**2.2. Dual wavelength retinal oximetry**

in the diabetic retina [35–40]. Studies point toward an increase in the number of leukocytes and increased leukocyte adhesion as a source of capillary occlusion and tissue nonperfusion [36, 41, 42]. Furthermore, it has been shown that there is tissue hypoxia in the diabetic retina since the choriocapillaris cannot adequately provide the inner retina with oxygen. Linsenmeier

in inner retina of diabetic (7.66 mmHg) compared to normal cats (16.42 mmHg) (**Figure 1**) [2]. These studies showed that hypoxia was evident early in disease progression, prior to observation of angiopathy, microaneurysms, hemorrhages and capillary dropout. Studies using magnetic resonance imaging (MRI) have also shown that there is a decrease in oxygen levels in galactosemic rats before retinal lesions appear [43]. These studies support the hypothesis that hypoxia may be a driving force in DR progression, rather than an outcome of other factors. Consistent with this hypothesis, hypoxia itself has been shown to stimulate production of a number of proangiogenic factors such as vascular endothelial growth factor (VEGF), one of the predominant targets of many therapeutic interventions in DR [44–48]. Steffanson et al. provided further evidence that oxygen delivery plays a crucial role in DR progression when they observed increased oxygen tension in the areas of the retina that had undergone panretinal photocoagulation compared to untreated areas [15]. This finding supports therapeutic strategies in DR which aim to restore normal oxygen supply in order to normalize disease.

Interestingly, although hypoxia has long been hypothesized as a potential driver of DR, it must be noted that there are a number of studies that were unable to detect the presence of hypoxia in the diabetic retina. Some studies in diabetic mice show that there is an initial decrease in retinal blood flow between three to 4 weeks due to arteriolar constriction, however arteriolar diameter and blood flow return to normal measurements at later time points of diabetes (12 weeks) [49–52]. Despite the initial decrease in blood flow these studies did not find any evidence of hypoxia in these animals at either 3 or 12 weeks of diabetes [53–55]. This may indeed be accounted for by compensatory vascular mechanisms in the rodent retina, such as autoregulation which may counteract early hypoxia in these species, and that onset of hypoxia may only occur at very late time points (>1.5 years) that are beyond typical published experimental endpoints in these models.

We are therefore in concurrence with a number of investigators in the ophthalmic and clinical research who identify retinal hypoxia as a significant mediator of initiation and progression of DR. Along with other researchers, we have sought to develop and translate strategies for optical detection of early retinal oxygen imbalance in patients to facilitate earlier clinical inter-

Oxygen-sensitive microelectrodes have long been considered the gold standard for measure-

highly invasive nature of the measurement, as it requires a direct puncture of the retinal tissue which prevents its use in clinics. Furthermore, oxygen imbalances in retinal vascular diseases

in tissues including the retina [2, 56–60]. While this technique gives an accurate

, there are many drawbacks, most importantly the

ventions and improved outcomes to reduce risk of future vision loss.

**2.** *In vivo* **imaging of hypoxia in diabetic retinopathy**

**2.1. Overview of hypoxia sensing and imaging technologies**

and direct measurement of retinal tissue PO2

ment of PO2

in the retinas of long term

was significantly lower

et al. used oxygen-sensitive microelectrodes to directly measure PO2

50 Early Events in Diabetic Retinopathy and Intervention Strategies

diabetic and non-diabetic cats (> 6 years) and showed that mean PO2

Retinal oximetry is a non-invasive technique used to measure the percent of hemoglobin oxygen saturation (SO2 ) in the retinal vasculature. Oximetry is based on the principle that oxygenated (HbO<sup>2</sup> ) and deoxygenated hemoglobin (Hb) have a different light absorption spectra. The use of spectrophotometric measurements to determine oxygen levels in large retinal vessels was first described by Hickam et al. in 1963, however their method required an independent arteriolar SO2 measurement for external calibration [71]. Since this initial work, Delori [72], Beach [73], and Hardason et al. [74] have expanded the field by developing new techniques that decrease the invasiveness of retinal oximetry by eliminating the need for an 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 and blood flow within the retinal vasculature [75, 76].

occur in the small capillary beds of the retinal microvasculature. Furthermore, retinal oxim-

measurement of what is happening within the retinal tissue itself. Whether the changes in

retina itself cannot be confirmed with retinal oximetry alone. This has been shown in studies examining the correlation between regional differences in oxygen saturation versus lesion formation in patients with proliferative diabetic retinopathy and diabetic maculopathy [84]. The

indicating that there was decreased oxygen perfusion from the retinal vasculature, however

retinal lesions [84]. This implies that other factors such as local changes in the microcirculation and within the tissue itself play a significant role in lesion formation and DR progression.

In addition to dual wavelength oximetry, full spectral methods have also taken advantage

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

erative DR [91]. This was confirmed by a significant difference in the arteriovenous difference

Phosphorescence-lifetime imaging is another minimally invasive technique that can be used

within the retina. The use of oxygen-dependent quenching of phosphorescence

O2

between healthy individuals and patients with DR and determined

was significantly higher in patients with prolif-

concentration was first described by Vanderkooi

observed in diabetic patients actually correlates to regions of hypoxia within the

levels within the vasculature, but does not give a direct

Imaging of Hypoxia in Retinal Vascular Disease http://dx.doi.org/10.5772/intechopen.72252 53

was increased in diabetic patients compared to healthy individuals

in the large retinal vessels did not correlate with the areas of

absorption spectra. Here, rather than using distinct

, a continuous range of wavelengths

etry provides a measurement of SO2

study found that total SO2

**2.3. Full spectral imaging**

changes in Sa

to image PO2

that Sa O2 O2 and Sv O2

between the two groups [91].

**2.4. Phosphorescence-lifetime imaging**

as a method of optical measurement of O2

was significantly lower, while S<sup>v</sup>

of the differences between Hb and HbO<sup>2</sup>

isobestic and non-isobestic wavelengths to measure SO2

the regional differences in SO<sup>2</sup>

vascular SO2

Most studies have measured SO2 by using dual wavelength oximetry. Here, two images at distinct 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 in large retinal vessels that can be depicted numerically or as a color map on the fundus image [74].

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 vasculature of diabetic patients. A number of studies have measured changes in venous (Sv O2 ) and arterial (Sa O2 ) oxygen saturation in patients with mild, moderate, or severe non-proliferative DR, and also proliferative DR. These studies consistently report increased Sv O2 values as the 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 that Sa O2 increases with increased disease severity and these changes may not be present until 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 individuals [77]. Studies using FOS saw no significant difference in either S<sup>a</sup> O2 or Sv O2 between healthy 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 patients; however due to the conflicting reports on S<sup>a</sup> O2 levels in DR patients, the cause of this Sv O2 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 performed in patients. This technique gives accurate and reliable measurements of SO2 in the 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 occur in the small capillary beds of the retinal microvasculature. Furthermore, retinal oximetry provides a measurement of SO2 levels within the vasculature, but does not give a direct measurement of what is happening within the retinal tissue itself. Whether the changes in vascular SO2 observed in diabetic patients actually correlates to regions of hypoxia within the retina itself cannot be confirmed with retinal oximetry alone. This has been shown in studies examining the correlation between regional differences in oxygen saturation versus lesion formation in patients with proliferative diabetic retinopathy and diabetic maculopathy [84]. The study found that total SO2 was increased in diabetic patients compared to healthy individuals indicating that there was decreased oxygen perfusion from the retinal vasculature, however the regional differences in SO<sup>2</sup> in the large retinal vessels did not correlate with the areas of retinal lesions [84]. This implies that other factors such as local changes in the microcirculation and within the tissue itself play a significant role in lesion formation and DR progression.
