3D-konfokale Laser-Scanning-Mikroskopie der kornealen epithelialen Nervenstruktur

Zusammenfassung

Hintergrund: Entwicklung und Beurteilung einer neuen Methode zur In-vivo-Charakterisierung der epithelialen Nervenstruktur. Methode: Die anteriore Hornhaut von Probanden wurde mit der 3D-konfokalen Laser-Scanning-Mikroskopie (HRT II + RCM) untersucht, wobei die erhaltenen optischen Schnitte dreidimensional rekonstruiert werden (AMIRA 3.1). Ergebnisse: Die räumliche Anordnung von Epithel, Nerven und Keratozyten der Hornhaut kann mit der entwickelten Methode in vivo dargestellt werden. Die 3D-Rekonstruktion liefert eine Vorstellung der räumlichen Architektur kornealer Nerven: Dicke Fasern steigen aus dem subepithelialen Plexus auf. Ein verzweigtes Netzwerk von dünneren Fasern verläuft exakt parallel zur Bowmann-Membran im subepithelialen Plexus. Äste, die in die superfiziale Epithelzellschicht aufsteigen, können nicht dargestellt werden. Alterationen nach refraktiven Eingriffen können beobachtet werden. Als limitierend wirkt sich die begrenzte Scantiefe von 40 - 50 µm aus. Schlussfolgerungen: Die 3D-konfokale Laser-Scanning-Mikroskopie erlaubt eine Visualisierung und Analyse der räumlichen Struktur der kornealen Nerven im Epithels und Stroma. Die entwickelte Methode liefert eine Basis für weitere Studien von Alterationen der kornealen Nervenstruktur nach refraktiven Eingriffen.

Abstract

Purpose: The aim of this study was the evaluation of a technology for in vivo visualization of distribution and morphology of corneal nerves by means of 3D confocal laser scanning microscopy (3D-CLSM). Method: The anterior corneas of four human volunteers were examined by an in-house developed confocal laser scanning microscope based on a commercially available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany). Raw stacks were converted using ImageJ (NIH, USA) for 3D-reconstruction using AMIRA 3.1 (TGS Inc, USA). Results: The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. After 3D-reconstruction of volunteers’ corneas, volume rendering and selective oblique sections have been done to demonstrate the nerves in the central human cornea. 3D-imaging shows thick nerve bundles rising out of the deeper stroma. The nerves further divide, resulting in fibers that are arranged parallel to Bowman’s layer and are partly interconnected. Branches rising up to the superficial cell layer cannot be visualized. Wound healing following refractive surgery can be evaluated. Conclusions: 3D-CLSM allows in vivo visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes of the human cornea. The developed method provides a basis for further studies on the alterations of the cellular arrangement and epithelial innervation in corneal diseases. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

Konfokale Mikroskopie - Kornea - Nerven

Confocal microscopy - cornea basic science - nerve fiber layer

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