Modeling Diabetic Corneal Neuropathy in a 3D In Vitro Cornea System

Abstract

Diabetes mellitus is a disease caused by innate or acquired insulin deficiency, resulting in altered glucose metabolism and high blood glucose levels. Chronic hyperglycemia is linked to development of several ocular pathologies affecting the anterior segment, including diabetic corneal neuropathy and keratopathy, neovascular glaucoma, edema, and cataracts leading to significant visual defects. Due to increasing disease prevalence, related medical care costs, and visual impairment resulting from diabetes, a need has arisen to devise alternative systems to study molecular mechanisms involved in disease onset and progression. In our current study, we applied a novel 3D in vitro model of the human cornea comprising of epithelial, stromal, and neuronal components cultured in silk scaffolds to study the pathological effects of hyperglycemia on development of diabetic corneal neuropathy. Specifically, exposure to sustained levels of high glucose, ranging from 35 mM to 45 mM, were applied to determine concentration-dependent effects on nerve morphology, length and density of axons, and expression of metabolic enzymes involved in glucose metabolism. By comparing these metrics to in vivo studies, we have developed a functional 3D in vitro model for diabetic corneal neuropathy as a means to investigate corneal pathophysiology resulting from prolonged exposure to hyperglycemia.

Assembly and Application of a Three‐Dimensional Human Corneal Tissue Model

“…11666789001;optional) 15-ml polypropylene centrifuge tubes ELISA kit (e.g., for Substance P, CGRP, IL-1β, TNF-α, or MMP-9) a SP, substance P; CGRP, calcitonin gene-related peptide; IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor-alpha; MMP-9, matrix metalloproteinase-9. b The relative concentration ranges for each protein detected in conditioned medium in response to various chemical stimuli have been previously reported by our group (Deardorff et al, 2018;Wang et al, 2018).…”

“…In our previous work, we have applied different sensory sources, including dorsal root ganglions (DRGs) (Wang et al., ; Wang, Ghezzi, White, & Kaplan, ) and human induced neural stem cells (hiNSCs) (Deardorff et al., ; Wang et al., ), to promote corneal sensory innervation in co‐cultures of hCECs and hCSSCs. Utilization of hiNSCs has many advantages, including well‐defined differentiation protocols that generate a gradual transition to differentiated nerves with elongated neuronal fibers (Cairns et al., ; Deardorff et al., ). Herein, we describe the application of hiNSCs in this system and detail culture conditions for isolation, subculture, and differentiation protocols.…”

“…This approach allows one to dissect the contribution of specific cell types to physiological and disease processes, which may be difficult to identify in a complex animal model. Using the described corneal tissue model, we have previously reported characterization of activation of pain‐related signaling pathways in response to chemical irritation (Wang et al., ) and nerve loss following hyperglycemia (Deardorff et al., ), two biological events with endpoints parallel to in vivo outcomes. A key advantage of this corneal tissue model system is the compartmentalization of individual cell populations into separable scaffolds, i.e., with the epithelium seeded on the anterior silk film, the stroma seeded on posterior silk films, and the sensory nerves seeded in the peripheral silk sponge.…”

Association between Diabetes and Keratoconus: A Retrospective Analysis

“…Furthermore, though a report suggesting that acute exposure to elevated glucose alone may not promote altered keratocyte marker expression, e.g . keratocan and lumican, by healthy corneal stromal stem cells in vitro 41 , stimulation with chronic hyperglycemia may promote a differential response in corneal fibroblasts isolated from DM patients due to permanent mitochondrial damage and increased ECM thickness 22 . We posit that the epigenetic changes that occur during prolonged DM 42,43 may give rise to the characteristic defects in corneal structure, including differences in ECM composition 22,23 .…”

Three-Dimensional Human Cell Culture Models to Study the Pathophysiology of the Anterior Eye

“…Combination of a stromal model with endothelial cells and the presence of Descemet's membrane [91] Fabrication of tissue-engineered endothelial grafts using decellularized corneal stroma as scaffolds [92] Corneal equivalents Development of the first 3D model of the whole human cornea [93] 3D corneal model using human primary cells and the "self-assembly approach" (no use of exogenous material) [94] 3D corneal model with epithelium, stroma, and innervation [99][100][101][102]…”