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Samuel Hanlon, Nancy Shenoi, Paul Harris, Paul Landry, Ali Behzad, Evelyn Brown, Margaret Gondo, Alan Burns; Keratocyte-Keratocyte Translamellar Connectivity in the Mouse Cornea is Revealed using a Novel 3-D Ultrastructural Approach. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5238.
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© ARVO (1962-2015); The Authors (2016-present)
Like the human cornea, mouse stromal keratocytes lie between the collagen lamellae and make extensive lateral connections with one another forming layers essentially parallel with the corneal surface. This parallel intralamellar network arrangement is important to the keratocytes as it allows for cell-cell communication via gap junctions. The extent to which keratocytes form transverse (i.e., translamellar) connections is unclear. The purpose of the present study was to use a novel imaging strategy to reconstruct the ultrastructural 3-D arrangement of the keratocyte network and specifically evaluate the distribution of translamellar keratocyte-keratocyte connections.
Corneas from C57BL/6 mice between the ages of 8-12 weeks were fixed, heavy metal contrasted and embedded in resin blocks for transverse serial block-face sectioning using a Gatan 3view microtome system mounted in an FEI Quanta FEG 200 scanning electron microscope. Stacks of 375-700 serial Z images (at 100 nm intervals; XYdimensions 28x28 um) were obtained from the stroma in the limbus, paralimbus, and central cornea. Amira 5.2 software was used to segment keratocytes for 3-D image reconstruction.
Segmented volumes revealed extensive intralamellar contact between keratocytes. Translamellar contact was rare in the central cornea region with only one translamellar contact observed in a total of five separate reconstructions. In the paralimbus and limbus regions, translamellar contacts were more common (2.7x105±1.3x105 and 6.6x105±2.2x105 per mm3, respectively) suggesting there may be as many as 1.2x105 keratocyte-keratocyte translamellar contacts in a single mouse cornea (est. 0.4mm3).
Collectively, the 3-D data obtained by serial block-face sectioning show for the first time that in the mouse, interlamellar keratocyte layers oriented parallel to the corneal surface are indeed linked by translamellar keratocyte-keratocyte connections, but this occurs predominantly at the peripheral cornea. We propose a model whereby keratocytes form a single contiguous 3-D network, rather than a series of independent parallel networks, with a higher degree of translamellar connectivity than previously appreciated.
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