control subjects were consecutive patients seen at the hospital among individuals without diabetes and any retinal or eye pathological conditions .
individuals were excluded from participation if they were aged > 70 years , were of nonwhite ethnic background , had a history of epilepsy or glaucoma , had previous vitreal surgery , and/or had a cataract on examination .
all participants and control subjects had a standardized clinical examination , measurement of blood chemistry , retinal photographs , and assessment of flicker - induced vasodilation using the dynamic vessel analyzer ( dva ; imedos , jena , germany ) .
tenets of the declaration of helsinki were followed , institutional review board approval was granted , and written informed consent was obtained from all participants .
the dva measures retinal vessel dilation in response to diffuse luminance flicker ( 12 ) .
the participant focused on the tip of a fixation bar within the retinal camera while the fundus was examined under green light .
an arteriole and venule segment between one - half and two disc diameters from the margin of the optic disc were selected .
the mean diameters of the arterial and venous vessel segments were calculated and recorded automatically .
baseline vessel diameter was measured for 50 s , followed by a provocation with flicker light of the same wavelength for 20 s and then a nonflicker period for 80 s. this measurement cycle was repeated twice , with a total duration of 350 s / eye .
when the eye blinked or moved , the system automatically stopped the measurement and restarted it once the vessel segments were automatically reidentified .
retinal arteriolar and venular dilation in response to flicker light was calculated automatically by the dva software .
it was represented as an average increase in the vessel diameter in response to the flicker light during the three measurement cycles and was defined as the percent increase relative to the baseline diameter size .
in addition to quantifying the flicker - induced vasodilation , we assessed overall static arteriolar and venular diameter using a computer - assisted program .
details of the digital image preparation are described elsewhere ( 15 ) . in brief , diameters of the largest six arterioles and venules passing through the circular zone between one - half and one disc diameter away from the optic disc margin were summarized as the central retinal arteriolar equivalent and central retinal venular equivalent using the parr - hubbard formula further modified by knudtson and colleagues ( 15 ) .
fasting blood samples were drawn from participants at suburban pathology centers for measurement of fasting blood glucose level within 2 weeks of their eye testing .
all participants with diabetes were patients recruited from the diabetic eye clinics and were managed with oral hypoglycemic mediations and/or insulin .
control subjects ( individuals without diabetes ) had confirmed nondiabetic status based on a lack of history of diabetes and fasting glucose < 7.0
mmol / l ( 126 mg / dl ) . in participants with diabetes , diabetic retinopathy was graded from fundus photographs at the centre for eye research australia , by graders masked to clinical details . for each eye ,
a retinopathy severity score was assigned based on modification of the airlie house classification system ( 16 ) . for our analysis ,
levels 10 , 11 , and 12 were defined as no diabetic retinopathy , 14 to 20 as minimal nonproliferative diabetic retinopathy ( npdr ) , 31 and 41 as early to moderate npdr , and 5180 as severe npdr ( proliferative diabetic retinopathy ) . a detailed questionnaire was used to obtain participant information , including past medical history , current cigarette smoking , and the use of antihypertensive and lipid - lowering medications .
hypertension was defined as systolic blood pressure ( sbp ) > 140 mmhg , diastolic blood pressure ( dbp ) > 90 mmhg , or current use of antihypertensive medications .
dyslipidemia was defined as cholesterol > 5.5 mmol / l or triglyceride > 2.0 mmol / l or current use of lipid - lowering medications .
fasting blood samples were drawn from participants at suburban pathology centers for fasting blood glucose level , cholesterol and triglyceride levels , and a1c within 2 weeks of their eye testing .
induced retinal vasodilation between individuals with diabetes and control subjects and in individuals with diabetes between those with and without dr .
flicker - induced arteriolar / venular dilation was analyzed as percent increase over baseline diameter , both as a continuous measure and in categories ( tertiles ) .
multiple logistic regression models were constructed using the generalized estimating equation models to account for correlation between the right and left eyes and to assess the odds of diabetes ( vs. nondiabetic control subjects ) or diabetic retinopathy ( vs. no diabetic retinopathy among subjects with diabetes ) , comparing the lower versus upper tertiles of flicker light 
in addition , multiple linear regression models were used to estimate the mean difference in arteriolar and venular dilation .
we initially adjusted for age , sex , and fasting blood glucose level ( model 1 ) and further adjusted for duration of diabetes ( in analysis of diabetic patients ) , use of antihypertensive and lipid - lowering medications , current smoking status , sbp , and cholesterol and triglyceride levels ( model 2 ) .
analyses were performed in stata ( version 10.1 ; statacorp , college station , tx ) .
the dva measures retinal vessel dilation in response to diffuse luminance flicker ( 12 ) .
the participant focused on the tip of a fixation bar within the retinal camera while the fundus was examined under green light .
an arteriole and venule segment between one - half and two disc diameters from the margin of the optic disc were selected .
the mean diameters of the arterial and venous vessel segments were calculated and recorded automatically .
baseline vessel diameter was measured for 50 s , followed by a provocation with flicker light of the same wavelength for 20 s and then a nonflicker period for 80 s. this measurement cycle was repeated twice , with a total duration of 350 s / eye .
when the eye blinked or moved , the system automatically stopped the measurement and restarted it once the vessel segments were automatically reidentified .
retinal arteriolar and venular dilation in response to flicker light was calculated automatically by the dva software .
it was represented as an average increase in the vessel diameter in response to the flicker light during the three measurement cycles and was defined as the percent increase relative to the baseline diameter size .
in addition to quantifying the flicker - induced vasodilation , we assessed overall static arteriolar and venular diameter using a computer - assisted program .
details of the digital image preparation are described elsewhere ( 15 ) . in brief , diameters of the largest six arterioles and venules passing through the circular zone between one - half and one disc diameter away from the optic disc margin were summarized as the central retinal arteriolar equivalent and central retinal venular equivalent using the parr - hubbard formula further modified by knudtson and colleagues ( 15 ) .
fasting blood samples were drawn from participants at suburban pathology centers for measurement of fasting blood glucose level within 2 weeks of their eye testing . all participants with diabetes were patients recruited from the diabetic eye clinics and were managed with oral hypoglycemic mediations and/or insulin .
control subjects ( individuals without diabetes ) had confirmed nondiabetic status based on a lack of history of diabetes and fasting glucose < 7.0 mmol / l ( 126 mg / dl ) .
in participants with diabetes , diabetic retinopathy was graded from fundus photographs at the centre for eye research australia , by graders masked to clinical details . for each eye ,
a retinopathy severity score was assigned based on modification of the airlie house classification system ( 16 ) . for our analysis , levels 10 , 11 , and 12 were defined as no diabetic retinopathy , 14 to 20 as minimal nonproliferative diabetic retinopathy ( npdr ) , 31 and 41 as early to moderate npdr , and 5180 as severe npdr ( proliferative diabetic retinopathy ) .
a detailed questionnaire was used to obtain participant information , including past medical history , current cigarette smoking , and the use of antihypertensive and lipid - lowering medications .
hypertension was defined as systolic blood pressure ( sbp ) > 140 mmhg , diastolic blood pressure ( dbp ) > 90 mmhg , or current use of antihypertensive medications .
dyslipidemia was defined as cholesterol > 5.5 mmol / l or triglyceride > 2.0 mmol / l or current use of lipid - lowering medications .
fasting blood samples were drawn from participants at suburban pathology centers for fasting blood glucose level , cholesterol and triglyceride levels , and a1c within 2 weeks of their eye testing .
we compared flicker light  induced retinal vasodilation between individuals with diabetes and control subjects and in individuals with diabetes between those with and without dr .
flicker - induced arteriolar / venular dilation was analyzed as percent increase over baseline diameter , both as a continuous measure and in categories ( tertiles ) .
multiple logistic regression models were constructed using the generalized estimating equation models to account for correlation between the right and left eyes and to assess the odds of diabetes ( vs. nondiabetic control subjects ) or diabetic retinopathy ( vs. no diabetic retinopathy among subjects with diabetes ) , comparing the lower versus upper tertiles of flicker light 
in addition , multiple linear regression models were used to estimate the mean difference in arteriolar and venular dilation .
we initially adjusted for age , sex , and fasting blood glucose level ( model 1 ) and further adjusted for duration of diabetes ( in analysis of diabetic patients ) , use of antihypertensive and lipid - lowering medications , current smoking status , sbp , and cholesterol and triglyceride levels ( model 2 ) .
analyses were performed in stata ( version 10.1 ; statacorp , college station , tx ) .
selected characteristics of normal control subjects ( n = 103 ) , participants with diabetes ( n = 224 , 85 with type 1 and 139 with type 2 diabetes ) , and those with ( n = 144 ) and without ( n = 80 ) diabetic retinopathy are shown in table 1 .
mean  age was 56.5  11.8 years in subjects with diabetes and 48.0  16.3 years in control subjects .
the proportion of men was similar for participants with diabetes ( 41.6% ) and control subjects ( 39.4% ) . compared with nondiabetic control subjects ,
participants with diabetes were less likely to be current smokers but had higher bmi and were more likely to have hypertension , dyslipidemia , lower dbp , and lower total cholesterol levels . compared with those with type 1 diabetes , individuals with type 2 diabetes were older , had greater bmi , but a shorter duration of diabetes , and were more likely to have hypertension and dyslipidemia ( data not shown ) . in participants with diabetes , those with diabetic retinopathy had a longer duration of diabetes , had higher sbp , and were more likely to have hypertension .
in addition , participants with diabetes had wider static arteriolar diameter than nondiabetic control subjects , whereas those with diabetic retinopathy had wider retinal venules than those without ( table 1 ) .
participant characteristics ( age - adjusted means and proportions ) comparing participants with diabetes and normal control subjects , and , among participants with diabetes , those with and without diabetic retinopathy data are means unless stated otherwise
. means and proportions are adjusted for age ( set to mean age of 53.8 years old ) , except for age . * comparing those with diabetic subjects and normal control subjects , adjusted for age .
comparing those with and without diabetic retinopathy in those with diabetes , adjusted for age .
induced retinal vasodilation was reduced in participants with diabetes compared with that in control subjects ( table 2 ) .
induced arteriolar dilation was 1.43  2.10% in participants with diabetes and 3.46  2.36% in normal control subjects ( p < 0.001 after adjustment for age , sex , fasting glucose , cholesterol and triglyceride levels , use of antihypertensive and lipid - lowering medications , and current smoking status ) .
retinal arteriolar dilation was not significantly different by type of diabetes : 1.57% in those with type 1 and 1.24% in those with type 2 diabetes ( p = 0.98 ) .
induced venular dilation was 2.83  2.10% in individuals with diabetes and 3.98  1.84% in normal control subjects ( p < 0.001 after multivariable adjustment ) and again was not significantly different by type of diabetes : 2.84% in those with type 1 and 2.83% in those with type 2 diabetes ( p = 0.99 ) . mean differences in flicker light  induced vasodilation between participants with diabetes and normal control subjects and by grades of diabetic retinopathy severity in participants with diabetes * adjustment for age , sex , fasting cholesterol and triglyceride levels , use of antihypertensive and lipid - lowering medications , current smoking status , and fasting glucose ( for analysis of diabetic retinopathy severity ) .
table 3 shows that after multivariable adjustment , individuals with reduced flicker light  induced vasodilation were more likely to have diabetes ( odds ratios [ ors ] 19.7 and 8.1 , comparing the lowest versus the highest tertile of arteriolar and venular dilation , respectively ) . among participants with diabetes ,
those with reduced flicker induced  dilation were more likely to have diabetic retinopathy ( ors 2.2 and 2.5 , respectively , for arteriolar and venular dilation ) ( table 4 ) .
these associations persisted after further adjustment for static arteriolar / venular diameters ( tables 3 and 4 , model 3 ) .
associations between reduced flicker - induced arteriolar and venular dilation and diabetes data are ors ( 95% ci ) unless indicated otherwise .
model 2 : adjusted for covariates in model 1 plus diabetes duration , use of antihypertensive and lipid - lowering medications , current smoking status , sbp , and fasting cholesterol and triglyceride levels .
model 3 : adjusted for covariates in model 2 plus static retinal arteriolar or venular diameter .
associations between reduced flicker - induced arteriolar and venular dilation and diabetic retinopathy data are ors ( 95% ci ) unless indicated otherwise .
model 2 : adjusted for covariates in model 1 plus diabetes duration , use of antihypertensive and lipid - lowering medications , current smoking status , sbp , and fasting cholesterol and triglyceride levels .
model 3 : adjusted for covariates in model 2 plus static retinal arteriolar or venular diameter . *
the distribution of diabetic retinopathy severity was not significantly different between those with type 1 and type 2 diabetes ( p = 0.57 , data not shown ) .
however , the association of reduced flicker light  induced vasodilation with diabetic retinopathy was stronger in participants with type 1 diabetes ( arteriolar dilation or 3.1 [ 95% ci 1.18.5 ] ; venular dilation or 3.8 [ 95% ci 1.410.0 ] ) compared with those with type 2 diabetes ( arteriolar dilation or 1.8 [ 95% ci 0.84.0 ] ; venular dilation or 1.3 [ 95% ci 0.63.1 ] ) , although the interaction with type of diabetes was not statistically significant ( p value for interaction term : p = 0.50 for arteriolar dilation and p = 0.09 for venular dilation ) .
in this study , we demonstrated a reduction in flicker light  induced retinal arteriolar and venular dilation in individuals with diabetes compared with nondiabetic control subjects and , among individuals with diabetes , in those with retinopathy signs .
importantly , we showed that these associations were independent of major risk factors for either diabetes or diabetic retinopathy and independent of static measurements of retinal arterioles and venular diameters .
( 17 ) examined 26 healthy control subjects and 26 individuals with type 1 diabetes who had none or minimal npdr and were not receiving antihypertensive treatment , whereas mandecka et al .
( 14 ) examined 240 individuals with diabetes ( 68 with type 1 and 172 with type 2 diabetes ) and 58 control subjects .
both showed reduced flicker light vasodilation in those with diabetes ( compared with those without diabetes ) .
furthermore , mandecka et al . also demonstrated a reduction in flicker light vasodilation with increasing diabetic retinopathy severity , while controlling only for age , sex , and use of antihypertensive medications .
we have now shown that the relationship of flicker light  induced vasodilation and both diabetes and diabetic retinopathy are independent of major confounders and risk factors for diabetic retinopathy , including duration of diabetes and glycemic control . retinal neuronal stimulation by flicker light results in retinal vessel dilation .
this response probably reflects endothelial function ( 14 ) , given the documented role of nitric oxide in this flickering light  induced vasodilation ( 13,18,19 ) . in a study by dorner et al .
( 13 ) , n - monomethyl - l - arginine , an inhibitor of nitric oxide synthase , blunted this flicker - induced vasodilation in healthy individuals .
in addition , impaired response to flicker light stimulation in individuals with hypertension could be restored by angiotensin ii subtype 1 receptor blockade ( 20 ) . however , this finding has been documented only in individuals without diabetes . it was hypothesized previously that the decreased endothelial dysfunction in subjects with diabetes is associated with impaired nitric oxide action because of its inactivation resulting from increased oxidative stress and that abnormal nitric oxide metabolism is related to advanced diabetic microvascular complications ( 21 ) .
this hypothesis is supported by recent data demonstrating similar retinal arteriolar and venular dilation after a single sublingual dose of 0.8 mg nitroglycerin between 20 patients with insulin - treated diabetes with no or only mild npdr and 20 healthy age - matched control subjects ( 22 ) .
however , it is becoming increasingly clear that neuronal cells of the retina are also affected by diabetes , resulting in dysfunction and degeneration ( 23 ) , and diabetic retinopathy is a disease of both retinal neurons and microcirculation ( 24 ) . because retinal blood flow is coupled with neuronal activity ( 25 ) , reduced flicker light
induced vasodilation can thus also reflect neurodegeneration ( 17,24 ) . in our study , significantly reduced flicker light 
induced vasodilation was observed in diabetic subjects with diabetic retinopathy compared with those without diabetic retinopathy .
this relationship appeared to be stronger among individuals with type 1 diabetes than among those with type 2 diabetes , given the similar distribution of diabetic retinopathy severity between the two groups .
this observation could be due to longer diabetes duration in those with type 1 diabetes ( mean 22.1 years for type 2 diabetes vs. 12.6 years for type 2 diabetes ) , resulting in possibly a greater level of impairment of retinal vascular autoregulation ( 26 ) , endothelial damage ( 26 ) , or neurodegeneration ( 17,24 ) .
alternatively , the underlying mechanisms of diabetic retinopathy may be different in type 1 and type 2 diabetes .
the strengths of this study include quantitative measures of retinal vasodilation after flicker light stimulation , assessment of diabetic retinopathy from fundus photographs using standardized grading protocols , and one researcher ( t.t.n . ) performing all dva measurements . limitations of this study should also be noted .
first , the cross - sectional nature of the study provides no temporal information on the associations reported .
second , our findings are only applicable to individuals with diabetes who are aged 70 years .
thus , further longitudinal studies are needed to ascertain cause and effect and to correlate flicker - induced vasodilation with retinal neuronal functions using tests such as electroretinography . in summary
induced retinal vasodilation in individuals with diabetes and , among those with diabetes , in those with retinopathy signs .
these findings further support the concept that early endothelial dysfunction is a likely key pathophysiological mechanism that underlies diabetes and its microvascular complications .
 objectiveflicker light  induced retinal vasodilation may reflect endothelial function in the retinal circulation . 
 we investigated flicker light  induced vasodilation in individuals with diabetes and diabetic retinopathy.research design and methodsparticipants consisted of 224 individuals with diabetes and 103 nondiabetic control subjects . 
 flicker light  
 induced retinal vasodilation ( percentage increase over baseline diameter ) was measured using the dynamic vessel analyzer . 
 diabetic retinopathy was graded from retinal photographs.resultsmean  sd age was 56.5  11.8 years for those with diabetes and 48.0  16.3 years for control subjects . 
 mean arteriolar and venular dilation after flicker light stimulation were reduced in participants with diabetes compared with those in control subjects ( 1.43  2.10 vs. 3.46  2.36% , p < 0.001 for arteriolar and 2.83  2.10 vs. 3.98  1.84% , p < 0.001 for venular dilation ) . 
 after adjustment for age , sex , diabetes duration , fasting glucose , cholesterol and triglyceride levels , current smoking status , systolic blood pressure , and use of antihypertensive and lipid - lowering medications , participants with reduced flicker light  induced vasodilation were more likely to have diabetes ( odds ratio 19.7 [ 95% ci 6.559.1 ] , p < 0.001 and 8.14 [ 3.121.4 ] , p < 0.001 , comparing lowest vs. highest tertile of arteriolar and venular dilation , respectively ) . 
 diabetic participants with reduced flicker light  induced vasodilation were more likely to have diabetic retinopathy ( 2.2 [ 1.24.0 ] , p = 0.01 for arteriolar dilation and 2.5 [ 1.34.5 ] , p = 0.004 for venular dilation).conclusionsreduced retinal vasodilation after flicker light stimulation is independently associated with diabetes status and , in individuals with diabetes , with diabetic retinopathy . 
 our findings may therefore support endothelial dysfunction as a pathophysiological mechanism underlying diabetes and its microvascular manifestations . 