Nerve influence on the metabolism of type I and type II diabetic corneal stroma: an in vitro study

Abstract

Corneal innervation plays a major role in the pathobiology of diabetic corneal disease. However, innervation impact has mainly been investigated in the context of diabetic epitheliopathy and wound healing. Further studies are warranted in the corneal stroma-nerve interactions. This study unravels the nerve influence on corneal stroma metabolism. Corneal stromal cells were isolated from healthy (HCFs) and diabetes mellitus (Type1DM and Type2 DM) donors. Cells were cultured on polycarbonate membranes, stimulated by stable Vitamin C, and stroma-only and stroma-nerve co-cultures were investigated for metabolic alterations. Innervated compared to stroma-only constructs exhibited significant alterations in pyrimidine, glycerol phosphate shuttle, electron transport chain and glycolysis. The most highly altered metabolites between healthy and T1DMs innervated were phosphatidylethanolamine biosynthesis, and pyrimidine, methionine, aspartate metabolism. Healthy and T2DMs main pathways included aspartate, glycerol phosphate shuttle, electron transport chain, and gluconeogenesis. The metabolic impact on T1DMs and T2DMs was pyrimidine, purine, aspartate, and methionine. Interestingly, the glucose-6-phosphate and oxaloacetate was higher in T2DMs compared to T1DMs. Our in vitro co-culture model allows the examination of key metabolic pathways corresponding to corneal innervation in the diabetic stroma. These novel findings can pave the way for future studies to fully understand the metabolic distinctions in the diabetic cornea.

FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

“…One method is to supplement culture media with ascorbic acid (vitamin C), which normally promotes collagen secretion and deposition without promoting myofibroblast differentiation [ 5 , 16 , 33 , 34 , 47 , 81 , 82 ]. This hCF stroma-like 3D model and their self-assembled matrix, which mimics the corneal stroma during development [ 33 , 47 , 83 ], but also useful in investigating other pathologies affecting the corneal stroma, such as keratoconus [ 84 , 85 , 86 ], wound healing [ 33 , 87 ], and diabetes [ 88 , 89 , 90 ]. Collectively, these studies highlight that by increasing ECM dimensionality around hCFs, which can significantly influence cell proliferation and survival, mechano-responses, and their differentiation capacity [ 33 , 47 , 83 ].…”

Monocarboxylate Transporters: Role and Regulation in Corneal Diabetes

“…Primary human corneal fibroblasts were isolated from healthy, T1DM, and T2DM donor corneas, as previously reported [ 24 ]. Briefly, all human corneas were scraped to remove epithelium and endothelium, using sterile surgical scalpel blades [ 27 ]. The stromal tissue was then cut into small pieces, and explants were transferred into T25 flasks (4 or 5 pieces of ~2 × 2 mM per flask), followed by a 45-minute incubation at 37°C in 5% CO 2 to allow adherence.…”

“…Similar to the monocultures, all cocultures were maintained with VitC media for the duration of the experiments, as described previously [ 27 , 33 ]. At the end of week 1 (for week 2 timepoints/cultures) and week 3 (for week 4 timepoints/cultures), 12 k or 500 k SH-SY5Ys were seeded directly on top of the assembled 3D stromal constructs per well.…”

“…3D Constructs: Monocultures. HCFs, T1DMs, and T2DMs were seeded at a density of 1 × 10 6 /well on 0.4 μM polycarbonate membranes (Corning, Corning, NY, USA), as previously described [27,[30][31][32]. Cultures were maintained in EMEM, 10% FBS, and 1% AA and further stimulated by 0.5 mM stable vitamin C (VitC; 0.5 mM 2-O-α-Dglucopyranosyl-L-ascorbic acid, Sigma-Aldrich, St. Louis, MO, USA) in order to promote secretion and assembly of extracellular matrix (ECM).…”

“…Both nondifferentiated and differentiated SH-SY5Y neurons were tested. After 24 hours in culture, stromal cells and nondifferentiated SH-SY5Ys were fixed in 4% formaldehyde, as previously described [27,35]. Briefly, fixed cells were permeabilized and blocked with 3% milk for 1 h, followed by overnight incubation with primary antibody: SLC16A1/ MCT1(HPA003324; Sigma-Aldrich, St. Louis, MO, USA) Louis, MO, USA) 1 : 100.…”