**2.2. Autoregulation of retinal vessel diameter**

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

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

#### **2.3. Retinal blood flow**

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 pathological alterations that occur in the diabetic retina.

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 stages of NPDR, research has provided evidence that retinal blood flow increases above normal levels [28–30], which may arise from the increased demand caused by tissue hypoxia due to capillary basement membrane thickening and capillary occlusion [29, 32]. In proliferative disease, retinal blood flow is decreased again as measured with different techniques: Blair et al. used the dye dilution technique to measure the mean circulation time (MCT) calculated as the difference between the mean venous and arterial retinal passage times, which turned out to be statistically significantly longer in the eyes with proliferative DR (PDR) than in healthy eyes or eyes with NPDR [33]. Laser Doppler flowmetry (LDF), which measures blood flow at the optic nerve head (ONH), and color Doppler imaging, also showed a greater reduction in total retinal blood flow in patients with PDR than in patients with NPDR or healthy individuals [34, 35]. Recently, several groups have demonstrated the potential of Doppler OCT for assessing retinal blood flow in the diabetic eye. Doppler OCT can also detect volumetric blood flow and provide information about the structural anatomy. As shown with the techniques mentioned above, eyes with PDR had statistically significantly decreased retinal blood flow compared with normal eyes [36], especially those that had been treated with panretinal photocoagulation [28, 37, 38]. However, acute elevations in blood glucose can still trigger an increase in blood flow [26]. This finding suggests that the chronic hyperglycemic state in diabetes mellitus is associated with a reduction in retinal blood flow, but the retina still is able to respond to increased metabolic rates associated with acutely raised blood glucose by increasing retinal blood flow.
