the study protocol was approved by the ethics committee of the medical university of vienna and followed the guidelines set forth in the declaration of helsinki .
all patients signed written informed consent prior to inclusion and passed a screening examination including physical and ophthalmological examination during the 10 days before the study day .
a total of 80 individuals aged > 18 years were included in this observer - blinded , controlled , parallel - group study .
three groups were composed : group 1 included 20 patients with type 1 diabetes with no signs of diabetic retinopathy or mild nonproliferative diabetic retinopathy .
group 2 included 40 patients with mild essential systemic hypertension at rest and/or with serum cholesterol levels 0.65
hypertension was defined as a blood pressure meeting the criterion of hypertension grade 1 of the world health organization blood pressure classification , with systolic blood pressure ( sbp ) from 140 to 159 mmhg and diastolic blood pressure ( dbp ) from 90 to 99 mmhg .
blood pressure was measured at two different occasions in a sitting position . as a control ,
20 healthy subjects with sbp < 140 mmhg , dbp < 90 mmhg , serum cholesterol levels < 0.55 mmol / l , and normal ocular findings were included in group 3 .
care was taken that the control group was comparable in age and sex distribution to the patient groups .
further exclusion criteria for all subjects were ametropia > 3 dpt , other relevant ocular abnormalities , a clinically relevant illness prior to the study , pregnancy or lactation , and a patient or family history of epilepsy .
participants had to abstain from beverages containing alcohol or caffeine for 12 h before the study .
the diameters of one temporal retinal artery and vein between 1 and 2 disc diameters from the margin of the optic disc were continuously measured using the dynamic vessel analyzer ( dva ; imedos , jena , germany ) .
the dva comprises a fundus camera ( ff 450 ; carl zeiss meditec , jena , germany ) , a digital video camera , and a personal computer with analyzing software for the determination of retinal vessel diameters that are analyzed from digitized images .
after selection of the measurement location , the dva is able to follow the vessels during movements within the measurement window .
retinal vessel diameters were measured for 4 min . for the second minute , full - field flickering light with a frequency of 12.5 hz was used for stimulation by square 
wave pattern modulation of the fundus camera illumination at a contrast ratio of 25:1 . to measure fmd ,
each subject was in the supine position with the left arm supported on a foam block and a pneumatic cuff placed on the upper arm proximal to the measurement area .
a high - resolution ultrasound system with a 7.0-mhz transducer ( vivid seven pro ; ge vingmed ultrasound , horten , norway ) was used to measure the brachial artery diameter .
the probe was fixed in an adjustable swivel arm to maintain an identical position during the experiments .
the brachial artery was scanned in a longitudinal section proximal to its bifurcation , which was used as an anatomical marker .
baseline diameter of the brachial artery was assessed as the mean of 1 min of continuous measurement .
thereafter , the cuff on the upper arm was inflated to suprasystolic pressure ( 250 mmhg ) for 4.5 min .
all subjects were studied under dilated pupil after instillation with tropicamide ( mydriaticum  agepha-gtt ; agepha , vienna , austria ) . after a 20-min resting period in a sitting position , baseline measurements of arterial blood pressure and pulse rate were performed . thereafter , retinal vessel measurements including flicker stimulation were performed . intraocular pressure ( iop ) was measured after the flicker experiment .
iop was measured with a slit - lamp  mounted goldmann applanation tonometer ( haag - streit , bern , switzerland ) . before each measurement ,
sbp , dbp , and mean arterial blood pressures ( map ) were measured on the upper arm by an automated oscillometric device ( hp - cms patient monitor ; hewlett packard , palo alto , ca ) .
pulse rate was automatically recorded by the same unit from a finger - pulse oxymetric device .
baseline values were calculated as an average of the last 20 s before start of the flicker stimulation .
flicker response was calculated as an average of the last 20 s of the stimulation period .
flow - mediated dilatation of the brachial artery was expressed as percentage change of diameter measured 60 s after cuff deflation compared with baseline .
an anova model was used for significance testing of the retinal vessel response to flicker stimulation and to fmd over time within the groups as well as between the three groups .
pearson product - moment correlation coefficient was calculated to assess correlation between the variables . to adjust for multiple testing ,
for all calculations , a p value < 0.05 was considered as the level of significance .
the diameters of one temporal retinal artery and vein between 1 and 2 disc diameters from the margin of the optic disc were continuously measured using the dynamic vessel analyzer ( dva ; imedos , jena , germany ) .
the dva comprises a fundus camera ( ff 450 ; carl zeiss meditec , jena , germany ) , a digital video camera , and a personal computer with analyzing software for the determination of retinal vessel diameters that are analyzed from digitized images .
after selection of the measurement location , the dva is able to follow the vessels during movements within the measurement window .
full - field flickering light with a frequency of 12.5 hz was used for stimulation by square 
to measure fmd , each subject was in the supine position with the left arm supported on a foam block and a pneumatic cuff placed on the upper arm proximal to the measurement area .
a high - resolution ultrasound system with a 7.0-mhz transducer ( vivid seven pro ; ge vingmed ultrasound , horten , norway ) was used to measure the brachial artery diameter .
the probe was fixed in an adjustable swivel arm to maintain an identical position during the experiments .
the brachial artery was scanned in a longitudinal section proximal to its bifurcation , which was used as an anatomical marker .
baseline diameter of the brachial artery was assessed as the mean of 1 min of continuous measurement .
thereafter , the cuff on the upper arm was inflated to suprasystolic pressure ( 250 mmhg ) for 4.5 min .
all subjects were studied under dilated pupil after instillation with tropicamide ( mydriaticum  agepha-gtt ; agepha , vienna , austria ) . after a 20-min resting period in a sitting position ,
thereafter , retinal vessel measurements including flicker stimulation were performed . intraocular pressure ( iop ) was measured after the flicker experiment .
iop was measured with a slit - lamp  mounted goldmann applanation tonometer ( haag - streit , bern , switzerland ) . before each measurement
sbp , dbp , and mean arterial blood pressures ( map ) were measured on the upper arm by an automated oscillometric device ( hp - cms patient monitor ; hewlett packard , palo alto , ca ) . pulse rate was automatically recorded by the same unit from a finger - pulse oxymetric device .
baseline values were calculated as an average of the last 20 s before start of the flicker stimulation .
flicker response was calculated as an average of the last 20 s of the stimulation period .
flow - mediated dilatation of the brachial artery was expressed as percentage change of diameter measured 60 s after cuff deflation compared with baseline .
an anova model was used for significance testing of the retinal vessel response to flicker stimulation and to fmd over time within the groups as well as between the three groups .
pearson product - moment correlation coefficient was calculated to assess correlation between the variables . to adjust for multiple testing ,
for all calculations , a p value < 0.05 was considered as the level of significance .
was significantly increased compared with the other groups ( anova , p < 0.001 ) ( table 1 ) .
map and total cholesterol were increased in group 2 ( anova , p < 0.003 ) .
average baseline vessel diameters were slightly increased in type 1 diabetes ( anova , p = 0.018 ) .
a detailed summary of concomitant medication can be found in online appendix table a1 ( available at http://care.diabetesjournals.org/cgi/content/full/dc08-2130/dc1 ) .
baseline data of the three participating groups data are means  sd , unless otherwise indicated .
* significant differences ( p < 0.05 , anova ) . in the healthy group ,
stimulation with flicker light induced a vasodilatation of 7.0  2.3% in retinal arteries ( anova , time effect , p < 0.001 ) ( fig . 1 ) and a dilatation of 6.8  3.4% ( p < 0.001 ) in retinal veins . in patients with type 1 diabetes , retinal arterial diameters increased by 2.9  2.8% ( p < 0.001 ) and retinal veins by 4.6  2.0% ( p < 0.001 ) .
patients with systemic hypertension and/or hypercholesterolemia showed a vasodilatation of 4.3  2.8% ( p < 0.001 ) in retinal arteries and a vasodilatation of 6.0  2.4% ( p < 0.001 ) in retinal veins .
thus , flicker - induced dilatation was reduced in patients with type 1 diabetes and patients with systemic hypertension and/or hypercholesterolemia compared with healthy control subjects .
this effect was significant at a level of p < 0.001 for retinal arteries and at a level of p = 0.045 for retinal veins ( anova , effect between groups ) .
group means  sd . * significant differences ( p < 0.05 , anova ) .
fmd of the brachial artery was 4.3  3.0% in the healthy group ( anova , time effect , p < 0.001 ) ( fig .
1 ) . in both patients with type 1 diabetes and patients with systemic hypertension and/or hypercholesterolemia , fmd was significantly attenuated to 2.6  1.7% in group 1 ( anova ; time effect : p < 0.001 ; effect between groups : p = 0.045 ) and to 2.4  2.4% in group 2 ( time effect : p < 0.001 ; effect between groups : p = 0.045 ) . again
, fmd of the brachial artery was not significantly different between the two patients groups . a correlation between fmd and flicker - induced vasodilatation in retinal arteries ( r = 0.3 , p = 0.044 ) was found ( fig .
no correlation , however , was observed between fmd and flicker response in retinal veins ( data not shown ) .
flicker - induced vasodilatation was negatively correlated with plasma cholesterol levels ( r = 0.33 , p = 0.044 ) but not with age ( r = 0.33 , p = 0.08 ) ( fig .
there was also no significant correlation between fmd and age ( r = 0.35 , p = 0.081 ) or cholesterol ( r = 0.22 , p = 0.090 ) after p value adjustment .
given that patients with diabetes are known to have a reduced flicker response , the type 1 diabetic group has been excluded in the latter analyses .
correlation analysis between fmd and flicker response of retinal arteries ( r = 0.3 , p = 0.044 ) .
correlation analysis between flicker - induced vasodilatation and plasma cholesterol levels ( r = 0.33 , p = 0.044 ) ( a ) , between fmd and age ( r = 0.35 , p = 0.081 ) ( d ) , and vice versa ( r = 0.33 , p = 0.08 ) ( b ) ( r = 0.22 , p = 0.090 ) ( c ) .
in the healthy group , stimulation with flicker light induced a vasodilatation of 7.0  2.3% in retinal arteries ( anova , time effect , p < 0.001 ) ( fig . 1 ) and a dilatation of 6.8  3.4% ( p < 0.001 ) in retinal veins . in patients with type 1 diabetes , retinal arterial diameters increased by 2.9  2.8% ( p < 0.001 ) and retinal veins by 4.6  2.0% ( p < 0.001 ) .
patients with systemic hypertension and/or hypercholesterolemia showed a vasodilatation of 4.3  2.8% ( p < 0.001 ) in retinal arteries and a vasodilatation of 6.0  2.4% ( p < 0.001 ) in retinal veins .
thus , flicker - induced dilatation was reduced in patients with type 1 diabetes and patients with systemic hypertension and/or hypercholesterolemia compared with healthy control subjects .
this effect was significant at a level of p < 0.001 for retinal arteries and at a level of p = 0.045 for retinal veins ( anova , effect between groups ) .
fmd of the brachial artery was 4.3  3.0% in the healthy group ( anova , time effect , p < 0.001 ) ( fig .
1 ) . in both patients with type 1 diabetes and patients with systemic hypertension and/or hypercholesterolemia , fmd was significantly attenuated to 2.6  1.7% in group 1 ( anova ; time effect : p < 0.001 ; effect between groups : p = 0.045 ) and to 2.4  2.4% in group 2 ( time effect : p < 0.001 ; effect between groups : p = 0.045 ) . again
, fmd of the brachial artery was not significantly different between the two patients groups .
a correlation between fmd and flicker - induced vasodilatation in retinal arteries ( r = 0.3 , p = 0.044 ) was found ( fig .
no correlation , however , was observed between fmd and flicker response in retinal veins ( data not shown ) .
flicker - induced vasodilatation was negatively correlated with plasma cholesterol levels ( r = 0.33 , p = 0.044 ) but not with age ( r = 0.33 , p = 0.08 ) ( fig .
there was also no significant correlation between fmd and age ( r = 0.35 , p = 0.081 ) or cholesterol ( r = 0.22 , p = 0.090 ) after p value adjustment .
given that patients with diabetes are known to have a reduced flicker response , the type 1 diabetic group has been excluded in the latter analyses .
correlation analysis between fmd and flicker response of retinal arteries ( r = 0.3 , p = 0.044 ) .
correlation analysis between flicker - induced vasodilatation and plasma cholesterol levels ( r = 0.33 , p = 0.044 ) ( a ) , between fmd and age ( r = 0.35 , p = 0.081 ) ( d ) , and vice versa ( r = 0.33 , p = 0.08 ) ( b ) ( r = 0.22 , p = 0.090 ) ( c ) .
given that impaired endothelial function has been observed to be an early feature in several systemic and ocular vascular - related diseases , much attention has been paid to the development of methods to noninvasively assess endothelial function in humans . as one of the most widely used techniques , the ultrasound - based fmd has been shown to give a reliable estimate of endothelium - dependent vasodilatation ( 10 ) .
fmd is based on the capacity of blood vessels to self - regulate vascular tone in response to changes of shear stress caused by changes in blood flow .
this regulation is dependent on endothelium - derived no ( 14 ) and can therefore be used as a marker for endothelial function .
reduced fmd has been found in patients with mild systemic hypertension ( 15 ) , hypercholesterolemia ( 16 ) , and diabetes ( 17 ) , indicating for an impaired endothelial function in these patient groups .
additionally , it has been shown that fmd can predict future cardiovascular events ( 18 ) .
however , the technique of fmd is hampered by the limited spatial resolution of the ultrasound systems currently available .
in addition , measurement of fmd requires significant training and involves a subjective component when data are evaluated .
it has been shown that flicker response is significantly diminished in patients with glaucoma or diabetes ( 5,8 ) .
even more importantly , a reduced response has also been observed in patients with systemic hypertension , indicating a potential insight into vascular function in general ( 7 ) , because an increase of blood pressure or iop alone does not influence the flicker response ( 19,20 ) .
these results support the hypothesis that the observed changes reflect long - term alterations of the vasculature .
the hypothesis that flicker - induced vasodilatation may at least partially reflect endothelial function has also been encouraged by the observation that flicker - induced vasodilatation is mainly dependent on an intact no synthesis ( 9 ) .
endothelial dysfunction due to abnormal release or action of no is a well - recognized early feature of vascular damage , as it has been reported previously in vascular - related diseases like diabetes , hypercholesterolemia , systemic hypertension , and atherosclerosis ( 16,21,22 ) .
our findings of greater baseline vessel diameters in patients with diabetes are in good accordance with earlier studies ( 23 ) .
however , given that our measures were done only in one single artery and vein and not in all visible vessels , our data do not represent total cross - sectional retinal vessel diameters .
this result is again a hint that endothelial dysfunction is involved in reduced flicker - induced vasodilatation , because cholesterol and oxidized ldl in particular are clearly associated with endothelial cell dysfunction ( 24 ) and reduced bioavailability of no .
evidence has been provided that reduction of serum cholesterol increases fmd and may therefore be beneficial for endothelial functions ( 22,24 ) .
whether this also holds true for flicker - induced vasodilatation has yet to be clarified .
correlations between fmd and age or plasma cholesterol , as observed earlier ( 16 ) , and between flicker - induced vasodilatation and age failed to reach level of significance after adjustment for multiple testing .
our study was , however , not designed for these outcome analyses , and a larger sample size may be required to investigate these issues .
the present study provides evidence that in patients with type 1 diabetes and in patients with systemic hypertension and/or hypercholesterolemia , both fmd and flicker - induced vasodilatation are reduced compared with healthy volunteers .
however , our study failed to show a strong correlation between fmd and flicker - induced vasodilatation
. what could be the reason for the differing responses between the two vascular beds ?
whereas the diameter increase in fmd is caused directly by the augmented shear stress in the endothelium and the connected tissue , flicker response is basically the vascular answer to increased neural activity in the retina .
this may be of special importance in patients with diabetes or glaucoma , since it can not be ruled out that in these patients decreased neural activity may partially account for the decreased flicker response .
second , it has to be noted that the properties of the vascular beds investigated differ significantly . whereas flicker stimulation investigates arteries in an order of 150250 m ,
thus , the weak correlation between fmd and flicker may indicate that the stimulation answer in the conduit arteries and in the smaller retinal arteries do not carry the same information , although both are diminished in patients with endothelial dysfunction .
this phenomenon is also known from other experiments showing that fmd and endothelium - dependent vasodilatation assessed with an invasive technique that mainly reflects the endothelial function of resistance arteries are both independently related to the risk of coronary heart disease ( 25 ) .
flicker - induced vasodilatation may provide additional information to these techniques because of the smaller size of vessels assessed .
, it provides excellent reproducibility and sensitivity ( 12 ) . on the other hand ,
it is easily performed and quick , although pupil dilatation is required with the fundus camera used in the present experiments .
most importantly , the system does not include a subjective component once an optimal fundus image is achieved . as a limitation of the study , no information is available about blood nitrate concentration .
although none of the subjects under study was under nitrate medication , we can not fully exclude that a nitrate - rich diet may influence fmd or flicker - induced vasodilatation . in summary ,
our data indicate that in both patient groups with endothelial dysfunction as assessed with fmd , flicker responses are diminished .
the reason why no major correlation was found between fmd and flicker - induced vasodilatation needs to be the subject of further studies .
furthermore , whether flicker stimulation may also serve as a predictor for risk of systemic diseases , as it has been shown for fmd , has yet to be investigated in longitudinal studies .
the system is , however , a candidate for assessing endothelial function in clinical routine because it induces minimum discomfort to the subject , provides good reproducibility and sensitivity , and does not include a subjective component .
 objectiveflicker - induced vasodilatation is reduced in patients with vascular - related diseases , which has at least partially been attributed to endothelial dysfunction of retinal vessels . currently , the standard method to assess endothelial function in vivo is flow - mediated vasodilatation ( fmd ) . 
 thus , the present study was performed to investigate whether a correlation exists between flicker - induced vasodilatation and fmd in patients with known endothelial dysfunction and healthy subjects.research design and methodsin the present study , 20 patients with type 1 diabetes , 40 patients with systemic hypertension ( systolic blood pressure 140159 mmhg ; diastolic blood pressure 9099 mmhg ) and/or serum cholesterol levels 0.65 mmol / l , and 20 healthy control subjects were included . 
 the flicker response was measured using the dynamic retinal vessel analyzer . 
 fmd was determined using a high - resolution ultrasound system , measuring brachial artery diameter reactivity during reperfusion after arterial occlusion.resultsthe flicker response of both retinal arteries and veins was significantly reduced in the two patients groups . 
 likewise , fmd was significantly reduced in patients compared with healthy control subjects . however , only a weak correlation between flicker - induced vasodilatation and fmd was observed.conclusionsthe study confirms that flicker responses and fmd are reduced in the selected patient groups . whether the weak correlation between fmd and flicker is due to the different stimulation type , the different vascular beds measured , or other mechanisms has yet to be investigated 
 . 