Abstract

DO.02.06

Activated keratocytes in confocal and histological appearance

Hovakimyan M.^1,4, Stachs O.^1,4, Wree A.², Seiler T.⁵, Heisterkamp A.^3,4, Guthoff R. F.¹
Universität Rostock, Medizinische Fakultät, ¹Klinik und Poliklinik für Augenheilkunde, ²Institut für Anatomie; ³Laser Zentrum Hannover, Abteilung Lasermedizin & Biophotonik; ⁴DFG Sonderforschungsbereich Transregio 37; ⁵Institut für Refraktive und Ophthalmo-Chirurgie

Objective: One of the most promising new approaches to manage keratoconus is collagen crosslinking in which riboflavin is applied to the cornea followed by UV-light irradiation. Crosslinking is now in clinical application and special emphasis is taken to ensure safety and wound healing properties of this method. Now existing data about the time lapse of wound healing process in cornea after crosslinking varies widely, ranging from weeks to months.
Methods: A cornea from a crosslinked patient with keratoconus was obtained and fixed post-mortem one month following crosslinking and paraffin slides have been investigated using DAPI and H.E.-stainings as well as immunhistochemistry.
Results: DAPI and H.E.-stainings have shown anterior stroma lacking nuclei. Only a small number of very occasional nuclei were to find in anterior stroma. Our suggestion was that these rare nuclei probably belong to cells which begun to repopulate anterior stroma. To verify the possibility of these cells of being activated keratocytes, characterized as fibroblasts and myofibroblasts, immunhistochemistry for PCNA (proliferating cell nuclear antigen) and a-smooth muscle actin has been performed. No immunoreactivity has been detected for both antibodies, suggesting that at this time point the repopulation has not yet begun.
Conclusions: In our previous study we have shown using in vivo confocal microscopy that 2 weeks following crosslinking highly hyperreflective cells with visible processes were present in the anterior stroma, resembling in their confocal appearance activated keratocytes. The interpretation of their morphology, however, has been based largely on existing confocal evidences about activated keratocytes. The comparison of existing findings about the crosslinking, obtained with different techniques, varies very. To our best knowledge, only confocal imaging is not sufficient to characterise healing process after crosslinking. Experiments, leading to combined confocal microscopic and immunhistological imaging of the same cornea at the same time point will be performed, enabling the correlation of both approaches.

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