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Alan Robert Burns, Thao Do, Paul Landry, Ali Behzad, Samuel D Hanlon; Ultrastructural details of corneal nerve mitochondria revealed by serial block face imaging and 3D reconstruction. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3077.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelium is richly supplied with nerve fibers which are not only essential for sensation but also for maintaining the health/integrity of the epithelium and cornea as a whole. The plexus of nerves is richly invested with mitochondria. Mitochondrial dysfunction/degeneration has been described in neuropathies such as diabetic peripheral neuropathy (DPN). Standard transmission electron microcopy, when coupled with stereology, can be used to estimate mitochondrial number, volume, density, and distribution within nerve fibers, but is limited by the difficulty encountered in obtaining large numbers of serial sections. The purpose of this study was to explore serial block face imaging as a novel ultrastructural method for 3D reconstruction and analysis of corneal nerves.
Corneas from C57BL/6 mice between 8-12 weeks of age were fixed and embedded in resin blocks for histological sectioning. Serial block-face images (100 nm intervals with 500-1000 images/z-stack) in perfect registration were obtained using an ultramicrotome (Gatan 3View) system mounted within the base of a scanning electron microscope. From the z-stacks generated, stromal, basal, and epithelial corneal nerves were manually segmented, as were the mitochondria within the nerves. Amira 5.2 software was used for 3D reconstruction and to evaluate potential means for characterizing nerves/mitochondria and obtaining quantifiable data.
Large stromal nerves were easily recognized whereas small epithelial fibers were often difficult to discern and did not become evident until scrolling through the stack of images. Small basal nerve fibers from the horizontal plexus just anterior to the epithelial basement membrane made occasional connections through the basement membrane to stromal fibers. Vertical extensions of fibers from the basal plexus within the epithelial cells were relatively more common. Quantification of nerve length, mitochondrial surface area, volume density, and number within nerve fibers was easily accomplished with the Amira 5.2 software.
Reconstruction of serial block-face images not only provided, for the first time, the means to observe 3D arrangements of mitochondria within corneal nerves but also to obtain quantitative data. Serial block face imaging has the potential for documenting mitochondrial changes that accompany a variety of corneal pathologies.
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