Abstract
Purpose: :
Zebrafish, a fresh water fish belonging to the minnow family, is an important model organism for studying vertebrate development and genetics. Advantages of using zebrafish as a model include rapid development with high fecundity and well-established imaging and genetic tools. The purpose of this study is to explore the utility of zebrafish cornea as a model for human corneal development and disease.
Methods: :
All zebrafish were handled according to University of Southern California IACUC approved protocols. Sections of zebrafish cornea were stained with hematoxylin and eosin for light microscopy and with osmium for electron microscopy. Confocal microscopy was performed on sox10 promoter:green flourescent protein transgenic zebrafish embryos at 4 days post fertilization using Zeiss LSM 5 confocal microscope. Adult wildtype zebrafish were euthanized and placed immediately in ice water prior to optical coherence tomography (OCT) and specular microscopy imaging. The central corneal thickness in zebrafish was determined by counting the pixels in the OCT scan of the central cornea relative to the full image depth of the OCT imaging system (2048 pixels = 5.2 mm). Endothelial cell size and density were determined using HAI Labs EB-2000 xyz eyebank specular microscope and HAI/CAS EB cell analysis software.
Results: :
Zebrafish corneas, like human corneas, were found to have 3 layers, epithelium, stroma and endothelium. The endothelium was found to consist of a single cell layer, but the epithelium was the thickest layer in zebrafish corneas. Zebrafish corneal endothelium was shown to express green fluorescent protein driven by the sox10 promoter, a transcription factor expressed in cells of neural crest origin during early development, indicating neural crest origin of the endothelial cells. The average central corneal thickness in adult wildtype zebrafish was determined to be 54 ± 7 µm (n = 16). Zebrafish corneal endothelial cells, like human corneal endothelial cells, were arranged as a polygonal monolayer. The average endothelial cell size was determined to be 317 ± 12 µm2 (based on 35 areas counted), and the average endothelial cell density in adult wildtype zebrafish was determined to be 3045 ± 258 cells/mm2 (n = 18).
Conclusions: :
The results show that zebrafish and human corneas share structural and developmental similarities, and provide normative values for central corneal thickness and endothelial cell size and density. This study suggests zebrafish could serve as a suitable model for human corneal development and disease.
Keywords: cornea: basic science • anatomy