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Elena Koudouna, Eric Mikula, Donald J. Brown, Robert D Young, Carlo Knupp, Andrew J Quantock, James Jester; Chick corneal morphogenesis: A new role for keratocytes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1998. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recent embryonic chick corneal studies have identified "fibropositors/keratopodia" supporting the hypothesis that keratocytes direct collagen organization during development. This hypothesis is contrary to conventional wisdom, which indicated a key role of the corneal epithelium through deposition of an orthogonally arranged, rotating primary stroma. The purpose of this study was to test this novel keratocyte hypothesis by revisiting the morphologic and structural changes in the corneal stroma during development using state-of-the-art 3-D collagen imaging through second harmonic generation (SHG) microscopy.
Collagen fiber and cell organization was examined in developing chick corneas from embryonic day 8 (E8) through E19 (N=4-6) using en face SHG and confocal imaging combined with 3D-Fast Fourier Transform (FFTs) reconstructions and Image J Directionality analysis. Cross-sectional vibratome sections extending from limbus to limbus were also imaged.
Morphology and orientation of presumptive keratocytes changed dramatically during corneal development. On E8, keratocytes were randomly organized in the anterior-mid stroma, but tended to orient parallel to the choroid fissure in the posterior stroma. By E9, keratocytes became orthogonally arranged and showed a clockwise rotation in the anterior stroma. On E10 and thereafter, cells became progressively more orthogonally arranged showing greater rotation. Before complete keratocyte invasion (E8-E9), 3D reconstructions of collagen showed the primary stroma to be orthogonally organized with no angular displacement. Vibratome section imaging similarly showed no evidence of collagen rotation prior to complete cell invasion. Importantly, appearance of orthogonal, rotated collagen was evident on E10, after cells had fully invaded the corneal stroma and showed an orthogonal and rotational organization. During primary stroma deposition (E7-E10) there was a strong SHG signal in the epithelial layer that significantly decreased (p < 0.025) after keratocyte invasion and deposition of rotating collagen fibers.
Contradictory to earlier studies, rotation of collagen fibers in the anterior stroma occurred after deposition of primary stroma and complete invasion by keratocytes. Furthermore, rotation of collagen fibers was preceded by earlier rotation of keratocytes, suggesting that the adult corneal stroma is deposited and organized predominantly by keratocytes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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