June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Mechanisms of retinal cell swelling, volume sensing and mechanical excitotoxicity
Author Affiliations & Notes
  • Daniel Ryskamp
    Interdepartmental Program in Neuroscience, University of Utah School of Medicine, Salt Lake City, UT
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Andrew Jo
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Amber Frye-Gordon
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Shiwani Chauhan
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Tünde Molnár
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • David Krizaj
    Interdepartmental Program in Neuroscience, University of Utah School of Medicine, Salt Lake City, UT
    Department of Ophthalmology and Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Footnotes
    Commercial Relationships Daniel Ryskamp, None; Andrew Jo, None; Amber Frye-Gordon, None; Shiwani Chauhan, None; Tünde Molnár, None; David Krizaj, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3701. doi:https://doi.org/
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      Daniel Ryskamp, Andrew Jo, Amber Frye-Gordon, Shiwani Chauhan, Tünde Molnár, David Krizaj; Mechanisms of retinal cell swelling, volume sensing and mechanical excitotoxicity. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3701. doi: https://doi.org/.

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

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Abstract

Purpose: We sought to identify the molecular determinants of retinal cell volume regulation, volume sensing and cell survival by characterizing the biophysical, electrophysiological and cellular properties of osmoregulation in retinal neurons and glia. We identified the osmosensor, TRPV4, and characterized the signaling cascades through which osmotic stress differentially regulates the amplitude and kinetics of calcium signals in retinal ganglion cells (RGCs) and Müller glia.

Methods: Retinal cells from C57 mice were bathed in isotonic (300 mOsm) and hypotonic saline. Volume and Ca2+ changes were measured in cells loaded with calcein and fura-2. Apoptosis and cell survival were measured with TUNEL and Live/Dead assays. Force transduction was examined with a high-resolution mechanoclamp technique.

Results: Antibody labeling localized the cation channel TRPV4 to RGCs and Müller glia. During agonist or hypotonic stimulation (HTS), TRPV4 response properties in RGCs and Müller glia exhibited contrasting kinetics and Ca2+ dynamics. Acute HTS dose-dependently elevated Ca2+ in both cell types, whereas prolonged swelling triggered apoptosis. BAPTA-AM (Ca2+ chelator) and HC067047 (selective TRPV4 antagonist) suppressed apoptosis (e.g. 90% and 62% suppression in RGCs and glia by HC067047) and swelling. [Ca2+]i levels in Müller glia were selectively elevated by 5’6’-EET, an eicosatrienoic acid derivative of arachidonic acid, consistent with an indirect activation of glial TRPV4. Inhibition of phospholipase A2 (PLA2) reduced HTS-induced swelling and Ca2+ elevations in Müller glia but had no effect on volume regulation in RGCs. In contrast, mechanotransduction in RGCs was intrinsic and 2nd-messenger independent, as shown by high-speed pressure clamp recordings of stretch-activated cation currents from outside-out RGC patches (fast, pressure-dependent inward currents at 10-50 mmHg).

Conclusions: Although both Müller glia and RGCs transduce mechanical stimuli into a TRPV4-dependent cation influx, glial TRPV4 activation relies on a slow, indirect, PLA2-dependent force transduction pathway, whereas the neuronal TRPV4 channel is directly yet transiently activated by lipid bilayer deformation. Inhibition of TRPV4 was strongly neuroprotective in a model of retinal edema. This data has implications for our understanding of retinal volume regulation as well as the molecular events that mediate cytotoxic edema, ischemic injury and glaucoma.

Keywords: 505 edema • 531 ganglion cells • 603 Muller cells  
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