**2.5. Hypoxia-sensitive fluorescent probes**

et al. [92, 93]. At the time, a similar method using oxygen-dependent quenching of fluorescence, rather than phosphorescence, had already been established [94]. The use of fluorescence however, was limited by low sensitivity to oxygen and by the fact that the decay in fluorescence brightness is rapid, which meant that only fluorescence intensity and not lifetime could be measured. Intensity measurements are complicated by variables such as solution composition and absorption in the tissue. By using phosphorescence, Vanderkooi et al. were able to measure lifetime, rather than intensity, due to the much slower decay in brightness of phosphorescence compared to fluorescence [92, 93]. It was observed that phosphorescencelifetimes were directly dependent on oxygen concentration, with an increase in phospho-

lature of large animals such as cats and pigs [95–97], and later for smaller animals such as mice and rats [98–104]. More recently Shahidi et al. have made significant advances by using phosphorescence-lifetime to image oxygen tension within the retinal tissue itself [105, 106]. This minimally invasive technique requires an intravenous injection of a phosphor that can be imaged using an intensified CCD camera to provide a clear image of retinal arteries, veins

To date, many of the studies utilizing phosphorescence-lifetime have sought to establish the technique and examine changes in oxygen tension during normal physiologic processes such as retinal response to light stimulation [68, 104]. A limited number of studies have utilized phosphorescence-lifetime to study oxygen imbalances in ischemic retinal diseases. Studies in a mouse model of oxygen-induced retinopathy (OIR) have shown that although there was no significant difference in arterial or venous PO2 between control and OIR mice, the arteriovenous difference was significantly higher in OIR mice [107]. This was attributed to a decreased vascular network in these OIR mice resulting in greater oxygen extraction from the larger vessels [107]. Other investigators have examined whether phosphorescence-lifetime imaging can be used to detect regions of local hypoxia created by laser photocoagulation. In these studies, a laser was used to create small (75 μm) focal lesions within the capillary network of the mouse retina [100]. Upon imaging and analysis using an oxygen map of the laser burn and surrounding area they observed a circular lesion with a central area of hypoxia (< 7 mmHg) that extended approximately 150–200 μm outward from the initial laser injury [100]. After imaging the lesion again 1 hour later there was no evidence of leakage of the phosphor into the tissue [100]. This study indicates that phosphorescence-lifetime imaging is a useful tool for identifying focal areas of regional hypoxia. Further experiments are needed to confirm

whether these results translate into animal models of diabetic retinopathy.

The primary advantage of phosphorescence-lifetime imaging is that it is minimally invasive, requiring only an intravenous injection of a phosphor. These phosphors are readily available as nontoxic, water soluble forms so they can be easily dissolved in blood, providing further potential for use in a clinic. Furthermore, studies have shown that this technique is capable of identifying small areas of regional hypoxia created by focal lesions similar to those seen in DR [100]. This can be combined with traditional fundus photographs and fluorescein angiography to determine whether the areas of regional hypoxia correlate with the location of retinal lesions or leakage during the progression of DR. Finally, the information gathered can be displayed as an easy to read oxygen map showing retinal vascular function. The disadvantage of phosphorescence-lifetime imaging is similar to retinal oximetry in that it is a measurement of

decreased, as described by a Stern-Volmer relationship [92, 93]. This

in the retinal and choroidal vascu-

rescence signal as PO2

technique was modified for use *in vivo* to measure PO2

54 Early Events in Diabetic Retinopathy and Intervention Strategies

and even some capillaries with good spatial resolution.

Another technique for imaging oxygen imbalances in the diabetic retina is the use of hypoxiasensitive fluorescent probes. Hypoxia-sensitive compounds such as 2-nitroimidazoles are bioreduced by nitroreductases in hypoxic tissues (PO2 < 10 mmHg) which leads to the formation of adducts with thiol containing proteins [109–113]. These compounds were originally discovered and used for detecting hypoxic areas within tumors and were imaged by autoradiography [112, 114]. Shortly after, immunohistochemical analysis was made possible by the production of antibodies that recognized the adducts formed by the reduced 2-nitroimidazoles and showed that the fluorescence intensity correlated with the severity of hypoxia [110, 111, 115]. More recently, 2-nitroimidazoles, such as pimonidazole, have been used to detect areas of hypoxia in a number of retinal vascular diseases, including extensive studies in diabetic retinopathy. *Ex-vivo* studies in non-diabetic and diabetic mice and rats have found significantly increased pimonidazole labeling in the retinas of even short-term diabetic mice and rats compared to their non-diabetic counterparts [53, 116–119]. Furthermore, the pimonidazole labeling was confirmed by increased staining of hypoxia inducible factor-1α (HIF-1α) and decreased ganglion cell function measured by electroretinogram (ERG) [53, 116].

Work by our group has sought to develop clinically useful hypoxia sensitive imaging agents by conjugating FDA-approved fluorescein dyes to adduct forming 2-nitroimidazoles. In preliminary studies, fluorescein isothiocyanate (FITC) was conjugated to a 2-nitroimidazole containing reagent to create the HYPOX-1 probe, and also to pimonidazole to create HYPOX-2. Both HYPOX-1 and HYPOX-2 formed adducts leading to accumulation in a variety of hypoxic retinal cells and allowed for imaging with excellent signal-to-noise ratio *in vitro* [120]. Furthermore, these imaging agents were capable of detecting hypoxic areas *ex vivo* in the retinas oxygen induced retinopathy (OIR) mice with no apparent toxicity [120]. Following the success of these fluorescent imaging agents, a new probe, HYPOX-3, was developed in order to create an "on-off" imaging agent for hypoxia [121]. Here, a near-infrared (NIR) imaging agent was coupled to Black Hole Quencher 3 (BHQ3), which had been shown to quench NIR dyes by Fӧrster resonance energy transfer (FRET) [122]. Interestingly, BHQ3 features a hypoxia-sensitive azo-bond that is cleavable by azoreductases under hypoxic conditions [122, 123]. HYPOX-3 displayed high sensitivity and specificity in forming adducts in a variety of hypoxic retinal cells *in vitro* with no detectable toxicity [121]. The ability to detect hypoxia in retinal vascular disease animal models was examined using a laser-induced choroidal neovascularization (LCNV) mouse model. In RPE-choroid flatmounts, HYPOX-3 clearly identified hypoxic regions in LCNV mice and showed increased fluorescence around the lesion, with minimal fluorescence in control animals [121].

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 without providing actual values for PO2 , although the PO2 threshold for bioreduction and 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 diabetic retinopathy needs to be examined.

**3. Summary**

Attribution 4.0 International License.

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

**Figure 3.** HYPOX-4 mediated retinal imaging of laser-induced retinal vein occlusion (RVO) in the mouse. HYPOX-4 injected 2 hours post vein occlusion with an argon photocoagulator in tandem with rose Bengal photosensitization. The imaging agent accumulated within the venous occlusion site (A, arrowhead) and downstream capillary bed (B). Disclaimer: this figure has been adapted from the original article by Uddin et al. [125] under Creative Commons

Imaging of Hypoxia in Retinal Vascular Disease http://dx.doi.org/10.5772/intechopen.72252 57

**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.

**Figure 3.** HYPOX-4 mediated retinal imaging of laser-induced retinal vein occlusion (RVO) in the mouse. HYPOX-4 injected 2 hours post vein occlusion with an argon photocoagulator in tandem with rose Bengal photosensitization. The imaging agent accumulated within the venous occlusion site (A, arrowhead) and downstream capillary bed (B). Disclaimer: this figure has been adapted from the original article by Uddin et al. [125] under Creative Commons Attribution 4.0 International License.
