Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions

Area of Science:

• Ocular physiology and biomechanics
• Cellular mechanotransduction
• Glaucoma research

Background:

• Intraocular pressure (IOP) is regulated by aqueous humor (AH) outflow through the trabecular meshwork (TM) and Schlemm's canal (SC).
• Shear stress from AH flow is an emerging factor influencing TM and SC cell function.
• Understanding mechanosensitive pathways is crucial for managing IOP and glaucoma.

Purpose of the Study:

• To investigate the role of shear stress in regulating AH outflow and IOP.
• To elucidate cellular and molecular mechanisms in the eye's outflow pathway.
• To present a protocol for live-cell imaging under fluid flow.

Main Methods:

• Utilized time-lapse live-cell imaging with fluorescent protein sensors.
• Studied primary cultured TM and SC cells under controlled fluid flow conditions.
• Investigated the activation of mechanosensitive ion channels and nitric oxide production.

Main Results:

• Demonstrated shear stress activates mechanosensitive ion channels in TM cells.
• Showed shear stress induces nitric oxide production in SC cells, affecting outflow resistance.
• Identified autophagy and primary cilia as key mechanosensors in AH outflow regulation.

Conclusions:

• Shear stress plays a significant role in regulating AH outflow and IOP.
• Live-cell imaging provides real-time insights into fluid flow-cell interactions.
• This protocol facilitates further research into ocular mechanobiology and glaucoma therapies.