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Sonja Mertsch, Gerd Geerling, Stefan Schrader; Evaluation of corneal nerve regeneration in vitro using novel 3D models. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1387.
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© ARVO (1962-2015); The Authors (2016-present)
Spontaneous regeneration of the corneal nerve plexus after injury or chronic damage is often incomplete and the underlying mechanisms of regeneration are mostly unknown. Therefore, stimulation of corneal nerve regeneration could be a promising curative approach to treat diseases such as neurotrophic keratopathy as well as neurogenic induced dry eye syndrome. The aim of this study was to evaluate novel 3D in vitro models for corneal nerve regeneration based on tissue engineered materials in order to gain new insights into corneal nerve regeneration.
Dorsal root ganglion cells (DRGs) and corneas were obtained from 8 – 10 week old C57BL/6 mice (n = 12). DRGs were dissociated and 1 x 104 cells were seeded on laminin coated wells and either into or on top of plastic compressed collagen (PCC) gels (n = 6). Immunohistochemistry and qRT-PCR were performed to characterize molecules associated with neurite growth. Inhibitor experiments were conducted to identify the involvement of Rho kinase in neurite growth by addition of 100 µM Y27632 to the models. Total neurite length in 10 photos per experimental replicate was measured after βIII tubulin staining using NeuronJ Software. A WST-1 assay was used to evaluate cytotoxicity of Y27632.
QRT-PCR analysis revealed distinct expression of all factors in mouse cornea as well as in cultured DRGs. ROCK1 and ROCK2 showed high expression with delta cts of 6.07 ± 0.02 and 4.50 ± 0.04 in DRGs and 5.20 ± 0.02 and 4.26 ± 0.09 in the cornea. Measurement of neurite length in ROCK inhibitor (Y27632) treated and untreated groups showed a significant effect of Y27632 on neurite growth on PCC gels with lengths of 6.83 ± 0.43 mm in treated DRGs compared with 3.42 ± 0.84 mm in controls (p = 0.000004). Similarly, in the PCC gels, neurites grew to lengths of 5.17 ± 1.10 mm in Y27632 treated DRGs, but only 1.62 ± 0.21 mm in controls (p = 0.0001). The WST-1 assay revealed no influence of 100µM Y27632 on cellular metabolism.
The data showed that these new 3D models are suitable for evaluation of corneal nerve regeneration in vitro, and to test the efficacy of factors to improve corneal nerve regeneration. A better understanding of the underlying molecular mechanisms will help to develop new therapeutic concepts to treat impairment of corneal nerves in diseases such as neurotrophic keratopathy and neurogenic dry eye syndrome.
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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