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Forty right-handed [44] subjects (27 females; age 38±6 years, range 19–46 years; education 17±3 years), who had been diagnosed as having a ‘reaction to severe stress and an adjustment disorder’ according to the International Classification of Diseases (ICD-10, F43), were recruited from the Stress Research Institute at Stockholm University. The selection was limited to subjects who attributed their illness to prolonged work-related stress, after working 60 to 70 hours per week continuously over several years prior to the onset of symptoms. Inclusion criteria consisted of a characteristic symptom course of sleeplessness, diffuse aches, palpitations and fatigue, a subsequent onset of irritability, anxiety, memory and concentration problems, feeling of depersonalization, and reduced work capacity (confirmed by the employers) [8],[19]. All of the subjects attributed their symptoms to chronic stress and had no other known etiology for their distress. Subjects were also required to have had a symptom duration of at least one year (their histories of stress-related burnout symptoms ranged from 1.5 to 3.5 years), to have been on sick leave (≥50%) for stress-related symptoms for a minimum of 6 months before entering the study, and to have an average stress-burnout score of ≥3.0 on the Maslach Stress-Burnout Inventory – General Survey (MBI-GS) [45]. This 7-point rating scale, ranging from 0 (never) to 6 (daily), consists of three subscales: exhaustion (five items), cynicism (five items) and lack of professional efficacy (six items). When rating perceived stress, subjects were asked to take into consideration the last six months, and not only the actual time-point. The average scores for Scandinavian populations are around 2 for MBI-GS [4],[46]. Subjects were excluded if they had a previous history of psychosis, personality disorder, major or bipolar depression, alcohol or substance abuse, chronic fatigue, chronic pain, fibromyalgia, or neurological or endocrine disease. Those who had experienced prominent stress factors in their private life or a major traumatic event at any time in their life, including sexual abuse, were also excluded. No daily medication was allowed during the two months prior to the study, except contraceptives. According to a review of their pharmacological treatment histories, none of them had taken drugs that are known to affect brain structure (e.g., psychopharmaca). Subjects who were sleep deprived the night before the scan/testing procedures were rescheduled, in order to exclude the acute effects of sleep deprivation. Seventy healthy, right-handed, non-smoking volunteers (45 females; age 33±6 years, range 24–45 years; education 17±3 years) with no history of chronic stress or heredity for neuropsychiatric disorders comprised the control group. The patient and control groups had similar gender distributions, and both were predominately female to accord with the female-dominated epidemiology of the condition studied [4]. The two groups were matched for socioeconomic status assessed on the basis of years of education, type of occupation, and organizational position (employee, middle management, supervisor). The study was approved by the Ethics Committee at the Karolinska Institute and written informed consent was received from each participant. Before the interview, participants completed questionnaires in order to evaluate their stress symptoms and assess their previous life events [47]. In addition, the occurrence of major life events among the subjects was assessed through a clinical psychiatric interview based on the non-work-related items of the Holmes and Rahe Scale [48]. The participants were asked to answer yes or no to whether they had experienced any non-work- related stressful life events (e.g., death of a relative or spouse, recent divorce, forced family relocation). Subjects were excluded if they answered positively to having experienced such an event in their lives. Patients also received a medical screening (physical examination, test of thyroid and liver function). A structured interview, the Swedish version of the Mini-International Neuropsychiatric Interview, MINI [49] was performed, along with a test for depression using the Montgomery-Asberg Depression Rating Scale [50]. Although some subjects had high scores in the MADRS they did not fulfill the MINI criteria for depression, and were therefore not excluded. Out of the participants who matched the inclusion criteria, 8 subjects with occupational burnout and 9 controls failed to display a startle response to the probe. The results from the emotion regulation experiment and the correlation analyses with fMRI are therefore based on data from the remaining 32 subjects with occupational burnout (20 females; mean age = 37.6 years, SD = 6.5) and 61 controls (33 females; mean age = 30.9 years, SD = 6.7), whereas the analysis of resting state amygdala connectivity is based on the entire study group.
Salivary cortisol was sampled according to a previously established protocol [51]. Saliva sampling was chosen because the method is simple, non-invasive, and non- stressful; the samples are shown to readily reflect the levels of the free fraction of cortisol in plasma [52]. Participants were instructed carefully on how to collect their own salivary samples. Samples were collected seven times on an ordinary weekday using Salivette cotton rolls (Sarstedt, Rommelsdorf, Germany), which participants were instructed to place in the mouth for 2 minutes. The first sample was collected immediately upon awakening in the morning, irrespective of time. The second sample was collected 15 minutes later, before eating or brushing teeth, and the third sample was collected 15 minutes after that. The fourth sample was collected around noontime, before lunch. The fifth sample was collected at about 3 p.m., the sixth at 8 p.m., and the seventh at bedtime, after having rested in bed for 15 minutes, before falling asleep. The samples were frozen (−18°C) until analyzed. The levels of salivary cortisol were measured with radioimmunoassay using the Spectria (125I) coated tubes radioimmunoassay kit (Orion Diagnostica, FIN-02101 Espoo, Finland). The within-assay coefficients of variation ranged from 0.8 to 0.9, and those between assays never exceeded 10 percent. All samples from each group were analyzed simultaneously in duplicate.
Before the experiment, the participants were given written as well as verbal explanations of the task and instructions. Participants were informed that they would receive three different instructions during the experiment and that these instructions would be symbolically represented by three different arrows: (1) an upward arrow indicated that the participant should make an effort to reinforce the feelings that are elicited by the picture, so that he/she experiences the image as more emotionally charged (“up-regulate”); (2) a horizontal arrow indicated that the participant should focus on the feeling the picture elicits, without trying to manipulate the emotion (“maintain”); and (3) a downward arrow indicated that the participant should make an effort to down-regulate the feelings that the picture elicits, so that he/she experiences the image as less emotionally charged, or as neutral as possible (“down-regulate”). Participants were thoroughly informed of the importance of following the instructions during the experiment and not distracting themselves from their feelings by thinking of something else or by looking away from the image or closing their eyes. The subjects were however free to choose the strategy to regulate their emotion. The experiment began with a practice session during which the participants were first subjected to the auditory startle probe six times to allow for habituation to the sound. This was followed by twelve practice trials that mirrored the experimental procedure. After the practice session, the participants were asked to describe the strategy they had used to regulate emotion. None of the participants reported that they were confused about how to adopt a reappraisal strategy for the neutral and negative trials before or after completing the experimental task. An example of an experimental trial is shown in Figure 1. During each trial, the participant was presented with a picture for 5 s, which was then replaced by an instruction cue for 1 s. For negative pictures, participants were instructed to suppress (down-regulate), enhance (up-regulate) or maintain their emotional response. Based on previous work [42] and to avoid confusion due to ambiguous instructions (e.g., to suppress emotional reactions to neutral pictures), neutral pictures were only coupled with the instruction to maintain the emotional response. Immediately following the instruction cue, the same picture was presented again for 5 s, during which time the participants carried out the regulation instruction. During each trial, startle probes were presented 3 s after picture onset during the first (pre-instruction) and the second (post-instruction) picture-viewing phases. Lastly, the participants were given 4 s to rate on a scale of 1–7 how well they had managed to carry out the instructions. Between each trial, a fixation cross was presented for 4–6 s (mean 5 s). Each trial lasted for 20 s. There were 60 trials, and the entire testing session lasted approximately twenty minutes, with a 15-second pause after the first 30 trials. The presentation of pictures was synchronized with the monitor's refresh rate and presented with the software Presentation (Neurobehavioural systems, www.neurobs.com).
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0104550.g001 Overview of one experimental trial with the maintain instruction.Participants were presented with a picture, which was replaced by an instruction cue. For negative picture trials, this cue indicated whether the participants' task was to maintain (horizontal arrow), down- regulate (downward arrow) or enhance (upward arrow) their emotional response. Immediately following the instruction cue, participants implemented the regulation instruction while being exposed to the same picture again. Lastly, participants rated how well they managed to implement the regulation instruction on a scale of 1–7.
We selected three sets of 15 negative pictures and one set of 15 neutral pictures from the International Affective Picture Set (IAPS) [53]. Each of the three sets of negative pictures was assigned to one of the three task instructions (maintain, down-regulate, up-regulate), and this assignment was counterbalanced between participants (male and female controls, and patients). Pictures were selected to match valence and arousal scores of pictures used in a similar report [54].
Electromyographic recordings: response definition and data reduction: The eye-blink component of the startle response was measured through electromyographic (EMG) recordings of the left orbicularis oculi muscle using two miniature Ag/AgCl electrodes prepared with electrolyte gel. A third ground electrode was placed behind the left ear over the mastoid. Startle probes were 50-ms bursts of approximately 95-db[A] white noise with a near instantaneous rise time (<1 ms), delivered through sound-proof headphones (Bose AE21, Bose Co. Framingham, Massachusetts). The raw EMG signal was amplified and filtered through a 28–50 Hz bandpass filter, rectified and integrated with a time constant of 20 ms. Startle eye-blink magnitude (microvolts) was measured as the amplitude from onset to peak, and trials with excessive baseline activity or recording artifacts were rejected. To assess initial, unaltered startle responses, pre-instruction (Startle 1) startle scores for negative and neutral images were normalized using z-standardization to ensure that all participants contributed equally to the group means, as has been described previously [55]–[56]. The z-score calculation is a within-individual normalization, resulting in a distribution with an overall mean of 0 and a standard deviation of 1 for each participant. To assess the regulation of the startle response according to instruction, for each participant, we calculated the change in startle response by subtracting the raw startle 1 response from the raw startle 2 response separately for each instruction (maintain neutral, maintain negative, down-regulate negative, up-regulate negative) of the task. This way, we defined emotion regulation ability as the magnitude during emotion regulation controlling for baseline levels before the regulation cues.
Initial startle reactions were assessed in a 2×2 repeated measures analysis of variance (ANOVA) with Valence (Negative, Neutral) as a within-subjects variable and Group (Burnout, Control) as a between subjects variable. To test the hypothesis that burnout patients would differ from controls in their startle responses during down-regulation of negative emotion, we ran a 2×2 repeated measures ANOVA with Instruction (Down-regulate, Maintain) as the within-subject variable and Group (Burnout, Control) as the between-subject variable. As a control, we similarly assessed whether there were any group differences in startle responses during up-regulation of negative emotions in a 2 (Up-regulate, Maintain)×2 (Burnout, Control) repeated-measures ANOVA. Possible group difference in salivary cortisol levels was tested with a repeated measure ANOVA (p<.05).
MR experiments were carried out on a separate day to avoid contamination by possible effects of the emotional regulation tasks. Functional MRI time series data were collected from all of the participants at rest over 8 minutes in a 3 Tesla MR scanner Discovery 750 (GE Healthcare), using a 32-channel head coil. Resting fMRI blood oxygenation-level dependent (BOLD) data were acquired in a standard gradient echo-planar-imaging (EPI) acquisition, TR = 2.5 s, TE = 30 ms, flip angle = 90°, resolution = 3×3×3 mm, whole-head coverage. The participants were asked to lie with their eyes closed, to think of nothing in particular, and not to fall asleep. Structural brain images were acquired using a T1-weighted 3D brain imaging volume imaging sequence with whole-head coverage, TR = 7.91 s, TE = 3.06 s, flip angle = 12°, and resolution 1×1×1 mm. These structural images were used to aid the registration of the functional data into a common standard brain coordinate system (MNI152).
Seed region analysis is based on calculating cross-correlation coefficients of the time series in a particular seed region-of-interest (ROI) with all other voxels in the brain, which reveals the strength of functional connectivity with respect to this seed region [57]. The seed regions consisted of the right and left amygdala, and were delineated with the guidance of the WFU-pick atlas, and after adaptation to the gray matter template of our own population. The MNI coordinates for the amygdala seeds where (sphere of 5 mm radius, co-ordinate −22, −7 −19, and 22 −7 −19); the seed regions covered the amygdala, with the exception of the most medial 2 mm of the basomedial amygdala, which was excluded to avoid the susceptibility artifact that was detected in some subjects. Given the amygdala's pivotal role in stress perception, we first evaluated whether and how the functional connectivity from the amygdala seeds differed between patients and controls. We then used multiple regression analysis to investigate whether the degree of perceived stress interacted with the pattern of connectivity from the amygdala seed. Spatial preprocessing and statistical analysis of functional images were performed using SPM8 (Welcome Department of Cognitive Neurology). Functional images were slice-timed and realigned, and then registered to structural T1 SPGR (spoiled gradient) images for each subject. Next, the individual T1 SPGR images were segmented into gray matter, white matter, and cerebrospinal fluid, and the gray matter image was used to determine the parameters of normalization for the standard Montreal Neurological Institute gray matter template. The spatial parameters were then applied to the slice-timed and realigned functional volumes that were finally resampled to 2×2×2 mm voxels and smoothed with a 6-mm full-width at half-maximum kernel. Each voxel's time series was corrected for noise using standard resting-state low-pass filtering with a cut-off frequency of 0.1 Hz. In addition, voxel-wise multidimensional regression analysis was employed in a standardized manner to remove artifacts due to motion and changes in ventricle and white matter signals. This was done by adding six movement regressors obtained from rigid-body head motion correction (SPM 8 statistical package). Segmented WM (white matte) and CSF (cerebro spinal fluid) were used as ROI for correction of signals from non-gray matter tissue. To ensure that signals from WM and CSF ROIs did not contain signals from gray matter, these ROIs were superimposed on the individual EPIs and, when needed, adapted to the respective subject, based on intensity differences between white matter, gray matter, and ventricular regions. Global signal correction was not employed, as it has been reported that regression against the global signal may artificially introduce anticorrelations into fMRI data sets [58]. For each subject, the average fMRI time course within the seed region was used as the regressor of interest. Individual time series in each seed region were extracted with MarsBar toolbox (http://marsbar.sourceforge.net/). Each subject's seed region time course was then regressed voxel-wise against the subject's fMRI time course using the entire brain as search space. The t-values of the corresponding regression coefficients at each voxel were used as each subject's connectivity map.
Group comparisons between stressed subjects and healthy controls were carried out in SPM8 using one-way ANOVA, with p<.001 voxel threshold, FWE corrected at cluster level, p<.05, and controlling for age and gender, which were used as nuisance covariates.
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