Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes

The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells. TPMD-induced calcium increases (Ca⁺⁺_i) were measured in Ca⁺⁺-containing and Ca⁺⁺-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-α-glycyrrhetinic acid (18α-GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca⁺⁺ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca⁺⁺ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.