A pilot study to assess the effect of a three-month vitamin supplementation containing L-methylfolate on systemic homocysteine plasma concentrations and retinal blood flow in patients with diabetes.

Purpose: The aim of the present study was to investigate the effect of a three-month dietary supplementation with a methylfolate formulation on homocysteine plasma concentrations and ocular blood flow parameters in patients with diabetes. Methods: Twenty-four patients with diabetes received a dietary supplement (Oculofolin, Aprofol AG, Switzerland) containing 900 µg L­methylfolate (levomefolate calcium or [6S]-5-methyltetrahydrofolic acid, calcium salt), methylcobalamin, and other ingredients for three consecutive months. The patients' plasma homocysteine concentration and retinal blood flow were assessed at baseline and after three months of folate intake. Retinal blood flow was measured using a custom-built dual-beam Doppler optical coherence tomography (OCT) system. In addition, flicker-induced retinal vasodilatation was assessed by means of a commercially available dynamic vessel analyzer (IMEDOS, Jena, Germany). Results: Supplementation was well tolerated by all patients. After three months, plasma homocysteine concentration significantly decreased from 14.2 ± 9.3 to 9.6 ± 6.6 µmol/L (p < 0.001). In addition, a tendency toward an increased total retinal blood flow from 36.8 ± 12.9 to 39.2 ± 10.8 µl/min was observed, but this effect did not reach the level of significance (p = 0.11). Supplementation had no effect on retinal vessel diameter or flicker-induced vasodilatation. Conclusions: The present data show that a three-month intake of a dietary supplement containing methylfolate can significantly reduce blood homocysteine levels in patients with diabetes. This is of importance because higher homocysteine plasma levels have been found to be associated with an increased risk of vascular associated systemic diseases and eye diseases. Whether systemic methylfolate supplementation affects retinal perfusion must be studied in a larger population.

Age-Related Decline of Retinal Oxygen Extraction in Healthy Subjects.

Purpose: To investigate the age-dependence of total retinal blood flow and total retinal oxygen extraction in healthy subjects and determine their possible correlations with structural optical coherence tomography (OCT) parameters. Methods: This observational cross-sectional study consisted of 68 healthy subjects (mean ± SD age, 45.6 ± 16.3 years; 47% female). Total retinal oxygen extraction was calculated based on measurement of total retinal blood flow using bi-directional Doppler OCT and measurement of oxygen saturation using spectroscopic reflectometry. Retinal nerve fiber layer thickness was measured using OCT, and the total number of retinal ganglion cells was estimated based on a previous published model. Correlation of these parameters with age was studied and the association between structural OCT parameters and hemodynamic vascular parameters was calculated. Results: Both structural and vascular parameters showed a significant decline with increasing age. The correlation coefficients were between r = -0.25 and r = -0.41. Furthermore, structural and vascular parameters were significantly correlated with each other. The strongest association was found between the level of total retinal oxygen extraction and the number of retinal ganglion cells (r = 0.75, P < 0.001). Conclusions: We showed that there was an age-related decline of retinal oxygen extraction. Levels of retinal oxygen extraction are correlated to retinal nerve fiber layer thickness and number of retinal ganglion cells. Our data partially explain the wide inter-individual variability in retinal blood flow values in healthy subjects. Longitudinal studies are required to study the time course of vascular and neuronal loss in humans.

Retinal oxygen extraction in individuals with type 1 diabetes with no or mild diabetic retinopathy.

AIMS/HYPOTHESIS: The aim of this study was to compare retinal oxygen extraction in individuals with diabetes with no or mild non-proliferative diabetic retinopathy and healthy age- and sex-matched volunteers. METHODS: A total of 24 participants with type 1 diabetes and 24 healthy age- and sex-matched volunteers were included in this cross-sectional study. Retinal oxygen extraction was measured by combining total retinal blood flow measurements using a custom-built bi-directional Doppler optical coherence tomography system with measurements of oxygen saturation using spectroscopic reflectometry. Based on previously published mathematical modelling, the oxygen content in retinal vessels and total retinal oxygen extraction were calculated. RESULTS: Total retinal blood flow was higher in diabetic participants (46.4 ± 7.4 µl/min) than in healthy volunteers (40.4 ± 5.3 µl/min, p = 0.002 between groups). Oxygen content in retinal arteries was comparable between the two groups, but oxygen content in retinal veins was higher in participants with diabetes (0.15 ± 0.02 ml O2/ml) compared with healthy control participants (0.13 ± 0.02 ml O2/ml, p < 0.001). As such, the arteriovenous oxygen difference and total retinal oxygen extraction were reduced in participants with diabetes compared with healthy volunteers (total retinal oxygen extraction 1.40 ± 0.44 vs 1.70 ± 0.47 µl O2/min, respectively, p = 0.03). CONCLUSIONS/INTERPRETATION: Our data indicate early retinal hypoxia in individuals with type 1 diabetes with no or mild diabetic retinopathy as compared with healthy control individuals. Further studies are required to fully understand the potential of the technique in risk stratification and treatment monitoring. TRIAL REGISTRATION: ClinicalTrials.gov NCT01843114.

Ultrahigh-resolution OCT imaging of the human cornea.

We present imaging of corneal pathologies using optical coherence tomography (OCT) with high resolution. To this end, an ultrahigh-resolution spectral domain OCT (UHR-OCT) system based on a broad bandwidth Ti:sapphire laser is employed. With a central wavelength of 800 nm, the imaging device allows to acquire OCT data at the central, paracentral and peripheral cornea as well as the limbal region with 1.2 µm x 20 µm (axial x lateral) resolution at a rate of 140 000 A-scans/s. Structures of the anterior segment of the eye, not accessible with commercial OCT systems, are visualized. These include corneal nerves, limbal palisades of Vogt as well as several corneal pathologies. Cases such as keratoconus and Fuchs's endothelial dystrophy as well as infectious changes caused by diseases like Acanthamoeba keratitis and scarring after herpetic keratitis are presented. We also demonstrate the applicability of our system to visualize epithelial erosion and intracorneal foreign body after corneal trauma as well as chemical burns. Finally, results after Descemet's membrane endothelial keratoplasty (DMEK) are imaged. These clinical cases show the potential of UHR-OCT to help in clinical decision-making and follow-up. Our results and experience indicate that UHR-OCT of the cornea is a promising technique for the use in clinical practice, but can also help to gain novel insight in the physiology and pathophysiology of the human cornea.

Effect of Diffuse Luminance Flicker Light Stimulation on Total Retinal Blood Flow Assessed With Dual-Beam Bidirectional Doppler OCT.

Purpose: We assess the increase in total retinal blood flow (TRBF) induced by flicker stimulation of the human retina in vivo and investigate the flicker induced hyperemia by means of a vascular flow model of the retinal circulation to study neurovascular coupling (NC). Methods: In six healthy subjects, TRBF was measured before and during stimulation with diffuse luminance flicker. Blood flow velocities in retinal vessels were measured via dual-beam bidirectional Doppler Fourier-domain optical coherence tomography (FD-OCT), retinal vessel diameters were assessed based on FD-OCT phase data. This allowed for the calculation of TRBF before and during visual stimulation. Additionally, a mathematical flow model for the retinal vasculature was adapted to study the implications of diameter variations on retinal perfusion. Measured and simulated perfusion was compared to draw conclusions on the diameter variations in different layers of the vascular tree. Results: The measured mean baseline flow was 36.4 ± 6.5 µl/min while the mean flow during flicker stimulation was 53.4% ± 8.3 µl/min. The individual increase in TRBF during flicker stimulation ranged between 34% and 66%. The average increase in TRBF over all measured subjects was 47.6% ± 12.6%. Conclusions: Dual-beam bidirectional Doppler FD-OCT allowed quantifying NC in the human retina in vivo and may be a promising method for monitoring alterations in NC caused by various pathologies. The comparison of the measured data with the results obtained in the simulated vasculature indicates that the vasodilation induced by NC is more pronounced in smaller vessels.

Ocular Blood Flow Measurements in Healthy White Subjects Using Laser Speckle Flowgraphy.

PURPOSE: To assess the feasibility and reliability of Laser Speckle Flowgraphy (LSFG) to measure ocular perfusion in a sample of healthy white subjects and to elucidate the age-dependence of the parameters obtained. METHODS: This cross-sectional study included 80 eyes of 80 healthy, non-smoking white subjects of Western European descent between 19 and 79 years of age. A commercial LSFG instrument was applied to measure ocular blood flow at the optic nerve head (ONH) three successive times before and after pharmacological pupil dilation. The mean blur rate (MBR), a measure of relative blood flow velocity, was obtained for different regions of the ONH. Eight parameters of ocular perfusion derived from the pulse-waveform analysis of MBR including blowout time (BOT) and falling rate (FR) were also recorded. RESULTS: Artifact-free LSFG images meeting the quality criteria for automated image analysis were obtainable in 93.8% without pupil dilation and in 98.8% with pharmacological pupil dilation. Measurements of MBR showed excellent repeatability with intraclass correlation coefficients ≥ 0.937 and were barely affected by pupil dilation. The majority of pulse-waveform derived variables exhibited equally high repeatability. MBR-related blood flow indices exhibited significant age dependence (p<0.001). FR (r = 0.747, p<0.001) and BOT (r = -0.714, p<0.001) most strongly correlated with age. CONCLUSIONS: LSFG represents a reliable method for the quantitative assessment of ocular blood flow in white subjects. Our data affirms that the LSFG-derived variables FR and BOT may be useful biomarkers for age-related changes in ocular perfusion.

Measurements of Retinal Perfusion Using Laser Speckle Flowgraphy and Doppler Optical Coherence Tomography.

PURPOSE: This study evaluated the validity of retinal perfusion measurements using laser speckle flowgraphy (LSFG) by means of in vitro experiments and direct comparison with dual-beam Doppler optical coherence tomography (D-OCT) in a healthy Caucasian population. METHODS: The flow velocity of scattering solution pumped through a glass capillary was measured at 17 different flow velocities (range, 0.5-47 mm/s) using LSFG. The flow within the glass capillary was produced by a computer-controlled infusion pump. In vivo, three consecutive LSFG scans were obtained in 20 eyes of 20 healthy Caucasian subjects before and after pharmacological pupil dilation. Relative flow volume (RFV), the primary output parameter of LSFG, was comparatively validated relative to absolute measurements of retinal blood flow and velocity as obtained from D-OCT. RESULTS: In the in vitro experiments, RFV was found to saturate at a level of approximately 700 arbitrary units (au) or 23.5 mm/s of actual velocity. In vivo, RFV was in significant agreement with absolute blood flow measurements as obtained from D-OCT in arteries (r = 0.69, P = 0.001) and veins (r = 0.74, P < 0.001). However, linear regression analysis revealed significant positive zero offset values for RFV of 223.4 and 282.7 au in arteries and veins, respectively. CONCLUSIONS: Measurements of RFV were successfully obtainable, reproducible, and not influenced by pharmacological pupil dilation. Nevertheless, our data revealed flaws in the LSFG method of measuring retinal perfusion in Caucasians. Adjustment to the technique is required to address apparent issues with RFV, especially saturation effects with higher arterial flow rates. The present dataset may provide a valuable tool to do so. (Clinicaltrials.gov number NCT02582411).

Estimating Retinal Blood Flow Velocities by Optical Coherence Tomography.

IMPORTANCE: While optical coherence tomography (OCT) angiography has been considered to evaluate retinal capillary blood flow instead of fluorescein angiography, the reflectance pattern of blood vessels on structural OCT might also provide retinal capillary flow data in the absence of fluorescein angiography. This potential has been insufficiently explored, despite promising data concerning a possible relationship between the reflectance pattern of blood vessels and their perfusion velocity in a laboratory setting. OBJECTIVE: To evaluate the potential of retinal blood flow velocity estimation by structural OCT. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional observational study conducted from June to November 2015 at a tertiary clinical referral center. Sixty arteries (the superior and inferior temporal arteries) from 30 eyes of 30 patients (17 female, 13 male) were included in the study. MAIN OUTCOMES AND MEASURES: Based on the intraluminal contrast patterns of retinal arteries on OCT, 3 independent graders categorized the blood flow velocities as low, medium, or high. These results and the results from a software-based intraluminal contrast analysis were compared with the retinal blood flow velocities measured by video fluorescein angiography. RESULTS: Among the 30 eyes of 30 patients (mean [SD] age, 72.6 [12.3] years; 17 female, 13 male), 15 were controls without retinal occlusion, 6 had a branch retinal artery occlusion, and 9 had a central retinal artery occlusion. When discriminating between low flow velocities and medium or high flow velocities, the graders' sensitivity ranged from 88.2% to 100% (grader 1: 88.2%; 95% CI, 63.6%-98.5%; grader 2: 88.2%; 95% CI, 63.6%-98.5%; and grader 3: 100%; 95% CI, 69.8%-100%) and their specificity ranged from 97.6% to 100% (grader 1: 100%; 95% CI, 87.7%-100%; grader 2: 97.6%; 95% CI, 87.4%-99.9%; and grader 3: 100%; 95% CI, 87.7%-100%). The κ coefficients of the comparison between the 3 graders and the angiography were 0.77 (95% CI, 0.60-0.93; P < .001), 0.64 (95% CI, 0.44-0.83; P < .001), and 0.87 (95% CI, 0.74-0.99; P < .001). In the computer-based assessment, the contrast reduction of the intraluminal pattern could be numerically expressed in a specific coefficient in the model (I2, describing the angular change of the backscattering intensity in the model), which presented nonoverlapping intervals between low flow velocities and medium or high flow velocities (mean [SD] I2, 0.3 [5.3], 20.4 [6.4], and 21.7 [4.0], respectively). CONCLUSIONS AND RELEVANCE: This study suggests that a low retinal blood flow velocity reflects in a visually distinct contrast reduction of the intraluminal pattern of retinal vessels on OCT. Larger studies are required to assess the clinical benefits.

Measurement of Retinal Vascular Caliber From Optical Coherence Tomography Phase Images.

PURPOSE: To compare retinal vessel calibers extracted from phase-sensitive optical coherence tomography (OCT) images with vessel calibers as obtained from the Retinal Vessel Analyzer (RVA). METHODS: Data from previously published studies in 13 healthy subjects breathing room air (n = 214 vessels) and 7 subjects breathing 100% oxygen (n = 101 vessels) were used. Vessel calibers from OCT phase images were measured vertically along the optical axis by three independent graders. The data from RVA fundus images were corrected for magnification to obtain absolute values. RESULTS: The average vessel diameter as obtained from OCT images during normoxia was lower than from RVA images (83.8 ± 28.2 µm versus 86.6 ± 28.0 µm, P < 0.001). The same phenomenon was observed during 100% oxygen breathing (OCT: 81.0 ± 22.4 µm, RVA: 85.5 ± 26.0 µm; P = 0.001). Although the agreement between the two methods was generally high, the difference in individual vessels could be as high as 40%. These differences were neither dependent on absolute vessel size nor preferably found in specific subjects. Interobserver differences between OCT evaluators were much lower than differences between the techniques. CONCLUSIONS: Extracting vessel calibers from OCT phase images may be an attractive approach to overcome some of the problems associated with fundus imaging. The source of differences in vessel caliber between the two methods remains to be investigated. In addition, it remains unclear whether OCT-based vessel caliber measurement is superior to fundus camera-based imaging in risk stratification for systemic or ocular disease. (ClinicalTrials.gov numbers, NCT00914407, NCT02531399.).

Super-resolved thickness maps of thin film phantoms and in vivo visualization of tear film lipid layer using OCT.

In optical coherence tomography (OCT), the axial resolution is directly linked to the coherence length of the employed light source. It is currently unclear if OCT allows measuring thicknesses below its axial resolution value. To investigate spectral-domain OCT imaging in the super-resolution regime, we derived a signal model and compared it with the experiment. Several island thin film samples of known refractive indices and thicknesses in the range 46 - 163 nm were fabricated and imaged. Reference thickness measurements were performed using a commercial atomic force microscope. In vivo measurements of the tear film were performed in 4 healthy subjects. Our results show that quantitative super-resolved thickness measurement can be performed using OCT. In addition, we report repeatable tear film lipid layer visualization. Our results provide a novel interpretation of the OCT axial resolution limit and open a perspective to deeper extraction of the information hidden in the coherence volume.

Factors Determining Flicker-Induced Retinal Vasodilation in Healthy Subjects.

PURPOSE: The purpose of this study was to analyze factors determining retinal arterial and venous responses to stimulation with diffuse luminance flicker in healthy subjects. METHODS: We retrospectively analyzed results obtained in 374 healthy subjects who had previously participated in clinical studies in our department. A total of 153 subjects underwent a protocol in which flicker stimulation was delivered through the fundus camera at 8 Hz (protocol 1), separating measurement and stimulation light depending on the wavelength, and 221 subjects underwent a protocol in which diffuse luminance flicker was delivered at 12.5 Hz with high modulation depth (protocol 2). We investigated whether sex, systemic blood pressure, baseline vessel size, blood plasma concentration of fasting glucose and hematocrit, and serum concentration of cholesterol, triglycerides, creatinine and C-reactive protein influenced the retinal vascular response to flicker stimulation. RESULTS: Flicker responses in arteries and veins were more pronounced in protocol 2 than in protocol 1 (P < 0.001, each). In both of the protocols the vascular response to stimulation with diffuse luminance flicker was larger in smaller vessels (P between 0.001 and 0.016). In protocol 2 the retinal arterial flicker response was negatively associated with cholesterol serum levels (P = 0.033); in protocol 1, only a tendency toward this effect was observed (P = 0.056). CONCLUSIONS: The present analysis indicates that retinal arterial and venous responses to stimulation with diffuse luminance flicker depend on the way the stimulation is delivered through the fundus camera. In addition, the flicker response varied with vessel size, that is, the smaller the vessel width, the larger the flicker response. Finally, our data indicate that, even within the normal range, higher cholesterol serum levels are associated with lower hyperemic flicker responses.

Total Retinal Blood Flow in a Nonhuman Primate Optic Nerve Transection Model Using Dual-Beam Bidirectional Doppler FD-OCT and Microsphere Method.

PURPOSE: We validated noninvasive Doppler-optical coherence tomography (OCT) blood flow measurements against the terminal microsphere method in a surgical induced optic nerve transection nonhuman primate model. METHODS: In 6 nonhuman primates, total retinal blood flow (TRBF) was measured with a custom-built dual-beam bidirectional Doppler Fourier Domain (FD)-OCT. Peripapillary retinal nerve fiber layer thickness (RNFLT) was measured by Spectralis spectral-domain (SD)-OCT. Measurements were performed every 10 to 15 days before and after unilateral optic nerve transection (ONT) until RNFLT was reduced by more than 40% from baseline. Before the animals were killed, TRBF was measured using the microsphere technique. RESULTS: A significant correlation between all arterial and venous Doppler OCT TRBF measurements was found in ONT and contralateral control eyes (both P < 0.01, n = 6). The Bland-Altman analysis showed a bias of 0.57 in the ONT group and 0.02 in the contralateral control group. Also, excellent agreement was observed between Doppler OCT and microsphere measurements (P < 0.01, r = 0.976, bias = 0.54). After ONT, TRBF and RNFLT decreased by -51% ± 42% and -44% ± 2% (n = 5), respectively. In the contralateral control eyes, TRBF and RNFLT were unchanged. CONCLUSIONS: Very good accordance was found between TRBF measurements, obtained with dual-beam bidirectional Doppler FD-OCT and the microsphere method. It also was possible to monitor changes over time in TRBF after ONT with Doppler OCT. These findings highlight the accuracy and potential of noninvasive Doppler OCT to provide valuable information for detecting early changes in ocular disease in future.

Retinal oxygen extraction in humans.

Adequate function of the retina is dependent on proper oxygen supply. In humans, the inner retina is oxygenated via the retinal circulation. We present a method to calculate total retinal oxygen extraction based on measurement of total retinal blood flow using dual-beam bidirectional Doppler optical coherence tomography and measurement of oxygen saturation by spectrophotometry. These measurements were done on 8 healthy subjects while breathing ambient room air and 100% oxygen. Total retinal blood flow was 44.3 ± 9.0 µl/min during baseline and decreased to 18.7 ± 4.2 µl/min during 100% oxygen breathing (P < 0.001) resulting in a pronounced decrease in retinal oxygen extraction from 2.33 ± 0.51 µl(O2)/min to 0.88 ± 0.14 µl(O2)/min during breathing of 100% oxygen. The method presented in this paper may have significant potential to study oxygen metabolism in hypoxic retinal diseases such as diabetic retinopathy.

Blood flow velocity vector field reconstruction from dual-beam bidirectional Doppler OCT measurements in retinal veins.

In this paper, we demonstrate the possibility to reconstruct the actual blood flow velocity vector field in retinal microvessels from dual-beam bidirectional Doppler optical coherence tomography measurements. First, for a better understanding of measured phase patterns, several flow situations were simulated on the basis of the known dual beam measurement geometry. We were able to extract the vector field parameters that determine the measured phase pattern, allowing for the development of an algorithm to reconstruct the velocity vector field from measured phase data. In a next step, measurements were performed at a straight vessel section and at a venous convergence; the obtained phase data were evaluated by means of the new approach. For the straight vessel section, the reconstructed flow velocity vector field yielded a parabolic flow. For the venous convergence, however, the reconstructed vector field deviated from a parabolic profile, but was in very good accordance with the simulated vector field for the given vessel geometry. The proposed algorithm allows predictions of the velocity vector field. Moreover, the algorithm is also sensitive to directional changes of the flow velocity as small as <1°, thereby offering insight in the flow characteristics of the non-Newtonian fluid blood in microvessels.