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Pouriska Kivanany, Byron Weiss, Elias Choi, Kevin Lam, Nihan Yönet-Tanyeri, David Schmidtke, Matthew Petroll; Cell patterning in native and newly formed corneal ECM post lamellar keratectomy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1256.
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© ARVO (1962-2015); The Authors (2016-present)
The organized arrangement of the collagen lamellae plays a key role in corneal transparency. Following injury to the cornea, quiescent corneal keratocytes transform into fibroblasts and myofibroblasts. We have previously shown that following freeze-injury, migrating corneal fibroblasts align parallel to the stromal lamellae. In this study, we compare cell and extracellular matrix (ECM) patterning within and on top of the stroma following lamellar keratectomy (LK).
LK was performed on New Zealand White Rabbits. From 1 to 60 days after injury, rabbits were monitored using in vivo confocal microscopy and sacrificed to further investigate cell and matrix patterning. The corneal tissue was fixed and labeled with Alexa Fluor 488 phalloidin in situ, and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging. Cell patterning was also assessed in vitro, by plating corneal keratocytes on top of aligned fibrillar collagen substrates that were generated using a microfluidics approach.
Immediately following LK, cell death occurred in the native corneal stroma directly beneath the area of injury. At 7 and 21 days after LK, confocal and SHG results indicated that the alignment of fibroblasts repopulating this region was highly correlated with the orientation of the collagen lamellae. In contrast, cells on top of the native corneal stroma were randomly arranged, contained more prominent stress fibers, and secreted fibrotic ECM. Alpha smooth muscle actin (α-SMA) was also detected in this region, indicating the presence of myofibroblasts. At 60 days, cells and matrix on top of the stroma were co-aligned into lamellar-like structures. Corneal haze peaked at 21 days after LK, and returned to near baseline levels by 60 days. In culture studies, corneal keratocytes stimulated with platelet derived growth factor aligned parallel to unidirectional collagen fibers.
Based on these results, we hypothesize that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after corneal injury in the rabbit. Initially, cells on top of the native stroma align randomly and produce a disorganized ECM, since they do not have topographic cues to guide them. Over time, cells remodel this fibrotic ECM to produce a lamellar structure that is similar to the native corneal stroma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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