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A. Aracil, A. Sánchez, J. Gallar, C. Belmonte; An in vitro Preparation to Study the Interactions Between Trigeminal Neurons and Corneal Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3469.
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To develop a method to study at the cellular level, the interactions between trigeminal neurons and corneal epithelium cells.
SV-40 immortalized human corneal epithelial cells (Araki-Sasaki, et al., 1995) were cultured in 24 well plates until confluence. In the center of each well, a circular wound of 1 mm of diameter was made by gently touching the monolayer with a pipette tip. The area of the wound was photographed immediately after wounding and every 12 hours, until it was completely healed. Immediately after wounding, wells were cultured in: 1- a solution lacking fetal bovine serum, FBS (negative control). 2- in the presence of FBS (positive control), and 3- together with acutely dissociated trigeminal neurons of mice seeded in Costar Transwell® inserts, in a medium without FBS. Neurons of adult male C57BL/6, CGRP-KO (Salmon, et al, 1999) and Tac-1 KO (Zimmer, et al, 1998) mice were used.
The wounds made in the positive control wells healed completely in 48 hr. In contrast, wounds in the negative control wells did not change the area of the wound during this same period. Co-culture of corneal epithelial cells in the absence of FBS with trigeminal neurons induced wound healing. This effect occurred with neurons of all mice strain tested.
This preparation allows to study in vitro the interactions between trigeminal neurons and corneal epithelial cells and the influence of neuropeptides on corneal epithelial wound healing.
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