June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
In vivo live-cell imaging of human trabecular meshwork cells under laminar flow
Author Affiliations & Notes
  • Akhilesh Kumar
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Xingjuan Chen
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Michael Conwell
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Na Luo
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Alexander Obukhov
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Yang Sun
    Department of Ophthalmology, Indiana University, Indianapolis, IN
  • Footnotes
    Commercial Relationships Akhilesh Kumar, None; Xingjuan Chen, None; Michael Conwell, None; Na Luo, None; Alexander Obukhov, None; Yang Sun, NIH (F), American Glaucoma Society (R), Knights Templar Eye Foundation (R), Reeves Foundation (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3574. doi:https://doi.org/
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      Akhilesh Kumar, Xingjuan Chen, Michael Conwell, Na Luo, Alexander Obukhov, Yang Sun; In vivo live-cell imaging of human trabecular meshwork cells under laminar flow. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3574. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Elevated intraocular pressure (IOP) has been known to be a strong risk factor for glaucoma pathogenesis. The precise regulatory mechanisms of aqueous outflow by trabecular meshwork cells are unknown. In this study, we have developed live-cell imaging system to study the functional aspect of human trabecular meshwork cells (HTM) in response to different fluid shear stress.

Methods: We have developed a live-cell flow-chamber system to study primary and transformed human trabecular meshwork (HTM) cells under flow conditions. Intracellular calcium regulation was examined in HTM loaded with fura-2 AM, which was alternatively excited at 345 nm and 380 nm. The fluorescence intensity was recorded by using a TillPhotonics imaging system attached to an inverted Zeiss microscope and a charge-coupled device camera was used for calcium imaging. Fluorescence imaging experiments and the analysis of images were performed using Tillvision software.

Results: HTM cells produced a dose dependent increase of fura-2 fluorescence (indicative of [Ca2+]i) which reach maximum at flow rate of (10 dyn/cm2). These changes in homogeneous laminar shear stress may mimic the condition of live-cell conditions in human aqueous outflow. HTM cells revealed distinct actin cytoskeletal changes under unidirectional laminar flow which may significantly impact its ability to respond to its environment.

Conclusions: Our results indicate that application of in vivo live-cell imaging of HTM cells under laminar flow is a good physiologic model system for the study of cell behavior in response to different flow conditions. This will provide the cellular model to examine functional changes in normal and diseases TM cells.

Keywords: 439 calcium • 551 imaging/image analysis: non-clinical • 735 trabecular meshwork  
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