Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

Abstract

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. Elastic modulus as determined by AFM for each corneal layer was: epithelium 0.57 ± 0.29 kPa (mean ± SD), ABM 4.5 ± 1.2 kPa, anterior stroma 1.1 ± 0.6 kPa, posterior stroma 0.38 ± 0.22 kPa, DM 11.7 ± 7.4 kPa, and endothelium 4.1 ± 1.7 kPa. Biophysical properties, including elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species with markedly less intertwining observed in the rabbit versus human cornea. Given that substratum stiffness considerably alters corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data will allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.

Myofibroblasts

“…Muscular tissues are stiffer (10-15 kPa) (Engler et al, 2004;Majkut and Discher, 2012), and bone and teeth are obviously stiff (Ho et al, 2007). The different layers of the rabbit cornea have recently been assessed for their elastic modulus using the same method of nano/microindentation (Thomasy et al, 2014). Corneal epithelium, anterior basement membrane, anterior and posterior stroma, and endothelium were shown to have distinct elastic moduli that range between 0.5-5.0 kPa, with Descemet's membrane being the stiffest layer in the cornea of ~10 kPa (Thomasy et al, 2014).…”

“…The different layers of the rabbit cornea have recently been assessed for their elastic modulus using the same method of nano/microindentation (Thomasy et al, 2014). Corneal epithelium, anterior basement membrane, anterior and posterior stroma, and endothelium were shown to have distinct elastic moduli that range between 0.5-5.0 kPa, with Descemet's membrane being the stiffest layer in the cornea of ~10 kPa (Thomasy et al, 2014). Notably, the scar of tissue under repair or in fibrosis is always stiffer than the normal tissue's texture (Balestrini and Hinz, 2015).…”

Robust and Artifact-Free Mounting of Tissue Samples for Atomic Force Microscopy

“…To evaluate this method, we chose two previously studied tissue types, rabbit corneal stroma (RC) and human trabecular meshwork (HTM) (11,12). Both tissues are sensitive to hydration.…”

“…For each tissue type, we chose six normal donors and split the tissue into two groups. For the first group, the tissue samples were affixed to the dish using an extremely thin layer of glue, as previously described (11,12). For the second group, the tissues were affixed using the method described above (Figure 1), with 4 mm windows for RC and 1.5–2 mm windows for HTM.…”

Lamellar and fibre bundle mechanics of the annulus fibrosus in bovine intervertebral disc

“…It has been applied to investigate, for instance, cartilage [18,19], cornea [20], tendon and ligament [21,22] structure and mechanics, as well as structural disorder in intervertebral disc [23].…”