the study population consisted of 59 patients with type 1 diabetes ( 36 women and 23 men , median age 32.0 years , range limits 2051 years ) , recruited from the regional diabetological centre of our university and 30 healthy volunteers ( 15 women and 15 men , median age 31.5 years , range limits 2039 years ) . we included patients with minimal diabetes duration of 2 years and who were free of focal neurological deficits , respiratory tract disease ( at present and in history ) , and renal insufficiency . we excluded patients with past cerebrovascular events or head trauma , those who were pregnant , and those with incidents of hypoglycemia within 30 days before the study . current and past smokers were also excluded in both patients and control groups , as well as heavy drinkers and users of any hormonal therapy within 30 days before study . all examinations were performed at the same time of the day ( between 10:00 a.m. and 2:00 p.m. ) . all study subjects were asked to stop drinking coffee on examination day and to avoid sleep deprivation and fasting . the study protocol included history taking , neurological examination , extracranial and transcranial ultrasound , fundoscopy , and laboratory testing . the study protocol was approved by the medical ethics committee of the medical university of gdask ( nkebn/3/2005 ) . upon entry , patient history was obtained , including the information on past and current disorders as well as on comorbid conditions . focal neurological deficits were excluded by neurological examination performed by neurologists certified by the polish neurological society . diabetic neuropathy was diagnosed with the criteria of neuropathy symptoms score , based on patients ' complaints and neuropathic deficits found on neurological examination ( 14 ) . hypertension was diagnosed if two consecutive measurements of systolic and diastolic blood pressures exceeded 130 and 80 mmhg , respectively , or if antihypertensive medication was used . laboratory examinations in patients with type 1 diabetes included measurements of circadian urinary protein / microalbuminuria , total serum cholesterol , and a1c . the biochemical examinations were performed 1 month ( 7 days ) before transcranial doppler examination ( tcd ) . hyperlipidemia was diagnosed if total cholesterol and/or triglycerides exceeded 175 and 150 mg / dl , respectively , or if cholesterol / triglyceride - lowering medications were used . based on american diabetes association criteria , microalbuminuria was defined as excretion of 30300 mg albumin/24 h on two of three urine collections repeated at intervals of up to 6 months ( 15 ) . overt nephropathy  per the american diabetes association recommendations corresponded to protein excretion over 300 mg/24 h. in our patients , quantitative measurements of urine protein excretion in a sample obtained from 24-h urine collection were used to assess the severity of proteinuria . we included in the statistical analysis the measurement that was closest in time to the tcd study . retinopathy was recognized on fundoscopy performed by an ophthalmologist certified by the polish ophthalmic society . grading was made with the use of the stages of diabetic retinopathy of the american academy of ophthalmology . previous therapy with photocoagulation was also recognized as a marker of diabetic retinopathy ( 16 ) . the imt in both carotid arteries was measured using an aloka 5000 ultrasound machine ( aloka , japan ) equipped with a linear probe with central working frequency of 7.5 mhz and range limits of 510 mhz , after standard examination of both carotid arteries . during each examination , the distal 2-cm - long segment of the common carotid artery in two different ( anterior - posterior and lateral ) longitudinal projections the final value of imt was calculated as a mean of four measurements ( two projections , both sides ) . imt was measured offline using a semiautomatic method ( carotid measure system ) performed by one investigator ( k.b.k . the middle cerebral artery flow parameters were measured using the multidop t2 ultrasound machine ( dwl elektronische systeme , singen , germany ) , equipped with the 2-mhz pulse wave probe . velocity measurements were performed simultaneously in both middle cerebral arteries with the use of a two - channel monitoring kit : two probes 2-mhz pulse wave , the fixation band , and the monitoring program ( mf version 8.27 l ; dwl elektronische systeme ) . the physiological techniques of provoking cerebrovascular reactivity by changes of pco2 were applied according to the published standards ( 17,18 ) . during the co2 reactivity test , the co2 content in expired air ( end tidal co2 concentration ) was monitored continuously ( capnograph ; datex , normocap , finland ) . before and after the tests , the vasomotor reactivity reserve ( vmrr ) , expressed in percent change from baseline , and breath - holding index ( bhi ) were calculating according to the standard protocol published previously ( 17,18 ) . the median values of arithmetical means of velocity measurements at rest ( rest vmean ) , vmrr , bhi , pulsatility index ( pi ) , and resistance index ( ri ) of both middle cerebral arteries were used for further analyses . in nine healthy volunteers , the unbiased intraclass correlation coefficient for the results of two consecutive measurements of vmrr was considered acceptable ( intraclass correlation coefficient = 0.89 , p = 0.007 ) . all the analyses were performed with statistica , version 7.1 ( statsoft , 2005 ; sn abdp509753932ar ) . differences between groups were analyzed with the student 's t test in case of normally distributed variables ( type 1 diabetes duration ) or with mann - whitney u test in case of non  normally distributed variables ( age , bmi , diastolic and systolic blood pressure , heart rate , vmrr , bhi , pi , ri , end tidal co2 concentration , and imt ) . the  test was used to compare a proportion of females to males in groups . multivariable linear regression analysis was used to assess confounders of cvr in patients with type 1 diabetes . patient history was obtained , including the information on past and current disorders as well as on comorbid conditions . focal neurological deficits were excluded by neurological examination performed by neurologists certified by the polish neurological society . diabetic neuropathy was diagnosed with the criteria of neuropathy symptoms score , based on patients ' complaints and neuropathic deficits found on neurological examination ( 14 ) . hypertension was diagnosed if two consecutive measurements of systolic and diastolic blood pressures exceeded 130 and 80 mmhg , respectively , or if antihypertensive medication was used . laboratory examinations in patients with type 1 diabetes included measurements of circadian urinary protein / microalbuminuria , total serum cholesterol , and a1c . the biochemical examinations were performed 1 month ( 7 days ) before transcranial doppler examination ( tcd ) . hyperlipidemia was diagnosed if total cholesterol and/or triglycerides exceeded 175 and 150 mg / dl , respectively , or if cholesterol / triglyceride - lowering medications were used . based on american diabetes association criteria , microalbuminuria was defined as excretion of 30300 mg albumin/24 h on two of three urine collections repeated at intervals of up to 6 months ( 15 ) . clinical albuminuria or  overt nephropathy  per the american diabetes association recommendations corresponded to protein excretion over 300 mg/24 h. in our patients , quantitative measurements of urine protein excretion in a sample obtained from 24-h urine collection were used to assess the severity of proteinuria . we included in the statistical analysis the measurement that was closest in time to the tcd study . retinopathy was recognized on fundoscopy performed by an ophthalmologist certified by the polish ophthalmic society . grading was made with the use of the stages of diabetic retinopathy of the american academy of ophthalmology . previous therapy with photocoagulation was also recognized as a marker of diabetic retinopathy ( 16 ) . the imt in both carotid arteries was measured using an aloka 5000 ultrasound machine ( aloka , japan ) equipped with a linear probe with central working frequency of 7.5 mhz and range limits of 510 mhz , after standard examination of both carotid arteries . during each examination , the distal 2-cm - long segment of the common carotid artery in two different ( anterior - posterior and lateral ) longitudinal projections the final value of imt was calculated as a mean of four measurements ( two projections , both sides ) . imt was measured offline using a semiautomatic method ( carotid measure system ) performed by one investigator ( k.b.k . the middle cerebral artery flow parameters were measured using the multidop t2 ultrasound machine ( dwl elektronische systeme , singen , germany ) , equipped with the 2-mhz pulse wave probe . velocity measurements were performed simultaneously in both middle cerebral arteries with the use of a two - channel monitoring kit : two probes 2-mhz pulse wave , the fixation band , and the monitoring program ( mf version 8.27 l ; dwl elektronische systeme ) . the physiological techniques of provoking cerebrovascular reactivity by changes of pco2 were applied according to the published standards ( 17,18 ) . during the co2 reactivity test , the co2 content in expired air ( end tidal co2 concentration ) was monitored continuously ( capnograph ; datex , normocap , finland ) . before and after the tests , the vasomotor reactivity reserve ( vmrr ) , expressed in percent change from baseline , and breath - holding index ( bhi ) were calculating according to the standard protocol published previously ( 17,18 ) . the median values of arithmetical means of velocity measurements at rest ( rest vmean ) , vmrr , bhi , pulsatility index ( pi ) , and resistance index ( ri ) of both middle cerebral arteries were used for further analyses . the unbiased intraclass correlation coefficient for the results of two consecutive measurements of vmrr was considered acceptable ( intraclass correlation coefficient = 0.89 , p = 0.007 ) . all the analyses were performed with statistica , version 7.1 ( statsoft , 2005 ; sn abdp509753932ar ) . differences between groups were analyzed with the student 's t test in case of normally distributed variables ( type 1 diabetes duration ) or with mann - whitney u test in case of non  normally distributed variables ( age , bmi , diastolic and systolic blood pressure , heart rate , vmrr , bhi , pi , ri , end tidal co2 concentration , and imt ) . the  test was used to compare a proportion of females to males in groups . multivariable linear regression analysis was used to assess confounders of cvr in patients with type 1 diabetes . all patients and volunteers were free of coronary heart disease , diabetic foot , orthostatic hypotension , chronic renal insufficiency , or hemodynamically significant stenosis ( > 50% ) of extra- and intracranial cerebral arteries , as determined by physical check , biochemical analysis , and carotid and transcranial ultrasound , respectively . there was no significant difference regarding age , sex , bmi , blood pressure , and heart rate between patients and healthy control subjects ( table 1 ) . characteristics of patients with type 1 diabetes and healthy control subjects data are median ( range ) , arithmetic mean  sd , or percentage of patients . the median values of vmrr and bhi were significantly lower in patients than in the healthy control subjects . both groups did not differ regarding values of resting vmean , pi , and ri . the end tidal co2 concentrations did not differ between the groups , both during hyperventilation and breath holding . the median value of imt in patients was significantly higher in comparison to respective values in healthy control subjects ( table 2 ) . we found no significant correlations between vmrr or bhi values and age , diabetes duration , daily insulin demand , a1c concentrations , total serum cholesterol , triglyceride level , or imt in patients with type 1 diabetes . furthermore , no significant correlations between imt and vmrr or bhi were seen in healthy control subjects . comparison of the middle cerebral artery blood flow parameters and imt between patients with type 1 diabetes and healthy control subjects data are median ( range ) . etco2hv ,  end tidal co2 during hyperventilation ;  etco2 bh ,  end tidal co2 during breath holding . taking together both microalbuminuria and overt nephropathy into one group , an analysis showed a significant reduction of cvr values in the presence of diabetic nephropathy ( table 3 ) . after exclusion of two patients with overt nephropathy , vmrr remained impaired in patients with microalbuminuria when compared with individuals without microalbuminuria ( 71.0 vs. 87.5% , p = 0.03 ) . impact of vascular risk factors and comorbidities on cvr parameters in patients with type 1 diabetes data are median values . the median age in patients with nephropathy was 30 years ( minimum 27 years , maximum 45 years ) , while in patients without nephropathy , it was 32 years ( minimum 20 , maximum 51 ) . the age difference between the two groups was not significant ( p = 0.69 ) . mean duration of type 1 diabetes in patients with nephropathy ( 18.3  7.8 years ) was not different from the duration in patients without nephropathy ( 15.1  6.4 years , p = 0.24 ) . we found no significant differences of cvr values between the subgroups of patients with type 1 diabetes distinguished on the basis of the presence of diabetic retinopathy , neuropathy , hypertension , and hyperlipidemia . additionally , no differences regarding vmrr or bhi were found between groups of patients distinguished on the basis of use of statins or ace inhibitor / angiotensin receptor blocker ( table 3 ) . the multivariable regression analysis confirmed a significant inverse association between diabetic microalbuminuria / nephropathy and vmrr independently from other vascular risk factors or type 1 diabetes  related organ damage ( table 4 ) . results of multivariable linear regression analyses performed in a group of patients with type 1 diabetes to assess vmrr confounders serum concentrations in percent of a1c were taken . our study shows a reduction of vmrr and bhi in patients with type 1 diabetes , who were free of overt cerebrovascular events . however , cvr was not associated with diabetes course , presence of additional risk factors , biochemical parameters , and intima - media thickening . we revealed that the presence of diabetic nephropathy can indicate subclinical cerebrovascular pathology in patients with type 1 diabetes independently from other micro- and macroangiopathy evidence . our findings are in line with results of a previous study , which demonstrated a close relationship between indexes of cerebral microangiopathy ( pi and cvr ) and microalbuminuria in patients with type 2 diabetes ( 19 ) . few studies have reported an association between cvr and diabetic retinopathy in both type 1 and type 2 diabetes ( 7,13 ) . we also found a similar trend with borderline significance toward a cvr impairment in the presence of diabetic retinopathy . however , lack of statistical significance can probably be explained by type 2 error because of the small sample size . interestingly , the relationship between cvr and nephropathy in our patients was not influenced by hypertension , a finding that has also been described in former studies on type 1 and type 2 diabetes ( 6,13 ) . furthermore , we did not find any relationship between the use of antihypertensive or statin treatment and cvr in our patients , although statin and ace inhibitor treatments were reported as potential cvr confounders ( 20,21 ) . the lack of confounder effects can be explained by the relatively small sample size in our study . the lack of correlation between imt and cvr seen in our study has also been observed in patients with type 2 diabetes ( 13 ) . to our best knowledge , no published data exist on this correlation in patients with type 1 diabetes . however , recently published research showed negative correlation of cerebrovascular reactivity to l - arginine with imt . this approach may be an alternative method for analysis of cvr and imt relationship in patients with type 1 diabetes ( 22 ) . the provocation methods used in our study to estimate the reactivity of cerebral vessels ( hyperventilation and breath - holding tests ) have not been published yet for the adult patients with type 1 diabetes . advantages of this approach are the simulation of the natural cerebrovascular autoregulation mechanisms resulting from the use of a physiological method of evoking changes in pco2 , safety , noninvasiveness , and lack of side effects . the vmrr enabled us to assess a full range of vasodilation , instead of assessing only the upper limit obtained during the hypercapnia , as it was previously published in patients with type 1 diabetes ( 56,8 ) . the use of these tests allowed us to detect cvr impairment in the whole group of patients and not only in subgroups with longer duration of type 1 diabetes or with higher complications rate , as it was previously presented ( 57 ) . also , to avoid a possible influence of smoking and hormonal replacement therapy on cvr , we limited our study to nonsmokers and nonhormonal users ( 23,24 ) . in previous studies , the confounding effects of smoking and hormonal therapy were not controlled ( 6,13 ) . as a limitation of the presented study , we should mention that a relatively small sample size allowed us to detect only a single factor that has a significant impact on cvr in the examined group patients . the correlation between the presence of diabetic retinopathy and the low cvr values found in some studies could not be confirmed by our data ( 6,7 ) . however , both the univariate and multivariate analyses have shown statistically significant correlation between diabetic nephropathy and cvr impairment . this trend is also in agreement with previously published data ( 7,19 ) . despite a relatively large proportion of hyperlipidemic patients in our study , no influence of hyperlipidemia on cvr was found , similar to previous reports ( 6,25 ) . the presence of clinically asymptomatic cvr impairment in a group of patients with type 1 diabetes and peripheral microcirculatory dysfunction is an argument for early implementation of primary stroke prevention in these patients .
objectivecerebrovascular reactivity impairment was reported as a marker of cerebral microangiopathy in long - term type 1 diabetes . 
 intima - media complex thickening reflects early stages of macroangiopathy in type 1 diabetes . 
 the analysis of the relationship between these variables and other microangiopathic complications might serve as a beneficial indicator for early prophylaxis in these patients.research design and methodsvasomotor reactivity reserve ( vmrr ) and breath - holding index ( bhi ) of the middle cerebral artery were measured with transcranial doppler in 59 patients ( median age 32.0 years , range 2051 , 36 females ) with type 1 diabetes , without history of cerebrovascular events , and 30 healthy control subjects ( median age 31.5 years , range 2539 , 15 females ) . 
 the relationships between the presence of selected vascular complications of type 1 diabetes and biochemical parameters , intima - media thickness ( imt ) , and vmrr and bhi in patients were analyzed.resultsvmrr and bhi were lower in patients with type 1 diabetes when compared with healthy subjects ( 81.5 vs. 100% , p < 0.01 , and 1.6 vs. 2.2 , p = 0.04 , respectively ) , whereas imt was significantly higher in patients then in healthy control subjects ( 0.36 vs. 0.30 mm , p = 0.001 ) . 
 however , no association of imt with vmrr was found . 
 we found a significant reduction of vmrr and bhi in patients with diabetic nephropathy.conclusionsthe presence of diabetic nephropathy , but not imt , can be regarded as an indicator of cerebral microangiopathy severity in patients with type 1 diabetes .