**2.3. Full spectral imaging**

external calibration measurement, while at the same time increasing sensitivity, accuracy, and reproducibility. More recently investigators have advanced this technology to develop new systems such as the Flow Oximetry System (FOS) that are able to measure oxygen saturation

tinct wavelengths are taken simultaneously. A traditional fundus camera is attached to a beam splitter and digital camera in order to obtain digital images at multiple distinct wavelengths. One image is taken at an isobestic wavelength that is insensitive to differences in hemoglobin oxygen saturation. This is required for compensation against variables such as hematocrit, path length and light intensity that will not differ between the two images. In the image obtained from the isobestic wavelength there is no visual difference between oxygen saturated arteries compared to oxygen depleted veins. Simultaneously, a second image is taken at a wavelength that is sensitive to hemoglobin oxygenation. In this image there are clear differences between the oxygen saturated arteries and oxygen depleted veins. Software has now been developed to automatically detect blood vessels in order to help minimize other factors that contribute to

optical density. This has led to highly reproducible measurements of SO2

sels that can be depicted numerically or as a color map on the fundus image [74].

DR, and also proliferative DR. These studies consistently report increased Sv

als [77]. Studies using FOS saw no significant difference in either S<sup>a</sup>

patients; however due to the conflicting reports on S<sup>a</sup>

lature of diabetic patients. A number of studies have measured changes in venous (Sv

Since diabetes has been linked to abnormal oxygen distribution in the diabetic retina, retinal oximetry serves as a useful tool to examine changes in oxygen saturation in the retinal vascu-

severity of DR increases [77–83]. Interestingly, the results on whether arterial oxygen saturation changes during DR progression differ between studies. A number of studies have found

the patient develops proliferative diabetic retinopathy [78–83], while others saw no difference in arterial oxygen saturation between DR patients in any stage compared to healthy individu-

individuals and DR patients, however identified significant changes in arteriovenous difference [75]. Together, these data indicate that there is increased venous oxygen saturation in DR

formed in patients. This technique gives accurate and reliable measurements of SO2

retinal vasculature to help provide insight into the dynamics of oxygen perfusion and consumption in these patients. Furthermore, retinal oximetry has shown that laser photocoagulation helps to improve oxygen delivery to the retina, and has therefore proven to be a useful tool in identifying the mechanisms of current DR treatments [15]. Another advantage of retinal oximetry is that systems are commercially available. However, since the use of retinal oximetry is restricted to the large retinal vessels, this technique might not adequately detect many of the changes seen in DR progression such as microaneurysms and acellular capillaries that

 increase remains to be confirmed. This increase in venous oxygen saturation could be a result of decreased oxygen perfusion into the tissue which could lead to tissue hypoxia, but could also be a result of increased arterial oxygen saturation as observed in some reports, which could lead to subsequent increases in venous oxygen saturation with the same level of perfusion. The advantages of retinal oximetry are that it is a non-invasive procedure that easily be per-

) oxygen saturation in patients with mild, moderate, or severe non-proliferative

increases with increased disease severity and these changes may not be present until

O2

by using dual wavelength oximetry. Here, two images at dis-

in large retinal ves-

O2

O2 or Sv O2

levels in DR patients, the cause of this

O2 ) and

values as the

between healthy

in the

and blood flow within the retinal vasculature [75, 76].

52 Early Events in Diabetic Retinopathy and Intervention Strategies

Most studies have measured SO2

arterial (Sa

that Sa O2

Sv O2 O2

In addition to dual wavelength oximetry, full spectral methods have also taken advantage of the differences between Hb and HbO<sup>2</sup> absorption spectra. Here, rather than using distinct isobestic and non-isobestic wavelengths to measure SO2 , a continuous range of wavelengths between visible and near-infrared spectrum are transmitted for measurement. Schweitzer et al. first described the technique by illuminating the retina with a narrow slit (1.5 × 40 mm) of light and capturing the image using an imaging ophthalmospectrometer, which consisted of a fundus camera adapted with a spectrograph coupled to an intensified CCD camera for detection [85, 86]. This allowed for collection of the full spectral data in a narrow band in a single dimension. Since then, full spectral imaging has developed into hyperspectral imaging (HSI) with algorithms used to construct a two dimensional image in order to visualize the data as an oxygen map [87–89]. Whereas this process originally took several seconds due to sequential acquisition of many single-dimension images, new technology allows for enough images to be taken to cover a 15 degree field with good spatial resolution in only a few milliseconds [90]. Today, HSI has been further developed into hyperspectral computed tomographic imaging spectroscopy (HCTIS), which in addition to giving detailed oxygen saturation maps, can give information about changes in the retina such as lesions, perfusion, and pigment density [90, 91].

Full spectral imaging has been used to examine oxygen imbalances in a number of vascular diseases including age-related macular degeneration [85], arteriovenous occlusion [88], and glaucoma [87, 89]. A limited number of studies have utilized full spectral imaging to examine changes in oxygen saturation in diabetic retinopathy. Kashani et al. used HCTIS to examine changes in Sa O2 and Sv O2 between healthy individuals and patients with DR and determined that Sa O2 was significantly lower, while S<sup>v</sup> O2 was significantly higher in patients with proliferative DR [91]. This was confirmed by a significant difference in the arteriovenous difference between the two groups [91].

### **2.4. Phosphorescence-lifetime imaging**

Phosphorescence-lifetime imaging is another minimally invasive technique that can be used to image PO2 within the retina. The use of oxygen-dependent quenching of phosphorescence as a method of optical measurement of O2 concentration was first described by Vanderkooi 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 phosphorescence signal as PO2 decreased, as described by a Stern-Volmer relationship [92, 93]. This technique was modified for use *in vivo* to measure PO2 in the retinal and choroidal vasculature 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 and even some capillaries with good spatial resolution.

oxygen levels within the retinal vasculature rather than the retinal tissue itself. It must be noted

electrodes) and found comparable results, with only slight decreases in PO2 within the tissue [95, 108]. The second disadvantage to phosphorescence-lifetime imaging is primarily due to the lack of evidence in animal models of DR. Although it has been proven useful in a number of vascular ischemic diseases that share commonalities with DR it would be necessary to confirm the applicability of phosphorescence-lifetime imaging in animal models of DR before

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].

fluorescence around the lesion, with minimal fluorescence in control animals [121].

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

(using phosphorescence-lifetime) and a variety of tissues including the retina (using O2

proposing the technique as a potentially useful tool for determining PO2

measurements between the vasculature

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

micro-

55

levels in DR patients.

however, that a number of studies have compared PO2

**2.5. Hypoxia-sensitive fluorescent probes**

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 oxygen levels within the retinal vasculature rather than the retinal tissue itself. It must be noted however, that a number of studies have compared PO2 measurements between the vasculature (using phosphorescence-lifetime) and a variety of tissues including the retina (using O2 microelectrodes) and found comparable results, with only slight decreases in PO2 within the tissue [95, 108]. The second disadvantage to phosphorescence-lifetime imaging is primarily due to the lack of evidence in animal models of DR. Although it has been proven useful in a number of vascular ischemic diseases that share commonalities with DR it would be necessary to confirm the applicability of phosphorescence-lifetime imaging in animal models of DR before proposing the technique as a potentially useful tool for determining PO2 levels in DR patients.
