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Stephanie M. Evans, Ashwath Jayagopal; Imaging of Retinal Vascular Disease Using Hypoxia-Sensitive Contrast Agents. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5873.
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Retinal vascular diseases, including diabetic retinopathy and age related macular degeneration, are often associated with retinal hypoxia. Therefore, the ability to image hypoxic retinal tissue in vivo would be beneficial for improved clinical management of these diseases. For this purpose, a hypoxia-sensitive fluorescent contrast agent was developed and characterized for imaging of hypoxia in retinal tissue using established cell culture and animal models of retinal vascular disease.
Fluorescently-labeled, hypoxia-sensitive contrast agents were synthesized, purified chromatographically, and characterized by mass spectrometric and nuclear magnetic resonance analyses. To evaluate the utility of this contrast agent for imaging hypoxia, in vitro assays using Human Retinal Microvascular Endothelial Cells (HRMEC) and in vivo studies using mice with oxygen-induced retinopathy (OIR) or laser-induced choroidal neovascularization (LCNV) were performed to determine the hypoxia-associated sensitivity and specificity of this contrast agent. Fluorimetric and flow cytometric assays were performed on normoxia- or hypoxia-conditioned HRMEC exposed to contrast agents to measure contrast agent binding and uptake. OIR and LCNV animal models were intravenously or intraocularly-injected with the contrast agents and analyzed by in vivo and ex vivo retinal fluorescence imaging to determine specificity and sensitivity of the contrast agent. Pimonidazole hydrocholoride immunostaining was utilized to confirm specificity of the contrast agent for hypoxic tissue.
HRMEC conditioned under hypoxia for varying durations of time up to 24 hrs. exhibited dose-dependent fluorescence enhancement due to hypoxia-selective uptake of the contrast agent. In animal models, regions of tissue hypoxia staining positive for pimonidazole hydrochloride were also colocalized with contrast agent uptake, as indicated by in vivo and ex vivo imaging. Contrast agent accumulation in hypoxic tissue was detectable within 2 hrs. post-injection.
These studies support the feasibility of imaging hypoxic tissue in vivo using targeted contrast agents in conjunction with readily available retinal fluorescence imaging equipment. Hypoxia-sensitive contrast agents, if clinically translated, may be useful for early detection of retinal vascular diseases and monitoring of therapeutic response in patients.
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