December 2003
Volume 44, Issue 12
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Physiology and Pharmacology  |   December 2003
Flicker Light–Induced Vasodilatation in the Human Retina: Effect of Lactate and Changes in Mean Arterial Pressure
Author Affiliations
  • Gerhard Garhöfer
    From the Departments of Clinical Pharmacology,
  • Claudia Zawinka
    From the Departments of Clinical Pharmacology,
  • Karl-Heinz Huemer
    From the Departments of Clinical Pharmacology,
    Physiology, and
  • Leopold Schmetterer
    From the Departments of Clinical Pharmacology,
    Institute of Medical Physics, University of Vienna, Vienna, Austria.
  • Guido T. Dorner
    From the Departments of Clinical Pharmacology,
    Ophthalmology and the
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5309-5314. doi:10.1167/iovs.03-0587
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      Gerhard Garhöfer, Claudia Zawinka, Karl-Heinz Huemer, Leopold Schmetterer, Guido T. Dorner; Flicker Light–Induced Vasodilatation in the Human Retina: Effect of Lactate and Changes in Mean Arterial Pressure. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5309-5314. doi: 10.1167/iovs.03-0587.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Diffuse luminance flicker light increases retinal and optic nerve head blood flow in animals and humans, but the exact mechanisms that mediate increased flow have yet to be identified. In the current study, the effect of increased plasma lactate levels on flicker-induced vasodilatation in the retina was investigated in three independent studies in healthy humans.

methods. In the first study, plasma lactate concentrations were increased by bicycle exercise in 12 volunteers, and the change in retinal vessel diameter to 8-Hz square-wave flicker stimulation was measured with the Zeiss Retinal Vessel Analyzer (Carl Zeiss Meditec, Oberkochen, Germany). In a different study, sodium lactate was administered intravenously, and flicker responses were measured in 12 subjects. As a control experiment accounting for pressure increases induced by exercise, the effect of elevated ocular perfusion pressure on the flicker response was investigated during tyramine infusion (n = 12).

results. The increase in plasma lactate concentration during intravenous infusion from 1.3 ± 0.4 to 6.3 mmol/L and during dynamic exercise from 1.2 ± 0.3 to 9.4 mmol/L decreased flicker responses in retinal arteries from 5.3% ± 0.9% to 1.7% ± 0.6% (P < 0.001) and from 3.6% ± 0.6% to 2.0% ± 0.8% (P = 0.03), respectively. In contrast, an increase of mean blood pressure from 81 ± 3 to 92 ± 3 mm Hg after tyramine infusion had no significant effect on flicker-induced vasodilatation in retinal arteries and veins.

conclusions. The signaling between neuronal activity and flow response in the human retina is sensitive to changes in blood lactate levels, whereas changes in systemic blood pressure have no major effect. Whether an increased cytosolic redox impairment contributes to flicker-induced vasodilatation has yet to be clarified.

The importance of energy metabolism and increased neural activity in blood flow regulation has long been realized, since the early description of Roy and Sherrington 1 in a study of the brain. It is now generally accepted that the brain can vary its blood supply depending on local differences in functional activity and the resultant metabolic demands. In the retina, this mechanism for vascular regulation of increased neural activity and metabolic demands exists as well. This coupling between augmented neuronal activity and increased blood flow has been demonstrated in several animal 2 3 4 and human experiments. 5 6 7 8 9 In these studies, diffuse luminance flicker was found to be a strong stimulus to augment blood flow in retinal vessels as well as in the optic nerve head, indicating that the enhanced neural activity in the retina triggers the subsequent increase in blood flow. 
Whereas several mediators have been proposed for signaling blood flow regulation, 10 11 12 13 the sensor for the need for increased blood flow is still unknown. 13 On a cellular level, much emphasis has been put on factors involved in oxidative phosphorylation and glucose metabolism. Furthermore, it has recently been speculated that a decrease in the intracellular ratio of nicotinamide adenine dinucleotide (NAD+) to its reduced form NADH stimulates retinal blood flow. 14  
NADH acts as one of the major electron carriers in the cell. The cytosolic NADH-to-NAD+ ratio is of special importance, because it affects the activity of numerous cytoplasmic and mitochondrial enzymes that use NADH or NAD+ as a cofactor. These effects include alterations in fatty acid oxidation, increased prostaglandin synthetic activity, and increased free radical production. 15 The free cytosolic NADH/NAD+ ratio can be altered through oxidation of substrates as different as sorbitol, nonesterified free-fatty acid, or glucuronic acid pathway metabolites, among others. Increased flux through these pathways may result in increased production of NADH, faster than it can be reoxidized in the mitochondria, which in turn leads to an increased NADH/NAD+ ratio. 
In contrast to cytosolic reductive stress induced by increased oxidation coupled to reduction of NAD+ to NADH, reductive stress can also be caused by hypoxia. This is mainly a consequence of impaired reoxidation of NADH to NAD+ in the mitochondria. Thus, increased free cytosolic NADH/NAD+ ratios caused by reasons other than hypoxia have been termed pseudohypoxia. 15 However, both pathways result in an increased ratio of NADH/NAD+ and could therefore account for blood flow changes. 
Activation of the sorbitol pathway has been thought to increase NADH during hyperglycemia, resulting in increased blood flow. 16 Whether this is correct remains to be clarified. 17 However, the hypothesis that increased NADH levels are involved in blood flow regulation is supported by evidence from animal experiments, revealing that an increased blood lactate concentration can augment blood flow, which is reversible by the administration of pyruvate. 14 Furthermore, we recently demonstrated that intravenous administration of lactate, which is known to shift the NADH/NAD+ ratio toward NADH, 14 increases retinal blood flow in healthy subjects. 18  
In the present study we set out to investigate the effect of increased lactate concentrations on flicker light–induced vasodilatation in the human retina in two studies: First, bicycle exercise was used to increase blood lactate levels endogenously (study A), and, second, the blood lactate concentration was elevated by intravenous administration of sodium-lactate (study B). A third study (study C) was designed as a control experiment to investigate the effect of an increase in mean arterial pressure on the flicker response of retinal vessels. 
Methods
Subjects
The study protocol was approved by the Ethics Committee of Vienna University School of Medicine and followed the guidelines of the Declaration of Helsinki. Thirty-six healthy male nonsmoking subjects were included. Volunteers signed a written informed consent and passed a screening examination that included medical history and physical examination; 12-lead electrocardiogram; complete blood count; activated partial thromboplastin time and thrombin time; fibrinogen; clinical chemistry (sodium, potassium, creatinine, uric acid, glucose, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, alkaline phosphatase, total bilirubin, total protein); hepatitis A, B, and C and HIV serology; and urinalysis. Subjects were excluded if any abnormality was found as part of the screening, unless the investigators considered an abnormality clinically irrelevant. An ophthalmic examination was performed in each subject before the study day. Inclusion criteria were normal ophthalmic findings, ametropia of less than 3 D, and anisometropia of less than 1 D. In all subjects the right eye was studied. 
Experimental Paradigms
All three experiments were performed in a different study cohort. After a 10-minute resting period baseline measurements of ocular and systemic hemodynamics were performed. In all three experiments, measurements with the Zeiss Retinal Vessel Analyzer (RVA; Carl Zeiss Meditec, Oberkochen, Germany) were performed for 4 minutes, superimposed by a flicker light period (Fig. 1) . Sixty seconds of baseline measurement were followed by 60 seconds of flicker stimulation and another consecutive 120 seconds of recording. 
Experimental Designs
Study A.
In study A a bicycle ergometer exercise was performed to investigate the conditional increase in blood lactate and increased mean arterial pressure on the vessel flicker response in 12 volunteers. A time schedule is given in Figure 2 . After the subject was allowed a short resting period in sitting position, baseline flicker measurements with the RVA were performed, and blood lactate concentrations were determined. Then, tests similar to conventional clinical exercise tests were performed on a bicycle ergometer. Workload then started at 80 W and was increased (20 W, 30 seconds) until exhaustion or 6 minutes of exercise were performed. Care was taken to perform a maximum exercise test. Real time electrocardiogram and pulse rate were measured continually. Blood pressure was measured every 2 minutes during the whole experiment. Samples for the measurement of lactate were drawn from an earlobe 3, 10, and 20 minutes after exercise. Measurements with the RVA were performed at rest and 4, 11, and 21 minutes after exercise. 
Study B.
The effect of intravenously administered sodium-lactate (0.6 mol/L; Mayrhofer Pharmazeutika, Linz, Austria) was investigated in an open study design in 12 volunteers with observer-blinded analysis of the flicker response. Baseline measurements of flicker response and blood lactate concentration were performed. Then, sodium lactate infusion was titrated to increase blood lactate concentration to 6 mmol/L and subsequently to 9 mmol/L for at least 15 minutes, respectively. These blood lactate levels are within the physiological range and can be reached by untrained subjects after a short period of exercise. 19 At the end of each infusion step flicker measurements were performed with the RVA. 
Study C.
As a control experiment the effect of an increase in ocular perfusion pressure during normal lactate concentration was measured in 12 subjects. For this purpose tyramine (Clinalfa, Läufelingen, Switzerland) was administered intravenously (0 [saline], 20 μg/kg per minute, 40 μg/kg per minute, and 60 μg/kg per minute; 15 minutes per infusion step) in a placebo-controlled, double-blind, randomized study. At the end of each infusion step, the effect of flicker stimulation was measured with the RVA. A time schedule of study C is given in Figure 3
Tyramine releases noradrenaline from sympathetic neurons and increases systemic blood pressure, but does not alter retinal blood flow. 20 Tyramine is optimally suitable to investigate autoregulation in the retina, because retinal vessels lack sympathetic innervation. 21  
Zeiss Retinal Vessel Analyzer
The Zeiss RVA is a commercially available system that comprises a fundus camera (FF 450; Carl Zeiss Meditec, Jena, Germany), a video camera, a high-resolution video recorder, a real-time monitor, and a personal computer with vessel diameter analysis software. The RVA allows for the precise (1.3% coefficient of variation) determination of retinal vessel diameter with a time resolution of 25 readings/s. For vessel diameter measurements, the fundus was illuminated through an interference filter placed in the illumination path of the RVA. This filter transmitted light in the range of wavelengths between 567 and 587 nm. In this spectral range, the contrast between retinal vessels and the surrounding tissue is optimal. Retinal irradiance was approximately 220 μW · cm−2, which is approximately 50 times lower than the maximum level allowed for constant illumination of the retina at the wavelengths just mentioned. Because of its adaptive algorithm, the system is largely independent of alterations in luminance as induced, for instance, by slight eye movements. A typical recording of retinal arterial diameter during flicker stimulation using the RVA is presented in Figure 1
Flicker Light Stimulation
For flicker stimulation, a custom-built device was used that stimulated with light flashes at a frequency of 8 Hz. Flicker was generated by focusing the light of a 150-W halogen light source on a rotating sector disc, producing a square-wave light pattern with a modulation depth of 100%. Using an optical fiber, flicker stimuli were delivered to the eye through the illumination pathways of the fundus camera of the RVA. The flicker was centered in the macula with an angle of approximately 30° producing a retinal irradiance of 300 μW · cm−2 (approximately 260 lux). 
To spectrally separate the flicker light from that used to illuminate the fundus, a wavelength-separation technique was used. For flicker stimulation, white light in combination with a 550-nm low-pass cutoff filter was used to ensure that light with wavelengths below 550 nm would be used for flicker stimulation. To separate flicker light from the light illuminating the fundus, an interference filter with a center wavelength of 577 nm and a bandwidth of 10 nm (Laser Components, Olchingen, Germany) was placed in front of the light source of the fundus camera. This window was chosen because the contrast between blood vessels and the surrounding tissue is optimal in this spectral range. A second interference filter, which exactly matches the one in the illumination pathway was placed in front of the video camera, to ensure that the light used for flicker stimulation did not reach the CCD chip of the video camera, but was perceived by the subject under study. This setup ensured constant illumination conditions at the fundus image during flicker stimulation, a prerequisite for adequate vessel diameter determination. 8  
Measurement of Blood Lactate Levels
Blood lactate concentrations were measured with a reflectance photometer (Accusport; Roche Molecular Biochemicals, Mannheim, Germany) from blood samples drawn from the arterialized earlobe. The values obtained by Accusport have been shown to be comparable in accuracy, linearity, and reliability with those obtained by a laboratory reference method. 22 23  
Blood Pressure Measurement
Mean arterial blood pressures were measured on the upper arm by an automated oscillometric device (HP-CMS patient monitor; Hewlett Packard, Palo Alto, CA). Pulse rate was automatically recorded from a finger pulse oximeter. 
Statistics
Changes in ocular hemodynamic parameters were expressed as percent change over baseline values. Retinal vessel diameters were calculated as an average of the last 30 seconds of each measurement. Vessel diameter during flicker was calculated as an average of the last 30 seconds of light stimulation. Data are presented as the mean ± SEM. Repeated-measures ANOVA was used to compare flicker-induced vasodilatation between groups. The t-test was used for post hoc analysis, and the Bonferroni correction was applied for multiple testing. Correlation analysis was performed with a Spearman R test. P < 0.05 was considered the level of significance. Calculations were performed on computer (Statistica; Statsoft, Tulsa, OK). 
Results
Study A
As shown in Figure 4 , flicker light induced a vasodilatation of 5.3% ± 0.9% (from 115 ± 4 to 121 ± 4 μm; P < 0.001) in retinal arteries and 3.5% ± 2.1% (from 148 ± 5 to 154 ± 5 μm, P < 0.001) in retinal veins at baseline lactate concentration of 1.2 ± 0.3 mmol/L. The flicker response was blunted in retinal arteries after exercise (P = 0.018, ANOVA). A tendency toward decreased flicker responses was also seen in retinal veins after exercise, but this effect did not reach significance (P = 0.23, ANOVA). Three minutes after the end of bicycle exercise, blood lactate levels were increased to 13.8 ± 1.1 mmol/L (P < 0.001) and mean arterial blood pressure from 86 ± 2 mm Hg at baseline to 102 ± 6 mm Hg (P < 0.001). At this time point, arterial flicker responses tended to be reduced to 2.4% ± 1.0% (P = 0.03, NS, due to the Bonferroni correction). Retinal arterial diameters showed no change 3 minutes after exercise (before exercise: 118 ± 4 μm and after exercise 117 ± 4 μm; change −0.9% ± 0.3%, P < 0.5). The diameter of retinal veins also remained unchanged after exercise (before exercise: 146 ± 5 μm and after exercise: 146 ± 5 μm; change 0.0% ± 0.3%, P < 0.8). 
Ten and 20 minutes after exercise, at blood lactate concentrations of 9.9 ± 1.1 and 4.5 ± 0.6 mmol/L, respectively, the flicker response in retinal arteries was still reduced (1.7% ± 0.7%, P = 0.01, t-test; 3.2% ± 0.8%, P = 0.03, t-test). Retinal arterial diameters showed no change 10 minutes after exercise (118 ± 4 μm) and tended to increase 20 minutes after exercise (120 ± 4 μm). This effect, however, did not reach the level of significance (P = 0.08, ANOVA). Retinal venous diameters were 146 ± 6 and 147 ± 5 μm at these time points. (P = 0.95, ANOVA). Mean arterial blood pressure returned to baseline 10 (82.7 ± 3.2 mm Hg, P = 0.2) and 20 (82.7 ± 2.8 mm Hg, P = 0.2, t-test) minutes after exercise. 
Study B
No significant changes in mean arterial pressure and pulse rate were observed during the whole study (data not shown). Resting plasma lactate levels were 1.3 ± 0.4 mmol/L. Flicker-induced vasodilatation by 3.6% ± 0.6% (from 121 ± 5 to 126 ± 5 μm, P < 0.001) in retinal arteries and 2.2% ± 0.4% (from 147 ± 6 to 151 ± 6 μm, P < 0.001) in retinal veins at baseline conditions. Lactate infusion increased plasma lactate concentration to 6.3 ± 0.6 and 9.4 ± 0.4 mmol/L, respectively. At these lactate concentrations the flicker response was significantly reduced to 2.0% ± 0.8% and 2.3% ± 0.7% in retinal arteries (P = 0.03, ANOVA). In retinal veins, no significant change in the flicker response was observed (P < 0.23, ANOVA). 
The diameter of retinal arteries tended to increase after administration of sodium lactate from 120 ± 4 to 122 ± 4 μm (+1.5% ± 0.3%) and 123 ± 5 μm (+1.7% ± 0.2%). This increase, however, was not statistically significant (P = 0.15, ANOVA). Retinal venous diameters increased from 145 ± 6 to 146 ± 6 μm (+0.4% ± 0.2%) at baseline and to 148 ± 6 μm (+2.4% ± 0.3%, P = 0.01, ANOVA) after administration of lactate. 
Study C
Lactate plasma levels at baseline were 1.1 ± 0.5 mmol/L on the tyramine day and 1.1 ± 1.0 mmol/L on the placebo day. Lactate levels did not change during the experiment (data not shown). As shown in Figure 5 , tyramine increased MAP (81 ± 3 to 92 ± 3 mm Hg; P < 0.001). After administration of 20 μg/kg per minute, 40 μg/kg per minute, and 60 μg/kg per minute, tyramine retinal arterial diameters decreased by −0.8% ± 0.9%, −1.7% ± 0.9%, and −3.4% ± 1.2%, venous diameter decreased by −0.6% ± 1.1%, −1.7% ± 1.1%, and −3.4% ± 0.8%. This effect failed to reach the level of significance (arteries: P = 0.18, ANOVA; veins: P = 0.16, ANOVA). Further, tyramine had no significant effect on the vessel response to flicker stimulation (Fig. 5) . No significant change in mean arterial blood pressure and retinal vessel diameters was observed during placebo infusion. 
Test–retest reproducibility of flicker-induced vasodilatation was calculated for arteries and veins, using coefficients of variation (CV). Baseline values were compared with values of placebo infusion of study C. The analysis revealed a variability for the flicker responses in arteries within subjects of CV = 15.2% and CV = 20.3% for retinal veins. 
Using the data of all three studies (n = 36), we investigated whether the obtained flicker response is dependent on the baseline vessel diameters. A tendency toward increasing flicker responses with decreasing vessel diameters was observed, but this effect did not reach the level of significance (retinal arteries: R = −0.23; P = 0.12 retinal veins: R = −0.1; P = 0.5). 
Discussion
The experiments presented herein are the first to demonstrate that endogenously and exogenously increased blood lactate concentrations alter the responsiveness of retinal arteries to diffuse luminance flicker in humans. In study A, we demonstrated that increased lactate concentrations induced by exercise lead to diminished flicker responses in retinal arteries, in keeping with the results of study B which showed that intravenous administration of sodium lactate blunts flicker responses in the retina. The control experiment indicated that increased mean arterial blood pressure has no influence on flicker-induced vasodilatation. 
In most tissues, lactate is produced only under anaerobic conditions, as a byproduct of anaerobic glycolysis. In contrast, in vitro and in vivo experiments have found that retinal cells produce lactate, even in the presence of high oxygen concentration. 24 25 26 In humans, lactate production has been attributed to a combination of aerobic and anaerobic lactate formation. 26 However, one has to consider that substantial species differences exist regarding consumption of glucose and lactate in the retina, possibly because of their differing angioarchitecture. 
Stimulation of the photoreceptors and the ganglionic cell layer with flickering light has been reported to change substantially the retinal metabolism in a variety of studies. The glucose consumption of the inner retina in monkeys was enhanced, thereby affecting retinal blood flow. 10 Furthermore, in the isolated rabbit retina an increase in lactate formation was measurable during flicker stimulation. 27 In vivo experiments in cats revealed that up to 80% of glucose is metabolized by aerobic lactate production 26 and that in rabbits retinal glucose consumption and lactate formation increases by approximately 15% to 20% during flicker stimulation. 27 This strong increase in lactate formation was also demonstrated in a study by Ames et al., 28 in which flicker stimulation after a period of dark adaption resulted in n 27% increase in lactate paralleled by a 4% reduction in oxygen consumption. 
The present studies indicate that an increased lactate level, regardless of whether it is induced by strenuous exercise or intravenous administration, decreases vascular reactivity in response to flicker light. This seems to be of special interest because intravenous administration of sodium lactate has been shown to induce an intracellular redox impairment in several tissues including the retina. Because under steady state conditions, equilibria exist between cytosolic free NADH/NAD+ and intracellular lactate/pyruvate ratios, established by lactate dehydrogenase, 29 this allows experimental modulation of the cytosolic free NADH/NAD+ ratio. Thus, increasing plasma lactate levels leads to changes in the lactate/pyruvate ratios and drives reduction of NAD+ to NADH, which in turn leads to increased intracellular NADH levels. 29  
In turn, increased NADH levels may alter flicker responses in the retina. This hypothesis is supported by recent experiments performed in albino rats, where retinal blood flow was sensitive to plasma lactate levels, 14 an effect that was abolished by coadministration of pyruvate. Furthermore, we have recently demonstrated that intravenous administration of lactate results in an increase in the order of 30% in retinal blood flow in healthy subjects, whereas no changes in choroidal blood flow were observed. 18 This increase was mainly attributed to an increase of red blood cell velocity, with little change in retinal vessel diameters in accordance with the results of the present study. 
Alternatively, one could hypothesize that an increase in retinal blood flow, evoked by augmented blood lactate levels, as described recently, 18 is responsible for the diminished vascular response to flicker stimulation. In view of the fact that flicker-induced vasodilatation has been suggested to be a reaction to increased local metabolic demands, one could argue that increased blood flow, per se, could lead to local hyperperfusion and therefore to a diminished vascular response to flicker stimulation. This question cannot be answered definitely from the present experiment, because our measurements in the retina provide only information about retinal vessel diameter and the reactivity of larger retinal vessels to photic stimulation. In view of the fact that blood flow in a main retinal vessel is πD 2 V mean/4, where D is the vessel diameter and V is the mean velocity of the blood, a twofold increase in diameter translates to a fourfold change in blood flow. This emphasizes the importance of vessel diameter in estimating blood flow in the retina, but also raises the question of blood flow velocity. In principle, information about blood flow velocity could be gained using bidirectional laser Doppler velocimetry, allowing for calculation of total retinal blood flow. 30 However, obtaining reproducible flicker responses with this system is difficult, because excellent target fixation is required for these measurements. 
Increased blood lactate levels did not alter flicker responses in retinal veins, but significantly blunted flicker-induced vasodilatation in retinal arteries in both experiments. In view of the fact that vasodilatation of retinal veins is primarily a passive effect, data on the retinal microvasculature are needed to elucidate this behavior. Although a variety of methods have been proposed to measure retinal vessel size in vivo, 31 32 33 34 there is no appropriate technique available for assessment of the human retinal microvasculature. Vessels with diameters less than 70 μm contribute significantly to vascular resistance but cannot be measured because of the limited resolution of the RVA. This problem also applies to the other techniques currently available. 
To exclude any potentially confounding effect of changes in mean arterial pressure on the flicker response, a control study using tyramine infusion was performed. The results of this study are in keeping with previous work from our laboratory. 20 35 It has been shown that this dose of tyramine does not alter retinal blood flow, indicating retinal autoregulation. At least in equivalent increases in retinal perfusion pressure, this autoregulatory response does not appear to include vasoconstriction of larger vessels, but is due to effects in the retinal microvasculature. Thus, we conclude that moderately increased blood pressure does not influence flicker-light–induced vasodilatation. 
There is, however, evidence that under some circumstances even the “larger” retinal arteries and veins, as measured in our experiments, contribute to changes in vascular resistance. It appears that during a pronounced increase in blood pressure, as induced by heavy exercise, even larger retinal arteries and veins constrict. 36 In arteries it seems that this is an active process that increases vascular resistance and limits the pressure that is propagated toward the retinal microvessels. Furthermore, in a recent study it was demonstrated that flicker response is not significantly different in retinal trunk and branch vessels 8 but that the increase in retinal vessel diameters tends to be higher in branch than in trunk vessels. This finding has been supported by the results of the present study. To answer finally the question of the extent to which the flicker response in retinal arteries or veins is dependent on baseline diameters, a larger cross-sectional study is necessary. Nevertheless, these data indicate that changes in major retinal vessels, at least in part, also reflect changes in retinal microvasculature. 
A limitation of our study is that the flicker responses between different study parts have a considerable variation with respect to baseline. This can be explained by the rather high interindividual variation in the flicker response. Thus, in the current studies, each subjects acted as his or her own control. With respect to the interindividual variations, flicker responses have been calculated as changes versus baseline in the same subject. Furthermore, no correlation was observed between flicker response and baseline retinal vessel diameters. The reason for this different response profile has not been clarified yet, but due to the high intraindividual reproducibility and sensitivity of the RVA measurements 37 this should not have interfered with our study results. 
In conclusion, we have demonstrated that increased blood lactate concentrations blunts the response of retinal arterial vessels to flicker light stimulation. Furthermore, our findings confirm previous data that a short period of increased ocular perfusion pressure does not alter retinal blood flow and the response of retinal vessels to flicker stimulation. 
 
Figure 1.
 
Typical reading of the Zeiss Retinal Vessel Analyzer (Carl Zeiss Meditec, Oberkochen, Germany) showing the effect of diffuse luminance flicker on the diameter of the inferior temporal artery. Stimulation with flicker light is indicated.
Figure 1.
 
Typical reading of the Zeiss Retinal Vessel Analyzer (Carl Zeiss Meditec, Oberkochen, Germany) showing the effect of diffuse luminance flicker on the diameter of the inferior temporal artery. Stimulation with flicker light is indicated.
Figure 2.
 
Time schedule of study A.
Figure 2.
 
Time schedule of study A.
Figure 3.
 
Time schedule of study C.
Figure 3.
 
Time schedule of study C.
Figure 4.
 
Top: blood lactate concentration as measured before and after exercise. Bottom: flicker-induced vasodilatation of retinal arteries expressed in percent change from baseline. *P < 0.05 versus baseline. Results are presented as the mean ± SEM.
Figure 4.
 
Top: blood lactate concentration as measured before and after exercise. Bottom: flicker-induced vasodilatation of retinal arteries expressed in percent change from baseline. *P < 0.05 versus baseline. Results are presented as the mean ± SEM.
Figure 5.
 
Top: (□) tyramine; (▪) placebo. Data indicate mean arterial blood pressure at baseline and after administration of tyramine or placebo. *P < 0.05. Bottom: corresponding tyramine and placebo data depict the response of retinal arteries to stimulation with diffuse luminance flicker. Flicker responses are expressed as percentage change compared with baseline levels. Results are presented as the mean ± SD.
Figure 5.
 
Top: (□) tyramine; (▪) placebo. Data indicate mean arterial blood pressure at baseline and after administration of tyramine or placebo. *P < 0.05. Bottom: corresponding tyramine and placebo data depict the response of retinal arteries to stimulation with diffuse luminance flicker. Flicker responses are expressed as percentage change compared with baseline levels. Results are presented as the mean ± SD.
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Figure 1.
 
Typical reading of the Zeiss Retinal Vessel Analyzer (Carl Zeiss Meditec, Oberkochen, Germany) showing the effect of diffuse luminance flicker on the diameter of the inferior temporal artery. Stimulation with flicker light is indicated.
Figure 1.
 
Typical reading of the Zeiss Retinal Vessel Analyzer (Carl Zeiss Meditec, Oberkochen, Germany) showing the effect of diffuse luminance flicker on the diameter of the inferior temporal artery. Stimulation with flicker light is indicated.
Figure 2.
 
Time schedule of study A.
Figure 2.
 
Time schedule of study A.
Figure 3.
 
Time schedule of study C.
Figure 3.
 
Time schedule of study C.
Figure 4.
 
Top: blood lactate concentration as measured before and after exercise. Bottom: flicker-induced vasodilatation of retinal arteries expressed in percent change from baseline. *P < 0.05 versus baseline. Results are presented as the mean ± SEM.
Figure 4.
 
Top: blood lactate concentration as measured before and after exercise. Bottom: flicker-induced vasodilatation of retinal arteries expressed in percent change from baseline. *P < 0.05 versus baseline. Results are presented as the mean ± SEM.
Figure 5.
 
Top: (□) tyramine; (▪) placebo. Data indicate mean arterial blood pressure at baseline and after administration of tyramine or placebo. *P < 0.05. Bottom: corresponding tyramine and placebo data depict the response of retinal arteries to stimulation with diffuse luminance flicker. Flicker responses are expressed as percentage change compared with baseline levels. Results are presented as the mean ± SD.
Figure 5.
 
Top: (□) tyramine; (▪) placebo. Data indicate mean arterial blood pressure at baseline and after administration of tyramine or placebo. *P < 0.05. Bottom: corresponding tyramine and placebo data depict the response of retinal arteries to stimulation with diffuse luminance flicker. Flicker responses are expressed as percentage change compared with baseline levels. Results are presented as the mean ± SD.
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