in post-penetrating keratoplasty buttons

Background/aim To investigate the in vivo presence of a natural plane of separation at the pre-Descemet membrane (DM) level in post-penetrating keratoplasty (PK) eyes using confocal microscopy. Methods In vivo confocal microscopy was performed on the corneal stroma of 20 post-PK eyes and 20 keratoplasty-naive healthy control eyes. Patients were recruited from the cornea service of Ospedali Privati Forlì € Villa Igea' between June 2022 and February 2024. The study adhered to the Declaration of Helsinki and was approved by the local ethics committee. Main outcome measures were stromal cell density (SCD) and the pattern of cell distribution. Results Posterior SCD was significantly higher in post-PK eyes compared with controls (387±96 vs 219±30 cell/mm², p<0.0001). A distinct layer densely populated by keratocyte-like cells, corresponding to the last visible row of nuclei before the DM, was observed in post-PK eyes but not in controls. Nuclei of these cells appeared irregularly dispersed or grouped in clusters, and in some cases arranged in columns along a preferred direction. Conclusions A distinct pre-DM layer of keratocyte-like cells is present in post-PK eyes potentially contributing to the previously described natural plane of separation during repeat keratoplasties. Further studies are needed to clarify the origin and clinical implications of this finding.