White matter microstructural changes are related to cognitive dysfunction in essential tremor

Diffusion tensor imaging (DTI) studies have detected white matter microstructural changes in essential tremor (ET). However, it is still unclear whether these changes are related to cognitive deficits, which have been described in ET patients. DTI-derived fractional anisotropy, mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity measures were compared between 23 ET patients and 23 age-, gender-, and education-matched healthy individuals, using whole-brain tract-based spatial statistics. Correlations of white matter changes with scores obtained from a detailed neuropsychological assessment were subsequently examined. ET patients demonstrated increases in MD in the bilateral posterior corona radiata, bilateral superior longitudinal fasciculus, bilateral fornix (cres)/stria terminalis, genu and splenium of the corpus callosum, bilateral anterior and posterior limbs of internal capsule, bilateral retrolenticular region part of internal capsule, and left posterior thalamic radiation. Except for the genu of the corpus callosum, an increase in AD values was also found in these same tracts. Furthermore, increased MD and AD values in different white matter areas was negatively correlated with performance on language and verbal memory and positively with visuospatial ability. These correlations suggest that white matter changes might be involved in the pathogenesis of cognitive deficits in ET.

Participants. ET patients were consecutively recruited from October 2012 to July 2013 from the outpatient neurology clinics of the University Hospital "12 de Octubre" in Madrid (Spain) after obtaining an appropriate informed consent. Patients with history of dementia, stroke, epilepsy, head injury or serious medical illness were excluded. Furthermore, based on a detailed clinical mental status examination, we excluded patients with Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria for dementia 34 .
Two neurologists with expertise in movement disorders (J.B.-L. and J.P.R.), who were blinded to the MRI results, examined the patients and used the Fahn-Tolosa-Marìn tremor rating scale to assign a total tremor score (range = 0-144) 35 . Diagnoses of ET were assigned by the 2 neurologists (JB-L and JPR) using the Consensus Statement on Tremor by the Movement Disorder Society 36 . Furthermore, all ET patients had a normal [(123) I] FP-CIT single photon emission computed tomography scan. All eligible ET patients underwent a detailed videotaped neurological examination. Each videotape was reviewed by a senior neurologist specializing in movement disorders (E.D.L.) who re-assessed ET diagnosis using the Consensus Statement on Tremor by the Movement Disorder Society 36 . The ET patients were also followed at regular intervals (3 months, 6 months, or 12 months, based on clinical need) after the MRI procedure, and their clinical assessment, described above, was repeated. The mean duration of follow-up after the MRI procedure was 2.5 years (median = 2.7 years; range = 1.8-2.8 years).
HCs were recruited either from relatives or friends of the health professionals working at the University Hospital "12 de Octubre" of Madrid (Spain) or among the relatives of patients who came to the neurological clinics for reasons other than ET (e.g., headache, dizziness). None reported having a first-degree or second-degree relative with ET. Each control was examined by two neurologists (JB-L and JPR), who were blinded to the MRI results, to further rule out any neurological or other serious conditions, including movement disorders, dementia, stroke, epilepsy, or head injury.
Neuropsychological testing. All participants underwent a detailed neuropsychological assessment covering the domains of attention, executive function, verbal memory, visual memory, visuospatial ability, and language. Testing was performed by a trained neuropsychologist (V. P., see acknowledgments) using standardized procedures who was blinded to the clinical diagnosis as well as the MRI results. These tests have previously been described 20 . Depressive symptoms severity was measured by the original 17-item version of the Hamilton Depression Rating Scale 37 .
In ET patients, each raw score was transformed into a standardized Z score based on the mean and standard deviation (SD) calculated from the HCs, according to the formula: Z = (raw score -mean score)/SD. Within each domain, Z scores were averaged to yield six composite scores that assessed attention, executive function, verbal memory, visual memory, visuospatial ability, and language, respectively, and that were used in subsequent correlation analyses. Individual cognitive measures were grouped into the following cognitive domains 20 : white matter integrity in patients with ET is unknown, we decided that all patients continued taking medication for their disease -propranolol and/or primidone during the MRI procedures.
Patients and controls were positioned in the scanner and were told to relax with their eyes closed. They were immobilized with a custom-fit blue bag vacuum mold (Medical Intelligence, Inc.) to prevent motion artifacts. Earplugs and noise-reduction headphones were used to attenuate scanner noise.
DWI were pre-processed using FMRIB's Diffusion Toolbox (FDT, http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FDT/), part of FMRIB Software Library (FSL) 38,39 . Pre-processing consisted of correction for eddy current distortion and head motion using the EDDYCORRECT function and field map correction using FUGUE and PRELUDE functions. Non-brain tissue from the average b0 image was removed using the FMRIB's Brain Extraction Toolbox, BET. The brain mask was applied to the rest of the diffusion-weighted images. Next, the diffusion tensor was estimated for each voxel using the DTIFIT function via linear regression to derive FA, MD, AD and RD maps.
Subsequently, the TBSS package was used to perform voxel-wise analyses of whole-brain white matter measures (http://www.fmrib.ox.ac.uk/fsl/tbss/index.html) 40 . Briefly, individual FA images underwent nonlinear registration to the FMRIB58_FA template space and were averaged to create a mean FA image. This was then thinned to create a white matter tract "skeleton" using the default FA threshold of 0.2 to exclude non-white matter voxels. Each participant's aligned FA map was then projected onto this skeleton, resulting in an alignment-invariant representation of the central trajectory of white matter pathways for all subjects. This process was repeated for each subject's MD, AD and RD map using the individual registration and projection vectors obtained in the FA nonlinear registration and skeletonization. Voxel-wise differences in FA, MD, AD and RD values between ET patients and HC were tested using permutation-based inference for nonparametric statistical thresholding (FSL's "randomize" function) 41 and two-sample t-tests. The number of permutations was set to 5000 to allow robust statistical inference. Age, gender and total intracranial volume were entered into the analysis as confound regressors. For between-group comparisons, a family-wise error corrected threshold of p < 0.05 was selected using the randomize tool's threshold-free cluster enhancement (TFCE) option 42 . The white matter tracts were identified using the ICBM-DTI-81 white matter labels atlas included with FSL 43,44 . In addition, significant white matter clusters were identified by their coordinates in Montreal Neurological Institute convention and by their cluster size.
The images shown in the current paper were created using the FSLview tool from FSL, by overlapping the group-averaged white matter skeleton (blue) and the results from between-group comparisons (family-wise error corrected threshold of p < 0.05) (red-yellow) onto a standard T1 Montreal Neurological Institute template.

Correlation analyses.
Correlation analysis was performed to study the relationship between neuropsychological test scores and each of the DTI measures. Cognitive domains (attention, executive function, verbal memory, visual memory, visuospatial ability, and language) were used as covariates of interest in the framework of a general linear model. Also, age, gender, total intracranial volume, disease duration, and total tremor score were entered in the design matrix throughout the analysis. Statistical analysis was performed using the FSL Randomize Tool with 5000 permutations. TFCE was performed to enhance cluster-like structures. In correlation analyses, there were not any clusters or voxels with analysis corrected for multiple comparisons, so the correlation results with p < 0.01 (uncorrected for multiple comparisons) were reported as statistically significant.

Sample size and statistical analyses of clinical and neuropsychological data.
In several recent publications on suitable sample sizes for DTI studies, using tract-based spatial statistics, it has been reported that a group size of approximately 20 is sufficient 25,45 .
Statistical analyses for the clinical and neuropsychological measures were conducted using Statistical Package for the Social Sciences (SPSS) Version 22.0 (SPSS, IBM Corporation). Mean scores (age and neuropsychological variables) were compared using two independent sample t-tests for continuous and normally distributed data, and Mann-Whitney U test for non-normally distributed data, where appropriate. The χ2 test was used to analyze differences in sex distribution.

Results
Clinical and Neuropsychological testing results. As this study was nested within the NEUROTREMOR project (http://www.neuralrehabilitation.org/projects/neurotremor/), a project whose main aim was to validate technically, functionally and clinically, a novel system for understanding, providing diagnostic support, and remotely managing tremors, most the ET patients who were eligible refused to participate because of lack of time because the study would have required that they come to the hospital several times duringthe study for the performance of clinical, neurophysiological (magneto-electroencephalography and electromyography recordings), neuropsychological, and imaging evaluations. Given this constraint, of the 300 ET patients seen at outpatient neurology clinics of the University Hospital "12 de Octubre" in Madrid (Spain) from October 2012 to July 2013, only 47 were eligible for the study. Of these 47 ET patients who were eligible for the study, 26 had complete neuropsychological testing (see above) and an MRI procedure with TBSS data. Of these 26 ET patients, two had dystonic features upon review of their videotapes; therefore, these two were excluded. One was excluded from the final analyses because he developed incident Parkinson's disease during follow-up. None of the patients and controls were excluded because of neurological comorbidities or structural abnormalities on conventional MRI images.
According to Fazekas visual rating scale, all participants had a Fazekas score ≤ 1 (i.e., normal in the elderly) 46 . On the other hand, a strict criterion for head movement assessment was adopted (maximal absolute head movement less than 1.0 mm and 1.0° in the x, y, and z directions). Neither patients nor HCs were excluded from the analysis due to this criterion.
The final sample included 23 right-handed ET patients (12 women and 11 men) and 23 right-handed HC (12 women and 10 men). The 23 ET patients did not differ to a significant degree from the 23 controls in terms of age, sex, and educational level ( Table 1). The mean tremor duration was 22.9 ± 16.5 years and the mean tremor rating scale score was 30.1 ± 15.0 ( Table 1).
The results of neuropsychological testing are shown in Table 1. In several domains, ET patients' cognitive performance was significantly worse than that of the HC. These differences involved selected tests of attention, executive function, and language.

Comparison of DTI metrics between ET and HCs. Patients with ET demonstrated increases in MD in
the bilateral posterior corona radiata, bilateral superior longitudinal fasciculus, bilateral fornix (cres)/stria terminalis, genu and splenium of the corpus callosum, bilateral anterior and posterior limbs of internal capsule, bilateral retrolenticular region of internal capsule, and left posterior thalamic radiation. See Fig. 1 and Table 2 for more details. Interestingly, with the exception of the genu of the corpus callosum, AD values were also significantly increased in the same tracts in ET patients compared with HCs ( Fig. 2 and Table 3). We did not find any tract in which there was statistically significant increase of MD and AD values in HCs with respect to the ET patients. Finally, no significant differences in FA and RD were detected between groups at the P family-wise error-corrected < 0.05 level.
Correlation Analyses. The following correlations (uncorrected for multiple comparisons, P < 0.01) between diffusion measures statistically different at a P family-wise error-corrected < 0.05 level and cognitive domains scores were observed in the ET group: There was no statistically significant correlation between FA and any of the cognitive domains scores or clinical scores (i.e., disease duration and total tremor score) in the ET group.

Discussion
The present exploratory study used TBSS to analyze whole-brain white matter microstructure in non-demented ET patients, finding increased MD in several regions including the bilateral posterior corona radiata, bilateral superior longitudinal fasciculus, bilateral fornix (cres)/stria terminalis, genu and splenium of the corpus callosum, both internal capsules, and left posterior thalamic radiation. AD was also increased in the majority of the same tracts, suggesting tract degeneration in these regions 45 . To date, only a few studies investigating white matter changes in ET patients have used the whole-brain TBSS approach 25,27,33 . Our findings are in agreement with these previous TBSS studies 25,27,33 .
Furthermore, increased MD and AD values in different white matter areas (right superior cerebellar peduncle, left corticospinal tract, cerebral peduncles, the splenium of corpus callosum, parahippocampal gyri, and the left posterior thalamic radiation) was negatively correlated with performance on language and verbal memory (in other words, the highest AD and MD values -i.e., more white matter changes -, the worse cognitive performance). On the other hand, increased MD (in parahippocampal gyri), and AD values (in the right sagittal stratum, body of corpus callosum and the splenium of corpus callosum) was positively correlated with performance on visuospatial ability (indeed, the highest AD and MD values -i.e., more white matter changes -, the better cognitive performance). The exact mechanism linking white matter changes with specific cognitive domains in ET remains unclear. In the specific case of the positive correlations between increased MD and AD values in different white matter areas with visuospatial ability may reflect compensatory reorganization of neural circuits indicative of adaptive or extended neuroplasticity, thereby allowing ET patients to maintain the same level of cognitive performance (visuospatial ability) as HCs (see Table 1). However, further work is necessary in order to confirm this.
We recognize that correlation and causality are not the same. Demonstrating that the white matter changes in several regions preceded the cognitive changes would be an important step in establishing causality; however, in the absence of a prospective, longitudinal study, this is not possible. Nonetheless, it is more biologically plausible that the white matter changes resulted in the cognitive changes than viceversa. These regions (the superior cerebellar peduncle, left corticospinal tract, cerebral peduncles, corpus callosum, parahippocampal cortex, posterior thalamic radiation, and sagittal stratum) have been associated with cognition in some way. First, dysfunction of the frontal-thalamic-cerebellar circuitry is thought to be associated with subtle cognitive abnormalities in ET patients [9][10][11][12][13][14][15] . Since the superior cerebellar peduncle is involved in neural connectivity in the frontal-thalamiccerebellar circuitry, subtle disruption of the superior cerebellar peduncle may be involved in the neural circuit deficits associated with ET. Second, the corticospinal tract consists of major efferent projection fibers that connect the motor cortex to the brain stem and spinal cord 47 . These fibers converge in the corona radiata and continue through the posterior limb of the internal capsule to the cerebral peduncle on their way to the lateral funiculus 47 . Although the corticospinal tract arises primarily from the primary motor cortex, projections from other areas including the somatosensory, cingulate, and insular cortices are also represented 48   is likely involved in a variety of functions, including cognition. Third, the genu of the corpus callosum connects several areas of the default-mode cortical network, a structure involved in cognitive processes 49 . Previous research by our group and others has reported abnormal functioning of default mode network regions in ET 19,20 . Likewise, the splenium of the corpus callosum is rostral to a key default-mode cortical network hub, the posterior cingulate cortex 49 . Increased MD in the splenium of the corpus callosum has correlated with posterior cingulate cortex functional connectivity 50 . Second, the parahippocampal cortex, which has been associated with many cognitive processes, including visuospatial processing and episodic memory 51 , links the default-mode cortical network with the medial temporal lobe memory system 52 . Third, the posterior thalamic radiation might be also involved in intellectual performance 53 . In fact, thalamocortical circuit integrity has been found to differentiate individuals at high risk of developing Alzheimer's disease from healthy elderly subjects, supporting the hypothesis that neurodegenerative mechanisms are active years before the patient is clinically diagnosed with dementia 54 . Finally, decreased FA has been reported in the inferior longitudinal fasciculus, a component of the sagittal stratum, in patients with mild cognitive impairment 55 . Hence, changes of these whiter matter areas and its correlation with  Table 3. White matter clusters showing increased axial diffusivity in essential tremor vs. healthy control subjects. White matter clusters with significantly increased axial diffusivity (P family-wise error-corrected < 0.05) in essential tremor vs. healthy control subjects. Coordinates indicate the location of the cluster peak in Montreal Neurological Institute convention. cognitive domains in our study extends prior findings and suggests early involvement of the same in ET patients prior to the development of dementia 18 . The study was not without limitations. First, the sample size was relatively small. However, we could detect significant differences between ET patients and HC at stringent thresholds even with these smaller numbers. Notwithstanding, it would be important to replicate these findings in a larger sample. Second, the diagnosis of ET was based on clinical criteria and further supported by normal [(123) I]FP-CIT single photon emission computed tomography scan results. None of the ET patients had post-mortem assessments, so that it was not possible to determine whether they had the types of changes that have been reported in ET 56 . Finally, the results of our correlation analyses should be interpreted carefully because they were not controlled for multiple comparisons, which may lead to false-positives.
In closing, our findings indicate that ET patients had white matter changes mainly in the corona radiata, internal capsule, corpus callosum, and superior longitudinal fasciculus. Although the correlations between cognitive performance and diffusivity in distinct white matter areas suggests that white matter changes might be involved in the pathogenesis of cognitive deficits in ET, our findings should be considered only as a preliminary result, more for exploratory purposes than for a solid conclusion. Further additional studies with larger samples are required.