diffusion tensor tractography and neuropsychological assessment in patients with vitamin b12...

DIAGNOSTIC NEURORADIOLOGY

Diffusion tensor tractography and neuropsychologicalassessment in patients with vitamin B12 deficiency

Pradeep Kumar Gupta & Ravindra Kumar Garg & Rakesh Kumar Gupta &

Hardeep Singh Malhotra & Vimal Kumar Paliwal & Ram Kishore Singh Rathore &

Rajesh Verma & Maneesh Kumar Singh & Yogita Rai & Chandra Mani Pandey

Received: 31 July 2013 /Accepted: 28 November 2013 /Published online: 10 December 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractIntroduction Structural imaging of the brain does not demon-strate any changes in a vast majority of patients with vitaminB12 deficiency, even in advanced stages. In this study, weaimed to assess and correlate the functional integrity of thebrain fiber tracts using diffusion tensor tractography withneuropsychological examination in patients with vitaminB12 deficiency.Methods The studywas conducted at two tertiary care centers.Thirty-two patients with vitamin B12 deficiency were en-rolled and subjected to diffusion tensor tractography, as anextension of diffusion tensor imaging, and neuropsychologi-cal assessment. Tests of significance were done to detectchanges, pre- and post-vitamin B12 supplementation in the

diffusivity parameters (fractional anisotropy and mean diffu-sivity) and the neuropsychological test scores.Results Statistically significant changes were observed in thediffusivity parameters and the neuropsychological test scoresbetween the controls and the patients with vitamin B12defi-ciency in the pre- and post-treatment phases.Conclusions This is the first study to evaluate the diffusiontensor tractography (DTT) parameters in the light of clinicalneuropsychological assessment in patients with vitamin B12deficiency. Utilization of DTT parameters may antedate struc-tural changes and may quantify the neurocognitive deficits.

Keywords Magnetic resonance imaging . Diffusion tensorimaging . Diffusion tensor tractography . Folic acid .

Cognition

Introduction

Vitamin B12 deficiency is considered to be a serious publichealth issue and a common cause of macrocytic(megaloblastic) anemia and pancytopenia. Neurologic mani-festations arising from vitamin B12 deficiency include pares-thesias, sensory ataxia, peripheral neuropathy, optic neuritis,and cognitive impairment [1-5]. Vitamin B12 deficiencycauses demyelination of the corticospinal tracts and subacutecombined degeneration of the spinal cord involving the pos-terior and the lateral columns [1, 3, 6]. Neuroimaging studiesusing MRI of brain and spinal cord have shown a character-istic pattern of white matter degeneration in some cases ofvitamin B12 deficiency which is commonly seen in multiplesclerosis, such as extensive areas of T2 high-intensity signal inthe periventricular white matter [3, 7].

Vitamin B12 acts as a cofactor for methionine synthase, theenzyme that remethylates homocysteine to methionine byusing 5-methyltetrahydrofolate as a methyl donor [4]. It is

P. K. Gupta :R. K. Garg (*) :H. S. Malhotra :R. Verma :M. K. SinghDepartment of Neurology, King George’s Medical University,Uttar Pradesh Lucknow, India 226003e-mail: [email protected]

Y. RaiDepartment of Radiodiagnosis, Sanjay Gandhi Postgraduate Instituteof Medical Sciences, Lucknow, India

V. K. PaliwalDepartment of Neurology, Sanjay Gandhi Postgraduate Institute ofMedical Sciences, Lucknow, India

R. K. S. RathoreDepartment of Mathematics and Statistics,Indian Institute of Technology, Kanpur, India

C. M. PandeyDepartment of Biostatistics, Sanjay Gandhi Postgraduate Institute ofMedical Sciences, Lucknow, India

R. K. GuptaDepartments of Radiology and Imaging, Fortis Memorial ResearchInstitute, Gurgaon, India

Neuroradiology (2014) 56:97–106DOI 10.1007/s00234-013-1306-y

believed that vitamin B12 deficiency resulting in elevatedserum total homocysteine concentration provides additionalinformation on the existent deficiency [4, 8]. Homocysteineitself exerts a direct toxic effect on the neurons, as a result ofwhich vitamin B12 deficiency and hyperhomocysteinemiamay together contribute to or enhance the neurocognitive def-icits [5, 9]. Significant associations between cognitive impair-ment and adverse profiles of vitamin B12 and homocysteinehave been observed across many cross-sectional studies [5]; thefindings, however, have not been consistent. Magnetic reso-nance imaging (MRI) changes, abnormal evoked-potentialsand neurological syndromes in patients with vitamin B12deficiency suggest demyelination of the white matter in thespinal cord, optic nerves, and the optic tracts [3, 10, 11].

Structural organization of the white matter cannot be assessedby routine MR-based volumetric neuroimaging [12], which pro-vides information about the white matter injury but does notidentify the extent of injury in the specific tracts. Diffusion tensorimaging (DTI) based on restrictions in the random movement ofwater molecules by macromolecules and myelin can be utilizedto visualize brain white matter tracts. DTI has been extensivelyused in understanding normal brain development [13-15] and hasbeen shown to improve detection of abnormalities in variousneurologic disorders [15-21]. In most of the previous studies,region of interest (ROI) analysis has been used to quantify DTImetrics. ROI-based morphometric DTI analysis is limited to twodimensions (2D) and does not provide information about theintegrity of white matter fiber tracts. The eigenvectors and theeigenvalues, which encode the direction of diffusion, have beenused in diffusion tensor tractography (DTT) [22] to assess theintegrity of the fiber tracts (axonal orientation) between voxelsand thus to infer the paths of the fiber tracts in three dimensions(3D) [23]. The individual fiber bundles are defined by using atracking algorithm for three-dimensional segmentation. Recon-structed different fiber bundles may be evaluated visually or withqualitative assessment. Besides qualitative localization, DTTalsoallows individual neuronal pathways to be quantified acrossregions of the brain where manual segmentation is not possible.

In the present study, we analyzed fractional anisotropy andmean diffusivity values in various brain regions in adult withvitamin B12 deficiency and age/sex-matched controls. Ourprimary hypothesis states that white matter injury might bedetected by DTT in different fiber bundles which may correlatewith neuropsychological scores. This is the first study to eval-uate the DTT parameters in the light of clinical neuropsycho-logical assessment in patients with vitamin B12 deficiency.

Materials and methods

The patients, presenting with complaints of cognitive impair-ment or limb paraesthesias and detected having vitamin B12deficiency, were selected from the outdoor and the indoor

units of the Departments of Neurology, King George’s Med-ical University, Uttar Pradesh, Lucknow, India and SanjayGandhi Postgraduate Institute of Medical Sciences, Lucknow,India. Imaging and quantification of data was derived from theDepartment of Radiodiagnosis which was assessed during2010–2012. The study was approved by the InstitutionalResearch Ethics Committee of both the Institutes (ref. no.PGI/DIR/RC/914/2010 and 53 E.C.M. 11B/P3) and writteninformed consent was obtained from all the participants.

All the subjects underwent clinical evaluation along withdetailed neuropsychiatric test, MRI, and laboratory tests. Se-rum vitamin B12 was measured by electrochemiluminescencemethod (COBAS e411), utilizing the kits obtained fromRocheDiagnostics GmbH , Mannheim, Germany. The diagnosis ofvitamin B12 deficiency was based on a low serum vitaminB12 level (<200 pg/ml) [24]. Follow-up vitamin B12 levelswere additionally obtained for comparison; the matched con-trols were similarly evaluated.

It was ensured to the best possible extent that the subjectsor the matched controls were not receiving any form ofsupplementation. Dietary history was also assessed forcorrelation.

Clinical evaluation

The clinical evaluation at baseline, as well as at follow-up, ofthe vitamin B12-deficient subjects was done by a neurologistand a neuropsychologist. It focused on detailed sensory ex-amination which included assessment of crude touch, finetouch, pain, temperature, joint position, vibration, and theRomberg's test.

Neuropsychological tests were performed on the controlsand the patients, both before and after vitamin B12 supple-mentation. The neuropsychological tests included the trail-making test as well as the performance subset of the modifiedWechsler Adult Intelligence Scale (WAIS-P, adapted for theIndian population). The trail-making tests included the num-ber connection test (A , B) and the figure connection test (A ,B ) to assess the visual motor coordination, concentration,mental speed, memory alteration, and attention. WAIS-P in-cluded the picture completion, digit symbol, block design,picture arrangement, and object assembly. The picture com-pletion was employed to assess the visual recognition andidentification (long-term visual memory); digit symbol tomeasure sustained attention, concentration, and psychomotorspeed; block design to assess spatial visualization, nonverbalconcept formation, and visual–motor coordination and per-ceptual organization; picture arrangement to test the ability toplan, interpret, and anticipate social events within a culturalcontext; and object assembly to measure visual–motor inte-gration. In the trail-making tests, lower “raw” scores repre-sented a better performance, whereas in WAIS-P, a higher“scaled” score represented a better performance.

98 Neuroradiology (2014) 56:97–106

MRI protocol

All the patients underwent neuroimaging of the brain andspine. Conventional MRI and DTI data were acquired on a3-T Signa HDxt MRI scanner (General Electric, Milwaukee,WI, USA) using an eight-channel head coil. Conventionalimaging included T2-weighted fast spin-echo sequence withecho time (TE)/repetition time (TR)/number of averages(NEX) 70/9,200 ms/1, and T2-weighted fluid-attenuated in-version recovery (FLAIR) with TE/TR/NEX/inversion time(TI) 120/9,000/1/2,400 ms. All these images were acquiredwith a field of view (FOV) of 240×240 mm2 and 3 mm slicethickness without any interslice gap. 3D fast-spoiled gradientecho brain volumewith TE/TR/NEX/inversion time/flip angle3.32/8.4/1/400ms/13 with slice thickness of 1 mm and FOV=240 mm with image acquisition matrix=288×288 were per-formed. In addition, sagittal T2-weighted, T1-weighted,FLAIR, and axial T2-weighted MR images of thecervicodorsal spine were acquired to look for the B12deficiency-related changes in the spinal cord. The DTI datawas acquired with the use of a single-shot echo planar dualspin echo sequence with ramp sampling. The diffusion tensorencoding used was a vender supplied DTI scheme with 30uniformly distributed directions. DTI was performed in theaxial plane and had identical geometrical parameters. Thediffusion weighting b factor was set to 1,000 s/mm2=240×240 mm2, slice thickness=3 mm, interslice gap=0, and num-ber of slices=62.

White matter structure segmentation

The principal eigenvector field was segmented into stablevoxels with minimal e1 variation [25, 26]. Therefore, a voxelP (i , j , k ) is a member of the stable fiber mass (SFM), if thereis a neighboring voxel Q (x , y, z ) such that the principaleigenvectors e1s at P and Q point to each other. Mathemati-cally, it translates to the relation G [F (P)]=P, where F (P )=ROUND (P +e1(P )+0.5u ), u =(1, 1, 1), andG (Q )=ROUND(Q-e 1(Q )+0.5u ); the function ROUND stands for thecomponent-wise “integral part” operation. This method in-volves generation of a SFM, followed by segmentation ofthe volume by coloring the voxel P according to the valuesthe components (l , m , n ) of the vector-joining P and Q take:(±1, 0 ,0) red, (0, ±1, 0) green, (0, 0, ±1) blue, (±1, ±1, 0)yellow, (0, ±1, ±1) cyan, (±1, 0, ±1) magenta, and (±1, ±1, ±1)white. A nonstable voxel is gray, and a voxel with frac-tional anisotropy <0.15 remains black. Typical segmentedaxial, sagittal, and coronal SFM color maps were gener-ated; this method emphasizes on the region-of-interestselection for the standard tractography to point out a colorsegment inside a broader region of interest through asingle mouse click [25, 26].

Diffusion tensor tractography

Continuous tracking algorithm was used for fiber assignmentand reconstruction [22, 26]. Different white matter fiber bun-dles have characteristic mark segments on the SFM colormaps and are automatically and reproducibly reconstructedby providing these segments as regions of interest to the fiberassignment by a continuous-tracking algorithm [21, 22, 26].This reconstruction identifies the coordinates of specific whitematter tracts and investigates their anatomy. DTI measureswere calculated for the entire fiber. Fractional anisotropythreshold of 0.15 was used for fiber tracking. The centralsulcus was identified and marked on sagittal surface imagereconstructed by 3D surface rendering of the b0 image stack.By using 3D cross-connectivity between three image planes,the central sulcus was displayed on the axial images. Motorand sensory tract were identified from fiber generated bydrawing freehand ROIs on axial T2-weighted images nearthe brain's vertex on the precentral and postcentral gyri [21].In-house developed JAVA-based software was used to gener-ate white matter tracts. Rathore et al. has published a moredetailed description of the DTT method [26].

Data quantification

The major white matter fiber tracts included in this study wereanterior thalamic radiations, posterior thalamic radiations, su-perior thalamic radiations, medial lemniscus, middle cerebel-lar peduncle, corpus callosum, tapetum, cingulum, fornix,inferior longitudinal fascicules, superior longitudinal fascicu-lus, inferior fronto-occipital fasciculus, and motor and sensorypathways, which were generated and quantified by an in-house developed JAVA-based software [21, 25].

The generated bulk of the white matter fiber tracts wereassessed in all the three views (axial, coronal, and sagittal).The reconstruction of fibers (anterior thalamic radiations,posterior thalamic radiations, superior thalamic radiations,middle cerebellar peduncle, corpus callosum, cingulum, for-nix, inferior longitudinal fascicules, superior longitudinal fas-cicules, inferior fronto-occipital fasciculus, and motor andsensory pathways) is described in detail elsewhere [21, 25].In the case of medial lemniscus and tapetum, the mouse clickswere made on the axial SFM color map segments. Identifica-tion of medial lemniscus on the color maps was done at thelevel of pons and tapetum on either side of the corpuscallosum. To minimize the inter-observer variability, differentfiber tracts were generated by two observers with average ofeach parameter used in analysis.

Vitamin B12 supplementation

The patients were administered 1,000 μg of vitamin B12intramuscularly, after sensitivity testing daily, for 10 days,

Neuroradiology (2014) 56:97–106 99

followed by once a week for 4 weeks, and then once a month.The activity was supervised by a trained professional withmaintenance of log to assess the compliance.

Follow-up

All the patients were followed for a minimum period of2 months and all the above-mentioned clinical, neuropsycho-logical, and neuroimaging parameters, along with vitaminB12 level, were reassessed.

Statistical analysis

All the data analyses were performed by two authorsusing statistical software SPSS, version 16.0 (SPSS Inc,Chicago, IL, USA). Paired t test was performed toevaluate the differences in DTT parameters (fractionalanisotropy and mean diffusivity) quantified from DTTof the different white matter regions of the right hemi-sphere and those of the left hemisphere. Student's inde-pendent t test was performed to assess the changes inthe DTT parameters and the neuropsychological testscores between the vitamin B12-deficient and thehealthy controls. Paired t test was performed to com-pare changes in the DTT parameters as well as theneuropsychological test scores at baseline and follow-up. For multiple comparisons, depending upon the num-ber of variables, Bonferroni correction was applied andthe p value was adjusted accordingly to obtain aBonferroni-corrected value of <0.05. Correction by afactor of 1/14 was done while comparing DTT param-eters and neuropsychological test scores of the controlpopulation with those obtained from the pre- and post-treatment patients with vitamin B12 deficiency. Similar-ly, correction by a factor of 1/56 and 1/70 was appliedwhile comparing DTT parameters of the individualtracts with the trail making test and WAIS-P,respectively.

Results

The present study was carried out on 32 adult patients withvitamin B12 deficiency (male=26, female=6, mean age=36.66±13.55 years, and range=16–60 years) and age/sex-matched 32 healthy controls (male=22, female=10, meanage=31.03±10.21 years, and range=16–60 years). Therewere no significant differences in demographic variables be-tween patients and control. Eighteen patients in the studygroup were vegetarians while 17 patients were vegetarian inthe control group. There was no significant difference inbetween vegetarians and nonvegetarians in the two groupsunder study. There was no significant difference in DTT

measures collected from the right and the left hemispheresof the white matter tracts under study (except the corpuscallosum) which were pooled together for the purpose ofquantitative analysis.

Neurological and serum vitamin B12 level assessmentof patients pre- and post-treatment

Mean serum vitamin B12 level in the study group at the timeof enrolment was 148.43±36.55 pg/ml and in control groupwas 327.87±169.7 pg/ml. After 2 months of treatment, meanserum vitamin B12 in the study group improved to 1,047.7.±536.45 pg/ml. Paresthesias (81.25 % at baseline) decreased inthe post-treatment phase to 18.5 %, and impairment of thejoint position and vibration sense (100 % at baseline) de-creased to 22.22 % at follow-up. Five patients were lost tofollow-up.

Conventional MRI findings in patientspre- and post-treatment

Bilateral involvement of the posterior columns involving thedorsal cord was observed in only four patients, which resolvedcompletely after the treatment.

Quantitative analysis

Fractional anisotropy, mean diffusivity,and neuropsychological test scores

At baseline, significantly decreased fractional anisotropyvalues were observed in patients with vitamin B12 deficiencyas compared to healthy controls in anterior thalamic radia-tions, posterior thalamic radiations, superior thalamic radia-tions, corpus callosum, cingulum, fornix, and sensory tracts;however, medial lemniscus, middle cerebellar peduncle, tape-tum, inferior longitudinal fascicules, superior longitudinalfascicules, inferior fronto-occipital fasciculus, and motorfiber tracts did not show any significant changes. A sig-nificant increase in the mean diffusivity values were ob-served in medial lemniscus, corpus callosum, tapetum,fornix, inferior longitudinal fascicules, inferior fronto-occipital fasciculus, and sensory tracts in patients withvitamin B12 deficiency as compared to healthy controls;however, no significant changes were observed in anteriorthalamic radiations, posterior thalamic radiations, superiorthalamic radiations, middle cerebellar peduncle, cingu-lum, superior longitudinal fascicules, and motor fibertracts. Patient with vitamin B12 deficiency showed sig-nificantly lower neuropsychological test scores for picturecompletion, digit symbol, block design, picture arrange-ment, object assembly, as well as significantly higherscores for number connection test A, number connection


test B, and figure connection test B as compared tohealthy controls (Table 1).

Comparison of the baseline parameters with those obtainedafter vitamin B12 supplementation revealed that fractionalanisotropy value from anterior thalamic radiations, posteriorthalamic radiations, superior thalamic radiations, medial lem-niscus, middle cerebellar peduncle, corpus callosum, tapetum,cingulum, fornix, inferior longitudinal fasciculus, superiorlongitudinal fasciculus, inferior fronto-occipital fasciculus,

and motor and sensory tracts had increased significantly.Mean diffusivity values from the anterior thalamic radiations,posterior thalamic radiations, superior thalamic radiations,medial lemniscus, middle cerebellar peduncle, corpuscallosum, tapetum, cingulum, fornix, inferior longitudinalfascicules, and inferior fronto-occipital fasciculus had de-creased significantly at follow-up as compared to the baseline;however, superior longitudinal fasciculus and motor and sen-sory tracts did not showed any significant difference.

Table 1 Summary of the com-parison of DTT parameters (frac-tional anisotropy and mean diffu-sivity) and neuropsychologicalassessment of the control popula-tion with those obtained from thepre- and post-treatment patientswith vitamin B12 deficiency

ATR anterior thalamic radiations,BD block design, CC corpuscallosum, CG cingulum,DS digitsymbol, FCT figure connectiontest, FX fornix, IFO inferiorfronto-occipital fasciculus, ILFinferior longitudinal fasciculus,MCP middle cerebellar peduncle,ML medial lemniscus, NCTnumber connection test, OA ob-ject assembly, PA picture ar-rangement, PC picture comple-tion, PTR posterior thalamic ra-diations, SLF superior longitudi-nal fasciculus, STR superior tha-lamic radiations, TP Tapetum

*p =<0.05, Bonferroni corrected(“controls versus pretreatment”and “controls versus post-treat-ment”); correction done by a fac-tor of 1/14

**p =<0.05, Bonferroni corrected(“pretreatment versuspost-treatment”); correction doneby a factor of 1/14

Fractional anisotropy Control (mean ± SD) Pretreatment (mean ± SD) Post-treatment (mean ± SD)

ATR 0.40±0.016 0.37±0.025* 0.39±0.021**

PTR 0.44±0.022 0.42±0.018* 0.44±0.021**

STR 0.46±0.022 0.44±0.024* 0.45±0.023**

ML 0.46±0.029 0.44±0.024 0.46±0.031**

MCP 0.43±0.020 0.42±0.019 0.43±0.017**

CC 0.50±0.020 0.48±0.026* 0.48±0.029**

TP 0.46±0.022 0.44±0.037 0.45±0.037**

CG 0.39±0.022 0.37±0.020* 0.38±0.020**

FX 0.33±0.013 0.31±0.022* 0.32±0.021**

ILF 0.43±0.029 0.42±0.028 0.42±0.025**

SLF 0.42±0.018 0.41±0.022 0.42±0.019**

IFO 0.45±0.023 0.43±0.024 0.44±0.023**

Sensory 0.44±0.017 0.42±0.023* 0.43±0.021**

Motor 0.45±0.022 0.43±0.019 0.44±0.020**

Mean diffusivity

ATR 0.87±0.030 0.89±0.044 0.87±0.035**

PTR 0.88±0.026 0.90±0.035 0.88±0.033**

STR 0.83±0.020 0.44±0.041 0.82±0.030**

ML 0.99±0.068 1.10±0.097* 1.00±0.57**

MCP 0.85±0.039 0.90±0.080 0.86±0.072**

CC 0.93±0.035 0.97±0.050* 0.95±0.045**

TP 0.99±0.085 1.25±0.187* 1.06±0.122**

CG 0.90±0.044 0.93±0.072 0.90±0.026**

FX 1.45±0.195 1.70±0.28* 1.53±0.256**

ILF 0.83±0.030 0.91±0.081* 0.85±0.028**

SLF 82±0.043 0.87±0.098 0.82±0.050

IFO 0.86±0.030 0.89±0.052* 0.86±0.043**

Sensory 0.82±0.020 0.88±0.10* 0.83±0.030

Motor 0.81±0.021 0.86±0.94 0.82±0.39

Neuropsychological test

PC 14.94±1.39 12.20±1.64* 14.48±1.67**

DS 11.25±2.26 7.03±2.65* 9.33±2.61**

BD 12.78±1.29 9.16±2.19* 11.52±1.99**

PA 13.22±1.04 11.47±1.97* 12.26±2.06**

OA 11.25±1.16 9.72±2.04* 10.07±1.64*

NCTA 38.12±9.23 57.75±22.68* 47.52±17.33

NCT B 69.19±15.80 103.97±44.29* 84.77±36.04

FCTA 60.25±23.62 80.84±38.51 66.22±37.10

FCT B 81.34±25.59 127.56±42.29* 104.15±33.88**


Significant differences were observed in the various neuropsy-chological test scores such as picture completion, digit symbol,block design, picture arrangement, and figure connection test B,but not in object assembly, number connection test A, numberconnection test B, and figure connection test A (Fig. 1). Objectassembly was the only test where significant differences wereobserved on comparing the healthy controls with the pre- andpost-treatment cohort with vitamin B12 deficiency in terms offractional anisotropy and mean diffusivity (Table 1).

Correlation of DTT parameters with respect to individualtracts and neuropsychological subset test scores

A significant correlation (positive or negative, depending onthe test type: trail-making test or WAIS-P) was observedbetween the various tracts under study and neuropsychologi-cal subset test scores (Table 2). Among correlations listed inTable 2, medial lemniscus was the only tract associated withsignificant changes in both fractional anisotropy and meandiffusivity values, and corresponding directly with changes inthe subsets of WAIS-P.

Discussion

This study describes the effect of low serum vitamin B12levels on various white matter tracts due to alteration ofmicrostructural environment leading to decreased fractionalanisotropy and increased mean diffusivity as well asneurocognitive decline; which improved on follow-up aftertreatment with vitamin B12 (Fig. 1; Table 1).

In this study, we found that in patients with vitamin B12deficiency multiple white matter tracts were involved,resulting in decreased fractional anisotropy and increasedmean diffusivity. Corpus callosum and fornix were the mostimportant tracts showing reciprocating changes in the DTTparameters throughout the spectrum of controls to the patientsin the post-treatment phase. Except for the non-significancebetween the pre- and post-treatment mean diffusivity values,sensory tracts also reflected the changes well through all thephases. Medial lemniscus showed the best correlation with theneuropsychological subset scores across the DTT parametersand gelling especially well withWAIS-P. It is emphasized thatfurther studies are needed in these patients to analyze thepredilection of these particular white matter tracts and theircorrelation with clinical manifestations.

Vitamin B12 deficiency is known to affect centraland peripheral nervous system leading to defective for-mation of myelin sheath due to incorporation of abnor-mal fatty acids [27] and defective methylation of myelinbasic protein, a component of CNS myelin [28]; wide-spread effect is therefore expected in the patients suf-fering from vitamin B12 deficiency.

Two major biological mechanisms are proposed in theavailable literature which interpret cognitive decline associat-ed with vitamin B12 deficiency. First, vitamin B12 and folicacid are crucial factors for the methylation of homocysteineinto methionine. A deficiency of vitamin B12 or folic acidwould cause a rise in homocysteine concentrations. Increasedplasma concentrations of homocysteine have been associatedwith an increased prevalence of poor cognitive functions [29]and an increased risk of development of dementia andAlzheimer's disease [30]. Second, low serum concentrationsof vitamin B12 or folic acid might result in a reduced avail-ability of methyl groups in the brain. This can ultimately resultin the impaired formation of myelin, various neurotransmit-ters, and membrane phospholipids [31]. Several studies havereported that damage to white matter in the brain is associatedwith cognitive decline [32, 33]. There is much evidence thatB12 deficiency is associated with brain atrophy and causescognitive decline [32, 34].

There is growing evidence that poor vitamin B12 status is arisk factor for brain atrophy and possibly white matterhyperintensity volume [35]. Other studies have suggested thatB12 deficiency may be associated with frontal lobe damage[36, 37]. It was reported that low vitamin B12 status is apredictor of whole brain atrophy in community-dwelling el-derly [38]. Previous studies have shown that vitamin B12deficiency is associated with damage to white matter in spinalcord and in the brain, which has been attributed to damage tomyelin as a result of deficient methylation of myelin basicprotein [39]. In the present study, the decreased fractionalanisotropy, along with increased mean diffusivity in patientscompared to controls in different white matter pathways re-flects a net loss or disorganization of the structural barriers tomolecular diffusion of water in these pathways. Mean diffu-sivity reflects changes in cell density and extracellular space[40]. The increased mean diffusivity values in various whitematter pathways of the patient with vitamin B12 deficiencycompared to controls can be attributed to increased extracel-lular water contents. FA values have been found to be a betterindicator of fiber maturation in comparison toMD as it reflectsoverall growth of axonal bundle along with ongoingmyelination in addition to fiber bundle thickness [25].

�Fig. 1 Plot compares the DTT measures and neuropsychological testscores of patients with vitamin B12 deficiency, at pre- andpostsupplementation. Fractional anisotropy values (a), mean diffusivityvalues (b) and WAIS-P subset score (c) as well as Figure connection testB score (d) are shown on the tracts where significant difference (withBonferroni-corrected p value <0.05) were observed. ATR anteriorthalamic radiations, PTR posterior thalamic radiations, STR superiorthalamic radiations, ML medial lemniscus, MCP middle cerebellarpeduncle, CC corpus callosum, TP Tapetum, CG cingulum, FX fornix,ILF inferior longitudinal fascicules, SLF superior longitudinal fascicules,IFO inferior fronto-occipital fasciculus, PC picture completion, DS digitsymbol, BD block design, PA picture arrangement, WAIS-P WechslerAdult Intelligence Scale, Performance subset FCT figure connection test


In the present study, strong correlation was found betweenfractional anisotropy values quantified from whole tracts ofposterior thalamic radiations, medial lemniscus, corpuscallosum, fornix, superior longitudinal fascicules, and senso-ry, motor, and the neuropsychological test score. Fractionalanisotropy value in posterior thalamic radiations significantlycorrelated with digit symbol which assesses sustain attention,psychomotor speed, spatial visualization, visual–motor coor-dination, and ability to plan. This supports the previous report

that posterior thalamic radiations connect the thalamus to theoccipital lobe involved in visual and motor functions [41].Medial lemniscus significantly correlated with picture com-pletion, digit symbol, block design, and number connectiontest A in our study, which assess visual recognition, long-termmemory, psychomotor speed, and visual motor coordination.This single tract stands out robustly in its association with theneuropsychological subset scores with both the DTT param-eters. Our findings second the report that medial lemniscus

Table 2 Summary of correlations between the DTT parameters (fractional anisotropy a mean diffusivity) of the individual tracts and the neuropsy-chological subset test scores

Wechsler adult intelligence scale Trail making test

PC DS BD PA OA NCTA NCT B FCTA FCT B

Fractional anisotropy

ATR R 0.274 0.305 0.210 0.034 0.194 −0.202 −0.329 −0.220 −0.244PTR R 0.311 0.381 0.276 0.174 0.009 −0.221 −0.198 −0.195 −0.232STR R 0.198 0.191 0.108 0.106 0.185 −0.186 −0.236 −0.181 −0.218ML R 0.373 0.470 0.383 0.249 0.192 −0.383 −0.279 −0.309 −0.322MCP R 0.223 0.302 0.240 0.244 0.203 −0.089 −0.221 −0.163 −0.167CC R 0.219 0.386 0.321 0.106 0.253 −0.313 −0.426 −0.253 −0.258TP R 0.120 0.276 0.192 −0.061 0.184 −0.251 −0.298 −0.074 −0.220CG R 0.262 0.310 0.196 0.036 0.270 −0.151 −0.287 −0.167 −0.160FX R 0.308 0.374 0.303 0.071 0.268 −0.197 −0.343 −0.252 −0.240ILF R 0.196 0.309 0.124 0.086 0.235 −0.188 −0.146 −0.234 −0.161SLF R 0.253 0.341 0.247 0.057 0.137 −0.376 −0.335 −0.277 −0.246IFO R 0.104 0.188 0.138 −0.057 0.143 −0.181 −0.144 −0.034 −0.027Sensory R 0.361 0.522 0.526 0.334 0.307 −0.441 −0.440 −0.358 −0.484Motor R 0.314 0.375 0.309 0.199 0.188 −0.346 −0.407 −0.358 −0.353

Mean diffusivity

ATR R −0.151 −0.097 −0.041 0.039 0.004 −0.043 0.034 0.112 0.145

PTR R −0.250 −0.298 −0.254 −0.090 −0.193 0.250 0.224 0.136 0.199

STR R −0.158 −0.038 −0.044 0.083 −0.162 −0.028 0.037 −0.045 −0.010ML R −0.468 −0.511 −0.409 −0.208 −0.186 0.478 0.341 0.308 0.459

MCP R −0.333 −0.245 −0.379 −0.026 −0.173 0.395 0.347 0.367 0.310

CC R −0.243 −0.273 −0.241 −0.105 −0.255 0.282 0.344 0.219 0.254

TP R −0.250 −0.313 −0.340 −0.154 −0.267 0.441 0.350 0.144 0.387

CG R −0.152 −0.097 −0.097 −0.058 −0.022 −0.072 −0.070 0.074 0.097

FX R −0.267 −0.254 −0.213 −0.099 −0.266 0.274 0.314 0.232 0.314

ILF R −0.310 −0.312 −0.293 −0.061 −0.284 0.382 0.211 0.183 0.304

SLF R −0.238 −0.137 −0.153 −0.027 −0.042 0.293 0.193 0.151 0.198

IFO R −0.184 −0.218 −0.174 −0.058 −0.092 0.273 0.295 0.177 0.244

Sensory R −0.205 −0.283 −0.185 −0.195 −0.180 0.061 0.113 0.032 0.083

Motor R −0.207 −0.275 −0.168 −0.214 −0.198 0.071 0.124 0.091 0.135

ATR anterior thalamic radiations, BD block design, CC corpus callosum, CG cingulum, DS digit symbol, FCT figure connection test, FX fornix, IFOInferior fronto-occipital fasciculus, ILF inferior longitudinal fasciculus, MCP middle cerebellar peduncle, ML medial lemniscus, NCT numberconnection test, OA object assembly, PA picture arrangement, PC picture completion, PTR posterior thalamic radiations, SLF superior longitudinalfasciculus, TP Tapetum, STR superior thalamic radiations

R value (bold) Bonferroni-corrected p value <0.05; correction done by a factor of 1/70 for the Wechsler adult intelligence scale and 1/56 for the trailmaking test, R value (italics) uncorrected P <0.05. R value (normal): uncorrected P >0.05


plays a critical role in sensory function and skillful movement[42]. It is worth noting that the centrally placed tracts, corpuscallosum and fornix, depicted the changesmost consistently inour assessment which might be the area bearing the effect ofvitamin B12 deficiency earliest. Corpus callosum, which con-nects the left and right side of brain, allows transfer of motor,sensory, and cognitive information between the two hemi-spheres, and was significantly associated with digit symboland number connection test. Fornix, carrying the nerve fibersbetween hippocampus to the thalamus, significantly correlat-ed with digit symbol. Superior longitudinal fasciculus, locatedat the superiolateral side of the putamen and associating all thefour lobes [25], correlated with the number connection test.Sensory tracts significantly correlated with picture comple-tion, digit symbol, block design, figure connection test, andnumber connection test. After corpus callosum and fornix, itseems to be the most important tract to show changes consis-tently. This may have some bearing on the clinical findingsalso when we consider medial leminscus and the callosal andfornix fibers together with the sensory tracts. The sensorycortex pathways are involved in the planning and executionof movement. These include thalamocortical links with so-matosensory cortex, cor t icofugal project ions tovestibulospinal and reticulospinal tracts, projections to thebasal ganglia, direct pyramidal tracts projections to spinalcord, and associations with the cerebellum via corticopontinetracts, which suggest that sensory tracts are possibly moredamaged in these patients [21]. Motor tracts in our studysignificantly correlated with number connection test and fig-ure connection test. The present findings of our study are inagreement with the previous reports in which the white matterhyperintensity volume, cerebral infarcts, and total brain vol-ume have been related to performance of multiple cognitivedomains in various pathological conditions [43-46], as well asin vitamin B12 and folate deficiency states [38, 47].

Various methods are available for DTT quantification likedeterministic and probabilistic algorithms. For this study, de-terministic algorithm was implemented using principle eigenvector field segmentation methodology [26]. The selection ofROI is user independent which leads to an automatic, robust,and efficient methodology for tractography whereas othermethods are semi-automatic and user dependent. This hasbeen validated and hence appears appropriate for the presentstudy.

Conclusion

This is the first study to evaluate the DTT parameters in thelight of clinical neuropsychological assessment in patientswith vitamin B12 deficiency. Clinical as well as radiologicalimprovement following vitamin B12 supplementation sug-gests that the changes are reversible to a significant extent.

Changes in the DTT parameters may antedate the structuralchanges; as a corollary it may be stated that for a given clinicaldeficit, a specific tract might be involved.

Acknowledgments This study is supported by Ministry of Science &Technology Department of Biotechnology (BT/IN/German/04/RKG/2010), New Delhi, India.

Conflict of interest We declare that we have no conflict of interest.

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