**2.5. Retinal oxygen supply**

evaluation. In addition, intravenous fluorescein dye injections can occasionally cause adverse side effects, nausea/vomiting, urticaria and rarely, but critically, anaphylactic reactions in

Optical coherence tomography angiography (OCTA) is a further advance in retinal microvascular evaluation and may represent a significant breakthrough in ophthalmic imaging, especially in diabetes care. Intravenous injection of extrinsic fluorescent dye is no longer required with this technology, but the perfused capillary architecture is non-invasively visualized with erythrocyte motion as an intrinsic contrast. A recent study has demonstrated that shorter acquisition times and a higher number of motion artifact-free images can be achieved

Several features of early disruption of microvascular perfusion in the development and progression of DR have already been investigated and objectively quantified using OCTA. Diabetic macular ischemia, clinically defined as an enlargement and disruption of the foveal avascular zone (FAZ) and capillary dropout in adjacent parafoveal areas [44], is thought to have predictive potential for DR progression [45]. The considerable inter-subject variability in FAZ size even in healthy people and the large overlap in FAZ size between healthy individuals and patients with diabetes have to be considered though [46]. Hence, FAZ size alone was suggested to be a poor diagnostic variable [47], and qualitative FAZ assessment (e.g., with FAZ outline and regularity) may constitute a more reliable biomarker for the ischemic state of the macula in the diagnosis of DR, either complementary to or in

OCTA is also reproducible for the measurement of vessel density in healthy eyes and eyes with DR. Compared with a healthy control group, patients with diabetes but without DR were shown to feature reduced parafoveal and perifoveal vessel density, and intercapillary areas increase as DR progresses [47, 49, 50]. A more consistent and severe decrease in vessel density has been observed in the superficial capillary network than in the deep plexus in most studies [51, 52]. Accordingly, mean vessel density in the superficial retinal layer, being highly inversely correlated to best-corrected visual acuity (BCVA), has already been proposed to be the best marker for a reliable differentiation between healthy eyes and those with DR [53]. Similarly, the total avascular area in the central 5.5-mm-diameter area was shown to distinguish eyes with DR from control eyes with 100% sensitivity and specificity. It was, therefore, suggested that total avascular area may be an excellent biomarker in the diagnosis of DR [47].

Compared with FA, where the edges of non-perfused areas appear fuzzy or cannot be detected at all, OCT angiograms clearly delimit the border between sparse-capillary areas and dense-capillary areas in most cases [52, 54]. Choi et al. also found impairment of flow in the choriocapillaris at all stages of DR, supporting the concept that choriocapillaris alterations

OCTA color-coded perfusion density mapping enhances areas of low capillary perfusion density in the SCP, DCP and the choriocapillaris in patients with diabetes. Additional trend analysis has shown a statistically significant decrease in capillary perfusion density values as

healthy people [43].

using swept source technology [42].

14 Early Events in Diabetic Retinopathy and Intervention Strategies

place of a quantitative assessment [48].

may play a role in the pathogenesis of DR [55].

DR progressed [56].

Capillary non-perfusion and tissue ischemia are well-known hallmarks of diabetic retinopathy. While FA provides information about the anatomic state of retinal vessels, changes in retinal oxygenation reflect metabolic dysfunction. Oxygen saturation (SO2) in retinal vessels is a direct measure of retinal oxygen metabolism [18].

Using retinal oximetry, retinal SO2 can be measured non-invasively in major retinal arterioles and venules. The retinal oximeter records fundus images reflected from the retina at two different wavelengths, one being sensitive to oxygen (600 nm), and one being insensitive to oxygen (570 nm). An inverse linear relation between the optical density ratio measured at the two wavelengths and SO2 is assumed. Retinal oxygen saturation can be presented numerically and as a color saturation map [61]. Low variability as well as high reproducibility and repeatability have been shown for retinal oximetry measurements in healthy individuals and in diseased retinas [62–64]. Furthermore, there have already been a number of approaches to compile normative databases for retinal oximetry measurements in Caucasian [61] and multiethnic populations [65], to set a basis for comparability for future clinical trials. Age is the most important factor that should be accounted for in the interpretation of retinal oximetry measurements. Beside age and ethnicity, other demographic factors do not seem to influence retinal oximetry results markedly [61, 65, 66]. Additionally, no statistically significant difference in SO2 levels between patients with type 1 and type 2 diabetes could be observed [61].

patients respond better to the treatment than others. Interestingly, a recent study indicates that together with arterial blood pressure, SO2 in retinal arterioles may predict visual acuity and central retinal thickness (CRT) in patients with diabetic macular edema after anti-VEGF treatment [75]. Retinal laser treatment destroys retinal tissue and therefore reduces oxygen consumption in treated retinal areas, which in turn reduces hypoxia and the subsequent production of VEGF [76]. The effects of this treatment can be detected with retinal oximetry. A slight increase in SO2 in retinal venules and unchanged SO2 in retinal arterioles was measured immediately after treatment in patients with diabetic maculopathy and patients with PDR, resulting in reduced oxygen extraction. Three months after treatment, arteriolar and venular SO2 were both increased, but arteriovenous SO2 difference was unchanged compared with pretreatment levels [77]. A more recent study in patients with treatment-naive PDR suggested that pre-laser retinal SO2 was not able to predict immediate post-treatment activity of neovascularization, but post-treatment changes in SO2 were closely linked to disease activity 3 months after photocoagulation. Each 1% increase in retinal venular SO2 was independently associated with a 30% higher risk of increased PDR activity despite laser treatment. This implies that if photocoagulation is successfully performed, the amount of the hypoxic retinal tissue is decreased. In the adjacent vital retinal tissue, oxygen is extracted efficiently from retinal arteries, which lowers the venous SO2 and the arteriovenous SO2 levels [78]. Therefore, investigation of oxygen supply may be a potential non-invasive marker of angiogenic disease activity in the monitoring of the treatment response in DR. Prospective studies are under way

Potential Imaging Biomarkers in the Development and Progression of Diabetic Retinopathy

http://dx.doi.org/10.5772/intechopen.71747

17

to further validate retinal oximetry as a biomarker in DR.

**3. The identification of lesions in the retinal periphery**

Increasing evidence from research suggests that the first lesions in DR develop in the periphery of the retina and that these lesions are potentially associated with DR progression [79, 80]. The gold standard for determining the severity of DR is the extended modified Airlie House classification, which was first used in the Early Treatment Diabetic Retinopathy Study (ETDRS) in 1991 [81]. This rigorously standardized grading scale comprises 13 distinct levels, ranging from the absence of DR to the most severe manifestations of the disease localized in the central posterior 90° of the retina, representing approximately 30% of the entire retinal surface. The ETDRS grading scale is an established measure of disease activity and predictive of the risk of DR progression and visual loss over time [82]. However, due to imaging limitations, a systematic assessment of the retinal periphery was not feasible when the original ETDRS criteria were created. Therefore, the presence of pathologic features outside the 7-fields of ETDRS photography was not accounted for in this grading scale. With the advent of commercially available high-resolution ultrawide-field (UWF) scanning laser ophthalmoscopes, peripheral retinal lesions within and outside the area of the 7-standard ETDRS fields can now be evaluated [83]. Instead of 30° captured by a single ETDRS photo, these UWF imaging systems cover up to 200° in a single image, representing approximately 82% of the retinal area. Combining low-powered green (532 nm) and red (633 nm) laser light, a composite color image with a resolution of 14 μm can be acquired in just a quarter of a second. The

Oxygen saturation levels in retinal vessels seem to steadily increase with progressing severity of DR, even if it is not fully elucidated if both, arterioles and venules [67, 68], or solely venules are affected by this increase [69]. Compared with healthy individuals, the change in SO2 levels only becomes statistically significant at more advanced stages of severe NPDR or PDR. Some investigators support the concept that in earlier stages of DR, increased levels of SO2 are detected in retinal venules only, which stands for a decreasing oxygen extraction in these patients, whereas in patients with PDR, SO2 levels are also increased in retinal arterioles, resulting in unchanged levels of oxygen extraction [70].

The metabolic results reflected by retinal oximetry also seem to correlate with the extent of retinal ischemia measured in FA [67].

At first, the findings of increased oxygen saturation levels in patients with diabetes with or without DR seem to conflict with the traditional concept of DR being an ischemic disease. However, this observation can be explained by at least three mechanisms: (1) capillary nonperfusion and shunting (2) thickening of the basement membrane of capillary vessel walls, and (3) greater affinity of hemoglobin for oxygen [71]. Capillary non-perfusion in conjunction with the formation of shunt vessel is already known from histologic studies in the diabetic retina. In capillary shunting, while some vessels dilate, others constrict, leading to blood flow bypassing parts of the capillary network. Blood is then transported faster through these dilated preferential channels, resulting in a shortened arterio-venous passage time and therefore a reduced oxygen extraction time [72]. Further, with thickening of the capillary basement membranes, inevitably, oxygen diffusion from the blood to the retinal tissue is hindered as the transport distance increases [73]. All these mechanisms lead to a maldistribution of oxygen. Oxygen cannot be delivered to the retinal cells in these ischemic areas, which makes venular blood relatively hyperoxic and retinal tissue relatively hypoxic. As a compensatory response, oxygen demand will increase, and more blood will be directed to the tissue. Therefore, oxygenation in arterioles increases too [68].

Intraocular injections of substances inhibiting the production of vascular endothelial growth factor (VEGF), as well as laser treatment and vitrectomy are therapeutic for complications in advanced DR and all of them influence retinal oxygen metabolism.

The vitreous cavities of patients with PDR who have undergone vitrectomy have lower oxygen tension than those who do not have diabetes [74]. Anti-VEGF injections can reduce diabetic macular edema and retinal neovascularization leading to a gain in visual acuity in patients with diabetic maculopathy and/or PDR. The introduction of this treatment modality has considerably improved the visual rehabilitation for patients with DR, but still, some patients respond better to the treatment than others. Interestingly, a recent study indicates that together with arterial blood pressure, SO2 in retinal arterioles may predict visual acuity and central retinal thickness (CRT) in patients with diabetic macular edema after anti-VEGF treatment [75]. Retinal laser treatment destroys retinal tissue and therefore reduces oxygen consumption in treated retinal areas, which in turn reduces hypoxia and the subsequent production of VEGF [76]. The effects of this treatment can be detected with retinal oximetry. A slight increase in SO2 in retinal venules and unchanged SO2 in retinal arterioles was measured immediately after treatment in patients with diabetic maculopathy and patients with PDR, resulting in reduced oxygen extraction. Three months after treatment, arteriolar and venular SO2 were both increased, but arteriovenous SO2 difference was unchanged compared with pretreatment levels [77]. A more recent study in patients with treatment-naive PDR suggested that pre-laser retinal SO2 was not able to predict immediate post-treatment activity of neovascularization, but post-treatment changes in SO2 were closely linked to disease activity 3 months after photocoagulation. Each 1% increase in retinal venular SO2 was independently associated with a 30% higher risk of increased PDR activity despite laser treatment. This implies that if photocoagulation is successfully performed, the amount of the hypoxic retinal tissue is decreased. In the adjacent vital retinal tissue, oxygen is extracted efficiently from retinal arteries, which lowers the venous SO2 and the arteriovenous SO2 levels [78]. Therefore, investigation of oxygen supply may be a potential non-invasive marker of angiogenic disease activity in the monitoring of the treatment response in DR. Prospective studies are under way to further validate retinal oximetry as a biomarker in DR.
