Edgar Nagel

Use of the retinal vessel analyzer in ocular blood flow research

Acta Ophthalmol. 2010: 88: 717–722

Repeatability of Autofluorescence Lifetime Imaging at the Human Fundus in Healthy Volunteers

Abstract Purpose: We aim to evaluate the repeatability of a new fluorescence lifetime imaging (FLIM) technique which measures time-resolved autofluorescence to assess metabolism of the retina. Materials and methods: We performed FLIM with two spectral channels (channel 1: 490–560 nm and channel 2: 560–700 nm) on 10 healthy volunteers, with 10 replicates per volunteer. From the 30° fundus FLIM images, we selected three regions: the fovea, the optic disc and the papillo-macular bundle. For each channel in these regions, we determined an average multi-exponential approximation with three components, and the six resulting parameters, α1–α3 (amplitudes) and τ1–τ3 (fluorescence lifetimes), were analyzed in terms of the coefficient of variation (CV). Results: Repeatability was highest in the papillo-macular bundle, followed by the fovea and the optic disc. Repeatability was higher in channel 1 (mean CV of 7.9%) than in channel 2 (mean CV of 17.7%). The average CV for the diagnostically most relevant channel 1 and the most relevant parameters was as follows: τ1 (5.5%) and τ2 (4.7%) in the papillo-macular bundle, and τ1 (6.8%) and τ2 (6.9%) in the fovea. Conclusions: We demonstrated repeatability of FLIM measurement results within acceptable ranges of variation. Based on the detailed coefficients of variation, we derived recommendations for parameter ranges suitable for diagnostic applications.

Dorzolamide influences the autoregulation of major retinal vessels caused by artificial intraocular pressure elevation in patients with POAG: a clinical study.

Purpose: The study investigated whether dorzolamide influences the autoregulatory behavior of major retinal arterioles in glaucoma patients via a moderate perfusion pressure reduction. Methods: The study included one eye each of 12 untreated patients with a primary open-angle glaucoma (POAG) (age 60.8 ± 8.3, IOP 22.3 ± 6.5 mmHg). Changes in the diameter of a retinal artery segment before (120 s), during (100 s), and after (380 s) artificial IOP elevation to 38 mmHg for 100 s were recorded continuously by means of a Retinal Vessel Analyzer. The measurement was repeated after 4-week treatment with dorzolamide eye drops t.i.d. Results: Ocular perfusion pressure (mmHg) was reduced by the intraocular pressure (IOP) elevation from 58 (± 10) to 41 (± 11) in the pretreatment examination and from 60 (± 8) to 40 (± 8) posttreatment (differences between the examinations n.s.). Before IOP elevation, the arterial diameter was found to be +1.7 ± 3.5% greater in the posttreated eyes than in the pretreated eyes (p < 0.02). During IOP elevation, the arterial diameter decreased by −1.8% ± 3.8 in the pretreated eyes, whereas dilatation by +1.4% ± 2.5 was observed in the posttreated eyes (p = 0.02). At the end of the observation period following IOP elevation, the vessel diameter in the pretreated eyes had increased by +1.8% ± 4.2, whereas in the posttreated eyes it had decreased by −1.7% ± 3.0. On average, dorzolamide reduced IOP by −5.6 mmHg (p = 0.001). Conclusions: The arterial diameter dilatation during IOP elevation in dorzolamide-treated eyes could be an accelerated counter-regulation on the induced elevated IOP and could constitute an additional therapeutic effect.

Microstructural alterations of retinal arterial blood column along the vessel axis in systemic hypertension.

Purpose. Image analysis by the retinal vessel analyzer (RVA) observes retinal vessels in their dynamic state online noninvasively along a chosen vessel segment. It has been found that high-frequency diameter changes in the retinal artery blood column along the vessel increase significantly in anamnestically healthy volunteers with increasing age and in patients with glaucoma during vascular dilation. This study was undertaken to investigate whether longitudinal sections of the retinal artery blood column are altered in systemic hypertension. Methods. Retinal arteries of 15 untreated patients with essential arterial hypertension (age, 50.9 +/- 11.9 years) and of 15 age-matched anamnestically healthy volunteers were examined by RVA. After baseline assessment, a monochromatic luminance flicker (530-600 nm; 12.5 Hz; 20 s) was applied to evoke retinal vasodilation. Differences in amplitude and frequency of spatial artery blood column diameter change along segments (longitudinal arterial profiles) of 1 mm in length were measured and analyzed using Fourier transformation. Results. In the control group, average reduced power spectra (ARPS) of longitudinal arterial profiles did not differ when arteries changed from constriction to dilation. In the systemic hypertension group, ARPS during constriction, baseline, and restoration were identical and differed from ARPS during dilation (P < 0.05). Longitudinal arterial profiles in both groups showed significant dissimilitude at baseline and restoration (P < 0.05). Conclusions. The retinal artery blood column demonstrates microstructural alterations in systemic hypertension and is less irregular along the vessel axis during vessel dilation. These microstructural changes may be an indication of alterations in vessel wall rigidity, vascular endothelial function, and smooth muscle cells in this disease, leading to impaired perfusion and regulation.

Reproducibility of the Retinal Vascular Response to Flicker Light in Asians

Purpose: Dilation of retinal vessels in response to diffuse luminance flicker may reflect endothelial function. Although this has previously been shown to be reproducible in whites, there have been no similar data in Asians. We assess the reproducibility of repeated measurements of this response in Asians. Material and Methods: Healthy Asians (n = 33) with normal vision and no history of glaucoma, age-related macular degeneration, cataract, or retinal arterial/venous occlusion participated in this study. Repeated measures from the same subjects were taken 30–60 min apart using the Dynamic Vessel Analyser (DVA, IMEDOS, Jena, Germany). Modification was made to the shape of the light source for Asian participants. Correlations of the first and second measures were assessed using Pearson correlation (R2), and agreement between the two measures was shown using Bland–Altman plots. Results: After modification to the shape of the light source, almost perfect correlation was found between the 1st and 2nd measurements of baseline arteriolar (R2 = 0.95) and venular diameters (R2 = 0.98) of arteriolar maximum dilation (R2 = 0.85). Substantially high correlation between the 1st and 2nd measurements of venular maximum dilation was found (R2 = 0.80). Conclusions: Measurements of the dilation response of retinal vessels to diffuse luminance flicker an Asian sample using the DVA show high reproducibility for repeated measures over a short period of time. Such measurements may allow non-invasive quantification of endothelial function to study its association with systemic and ocular diseases.

Functional in vivo assessment of retinal artery microirregularities in glaucoma.

Purpose:  We investigated whether retinal branch arteries in healthy subjects, and non‐treated and treated primary open‐angle glaucoma (POAG) patients show irregular local patterns during dynamic reaction to acute increases of different magnitudes in intraocular pressure (IOP).

Effects of short term changes in the blood glucose level on the autofluorescence lifetime of the human retina in healthy volunteers

Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) provides in vivo metabolic mapping of the ocular fundus. Changes in FLIO have been found in e.g. diabetes patients. The influence of short term metabolic changes caused by blood glucose level changes on is unknown. Aim of this work is the detection of short-term changes in fundus autofluorescence lifetime during an oral glucose tolerance test. Methods: FLIO was performed in 10 healthy volunteers (29±4 years, fasting for 12h) using a scanning laser ophthalmoscope (30° fundus, 34μm resolution, excitation with 473nm diode laser with 70 ps pulses at 80 MHz repetition rate, detection in two spectral channels 500-560nm (ch1) and 560-720nm (ch2) using the timecorrelated single photon counting method). The blood glucose level (BGL) was measured by an Accu-Chek® Aviva self-monitoring device. Before and after a glucose drink (300ml solution, containing 75g of glucose (Accu-Chek® Dextrose O.G.T.), BGL and FLIO were measured every 15min. The FLIMX software package was applied to compute the average fluorescence lifetime τ on the inner ring of the ETDRS grid using a modified 3-exponential approach. Results: The results are given as mean ± standard deviation over all volunteers in ch1. Baseline measurement: BGL: 5.3±0.4 mmol/l, τ1: 49±6ps. A significant reduction (α=5%; Wilcoxon rank-sum test) in τ1 is detected after 15min (BGL: 8.4±1.1 mmol/l, τ1: 44±5ps) and after 90min (BGL: 6.3±1.4 mmol/l, τ1: 41±5ps). Results of ch2 show smaller reductions in the fluorescence lifetimes over time.

Agreement Between Eyes in Wide-Field Fluorescence Lifetime Imaging Ophthalmoscopy Measurements at the Human Retina in Healthy Volunteers

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new imaging technique for measuring the time-resolved in vivo autofluorescence generated by endogenous fluorophores in the ocular fundus. The aim is to assess the metabolism of the retina. Based on the fluorescence lifetime, different fluorescent compounds in the eye can be distinguished. Pathologic changes may be observed by detecting changes in the fluorescence lifetime. Until now, FLIO is performed using 30° fundus images. For certain diseases section of the fundus is of interest. In this work, observational clinical study in young healthy volunteers was performed to examine the usability of 55° wide-field fluorescence lifetime images. The time-resolved retina autofluorescence was measured (scanning laser ophthalmoscope: 55° of fundus, 62x62 µm2 per pixel; excitation: diode laser with pico-second pulses, 473nm, 80MHz repetition rate; detection: spectral channels 498-560nm (ch1) and 560-720nm (ch2), time-correlated single photon counting method) in both eyes of 11 healthy volunteers (28.7±3.6 years). Three repetitive measurements on different days within one week at a similar time have been performed. All subjects had a crystalline lens and an undilated pupil. A modified 3-exponential approach was applied to determine the mean fluorescence lifetimes tm. tm were computed on the ETDRS grid and in a 15x15 pixel region 25° superior to the fovea. The Wilcoxon rank-sum test was used to test for statistical significant differences between left and right eye. The coefficient of variation in the superior region is 5.6% (right) and 4.7% (left) in ch1 and 2.5% (right) and 2.1% (left) in ch2. No statistical significant differences have been found between right and left eye. FLIO measurements in young healthy volunteers using a 55° lens are repeatable in both eyes and show no significant differences. Spectral channel 2 should be preferred because of its lower variability. The data analysis was done with FLIMX (available at http://www.flimx.de).