Subjects and procedure
In this study,
40 cirrhotic patients were recruited with different grades of HE before getting Transjugular Intrahepatic Portosystemic Shunt (TIPS).
Laboratory parameters were obtained from all patients to assess the severity of liver disease.
The functional liver status was assessed by Child Pugh score [5],
and risk for mortality evaluated by MELD (model of end stage liver disease) score.
HE was diagnosed by clinical examination using the West Haven Criteria [6,7].
Forty age- and sex-matched healthy controls without any medical history of neurological disease served as control group.
Controls scored within 1.5 standard deviations of the age and education adjusted norm in all subtests of the CERAD (consortium to establish a registry for Alzheimer's disease) cognitive battery [8].
Neuropsychological Assessment
Patients underwent a comprehensive neuropsychological test battery assessing executive functioning/attention,
memory,
language and visuospatial abilities,
with a duration of 60 minutes.
The battery comprised the Trail-Making Test-B [9] and similarities (a subtest of Wechsler Adult Intelligence Scale – Fourth Edition,
WAIS-IV) [10] to evaluate primarily executive function.
The line drawing test (LDT) [11,12] was used to test psychomotor speed and accuracy.
The subtest logical memory of the Wechsler Memory Scale - Fourth Edition WMS-IV evaluated episodic memory.
All subjects performed computerized tests for attentional performance (TAP version 2.3 [13]) assessing alertness,
and divided attention.
Premorbid verbal intelligence was assessed with a German vocabulary test (WST) [14].
Our test battery contained three (TMT-A/B,
LDT) out of five tests defining the psychometric hepatic encephalopathy score (PHES) [15].
MRI data acquisition
All MRI examinations were performed using a 3 Tesla (T) system (TIM Verio,
Siemens Medical Solutions,
Erlangen,
Germany).
MRI sequences included high-resolution T1-weighted gradient echo sequence and spin-echo-based echo planar imaging sequence with diffusion gradients applied along 30 non-collinear directions (b=1000 s/mm2) for DTI.
To rule out any incidental pathological abnormalities,
a FLAIR-sequence and a blood sensitive T2* gradient echo sequence were acquired.
MRI data processing
T1 images were processed using Statistical Parametric Mapping (SPM8) and the VBM8-toolbox [16].
Segmentation into grey matter (GM) and white matter (WM) was followed by registering GM and WM partitions of each subject to Montreal Neurological Institute (MNI) standard space,
using the diffeomorphic DARTEL warping algorithm [17].
For the voxel-wise whole brain analyses,
warped grey matter maps were smoothed with a Gaussian smoothing kernel of 8mm full-width at half maximum.
The DTI datasets were processed with the FSL software package [18].
A diffusion tensor model was fitted at each voxel,
generating maps of fractional anisotropy (FA) and mean diffusivity (MD).
Tract-based spatial statistics (TBSS) was employed with a threshold of FA > 0.2 for the mean FA skeleton.
Statistical Analysis
Concerning neuropsychological data,
we derived standard scores (z-scores) from neuropsychological test raw scores,
based on test-specific age and gender adjusted normative data.
For comparisons of z-scores between the subgroups regarding HE stages we conducting series of univariate analyses of covariance (ANCOVA) with etiology as covariate.
The significance level was set to p<0.05.
Regarding voxel-wise GM volume,
group comparisons between healthy controls and subgroups were conducted using ANCOVA,
controlling for age and sex.
Results were considered significant passing the statistical threshold of p<0.05,
corrected for multiple comparisons by Family Wise Error correction (FWE).
To compare diffusion measures (DTI data) between cirrhosis patients and healthy controls as well as within patient's subgroups,
ANCOVAs were conducted considering age,
sex and etiology as covariates,
using permutation tests with 5000 permutations and threshold-free cluster enhancement (TFCE) implemented in the FSL 4.1 submodule Randomize [19].
We controlled for multiple comparisons by using the family-wise error (FWE) method (p<0.05).
Additionally,
maps of effect sizes (Cohen´s d) were generated.
In addition,
concerning VBM and DTI data,
group comparisons of regions of interest (ROIs) were performed by ANCOVAs with age and gender as covariates for both grey and white matter.
The relationship between ROIs of grey and white matter and neuropsychological test scores was evaluated using partial correlation analyses considering age and gender as covariates.
These analyses were conducted using SPSS V 18.0 with Bonferroni-Holm-correction for multiple comparisons (p<0.05).