Abstract
Purpose:
To determine the changes in the levels of selected mechanosensing channels (Piezo1-2, TASK1, TREK-1, TRPC1-3, TRPC6, TRPM2, TRPP2) in the trabecular meshwork (TM) of DBA/2J mice in normotensive versus hypertensive state.
Methods:
All studies were performed adhering to the ARVO statement for use of animals in vision research under IACUC approved protocols. DBA/2J mice of age 3-9 months (n=40 at each point) was used for these studies. Intraocular pressure (IOP) was measured using a TonoLab (Colonial Medical Supplies, NH). DBA/2J mice were categorized into normotensive (IOP ≤15mmHg; usually in mice with less than 7.5 months of age) and hypertensive (IOP range of 18-36mmHg) groups. Quantitative Western Blot and immunohistochemistry were carried out using commercial antibodies to these channels (as noted above). These channels were previously found in the DBA/2J TM by microarray and immunohistochemical analyses in our laboratory. Non-amplified IR-coupled secondary antibody was used for Western Blots. Confocal microscopy on a Leica SP5 microscope was used for quantification of immunohistochemical signals. Quantitative label free mass spectrometry was also carried out to validate the relative quantification of these channels.
Results:
Selected mechanosensing channels (Piezo1&2, TASK1, TREK-1, TRPC1-3, TRPC6, TRPM2, TRPP2) showed at least a 30% difference in levels of expression between normotensive and hypertensive states (normalized with beta-actin) determined by Quantitative Western Blot analysis and corroborated by other methods.
Conclusions:
Our investigation confirms the modulation of selected mechanosensing channels in TM tissue in different states of IOP.
Keywords: 735 trabecular meshwork •
568 intraocular pressure