Introduction

Human brain development is an ongoing process of maturation leading to significant changes in both its structural architecture and functional organization. Maturation of white matter connectivity is an important factor in cognitive, linguistic, behavioural, emotional and motor development throughout childhood, adolescence and into adulthood [1]. Among white matter tracts, the association fibres interconnect functionally specialized areas of the cortex and are extremely important in informing different brain regions of ongoing neuronal processing thus allowing integration of different brain activities [2]. While efficient organization of these fibres is essential for optimal cognitive, linguistic and learning performance [3], aberrations in the normal development of these tracts could underlie certain psychopathologies [4,5,6]. Consequently, identifying normal developmental brain trajectory contributes to better understanding of neurodevelopmental disorders and other disabilities.

Diffusion tensor imaging is an innovative technique that enables in vivo analysis of white matter (WM) microstructure by measuring several biomarkers of anisotropy (fractional anisotropy, FA) and diffusivity (Trace; axial diffusivity, AD; radial diffusivity, RD). Studies of WM development have demonstrated opposite trends of increasing FA and decreasing mean diffusivity in several brain regions throughout infancy, childhood and adolescence, reflecting increase in fibre density and/or myelination [7, 8]. Most of these studies were based on region of interest analysis (ROI), voxel-based morphometry (VBM) or tract-based spatial statistics (TBSS). A great limitation of ROI is the possible error related to including other grey or white matter structures or even cerebrospinal fluid together with the examined white matter regions [8]. VBM and TBSS have the advantage of the ability to execute a whole brain analysis. However, registration and spatial normalization are required, and the data should be interpreted cautiously especially in groups with more heterogeneous anatomy such as children [9,10,11]. ROI approach and VBM have limited ability to examine white matter tracts along their entire course. On the contrary, diffusion tensor tractography (DTT) method is a more specific approach compared to ROI-based analysis or VBM. By DTT, only the tract of interest is targeted rather than performing coordinate-based measurement of white matter irregularities between groups. It allows the researchers to compare equivalent fibre connections across individuals [12,13,14].

Despite the great interest in studying WM development in neonatal period, infancy and in adolescence [7, 15,16,17,18,19,20,21,22], there are few papers in the scientific literature studying age-related white matter changes in younger adults [23,24,25,26,27]. To the best of our knowledge, few papers investigated the association fibres through fibre tract reconstruction [22, 24, 26, 28]. Furthermore, little is known about the AD and RD, as most of the scientific literature has focused on FA and mean diffusivity [15, 19,20,21].

We hypothesised that there may be age-related changes in the association tracts during late childhood and adulthood, in addition lateral asymmetry and age-related differences may be encountered. The aim of this work is to study the age, gender and lateral asymmetry-related white matter changes of long association tracts throughout late childhood, adolescence and into adulthood using DTT.

Materials and methods

Participants

From 2014 to 2016, a cross-sectional study was conducted after approval of the local institutional review board. All volunteers gave informed consent; both child assent and parent consent were obtained for volunteers under 21 years.

Participants included 44 healthy subjects, 7–45 years of age (23 males and 21 females; mean age = 19 years). In the whole sample, there were no mean age differences between the two genders (p = 0.53). According to the WHO project on data set for ageing in Africa, 50 years of age and older were used, as the general definition of an older person and so the upper limit of younger adult were 50 years (http://www.who.int/healthinfo/survey/ageingdefnolder/en/). Participants who fit the inclusion criteria and recruited at the study period were enrolled in the study.

Detailed history taking followed by general and neurological examination was performed for all volunteers. During the interview, special concern was addressed to the developmental history, history of psychiatric disorders and scholastic performance. Patients with abnormal development and/or disabilities with poor scholastic achievements were excluded. Patients with psychiatric disorders, neurologic disease or brain injury were excluded as well. In addition, for ethical considerations, patients needing sedation were also excluded. All subjects were right-handed native Egyptians of similar socioeconomic standard. The same MRI scanner was used for all participants.

MRI protocol

Participants were scanned with a 1.5 T scanner (Achieva, Philips Healthcare, Eindhoven, The Netherlands) using 8-channel SENSE head coil (SENSE acceleration factor of 8). All participants were asked to remain quiet during scanning. Foam bads and ear plugs were used to reduce noise and head motion. The DTI data were acquired using single shot echo-planar sequence. In addition to baseline b0 image, diffusion-weighted images were acquired in 32 non-collinear direction (DTI high protocol). Seventy axial slices with 2 mm slice thickness were positioned parallel to the anterior-posterior commissure line. The field of view was 230 mm × 230 mm and in-plain resolution = 2.5 × 2.5 mm2. FLAIR (TR/TE/IT: 11,000/130/2800) and 3D SPGR (TR/TE/FA: 22/9/30) were obtained in the axial plane. Slice thickness was 5 and 1.6 mm, respectively. Participants with scans that showed head motion artefact were excluded from the study (two participants).

Image analysis

The work station was Windows-operated personal computer. DTI and conventional MRI sequences of each individual subject were analysed offline using DTI Studio software produced by this laboratory (H. Jiang and S. Mori, Johns Hopkins University and Kennedy Krieger institute, http://godzilla.kennedykrieger.org or http://lbam.med.jhmi.edu) [29]. The images were first visually inspected for apparent artefact. The raw diffusion-weighted images were then co-registered to b0 images and corrected for small subject motion using automatic image registration (AIR) tool. FA and colour FA and different diffusivity indices RD, AD and Trace maps were calculated in the native space. RD is calculated by averaging the two main eigenvalues. AD represents the diffusivity along the main eigenvector. Trace is calculated by summation of diffusion in three directions (λ1 + λ2 + λ3). Trace is a measure of overall diffusion, and it is proportional to the mean diffusion [(λ1 + λ2 + λ3)/3] [30, 31].

Tractography

Tractography was performed using two ROI approach according to the previously described protocol with high reproducibility which was adopted by Wakana et al. using the Fibre Assignment by Continuous Tracking (FACT) method [14]. The following tracts were extracted for both hemispheres: uncinate fasciculus, inferior longitudinal fasciculus (ILF), inferior fronto-occipital fasciculus (IFOF), and superior longitudinal fasciculus (SLF) and its arcuate fasciculus (which was described as the temporal component of SLF by Wakana et al. [14]) (Fig. 1): All these tracts represent long association tracts connecting brain regions from different lobes. The average FA, RD, AD and Trace were calculated for each tract. Absolute tract volume (number of voxels that fibres go) and density (mean number of fibres/voxel) were also calculated.

Fig. 1
figure 1

Illustrative figure displaying 3D views of reconstructed long association tracts overlaid on fractional anisotropy images. a Uncinate fasciculus. b Inferior fronto-occipital fasciculus. c Inferior longitudinal fasciculus. d Superior longitudinal fasciculus. e Arcuate fasciculus

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Statistical analysis

Kolmogorov-Samirnov and Shapiro-Wilk tests were performed first to test for the normal distribution of the data along with analysis of the normal Q-Q plot. Correlation analysis was performed for FA, RD, Trace, AD, volume and density versus age within 7- to 45-year age range using Pearson correlation test. In addition, each tract values were compared between both hemispheres using paired Student’s t test among whole population. To study gender effects, FA, diffusivity indices, volume and density of the tracts were compared between the females and males among the whole population using unpaired Student’s t test. Statistical analysis was performed first without corrections for multiple comparisons, and then we applied false discovery rate (FDR) to reduce the potential type 1 error from multiple comparisons. A p value less than 0.01 (with FDR correction) was considered significant.

Results

Correlation analysis (Table 1)

The majority of the examined tracts (SLF and IFOF of both hemispheres, and the arcuate fasciculus, uncinate fasciculus and ILF of the left hemisphere) followed a common pattern of metric changes with age. This pattern was characterized by significant increase in FA and reduction of RD and Trace with no significant changes in AD (Fig. 2). The right arcuate fasciculus showed similar pattern but without significant changes in FA. The right uncinate and right ILF fasciculus demonstrated significant reduction in RD, Trace and AD, with and without significant increase in FA, respectively (Fig. 2).

Table 1 Correlation of the DTI data with age
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Fig. 2
figure 2

Scatter plot showing maturation of long association tracts with age in terms of FA and diffusivity indices. a Data of the left ILF is chosen to represent the predominant pattern that involves seven of the examined ten tracts. It shows significant increase in FA accompanied by reduction in RD, Trace without significant AD changes. b The right arcuate fasciculus shows significant reduction in RD, Trace without significant changes in FA or AD. c The right uncinate fasciculus shows significant increase in FA accompanied by reduction in RD, Trace, AD. d The right ILF shows significant reduction in RD, Trace, AD without significant FA changes. AD axial diffusivity, FA fractional anisotropy, RD radial diffusivity, Trace Trace value, ILF left inferior longitudinal fasciculus, Rt right, Lt left

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RD and Trace showed significant negative correlation with age in all tracts. Increase of FA was seen in all tracts that were significant in all but two tracts. Decrease of AD with increasing age was significant in two tracts only (right uncinate and right ILF). Significant increase of absolute tract volume was seen in left SLF, left ILF and RT IFOF. No significant density changes were documented with age.

Hemispheric lateralization among all participants (Table 2)

The uncinate fasciculus on the left hemisphere exhibited significantly higher FA, lower Trace and RD compared to the right, with trend towards significantly lower AD. The left ILF showed trends towards significant lower AD and Trace. The left IFOF showed trend towards significant lower Trace.

Table 2 Differences of DTI parameters between both hemispheres
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Gender effects among all participants

The right SLF exhibited lower AD in females compared to males that turned out to be insignificant after correction of multiple comparisons (p value > 0.01 with FDR correction). Otherwise, there were non-significant gender-related effects on DTI biomarkers in most of the tract studies.

Discussion

The development of cognitive and linguistic functions during childhood depends on many neuroanatomical maturation processes which continue throughout adolescence into adulthood [32]. This tractography study of 44 healthy subjects tracking long association fibres has demonstrated maturation changes in the brain through late childhood, adolescence and into adulthood.

We found age-related increases in FA in white matter tracts with concomitant reduction in RD, Trace, and to lesser extent AD values. Our results were consistent with previous tractography studies [22, 24, 26, 28], as well as VBM studies [8, 18, 25], ROI-based analysis [19, 23, 27, 33] and TBSS [8, 25] despite technical differences.

White matter maturation passes into three phases. The first phase that occurs in late intrauterine life is characterized by progressive organization of fibres into fascicles leading to increased FA, and AD and reduction of RD with no changes in mean diffusivity. The second phase is linked to decrease brain water content and an increase in membrane density. There is proliferation of oligodendrocytes linage precursors along with neurofilaments and microtubules with reduction of AD, RD and mean diffusivity without significant changes in FA. The last phase of true fibre myelination is characterized by ensheathment of oligodendroglial processes around axons. It shows progressive increase in FA with concomitant reduction of RD and mean diffusivity with no changes in AD [34,35,36]. In addition, diffusivity changes are proposed to appear earlier than anisotropy changes [34].

The current study revealed a predominant pattern of white matter maturation (involves seven of the examined ten tracts) showing increased FA, with concomitant reduction in RD and Trace with no significant AD changes. RD measurements reflect myelin changes [30], while FA can be related to several histological characteristics, such as axonal density and diameter, and degree of myelination, and the increase of FA is related to increase in the connectivity of white matter bundles [37]. This pattern likely reflects ongoing myelination corresponding to the third phase of white matter maturation. The right arcuate fasciculus showed similar changes with age but without significant FA changes. This could represent subtle changes in myelination that were not sufficient to induce FA changes (as diffusivity changes precede FA changes). The remaining two tracts, the right uncinate fasciculus and right ILF, exhibited reduction in AD, Trace and RD with or without significant increase in FA, respectively. AD measures diffusion along the principle axis of diffusion ellipsoid, and it is principally indicating axonal status. It was postulated that the reduction of AD with age may result from increased calibre of axons with reduction of the inter-axonal space allowing fibres to be less straight. Also, the decrease in AD may be related to the growth of neurofibrils and glial cells [7, 8, 33]. The patterns of these two tracts could represent transitional stages in the maturation process (probably corresponding in part to the second phase of maturation) before proceeding into the last phase of maturation in which myelination is the predominant pattern with no significant changes in AD. This may signify relatively more delay in maturation of the involved tracts (uncinate fasciculus and ILF of the right hemisphere). Interestingly, opposite patterns of age-related degeneration in white matter microstructure in older adults were described by Burzynska et al. and Bennett et al. (showing reduction of FA with increase in diffusivity indices) [38, 39]. We assume that the observed white matter maturation patterns in our younger cohort could represent maturation processes that would be reversed with advanced age.

These changes of anisotropy and diffusivity can support the notion that much of the complex cognitive functions (as processing speed, voluntary response inhibition and working memory) continue maturation during late adolescence and adulthood [32, 40, 41]. Also, it was reported that the increase in FA and reduction in mean diffusivity were correlated to measures of cognitive function such as IQ [42]. Continued white matter maturation with progressive myelination to post-adolescent period may be necessary to increase efficiency of neuronal communication. This yields constant latencies between brain regions across different ages to compensate for brain growth [43]. In addition, post-adolescent training and life experiences may have implications on brain development leading to white matter microstructural changes [44].

FA changes were more significantly correlated with age in the right uncinate fasciculus, IFOF and left ILF. The occipito-frontal cortical regions connect through two major pathways: the direct pathway through the IFOF that connects the occipital, posterior temporal and oribito-frontal areas; and the indirect pathway of the ILF which connects the occipito-temporal regions and joins the uncinate fasciculus anteriorly to relay information in the orbito-frontal areas. Significant correlations were found between the IFOF, ILF and uncinate fasciculus reflecting functional similarity [45]. In addition to their role in higher cognitive functions (social cognition, attention, visuospatial processing, and reading, semantic processing) [46], these tracts are among important tracts that constitute the ventral stream of language pathway [47]. Moreover, the protracted maturation of uncinate fasciculus may have functional contribution to the orbitofrontal cortex which represents a key region in reward processing with its possible implications on development and reward modulation and cognitive control [48].

Less appreciable correlations with age (although they were significant) were seen in SLF and arcuate fasciculus bilaterally. The SLF was reported to undergo more significant maturation before the age of 6 years (younger age than that included in the present study). This maturation supports a basic proficiency in language and auditory functions which are essential for the development of higher cognitive skills such as reading [49]. Moreover, previous DTT measures of the SLF and arcuate fasciculus were found to be significantly correlated with inhibitory control, fine motor speed and expressive language functions [50].

Prior brain volumetric analysis research revealed constancy of total brain volume through late childhood to adulthood, with opposite trend of changes regarding the white and grey matter [24, 26]. Although there is progressive increase in total white matter volume with age [24, 26], we have found progressive increase in the absolute volume of the left SLF, left ILF and RT IFOF only, so the overall changes in whole brain white matter volume may be related to increases in some pathways selectively, rather than potentially as a global effect.

Using tractography, we have found that the left cerebral hemisphere demonstrated higher FA of the uncinate fasciculus. This is consistent with previous documents that reported left greater than right asymmetry [20, 51,52,53]. In addition, we have found reduction in Trace and RD. To our knowledge, hemispheric asymmetry in terms of RD and Trace was not previously documented. Interestingly, significant correlation between structural lateralization of the white matter tracts and the left-right difference in functional activation has been identified. It was reported that fronto-temporal connections via the uncinate fasciculus and IFOF were stronger on the left side [54]. The left uncinate fasciculus has been linked to semantic memory that is used for remembering facts and concepts and is closely integrated with language processing. Patients with semantic dementia have lower uncinate fasciculus FA values than control group [55]. Moreover, the decrease in the left ward lateralization of the FA values of the uncinate fasciculus has been linked to autism spectrum disorder [56].

Similar to other reports studying association fibres [22, 26, 28], there was limited gender effect in the present study, and other researchers found that most of the gender-related differences were related to tracts involved in connectivity between the two hemispheres such as corpus callosum [57, 58]. Other reports found sexual dimorphism in terms of FA and or mean diffusivity of the superior fronto-occipital fasciculus, cingulum, uncinate fasciculus and corticospinal tracts [1, 26].

Although we could find statistically significant correlations with age for FA, and other diffusivity indices as well as hemispheric lateralization, our study is limited by relatively small number of participants with few subjects per age. Our study was cross-sectional including subjects of different ages. Longitudinal studies with larger number of participants may reveal more accurate results. Correlation of various DTI parameters with cognitive function was not addressed. Further studies that correlate FA and diffusivity indices with cognitive function in different ages could be of value in studying white matter maturation processes and how they influence the different aspects of intelligence. Finally, some of the limitations were related to diffusion tensor tractography and measured biomarkers. Crossing fibres have remarkable effect on anisotropy and tractography results. In addition, equating FA as a marker of white matter integrity is an oversimplified interpretation because FA cannot separate individual microscopic contributions (myelination, density and axonal diameter). However, complementary diffusion measures such as axial and radial diffusivities, in addition to anisotropy, have been implemented to yield more comprehensive picture of different elements of white matter microstructures. Furthermore, tractography using manually delineated ROIs implies that tractography results are observer dependant. However, we strictly followed a high reproducibility protocol described by Wakana et al. [14] to overcome this limitation.

To summarize, in this study, we found maturation of the long association tracts through late childhood, adolescence and into adulthood. Seven of the association tracts followed a predominant pattern of white matter maturation demonstrating progressive increases in FA and concurrent decreases in RD and Trace with no AD changes; the remaining three tracts exhibited different patterns that probably suggest different biological changes and may represent stages in a cascade of age-related maturation in white matter microstructure. Left hemispheric dominance regarding the FA and diffusivity indices was demonstrated in uncinate fasciculus, and to lesser extent ILF and IFOF. To the extent of our knowledge, hemispheric lateralization in terms of RD, AD and Trace was not previously documented.