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Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts.

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.

SUBMITTER: Yam GH

PROVIDER: S-EPMC5369053 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts.

Yam Gary Hin-Fai GH Williams Geraint P GP Setiawan Melina M Yusoff Nur Zahirah Binte M NZ Lee Xiao-Wen XW Htoon Hla Myint HM Zhou Lei L Fuest Matthias M Mehta Jodhbir S JS

Scientific reports 20170328

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 sam ...[more]

PMID: 28349952

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Project description:BackgroundHuman corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth.MethodsThree placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry.ResultsAME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures.ConclusionAME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Project description:Neurotrophic Keratopathy (NK) is a neurodegenerative corneal disease that results in diminished corneal sensation. Previous studies have found that Cenegermin 0.002%, a recombinant human nerve growth factor (rhNGF), improves corneal epithelial healing in stage 2 and 3 NK patients. However, rhNGF effect on corneal sensation and nerve regeneration has not been well established. Thus, this study aims to analyze the effect of rhNGF on corneal nerve regeneration using in vivo confocal microscopy (IVCM) and on corneal sensitivity in NK patients. This is a retrospective, longitudinal, case-control study that included patients with NK, treated with rhNGF for at least 4 weeks, with pre- and post-treatment IVCM images available for analysis. Chart reviews were conducted documenting prior medical and surgical history, clinical signs and symptoms, and corneal sensation using Cochet-Bonnet esthesiometry. Corneal nerve parameters were assessed by IVCM. Sex- and age-matched reference controls were selected from a database of healthy subjects for comparison. The study included 25 patients, with 22 (88%) stage 1, two (8%) stage 2, and 1 (4%) stage 3 NK patients, with a median age of 64 years (range: 30-93 years). Total, main, and branch nerve densities [median (range) in mm/mm2] were lower in the NK group pre-treatment [2.3 (0.0-21.1); 1.7 (0.0-13.0); 0.5 (0.0-10.2); respectively] vs. controls [22.3 (14.9-29.0); 10.1 (3.2-15.4); and 12.1 (6.2-18.4), (p < 0.0001 for all), respectively]. Post-treatment nerve densities increased compared to pre-treatment to 5.3 (0.0-19.4, p = 0.0083) for total, 3.5 (0.0-13.2, p = 0.0059) for main, and 2.0 (0.0-10.4, p = 0.0251) for branch nerves, but remained lower than controls (p < 0.0001 for all). Corneal sensation increased from 2.3 ± 1.1 cm pre-treatment to 4.1 ± 1.4 cm post-treatment (p = 0.001). Median best corrected visual acuity significantly increased following rhNGF treatment from 0.4 (0.0-1.6) to 0.12 (-0.1 to 1.6) (p = 0.007). Patients with NK treated with at least 4 weeks of rhNGF, showed a significant increase in corneal nerve densities after treatment. A significant increase in corneal sensation, as well as best corrected visual acuity, was observed following treatment.

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Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts.

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Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts.

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