June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Ex vivo analysis of mast cell degranulation in human donor choroids
Author Affiliations & Notes
  • Imran Ahmed Bhutto
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Tomohiro Niizawa
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Alexandra Pado
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Erin McDonnell
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Gerard A Lutty
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Malia Michelle Edwards
    Ophthalmology, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Imran Bhutto None; Tomohiro Niizawa None; Alexandra Pado None; Erin McDonnell None; Gerard Lutty None; Malia Edwards None
  • Footnotes
    Support  NIH EY016151 (ME & GL); NIH EY03144 (ME); P30EY001765 (Wilmer)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3029 – F0400. doi:
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    • Get Citation

      Imran Ahmed Bhutto, Tomohiro Niizawa, Alexandra Pado, Erin McDonnell, Gerard A Lutty, Malia Michelle Edwards; Ex vivo analysis of mast cell degranulation in human donor choroids. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3029 – F0400.

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

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Abstract

Purpose : We have previously demonstrated that mast cells are increased in number and there is increased mast degranulation in submacular choroids in all stages of age-related macular degeneration (AMD). We also recently reported ex vivo and in vivo rat models in which a snake venom-like compound, 48/80, stimulates degranulation of choroidal mast cells. Moreover, both oral and topical administration of ketotifen (KTF), a known mast cell stabilizer, reduced mast cell degranulation in the rat model. This study was designed to determine whether human choroid explants can be used as an ex vivo model to study mast cell degranulation and screen potential therapies. In addition, this study investigated the ability of C-reactive protein (CRP), which is elevated in AMD, to stimulate mast cell degranulation.

Methods : Human donor eyes (N=4) were received on wet ice within 30 hrs postmortem. Retinas were isolated from the choroid/RPE complex. The RPE/choroid complex adjacent to the submacula was dissected into pieces. The choroid pieces incubated for 3 hrs at 37°C in either Dulbeccos modified Eagle’s media (DMEM) (control), 48/80 (300µg/ml), 48/80 + KTF (300µg/ml), or hCRP (2, 4, and 10 ng/ml). The RPE were then removed with 1% EDTA. Choroids were then fixed in 2% PFA in cacodylate buffer at 4°C overnight and stained for non-specific esterase (NSE) activity. Pigments were bleached with 30% hydrogen peroxide and NSE-stained choroids were imaged. Mast cells were counted using Image J. The percent of degranulated mast cells was calculated in 20x images (4 random fields per piece).

Results : 48/80 stimulated mast cell degranulation in human choroid explants. Moreover, this stimulation was significantly reduced by KTF. CRP also increased mast cell degranulation in a dose-dependent manner, reaching significance at 4 and 10ng/ml. At the 10 ng/ml CRP dose, 43.5% of mast cells were degranulated compared to 15% in the control.

Conclusions : Similar to previous observations in rats, KTF can prevent 48/80-induced mast cell degranulation in human donor eyes. CRP, at physiological concentrations, significantly stimulates mast cell degranulation ex vivo. The ability to study mast cell degranulation ex vivo in human donor choroids may prove useful in testing therapies.

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

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