July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Making eyes invisible to make things more visible: An optimized tissue clearing approach for three-dimensional imaging and high-throughput analysis of whole rodent eyes
Author Affiliations & Notes
  • Akshay Gurdita
    Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Philip Nickerson
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Neno Pokrajac
    Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Arturo Ortin-Martinez
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Samuel Tsai
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Lacrimioara Comanita
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Nobuhiko Tachibana
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Zhongda Chris Liu
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
  • Danian Chen
    Lunenfeld-Tanenbaum Research Institute Mount Sinai Hospital, Toronto, Ontario, Canada
  • Rod Bremner
    Lunenfeld-Tanenbaum Research Institute Mount Sinai Hospital, Toronto, Ontario, Canada
  • Valerie Wallace
    Donald K Johnson Institute, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada
    Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
  • Footnotes
    Commercial Relationships   Akshay Gurdita, None; Philip Nickerson, None; Neno Pokrajac, None; Arturo Ortin-Martinez, None; Samuel Tsai, None; Lacrimioara Comanita, None; Nobuhiko Tachibana, None; Zhongda Chris Liu, None; Danian Chen, None; Rod Bremner, None; Valerie Wallace, None
  • Footnotes
    Support  This work was supported by operating grants to V. A. Wallace from Brain Canada, Foundation for Fighting Blindness, Ontario Institute of Regenerative Medicine and Krembil Foundation.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5990. doi:
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      Akshay Gurdita, Philip Nickerson, Neno Pokrajac, Arturo Ortin-Martinez, Samuel Tsai, Lacrimioara Comanita, Nobuhiko Tachibana, Zhongda Chris Liu, Danian Chen, Rod Bremner, Valerie Wallace; Making eyes invisible to make things more visible: An optimized tissue clearing approach for three-dimensional imaging and high-throughput analysis of whole rodent eyes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5990.

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

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Abstract

Purpose : The mouse eye serves as a useful tool to model a variety of pathologies and processes related to development, neurological disorders, regenerative therapies, vascular angiogenesis and tumorigenesis. Light sheet fluorescence microscopy allows for high-throughput analysis of optically cleared tissue samples, however established clearing methods do not address challenges posed by tissues such as the eye, which contains pigments that mitigate imaging accessibility. Thus, we have developed an optimized tissue clearing protocol for the rodent eye and have applied it to the analysis of three-dimensionally relevant features.

Methods : Mouse eyes were dissected, permeabilized, fixed and depigmented prior to clearing using an optimized version of the CUBIC clearing protocol. Imaging using the LaVision UltraMicroscope II light sheet, and analysis with Imaris 9.2.0 was performed on photoreceptor transplanted, retinoblastoma tumor, and Norrie disease eyes.

Results : We demonstrate using our optimized protocol that pigmented rodent eyes can undergo tissue clearing and subsequent high-throughput analysis using a light sheet fluorescent microscope. We demonstrate that our method provides single-cell resolution of retinal cells and allows for rapid confirmation of a successful sub-retinal injection and analysis of the topographic distribution of deposited cells. We have also applied our method for high-throughput analysis of the 3D topographical distribution and volume of retinoblastoma tumors. The 3D volumetric analysis of the inner retinal vasculature and choroidal vasculature may also be determined and compared to models with vasculature defects.

Conclusions : Our tissue clearing, depigmentation and staining protocol is a more efficient and effective method, than traditional sectioning methods, to study retinal transplantation, tumorigenesis, vasculature and other processes in the eye. This method of analysis may also provide new information, previously not attainable due to artifacts or limitations present in tissue processing.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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