June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Isolation of trabecular meshwork (TM) RNA and analysis of gene expression following serial bevacizumab injections in rat eyes.
Author Affiliations & Notes
  • Eliesa Ing
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • William Cepurna
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Diana C Lozano
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Virginia O'Callahan
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Yong-Feng Yang
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Fountane Chan
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Kate E Keller
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • John C Morrison
    Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Eliesa Ing None; William Cepurna None; Diana Lozano None; Virginia O'Callahan None; Yong-Feng Yang None; Fountane Chan None; Kate Keller None; John Morrison None
  • Footnotes
    Support  R21 EY033073, R01 EY019643, R01 EY010145, P30 EY010572 from the National Institutes of Health (Bethesda, MD), and an unrestricted departmental funding grant from Research to Prevent Blindness (New York, NY)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2703 – A0067. doi:
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    • Get Citation

      Eliesa Ing, William Cepurna, Diana C Lozano, Virginia O'Callahan, Yong-Feng Yang, Fountane Chan, Kate E Keller, John C Morrison; Isolation of trabecular meshwork (TM) RNA and analysis of gene expression following serial bevacizumab injections in rat eyes.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2703 – A0067.

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

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Abstract

Purpose : The mechanism by which repeated intravitreal injections of the anti-VEGF drug beviczimab can produce chronically elevated intraocular pressure (IOP) is poorly understood. Here we describe a novel method for rat TM dissection, RNA isolation, and preliminary findings of TM gene expression after serial beviczimab injections.

Methods : Twelve Brown Norway male retired breeder rats were randomly assigned to receive 3 uniocular, pars plana intravitreal injections of either 0.20 µL bevacizumab (0.005 mg, n=6) or 0.20 µL balanced salt solution plus (BSS+ control, n=6) once per week under isoflurane anesthesia. Fellow eyes were untreated. Awake IOP (10 measurements per eye) was monitored three times per week prior to the start of injections and until the animals were sacrificed. One week after the third injection, animals were sacrificed, followed by immediate enucleation of both experimental and uninjected eyes. Anterior segments were first cut into quarters, and the iris and ciliary body removed with fine jewelers forceps to expose the TM. Using these same forceps, the TM was gently pealed off, revealing the outer wall of Schlemm’s canal. TM tissue was placed in Trizol with the aid of a bent 30g needle and RNA was isolated using glycogen as a carrier. After pre-amplification, RT2 Profiler PCR Arrays (Qiagen) were used to quantitate extracellular matrix and adhesion molecule-related gene expression.

Results : Linear regression indicated that IOP did not change in the experimental (P=.47) or control eyes (P=.48) over the 4 week experiment. We were successfully able to extract RNA from rat TM (n=24) obtained with the above technique (mean=77.6 ng/ml ± 38.1). In PCR arrays, Mmp11 was signficiantly downregulated in the TMs of beviczimab-injected eyes compared to fellow eyes (-2.97 fold, p = 0.046), but not in BSS+-injected eyes. Spp1 was significantly down-regulated in BSS+ eyes (-1.59 fold, p=0.031), but one of the fellow eye samples was unusable.

Conclusions : We can successfully isolate sufficient RNA from rat TM tissue for gene expression analysis. Mmp11 degrades α1-antitrypsin and IGFBP1, but has limited proteolytic activity on classical extracellular matrix molecules. Further work is needed over longer time periods to understand better the chronic effects of anti-VEGF injections on TM gene expression and IOP in this promising model.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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