July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
In vitro and ex vivo models for evaluating drugs that quiesce mast cells, models for mast cell degranulation in geographic atrophy choroid
Author Affiliations & Notes
  • Shuntaro Ogura
    Department of Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
  • Rajkumar Baldeosingh
    Department of Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
  • Manasee Gedam
    Department of Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
  • Malia Michelle Edwards
    Department of Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
  • Gerard A Lutty
    Department of Ophthalmology, Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Shuntaro Ogura, None; Rajkumar Baldeosingh, None; Manasee Gedam, None; Malia Edwards, None; Gerard Lutty, None
  • Footnotes
    Support  NIH grants EY016151 (GL) and EY01765 (Wilmer) and an RPB Unrestricted Grant (Wilmer).
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3471. doi:
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      Shuntaro Ogura, Rajkumar Baldeosingh, Manasee Gedam, Malia Michelle Edwards, Gerard A Lutty; In vitro and ex vivo models for evaluating drugs that quiesce mast cells, models for mast cell degranulation in geographic atrophy choroid. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3471.

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

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Abstract

Purpose : Our pervious studies suggest that degranulation (DG) of resident mast cells (MCs) are considered as a trigger of geographic atrophy (GA). The purpose of this study was to develop rapid in vitro and ex vivo assays to evaluate drugs to reduce MC DG.

Methods : RBL-2H3 cells were used for in vitro assays and excised rat choroids for ex vivo. In both models, MCs were stimulated with either compound 48/80 or calcium ionophore (CI), and co-treated with two commercially available MC stabilizers, ketotifen fumarate (KTF) and cromolyn sodium (CS). Percentage of beta-hexosaminidase release compared to triton control was assessed for MC DG in vitro. Choroidal MCs were stained with non-specific esterase and the percentage of MC DG was evaluated ex vivo. Macrophages (MPs) were co-stained with an anti-Iba1 antibody and their cell volume and sphericity were measured. Significance of the drug effect was determined with ANOVA analysis.

Results : DG of RBL-2H3 cells was reduced 50.4±8.6% (48/80 stimulation), 43.8±7.9% (CI) when they were treated with KTF, and 42.2± 16.6% (48/80) and 39.4±8.8% (CI) with CS (p < 0.01). Ex vivo, both 48/80 and CI stimulation led to significant choroidal MC DG (%DG with 48/80; 68.8±8.1%, CI; 73.1±8.4%), compared to control (16.8±3.7%) (p < 0.01). MC DG was significantly inhibited by treatment with either KTF [48/80+KTF, 11.0±4.9%; CI+KTF, 19.0±5.5% (p < 0.01)] or CS [48/80+CS, 16.0±6.2%; CI+CS, 15.2±6.0% (p < 0.01)]. MP volume was decreased with either stimulation [48/80, 378.7±141.1 μm3; CI, 358.9±111.6 μm3 (p < 0.01)] compared to control (584.5±170.3 μm3) and restored with treatments [48/80+KTF, 621.3±306.1 μm3; 48/80+CS, 554.7±252.4 μm3; CI+KTF, 561.0±226.5 μm3; CI+CS, 611.1±190.5 μm3 (p < 0.01)]. MP sphericity was increased with stimulation [48/80, 0.84±0.09; CI, 0.86±0.09 (p < 0.01)] compared to control (0.66±0.09), and was lowered with treatments [48/80+KTF, 0.71±0.11; 48/80+CS, 0.65±0.10; CI+KTF, 0.67±0.13; CI+CS, 0.64±0.10 (p < 0.01)].

Conclusions : MC quiescence by drugs prevented MP activation (increased sphericity, decreased volume), indicating that MC cytokine release was prevented and our stimuli act only on MCs not MPs. Our ex vivo model system will enable rapid screening of drugs targeting MCs and subsequent ocular inflammation in GA. Also, the ex vivo model is more representative of drug effects on choroidal MCs specifically.

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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