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Gary Hin-Fai Yam, Geraint P Williams, Matthias Fuest, Xiao-Wen Lee, Lei Zhou, Jodhbir Mehta; Nerve regeneration by human corneal stromal keratocytes (CSKs) and stromal fibroblasts (SFs). Invest. Ophthalmol. Vis. Sci. 2017;58(8):1173.
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© ARVO (1962-2015); The Authors (2016-present)
Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves, resulting in desensitization and neuro-epitheliopathy. After injury, surviving CSKs are activated to become SFs for wound healing. Here, we studied how these 2 phenotypically and functionally different cell types influence nerve regeneration.
Human CSKs and SFs were cultured from same donor stroma (n=6). Primary cells were characterized to express respective markers for CSK (keratocan, lumican, ALDH3A1) and SF (Thy1, 5B5). Serum-free media were conditioned, concentrated and applied to an in vitro chick dorsal root ganglion model. The extent of neurite outgrowth at 72 hours was quantified by the validated concentric circle intersection count method. The protein composition of conditioned media (CM) was profiled by Bio-Plex Human Cytokine assay and nanoLC-MS/MS with Enriched Gene Ontology (GO) Term analysis (Database for Annotation, Visualization and Integrated Discovery DAVID v6.8).
SFCM significantly promoted neurite outgrowth in a dose-dependent manner, compared to CSKCM. An array of neurotrophic and pro-inflammatory proteins were detected in SFCM, including IL8 (P=0.004, Mann-Whitney U test), IL15 (P<0.01), MCP1 (P=0.01), Eotaxin (P<0.01) and RANTES (P<0.01), but not in CSKCM. MS analysis followed by ProteinPilot search against InterPro human protein database identified >130 proteins in SFCM versus 49 in CSKCM. A list of proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing.
SFCM induced neurite outgrowth dose-dependently while CSKCM had negligible effect, suggesting that quality of nerve regeneration could be affected by SF density. LASIK photoablation induces CSK loss in the interface and retroablation zone. Delayed repopulation by SFs that have migrated from the para-ablation region will postpone nerve regrowth. Early repopulation of viable CSKs, e.g. following small incision lenticule extraction, provides a source of SFs that can contribute to more efficient nerve regeneration and quicker recovery of corneal sensation.
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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