Abstract
Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density.
Highlights
During LASIK (Laser-assisted In situ Keratomileusis), a hinged corneal flap is created, by either a microkeratome or a femtosecond laser, and the exposed stromal bed is ablated by an excimer laser[8]
Using 12 dorsal root ganglion (DRG) incubated with medium containing 50 ng/ml nerve growth factor (NGF) for 72 hours, the immunofluorescence of TuJ1 images were converted into grey-scale and edges were enhanced (Fig. 1A and B)
The neurite extension profile plotted with the number of intersection against distance from DRG centre showed a reduction in neurites with increasing distance from DRG center, with an average of 57.7 ± 21.1 at 500 μmto 2.3 ± 4.5 intersections at 1500 μm (Fig. 1D)
Summary
During LASIK (Laser-assisted In situ Keratomileusis), a hinged corneal flap is created, by either a microkeratome or a femtosecond laser, and the exposed stromal bed is ablated by an excimer laser[8]. The post-operative reduction of nerve fiber density in the ablation zone has been reported to be as high as 80% compared to pre-operative levels[11] This axotomy leads to reduced corneal sensation, blink rates, lowered basal and reflex tearing and increased ocular surface exposure, that can result in LASIK-induced neurotrophic epitheliopathy. The surviving CSKs transit into activated stromal fibroblasts (SFs), which are proliferative, migratory and actively producing repair-type ECM components, including fibronectin, proteinases and cell-ECM adhesion molecules (α5-integrin)[7,31] They produce chemokines that attract inflammatory cells from the limbal blood supply and tear film to the corneal stroma where these incoming cells scavenge the cellular debris[32]. The potential secretory neuropeptides and factors that are involved in neurite growth were identified by proteomics and Bio-Plex cytokine assay
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