**2.4. The capillary network**

The population-based Multi-Ethnic Study of Atherosclerosis (MESA) study showed that arteriolar calibers are dilated in patients with diabetes [13], whereas other researchers claim that

The discrepancy between results of different studies may be due to differences between the study cohorts in demographic (e.g., distribution in age) and metabolic traits including blood glucose levels, duration of diabetes, and cardiovascular risk factors (such as hypertension/ hyperlipidemia) as well as differences in sample size, follow-up period, and the methods applied. Before retinal vascular caliber assessment can be used as a biomarker in clinical prac-

Besides a "static" measurement of retinal vessel diameter, "dynamic" changes in the diabetic retinal vasculature can be assessed too. The potential for an efficient diameter change in order to adjust blood flow according to changes in arterial blood pressure (pressure autoregulation) and retinal metabolism (metabolic autoregulation) is reduced in the early stages of DR [17]. Vasoactive molecules activate pericytes and smooth muscle cells to regulate the capillary diameter [18]. A dysfunction in pressure autoregulation of retinal arterioles implies that changes in the arterial blood pressure are directly transmitted to the retinal microcirculation [19]. The fact that pressure autoregulation decreases with increasing severity of DR highlights

tice, age-, sex-, body size-, and blood pressure-specific normative data are required.

the destructive effect of arterial hypertension on the retinal microcirculation [17, 20].

dysfunction in the retinal microcirculation that clinicians could follow non-invasively.

Besides measurement of retinal vessel caliber, numerous other techniques such as laser Doppler velocimetry, laser Doppler flowmetry (LDF), fluorescein angiography (FA), color Doppler, and Doppler optical coherence tomography (OCT) imaging have been proposed for quantifying retinal blood flow in patients with diabetes [26–30]. Contradicting results concerning retinal blood flow have been published. This may reflect the complexities of the

Most studies suggest that in patients without or with mild non-proliferative DR (NPDR), retinal blood flow is reduced [26, 27]. Evidence from animal studies in streptozotocin-treated rats also suggests decreased retinal blood flow in the very early stages of DR [31]. In more severe

Luminance flicker stimulation is an example to test the capability of retinal vessels to adapt perfusion to changes in retinal metabolism. Exposure to flickering light stimulates retinal neuronal cells to release local vasodilating metabolites, most importantly nitric oxide [21], which consequently leads to retinal vasodilatation. This results in an increase in retinal blood flow in healthy individuals [22]. Several studies have reported that the flicker light-induced vasodilation is reduced in patients with diabetes [17, 23, 24] and even in patients with prediabetes [25], being equivalent in magnitude to patients with manifest diabetes. Thus, monitoring retinal vascular reactivity may provide an early marker of autoregulation and endothelial

arteries tend to constrict in diabetes [14, 16].

12 Early Events in Diabetic Retinopathy and Intervention Strategies

**2.2. Autoregulation of retinal vessel diameter**

**2.3. Retinal blood flow**

pathological alterations that occur in the diabetic retina.

The structure of the retinal capillary network is unique. It has to feed one of the highest metabolically active tissues while limiting the extent of the vascular beds to a minimum in order to prevent optical interference to the photoreceptors [39]. The inner retina is perfused by four interconnected capillary plexi that include the peripapillary capillary plexus which is found in the retinal nerve fiber layer (RNFL) adjacent to the optic nerve head (ONH), the superficial capillary plexus in the ganglion cell layer (GCL), as well as an intermediate (ICP) and a deep capillary plexus (DCP), which are located at the two borders of the inner nuclear layer (INL) [40]. Currently most segmentation algorithms display the ICP and DCP as one capillary layer. The three vascular layers unite in the center of the macula to form a terminal capillary ring surrounding the foveal avascular zone (FAZ). The outer retina and the photoreceptors are dependent on blood supplied by diffusion from the choriocapillaris. The early changes in capillary architecture and perfusion in patients with diabetes have not yet been definitely established, as assessing the human retinal microvasculature *in vivo* is very difficult due to its small size and low optical contrast.

FA, introduced in 1961, has been the gold standard imaging technique for assessing the retinal capillary network [41]. The value of this imaging modality is undeniable, but so are its limitations. First, dye leakage and the superimposition of capillary beds from the different retinal layers into a single two-dimensional image hinder a proper differentiation between the superficial and deep capillary plexi [42]. Furthermore, FA is a time-consuming and invasive technique which does not render it optimal for DR screening or frequent longitudinal evaluation. In addition, intravenous fluorescein dye injections can occasionally cause adverse side effects, nausea/vomiting, urticaria and rarely, but critically, anaphylactic reactions in healthy people [43].

OCTA techniques have also been used to study the development and progression, as well as the treatment response of clinically visible signs of DR. Microaneurysms can be identified in OCTA, but with a significantly lower sensitivity compared with conventional FA [52]. Nevertheless, OCTA provides additional information about their originating capillary plexus. Significantly, more microaneurysms were found in the intermediate/deep capillary plexus than in the superficial one [54, 57]. Additionally, it has been proposed that OCTA is more useful to evaluate clinically active microaneurysms, which are a major cause of diabetic macular edema (DME) [58]. Intraretinal microvascular abnormalities (IRMA), on the other

Potential Imaging Biomarkers in the Development and Progression of Diabetic Retinopathy

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

15

The significance of the individual evaluation of the integrity of the deep capillary plexus, impossible with FA alone, is further supported as macular outer retinal changes on spectraldomain OCT (SD-OCT) correspond to areas of capillary non-perfusion at the level of the DCP in patients with DR. The spectrum of outer retinal alterations encompassed different degrees of thinning of the outer nuclear layer (ONL), disruption of the photoreceptor lines, and focal

Diagnosis of retinal neovascularization on FA depends on identifying characteristic pathologic vessels with profuse leakage in late angiographic phases. With OCTA, spots of neovascularization that were not identified with FA were visualized as an abnormal flow signal above the inner limiting membrane, which may further help in the identification of patients

Certainly, there are limitations to the OCTA systems in their current state that have to be acknowledged including the incidence of motion artifacts and the relatively small field of view [41], but these can be improved with future development efforts [60]. In summary, OCTA enables the visualization of early microvascular perfusion abnormalities representing imminent DR development and simultaneous monitoring of the treatment response of pathognomonic lesions of DR. It could therefore provide clinicians and scientists in clinical trials with valuable and reliable biomarkers, using an imaging technology that is safely toler-

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

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

hand, were well detected by both FA and OCTA [54].

photoreceptor layer thinning [59].

requiring treatment [47, 55].

ated by patients.

**2.5. Retinal oxygen supply**

is a direct measure of retinal oxygen metabolism [18].

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 using swept source technology [42].

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 place of a quantitative assessment [48].

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

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 DR progressed [56].

OCTA techniques have also been used to study the development and progression, as well as the treatment response of clinically visible signs of DR. Microaneurysms can be identified in OCTA, but with a significantly lower sensitivity compared with conventional FA [52]. Nevertheless, OCTA provides additional information about their originating capillary plexus. Significantly, more microaneurysms were found in the intermediate/deep capillary plexus than in the superficial one [54, 57]. Additionally, it has been proposed that OCTA is more useful to evaluate clinically active microaneurysms, which are a major cause of diabetic macular edema (DME) [58]. Intraretinal microvascular abnormalities (IRMA), on the other hand, were well detected by both FA and OCTA [54].

The significance of the individual evaluation of the integrity of the deep capillary plexus, impossible with FA alone, is further supported as macular outer retinal changes on spectraldomain OCT (SD-OCT) correspond to areas of capillary non-perfusion at the level of the DCP in patients with DR. The spectrum of outer retinal alterations encompassed different degrees of thinning of the outer nuclear layer (ONL), disruption of the photoreceptor lines, and focal photoreceptor layer thinning [59].

Diagnosis of retinal neovascularization on FA depends on identifying characteristic pathologic vessels with profuse leakage in late angiographic phases. With OCTA, spots of neovascularization that were not identified with FA were visualized as an abnormal flow signal above the inner limiting membrane, which may further help in the identification of patients requiring treatment [47, 55].

Certainly, there are limitations to the OCTA systems in their current state that have to be acknowledged including the incidence of motion artifacts and the relatively small field of view [41], but these can be improved with future development efforts [60]. In summary, OCTA enables the visualization of early microvascular perfusion abnormalities representing imminent DR development and simultaneous monitoring of the treatment response of pathognomonic lesions of DR. It could therefore provide clinicians and scientists in clinical trials with valuable and reliable biomarkers, using an imaging technology that is safely tolerated by patients.
