**3. Summary**

**Figure 2.** Imaging of HYPOX-4 in a mouse model of oxygen-induced retinopathy (OIR). Fundus and fluorescein channel *in vivo* images in OIR mice at P13 indicate accumulation of imaging probe in central, avascular hypoxic regions (A, B), which was not reflected by imaging in room air-reared age-matched controls (C, D). Findings in the OIR model correlated with microscopic imaging of retinal flatmounts (E, F, merged in G). Likewise, *ex vivo* analysis of room air control retinal flatmounts confirmed lack of HYPOX-4 accumulation in healthy, fully vascularized retinas (H). Disclaimer: This figure has been adapted from the original article by Uddin et al. [124] under Creative Commons Attribution 4.0 International License.

Due to the pharmacokinetics of HYPOX-1, -2, and -3, a new probe was designed with goal of creating an imaging agent for use *in vivo* with a potential for clinical application. This new probe, HYPOX-4, was characterized for *in vitro* and *in vivo* use and compared to immunostaining of pimonidazole-adducts [124, 125]. *In vitro,* HYPOX-4 displayed increasing fluorescence with decreasing oxygen concentration in a variety of different retinal cell lines [124]. *Ex vivo*, HYPOX-4 successfully identified avascular regions in the retinal flatmounts of OIR mice [124] (**Figure 2**) and hypoxic regions downstream of the occluded vein in the retinas of laserinduced retinal vein occluded (RVO) mice [125] (**Figure 3**). Using a micron IV imaging system, HYPOX-4 was then used for *in vivo* imaging of hypoxia in both the OIR and RVO mice. In both models, HYPOX-4 clearly identified areas of hypoxia *in vivo* [124, 125]. HYPOX-4 had no effect on proliferation (as measured by BrdU assay), toxicity (TUNEL), or function (ERG) [124].

The advantages to these hypoxia sensitive fluorescent probes are that they can be conjugated to already FDA approved fluorescent dyes and they allow for direct imaging of hypoxia within the retinal tissue, rather than the microvasculature. Furthermore, studies in the OIR mice have shown they are capable of detecting hypoxia in diseases where there is oxygen imbalance in the entire retina, while the RVO model has shown that they are also capable of detecting regional, focal hypoxia downstream of either a single or double vein occlusion. This alone makes these probes particularly useful in diseases such as DR where there is likely capillary occlusion leading to localized hypoxia within the retinal tissue. A disadvantage of these hypoxia sensitive fluorescent probes are that they only give an image of hypoxic areas

, although the PO2

adduct formation is well characterized. Furthermore, these probes have been used in OIR and LCNV models to show their ability to identify focal hypoxia; however their use in models of

threshold for bioreduction and

without providing actual values for PO2

56 Early Events in Diabetic Retinopathy and Intervention Strategies

diabetic retinopathy needs to be examined.

Hypoxia has been shown to play a significant role in DR progression. Hypoxia stimulates the production of a number of different pro-inflammatory cytokines (IL-1beta, TNF-a, ICAM-1) [7, 126, 127] and growth factors (VEGF and PDGF) [45, 47, 128, 129]that lead to neovascularization, increased vascular permeability and cell death. Studies have found that treatments such as laser photocoagulation provide benefits by restoring oxygen tension in the diabetic retina [15]. Furthermore, studies have indicated that oxygen imbalance actually precedes many of the pathological events that occur throughout the progression of diabetic retinopathy [2, 43, 130]. Therefore, early detection of hypoxic regions in the diabetic retina can potentially help clinicians choose appropriate treatment strategies before irreversible damage has already occurred.

New advances in imaging strategies allow for optical measurement the of oxygen levels *in vivo*. Oxygen sensitive microelectrodes have been the gold standard for direct measurement of oxygen levels in the retinal tissue, however the measurement is highly invasive and unable to consistently identify small areas of focal hypoxia. Together these factors prevent oxygen sensitive microelectrodes from being used in DR patients. More recently, less invasive techniques such as retinal oximetry, phosphorescence-lifetime imaging and hypoxia sensitive fluorescent probes have been developed in an effort to detect oxygen imbalances and allow for optical identification of hypoxic regions *in vivo*. Retinal oximetry and phosphorescence-lifetime have been used primarily to measure oxygen saturation in the retinal vasculature. These methods have been used in a number of different animal models and have shown that they can successfully identify regions of focal hypoxia surrounded by predominantly normoxic tissue, similar to what is hypothesized in DR. Hypoxia sensitive fluorescent probes differ from these techniques in that they detect hypoxic regions within the retina itself, rather than the microvasculature. These probes have been developed by the conjugation of fluorescein dyes, such as FITC, to 2-nitroimidazoles. These 2-nitroimidazoles are bioreduced by nitroreductases in under hypoxic conditions, causing them to aggregate within the hypoxic cells. A number of these hypoxia sensitive fluorescent probes have been developed and characterized for *in vitro, ex vivo,* and *in vivo* use with low toxicity.

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The imaging techniques reviewed here have all been shown to optically identify regions of focal hypoxia *in vivo.* Clinically, these techniques can help to give an accurate depiction of oxygen imbalances within the diabetic retina before retinal pathologies are detectable and may therefore guide future treatment strategies in DR patients.
