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Danielle Robertson, Matthew Petroll, Meifang Zhu, Vindhya Koppaka; Three-dimensional volumetric reconstruction of the mouse diabetic cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5441.
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Mouse models of diabetes are routinely used to investigate the effects of hyperglycemia in vivo. The purpose of this project was to investigate the use of three-dimensional volumetric imaging of the subbasal nerve plexus and associated corneal epithelial nerve fibers to allow for quantitative assessment of corneal nerve changes in a diabetic mouse model; and to correlate alterations in corneal nerves with total and sublayer thickness changes in vivo.
Type I diabetes was induced in male C57BL/6 mice using the Animal Models of Diabetic Complications Consortium high dose streptozotocin (150 mg/kg body weight) protocol. Mice were evaluated for corneal nerve and epithelial changes at 6 and 12 weeks. Age-matched vehicle treated mice were used as controls. Serum glucose levels and body weight were assessed at 6 and 12 weeks. A serum glucose level greater than 300 mg/dl was diagnostic of diabetes. Total corneal and sublayer thickness changes were assessed using quantitative three-dimensional in vivo confocal microscopy (q3D-IVCM). Corneas were then excised and stained with anti-beta-tubulin III and labeled with a FITC-conjugated secondary antibody. Nuclei were counter-stained with propidium iodide. Whole mount corneas were imaged on a laser scanning confocal microscope. Image stacks were reconstructed three-dimensionally and analyzed using MetaMorph and IMARIS software.
Using q3D-IVCM, mean corneal thickness in control mice was 111.5 ± 11.2 µm. Sublayer thickness values were 73.2 ± 5.2 µm and 38.3 ± 6.5 µm, for the stroma and epithelium, respectively. Volumetric reconstruction of corneal nerves demonstrated that epithelial nerve fibers extending from the subbasal plexus turn and course horizontally between superficial epithelial cell layers prior to termination with occasional branching and anastomoses detected. In STZ-treated mice, significant changes in serum glucose levels and body weight were evident. Alterations in corneal epithelial nerves and the subbasal nerve plexus were seen as early as 6 weeks.
Three-dimensional volumetric reconstruction of the mouse cornea allows for quantitative assessment of corneal tissue and nerve changes and represents a novel technique for assessing diabetic-induced changes in the mouse cornea.
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