June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
In Vivo Imaging of Retinal Hypoxia
Author Affiliations & Notes
  • Ashwath Jayagopal
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN
  • Md Imam Uddin
    Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN
  • Footnotes
    Commercial Relationships Ashwath Jayagopal, Vanderbilt University Medical Center (P); Md Imam Uddin, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 31. doi:
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      Ashwath Jayagopal, Md Imam Uddin; In Vivo Imaging of Retinal Hypoxia. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):31.

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

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Purpose: Hypoxia has been associated with initiation and progression of many retinal diseases. Technologies for imaging retinal hypoxia are needed to improve clinical management of these diseases by enabling early detection, monitoring of disease progression, and response to therapy. In this work we developed and characterized a hypoxia-selective fluorescein based optical imaging probe for detection of retinal hypoxia in several cell culture and animal models using in vivo retinal fluorescence imaging.

Methods: Sensitivity, specificity, and safety of fluorescein-based hypoxia sensitive imaging probes were characterized in R28 rat retinal neuronal cell lines, human Muller cells, and human RPE cells, as well as mouse models of laser-induced choroidal neovascularization (LCNV), retinal vein occlusion (RVO), and oxygen-induced retinopathy (OIR). Cell culture studies included FACS analysis of uptake and toxicity, confocal microscopy uptake studies, and gel electrophoresis/blotting to identify hypoxia probe retention in hypoxic vs. normoxic cell lysates. Animal models were imaged using an in vivo retinal fluorescence imaging system and tissues were dissected and stained to confirm probe uptake ex vivo, colocalization with hypoxic or avascular retinal tissue regions, as well as toxicity (TUNEL, caspase-3 staining).

Results: Cells conditioned under hypoxia exhibited dose-dependent fluorescence enhancement due to selective cellular retention of imaging probes. Colocalization with Hypoxyprobe immunostaining and Western blot analysis of imaging probes in lysates further confirmed hypoxia selectivity. In LCNV, RVO, and OIR animal models, hypoxia imaging probes colocalized with hypoxic regions of tissue identifed by immunofluorescence staining. Signal to noise ratios of these imaging probes exceeded 10:1 in several disease models. Imaging probes were well tolerated as indicated by BrdU, TUNEL, Caspase-3, and FACS assays.

Conclusions: We have developed a promising in vivo imaging probe for detection of hypoxic retina using noninvasive fluorescence imaging equipment. These probes are biocompatible and sensitive, and complement existing technologies for measuring retinal vascular PO2 and blood flow. Furthermore, hypoxia imaging probes described here are readily useful for elucidating the role of hypoxia in retinal disease in preclinical studies.


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