Altered locus coeruleus functional connectivity in migraine without aura based on resting-state functional magnetic resonance imaging

2.1 Participants

A cross-sectional analysis was conducted between May 2018 and June 2023 and included 55 individuals diagnosed with MwoA and 50 HCs. This study was approved by the Human Research Ethics Committee of Nanjing First Hospital (No. KY20200301-16), and written informed consent was obtained from all participants before MRI scanning. Participants were recruited from the Department of Neurology and the Department of Pain Outpatient Clinic of Nanjing First Hospital. The inclusion criteria for patients with MwoA were based on the International Classification of Headache Disorders, Third Edition, beta version (ICHD-3 beta) [2]. Exclusion criteria included a history of neurological or psychiatric disorders, serious systemic diseases such as cancer, excessive use of psychoactive medications, and any contraindications to MRI. To minimize the potential effects of pain and medication on blood oxygen level-dependent (BOLD) signal fluctuations, all patients were required to be free of headache attacks and medication use for at least 72 hours before scanning. In addition, patients were followed up 3 days after scanning to confirm that they had remained migraine-free throughout this period. Moreover, to reduce the potential effects of hormonal fluctuations on cortical excitability, female participants were scanned during the mid-cycle phase, and women who were pregnant or breastfeeding were excluded.

Age-, sex-, and education-matched HCs were recruited from the local population. These individuals had no personal or family history of migraine or other headache disorders and either experienced no headaches or had only infrequent tension-type headaches, defined as fewer than one episode per month. Participants who had received any treatments that could potentially affect the central nervous system were excluded from the study.

All participants were right-handed according to self-report. Clinical and neuropsychological assessments were completed before MRI scanning. All participants completed the Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS), and Montreal Cognitive Assessment (MoCA). In addition, patients with MwoA completed the Headache Impact Test-6 (HIT-6) and Migraine Disability Assessment (MIDAS). Anxiety and depressive symptoms were assessed using the SAS and SDS, respectively. Both instruments are self-report scales developed by Zung [13, 14], and they were completed by the participants themselves under the guidance of trained researchers to ensure the completeness and accuracy of the responses. Higher SAS and SDS scores indicate more severe anxiety and depressive symptoms.

2.2 MR image acquisition

A 3.0 T MRI scanner (Ingenia, Philips Medical Systems, Best, The Netherlands) equipped with an 8-channel head coil was used in this study. All participants underwent MRI scanning during the interictal period and were not taking preventive medications. Functional images were acquired in the axial plane using a gradient echo-planar imaging sequence. During resting-state fMRI acquisition, participants were instructed to remain still, keep their eyes closed, stay awake, and avoid engaging in any specific thoughts.

Resting-state functional images were acquired using the following parameters: repetition time (TR), 2000 ms; echo time (TE), 30 ms; flip angle (FA), 90°; 36 slices; slice thickness, 4 mm; no interslice gap; field of view (FOV), 240 × 240 mm; and acquisition matrix, 64 × 64. The total acquisition time for rs-fMRI was 8 min 8 s. As this study was based on a retrospective whole-brain resting-state fMRI dataset acquired for conventional whole-brain analysis rather than LC-optimized brainstem imaging, the spatial resolution was not specifically tailored for precise delineation of the locus coeruleus. High-resolution three-dimensional T1-weighted structural images were acquired using a turbo fast-echo sequence with the following parameters: TR, 8.1 ms; TE, 3.7 ms; 170 slices; slice thickness, 1 mm; no interslice gap; FA, 8°; acquisition matrix, 256 × 256; and FOV, 256 × 256 mm. Fluid-attenuated inversion recovery (FLAIR) images were also acquired to exclude structural abnormalities, using the following parameters: TR, 7000 ms; TE, 120 ms; 18 slices; slice thickness, 6 mm; interslice gap, 1.3 mm; FA, 110°; and voxel size, 0.65 × 0.95 × 6 mm³. After visual inspection of structural MRI scans, participants with brain tumors, cerebrovascular disease, hydrocephalus, or obvious white matter hyperintensities were excluded from further analysis.

2.3 MR image processing

The present study was based on a retrospective whole-brain resting-state fMRI dataset, and a conventional standardized preprocessing pipeline was applied to all participants to ensure methodological consistency and comparability in group-level analyses. Image preprocessing was performed using Statistical Parametric Mapping (SPM12; www.fil.ion.ucl.ac.uk/spm/software/spm12/) and the Conn toolbox (version 18b) [15], implemented in MATLAB R2013b (MathWorks, Natick, MA, USA). The preprocessing procedures included realignment and unwarping, slice-timing correction, segmentation into gray matter, white matter, and cerebrospinal fluid (CSF), normalization to the Montreal Neurological Institute (MNI) standard space, and spatial smoothing with an 8-mm full-width at half-maximum (FWHM) Gaussian kernel. To reduce the effects of head motion and other artifacts, ART-based scrubbing implemented in the Conn toolbox was used to identify outlier volumes. The motion threshold was set at 3 mm, and the global signal threshold was set at Z = 9. Nuisance variable regression was conducted, with the first five principal components from the segmented white matter and CSF removed from the signal. The six motion realignment parameters, their first-order derivatives, and the outlier volumes detected during the scrubbing procedure were similarly regressed out of the signal. The data then underwent linear detrending followed by bandpass filtering between 0.01 and 0.08 Hz. Although this conventional preprocessing pipeline is widely used in whole-brain rs-fMRI studies, it may be suboptimal for a structure as small as the LC and may therefore reduce anatomical specificity in this region.

Seed-to-voxel FC analysis was performed to investigate whole-brain FC patterns associated with the LC. Bilateral LC masks were defined according to Bär et al. [16]. Specifically, the left LC was defined as a 4 × 6 × 10 mm region centered at the MNI coordinates −5, −34, −21, and the right LC was defined as a 4 × 6 × 10 mm region centered at 7, −34, −21 (Fig. 1, Ref. [16]). These previously published atlas-based coordinates were used to promote anatomical consistency across participants in this retrospective dataset, in which subject-specific LC delineation was not available. Separate left and right LC masks were used as seed regions for subsequent FC analyses. Individual seed-to-voxel maps were generated for each participant using the LC seeds. Two-sample t-tests were performed within a default whole-brain mask to identify group differences in LC-related FC between patients with MwoA and HCs. A voxel-level statistical threshold of p < 0.01 was applied, followed by cluster-level correction using Gaussian random field theory with a two-tailed threshold of p < 0.05. Using RESTplus, significant positive clusters were transformed into binary masks, and mean FC strength values were subsequently extracted from these regions based on the z-maps. In light of the spatial resolution and preprocessing characteristics of the present dataset, these FC results were interpreted as reflecting LC-centered connectivity alterations at the network level rather than definitive abnormalities confined exclusively to the LC proper.

Fig. 1.The bilateral Locus Coeruleus seed masks. Masks were defined according to Bär (Bär et al. [16], 2016) (combined left and right LC; left LC seed was defined as 4 × 6 × 10 mm centered at MNI-coordinates −5, −34, −21, and right LC seed was defined as 4 × 6 × 10 mm centered at MNI-coordinates 7, −34, −21). Blue, left locus coeruleus; Red, right locus coeruleus.

2.4 Statistical analysis

Statistical analyses of demographic and clinical data were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as the mean ± standard deviation (SD), while categorical variables were expressed as counts. The normality of continuous variables was assessed using the Shapiro-Wilk test before statistical analysis. Between-group differences in demographic and clinical characteristics were assessed using independent-samples t-tests for continuous variables and chi-square tests for categorical variables. A value of p < 0.05 was considered statistically significant. Brain regions showing significant between-group differences were then selected for subsequent analyses of the associations between FC alterations and clinical variables in the MwoA group. Subsequently, average z-values were calculated for each participant within the aberrant FC region mask. To further explore the clinical significance of altered LC-related FC, Pearson correlation analyses were performed between these mean z-values and clinical variables only in the MwoA group, including migraine attack frequency, disease duration, SAS scores, and SDS scores, with adjustment for age, sex, and educational background. Finally, region-of-interest (ROI)-based group comparisons were performed using analysis of covariance (ANCOVA), followed by Bonferroni-corrected post hoc analyses where appropriate. These analyses were intended to examine within-patient dimensional associations rather than symptom-brain relationships in HCs; therefore, parallel correlation analyses were not performed in the HC group.

In addition, ROC curve analysis was performed in SPSS to evaluate the ability of FC values from brain regions showing significant between-group differences to discriminate patients with MwoA from HCs. For each ROC curve, sensitivity and specificity were calculated across all possible cutoff values of the corresponding FC measure, and the optimal cutoff value was determined according to the maximum Youden index. The area under the curve (AUC), sensitivity, and specificity were then reported accordingly. Sex, age, years of education, and mean gray matter volume were included as covariates in the group comparison analyses from which the altered FC values were derived. Due to the relatively limited sample size, no external validation set or cross-validation procedure was applied in the present study. Therefore, the ROC analysis should be considered exploratory, and the corresponding classification performance should be interpreted with caution.

3.1 Demographic and clinical characteristics of participants

The demographic characteristics and clinical assessment results of all participants are summarized in Table 1. There were no significant differences between patients with MwoA and HCs in age, sex distribution, years of education, MoCA score, SAS score, or SDS score, as assessed using the chi-square test for sex and two-tailed independent-samples t-tests for the remaining variables (all p > 0.05). These findings indicate that the two groups were well matched in regard to the major demographic and baseline neuropsychological variables.

3.2 Resting-state functional connectivity maps of the LC seed regions

The resting-state FC maps of the bilateral LC seed regions in the MwoA and HC groups are shown in Fig. 2. In both groups, the left and right LC demonstrated distributed functional connectivity with widespread cortical and subcortical areas, including frontal, temporal, parietal, occipital, and cerebellar regions. This connectivity pattern was broadly consistent with the extensive anatomical and functional projections of the LC.

Fig. 2.Resting-state functional connectivity maps of the bilateral Locus Coeruleus. The Migraine patients, n = 55; the healthy controls, n = 50. L, left; R, right; LC, Locus Coeruleus.

3.3 Between-group differences in LC-centered FC

Compared with HCs, patients with MwoA showed abnormal LC-centered FC in several brain regions (Table 2 and Fig. 3). For the left LC seed, patients with MwoA exhibited significantly increased FC with the right superior temporal gyrus (STG) and left cerebellar posterior lobe (CPL), whereas significantly decreased FC was observed with the right superior frontal gyrus (SFG) (p < 0.05).

Fig. 3.Between-group differences in functional connectivity. Altered functional connectivity between the bilateral Locus Coeruleus and whole-brain regions in patients with migraine without aura (MwoA) compared with healthy controls. L, left; R, right; LC, Locus Coeruleus; SFG, Superior Frontal Gyrus; IOG, Inferior Occipital Gyrus; STG, Superior Temporal Gyrus; CPL, Cerebellar Posterior Lobe.

For the right LC seed, patients with MwoA showed significantly increased FC with the left inferior occipital gyrus (IOG) and left STG, while significantly decreased FC was again observed with the right SFG (p < 0.05). These findings indicate that MwoA is associated with altered LC-centered connectivity involving temporal, occipital, cerebellar, and prefrontal regions, with partly distinct but overlapping patterns between the left and right LC seeds.

3.4 Associations with the clinical characters

To explore the clinical relevance of LC-related FC abnormalities, mean z-values were extracted from all significant clusters showing altered FC with the LC (Table 2; Figs. 2,3). Pearson correlation analyses were then performed within the MwoA group between these extracted FC values and clinical measures, including migraine attack frequency, disease duration, SAS scores, and SDS scores.

Most altered FC measures were not significantly correlated with the examined clinical variables. However, two significant positive correlations were identified. FC between the right LC and left IOG was positively correlated with SDS score (p = 0.011, r = 0.344), suggesting a potential association between LC-occipital connectivity and depressive symptom burden in patients with MwoA. In addition, FC between the left LC and right STG was positively correlated with disease duration (p = 0.012, r = 0.341) (Fig. 4).

Fig. 4.Correlations between the functional connectivity of the Locus Coeruleus and the clinical characteristics. (A) Positive correlation between SDS score and FC of the right LC–left IOG connection (p = 0.011, r = 0.344). (B) Positive correlation between disease duration and FC of the left LC–right STG connection (p = 0.012, r = 0.341). L, left; R, right; LC, Locus Coeruleus; SFG, Superior Frontal Gyrus; IOG, Inferior Occipital Gyrus; STG, Superior Temporal Gyrus; CPL, Cerebellar Posterior Lobe; SDS, Self-Rating Depression Scale; FC, functional connectivity.

3.5 ROC analysis for differential diagnosis

ROC analyses were performed using FC values extracted from the abnormal clusters to evaluate their ability to distinguish patients with MwoA from HCs. The AUC values for all examined clusters were ≥0.696, and the corresponding AUC, sensitivity, and specificity values are presented in Fig. 5. Among these altered connectivity measures, FC between the right LC and right SFG showed the strongest discriminatory performance, with a sensitivity of 81.82%, a specificity of 68.00%, and an optimal resting-state FC cutoff value of −0.012.

Fig. 5.ROC analysis for differential diagnosis. The ROC curves of significant ROI of LC FC in the MwoA group and the HC group. ROC, receiver operating characteristic; ROI, region of interest; LC, Locus Coeruleus; FC, functional connectivity; MwoA, Migraine without aura; HC, healthy control; L, left; R, right; STG, Superior Temporal Gyrus; CPL, Cerebellar Posterior Lobe; SFG, Superior Frontal Gyrus; IOG, Inferior Occipital Gyrus.