**1. Introduction**

#### **1.1. Oxygen supply and consumption in the healthy retina**

The retina is one of the most metabolically active sites of the entire body and is therefore dependent on a consistent supply of oxygen and other nutrients. In order to meet these metabolic

demands, the retina requires two distinct blood supplies, the inner retinal circulation and choroidal circulation. The inner retinal circulation stems from the central retinal artery which enters the retina near the optic disc. From there, it branches to form the deep and superficial retinal capillary plexuses. In a healthy individual, these blood vessels are found only in the peripheral retina and do not enter into the avascular fovea. The central retinal artery is responsible for supplying the inner retina with oxygen and nutrients and receives about 20–30% of the blood flow to the retina [1]. The second blood supply is the choroidal circulation. The choroidal circulation is a dense network of capillaries located just posterior to the retinal pigment epithelium (RPE) cell layer and is responsible for supplying the outer retina (RPE and photoreceptors) with oxygen. Due to the high metabolic demand of the photoreceptors the choroid receives the majority (65–85%) of the blood that is supplied to the retina [1].

Studies in cats have used oxygen sensitive microelectrodes to measure oxygen tension (PO2 ) in the various layers of the healthy retina. These studies have shown that oxygen levels are highest (≈60 mmHg) in the rod outer segment layer due to their close proximity to the oxygen saturated choroid (**Figure 1**) [2]. Oxygen tension drops to nearly 0 mmHg in the outer nuclear layer, indicating that the oxygen that is perfused from the choroidal circulation is consumed almost entirely by the photoreceptors during visual phototransduction [2]. Moving inward, PO2 climbs gradually in the inner retina due to the inner retinal circulation, with two small spikes in PO2 occurring in the deep (≈20 mmHg) and superficial (≈25 mmHg) retinal capillary plexuses [2]. Therefore, any vascular changes, especially in the inner retinal circulation can lead to tissue hypoxia since the choroidal circulation cannot adequately supply oxygen to the inner retina. Because of this, perturbations in oxygen supply play a significant role in many of the most common vision threating diseases including age-related macular degeneration (AMD) [3–6], glaucoma [7, 8], retinopathy of prematurity [9–11], and diabetic retinopathy (DR) [2, 12–15].

## **1.2. Hypoxia in diabetic retinopathy**

Hypoxia has been implicated as a potential key contributor to the pathogenesis of many retinal diseases, including diabetic retinopathy (DR). The cellular hypoxia response is transcriptionally regulated by hypoxia inducible factors (HIFs) [16, 17], heterodimeric complexes comprising oxygen-sensing HIF1/2/3α subunits and HIFβ. The HIF alpha subunits share common features, although HIF3α has a distinct structure and is found in multiple variants which exert different transcriptional outcomes [18, 19]. Under normoxic conditions, proline residues in the oxygen-dependent degradation domain of HIFα are modified by oxygendependent prolyl hydroxylases (PHD) [20], creating a binding site for the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex [21, 22]. HIFα bound by VHL is targeted for proteasomal destruction [23, 24], thus preventing transcriptional activity. However, during hypoxia HIFα proline hydroxylation is abrogated, stabilizing the protein. Transcriptional activity of HIF1α and HIF2α is also promoted during hypoxia, as hydroxylation of a key asparagine residue located in the transactivation domain is prevented, promoting interaction between HIF and the p300/ CBP transcriptional co-activator complex [25]. HIF3α, which lacks the key asparagine residue, is not subject to this regulatory mechanism [18]. The HIFα-HIFβ complex can activate transcription of genes with promoters featuring hypoxia response elements (HRE) including VEGF and erythropoietin (EPO).

Although regulatory mechanisms are similar between HIF1α and HIF2α, expression of the proteins has been suggested to be confined to distinct cellular populations in the ischemic inner retina [26]. Expression of both HIF1α and HIF2α is temporally correlated with VEGF expression during retina ischemia [26, 27], and HIF2α haploinsufficiency has been shown to reduce pro-angiogenic factor expression and neovascularization in the oxygen-induced retinopathy (OIR) model [28]. Interestingly, PHD-dependent HIF1α degradation is also regulated by citric acid cycle intermediates such as succinate [29], which accumulate during hypoxia as oxygen tension is insufficient to support oxidative phosphorylation, leading to feedback inhibition of citric acid cycle enzymes [30]. Succinate inhibits PHD activity, further stabilizing HIF1α when cellular oxidative metabolism is compromised [29]. Succinate is also thought to have an addition role in the hypoxic retina, binding and signaling through the G protein coupled receptor 91 (GPR91) [31]. GPR91 regulates VEGF production in retinal ganglion cells via mitogen-activated protein kinase and prostaglandin signaling [32, 33] which contributes to the neovascular response following hypoxia [34].

figure has been reproduced with permission from original article by Linsenmeier et al. [2].

**Figure 1.** PO2 gradient across the retinal layers in a healthy (60 W2) and diabetic (all other tracings) cat. The tracing of the healthy cat shows normal oxygen perfusion in the choriocapillaris and inner retina. The diabetic cats show evidence of decreased oxygen perfusion in the inner retina and inadequate compensation by the choriocapillaris. Disclaimer: This

Imaging of Hypoxia in Retinal Vascular Disease http://dx.doi.org/10.5772/intechopen.72252 49

Some of the hallmarks of DR progression which include the formation of acellular capillaries, capillary occlusion and associated nonperfusion could lead to this cellular hypoxia response

demands, the retina requires two distinct blood supplies, the inner retinal circulation and choroidal circulation. The inner retinal circulation stems from the central retinal artery which enters the retina near the optic disc. From there, it branches to form the deep and superficial retinal capillary plexuses. In a healthy individual, these blood vessels are found only in the peripheral retina and do not enter into the avascular fovea. The central retinal artery is responsible for supplying the inner retina with oxygen and nutrients and receives about 20–30% of the blood flow to the retina [1]. The second blood supply is the choroidal circulation. The choroidal circulation is a dense network of capillaries located just posterior to the retinal pigment epithelium (RPE) cell layer and is responsible for supplying the outer retina (RPE and photoreceptors) with oxygen. Due to the high metabolic demand of the photoreceptors the choroid

receives the majority (65–85%) of the blood that is supplied to the retina [1].

**1.2. Hypoxia in diabetic retinopathy**

48 Early Events in Diabetic Retinopathy and Intervention Strategies

including VEGF and erythropoietin (EPO).

Studies in cats have used oxygen sensitive microelectrodes to measure oxygen tension (PO2

entirely by the photoreceptors during visual phototransduction [2]. Moving inward, PO2

the various layers of the healthy retina. These studies have shown that oxygen levels are highest (≈60 mmHg) in the rod outer segment layer due to their close proximity to the oxygen saturated choroid (**Figure 1**) [2]. Oxygen tension drops to nearly 0 mmHg in the outer nuclear layer, indicating that the oxygen that is perfused from the choroidal circulation is consumed almost

gradually in the inner retina due to the inner retinal circulation, with two small spikes in PO2 occurring in the deep (≈20 mmHg) and superficial (≈25 mmHg) retinal capillary plexuses [2]. Therefore, any vascular changes, especially in the inner retinal circulation can lead to tissue hypoxia since the choroidal circulation cannot adequately supply oxygen to the inner retina. Because of this, perturbations in oxygen supply play a significant role in many of the most common vision threating diseases including age-related macular degeneration (AMD) [3–6], glaucoma [7, 8], retinopathy of prematurity [9–11], and diabetic retinopathy (DR) [2, 12–15].

Hypoxia has been implicated as a potential key contributor to the pathogenesis of many retinal diseases, including diabetic retinopathy (DR). The cellular hypoxia response is transcriptionally regulated by hypoxia inducible factors (HIFs) [16, 17], heterodimeric complexes comprising oxygen-sensing HIF1/2/3α subunits and HIFβ. The HIF alpha subunits share common features, although HIF3α has a distinct structure and is found in multiple variants which exert different transcriptional outcomes [18, 19]. Under normoxic conditions, proline residues in the oxygen-dependent degradation domain of HIFα are modified by oxygendependent prolyl hydroxylases (PHD) [20], creating a binding site for the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex [21, 22]. HIFα bound by VHL is targeted for proteasomal destruction [23, 24], thus preventing transcriptional activity. However, during hypoxia HIFα proline hydroxylation is abrogated, stabilizing the protein. Transcriptional activity of HIF1α and HIF2α is also promoted during hypoxia, as hydroxylation of a key asparagine residue located in the transactivation domain is prevented, promoting interaction between HIF and the p300/ CBP transcriptional co-activator complex [25]. HIF3α, which lacks the key asparagine residue, is not subject to this regulatory mechanism [18]. The HIFα-HIFβ complex can activate transcription of genes with promoters featuring hypoxia response elements (HRE)

) in

climbs

**Figure 1.** PO2 gradient across the retinal layers in a healthy (60 W2) and diabetic (all other tracings) cat. The tracing of the healthy cat shows normal oxygen perfusion in the choriocapillaris and inner retina. The diabetic cats show evidence of decreased oxygen perfusion in the inner retina and inadequate compensation by the choriocapillaris. Disclaimer: This figure has been reproduced with permission from original article by Linsenmeier et al. [2].

Although regulatory mechanisms are similar between HIF1α and HIF2α, expression of the proteins has been suggested to be confined to distinct cellular populations in the ischemic inner retina [26]. Expression of both HIF1α and HIF2α is temporally correlated with VEGF expression during retina ischemia [26, 27], and HIF2α haploinsufficiency has been shown to reduce pro-angiogenic factor expression and neovascularization in the oxygen-induced retinopathy (OIR) model [28]. Interestingly, PHD-dependent HIF1α degradation is also regulated by citric acid cycle intermediates such as succinate [29], which accumulate during hypoxia as oxygen tension is insufficient to support oxidative phosphorylation, leading to feedback inhibition of citric acid cycle enzymes [30]. Succinate inhibits PHD activity, further stabilizing HIF1α when cellular oxidative metabolism is compromised [29]. Succinate is also thought to have an addition role in the hypoxic retina, binding and signaling through the G protein coupled receptor 91 (GPR91) [31]. GPR91 regulates VEGF production in retinal ganglion cells via mitogen-activated protein kinase and prostaglandin signaling [32, 33] which contributes to the neovascular response following hypoxia [34].

Some of the hallmarks of DR progression which include the formation of acellular capillaries, capillary occlusion and associated nonperfusion could lead to this cellular hypoxia response 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 et al. used oxygen-sensitive microelectrodes to directly measure PO2 in the retinas of long term diabetic and non-diabetic cats (> 6 years) and showed that mean PO2 was significantly lower 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.

such as DR originate largely from capillary occlusion and local changes in the retinal vasculature. These occlusions are likely to create small areas of regional hypoxia rather than an entirely hypoxic retinal tissue. Since oxygen sensitive microelectrodes provide point measurements that depend on the placement of the electrode this method will likely miss areas of focal hypoxia that are surrounded by large areas of normoxic tissue unless multiple measurements are made. Other methods such as MRI have been used to provide insight into oxygen distribution within the retina and are less invasive than oxygen-sensitive microelectrodes. The advantages of MRI are that it is minimally invasive, offers a large field of view, and has no depth limitation. These MRI techniques are often based on the blood oxygenation level-dependent (BOLD) contrast that was first described by Ogawa et al. which rely on natural differences in MR signal between deoxygenated hemoglobin versus oxygenated hemoglobin [61–63], but can also utilize exogenous contrast agents for increased sensitivity as seen in Dynamic Contrast-Enhanced MRI (DCE-MRI) [64]. The use of MRI to detect changes in oxygen levels was first used in the brain but has since been adapted to visualize oxygen fluctuations in the diabetic retina. Berkowitz et al. have used MRI to show increases in blood retinal barrier permeability in rats after 8 months of diabetes [64] and changes in retinal oxygenation in galactosemia-induced diabetic-like retinopathy [43]. The primary limitation of MRI is that the information is typically displayed as either a cross section of the eye or single slice heat maps which are then pieced together to give an overview of the retina [43, 64–67]. This severely limits the techniques ability to provide the adequate resolution required to identify small regions of focal hypoxia in the diabetic retina. Laser Doppler is another method that has been established to determine blood flow within

the retinal vasculature but does not provide information on oxygen PO2

ability to detect areas of focal hypoxia in the diseased retina.

**2.2. Dual wavelength retinal oximetry**

oxygen saturation (SO2

independent arteriolar SO2

oxygenated (HbO<sup>2</sup>

have emerged as potential diagnostic tools for early detection of DR.

vasculature or tissue [68–70]. More recently however, a number of minimally invasive techniques have been established and adapted to measure oxygen tension optically and identify areas of regional hypoxia in the retina. These techniques take full advantage of the unique anatomy of the eye, which unlike other organs is readily accessible and easy to image due to the naturally transparent front of the eye. Methods such as dual wavelength and full spectral retinal oximetry, and also phosphorescence lifetime imaging all provide information on oxygen levels in the retinal vasculature. Other techniques such as hypoxia sensitive fluorescent probes can help to image hypoxic regions within the retinal tissue itself. These techniques are prime candidates for use in a clinical setting due to their minimally invasive nature and their

This review will examine the advantages and disadvantages of the imaging techniques that

Retinal oximetry is a non-invasive technique used to measure the percent of hemoglobin

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

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

) in the retinal vasculature. Oximetry is based on the principle that

measurement for external calibration [71]. Since this initial work,

) and deoxygenated hemoglobin (Hb) have a different light absorption

within the retinal

Imaging of Hypoxia in Retinal Vascular Disease http://dx.doi.org/10.5772/intechopen.72252 51

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