May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
The Effect of Increased Hydrostatic Pressure on the Survival of Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • S. Vinjamaram
    Ophthalmology, University Of Florida HSC, Jacksonville, FL, United States
  • K.V. Chalam
    Ophthalmology, University Of Florida HSC, Jacksonville, FL, United States
  • V.A. Shah
    Ophthalmology, University Of Florida HSC, Jacksonville, FL, United States
  • Footnotes
    Commercial Relationships  S. Vinjamaram, None; K.V. Chalam, None; V.A. Shah, None.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1735. doi:
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      S. Vinjamaram, K.V. Chalam, V.A. Shah; The Effect of Increased Hydrostatic Pressure on the Survival of Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1735.

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

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Abstract

Abstract: : Purpose: To study the effect of increased hydrostatic pressure on the Retinal pigment epithelial cell viability. Pressure is a crucial component of the cellular environment, and it is important to understand the cell susceptibility due to the alteration in this component. Methods: Retinal Pigment epithelial cells (RPE) were cultured using Dulbeccos high glucose modified eagles medium. After the cells reached 80% confluence they were subjected to increased hydrostatic pressure for 24 hours with the help of a reservoir containing the medium at about 20 cm from the level of the culture flask. The cells were maintained in the microscope incubation chamber at 5% CO2. Cell survival was determined by using a Live/Dead viability kit (Molecular Probes) after 24 hour exposure to hydrostatic pressure. Cells were washed gently with D-PBS once and incubated with 6µ M ethidium homodimer-1 and 2 µ M calcein AM at 37° C for 20 minutes. Live cells were distinguished by the esterase activity, as determined by the enzymatic conversion of the virtually nonfuorescent cell-permeant calcein AM to the intensely fluorescent calcein. Calcein is retained within live cells and produces intense green fluorescence. Ethidium enters cells that have damaged membranes, binds the nucleic acid and thereby produces bright red fluorescence in dead cells. The number of live cells and dead cells in four randomly chosen fields were counted and compared. Cells grown under normal conditions without the increased hydrostatic pressure were used as controls. Results: The number of dead cells increased significantly over time, especially after the first 24 hours. The control group had less number of dead cells compared to the experimental group, this variance was observed soon after the hydrostatic pressure was applied and after 24 hours. The difference in cell viability, between the experimental and the control group was statistically significant. Conclusions: Elevated hydrostatic pressure has a significant effect on the RPE cell viability. Increased hydrostatic pressure augments RPE cell death, especially after first 24 hours.

Keywords: cell death/apoptosis • retinal pigment epithelium • intraocular pressure 
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