June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Isolation and characterization of peripapillary scleral fibroblast, in normal and glaucomatous canine eyes
Author Affiliations & Notes
  • Oluwabunmi Titilope Ojo
    Veterinary Clinical sciences, Purdue University, West Lafayette, Indiana, United States
  • Abhijit Mukhopadhyay
    Veterinary Clinical sciences, Purdue University, West Lafayette, Indiana, United States
  • Sunjun Jung
    Veterinary Clinical sciences, Purdue University, West Lafayette, Indiana, United States
  • Andras M Komaromy
    Small Animal Clinical Sciences, Michigan State University, East Lansing, Michigan, United States
  • Shin Ae Park
    Veterinary Clinical sciences, Purdue University, West Lafayette, Indiana, United States
  • Footnotes
    Commercial Relationships   Oluwabunmi Ojo None; Abhijit Mukhopadhyay None; Sunjun Jung None; Andras Komaromy None; Shin Ae Park None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4714. doi:
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      Oluwabunmi Titilope Ojo, Abhijit Mukhopadhyay, Sunjun Jung, Andras M Komaromy, Shin Ae Park; Isolation and characterization of peripapillary scleral fibroblast, in normal and glaucomatous canine eyes. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4714.

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

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Abstract

Purpose : Increase in intraocular pressure causes changes to the essential components of the sclera, the scleral fibroblasts, and their extracellular matrix. This affects the biomechanical response of the optic nerve head to elevated intraocular pressure which translates to glaucomatous optic neuropathy. Dogs have been proven to be useful models in the study of glaucoma as they have similar eye anatomy as humans and can also naturally develop glaucoma as in humans. There are also similarities in the pathogenesis of canine and human glaucoma, and human glaucoma treatments have been found to be effective in dogs. The purpose of our study was to isolate, culture and characterize with immunocytochemistry the sclera fibroblasts of normal and glaucomatous canine eyes.

Methods : Freshly enucleated normal (n=5) and glaucomatous (n=5) canine eyes were obtained and used for primary cell culture. At 37°C and 5% CO2 humidified incubator, peripapillary sclera (PPS) explants were cultured using their specific media. Sclera fibroblasts were fixed and stained with vimentin, laminin, CD45 and AF488 primary antibodies, and their respective secondary antibodies. Immunofluorescence microscopy for visualization of the proteins was done with Nikon Eclipse Ni Microscope using the NIS Elements imaging system.

Results : We were able to successfully extract and culture primary fibroblasts from the peripapillary scleral tissues of canine eyes with and without glaucoma. Consistent with the immunostaining results in humans, the canine scleral fibroblasts were positive for vimentin as a specific fibroblast marker and also positive for laminin, an extracellular marker. The cells were however negative for cytokeratin and CD45 as expected. No marked differences were observed between normal and glaucomatous eyes based on their immunocytochemical characteristics.

Conclusions : This is the first study describing the primary culture of canine scleral fibroblasts. We believe the techniques will be useful for future investigations to understand the cellular and molecular changes with glaucoma development in dogs as a large animal model for naturally occurring glaucoma. Future studies planned in our lab include proteomic analysis and biomechanical tests of established cells from the eyes with different types and stages of glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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