July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Effect of TGF-β2 on outflow facility and gene expression in anterior segments of calf eyes
Author Affiliations & Notes
  • Enhua H Zhou
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Michael Paolucci
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Christopher Wilson
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Fjodor Merkuri
    University of Massachusetts Lowell, Lowell, Massachusetts, United States
  • Tom Vollmer
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Katherine Prosen
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Katherine Thompson
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Bret Benton
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Brett Thibodeaux
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Yan Wong
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Valentin Sluch
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Ted Manley
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Dennis S Rice
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Amy Chen
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Ganesh Prasanna
    Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Enhua Zhou, Novartis Institutes for BioMedical Research (E); Michael Paolucci, Novartis Institutes for BioMedical Research (E); Christopher Wilson, Novartis Institutes for BioMedical Research (E); Fjodor Merkuri, None; Tom Vollmer, Novartis Institutes for BioMedical Research (E); Katherine Prosen, Novartis Institutes for BioMedical Research (E); Katherine Thompson, Novartis Institutes for BioMedical Research (E); Bret Benton, Novartis Institutes for BioMedical Research (E); Brett Thibodeaux, Novartis Institutes for BioMedical Research (E); Yan Wong, Novartis Institutes for BioMedical Research (E); Valentin Sluch, Novartis Institutes for BioMedical Research (E); Ted Manley, Novartis Institutes for BioMedical Research (E); Dennis Rice, Novartis Institutes for BioMedical Research (E); Amy Chen, Novartis Institutes for BioMedical Research (E); Ganesh Prasanna, Novartis Institutes for BioMedical Research (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4711. doi:
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      Enhua H Zhou, Michael Paolucci, Christopher Wilson, Fjodor Merkuri, Tom Vollmer, Katherine Prosen, Katherine Thompson, Bret Benton, Brett Thibodeaux, Yan Wong, Valentin Sluch, Ted Manley, Dennis S Rice, Amy Chen, Ganesh Prasanna; Effect of TGF-β2 on outflow facility and gene expression in anterior segments of calf eyes. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4711.

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

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Abstract

Purpose : Intraocular pressure (IOP) elevation in primary open angle glaucoma patients is believed to be due to an increase in outflow resistance through the trabecular meshwork (TM) and Schlemm’s canal. To understand long-term regulation of outflow resistance, anterior segment perfusion provides a valuable model. Here we report the development of an anterior segment perfusion assay that can evaluate 20 samples in parallel for up to 2 weeks.

Methods : Calf anterior segments (calfAS) were dissected and mounted onto a perfusion apparatus with a clamping ring. The apparatus was 3-D printed and optimized to fit calfAS. IOP was controlled by adjusting the reservoir height and flow rate was measured with flow sensors (Zhou et al. IOVS 2017). An effective antibiotic cocktail was identified to prevent bacterial contamination. Cell viability was assessed using a live-dead stain and lactate dehydrogenase (LDH) release. To evaluate the effects of TGF-β2, media containing vehicle or two doses of TGF-β2 (40 or 400 ng/ml TGF-β2 in 300 µl) was injected once daily for 5 days. Soluble fibronectin concentration was evaluated using ELISA. Expression of extracellular matrix genes was evaluated using real time qPCR in excised TM.

Results : After 2 weeks of perfusion, TM cells remained mostly viable. Minimal LDH release was detected in the perfusate. As a positive control, an injection of 30 µM Y-39983 (a ROCK inhibitor) increased outflow facility by 56 ± 24% (mean ± SD, n = 6-8, p < 0.01) after 8 hours of treatment. TGF-β2 treatment for 5 days at both doses reduced outflow facility by up to 30% relative to contralateral eyes receiving vehicle control. This reduction was accompanied by a 2-3 fold increase in soluble fibronectin concentration and increases in the transcript levels of fibronectin and alpha-smooth muscle actin (α-SMA).

Conclusions : We have extended our compact eye perfusion system to perform long-term perfusion of anterior segments and have demonstrated its utility by TGF-β2 and ROCK inhibition treatment. This assay enables the identification of molecules and pathways that chronically modify the outflow pathway to achieve a long lasting impact on IOP.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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