Injury, Inflammation, and the Effect of the Metabolic Syndrome on the Mouse Cornea
Hargrave, Aubrey Christine
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PURPOSE: The cornea is susceptible to injury and internal systemic damage that can affect its transparency, and subsequently, vision quality. The health of the cornea, the most innervated tissue in the body, depends on functional and intact corneal nerves. The purpose of this dissertation was to characterize changes in the mouse cornea under two distinct conditions: 1) injury (epithelial abrasion) and 2) systemic inflammation (high fat diet-induced obesity accompanied by metabolic syndrome). The role of the limbal mast cell after abrasion was evaluated in the context of epithelial wound healing, and nerve and keratoctye regeneration. In mice fed a high fat diet (HFD), morphological and functional changes in the corneal epithelium and nerves were analyzed in the uninjured cornea, as well as associated changes in cytokine expression. Finally, the effect of diet-reversal (return to normal diet (ND) consumption) on corneal pathology was evaluated. METHODS: For mast cell studies, C57BL/6 and mast-cell deficient KitW-sh/W-sh mice underwent a 2mm corneal abrasion; some C57BL/6 mice were treated with a mast cell stabilizer (cromolyn or ketotifin) prior to the injury. For obesity studies, 6-week (w) old male C57BL/6 mice were fed a HFD (42% Kcal milk fat) for 5, 10, or 15w. Controls were age-matched mice on a ND. Diet reversal (DR) mice were fed a HFD for 5w, followed by a ND for 5 or 10w. Cornea homogenates were collected after 5, 10, or 15w of HFD feeding. At 10w, some corneas were incubated in cell culture medium for 6h. Homogenates were analyzed for cytokine expression using a 32-plex Luminex assay. Corneal sensitivity was measured using a Cochet Bonnet aesthesiometer. Excised corneas were prepared for plastic sectioning to evaluate epithelial thickness. Flat-mount corneas were prepared for immunofluorescence microscopy to evaluate wound healing (leukocyte extravasation, epithelial recovery, wound closure rate), nerve density and morphology, or keratocyte density using MATLAB software or stereology. Data were analyzed using a two-tailed t-test or a one-way ANOVA with a Tukey or Dunnett post-test. A p-value ≤ 0.05 was considered significant. RESULTS: Prior to injury, corneal parameters between adult C57BL/6 and KitW-sh/W-sh mice were similar. During the first 36h post-injury, KitW-sh/W-sh and wild-type mice treated with mast cell stabilizers showed reduced limbal vessel dilation, platelet extravasation, and epithelial cell division. Wound closure and neutrophil recruitment were delayed in the KitW-sh/W-sh mice. At 96h post-injury, wild-type and KitW-sh/W-sh mice showed similar levels of epithelial restratification. Long-term nerve and keratocyte recovery (4-16w) was similar between the two groups of mice. After 5w of HFD consumption, corneal sensitivity and the number of central vertical nerves declined. After 10w of HFD, corneal nerve density and central epithelial thickness declined, and inflammatory mediator expression increased. Similarly, inflammatory mediator expression increased in supernatants collected from 10w HFD excised corneas cultured for 6h. Remarkably, diet reversal for 5w restored corneal sensitivity, nerve density, epithelial thickness, and inflammatory mediator expression in excised corneas to the level of ND mice. Inflammatory mediators remained upregulated in the cultured corneas and supernatants following DR. CONCLUSION: In the mouse, limbal mast cells are necessary for early and efficient wound closure following a corneal abrasion. However, long-term recovery of epithelial nerves and keratocytes is not dependent on mast cells. The contribution of limbal mast cells to early wound healing likely reflects their ability to amplify the inflammatory response and enhance leukocyte recruitment, which is necessary for efficient wound closure. Consumption of a HFD is associated with pathological changes in corneal nerve and epithelial morphology and function, along with an increase in expression of inflammatory mediators. Importantly, these HFD-induced changes are largely reversed or prevented by resuming a normal diet.