children between 3 and 10 years of age with type 1 diabetes for at least 6 months and healthy controls subjects were recruited .
all procedures were approved by the institutional review board and all parents or guardians signed informed consents . children over 7 years of age signed assents ( clinicaltrials.gov study nct # 00449891 ) . diabetes history , time - weighted average hba1c ( calculated using the trapezoid rule   for all hba1c values from the time of diagnosis ) , cognitive testing , and mri training were completed as previously described ( 12 ) .
subjects completed the wechsler preschool and primary scale of intelligence iii ( wppsi - iii ) or wechsler intelligence scale for children iv ( wisc - iv ) based on their age .
all subjects were included in the full - scale intelligence quotient ( fsiq ) scores analysis , but the younger subjects were excluded from the wisc - iv score analyses . the time - weighted hba1c value is used for all data analyses .
bg values were between 80 and 250 mg / dl before the start of any neurocognitive testing or mri scan .
we specifically examined neurocognitive skills associated with wm function using tasks specifically designed for children , including subtests from the wisc - iv ( block design digit span , picture concepts , vocabulary , letter - numbering sequencing , matrix reasoning , and comprehension ) and the developmental neuropsychological assessment ( nepsy ) ( design copy , phonological processing , memory of faces , tower , auditory attention and response , speeded naming , arrows , delayed memory of faces , comprehension of instructions , imitating hand positions , and narrative memory ) .
subjects underwent high - resolution structural mri scans using a general electric signal 1.5 tesla imaging system .
coronal three - dimensional volumetric spoiled grass gradient recalled series were acquired with the following scan parameters : repetition time ( tr ) 35 ms , echo time ( te ) 6 ms , flip angle 45 , 1.5 mm thick , 0-mm gap , number of excitations 1 , field of view ( fov ) 24 cm , and a 256  192 matrix size for 124 contiguous slices ( scan time : 14 min ) .
imaging parameters for the diffusion - weighted sequence were as follows : fov , 24 cm ; matrix size , 128  128 ; tr / te 5,400/min ; 43 axial - oblique slices ; slice thickness , 3.2 mm / no skip .
diffusion gradient duration was  = 32 ms and diffusion weighting was b = 850 s / mm , and b = 0 as reference images .
diffusion - weighted images were corrected for eddy current distortions and head motion using an affine transformation of automated image registration ( 16 ) .
we excluded six subjects ( four with diabetes , two control subjects ) from further analysis because of significant artifacts .
the remaining images were averaged and the pixel intensities of the multiple diffusion - weighted images were then fitted to obtain the six elements of the symmetric diffusion tensor .
scalars such as fractional anisotropy ( fa ) , axial diffusivity ( ad ) , and radial diffusivity ( rd ) were calculated using dtistudio ( https://www.mristudio.org/ ) .
anisotropy within a given wm voxel is determined by fiber diameter and density , degree of myelination , extracellular diffusion , interaxonal spacing , and intravoxel fiber -tract coherence .
ad is the diffusivity of water molecules along the main axis of diffusion within a voxel ( vector with the largest eigenvalue ) , and rd is the mean of the diffusivities perpendicular to the vector with the largest eigenvalue .
ad is thought to reflect fiber coherence , whereas rd is thought to represent fiber integrity and myelination ( 17,18 ) .
first , fa images from each subject were aligned into a common space using nonlinear and linear registrations .
a skeletonization algorithm was applied to the group mean fa image to define a group template of the lines of maximum fa , thought to correspond to centers of wm tracts .
the original registration parameters of the fa were then applied to the ad and rd images .
fa , ad , and rd data projected onto the skeleton were fed into voxel - wise cross - subject statistics ( p < 0.05 ) using  randomise  ( v. 2.1 in fsl4.1 ) , a permutation program used for inference ( thresholding ) on statistic maps when the null distribution is not known ( 19 ) .
all analyses were corrected for multiple comparisons ( family - wise error ) and used threshold - free cluster enhancement ( 20 ) with default parameters .
statistical analyses of behavioral and cognitive data were performed using spss software version 13 ( spss inc . ,
index and specific subtest scores were compared between diabetes and hc subjects using t tests . among those with diabetes
, linear correlations were performed between wisc - iv and nepsy subtest scores and hba1c and number of seizures all scores are reported as mean values and standard deviation .
we further analyzed correlations between cognitive data , time - weighted hba1c , age at diagnosis , duration of illness , history of seizures , and wm structure ( fa , ad , and rd ) using tbss .
diffusion - weighted images were corrected for eddy current distortions and head motion using an affine transformation of automated image registration ( 16 ) .
we excluded six subjects ( four with diabetes , two control subjects ) from further analysis because of significant artifacts .
the remaining images were averaged and the pixel intensities of the multiple diffusion - weighted images were then fitted to obtain the six elements of the symmetric diffusion tensor .
scalars such as fractional anisotropy ( fa ) , axial diffusivity ( ad ) , and radial diffusivity ( rd ) were calculated using dtistudio ( https://www.mristudio.org/ ) .
anisotropy within a given wm voxel is determined by fiber diameter and density , degree of myelination , extracellular diffusion , interaxonal spacing , and intravoxel fiber -tract coherence .
ad is the diffusivity of water molecules along the main axis of diffusion within a voxel ( vector with the largest eigenvalue ) , and rd is the mean of the diffusivities perpendicular to the vector with the largest eigenvalue .
ad is thought to reflect fiber coherence , whereas rd is thought to represent fiber integrity and myelination ( 17,18 ) .
first , fa images from each subject were aligned into a common space using nonlinear and linear registrations .
a skeletonization algorithm was applied to the group mean fa image to define a group template of the lines of maximum fa , thought to correspond to centers of wm tracts .
the original registration parameters of the fa were then applied to the ad and rd images .
fa , ad , and rd data projected onto the skeleton were fed into voxel - wise cross - subject statistics ( p < 0.05 ) using  randomise  ( v. 2.1 in fsl4.1 ) , a permutation program used for inference ( thresholding ) on statistic maps when the null distribution is not known ( 19 ) .
all analyses were corrected for multiple comparisons ( family - wise error ) and used threshold - free cluster enhancement ( 20 ) with default parameters .
statistical analyses of behavioral and cognitive data were performed using spss software version 13 ( spss inc . ,
chicago , il ) . index and specific subtest scores were compared between diabetes and hc subjects using t tests . among those with diabetes
, linear correlations were performed between wisc - iv and nepsy subtest scores and hba1c and number of seizures all scores are reported as mean values and standard deviation .
we further analyzed correlations between cognitive data , time - weighted hba1c , age at diagnosis , duration of illness , history of seizures , and wm structure ( fa , ad , and rd ) using tbss .
we approached 81 subjects with type 1 diabetes : 35 by e - mail or phone and 46 directly in the clinic .
likewise , 30 healthy control subjects , mostly siblings or friends of our subjects , were directly approached in person .
forty - five subjects ( n = 27 diabetes , n = 18 healthy control subjects ) completed the neurocognitive testing , and 42 ( n = 26 diabetes , n = 16 healthy control subjects ) of them completed dti scans .
six subjects ( n = 4 diabetes , n = 2 healthy control subjects ) were excluded from further analysis because of significant image artifacts .
the data presented in this manuscript are restricted to the subset of subjects who completed both the neurocognitive testing and the dti scans ( table 1 ) .
wppsi - iii and wisc - iv results were very similar ( p = 0.38 ) for fsiq score for subjects with diabetes ( wppsi - iii 109  4.2 ; wisc - iv 108.4  14.7 ) and healthy control subjects ( wppsi - iii 107  11.0 ; wisc - iv 112.7  13.5 ) . there were no statistically significant differences in general cognitive ability in regard to executive function , processing speed , memory / attention , and motor domains between subjects with diabetes and healthy controls ( all p values > 0.05 ) .
however , calculations of the effect size are approximately 0.30 ( cohen s d ) , suggesting that there may be a modest difference between the two groups wisc scores . there were no significant between - group differences on nepsy scores ( all p values > 0.05 ) . within the diabetes group ,
higher hba1c levels were correlated with lower overall intellectual functioning measured by fsiq ( r = 0.215 , p = 0.03 ) ( fig .
1 ) . there was a nearly statistically significant relationship between greater seizure occurrence and lower wisc - iv verbal comprehension index scores ( r = 0.173 , p = 0.054 ) , as well as with hba1c levels and wisc - iv digit span ( r = 0.214 , p = 0.053 ) .
lastly , greater seizure number was predictive of lower wisc - iv processing speed index scores ( r = 0.402 , p < 0.01 ) .
greater hba1c levels predicted lower overall intellectual functioning measured by fsiq in children with type 1 diabetes ( r = 0.215 , p = 0.03 ) .
compared with controls , children with diabetes had lower ad values ( p = 0.046 covaried for age and gender ) in the internal capsule , body of the corpus callosum ( cc ) , right cingulate gyrus , right thalamus , right superior temporal gyrus , and posterior parietal lobe ( fig .
2 ) . there were no significant differences between groups in fa and rd . within the diabetes group
, there was a statistically significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) ( fig .
specifically , this correlation was seen in the inferior fronto - occipital fasciculi , uncinate fasciculi , subgenual wm , anterior forceps , right internal capsule , superior middle and inferior temporal gyri , splenium of the cc , superior longitudinal fasciculi , and occipital wm . a negative correlation between hba1c values and fa approached significance ( p = 0.057 ) in the right internal capsule , the anterior forceps , inferior fronto - occipital fasciculi , and splenium of the cc .
regions of significant reductions in ad ( shown in yellow ) in children with type 1 diabetes as compared with healthy controls subjects , shown in serial images in the axial orientation .
( a high - quality digital representation of this figure is available in the online issue . ) regions of significant positive correlation between hba1c values and rd ( shown in light blue ) within the type 1 diabetes group , shown in serial images in the axial orientation .
group differences were  thickened  for visualization purposes ( shown in dark blue ) .
( a high - quality digital representation of this figure is available in the online issue . ) within - group analysis of those with type 1 diabetes detected a positive correlation between fa and wisc coding ( p = 0.034 ) and digit span ( p = 0.028 ) subtests , and positive correlations between fa and wisc fsiq ( p = 0.064 ) and fa and wisc ( p = 0.058 ) processing speed approached significance .
higher fa values correlated with the subjects abilities to focus attention and quickly scan , discriminate between , and sequentially order visual information .
there was a significant negative correlation between rd and wisc fsiq ( p = 0.022 ) and rd and nepsy auditory attention score ( p = 0.02 ) .
the negative correlation between rd and nepsy attention / executive function approached statistical significance ( p = 0.067 ) .
wppsi - iii and wisc - iv results were very similar ( p = 0.38 ) for fsiq score for subjects with diabetes ( wppsi - iii 109  4.2 ; wisc - iv 108.4  14.7 ) and healthy control subjects ( wppsi - iii 107  11.0 ; wisc - iv 112.7  13.5 ) . there were no statistically significant differences in general cognitive ability in regard to executive function , processing speed , memory / attention , and motor domains between subjects with diabetes and healthy controls ( all p values > 0.05 ) .
however , calculations of the effect size are approximately 0.30 ( cohen s d ) , suggesting that there may be a modest difference between the two groups wisc scores .
there were no significant between - group differences on nepsy scores ( all p values > 0.05 ) . within the diabetes group ,
higher hba1c levels were correlated with lower overall intellectual functioning measured by fsiq ( r = 0.215 , p = 0.03 ) ( fig .
1 ) . there was a nearly statistically significant relationship between greater seizure occurrence and lower wisc - iv verbal comprehension index scores ( r = 0.173 , p = 0.054 ) , as well as with hba1c levels and wisc - iv digit span ( r = 0.214 , p = 0.053 ) .
lastly , greater seizure number was predictive of lower wisc - iv processing speed index scores ( r = 0.402 , p < 0.01 ) .
greater hba1c levels predicted lower overall intellectual functioning measured by fsiq in children with type 1 diabetes ( r = 0.215 , p = 0.03 ) .
compared with controls , children with diabetes had lower ad values ( p = 0.046 covaried for age and gender ) in the internal capsule , body of the corpus callosum ( cc ) , right cingulate gyrus , right thalamus , right superior temporal gyrus , and posterior parietal lobe ( fig .
2 ) . there were no significant differences between groups in fa and rd . within the diabetes group , there was a statistically significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) ( fig .
specifically , this correlation was seen in the inferior fronto - occipital fasciculi , uncinate fasciculi , subgenual wm , anterior forceps , right internal capsule , superior middle and inferior temporal gyri , splenium of the cc , superior longitudinal fasciculi , and occipital wm . a negative correlation between hba1c values and fa approached significance ( p = 0.057 ) in the right internal capsule , the anterior forceps , inferior fronto - occipital fasciculi , and splenium of the cc .
regions of significant reductions in ad ( shown in yellow ) in children with type 1 diabetes as compared with healthy controls subjects , shown in serial images in the axial orientation .
( a high - quality digital representation of this figure is available in the online issue . ) regions of significant positive correlation between hba1c values and rd ( shown in light blue ) within the type 1 diabetes group , shown in serial images in the axial orientation .
group differences were  thickened  for visualization purposes ( shown in dark blue ) .
( a high - quality digital representation of this figure is available in the online issue . )
within - group analysis of those with type 1 diabetes detected a positive correlation between fa and wisc coding ( p = 0.034 ) and digit span ( p = 0.028 ) subtests , and positive correlations between fa and wisc fsiq ( p = 0.064 ) and fa and wisc ( p = 0.058 ) processing speed approached significance .
higher fa values correlated with the subjects abilities to focus attention and quickly scan , discriminate between , and sequentially order visual information .
there was a significant negative correlation between rd and wisc fsiq ( p = 0.022 ) and rd and nepsy auditory attention score ( p = 0.02 ) .
the negative correlation between rd and nepsy attention / executive function approached statistical significance ( p = 0.067 ) .
neuroimaging techniques have been used to further our understanding of how medical diseases affect brain structure ( 21,22 ) . in our study
, we examined how type 1 diabetes affects wm microstructure in young children and the resultant effects on neurocognitive performance .
although there were no differences in cognitive test scores between the two groups , we did find that in those with diabetes , higher hba1c levels were correlated with lower fsiq scores .
also , we describe differences in wm microstructure between those with diabetes and controls , especially in the frontal and temporal regions , and that these differences were more apparent among those with higher hba1c levels . neuroimaging studies in young children with type 1 diabetes have been limited to date ( 12,23,24 ) , and understanding the impact of diabetes on neurodevelopment is still based largely on inferences drawn from adult neurocognitive and neuroimaging data ( 25 ) .
dti provides a valuable tool to determine the relationship between wm connectivity and cognitive performance and allows additional , fine - grained investigation of wm microstructure .
based on adult studies , abnormal wm ( lower fa ) has been reported in four major brain regions and is thought to be responsible for neurocognitive deficits involving mental processing speed , attention , and executive functioning skills such as mental flexibility ( 13,26 ) .
however , it is unknown when these wm differences developed or whether they are directly attributed to the course of the diabetes .
although we did not find significant differences in fa between those with diabetes and controls , this may be due to the young age of our participants , resulting in lack of diabetes - related complications and shorter duration of the disease . despite this
, we did see a negative correlation between hba1c and fa ; although this did not reach statistical significance , larger future studies may indicate a clearer connection .
ad and rd are indices that complement fa in providing additional information and increased understanding about the underlying wm and neuronal integrity . in this study ,
children with type 1 diabetes had lower ad values , a measure associated with less axonal coherence ( 27,28 ) . within this group
, there also was a significant , widespread positive correlation between hba1c and rd , suggesting that higher bg values may affect fiber myelination or the permeability of axonal membranes ( 14 ) .
parents and providers fear hypoglycemia in young children at the expense of hyperglycemia ( 29 ) and accept chronically elevated bg values as reflected by the american diabetes association guidelines where there is a higher hba1c target in young children ( 30 ) .
hypoglycemia has been traditionally thought to be the cause of the neurocognitive deficits ; however , two longitudinal pediatric studies show that history of severe hypoglycemia alone could not explain the neurocognitive differences from healthy controls ( 31,32 ) .
although we found a near - significant relationship between history of seizure occurrence and selected neurocognitive measures , the parents reaction to the hypoglycemia , resulting in a period of chronic hyperglycemia , may be a confounding factor .
therefore , the discrepancy between the between - group analysis ( which revealed significant differences in ad ) and the within - group correlation of wm structure ( as reflected by rd and hba1c levels ) could possibly be explained by other diabetes - related factors such as hyperglycemia , perhaps leading to increased nonenzymatic glycosylation .
hyperglycemia has a number of proposed mechanisms to cause wm changes , including nonenzymatic glycosylation and activation of the sorbitol pathway .
myelin has lysine residues that are specifically susceptible to nonenzymatic glycosylation ( 33 ) . in animal models of diabetes , advanced glycosylation end products and the receptor for advanced glycosylation end products
are both increased in the hypothalamus ( 34 ) and associated with significant loss of wm ( 35 ) .
activation of the sorbitol pathway has been seen in 1-month - old rats when subjected to chronic hyperglycemia , leading to reductions in dendritic branching and spine density , and subsequent significant increases in brain sorbitol and inositol , when compared with rats with hypoglycemia ( 36 ) .
therefore , the effect of hyperglycemia in young children may be particularly relevant to wm microstructure and in the resulting neurocognitive implications
. a history of diabetic ketoacidosis ( dka ) may be another cause of axonal injury and be reflected in changes in ad .
( 37 ) of children between ages 7 and 16 years found that children with a history of type 1 diabetes and dka perform poorly on tests of memory function compared with children with a history of type 1 diabetes but not dka .
the memory deficits seem to be specifically related to the hippocampus , a region of the brain that is particularly sensitive to episodes of hypoxia or ischemia . in our study , seven of the children with diabetes had presented with dka at onset and no one experienced dka after diagnosis .
we did not observe a significant correlation between those who experienced dka and fa or ad or rd , although our small number of subjects do not allow for valid inferences .
between - group differences in ad and rd occurred most prominently in the temporal and parietal regions of the brain in our study .
positron emission tomography studies in healthy adults show that the temporal and frontal regions are most vulnerable to hypoglycemia and hyperglycemia ( 5,28 ) .
young children with diabetes frequently experience glycemic variability ( 3 ) , resulting in more fluctuations in brain glucose levels and impacting brain glucose metabolism .
therefore , it is not surprising to find that the most prominent areas of wm microstructural changes in children with type 1 diabetes also occur in these regions .
although the study did not find any particular neurocognitive variation that correlated with either the temporal or frontal lobe function at this time , it will be intriguing to see if the microstructural changes on dti predict temporal and frontal lobe  related neurocognitive changes over time .
perhaps decreased episodes of glycemic variability in these regions prevent memory loss , improve attention span , or decrease the comorbidity of depression in children with diabetes as they age .
it will also be important to elucidate if wm microstructural patterns predict adverse neurocognitive changes over time , and to develop clinical interventions to prevent such changes .
both the small sample size and the high heterogeneity in performance on neurocognitive testing in this age group may have not allowed for detection of the cognitive differences .
the study was not adequately powered to detect subtle or even moderate wm neurocognitive measures between those with type 1 diabetes and controls .
also , subjects in this study completed different wechsler intelligence scales because of their ages .
although not ideal , there is still a strong correlation between the two wechsler intelligence scales .
the correlation between the two fsiq scores ( r = 0.89 ) ( 38 ) is nearly as high as the wisc - iv fsiq test - retest correlation and wppsi - iii fsiq test - retest correlations ( r = 0.93 and r = 0.92 , respectively ) , suggesting they measure similar constructs . finally , because of the age range of our sample , the subjects in the study are undergoing brain development at varying rates .
however , the examination of wm microstructure variations in this age range is important and makes this study novel .
wm changes and neurocognitive differences reported in adults with long - term type 1 diabetes likely begin in childhood .
an association between early age of onset of disease ( generally before the age of 6 years ) and neurocognitive deficits has been the most consistent finding in the pediatric literature ( 1,25 ) . as northam
et al . ( 25 ) recently summarized , chronic hyperglycemia , occurrence of diabetic ketoacidosis , bg fluctuations , and hyper- or hypoinsulinism all may impact the neurodevelopment of young children with type 1 diabetes .
we suggest that there are wm microstructure differences between young children with type 1 diabetes and healthy controls after a short duration of diabetes , and support larger , longitudinal studies to confirm these changes .
knowledge of when these changes begin will impact our understanding of diabetes and the brain , identify the mechanisms of neuronal injury , and modify management of diabetes to minimize the chances of injury .
traditionally , the management of type 1 diabetes in young children has been to avoid hypoglycemia ; however , we may need to tolerate less hyperglycemia during childhood as well .
 objectiveto detect clinical correlates of cognitive abilities and white matter ( wm ) microstructural changes using diffusion tensor imaging ( dti ) in young children with type 1 diabetes.research design and methodschildren , ages 3 to < 10 years , with type 1 diabetes ( n = 22 ) and age- and sex - matched healthy control subjects ( n = 14 ) completed neurocognitive testing and dti scans.resultscompared with healthy controls , children with type 1 diabetes had lower axial diffusivity ( ad ) values ( p = 0.046 ) in the temporal and parietal lobe regions . 
 there were no significant differences between groups in fractional anisotropy and radial diffusivity ( rd ) . within the diabetes group , there was a significant , positive correlation between time - weighted hba1c and rd ( p = 0.028 ) . a higher , time - weighted hba1c value was significantly correlated with lower overall intellectual functioning measured by the full - scale intelligence quotient ( p = 0.03).conclusionschildren with type 1 diabetes had significantly different wm structure ( as measured by ad ) when compared with controls . 
 in addition , wm structural differences ( as measured by rd ) were significantly correlated with their hba1c values . 
 additional studies are needed to determine if wm microstructural differences in young children with type 1 diabetes predict future neurocognitive outcome . 
RESEARCH DESIGN AND METHODS
Diffusion-weighted scans preprocessing
Tract-based spatial statistics (TBSS) analysis
Statistical analysis
RESULTS
Cognitive testing
TBSS analysis
DTI and neurocognitive correlations
CONCLUSIONS