Reproducibility of the retinal vascular response to flicker light in Asians

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Reproducibility of the retinal vascular response to flicker light in Asians

NGUYEN, Thanh Tam, et al.

Abstract

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.

NGUYEN, Thanh Tam, et al . Reproducibility of the retinal vascular response to flicker light in Asians. Current Eye Research , 2009, vol. 34, no. 12, p. 1082-8

DOI : 10.3109/02713680903353764 PMID : 19958128

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Current Eye Research

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Reproducibility of the Retinal Vascular Response to Flicker Light in Asians

Thanh T. Nguyen, Andreas J. Kreis, Ryo Kawasaki, Jie Jin Wang, Bernd-U.

Seifert, Walthard Vilser, Edgar Nagel & Tien Y. Wong

To cite this article: Thanh T. Nguyen, Andreas J. Kreis, Ryo Kawasaki, Jie Jin Wang, Bernd- U. Seifert, Walthard Vilser, Edgar Nagel & Tien Y. Wong (2009) Reproducibility of the Retinal Vascular Response to Flicker Light in Asians, Current Eye Research, 34:12, 1082-1088, DOI:

10.3109/02713680903353764

To link to this article: http://dx.doi.org/10.3109/02713680903353764

Published online: 03 Dec 2009.

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1082

INTRODUCTION

The retina can be viewed directly and non-invasively, allowing an assessment of the human microvascula- ture in vivo. Recent studies show that structural reti- nal microvascular changes, such as the presence of

retinopathy or variations in retinal vascular caliber, are predictive of a range of systemic and ocular conditions, such as diabetes, stroke, coronary heart disease and heart failure, and diabetic retinopathy.^1–7

It has been hypothesized that dilation of retinal vessels in response to diffuse luminance flicker may reflect intrinsic endothelial function,⁸ mediated possibly by nitric oxide.^9–12 Thus, such measurements may allow in vivo quantification of retinal endothelial function, which may help in understanding the physiological and pathological alterations of retinal

ISSN: 0271-3683 print/ 1460-2202 online DOI: 10.3109/02713680903353764

ORIGINAL ARTICLE

Reproducibility of the Retinal Vascular Response to Flicker Light in Asians

Thanh T. Nguyen

¹

, Andreas J. Kreis

¹

, Ryo Kawasaki

¹

, Jie Jin Wang

^1,2

, Bernd-U. Seifert

³

, Walthard Vilser

³

, Edgar Nagel

⁴

, and Tien Y. Wong

^1,5

1Centre for Eye Research Australia, University of Melbourne, Australia

2Centre for Vision Research, Westmead Millennium Institute, University of Sydney, Australia

3IMEDOS, Jena, Germany

4Ophthalmic Private Practice, Rudolstadt, Germany

5Singapore Eye Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

ABSTRACT

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.

KEYWORDS: Diffuse Luminace Flicker; Reproducibility; Retinal Vascular Imaging; Auto regulation, Vasodilation

Received 22 March 2009; accepted 20 September 2009 Correspondence: Thanh T Nguyen, Center for Eye Research Australia, University of Melbourne, 32 Gisborne Street, Victoria 3002, Australia. E-mail: bittet@yahoo.com

22 March 2009 16 September 2009 20 September 2009

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Flicker-Light Retinal Vasodilation Is Reproducible 1083

microcirculation in various ocular and systemic diseases.

The Dynamic Vessel Analyser (DVA) has previously been used to measure the retinal vessel dilation in response to diffuse luminance flicker. Previous clinical studies using the DVA have shown the impaired dilation to this flicker, reflecting possibly endothelial dysfunction, is associated with systemic diseases,¹³ including hypertension⁸ and diabetes,¹⁴ and ocular diseases, such as diabetic retinopathy¹⁴ and glaucoma.¹⁵ However, although DVA has previously been shown to be reproducible in whites,¹⁶ there have been no similar data in groups/races other than whites, including Asians. In this study, we aim to assess the reproducibility of the retinal vessel dilation in response to flicker-light, as measured by the DVA in Asians and compare the corresponding reproducibility reported in whites.

MATERIALS AND METHODS

We included 33 Asian participants from the Cen- ter for Eye Research Australia, University of Mel- bourne, Australia. The mean age is 34.1 (+13.3) years, and women accounted for 58% of the sample.

The study was conducted in accordance with the Declaration of Helsinki and informed written con- sent was obtained from each participant. Eligibility criteria were normal visual function and no pathological findings upon slit lamp and fundus examination. Furthermore, the participants did not have a history of systemic or ocular diseases. Participants were not fasted. Measurements were obtained by one person (Thanh Nguyen).

DVA

Examination was conducted in a dark room. The participant focused on the tip of a fixation bar within the camera while the fundus was examined under green light. An arteriolar and venular segment of approximately 1.5 mm in length, between half and 2 disc diameters from the margin of the optic disc were selected (see Figure 1). The diameter of the vessel segments was calculated automatically. Baseline vessel diameter was measured for 50 sec, followed by a provocation with flicker light of the same wavelength for 20 sec, and then a non-flicker period for 80 sec. This measurement cycle was repeated twice, with a total duration of 350 sec. When the eye blinked, the vessel segments were not measured and measurement restarted once the vessel segments were automatically re-identified.

The measurements were assessed to ensure quality after each test. Good quality readings were those with minimal missing values and the diameter dilation pat- terns were similar over the three cycles (Figure 2A). If a cycle is of poor reading, then it was removed from the analysis. Retinal arteriolar and venular dilation in response to flickering light was calculated automatically by the DVA software, and defined as the average maximal increase in diameter of the three cycles during flicker-light stimulation. Manual calculation of the flicker-induced dilation was performed if there was only one cycle with good reading.

One eye was measured for each participant, and the same eye was re-measured 30–60 min later.

Repetition mode was used during re-measurement to ensure that the same vessel segments were measured.

Modification of Shape of the Light Source We have modified the shape of the light source of the DVA from a circular shape to a bandage-like shape to reduce the reflection of the light from the eyelids (Figure 3). This was necessary for Asians due to their relatively low vertical height of the palpebral fissure.

Statistical Analysis

Correlation of the 1st and 2nd measures were assessed by Pearson correlation (R²) (Stata, Version 10.0, Stata Corp., College Station, TX, USA). R² values less than 0.40 represent poor to fair correlation, between 0.61 and 0.80 represent substantially high correlation, and 0.81 to 1.00 represent almost perfect correlation.¹⁷

V A

FIGURE 1 An example of the measurement locations—

arteriolar (A) and venular (V) segments.

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1084 T. T. Nguyen et al.

Current Eye Research

Bland–Altman plots were used to assess agreement between the 1st and 2nd measurements.¹⁸

RESULTS

Figure 4 shows an example of the improvement of the readings after modification of the light source in a healthy 27-year-old Asian female. After modification, we can see proper flicker light induced dilation and a reduction of the missing data. The measurements shown were from different vessels (but of the same subject).

Measurements (from one session) from two Asians are unable to be evaluated due to poor quality and are excluded. Table 1 and Figure 5 show the correlation of the 1st and 2nd measurements of arteriolar/

venular diameters at baseline and the maximum dilation. Almost perfect correlation is found between the 1st and 2nd measurements of baseline arteriolar (0.95) and venular (0.98) diameters and of arteriolar maximum dilation (0.85). Substantially high correlation between the 1st and 2nd measurements of venular maximum dilation was found (0.80).

Figure 6 shows the Bland–Altman plots of the differences between the 1st and 2nd measurements against the mean of the two measurements. The mean difference between the 1st and 2nd measurements of arteriolar maximum dilation is 0.0%, and only 1 of 31 subjects (3.2%) is outside the 95% limits of agreement. The mean difference between the 1st and 2nd measurement of venular maximum dilation is 0.2%, and 2 of 31 (6.5%) are outside the 95% limits of agreement.

100 120 140 160 180 200

D (ME)

Zeit (s)350 Zeitverlauf

Flickering periods

0 35 70 105 140 175 210 245 280 315

FIGURE 2 Real time tracing of the retinal venular (top) and arterial (bottom) diameters (y-axis is the diameter in Measure- ment Units which correspond to µm in the Gullstrand’s eye, and the x-axis is the time in seconds). The tracing demonstrated an increase in the vessel diameters after each flickering period.

FIGURE 3 Shape of the light source: Pre- (left) and post- modification (right).

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DISCUSSION

Although DVA has previously been shown to be reproducible in whites,¹⁶ there have been no similar data in Asians. In this study, we show that the measurement of retinal vessels to flicker using the DVA has high reproducibility for repeated measures over a short period of time in Asians, similar to the reliability level previously obtained in whites¹⁶ with the use of the band-shape light source.

Recent studies showed that retinal vascular caliber sizes differed between whites and Asians, and thus speculated that the differences could have been resulted from different contrast of retinal background relatively to the retinal vessels between Asian and Cau- casian eyes.^19,20 Our findings did not provide evidence supporting such a speculation but documented that the amount of light exposed to the retina was likely influenced by the different vertical heights of the palpebral fissure between Asian and Caucasian eyes.

After modification

Before modification

Flickering periods

200 180 160 140 120 100

300 250 200 150 100 50

0 35 70 105 140 175 210 245 280 315Time(s)350

0 35 70 105 140 175 210 245 280 315Time(s) 350

FIGURE 4 An example of the improvement of the readings after modification of the light source in a healthy 27-year-old Asian female. After modification, we can see proper flicker light induced dilation and a reduction of the missing data. The measure- ments shown were from different vessels (but of the same subject). Again, the y-axis is the diameter in Measurement Units which correspond to µm in the Gullstrand’s eye, and the x-axis is the time in seconds. In both situations described below, the venules are wider than the arterioles.

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Current Eye Research TABLE 1 Mean ± standard deviation of retinal vessel baseline diameters and maximum dilation of 1st and 2nd measurements, and Pearson correlation statistics (R²)

Measurement 1st measurement 2nd measurement Difference R²

Arteriole

Baseline diameter* 117.6 ± 12.6 117.7 ± 11.3 -0.1 + 3.9 0.95

Maximum dilation (%)† 3.9 ± 1.8 3.9 ± 1.9 0.0 + 1.0 0.85

Venule

Baseline diameter* 149.5 ± 18.4 149.9 ± 18.4 -0.5 + 3.7 0.98

Maximum dilation (%)† 5.4 ± 2.1 5.1 ± 2.2 0.2 + 1.3 0.80

*Measurement Units which correspond to µm in the Gullstrand’s eye.

†In % to individual baseline.

75 100 125 150 175

2nd measurement (micrometer)

75 100 125 150 175

1st measurement (micrometer) Baseline arteriolar diameter

Asian

75 100 125 150 175 200

2nd measurement (micrometer)

75 100 125 150 175 200

1st measurement (micrometer) Baseline venular diameter

Asian

0 5 10 15

2nd measurement (%)

0 5 10 15

1st measurement (%) Maximum dilation of arteriole

Asian

0 5 10 15

2nd measurement (%)

0 5 10 15

1st measurement (%) Maximum dilation of venule

Asian

FIGURE 5 Plots of the 1st vs. 2nd measurements for baseline vessel diameters (Measurement Units which correspond to µm in the Gullstrand’s eye) and maximum dilation of retinal arteriole and venule. Solid lines represent linear regression, and dashed lines are lines of equality.

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The reproducibility was improved substantially, with changing the shape of the light source, to be similar to that previously reported from whites.¹⁶ As the DVA has wide application potential, the modification of the light source and our study results have important implication for a wider application of this testing.

The endothelium plays a critical role in vascular physiology, and endothelial dysfunction has been suggested to play a pivotal role in the pathogenesis of cardiovascular diseases²¹ and ocular diseases, such as diabetic retinopathy.¹⁴ A major obstacle to clinical research of endothelial dysfunction is the difficulty in

assessing it in vivo. Most measurements of endothe- lial function are time consuming and require highly specialized personnel and equipment. In contrast, the DVA doesn’t require much training, and the measurement can be performed in less than 15 min. The DVA quantifies non-invasively retinal vessel dilation in response to flickering light,⁸ which is believed to reflect endothelial function, given the documentation of the role of nitric oxide played in this flickering light induced retinal vasodilation.^9–12

The strength of this study is to extend the application of DVA beyond whites and confirm the same

−5

−2.5 0 2.5 5

Difference [1st] - [2nd]: arteriolar max dilation (%)

0 5 10

Average of 1st and 2nd measurement: arteriolar max dilation (%) 1/31 = 3.23% outside the limits of agreement

Mean difference 0.044 95% limits of agreement ( 2.060,-1.971)

−5

−2.5 0 2.5 5

Difference [1st] - [2nd]: venular max dilation (%)

0 5 10

Average of 1st and 2nd measurement: venular max dilation (%) 2/31 = 6.45% outside the limits of agreement

Mean difference 0.217 95% limits of agreement ( 2.847,-2.414) Venular maximum dilation

Arteriolar maximum dilation

FIGURE 6 Bland–Altman plots of the differences between the 1st and 2nd measurements against the average of 1st and 2nd measurement of. (Dashed line represents mean difference between 1st and 2nd measurements; gray area represents mean difference ±2 standard deviation.)

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Current Eye Research

measurement validity of the DVA in Asians as that in whites. Possible limitations of this study include that the repeated measurements are taken over a short period of time, and our results are only applicable to healthy persons with healthy eyes and normal visual function. Furthermore, we are unable to say whether the variability between the readings is due to the technical limitations of the DVA machine or the biological/physiological variability in a human subject. In addition, we are uncertain whether this modification will also improve the results of DVA assessment in whites. This will be an area of future research.

In conclusion, we have demonstrated a high reproducibility of repeated measurements over a short period of time using the DVA to assess retinal vessel dilation in response to flickering light in Asians. The response of retinal vessels to diffuse luminance flicker has been suggested to reflect endothelial function.

Thus, such measurements may allow non-invasive quantification of endothelial function to study its association with systemic and ocular diseases. Our findings assure this DVA technique is not only applicable to whites but Asians.

ACKNOWLEDgEMENTS

Declaration of interest: Bernd-U Seifert and Walthard Vilser are employees of Imedos as indicated. The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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