Observation of Corneal Wound Healing and Angiogenesis Using Low-Vacuum Scanning Electron Microscopy

Purpose: Wound healing processes in a rat corneal alkali burn model were observed using low-vacuum scanning electron microscopy (LV-SEM), a new observation method that can use paraffin sections for light microscopic immunostaining. Methods: Injured cornea was observed under immunohistochemistry, LV-SEM, and transmission electron microscopy. In LV-SEM, periodic acid-methenamine silver staining was used to observe collagen and platinum blue staining was used to observe vascular endothelial cells. Analyses of the messenger RNA expression involved in neovascularization processes after wound creation were also performed. Results: LV-SEM depicted progression of corneal wound healing in a stereoscopic fashion. In neovascularization processes after wound creation, LV-SEM with osmification clearly demonstrated detachment of pericytes from the vascular endothelial cells, in association with up-regulation of angiopoietin-2 messenger RNA expression. Conclusions: LV-SEM enables high magnification observation of paraffin sections used for immunohistochemistry. LV-SEM provides easy, detailed observations and offers a promising new observational modality in the field of ophthalmology. Translational Relevance: Highmagnification analysis was easily available using LV-SEM with conventional paraffin sections for light microscopy.