June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Aqueous Angiography Based Comparison of High- and Low-Flow Regions for Alpha-Smooth Muscle Actin (α-SMA) and Versican in Post-Mortem Human Eyes.
Author Affiliations & Notes
  • Sindhu Saraswathy
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
  • Thania Bogarin
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles, Los Angeles, California, United States
  • Chirayu Mohindroo
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
  • Brian Alan Francis
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
  • James C Tan
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles, Los Angeles, California, United States
  • David R Hinton
    Pathology, University of Southern California, Los Angeles, California, United States
  • Robert N Weinreb
    University of California, San Diego, La Jolla, California, United States
    Shiley Eye Institute, San Diego, California, United States
  • Alex S Huang
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Sindhu Saraswathy, None; Thania Bogarin, None; Chirayu Mohindroo, None; Brian Francis, Allergan (F), Aquesys (F), BVI Endooptiks (C), Diopsys (F), Infocus (F), Lumenis (F); James Tan, None; David Hinton, None; Robert Weinreb, Aerie Pharmaceutical (C), Alcon (C), Allergan (C), Bausch & Lomb (C), Carl Zeiss Meditec (F), Eyenovia (C), Forsight Vision V Sensimed (C), Genentech (F), Heidelberg Engineering (F), Optovue (F), Quark (F), Topcon (F), Unity (C); Alex Huang, Allergan (C), Heidelberg Engineering (F)
  • Footnotes
    Support  Supported by National Institutes of Health, Bethesda, Maryland (K08EY024674 [ASH]); AGS Young Clinician Scientist Award 2015 [ASH]; Research to Prevent Blindness Career Development Award 2016 [ASH]; and an unrestricted grant from Research to Prevent Blindness (New York, NY).
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3479. doi:
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      Sindhu Saraswathy, Thania Bogarin, Chirayu Mohindroo, Brian Alan Francis, James C Tan, David R Hinton, Robert N Weinreb, Alex S Huang; Aqueous Angiography Based Comparison of High- and Low-Flow Regions for Alpha-Smooth Muscle Actin (α-SMA) and Versican in Post-Mortem Human Eyes.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3479.

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

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Abstract

Purpose : Here, aqueous angiography was used to identify low- and high-flow regions to enrich for biological protein differences that were probed for using various methods.

Methods : Post-mortem eyes (San Diego and One Legacy eye bank) were obtained with no history of glaucoma, within 48 hours of death. The eyes were perfused with Balanced Salt Solution (BSS) followed by tracer (fluorescein [2.5%] or indocyanine green [0.4%]) diluted in BSS with a pressure of ~10 mm Hg. After angiographic imaging with the Spectralis HRA+OCT (Heidelberg Engineering), eyes were fixed with 4% paraformaldehyde or prepared for western blot. Wedges including the angle were cut from angiographically positive and negative regions, paraffin embedded, and processed for sectioning (~5 microns). The sections were immunostained and viewed under a Keyence BZ-X700 digital imaging microscope (IF). Separately, the TM from positive and negative regions were dissected and processed for western blot (WB). Antibodies were used against α-SMA and versican with GAPDH as a WB control.

Results : α-SMA levels were increased in low-flow regions by WB (3.23-fold from 2 eyes) and IF (4.49-fold; n = 8 sections of low- and high-flow from 2 eyes; p < 0.001). By WB, in a small sample (n = 2 eyes) versican was also increased (5.46-fold).

Conclusions : Immunofluorescence and WB analyses of target proteins after aqueous angiography-based segregation of high- and low-flow AHO regions demonstrated increased protein levels of α-SMA and versican. Elevated levels of α-SMA have been implicated in glaucomatous trabecular meshwork and increased levels of versican have been seen in low-flow trabecular meshwork regions in micro-bead based outflow studies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Figure 1. TM from positive and negative regions were dissected out and processed for western blot analysis against antibodies specific for α-SMA with GAPDH as a WB control.α-SMA levels were increased in low flow regions by 3.23 fold by WB. LF = low-flow and HF = high-flow.

Figure 1. TM from positive and negative regions were dissected out and processed for western blot analysis against antibodies specific for α-SMA with GAPDH as a WB control.α-SMA levels were increased in low flow regions by 3.23 fold by WB. LF = low-flow and HF = high-flow.

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