March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Overstimulation of TRPV4 in vivo Induces Selective Apoptosis of Retinal Ganglion Cells. An Acute in vivo Experimental Model for Glaucoma
Author Affiliations & Notes
  • amber m. frye
    Moran Eye Institute, The University of Utah, Department of Opthalmology & Visual Sciences, Salt Lake City, Utah
  • Daniel Ryskamp
    ophthalmology, The University of Utah, Salt Lake City, Utah
  • Shiwani Chauhan
    ophthalmology, The University of Utah, Salt Lake City, Utah
  • Andrew Jo
    ophthalmology, The University of Utah, Salt Lake City, Utah
  • David Krizaj
    ophthalmology, The University of Utah, Salt Lake City, Utah
  • Footnotes
    Commercial Relationships  amber M. frye, None; Daniel Ryskamp, None; Shiwani Chauhan, None; Andrew Jo, None; David Krizaj, None
  • Footnotes
    Support  NIH EY13870
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6944. doi:
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      amber m. frye, Daniel Ryskamp, Shiwani Chauhan, Andrew Jo, David Krizaj; Overstimulation of TRPV4 in vivo Induces Selective Apoptosis of Retinal Ganglion Cells. An Acute in vivo Experimental Model for Glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6944.

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Abstract

Purpose: : To investigate the relationship between stimulation of TRPV4, a mechanosensitive calcium-permeable plasma membrane channel and viability of retinal ganglion cells (RGC). TRPV4 is expressed in all RGCs, the output retinal neurons that represent a primary target in glaucoma. While the molecular mechanisms that cause glaucoma have not yet been conclusively established, mechanical stimulation through elevated intraocular pressure represents a primary risk factor. We therefore hypothesize that excessive stimulation of mechanosensitive ion channels such as TRPV4, produces a morphological and functional phenotype that mimics glaucoma.

Methods: : C57BL/6J mice were intravitreally injected with GSK1016790A (GSK) and transcardially perfused with PBS followed by 4% PFA. Retinas were removed, flattened, and radially cut into wholemount preparations. Immunohistochemistry with NeuN-AlexaFluor 488 (NeuN 1:500) conjugates was performed. Quadrants of retinal wholemounts were imaged with a confocal microscope and the number of RGC’s present was quantified.

Results: : Mice injected with 2μL of 75μM GSK, a selective agonist of TRPV4, displayed 50% reduction of the density of RGC somata (RGCs/mm2) compared to contralateral vehicle controls. This effect was dose-dependent, as a 7.5 μM GSK injection only reduced the density of RGCs by 25%. Injection with PBS had no effect on RGC density compared to un-injected eyes. Additionally, injecting GSK into the posterior chamber did not affect intraocular pressure (IOP) Calcium imaging of fura-2-loaded retinal cells 24 hours post-injection revealed that the injection of GSK eliminated RGCs with sustained and/or regenerative responses to GSK. In vitro experiments show that 1 hour incubation with GSK elicited apoptosis in a significant (60%) fraction of RGCs while having little effect on putative amacrine cells and photoreceptors, which lack TRPV4 expression. The proapoptotic action of GSK was antagonized by the nonselective TRP antagonist Ruthenium Red (p<0.001)

Conclusions: : We conclude that excessive activation of the mechanosensitive TRPV4 channel adversely impacts RGC survival under in vivo and in vitro conditions. This suggests that this channel, targeted by sustained increases in intraocular pressure, could injure cells in chronic glaucoma and TRPV4 may serve as a therapeutic target.

Keywords: apoptosis/cell death • ganglion cells • ion channels 
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