June 2020
Volume 61, Issue 7
ARVO Annual Meeting Abstract  |   June 2020
Effects of Endothelin-1 on Resistance and Flow Patterns in Conventional Outflow Pathway of Perfused Mouse Eyes
Author Affiliations & Notes
  • Todd Fleming
    Duke University School of Medicine, Durham, North Carolina, United States
  • Fiona McDonnell
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Joseph Sherwood
    Bioengineering, Imperial College London, United Kingdom
  • Daniel Stamer
    Ophthalmology, Duke Eye Center, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Todd Fleming, None; Fiona McDonnell, None; Joseph Sherwood, None; Daniel Stamer, None
  • Footnotes
    Support  5P30EY005722-34, Research to Prevent Blindness Unrestricted Grant, and 5RO1EY022359-08
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4626. doi:
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      Todd Fleming, Fiona McDonnell, Joseph Sherwood, Daniel Stamer; Effects of Endothelin-1 on Resistance and Flow Patterns in Conventional Outflow Pathway of Perfused Mouse Eyes. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4626.

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

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Purpose : The distal portion of the conventional outflow (CO) pathway generates 25-50% of total outflow resistance. However, regulatory regions of distal vessels (DV) in the CO pathway, and their dynamic contribution to intraocular pressure (IOP) are poorly understood. We set out to develop, and test a model to study the role and location of vasoactive control points responsible for regulation of CO resistance in DVs. We used endothelin-1 (ET-1) as a tool compound to investigate effects on CO resistance and DV behavior.

Methods : To mark active flow passageways, enucleated eye pairs from 8 adult C57BL/6 mice were perfused ex vivo with media containing 10 µM fluorescein tagged soy bean agglutinin (SBA) lectin using iPerfusion. In a masked fashion, one eye of each pair served as control, and perfusion media in the contralateral eye also contained 10 nM ET-1. After perfusion was complete, eyes were emersion fixed in 4% paraformaldehyde (PFA) for 1 hour before transfer to 0.4% PFA until further processing. All eyes were probed with antibodies specific for alpha smooth muscle actin (αSMA), and platelet endothelial cell adhesion molecule (PECAM) to localize vasoactive cells surrounding blood vessels. The DVs were visualized on confocal microscopy with an inverted Nikon ECLIPSE Ti2-E microscope. Data are presented as mean±SD, the Student’s t-test was used to assess statistical significance between groups.

Results : The mean outflow facilities (inverse of outflow resistance) in the control and experimental groups were 7.4 ± 2.3 and 4.2 ± 0.78 nl/min/mmHg, respectively. Thus, ET-1 was active, increasing outflow resistance by 46% (p = 0.0034). In control eyes, SBA lectin was present in DVs, evident within collector channels, aqueous venous plexus, and scleral veins. Lectin label was found equally within DVs containing low and high density of αSMA. In experimental eyes, however, lectin label was mainly localized to Schlemm's Canal (SC) circumferentially with reduced labeling beyond collector channels that contained high expression of αSMA.

Conclusions : We have developed and optimized a mouse model to localize critical regions in the DVs responsible for creating outflow resistance. We found that increased outflow resistance, caused by ET-1, correlated with increased retention of SBA lectin in SC and lower perfusion through the collector channels and aqueous venous plexus of the distal outflow pathway.

This is a 2020 ARVO Annual Meeting abstract.


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