Abstract
Purpose :
Examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (Ca++ Wvs) in control and diabetic mouse and human corneas.
Methods :
A multiphoton microscope was used to create TPMDs in single cultured mouse (MCEC) and primary human (HCEC) cornea cells, ex vivo and in vivo control and diabetic mouse corneal cells, ex vivo human cornea rims, and primary human diabetic stromal cells within a 3-D matrix. The Ca++ source for Ca++ waves were explored using Ca++ channel inhibitors. TPMDs were also studied in ex vivo and in vivo control and diabetic MCEC after mild eye rubbing over a closed lid. Glass bead rolling-induced TPMDs were studied in MCEC and HCEC, with actin dynamics following glass bead rolling being studied using light sheet microscopy. Confocal microscopy was used to study MCEC actin architecture following live mouse eye rubbing-induced TPMDs. CEC TPMDs were observed in all models.
Results :
Laser-induced Ca++ Wvs were observed in MCEC and HCEC, ex vivo control and diabetic mouse corneas and human corneal rims, and in live mouse CEC. The SR Ca++ ATPase inhibitors thapsigargin and CPA reduced Ca++ Wvs in MCEC and HCEC. The TRP V1 antagonists AMG 9810 and BCTC reduced Ca++ Wvs in MCEC but not HCEC. Ca++ free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) had no effect on CEC Ca++ Wvs. Eye rubbing produced more TPMDs in diabetic MCEC as compared to controls, although diabetic ex vivo MCEC had reduced Ca++ Wv activity. Diabetic HSC within a 3D matrix also had reduced Ca++ Wvs. MCEC and HCEs had denser cortical actin and stress fibers following TPMD induction compared to controls, with thapsigargin preventing stress fiber formation.
Conclusions :
This is the first demonstration of TPMDs and associated Ca++ Wvs in CEC. TPMDs were created by eye rubbing, a routine mechanical stress placed on CEC, indicating that TPMDs and Ca++ Wvs are likely common in CEC. Pharmacologic studies indicate that intracellular Ca++ stores are the primary Ca++ source for CEC Ca++ Wvs, with TRPV1 channels also providing a Ca++ source in MCEC. CEC Ca++ Wvs are not influenced by gap junctions or ATP. Diabetic CEC membranes are more susceptible to TPMDs, but have a reduced Ca++ Wv response. Diabetic HSCs also have a reduced Ca++ Wv response. MCEC and HCEC have denser actin filament structures and stress fibers following TPMD induction.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.