Participants

The present study included 12 patients who fulfilled the American College of Rheumatology criteria for FM [18], and were recruited from the National University Hospital. In addition, 11 CRPS patients who fulfilled the International Association for the Study of Pain criteria for CRPS (Budapest criteria) and had been also recruited in the authors’ previous study [17] were included. Finally, 15 individuals, who were of comparable age and gender with the FM and CRPS patients, and exhibited no pain or neurological symptoms, were used as healthy controls. The subjects used as the healthy controls were the same used in a previous CRPS study [17]. 12 patients with FM and additional control subjects were recruited using Internet advertisements for 7 months. The number of study participants was based on a sample size calculation for a comparison of distribution volume ratio (DVR) between groups. Effect size was estimated from available PK11195 pilot study [19]. In addition, we used G*power 3.1.9.2 (Heinrich Heine, Universität Düsseldorf, Germany) program [20] with unpaired t test, an 80% power while controlling type I error rate at 5%. The computed sample size was 11 subjects for each group, so we planned to recruit 15 subjects for each group considering potential withdrawal of subjects.

Subjects who exhibited high levels of high-sensitivity C-reactive protein (hs-CRP) or leukocytosis were excluded. The inclusion criteria for FM subjects were as follows: diagnosis of FM; age between 21 and 63 years; and not taking benzodiazepine or could discontinue benzodiazepine medication 2 weeks before the study. Individuals with a major neuropsychiatric disorder before the diagnosis of FM, a neurological disease (cerebrovascular disease or brain tumor), a history of brain trauma, high levels of hs-CRP or leukocytosis, as well as those who could not undergo the PET/magnetic resonance imaging (MRI) were excluded. Sensory and affective dimensions of current pain were assessed using the McGill pain Questionnaire Short-Form (SF-MPQ), which contains 11 McGill Pain Questionnaire-Sensory (MPQ-S) and 4 McGill Pain Questionnaire-Affective (MPQ-A) pain items [21]. Stress levels in the FM patients were assessed using the Stress Response Inventory (SRI), which consists of 39 items (score range: 0–156) categorized into 7 factors: fatigue, tension, frustration, anger, depression, somatization, and aggression [22]. The post-traumatic stress disorder (PTSD) Check List (PCL) assesses the development of PTSD in respondents after exposure to a traumatic event [23]. This study was approved by the Institutional Review Board (IRB) at the Seoul National University Hospital (IRB No. 1703-138-841). All data were obtained under written informed consent granted by all subjects after a full explanation of the experimental methods.

Synthesis of [¹¹C]-(R)-PK11195

[¹¹C]-(R)-PK11195 was synthesized using a previously reported method with a few modifications [24]. The precursor and the cold-standard of [¹¹C]-(R)-PK11195 were purchased from ABX (Radeberg, Germany); the other chemicals were obtained from Sigma-Aldrich Korea (Kyunggi-do, Korea). Briefly, (R)-N-desmethyl-PK11195 (the precursor of [¹¹C]-(R)-PK11195, 1 mg, 2.8 μmol) and sodium hydride (60% in mineral oil, 7 mg, 486 μmol) were dissolved in 500 μL of dimethyl sulfoxide in a reaction vial. [¹¹C]CH₃I was bubbled into the reaction vial for 10 minutes, and the reaction mixture was allowed to proceed for an additional 5 minutes. The reaction mixture was purified by preparative high-performance liquid chromatography (HPLC) (Xterra C18 column, 10 × 250 mm², 10 μm, Waters Corp.; Milford, MA) with the eluent (water:ethanol  =  55:45, 5 mL/min). The final product solution was reconstituted with 10 mL of a saline solution. The purity of the final product was checked by analytical HPLC (YMC-Triart C18 column, 4.6 × 100 mm², 3 μm, YMC Co., Ltd.; Kyoto, Japan) with the eluent (water:MeCN  =  40:60, 1 mL/min). The radioactivity of [¹¹C]-(R)-PK11195 was 5.7 ± 1.8 GBq (156.7 ± 51.3 mCi [n  =  10]); the specific activity was 215.2 ± 93.7 GBq/μmol (5.8 ± 2.5 Ci/mol [n  =  10]). The total synthesis time, including formation and purification, was less than 40 minutes from the end of bombardment.

PET/MR image acquisition

Each participant underwent a 60 min dynamic [¹¹C]-(R)-PK11195 PET scan using a PET/MRI scanner (Biograph mMR, Siemens Healthcare GmbH, Erlangen, Germany). PET was performed using a spatial resolution of 4.4 mm at 1 cm and 5.2 mm full-width at half-maximum (FWHM), with a 10 cm offset from the center of the transverse field of view. The details were described in the authors’ previous study [17].

Quantification of binding

A parametric image of the DVR for each participant was generated using relative equilibrium-based graphical analysis with a reference-region input [25]. For the reference region, the region of interest (ROI) of the cerebellum was delineated on a simultaneously acquired sagittal T1-weighted MR image. For the extraction of ROI mean values and subsequent statistical analyses, the individual DVR images were spatially normalized by registering the MR images to the MNI152 template using statistical parametric mapping (SPM) (SPM8; www.fil.ion.ucl.ac.uk/spm). The ROI DVR values were then extracted from the normalized DVR images using population-based probability maps [26, 27], the details of which are described in the authors’ previous study [17].

Statistical analysis

Statistical analyses of ROI data were performed using SPSS version 21.0 (IBM Corporation, Armonk, NY, USA). The differences in DVR of [¹¹C]-(R)-PK11195 in ROI between FM and healthy controls was assessed using analysis of covariance (ANCOVA) controlling for any effects of gender. The differences in DVR in ROI between patients with CRPS and healthy controls were assessed using ANCOVA controlling for the duration effect. The differences in DVR of [¹¹C]-(R)-PK11195 in ROI between patients with FM and CRPS were assessed using a two-tailed Student’s t-test. If normality was not satisfied, Mann–Whitney U test was used. Pearson’s correlation analysis was used to evaluate the association between psychological test scores and the DVR of [¹¹C]-(R)-PK11195. Differences with P < 0.05 were considered to be statistically significant in the ROI analysis.

To assess group differences at the voxel level, statistical inferences were further performed on the spatially normalized DVR images using SPM8 after 10 mm Gaussian smoothing to reduce the image registration error. ANCOVA was applied for a comparison between FM and healthy controls with gender as a covariate, and between CRPS and healthy controls with duration as a covariate. A two-sample t test was used to compare patients with CRPS and FM. The resulting maps were thresholded at the P = 0.01 level, without correction for multiple comparisons.

Study participants and basic information

Twelve FM, 11 CRPS patients, and 15 healthy control subjects completed all study procedures. Demographic and clinical characteristics of the subjects were summarized in Table 1. Age, gender, and education level were not significantly different among the groups. We exhibited CNS medication information for patients’ therapies in S1 Table. FM patients were taking various CNS medications, including opioids (N = 4), anticonvulsants (N = 10), antidepressants (N = 8), antipsychotics (N = 6), anxiolytics (N = 4), antimigraine agent (N = 3), antiparkinson agent (N = 2) and benzodiazepine (N = 1). CRPS patients were also taking CNS medications, including opioids (N = 7), anticonvulsants (N = 7), antidepressants (N = 7), anxiolytics (N = 1), and benzodiazepine (N = 6).

Table 1

Participant`s demographic characteristics.

	FM patients	CRPS patients	Healthy controls	p-value
N	12	11	15
Age	41.7±14.0	40.9±8.8	41.3±6.6	p = 0.929, p = 0.907
Gender	5M, 7F	8M, 3F	10M, 5F	p = 0.182, p = 0.543
Education(years)	14.4±2.6	14.8±2.4	16.2±2.0	p = 0.054, p = 0.120
Duration of illness(years)	4.4 ± 3.1	8.9 ± 5.3	N/A

Data are mean ± standard deviation. Bold p shows p-value between FM and healthy controls. Non-bold p shows p-value between CRPS and healthy controls. FM: Fibromyalgia, CRPS: Complex Regional Pain Syndrome; M = male; F = female.

Differences in neuroinflammation between FM and healthy controls

The voxel-wise ANCOVA, controlled for the effect of gender, revealed significant differences in neuroinflammation (i.e., DVR of [¹¹C]-(R)-PK11195) between the FM and healthy control group. The FM group exhibited decreased [¹¹C]-(R)-PK11195 binding in the medulla (p < 0.005) and the left superior temporal gyrus (p < 0.005) compared with the healthy control group (Fig 1), but an increased [¹¹C]-(R)-PK11195 DVR in the left pre- and post-central gyri (p < 0.001), the right postcentral gyrus (p < 0.005), the left superior parietal and superior frontal gyri (p < 0.005), and the left precuneus (p < 0.01) (Fig 1). Additionally, ROI analysis controlling for the effect of gender revealed lower binding in the medulla (p = 0.001) and left amygdala (p = 0.020), but higher binding in left medial frontal (p = 0.036) in FM patients compared with that in healthy controls.

Fig 1

Differences in neuroinflammation indexed as DVR of [¹¹C]-(R)-PK11195 between FM and healthy controls.

Analysis of covariance (ANCOVA) with gender covariate, uncorrected p < 0.01.

Differences in neuroinflammation between CRPS patients and healthy controls

When accounting for the duration effect in comparing the CRPS and healthy control groups, abnormally higher [¹¹C]-(R)-PK11195 binding levels in the CRPS group was detected in the left superior, middle and medial frontal gyri (p < 0.01, p < 0.01, and p < 0.001, respectively), the left inferior and superior parietal gyri, the left precuneus (p < 0.01), the left caudate (p < 0.01), the right putamen (p < 0.01), and the right superior, middle, and medial frontal gyri (p < 0.005) (Fig 2). Additionally, using ROI analysis while controlling for the duration effect in the comparison of CRPS and healthy control groups, a higher [¹¹C]-(R)-PK11195 DVR was found in the in right insula (p = 0.033), left thalamus (p = 0.042), left precentral gyrus (p = 0.027), right and left postcentral (p = 0.018, p = 0.025, respectively), right and left middle frontal (p = 0.001, p = 0.004), right and left medial frontal (p = 0.009, p = 0.006), right and left superior frontal (p = 0.011, p = 0.010), left lateral orbitofrontal (p = 0.029), left superior parietal (p = 0.035), right and left caudate head (p = 0.014, p = 0.013), right and left nucleus accumbens (p = 0.007, p = 0.020), left putaman (p = 0.004), left caudate (p = 0.019), left globus pallidus (p = 0.050), and right superior marginal (p = 0.012) in CRPS patients.

Fig 2

Differences in [¹¹C]-(R)-PK11195 DVR between CRPS patients and healthy controls.

Analysis of covariance (ANCOVA) with duration covariate, uncorrected p < 0.01.

Differences in neuroinflammation between FM and CRPS patients

Higher neuroinflammation levels in the left pre- and post-central gyri were found in the FM group compared with the CRPS group (p < 0.001 and p < 0.005, respectively) (Fig 3). On the other hand, higher binding levels in the CRPS group were observed in the medulla, left insula, left thalamus, left superior temporal gyrus, the right and left putamen, and the right and left medial orbital gyri compared to that of the FM group (p < 0.01) (Fig 3). Additionally, ROI analysis revealed higher [¹¹C]-(R)-PK11195 binding in the right and left thalamus (p = 0.023, p = 0.008) right and left putamen (p = 0.008, p = 0.046), right and left caudate (p = 0.012, p = 0.029), right and left caudate head (p = 0.005, p = 0.034) and right globus pallidus (p = 0.037) of CRPS patients compared with that of the FM group.

Fig 3

Differences in DVR of [¹¹C]-(R)-PK11195 between FM and CRPS patients.

Two sample t-test, uncorrected p < 0.01.

Correlations between neuroinflammation and psychological symptoms in FM patients

In patients with FM, there were positive correlations between the DVR of [¹¹C]-(R)-PK11195 in the left medial frontal (r = 0.584, p = 0.046), left superior parietal (r = 0.681, p = 0.015) and left amygdala (r = 0.655, p = 0.021) and the affective pain score (MPQ-A) (Fig 4A). In patients with FM, there were positive correlations between the DVR of [¹¹C]-(R)-PK11195 in the left medial frontal (r = 0.699, p = 0.011), left superior frontal (r = 0.641, p = 0.025) and left amygdala (r = 0.808, p = 0.001) and SRI stress scores (Fig 4B). Furthermore, the DVR of [¹¹C]-(R)-PK11195 in the left amygdala demonstrated a positive correlation with PTSD scores (r = 0.695, p = 0.012) in FM patients (Fig 4C).

Fig 4

Correlations between affective pain, stress and PTSD and neuroinflammation ([¹¹C]-(R)-PK11195 DVR) in FM.

(A-C) LMF: left medial frontal, LSF: left superior frontal, LSP: left superior parietal, LA: left amygdala. MPQ-A: McGill pain Questionnaire-Affective, SRI: Stress Response Inventory, PTSD: posttraumatic stress disorder. *p < 0.05, ** p < 0.01.