We analyzed the study cohort data dividing the macula into quadrants. We used only quadrants with ≥3 segments of arteries or veins for the analysis. When comparing the average arterial velocity in each quadrant, we did not find a significant difference either in the healthy or DM group (P = 0.17 and 0.45 for healthy group and DM group, respectively, analysis of variance). Similarly, no difference was detected in the quadrant analysis in veins (P = 0.35 and 0.27 for healthy group and DM group, respectively, analysis of variance). Interestingly, when we analyze the data divided into halves, including this time any hemiretina with ≥3 segments of arteries or veins, we found higher arterial velocity in the nasal hemiretina compared with the temporal region in the healthy group (4.38 vs. 3.88 mm/second, P = 0.007). A similar difference was evident in the DM group (5.07 vs. 3.94 mm/second for nasal and temporal sides, respectively, P = 0.003). Venous velocity was also higher nasally than temporally in healthy subjects (3.07 vs. 2.80 mm/second, P = 0.01), but such differences were not observed in veins of DM patients. Dividing the data attitudinally to superior and inferior halves did not show any differences in both groups. Regional comparison between healthy subjects and DM patients according to vertical and horizontal halves showed significantly higher velocities in veins in all areas and in arteries only in nasal and inferior hemiretina (gender and age-adjusted model).
To examine whether the velocity in macular vessels is different from that in the periphery, we compared the velocity of similarly sized vessels in the periphery and the macula. Our sample size (eight experiments in two healthy volunteers) did not allow statistical analysis, although we saw a trend for higher arterial velocity in the macula. We calculated vessel density (the total length of visible vessels in pixels divided by the total number of pixels in the images) and found a marginally lower density in the diabetic patients compared with healthy subjects (P = 0.05).
Correlation Between Blood Flow Velocity to Physiologic and Pathologic Parameters
In the DM group, velocities in neither arteries nor veins correlated significantly with BP, while in healthy subjects, we found a significant correlation between average arterial blood flow velocity and BP (systolic BP: r = 0.3, P = 0.04, and diastolic BP: r = 0.4, P = 0.009). Such a correlation was apparent in all arterial categories (small: systolic BP: r = 0.3, P = 0.04, and diastolic BP: r = 0.5, P < 0.001; large: systolic BP: r = 0.4, P = 0.02, and diastolic BP: r = 0.3, P = 0.05). Venous velocity correlated significantly only to systolic BP in the large veins category (r = 0.3, P = 0.02).
The average heart rate did not correlate with average velocity of either the healthy or the DM group. We assessed the relationship between retinal blood flow velocity and heart rate in individual participants by attempting to correlate the heart rate recorded by the instrument in parallel with each velocity measurement. For each participant, we obtained a series of three separate paired measurements of heart rate and flow velocity. Each value was normalized by the corresponding subject's average. We found a positive correlation between the heart rate and both arterial and venous velocities in the healthy group (r = 0.4, P < 0.0001, for both arteries and veins; Figure 3A). In the DM patients, a small correlation exists only with the arterial velocity and not with the venous velocity (r = 0.4, P = 0.0008, for arteries, and r = 0.06, P = 0.6, for veins; Figure 3B).
In the DM group, we did not detect a significant correlation between velocity values of either arteries or veins and the duration of diabetes. Neither did the glucose and HbA1C levels and the body mass index correlate with velocity values in this group.
The primary result of this study is finding an increased retinal blood flow velocity in secondary–tertiary arterial and venous branches in patients with adult-onset DM and apparently normal retina compared with healthy subjects. Previous studies presented controversial results regarding the existence and nature of hemodynamic changes in patients with preretinopathy DM using different measurement instruments and modalities.13 These instruments differ from the RFI not only in the method of measuring the retinal blood flow velocity but also in the location and type of vessels studied. Intravessel measurement location also varies; some techniques measure centerline velocity (laser Doppler velocimetry, RFI, and blue-field simulation technique), while video fluorescein angiography measures flow of dye in the plasma that tends to flow in the periphery of the vessels.13 Scanning laser Doppler flowmetry (Heidelberg retina flowmeter) measures flow and velocity in capillaries and has found an increase in velocity and flow in the papillomacular region in patients with both Type I and Type II diabetes14,15 similar to our findings. Blue-light entoptic phenomenon enables measurement of retinal blood flow velocity in vessels in a similar location to the RFI but is tracing leukocyte movement and not erythrocytes. In one study, measurements in preretinopathy Type I diabetic patients showed, like in the current study, higher velocity in macular vessels16; however, another study did not find differences from healthy subjects.17 Measuring velocity in major retinal vessels, much larger in caliber than the RFI, with bidirectional laser Doppler velocimetry found an increase in the total retinal blood flow in veins of Type I diabetic patients18 but no change in velocity or flow in arteries in both Type I and Type II diabetes.19,20 Increased blood flow velocity has also been detected by shorter fluorescein mean circulation time in preretinopathy diabetic patients,21 although other studies did not detect such a change.22 In insulin-dependent DM, changes in the opposite direction were detected with this technique.23 It may be that the diabetic process have a different effect in central vessels compared with the peripheral retina or that the cause of early vascular disease in Type 2 diabetes is different than that in Type 1 diabetes.
In principle, the increase in blood velocity reported here might be secondary to immediate hyperglycemic state or because of long-standing retinal damage. About the first possibility, previous studies demonstrated increased retinal blood flow velocity during acute elevation in blood glucose (to 300 mg/dL) in diabetic patients without retinopathy.23,24 In contrast, others have shown negative correlation of actual glucose levels with blood flow.14 In our study, we did not detect a correlation between the velocity and glucose levels, probably because of comparatively well-controlled glucose levels (average, 137 ± 48.3 mg/dL). The patients' blood glucose levels were maintained comparatively low, according to modern guidelines established since the Diabetes Control and Complications Trial25,26 showed that intensive therapy reduces the risk of developing retinopathy. When we directly compared the velocity between patients with glucose level <130 mg/dL and those above that level (6 eyes of 4 patients), there was no significant difference in neither the arterial nor the venous velocity. This seems to rule out the possibility that increased blood glucose level as the sole or primary reason for the higher velocities we found.
Considering other causes, the increased velocity found in the DM group might reflect counteracted perfusion abnormalities in diabetic patient retina, stimulated, for example, by changes in blood rheologic properties or increased vascular resistance. In diabetic patients, there is increased aggregation and reduced deformability of erythrocytes, with increased plasma viscosity,11,12 translating to increased capillary resistance. Vascular resistance can result also from multiple molecular changes associated with long-term hyperglycemia and endothelial dysfunction. Many of these pathways are interrelated and may be simultaneously activated in retinal cells.27 Some known vasoconstrictor effectors are related to diabetic changes, like increased expression of endothelin-128 and overactivation of protein kinase C.13 Other vasodilatory mechanisms were identified as well, like endothelin-1 resistance, inhibition of calcium influx channel in smooth muscle cells, tissue hypoxia,29 and increased activity of nitric oxide synthase.30 In addition, in diabetes, there is increased leukocyte adhesion to endothelium, which is caused by increased expression of adhesion molecules31 and is associated with endothelial dysfunction.32 Indeed, in vivo studies found elevated levels of markers of endothelial dysfunction in patients with DR (soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1).33 However, studies mimicking retinal capillary obstruction by leukocytes did not detect an effect on retinal blood flow.32 The most physiologically plausible scenario consistent with the findings reported here is that arteries widen in response to impaired capillary perfusion, while venous diameter remains relatively constant. An increase in the arterial:venous diameter ratio is implied by our finding of a greater relative increase in venous velocity (31%) compared with arterial velocity (15%). Excluding an increase in BP, this also implies increased flow volume. Either loss of feedback linearity or capillary resistance inhomogeneity could produce this overcompensation. These changes could join a vicious cycle, according to the hemodynamic hypothesis34,35 that increased blood flow in diabetic patients induces further endothelial damage because of increased shear stress.36 The decreased vessel density in early diabetes that was found here was reported previously.37
In both groups, a higher velocity was found in nasal arteries compared with arteries in the temporal half of the macula. This can be a result of more proximal location manifesting in higher pressure and velocity. The same phenomenon was found in the veins of the healthy group but not in the diabetic group, which probably reflects the higher intersubject variability in the diabetic group related to different stages of the disease. Regional comparison between healthy and diabetic patients was significant in any hemiretina analyzed. In arteries, the regional difference between the groups was observed in the nasal and not in the temporal hemiretina. The nasal hemiretina is the faster half in both groups, which can highlight differences. The intergroup differences were also apparent in the inferior but not in the superior half, which may be related to the small region of interest when imaging with 20°, which can cause asymmetrical number of segments that increase the data variability when viewing small sections. Regional differences in velocity, as shown here, might contribute to the contradictory results obtained by different instruments. It may be that physiologic and pathologic processes affect these central vessels in a different way than the more peripheral retina.
A second independent finding of this study is that the retinal blood flow velocity values in the diabetic group were not correlated to BP, whereas in the healthy population, most values were. Although we found a reduced correlation in the diabetic group compared with the healthy group, this does not imply that a fundamental dependency is lost. One possibility is that the dependency relationship itself changes as diabetes develops, so that statistical significance is obscured by uncontrolled factors between patients, such as the progress of the disease. In addition, the correlation of blood flow velocity to heart rate was evident in the healthy group, but only arterial velocity was correlated to heart rate in the DM group. The correlation of velocity with heart rate was calculated for each subject comparing three sets of images. The primary confounding factor that remains is blood vessel diameter changes that are not directly measured. If blood flow volume increases, the velocity in veins, which are relatively static, should be more affected than that in arteries. This also explains how the correlation between venous velocity and heart rate may be lost in the diabetic group, while arterial velocity remains correlated to heart rate. The range of blood velocity variations may be larger or at least more irregular in diabetic patients, causing loss of correlation between venous velocity and heart rate in this group.
There was a higher proportion of subjects with systemic hypertension in the DM group. Nevertheless, when controlling for the effect of differences in patients' characteristics by the statistical model, the velocity comparison was consistent, showing significantly higher velocities in diabetic patients compared with control subjects. Diabetic patients, even with coexisting hypertension, were found to have similar caliber19,38 or even wider39 vessels compared with healthy subjects. Despite the higher rate of hypertensive patients, the actual diastolic BP was lower in the DM group; therefore, the same correlation observed between BP and blood flow velocity in healthy subjects could not explain the velocity rise found in the diabetic group.
In this study, we found increased velocity in preretinopathy patients compared with healthy subjects, while we previously found decreased velocity in nonproliferative diabetic retinopathy patients.10 Thus, the patient to healthy blood flow velocity relationship reverses during the development of morphologic alterations in the retina, as arteries reach the end of their compensating range, or capillary resistance assumes dominance in determining flow volume. In longitudinal studies,40,41 decreasing blood flow velocity over time was found in some but not in all diabetic patients. Extending this cross-sectional study to follow this cohort of patients over time with periodic noninvasive evaluations is warranted to study how such retinal blood flow velocity changes are related to the rate of progression and risk of blind-threatening consequences. Additionally, the RFI gives information on velocity only and not on flow volume, which is a weakness of the current study. Improvements in resolution and algorithm are under way and should allow getting volume information.
The ability to measure blood flow velocity simultaneously in a large number of vessels allows us to compare simultaneous changes in the arterial and venous compartments. Another unique characteristic of this method is the ability to measure velocity from secondary and tertiary branches of the retinal vasculature. The measurements are noninvasive, and therefore, frequent monitoring can be performed to detect transition to DR. The instrument used in the study offers measurements of the speed of blood cells moving in the smaller arterioles and venules of the retinal vasculature. It does not, however, measure the diameters of these vessels, so that the actual blood flows are not known. Nevertheless, there is clinical importance of finding a parameter with abnormal values in patients, especially in patients with no apparent retinal pathology.
To conclude, here we found an increased flow velocity through arteries and veins in the diabetic retina that otherwise appears normal. The question of whether these hemodynamic changes are an early marker of pathology in the vascular function of the eye or even contributing factors for microangiopathy warrants further explorations. Identifying such changes, and administrating modifying factors like meticulous glycemic control, might reverse these changes and prevent or defer irreversible sight-threatening changes to occur.
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Keywords:© The Ophthalmic Communications Society, Inc.
retinal blood flow velocity; diabetes mellitus; diabetic retinopathy; imaging.