July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Effects of the Na-K ATPase blocker digoxin on microglia structure and function in real time in the mouse retina.
Author Affiliations & Notes
  • Ethan D Cohen
    Office of Sci & Eng Labs/CDRH, FDA, Silver Spring, Maryland, United States
  • Roslyn J Brehl
    University of Minnesota, Minneapolis, Minnesota, United States
  • Haohua Qian
    NEI, NIH, Bethesda, Maryland, United States
  • Lin Xu
    CDER/OTS/OCP/DARS, FDA, Silver Spring, Maryland, United States
  • Nikita Kedia
    Engineering, University of Maryland College Park, Silver Spring, Maryland, United States
  • Huy Vo
    Engineering, George Mason Univ., Fairfax, Virginia, United States
  • Zhuolin Liu
    Office of Sci & Eng Labs/CDRH, FDA, Silver Spring, Maryland, United States
  • Katherine Shea
    CDER/OTS/OCP/DARS, FDA, Silver Spring, Maryland, United States
  • Daniel Hammer
    Office of Sci & Eng Labs/CDRH, FDA, Silver Spring, Maryland, United States
  • Joseph Hanig
    CDER/OPQ/OTR, FDA, Silver Spring, Maryland, United States
  • Footnotes
    Commercial Relationships   Ethan Cohen, None; Roslyn Brehl, None; Haohua Qian, None; Lin Xu, None; Nikita Kedia, None; Huy Vo, None; Zhuolin Liu, None; Katherine Shea, None; Daniel Hammer, None; Joseph Hanig, None
  • Footnotes
    Support  FDA Office of the Chief Scientist
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4015. doi:https://doi.org/
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    • Get Citation

      Ethan D Cohen, Roslyn J Brehl, Haohua Qian, Lin Xu, Nikita Kedia, Huy Vo, Zhuolin Liu, Katherine Shea, Daniel Hammer, Joseph Hanig; Effects of the Na-K ATPase blocker digoxin on microglia structure and function in real time in the mouse retina.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4015. doi: https://doi.org/.

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

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Abstract

Purpose : Microglia are sensitive indicators of retinal damage, however, it is unclear whether they can identify retinotoxins. The mouse retina contains 2 layers of microglia. We examined how the α2/α3-subunit selective Na-K ATPase blocker digoxin altered microglia morphology and retinal structure, in real time using a new eyecup preparation and confocal microscopy.

Methods : We used a CX3CR1 GFP (green fluorescent protein) transgenic mouse to track microglia in the retina in real time using confocal microscope Z-stack images. In some cases, histology was also performed to validate the changes observed in the microglial population with changes in the retinal structure. Mouse retina microglia were imaged using a miniature eyecup chamber, and superfused with oxygenated Ames Ringer at 35°C. To examine for cell death, the nuclear binding dye 7-amino-actinomycin D (7AAD) was used to label dead cells.

Results : To examine the microglial response, digoxin was bath applied (3-10μM) to the retina for 10 minutes, followed by a 30min washout period and nuclear dye labeling for cell death. In each case examined, the first effect of digoxin (3μM) was a 57±11% (mean±std. dev.) decline of fluorescence in the processes of the outer retinal microglia from their pre drug levels, while the inner microglia fluorescence declined only 14 ±12% (n=7 retinas) (P<0.001). Histological data suggested this inner/outer fluorescence difference may be due to increased optical scatter by the swollen processes of inner retinal cells in digoxin. Digoxin had a curiously delayed action on the mouse retina, with the peak response some 20 minutes after the drug washout. By 30min, there was a retraction of the fine processes of inner retinal microglial indicative of activation. After digoxin, 7AAD labeled small nuclei in the ganglion cell layer and the microglia (as previously reported). Histological analysis of digoxin-applied retinae showed damage to the middle of the inner nuclear layer and swelling in the IPL. Preliminary data using microscopic OCT showed a similar pattern.

Conclusions : Microglia are sensitive indicators of retinal injury by toxins. The loss of fluorescence by the outer microglia in digoxin is suggestive of inner retinal tissue swelling causing optical scatter in confocal images, and suggest microglia morphological analysis may be useful in neurotoxin assays of whole retinal tissue.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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