Dynamic Responses in Retinal Vessel Caliber With Flicker Light Stimulation and Risk of Diabetic Retinopathy and Its Progression

Abstract

Purpose: This study investigated the associations between the responses of retinal vessels to flickering light and the incidence and progression of diabetic retinopathy (DR).

Methods: A prospective cohort study of adult subjects with diabetes mellitus. The dynamic vessel analyser (DVA) was used to measure retinal vascular dilatation in response to diffuse illuminance flicker. Diabetic retinopathy was graded from retinal photography at baseline and at 1 year. Incident DR and two-step change in DR were analyzed.

Results: There were 276 subjects in total, with a mean age of 59.8 ± 8.9 years. The majority were male (73%) and the mean glycated hemoglobin A1c (HbA1c) level and mean duration of diabetes were 7.7 ± 1.4% and 14.0 ± 10.5 years, respectively. After adjustments for age, sex, smoking, duration of diabetes, HbA1c, hypertension, and hyperlipidemia, the responses of retinal arterioles to flicker stimulation were lower in subjects with incident DR (P = 0.048). Subjects with greater arteriolar dilatory responses were less likely to have DR progression (odds ratio [OR] 1.85, [95% confidence interval [CI] 1.33–2.56], P = 0.012, per SD decrease). Subjects with greater venular dilatory responses were also less likely to have DR progression (OR 1.89, [95% CI 1.35–2.63], P = 0.003, per SD decrease). There were no significant associations between arteriolar or venular dilation response and incident proliferative DR (PDR) and DR regression.

Conclusions: Reduced retinal arteriolar and venular dilatory responses to flickering light are associated with risk of DR progression at 1 year in adult patients with diabetes.

In persons with diabetes, the risk of developing diabetic retinopathy (DR) and the risk of DR progression have been clearly shown to be associated with traditional risk factors, such as diabetes duration, glycemic control, and blood pressure.^1–3 Nevertheless, these factors account for only 40% to 60% of the variability in DR severity between persons with diabetes.⁴ Unknown risk factors and pathways are likely to also account for an individual's risk of DR.^4,5 

The pathogenesis of DR is complex. While primarily thought of as a microangiopathy, there is now increasing evidence that neurodegeneration pathways may play a critical role in early stages of DR, possibly preceding the classic microvascular lesions of microaneurysms and retinal hemorrhages.^6–8 Observations of thinner inner retinal layers in the macula of type 2 diabetic patients with minimal DR have long been reported,^9–11 supporting the concept that early DR includes a neurodegenerative component. Furthermore, the neurovascular interface in the retina likely plays a critical role, and studies show neurovascular coupling, modulated by nitric oxide (NO) levels and perhaps reflecting retinal vascular endothelium function, is one of the pathways in the development of DR.^{6–8,12–15} However, measuring neurovascular changes in vivo has been limited in clinical settings. The dynamic vessel analyser (DVA; IMEDOS, Jena, Germany) measures retinal vascular dilatation in response to diffuse illuminance flicker, based on the observation that retinal vessel diameters increase in response to stimulation with diffuse luminance flicker.^16–19 This response may represent a form of functional hyperemia, mediated by neurovascular coupling and NO levels.^20–23 Impaired retinal arteriolar and venular dilation responses to flicker stimulation have been demonstrated in subjects with prediabetes and diabetes in comparison with nondiabetic subjects.^13,24–30 Limited data have also shown associations of retinal responses with DR and DR severity in persons with diabetes, supporting the concept that DR may be related to underlying endothelial dysfunction. For example, in a cross-sectional study, Nguyen and colleagues²⁹ demonstrated that diabetic subjects with reduced flicker light–induced vasodilation were more likely to have DR. Mandecka²⁷ has similarly shown a continuous reduction in flicker responses with increasing DR severity. Likewise, in our previous cross-sectional study of 279 Asian subjects with diabetes, we have shown that retinal vessel responses to flicker light are diminished in subjects with DR, and decrease progressively with increasing DR severity.³¹ 

However, there are no prospective data on the possible associations between dynamic retinal vessel responses and DR incidence and progression. The aim of this study was to assess prospectively the associations between retinal vessel diameter responses to flickering light measured using the DVA at baseline and the incidence and progression of DR in type 2 diabetes after 1 year. Our primary hypothesis is that reduced retinal arteriolar and venular dilatory responses to flickering light, are associated with incidence and progression of DR. 

Methods

This was a prospectively designed cohort study. We recruited adult subjects with type 2 diabetes from the DR screening clinics at a tertiary eye care center. All subjects were of Chinese ethnicity.³¹ At the baseline examination, the subjects were evaluated for demographic and systemic risk factors, and underwent DVA testing and retinal photography. The subjects were recalled after 1 year for repeat retinal photography to assess DR incidence or progression. Detailed interviewer-administered questionnaires were used to collect relevant sociodemographic data and medical history at the baseline visit. 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-density lipoproteins [HDL], low-density lipoproteins [LDL] cholesterol), glycated hemoglobin A1c (HbA1C), creatinine, and glucose. 

In total, 276 subjects were included. The mean age of the subjects was 59.8 ± 8.9 years, the majority was male (73%) and the mean HbA1c level and mean duration of diabetes were 7.7 ± 1.4% and 14.0 ± 10.5 years, respectively. There were 15 subjects with type 1 and 216 subjects with type 2 diabetes. All of the type 1 diabetics were on insulin therapy, while 48 of the type 2 diabetics were receiving insulin (Table 1). 

Table 1

 

Baseline Characteristics of Subjects by Diabetic Retinopathy Outcomes at 1 Year

 

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 institutional review board of the Singapore Eye Research Institute approved the study. 

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.^31,32 Briefly, after pupil dilation, two retinal photographs, centered at the optic disc and macula, were obtained from each eye of the participants using a digital retinal camera (Canon CR-DGi with a 10-D SLR back; Canon, Tokyo, Japan). Trained, masked graders at the Singapore Eye Research Institute then graded photographs. 

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 (IRMA), hard exudates (HE), venous beading, and new vessels.³³ For each eye, a retinopathy severity score was assigned according to a scale modified from the Airlie House classification system.^33,34 The DR level for each participant was then derived by concatenating the levels for the two eyes, giving greater weight to the eye with the more severe grade.³⁴ This grading scheme has 15 levels of severity. If an eye was ungradable, it was presumed to have an equivalent grade to the fellow eye. 

The main outcomes were defined as follows. Incident DR was defined as the presence of any DR (level 21/<21 or worse) at 1 year in subjects with no DR (level 10/10) at baseline. Incident proliferative DR (PDR) was the presence of PDR (level 60+/<60+ or worse) at 1 year in subjects without PDR at baseline (less than level 60+/<60+). Diabetic retinopathy progression was defined as any two level or greater worsening of DR at 1 year, while DR regression was any two level or greater improvement in DR at 1 year. 

Assessment of Retinal Vessel Responses Using the Dynamic Vessel Analyzer

All subjects were prepared by being asked to abstain from no alcohol for 48 hours and from no cardioactive medications for four half-lives or a maximum of 48 hours. No tea/coffee was allowed for 24 hours, no exercise for 12 hours, and no food for 12 hours. No smoking was allowed on the morning of the study and all studies were done between 8 AM and 11 AM. 

The DVA 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.^16,19,31 Subjects were rested for 15 minutes before examination. 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 one-half and two 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 at 12.5 Hz 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. The DVA software calculates retinal arteriolar and venular dilatation in response to flickering light automatically. 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.³¹ 

Statistical Analyses

Descriptive data are presented as mean (SD) for continuous variables or number (percentages) of participants for categorical variables. Multivariable cox proportional models were constructed with the DR outcome as the dependent variables to examine hazard ratios (HRs) with 95% onfidence interval (CI) between arteriolar and venular dilation. 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. Proportional hazard assumption was confirmed for all predictors with Schoenfeld's residuals. 

We regarded P values of less than 0.05 from two-sided tests as statistically significant. All statistical analyses were performed using STATA (Version 12; StataCorp, College Station, TX, USA). 

Results

The distribution of DR severity at baseline was no DR in 219 (43.98%) eyes, minimal nonproliferative diabetic retinopathy in 63 (12.65%) eyes, mild NPDR in 74 (14.86%) eyes, moderate NPDR in 104 (20.88%) eyes, severe NPDR in 20 (4.02%) eyes, and PDR in 18 (3.61%) eyes. 

The incidence of DR at 1 year was 19 of 90 subjects (21.1%) and the incidence of PDR was 8 of 253 subjects (3.1%). The number of subjects with two step or greater progression of DR at 1 year was 31 of 276 (11.2%), and the number of subjects with two step or greater regression of DR at 1 year was 36 of 127 subjects (28.3%). 

When stratified by quartiles, in univariate analyses there were no associations between incident DR and either arteriolar or venular dilation. However, in multivariate analyses adjusting for age, sex, smoking status, HbA1c, hypertension, and hyperlipidaemia, reduced arteriolar dilation was significantly associated with incident DR (P = 0.048). There were no significant associations between arteriolar or venular dilation and incident PDR (Table 2). 

Table 2

 

Associations Between Changes in Retinal Vessel Diameter With Flicker Light and Diabetic Retinopathy Incidence and Progression

 

In univariate analyses, subjects in the lowest quartile of arteriolar dilation were more likely to have DR progression (P = 0.009). This relationship persisted in multivariate analyses adjusting for age, sex, smoking status, HbA1c, hypertension, and hyperlipidaemia, (odds ratio [OR] 1.85, [95% CI, 1.33–2.56], P = 0.012, per SD decrease). In univariate analyses, subjects in the lowest quartile of venular dilation were more likely to have DR progression (P = 0.009). This relationship persisted in multivariate analyses adjusting for age, sex, smoking status, HbA1c, hypertension, and hyperlipidaemia (OR 1.89, [95% CI, 1.35–2.63], P = 0.003, per SD decrease). There were no significant associations between arteriolar and venular dilation and DR regression (Table 2). Additional multivariate models controlling for age, sex, smoking status, HbA1c, diabetes duration, hypertension, and hyperlipidaemia confirmed the associations between arteriolar and venular dilation and DR progression (HR 1.43 [95% CI 1.02–2.00], P = 0.04 and 1.87 [1.34–2.60], P < 0.001, respectively; Tables 3, 4). 

Table 3

 

Associations of Arteriolar Dilation Response to Flickering Light With Diabetic Retinopathy Progression in Multivariate Adjusted Models

 

Table 4

 

Associations of Venular Dilation Response to Flickering Light With Diabetic Retinopathy Progression in Multivariate Adjusted Models

 

Representative DVA tracings from subjects with DR progression are shown in the Figure. 

Figure

 

Dynamic vessel analyser tracings from (A) subject without retinopathy showing robust dilatory responses in both arterioles (upper panel) and venules (lower panel) to flicker light. Tracings in (B) and (C) from subjects with three-step progression of diabetic retinopathy at 1 year showed diminished dilatory responses.

Discussion

Our prospective study in a cohort of persons with type 2 diabetes demonstrates an association between reduced baseline arteriolar and venular dilatory responses to flickering light and increased risk of DR and progression at 1 year. Reduced arteriolar and venular dilatory responses were consistently associated with two step or greater progression of DR at 1 year. Reduced arteriolar dilation was associated with DR incidence at 1 year. There were no associations between arteriolar or venular dilation responses and incident PDR or DR regression. Our study provides the first prospective evidence that reduced retinal arteriolar and venular dilatory responses to flickering light, are associated with DR progression in type 2 diabetes. 

The retinal vasculature has been suggested to reflect early microvascular changes in diabetes. In this regard, assessment of structural parameters of retinal vessel caliber and geometry (e.g., tortuosity), typically from retinal photographs, have been also shown to be associated with and have predictive value in the assessment of both macro- and microvascular complications of diabetes.^35–40 Interest has now turned to functional/dynamic retinal vessel assessments in the hope that they may provide further insights and greater predictive value. Multiple ways to study these pathways using different methods have been explored. For example, Lorenzi^41–43 has shown that abnormal myogenic responses of retinal vessels measured using laser Doppler flowmetry are precursors to clinical DR and may predict accelerated DR development. Other authors have used magnetic resonance imaging to quantify retinal oxygenation and shown it to predict DR risk.^44–47 However, there is insufficient data on the use of these novel methods for clinical screening at this time. The DVA is designed to measure vascular reactivity to a flickering light stimulus in vivo, and changes in the DVA responses have been shown in subjects with prediabetes and diabetes.^24,30,48 A study by Mandecka²⁸ has even shown that in diabetic patients without DR, reductions in the dynamic vascular response were demonstrable in the absence of discernible differences in static vessel measurements. These findings hint at the possibility that dynamic vessel changes may precede static measures, with potentially greater clinical utility in microvascular risk management. 

Limited data on the associations with DR exist, with no longitudinal data published to date. Nguyen²⁹ investigated the DVA responses in a cohort of 103 healthy Caucasian controls and 224 patients with diabetes. Subjects with diminished arteriolar and venular responses were more likely to have DR (OR, 2.2 and 2.5, respectively). Mandecka²⁷ evaluated 53 healthy volunteers, 68 type 1 diabetic patients, and 172 type 2 diabetic patients from a predominantly Caucasian population. Flicker responses in both arterioles and venules diminished continuously with increasing severity of DR. In a small cohort of 18 subjects with nonproliferative DR, Hammer⁴⁹ measured dynamic vessel changes concurrently with oxygen saturation, and found that subjects with DR had reduced dynamic vessel responses and correspondingly reduced oxygen saturation. In our Asian cohort, the baseline cross-sectional analyses were generally in agreement with the published data, with progressive diminishment in both arteriolar and venular dilatory responses seen with increasing severity of DR.³¹ Our longitudinal data showing associations between reduced arteriolar and venular dilatory responses and DR progression provide further support for the association between DVA responses and DR. Further study may define the clinical role of the DVA in identifying patients who might benefit from interventions to prevent DR progression. 

The association between reduced flicker responses and increased severity and progression of DR is biologically plausible, implicating progressive impairment of vasculo-regulatory mechanisms as a factor in disease progression. This association may reflect various mechanisms, including endothelial dysfunction, impaired autoregulation, as well as dysfunctional neurovascular coupling.³¹ Neurovascular coupling refers to the changes in vessel caliber and blood flow induced by neuronal stimuli. There is increasing interest in neurodegeneration as an early step in DR pathogenesis.^{6-8,12,15,50,51} It has been shown that thinning of the photoreceptor, ganglion cell and nerve fiber layers occurs in diabetic patients without clinically evident DR.^9–11,14,50 Direct, functional evidence of neurologic dysfunction has come from electrophysiological studies. Lecleire-Collet, et al.²⁶ correlated the DVA responses in normotensive patients with diabetes but no DR with electroretinography (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, Lasta¹³ measured the pattern ERG responses in type 1 diabetes patients and found that diminished DVA responses occurred before pattern ERG abnormalities became evident. These contrasting findings have been interpreted as indicating that in later stages of DR, reduced neural function could contribute to the impaired DVA responses. There is also increasing recognition that endothelial cell dysfunction may be an early step in the pathogenesis of diabetes and its complications,^52–57 and limited evidence indicates that DVA responses may be regulated by endothelial function. Pemp⁵⁸ measured DVA responses as well as flow-mediated vasodilatation (FMD), the current gold standard index of endothelial dysfunction, in diabetic subjects. Both DVA responses and FMD were reduced, with a weak correlation demonstrated between the two. 

The strengths of our study include its prospective design, a relatively large clinical cohort, and 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 reproducible results.¹⁹ General limitations of our study include the possibility of loss to follow-up bias. The relatively short follow-up period of a year also limited our ability to detect DR incidence, although it did not limit the ability to detect DR progression. It has also recently been shown that responses to flickering light vary according to the stimulation procotol, and are dependant on the vessel size as well.⁵⁹ As such, although the measurement protocol was standardized to include an arteriolar and venular segment between one-half and two disc diameters from the margin of the optic disc, variability in vessel size may have affected the results. 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, we demonstrated that reduced retinal arteriolar and venular dilatory responses to flickering light, are associated with a higher likelihood of DR progression at 1 year in adult patients with diabetes. Further study may help to define the clinical value of the DVA in predicting DR progression, or in determining its utility as a tool for monitoring therapeutic efficacy. 

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; T.Y. Wong, None