Neovascularization (NV) is a common complication in age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP) and retinal vein occlusion (RVO). Retinal hypoxia plays an important role during NV development and progression. Thus, in vivo molecular imaging of retinal hypoxia at an early stage could be an important predictive tool assessing the risk of NV development and progression. To this end, the PI has developed HYPOX-4, a highly sensitive molecular imaging probe capable of detecting retinal hypoxia in vivo in animal models of ROP and RVO. In this proposed study, quantitative assessment of HYPOX-4 fluorescence intensities measured by computational methods, correlating with levels of retinal hypoxia will be assessed to create a predictive tool for retinal NV development and progression. Current methods for the measurement of tissue oxygen tension include nuclear magnetic resonance, retinal oximetry, phosphorescence lifetime imaging, doppler optical coherence tomography (D-OCT), and visible-light OCT. Although their application has provided a clearer understanding of the vascular oxygen supply and metabolism in the retina, none of these imaging methods have been used successfully to measure retinal hypoxia in vivo within avascular retina or in ischemic tissues. Pimonidazole-adduct immunohistochemistry is the common method to study tissue hypoxia, but this technique is limited by it's exclusive ex vivo method of examination. Thus, it is not useful for ophthalmic clinical in vivo applications. To this end, HYPOX-4 (developed by the applicant) will be used to address the limitations of indirect or invasive hypoxia detection technologies.
In Aim 1 and 2, HYPOX-4 will be tested to determine graded levels and temporal profiles of retinal hypoxia depicting the onset, evolution and resolution of NV in mouse model of oxygen-induced retinopathy (OIR) in vivo as well as ex vivo. Hypoxia profiles will be further correlated with the expression of hypoxia-associated biomarkers including HIF-1?, CAIX and VEGFR2. The standard pimonidazole-adduct ex vivo immunostaining and phosphorescence lifetime imaging will be performed in parallel experiments to confirm the temporal and graded retinal-hypoxia profiles and tissue oxygen pressures.
In Aim 3, HYPOX-4 will be evaluated for biodistribution, safety and toxicity in this mouse OIR and room air (RA) control animals. In this proposal, the applicant (Dr. Uddin), experts in molecular imaging methods in the context of retinal vascular diseases and other types of vascular dysfunction, will collaborate with Dr. John Penn, Dr. Manaz Shahidi and Dr. Marnett, experts in biology of animal models and ocular angiogenesis to demonstrate the utility of this new hypoxia sensitive probe for the early detection of retinal hypoxia. These studies have significant potentials for advancing the implementation of molecular imaging technologies in preclinical and clinical settings, and to establish a novel method to investigate hypoxia as a component of retinal vasculopathies.

Public Health Relevance

Neovascularization (NV) is a common complication in retinal vascular diseases. Retinal hypoxia plays an important role in NV development and progression. In this proposed study, a novel in vivo molecular imaging method will be established as a diagnostic tool to predict the risk of NV development by correlating the levels of retinal hypoxia measured by in vivo molecular imaging probe, HYPOX-4. This study will facilitate the early detection of molecular changes in the retina before the onset of proliferative retinopathy.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Neuroscience and Ophthalmic Imaging Technologies Study Section (NOIT)
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Shen, Grace L
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Vanderbilt University Medical Center
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