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Colton Heinrich, Ali Behzad, John D Goosey, Alan Robert Burns, Jan P G Bergmanson; 3-D Imaging of Epithelial-Stromal Interactions Found in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4202.
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Keratoconus (Kc) is a disease where the cornea becomes thinner, ectatic, and scarred. The basement membrane (BM), anterior limiting lamina (ALL), and anterior stromal lamellae are altered or lost throughout the course of the disease. While epithelial and stromal involvement is thought to contribute to disease initiation and progression, the nature of this interaction is unclear. The purpose of this study was to use serial block face scanning electron microscope (SEM) imaging and computerized 3-D image reconstruction to evaluate the pathophysiology of Kc.
A penetrating keratoplasty was performed on a 47 year old white male diagnosed with Kc four years prior. The surgically removed Kc corneal button was fixed, processed, and embedded in resin blocks for transverse serial block-face imaging using a Gatan 3 View system mounted in an FEI Quanta FEG 200 SEM, using an established protocol. A stack of 165 serial images separated by 100 nm increments and measuring 49 μm X 49μm in XY was obtained from the central anterior cornea. Amira 5.4.3 software was used to segment the anterior cornea for 3-D image reconstruction.
Following 3-D reconstruction, epithelial and stromal interactions were evident. Two keratocytes were observed to project anteriorly and engage with epithelial cells located in the basal epithelium. Likewise, complementary basal epithelial cell protrusions were seen to extend toward the keratocytes.
The novel Gatan 3 technology can provide unique and useful data on Kc pathophysiology. The 3-D reconstructions obtained by serial block-face imaging demonstrate for the first time in Kc that there are interactions between the stroma and epithelium. This observation warrants further study as it will be important to establish the extent and consequence of this pathological tissue interaction.
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