During a sample period of 22 months, 50 patients with UWS were screened. Twenty of them had to be excluded due to medical or other reasons (magnetic resonance imaging [MRI] or medical exclusion criteria, n = 6; palliative care or death, n = 5; discharged from hospital, n = 3; refusal of informed consent, n = 6). Thirty UWS patients fulfilling the inclusion criteria underwent the fMRI examination (16 males, mean age 48.4 ± 15.5 years, range 16–72) as well as 15 healthy participants (eight males, mean age 42.4 ± 11.8 years) (Table 1).
Table 1. Clinical characteristics of patients
|No||Scanner||Sex||Age||Etiology||Time interval in months||CRS-R||CRS-R subscores: auditive–visual–motor–oromotor–communication–arousal||Atrophy|
All patients were of nontraumatic etiology, including hypoxic encephalopathy (n = 25), subarachnoid or intracerebral hemorrhage (n = 4) and encephalitis (n = 1). Patients' morphologic information provided by T1-weighted scans was assessed using a scale developed by Galton et al. (2001) and Bekinschtein et al. (2011) (from 0 = no atrophy to 4 = very severe atrophy). The degree of atrophy was evaluated by three experienced raters who were blind concerning the identity of patients. The mean degree of atrophy was 3.1 (±0.9) and the value of the Coma Recovery Scale was on average 5.4 (±1.4).
The diagnosis was made on the basis of careful, repeated clinical examination including the Coma Recovery Scale – Revised (CRS-R) (Giacino et al. 2004).
Twenty-six patients underwent a CRS-R examination within the first week of their stay and then every 2 weeks. Within the week before the MR scan, another CRS-R score was determined, which went into our analysis.
In four patients, the standardized examination according to the CRS-R was not possible. They had to be transported over a long distance and were directly brought to the scanning center. All of them were chronic patients. Their diagnoses have been verified by their attending physicians.
Exclusion criteria for healthy participants were the history of head trauma, neurological diseases, or any chronic illness. Exclusion criteria for all participants were any contraindication to fMRI. The participants' legal guardians gave written informed consent. The study was approved by the ethical committee of the University of Tuebingen and conducted in accordance with the Declaration of Helsinki.
An alternating block design (three noxious stimulation blocks, three baseline blocks) was performed. Each block consisted either of 60 noxious stimuli (1/sec) or a 60-sec baseline rest interval. The nociceptive experience was elicited by an electrical stimulus (5 mA, 200 msec) at the left index finger using the DS7A HV Constant Current Stimulator from Digitimer. The HC group evaluated the electrical stimuli as moderately painful (mean 3.93; SD = 1.28) on a visual analogue scale (VAS) from 0 (no pain) to 10 (worst pain imaginable). The moderate pain stimulation was used for ethical reasons. Another group of 16 healthy individuals (seven males, mean age 25.7 [SD = 4.41]), who did not participate in the fMRI experiment, additionally evaluated the valence (mean 7.81, SD = 0.91 on the scale from 1 = very pleasant to 9 = very unpleasant) and arousal (mean 7.31, SD = 1.54 on the scale from 1 to 9) of the same stimuli.
The examination was always accompanied by a physician. The patient's vital signs (heart rate, oxygen saturation) were monitored continuously.
Image acquisition and statistical analysis
Blood oxygenation level-dependent (BOLD) images were obtained at two imaging centers (Bad Aibling and Tuebingen, Germany) in order to avoid unnecessary patient transportation. In Bad Aibling, where 22 patients were examined, data were collected using a 1.5 Tesla MRI scanner (TIM Symphony; Siemens Medical Systems, Erlangen, Germany) system equipped with a 12-channel head coil. Changes in BOLD T2*-weighted MR signal were measured using a gradient echo-planar imaging (EPI) sequence (TR = 3410 msec, TE = 50 msec, FoV = 192 mm, flip angle = 90°, 64 × 64 matrix, 36 slices covering the whole brain, slice thickness 3.0 mm, no gap, voxel size 3 × 3 × 3 mm). A T1-weighted anatomical image was additionally acquired for each subject to allow anatomical localization (TR = 2300 msec, TE = 2.98 msec, 160 slices, voxel size 1.0 × 1.0 × 1.1 mm). In Tuebingen, imaging was performed on a 3 T Siemens Trio scanner. After a T2*-weighted acquisition (TR = 2380 msec, echo time = 25 msec, FoV = 210 mm, flip angle = 90°, 64 × 64 matrix, 40 slices covering the whole brain, slice thickness 3 mm, no gap, voxel size 3.3 × 3.3 × 3.0 mm), anatomical images were obtained using the MP-RAGE sequence (repetition time = 2300 msec, echo time = 2.98 msec, 160 slices, slice thickness = 1 mm, voxel size 1.0 × 1.0 × 1.1 mm).
Magnetic resonance imaging scans of the 15 healthy subjects were acquired in Bad Aibling using the above-mentioned 1.5 T Siemens Symphony MR Scanner and the same imaging parameters.
Image processing and statistical analysis were conducted using Statistical Parametric Mapping (Friston et al. 1995) version 8 (Wellcome Department of Cognitive Neurology, London, UK; http://www.fil.ion.ucl.ac.uk/spm/software/spm8/). Preprocessing included realignment, coregistration, segmentation, and spatial normalization (template of Montreal Neurological Institute [MNI]). Then, a Gaussian filter of 8-mm full width at half maximum was applied to smooth the data spatially.
For the statistical analysis of regional differences in brain activation, painful stimulation and resting condition were input into the categorical general linear model design at the subject level (Friston et al. 1995). Contrasts between pain and baseline conditions were computed for each subject. In addition, main effects were computed using one-sample t-tests for each group (UWS, HC) separately. The probability threshold was set at P < 0.05, corrected for family-wise errors (FWE) for whole-brain analysis.
In addition, region of interest (ROI) analyses were performed for pain-related brain areas on the individual level, such as the ACC, insula, S1, S2, thalamus, and cerebellum using automated anatomical labeling masks (Tzourio-Mazoyer et al. 2002) and the WFU Pickatlas (Maldjian et al. 2003). ROI analyses were applied in HCs and patients. The ROIs were superimposed onto each patient's T1 image with manual adjustments to those anatomical landmarks if necessary (Bekinschtein et al. 2011). A significance level of P < 0.05 (FWE corrected) was used.
For comparison between UWS and HC, several chi-squared tests were applied. Their significance was corrected by the number of the tests using the Bonferroni–Holm correction procedure (Holm 1979).