August 2014
Volume 55, Issue 8
Free
Clinical and Epidemiologic Research  |   August 2014
Dynamic Responses in Retinal Vessel Caliber With Flicker Light Stimulation in Eyes With Diabetic Retinopathy
Author Affiliations & Notes
  • Laurence S. Lim
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Lieng H. Ling
    Cardiac Department, National University Heart Centre, Singapore
    Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Peng Guan Ong
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
  • Wallace Foulds
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
  • E. Shyong Tai
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
    Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Edmund Wong
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
  • Shu Yen Lee
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
  • Doric Wong
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
  • Chui Ming Gemmy Cheung
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Tien Yin Wong
    Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS Graduate Medical School, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Correspondence: Laurence S. Lim, Singapore National Eye Centre, 11 Third Hospital Avenue, Singapore 168751; llimshen@gmail.com
Investigative Ophthalmology & Visual Science August 2014, Vol.55, 5207-5213. doi:10.1167/iovs.14-14301
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      Laurence S. Lim, Lieng H. Ling, Peng Guan Ong, Wallace Foulds, E. Shyong Tai, Edmund Wong, Shu Yen Lee, Doric Wong, Chui Ming Gemmy Cheung, Tien Yin Wong; Dynamic Responses in Retinal Vessel Caliber With Flicker Light Stimulation in Eyes With Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2014;55(8):5207-5213. doi: 10.1167/iovs.14-14301.

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

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Abstract

Purpose.: This study investigated the responses of retinal vessels to flickering light in diabetic patients with various stages of diabetic retinopathy (DR).

Methods.: This cross-sectional observational study evaluated adult subjects with diabetes mellitus. The Dynamic Vessel Analyzer (DVA) was used to measure retinal vascular dilatation in response to diffuse illuminance flicker. Diabetic retinopathy was graded from retinal photography.

Results.: There were 279 subjects in total, with a mean age of 59.9 ± 9.2 years. The majority were male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7% ± 1.4% and 13.9 ± 10.4 years, respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension, and hyperlipidemia, the responses of both retinal arterioles and venules to flicker stimulation decreased continuously with increasing stages of diabetic retinopathy (P = 0.008 and <0.001, respectively). Subjects with reduced arteriolar dilation responses were more likely to have any DR (odds ratio, OR, 1.20, [95% confidence interval (CI), 1.01–1.45], P = 0.045, per SD decrease). Subjects with reduced venular dilation responses were more likely to have any DR, moderate DR, or vision-threatening DR (OR: 1.27 [1.04–1.53], P = 0.02; OR: 1.27 (1.06–1.49), P = 0.007; and OR: 1.51 (1.14–1.50), P = 0.002; per SD decrease, respectively).

Conclusions.: The responses of retinal arterioles and venules to flickering light are reduced in subjects with DR, and decrease progressively with more severe stages of DR.

Introduction
Diabetic retinopathy (DR) is a leading cause of visual loss and blindness worldwide, affecting more than 100 million persons, with sight-threatening disease affecting 25 million persons. 1,2 While clear associations with the traditional risk factors such as diabetes duration, glycemic control, and blood pressure have been established, these factors have been shown to account for only 40% to 60% of the variability in DR severity. 3 Novel risk factors and pathways are likely to also account for the development of DR. 3,4  
The Dynamic Vessel Analyzer (DVA) measures retinal vascular dilatation in response to diffuse illuminance flicker. Human studies have demonstrated that retinal vessel diameters increase in response to stimulation with diffuse luminance flicker. 58 This response represents a form of functional hyperemia mediated by neurovascular coupling, and may be modulated by nitric oxide (NO) levels. 912 Impaired retinal arteriolar and venular dilation responses to flicker stimulation have been shown in subjects with diabetes, 1320 but data on the associations with DR and DR severity are limited. Nguyen 19 demonstrated that diabetic subjects with reduced flicker light–induced vasodilation were more likely to have DR, while only one other study has investigated the relationship between flicker responses and DR severity. 17  
The aim of this study was to assess the associations between retinal vessel diameter responses to flickering light measured using the DVA and the presence and severity of DR. 
Methods
This was a prospectively designed, cross-sectional observational study. Adult subjects with diabetes were recruited from the DR screening clinics at a tertiary eye care center. All subjects were of Chinese ethnicity. 
All study procedures were performed in accordance with the tenets of the Declaration of Helsinki as revised in 1989. Written informed consent was obtained from the subjects, and the study was approved by the Institutional Review Board of the Singapore Eye Research Institute. 
Assessment of Diabetic Retinopathy
Retinal photography was performed following a standardized protocol, the details of which have been described in other publications from our center. 21 Briefly, after pupil dilation, two retinal photographs, centered at the optic disc and macula, were obtained from both eyes of each participant using a digital retinal camera (Canon CR-DGi with a 10-D SLR back; Canon, Inc., Tokyo, Japan). Photographs were then graded by trained, masked graders at the Singapore Eye Research Institute. 
Diabetic retinopathy was considered present if any characteristic lesion as defined by the Early Treatment Diabetic Retinopathy Study (ETDRS) severity scale was present: microaneurysms (MA), hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading, and new vessels. 22 For each eye, a retinopathy severity score was assigned according to a scale modified from the Airlie House classification system. 22 Macular edema (ME) was defined by hard exudates in the presence of MA and blot hemorrhage within 1 disc diameter from the foveal center or the presence of focal photocoagulation scars in the macular area. Clinically significant macular edema (CSME) was considered present when the ME was within 500 mm of the foveal center or if focal laser photocoagulation scars were present in the macular area. Retinopathy severity was categorized as minimal nonproliferative diabetic retinopathy (NPDR; ETDRS levels 15–20); mild NPDR (level 35); moderate NPDR (levels 43–47); severe NPDR (level 53); and proliferative retinopathy (level > 60). 
We defined three additional outcomes for analyses. Any DR was defined as minimal DR or worse. Moderate DR was defined as moderate NPDR or worse and vision-threatening retinopathy as the presence of severe NPDR, proliferative retinopathy, or CSME using the Eye Diseases Prevalence Research Group definition. 23 If an eye was ungradable, it was excluded from that particular analysis. 
Assessment of Retinal Vessel Responses Using the Dynamic Vessel Analyzer
The DVA (DVA; Imedos Systems UG, Jena, Germany) is a commercially available system consisting of a fundus camera, video camera, real-time monitor, and a personal computer with analysis software. The DVA measures retinal vessel dilatation in response to diffuse luminance flicker. 5,8 Examinations are conducted under half-lighting conditions. The subject focuses on the tip of a fixation bar within the retinal camera while the fundus is examined under red-free (green) light. An arteriolar and venular segment between 0.5 and 2 disc diameters from the margin of the optic disc is selected. The mean diameter of the arterial and venous vessel segments are calculated and recorded automatically. Baseline vessel diameter is measured for 50 seconds, followed by a provocation with flickering light of the same wavelength for 20 seconds, and then a nonflicker period for 80 seconds. This measurement cycle is repeated twice, with a total duration of 350 seconds per eye. The system automatically stops the measurement when the subject blinks or the eye moves, and restarts it once the vessel segments are automatically reidentified. Retinal arteriolar and venular dilatation in response to flickering light are calculated automatically by the DVA software. The responses are represented as an average increase in the vessel diameter in response to the flickering-light during the three measurement cycles, and defined as the percentage increase relative to the baseline diameter size. We analyzed the arteriolar dilation and constriction responses as well as the venular dilation responses (Fig.). 
Figure
 
Dynamic vessel analyzer traces from (A) subject without retinopathy showing dilatory responses in both arterioles (upper panel) and venules (lower panel) to flicker light, and (B) subject with diabetic retinopathy showing diminished dilatory responses.
Figure
 
Dynamic vessel analyzer traces from (A) subject without retinopathy showing dilatory responses in both arterioles (upper panel) and venules (lower panel) to flicker light, and (B) subject with diabetic retinopathy showing diminished dilatory responses.
Other Study Evaluations
Detailed interviewer-administered questionnaires were administered to collect relevant sociodemographic data and medical history. Data collected included country and state of birth, marital status, education, occupation, and current housing status, participants' lifestyle factors, history of smoking, eye symptoms, use of spectacles, falls and fractures, current medications, systemic medical and surgical history, and family history of eye diseases. 
Fasting venous blood sample were collected for biochemistry tests including serum lipids (total cholesterol, high- and low-density lipoprotein cholesterol), glycosylated hemoglobin A1c (HbA1C), creatinine and glucose. 
Statistical Analyses
Models were based on analyses by eyes using generalized estimating equation (GEE) models to account for correlations between the two eyes. Multivariate logistic regression models were constructed with the DR outcomes as the dependent variables to assess the relationship with arteriolar and venular dilation as independent variables. Initial adjustments were made for age and sex. Smoking, mean duration of diabetes, glycated hemoglobin, hypertension, and hyperlipidemia were added in a second multivariate model. We regarded P values < 0.05 from 2-sided tests as statistically significant. All statistical analyses were performed using commercial software (STATA version 12; StataCorp LP, College Station, TX, USA). 
Results
In total, 558 eyes of 279 subjects were included. The mean age of the subjects was 59.9 ± 9.2 years, the majority were male (73%), and the mean HbA1c level and mean duration of diabetes were 7.7 ± 1.4% and 13.9 ± 10.4 years, respectively. There were 15 subjects with type 1 and 216 subjects with type 2 diabetes, respectively. All of the type 1 diabetics were on insulin therapy, while 48 of the type 2 diabetics were receiving insulin. 
The distribution of DR severity was no DR in 233 (42%) eyes; minimal NPDR in 66 (12%) eyes; mild NPDR in 83 (15%) eyes; moderate NPDR in 127 (23%) eyes; severe NPDR in 21 (4%) eyes; and proliferative DR in 28 (5%) eyes (Table 1). 
Table 1
 
Baseline Characteristics of Study Participants (Eyes) by DR Grade
Table 1
 
Baseline Characteristics of Study Participants (Eyes) by DR Grade
Total eyes, n = 558 No DR, n = 233 Minimal NPDR, n = 66 Mild NPDR, n = 83 Moderate DR, n = 127 Severe DR, n = 21 Proliferative DR, n = 28 P Value Trend
Mean age, y (SD) 59.9 (9.22) 61.1 (9.09) 60.2 (8.31) 58.7 (9.84) 59.2 (8.74) 55.7 (10.5) 58.2 (10.6) <0.001
Mean duration of diabetes, y (SD) 13.9 (10.4) 12.0 (10.4) 14.2 (10.1) 16.2 (9.6) 14.9 (10.5) 14.0 (9.06) 17.6 (10.5) <0.001
Mean HbA1c, % (SD) 7.73 (1.38) 7.26 (1.18) 7.68 (1.08) 8.03 (1.20) 8.05 (1.61) 9.09 (1.35) 8.40 (1.57) <0.001
Female sex, n (%) 152 (27.24) 65 (27.9) 17 (25.76) 24 (28.92) 34 (26.77) 5 (23.81) 7 (25) 0.711
Smoking, n (%) 0.232
 Never 371 (66.49)
 Current 78 (13.58) 23 (9.87) 7 (10.6) 17 (20.5) 22 (17.3) 4 (19.1) 5 (17.9)
 Past 109 (19.53) 48 (20.6) 13 (19.7) 13 (15.7) 25 (19.7) 4 (19.1) 6 (21.4)
Hypertension, n (%) 420 (76.36) 179 (78.2) 48 (73.9) 57 (69.5) 100 (78.7) 16 (76.2) 20 (76.9) 0.832
Hyperlipidemia, n (%) 377 (67.56) 158 (67.8) 48 (72.7) 53 (63.9) 84 (66.1) 12 (57.1) 22 (78.6) 0.93
In age-sex adjusted models, the arteriolar dilatory responses showed a U-shaped relationship with increasing severity of DR. These relationships persisted with multivariate adjustment for smoking, mean duration of diabetes, glycosylated hemoglobin, hypertension, and hyperlipidemia. The mean arteriolar dilatory responses were 1.82%, 1.37%, 0.75%, 0.88%, 1.15%, and 1.67% for eyes with no DR, minimal NPDR, mild NPDR, moderate NPDR, severe NPDR, and proliferative DR respectively (P = 0.008). In age-sex adjusted models, the venular dilatory responses showed a progressive declining trend with increasing severity of DR These relationships persisted with multivariate adjustment. The mean venular dilatory responses were 3.50%, 2.89%, 2.64%, 2.17%, 1.87%, and 1.64% for eyes with no DR, minimal NPDR, mild NPDR, moderate NPDR, severe NPDR and proliferative DR, respectively (P < 0.001). There were no consistent relationships seen for arteriolar constriction (Table 2). 
Table 2
 
Associations Between Changes in Retinal Vessel Diameter With Flicker Light and DR Grade
Table 2
 
Associations Between Changes in Retinal Vessel Diameter With Flicker Light and DR Grade
Total DR Cases, Eyes No DR, n (%) Minimal NPDR, n (%) Mild NPDR, n (%) Moderate DR, n (%) Severe DR, n (%) Proliferative DR, n (%) P Value*
558 233 (41.8) 66 (20.3) 83 (25.5) 127 (39.1) 21 (6.46) 28 (8.62)
Arteriolar dilation, μm Age-sex adjusted 1.77 (1.47, 2.07) 1.38 (0.82, 1.94) 0.82 (0.32, 1.32) 0.82 (0.41, 1.22) 1.13 (0.13, 2.13) 1.46 (0.59, 2.32) 0.002
Multivariate adjusted† 1.82 (1.5, 2.15) 1.37 (0.80, 1.95) 0.75 (0.21, 1.28) 0.88 (0.46, 1.31) 1.15 (0.10, 2.21) 1.67 (0.72, 2.62) 0.008
Venular dilation, μm Age-sex adjusted 3.57 (3.28, 3.87) 2.88 (2.32, 3.43) 2.57 (2.07, 3.06) 2.11 (1.71, 2.51) 1.83 (0.85, 2.82) 1.48 (0.63, 2.33) <0.001
Multivariate adjusted* 3.50 (3.18, 3.82) 2.89 (2.33, 3.46) 2.64 (2.11, 3.17) 2.17 (1.75, 2.59) 1.87 (0.82, 2.91) 1.64 (0.70,2.58) <0.001
When stratified by quartiles, the subjects in the lower quartiles of arteriolar dilation responses were more likely to have any DR than those in the higher quartiles after multivariate adjustment (P = 0.02). Subjects with reduced arteriolar dilation responses were more likely to have any DR (odds ratio [OR], 1.20 [95% confidence interval, (CI) 1.01–1.45], P = 0.045, per SD decrease). Subjects in the lower quartiles of venular dilation responses were also more likely to have any DR, or vision-threatening DR (P = 0.009 and 0.02, respectively). Subjects with reduced venular dilation responses were more likely to have any DR (OR: 1.27 [1.04–1.53] per SD decrease, P = 0.02), moderate DR (OR: 1.27 [1.06–1.49] per SD decrease, P = 0.007) or vision-threatening DR (OR: 1.51 [1.14–1.50] per SD decrease, P = 0.002) (Table 3). 
Table 3
 
Associations of DVA Indices With DR in Multivariate Adjusted Models
Table 3
 
Associations of DVA Indices With DR in Multivariate Adjusted Models
Any DR Moderate DR Vision Threatening DR
Age-Sex Adjusted Multivariate Adjusted Age-Sex Adjusted Multivariate Adjusted Age-Sex Adjusted Multivariate Adjusted
OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Arteriolar dilation
 Q1 (−11.2 to −0.1) Referent Referent Referent Referent Referent Referent
 Q2 (0 to 0.8) 0.82 (0.58, 1.16) 0.79 (0.52, 1.21) 0.72 (0.51, 1.01) 0.72 (0.49, 1.07) 0.59 (0.26, 1.33) 0.57 (0.21, 1.51)
 Q3 (0.9 to 2.3) 0.67 (0.44, 1) 0.59 (0.36, 0.98) 0.66 (0.45, 0.96) 0.70 (0.46, 1.06) 0.50 (0.22, 1.14) 0.46 (0.17, 1.23)
 Q4 (2.4 to 11.8) 0.60 (0.39, 0.91) 0.58 (0.34, 0.96) 0.58 (0.36, 0.92) 0.67 (0.42, 1.09) 0.65 (0.27, 1.60) 0.81 (0.30, 2.16)
 Per SD increase; P 0.85 (0.73, 0.99); 0.042 0.83 (0.69, 0.99); 0.045 0.95 (0.81, 1.10); 0.495 0.98 (0.83, 1.16); 0.853 1.03 (0.74, 1.41); 0.878 1.13 (0.78, 1.63); 0.522
P value for trend 0.009 0.015 0.015 0.094 0.324 0.592
Venular dilation
 Q1 (−12.6 to 13) Referent Referent Referent Referent Referent Referent
 Q2 (1.4 to 2.5) 0.77 (0.52, 1.14) 0.68 (0.43, 1.07) 0.89 (0.59, 1.32) 0.94 (0.61, 1.45) 0.36 (0.17, 0.81) 0.38 (0.18, 0.81)
 Q3 (2.6 to 4.1) 0.76 (0.50, 1.16) 0.72 (0.44, 1.19) 0.82 (0.54, 1.24) 0.86 (0.54, 1.38) 0.70 (0.36, 1.38) 0.74 (0.35, 1.54)
 Q4 (4.2 to 14.4) 0.43 (0.27, 0.68) 0.46 (0.27, 0.78) 0.54 (0.35, 0.82) 0.60 (0.37, 0.97) 0.24 (0.09, 0.63) 0.22 (0.07, 0.66)
 Per SD increase; P 0.75 (0.62, 0.90); 0.002 0.79 (0.65, 0.96); 0.016 0.76 (0.65, 0.90); 0.001 0.79 (0.67, 0.94); 0.007 0.63 (0.46, 0.85); 0.003 0.66 (0.50, 0.86); 0.002
P value for trend 0.001 0.009 0.009 0.059 0.008 0.017
Discussion
Our study in a cohort of persons with diabetes demonstrates reduced arteriolar and venular dilatory responses to flickering light in subjects with DR progressive diminishment in both arteriolar and venular dilatory responses was seen with increasing severity of DR impaired arteriolar dilation responses were most consistently associated with the presence of any DR, while impaired venular dilatory responses were associated with any DR, moderate DR, and vision-threatening DR. 
Human studies have demonstrated that retinal vessel diameters increase in response to stimulation with diffuse luminance flicker. 68 The DVA has been specifically designed to measure this response, and Polak et al. 7,8 have demonstrated its sensitivity and reproducibility in this context. In a cohort of healthy young volunteers, the sensitivity for detecting changes in retinal arterial and venous caliber in response to sodium nitroprusside, an NO donor, was between 3% and 7%, and the intraclass correlation coefficients were between 0.96 and 0.98. Changes in the DVA responses have been shown in subjects with prediabetes and diabetes. Lott 13 compared DVA responses in 22 subjects with prediabetes, 25 subjects with type 2 diabetes, and 19 healthy controls. Both prediabetic and diabetic individuals had diminished arteriolar and venular vasodilator responses compared with normal subjects. Similarly, it has been shown that flicker responses are comparably diminished in children and adolescents who have type 1 diabetes mellitus or who are obese. 20  
While associations between the DVA responses and diabetes have been evaluated in many studies, limited data on the associations with DR exist. Nguyen 19 investigated the DVA responses in a cohort of Caucasian subjects consisting of 103 normal controls and 224 patients with diabetes, of whom 37% had DR. Subjects with diabetes had reduced retinal arteriolar and venular dilatory responses compared with normal controls. Subjects with diminished arteriolar and venular responses were more likely to have DR (odds ratios of 2.2 and 2.5, respectively). Mandecka 17 evaluated 53 healthy volunteers, 68 diabetic patients who were type 1, and 172 diabetic patients who were type 2 from a predominantly Caucasian population. After adjustments for age, sex, and antihypertensive treatment, retinal vasodilator responses were significantly diminished in patients with type 1 and type 2 diabetes compared with healthy volunteers, while retinal venular dilation responses were significantly diminished in patients with type 2 diabetes compared with healthy volunteers. The flicker responses of both arterioles and venules showed continuously diminishing responses with increasing severity of DR arteriolar constriction responses; but did not show consistent relationships with either diabetes or DR. Our results in an Asian population are generally in agreement with the results of these studies, with more consistent associations demonstrated for venular dilation. The progressive diminishment of flicker responses with increasing severity of DR is biologically plausible, indicating progressive impairment of vasculoregulatory mechanisms with more severe DR. 
The association between impaired vascular dilatory responses and DR may be attributed to the combined mechanisms of endothelial dysfunction, impaired autoregulation, and dysfunctional neurovascular coupling. Lott 15 has shown that flicker-induced vasodilation is inversely correlated with hyperoxia-induced vasoconstriction in subjects with type 2 diabetes, suggesting a common autoregulatory pathway. Neurovascular coupling refers to the changes in vessel caliber and blood flow induced by neuronal stimuli. The mechanisms underlying neurovascular coupling are complex and incompletely understood. However, current evidence suggests that glial cells are mediators of vascular responses in the retina, and NO functions as a modulator of these responses. 912 It has been shown that both glial stimulation and light stimulation are able to induce changes in vascular caliber. 10 In turn, nitric oxide influences these responses, possibly by modulating the production of arachidonic acid derivatives. 10,11 Metea 10 has shown that NO can reverse the dilatory responses of arterioles to light and cause constriction instead in the isolated rat wholemount retina. In vivo, Dorner 6 has demonstrated that NO regulates both resting vascular caliber and the responses of retinal vessels to flickering light. In a cohort of 12 healthy young subjects, an infusion of an NO synthase inhibitor reduced resting arteriolar and venular diameter, and also attenuated the dilatory responses of arterioles and venules to flickering light. In diabetic rats, attenuated light and glial-induced vasodilation has been correlated with NO synthase upregulation. 24 The reduced flicker–induced responses seen in subjects with more severe DR may thus be mediated by localized perturbations in NO levels. Lecleire-Collet et al. 16 investigated the DVA responses in normotensive patients with diabetes without DR and correlated them with ERG findings. Vasodilatory responses were found to be impaired, and correlations were found between flicker-induced arteriolar vasodilation and the amplitude and implicit time of the N95 wave of pattern ERG, the b-wave implicit time of rod ERG, and the oscillatory potentials. In contrast, Lasta 14 correlated the pattern ERG responses with DVA responses in type 1 diabetes patients and found that diminished DVA responses occurred before pattern ERG abnormalities became evident. However, the authors acknowledged several methodological differences between the studies and suggested that in later stages of DR, reduced neural function could still contribute to the impaired DVA responses. These findings have attempted to establish a link between DVA responses and inner retinal neural dysfunction. This relationship may simply reflect parallel changes in the retinal vessels and retinal neurons that occur in diabetes, or may indicate that the retinal vessel dilatory responses to flickering light are subject to neurovascular regulation. Consistent with theories that DR is a neurodegenerative disease, 25 it has been shown that reductions in flickering light–induced vasodilatory responses can be demonstrated in subjects with well-controlled diabetes before the onset of DR. 16,18  
There is increasing recognition that endothelial cell dysfunction is an early step in the pathogenetic pathways for diabetes and its complications, and may thus constitute a novel risk factor for DR. 2530 While studies show endothelial dysfunction may precede the development of diabetes, 3133 and is linked to the development of macrovascular complications including cardiovascular diseases, 34,35 there are fewer consistent associations with microvascular complications and DR. 27,30 The current evidence suggests that DVA responses may only partially be mediated by endothelial dysfunction. Pump-evaluated DVA responses as well as flow-mediated vasodilatation (FMD), the standard measure of endothelial dysfunction, in diabetic subjects. 36 Both DVA responses and FMD were reduced, but only a weak correlation was demonstrated between the two measures. 
The strengths of our study include a relatively large cohort, the standardized assessments of DR severity from retinal photography as well as systemic covariates. The DVA device is also notable for being operator independent and for producing highly reproducible results. 8 General limitations of our study include the possibility of selection bias, and the cross sectional design which restricts inferences of causality. We also did not analyze type 1 and type 2 diabetes subjects separately due to the small proportion of subjects with type 1 diabetes, and responses have been shown to differ between these groups of patients. 17 It should also be noted that while the DVA provides data on vessel caliber, it does not give an accurate indication of blood flow. Changes in arteriolar and venular caliber may not reflect changes in the microcirculation. As such, the relevance of our results with the DVA to DR, which is primarily a microangiopathy, needs cautious interpretation. 
In conclusion, our study has shown that retinal arteriolar and venular dilatory responses are reduced in subjects with DR and this response is further reduced in more severe DR. These findings may reflect endothelial dysfunction or disordered neuroregulation of retinal vascular tone in eyes with DR. 
Acknowledgments
Supported by National Medical Research Council Grant R710/60/2009. 
Disclosure: L.S. Lim, None; L.H. Ling, None; P.G. Ong, None; W. Foulds, None; E.S. Tai, None; E. Wong, None; S.Y. Lee, None; D. Wong, None; C.M.G. Cheung, None; T.Y. Wong, None 
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Figure
 
Dynamic vessel analyzer traces from (A) subject without retinopathy showing dilatory responses in both arterioles (upper panel) and venules (lower panel) to flicker light, and (B) subject with diabetic retinopathy showing diminished dilatory responses.
Figure
 
Dynamic vessel analyzer traces from (A) subject without retinopathy showing dilatory responses in both arterioles (upper panel) and venules (lower panel) to flicker light, and (B) subject with diabetic retinopathy showing diminished dilatory responses.
Table 1
 
Baseline Characteristics of Study Participants (Eyes) by DR Grade
Table 1
 
Baseline Characteristics of Study Participants (Eyes) by DR Grade
Total eyes, n = 558 No DR, n = 233 Minimal NPDR, n = 66 Mild NPDR, n = 83 Moderate DR, n = 127 Severe DR, n = 21 Proliferative DR, n = 28 P Value Trend
Mean age, y (SD) 59.9 (9.22) 61.1 (9.09) 60.2 (8.31) 58.7 (9.84) 59.2 (8.74) 55.7 (10.5) 58.2 (10.6) <0.001
Mean duration of diabetes, y (SD) 13.9 (10.4) 12.0 (10.4) 14.2 (10.1) 16.2 (9.6) 14.9 (10.5) 14.0 (9.06) 17.6 (10.5) <0.001
Mean HbA1c, % (SD) 7.73 (1.38) 7.26 (1.18) 7.68 (1.08) 8.03 (1.20) 8.05 (1.61) 9.09 (1.35) 8.40 (1.57) <0.001
Female sex, n (%) 152 (27.24) 65 (27.9) 17 (25.76) 24 (28.92) 34 (26.77) 5 (23.81) 7 (25) 0.711
Smoking, n (%) 0.232
 Never 371 (66.49)
 Current 78 (13.58) 23 (9.87) 7 (10.6) 17 (20.5) 22 (17.3) 4 (19.1) 5 (17.9)
 Past 109 (19.53) 48 (20.6) 13 (19.7) 13 (15.7) 25 (19.7) 4 (19.1) 6 (21.4)
Hypertension, n (%) 420 (76.36) 179 (78.2) 48 (73.9) 57 (69.5) 100 (78.7) 16 (76.2) 20 (76.9) 0.832
Hyperlipidemia, n (%) 377 (67.56) 158 (67.8) 48 (72.7) 53 (63.9) 84 (66.1) 12 (57.1) 22 (78.6) 0.93
Table 2
 
Associations Between Changes in Retinal Vessel Diameter With Flicker Light and DR Grade
Table 2
 
Associations Between Changes in Retinal Vessel Diameter With Flicker Light and DR Grade
Total DR Cases, Eyes No DR, n (%) Minimal NPDR, n (%) Mild NPDR, n (%) Moderate DR, n (%) Severe DR, n (%) Proliferative DR, n (%) P Value*
558 233 (41.8) 66 (20.3) 83 (25.5) 127 (39.1) 21 (6.46) 28 (8.62)
Arteriolar dilation, μm Age-sex adjusted 1.77 (1.47, 2.07) 1.38 (0.82, 1.94) 0.82 (0.32, 1.32) 0.82 (0.41, 1.22) 1.13 (0.13, 2.13) 1.46 (0.59, 2.32) 0.002
Multivariate adjusted† 1.82 (1.5, 2.15) 1.37 (0.80, 1.95) 0.75 (0.21, 1.28) 0.88 (0.46, 1.31) 1.15 (0.10, 2.21) 1.67 (0.72, 2.62) 0.008
Venular dilation, μm Age-sex adjusted 3.57 (3.28, 3.87) 2.88 (2.32, 3.43) 2.57 (2.07, 3.06) 2.11 (1.71, 2.51) 1.83 (0.85, 2.82) 1.48 (0.63, 2.33) <0.001
Multivariate adjusted* 3.50 (3.18, 3.82) 2.89 (2.33, 3.46) 2.64 (2.11, 3.17) 2.17 (1.75, 2.59) 1.87 (0.82, 2.91) 1.64 (0.70,2.58) <0.001
Table 3
 
Associations of DVA Indices With DR in Multivariate Adjusted Models
Table 3
 
Associations of DVA Indices With DR in Multivariate Adjusted Models
Any DR Moderate DR Vision Threatening DR
Age-Sex Adjusted Multivariate Adjusted Age-Sex Adjusted Multivariate Adjusted Age-Sex Adjusted Multivariate Adjusted
OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Arteriolar dilation
 Q1 (−11.2 to −0.1) Referent Referent Referent Referent Referent Referent
 Q2 (0 to 0.8) 0.82 (0.58, 1.16) 0.79 (0.52, 1.21) 0.72 (0.51, 1.01) 0.72 (0.49, 1.07) 0.59 (0.26, 1.33) 0.57 (0.21, 1.51)
 Q3 (0.9 to 2.3) 0.67 (0.44, 1) 0.59 (0.36, 0.98) 0.66 (0.45, 0.96) 0.70 (0.46, 1.06) 0.50 (0.22, 1.14) 0.46 (0.17, 1.23)
 Q4 (2.4 to 11.8) 0.60 (0.39, 0.91) 0.58 (0.34, 0.96) 0.58 (0.36, 0.92) 0.67 (0.42, 1.09) 0.65 (0.27, 1.60) 0.81 (0.30, 2.16)
 Per SD increase; P 0.85 (0.73, 0.99); 0.042 0.83 (0.69, 0.99); 0.045 0.95 (0.81, 1.10); 0.495 0.98 (0.83, 1.16); 0.853 1.03 (0.74, 1.41); 0.878 1.13 (0.78, 1.63); 0.522
P value for trend 0.009 0.015 0.015 0.094 0.324 0.592
Venular dilation
 Q1 (−12.6 to 13) Referent Referent Referent Referent Referent Referent
 Q2 (1.4 to 2.5) 0.77 (0.52, 1.14) 0.68 (0.43, 1.07) 0.89 (0.59, 1.32) 0.94 (0.61, 1.45) 0.36 (0.17, 0.81) 0.38 (0.18, 0.81)
 Q3 (2.6 to 4.1) 0.76 (0.50, 1.16) 0.72 (0.44, 1.19) 0.82 (0.54, 1.24) 0.86 (0.54, 1.38) 0.70 (0.36, 1.38) 0.74 (0.35, 1.54)
 Q4 (4.2 to 14.4) 0.43 (0.27, 0.68) 0.46 (0.27, 0.78) 0.54 (0.35, 0.82) 0.60 (0.37, 0.97) 0.24 (0.09, 0.63) 0.22 (0.07, 0.66)
 Per SD increase; P 0.75 (0.62, 0.90); 0.002 0.79 (0.65, 0.96); 0.016 0.76 (0.65, 0.90); 0.001 0.79 (0.67, 0.94); 0.007 0.63 (0.46, 0.85); 0.003 0.66 (0.50, 0.86); 0.002
P value for trend 0.001 0.009 0.009 0.059 0.008 0.017
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