Spatial relationship between histological staining intensity and corneal stiffness variations: Insights from AFM indentation in infant African green monkeys.

Abstract

This study investigated the spatial variations of mechanical properties and microstructure in the cornea using atomic force microscopy (AFM) indentation tests and histological analysis. Corneal samples were collected from three infant African green monkeys, approximately 6 months old. Hematoxylin and eosin (H&E) staining was performed on corneal cross-sections to examine microstructure and quantify staining intensity. AFM indentations were conducted to quantify stiffness variations through pathline scanning and 16 × 16 stiffness mappings. Results showed that the corneal microstructure transitions from thinner, denser lamellae in the anterior layer to thicker, looser lamellae in the posterior layer. Stiffness variations along pathlines in the central, paracentral, peripheral, and limbus regions correlate positively with the corresponding staining intensities. The average stiffness across all samples was highest at the central anterior cornea (392.6 ± 118.4 kPa) and anterior limbus (645.4 ± 158.1 kPa). Additionally, both the anterior and posterior layers showed higher stiffness than the middle layer, except in the central region. AFM stiffness maps further revealed the layered structure of the lamellae. The stiffness variations between layers may result from different orientations of collagen fibrils in each lamellae. These observations were expected to provide valuable insights into corneal microstructure and mechanical properties variations during the progression of corneal diseases, aiding in the design of optimal artificial corneas. While this study focuses on infant monkey eyes, further testing across different age and sex groups is needed to refine these observations.