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D. Meller, A. Schröder, K.P. Steuhl, C. Theiss; Neurotrophic Effect of Amniotic Membrane on Neuronal Cell Cultures: An in vitro Model to Study Underlying Action Mechanisms of Amniotic Membrane in the Treatment of Neurotrophic Keratopathy . Invest. Ophthalmol. Vis. Sci. 2005;46(13):875.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Transplantation of amniotic membrane (AMT) has been successfully applied to promote corneal wound healing in neurotrophic ulcers with different aetiologies. In this line, healing of corneal surface after AMT correlated clinically with a partial recovery of corneal sensitivity. Moreover, AMT compared to conventional treatment strategies accelerated axonal sprouting in an experimental animal model of keratitis induced by Herpes virus. In this study, we investigated potentially underlying neurotrophic action mechanisms of amniotic membrane (AM). Methods: Organ–typical cell cultures of dorsal root ganglion (DRG) neurons were gained from 10–day–old chick embryos and cultured with MEM on the stromal or epithelial side of intact AM for 3 to 5 days. In an additional group AM was pretretead with Dispase for 15 min at 37°C in order to remove the amniotic epithelium. Afterwards, DRG neurons were cultured in the same manner on the exposed basement membrane side of AM. Sprouting of neuronal axons was screened with monoclonal antibodies against cytoskeletal proteins such as neurofilament (NF–M) and tubulin (Tub). Finally, the specimens were analyzed with a confocal laser scanning microscope (LSEM 501, Zeiss). Results: DRG neurons cultured on the stromal or basement membrane side of AM exhibit within few days the formation of numerous NF–M– und Tub–positive axonal neurites. These typically run a radial fashion and are arranged partially in nerve bundles. However, axonal sprouting of DRG neurons is drastically inhibited when cultured directly on the amniotic epithelium of AM. Conclusions: The basement membrane and the stromal side of AM promote in vitro extensively the outgrowth of axonal neurites. However, a substantial inhibition of axonal sprouting is induced by the amniotic epithelium. If soluble, neurotrophic growth factors and/or adhesion–molecules mediated mechanisms are enrolled in these events and thereby potentially promote wound healing in neurotrophic keratopathy, will be analyzed in further studies.
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