Abstract
Purpose :
We previously described that a single transient plasma membrane disruption (TPMD) induces calcium waves in corneal keratocytes (Chen, et. al, 2019 ARVO, #B0211). The purpose of this study was to determine whether diabetes has an influence on Ca++i transmission in corneal stromal cells responding to TPMD-induced stimulation and to further characterize the source of calcium for these calcium waves.
Methods :
Corneal stromal cells were cultured from mouse (MCSC) and human corneas (HCSC) including control and type 1 and type 2 human diabetic corneas. HCSCs were also cultured in three-dimension (3D) as previously described (Sharif, et. al, 2018). A single 9 mm TPMD was produced using a multiphoton microscope in Cal-520-AM fluorescent dye-loaded cells. TPMD-induced cytoplasmic calcium (Ca++i) increases were measured in cells exposed to the selective TRPV1 and TRPM8 antagonists AMG 9810 and AMTB, respectively, with the Ca++ chelator BAPTA-AM, and with ryanodine to block SR Ca++ release. Cell fluorescence intensity was recorded over time, and the number of neighboring cells responding to the single-cell TPMD was counted (cell number), as was the area under the fluorescence versus time curve. Experiments on diabetic 3D-cultured HCSCs and a control group were performed and analyzed by a masked investigator.
Results :
TPMD resulted in calcium waves in both targeted and non-wounded neighboring cells. The number of neighboring type 1 diabetic cells with Ca++ waves was reduced, and the fluorescence area was reduced in both type 1 and type 2 diabetic cells compared to control. BCTC (a general TRPV1, TRPM88 inhibitor), AMG 9810, and AMTB had no effect on cell number in HCSC or MCSC, although AMG 9810 reduced the number of neighboring responding cells in MCSC. In Ca++-containing medium, ryanodine, and BAPTA-AM showed inhibited cell numbers in HCSC and MCSC, although ryanodine increased the cell number in MCSC.
Conclusions :
We have demonstrated reduced TPMD-induced Ca++i wave activity in 3D diabetic cultured HCSCs. The source of increased Ca++i is both intra-and extra-cellular. The TRPM8 channel does not appear to be involved in the response, while the TRPV1 channel may have a small influence.
This is a 2020 ARVO Annual Meeting abstract.