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Thomas Stahnke, Bhavani S Kowtharapu, Andreas Wree, Rudolf Guthoff, Klaus-Peter Schmitz, Anselm Juenemann, Oliver Stachs; Role of neurotrophic factors in corneal stromal wound healing. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1306.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal keratocytes have a significant capability of healing the wounded cornea throughout the life. During corneal wound repair, coordinated interactions and bi-directional communication between epithelial cells and stromal keratocytes contributes to tissue reorganisation and wound healing. Previously, we reported that cultured trigeminal ganglion neurons, in the presence of corneal epithelial cells, show an increase in the expression of substance P (SP). In this context, the present study is designed to investigate the effects of neurotrophic factors, such as nerve growth factor (NGF) and SP in stromal wound healing.
Primary human corneal fibroblasts (CF) from the human cornea were cultured by using an explant culture method. The purity of CFs in culture was confirmed by vimentin antibody immune staining whereas the transformation of CFs into “repair-phenotype” after scratch assay was analyzed by staining with alpha-smooth muscle actin (α-SMA). Reactive oxygen species (ROS) generation in the presence of SP and NGF was measured by using CellROX oxidative stress detection dyes and western blotting analyses were performed on the respective cell lysates to study the differences in the expression of extracellular matrix (ECM) proteins and signaling pathway components.
Differences in the generation of ROS were observed after treatment of CFs with SP and NGF with a substantial decrease in the presence of SP. Even though individual treatment of CFs with SP for 72 hours could not induce the “repair-phenotype” as observed by α-SMA immunocytochemistry, we observe differences in the expression of vinculin and significant morphological changes as evidenced by elongated cell morphology and cytoskeleton. Furthermore, differences in the ECM component fibronectin and activation of MAPK signaling pathway could be observed by western blotting.
Our results display changes in ECM components, activation of MAPK signaling pathway and differences in the generation of ROS after treatment of CFs with neurotrophic factors which may suggest a paracrine consequence of the released neurotrophic factors from the corneal epithelial cells on stromal keratocytes during wound healing.
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