Altered Cerebral Iron Over the Whole Brain in Chronic Migraine Using Voxel-Based Quantitative Susceptibility Mapping

Background: The previous documents demonstrated that iron deposition was identied in brain deep nuclei and periaqueductal gray matter region in chronic migraine (CM), and less is known about the cerebral iron deposition in CM. The aim of this study is to investigate the cerebral iron deposition in CM using an advanced voxel-based quantitative susceptibility mapping. Methods: A multi-echo gradient echo MR sequence was obtained from 14 CM patients and 28 normal controls (NC), and quantitative susceptibility mapping images were reconstructed and voxel-based analysis was performed over the whole cerebrum. The susceptibility value of all the positive brain regions was extracted and correlation was calculated between the susceptibility value and the clinical variables. Results: The brain regions with increased susceptibility value in CM patients located in right precuneus, dorsolateral superior frontal gyrus, postcentral gyrus, cuneus, insula and left postcentral gyrus compared with NC. The correlation analysis demonstrated that a positive correlation was identied between susceptibility value of all the positive brain regions and VAS score. Conclusion: The current study demonstrated increased cerebral iron deposition presented in chronic patients, which suggested that increased cerebral iron deposition might play a role in the migraine chronicization.


Introduction
Chronic migraine (CM) is de ned as at least 15 days of headache per month for more than 3 months, including at least eight days a month on which the headache feature and associated symptoms are fully developed migraine attacks [1]. It is estimated that CM affects 1.4-2.2% of the general population globally [2] and around 3% of episodic migraine (EM) patients evolve to CM each year [3]. The etiopathogenesis of migraine are hypothesized to be various processes and mechanisms involving activation of the trigeminal nerve system, abnormality of brain structure and function, vasoconstriction and vasodilation, and cortical spreading depression [4], among which the cerebral cortices serve a vital role. Structural and functional alterations of cerebral cortices have been widely reported in migraine patients [5]. Cortical hyperexcitability and aberrant resting-state brain activity may underlie the cascade of migraine attacks [6]. Interfering with cortical activity can affect pain perception, while pain-related cortical regions undergoing changes in synaptic plasticity can generate pain perception even with no detectable sensory input from the periphery [7].
Increased iron deposition in the brain has been described in migraine patients. Different MRI techniques have been used to measure brain iron concentrations in migraine, including susceptibility weighted imaging (SWI), T2 gradient echo imaging, T2 turbo spin echo sequence, transverse relaxation rates R2, and quantitative T2* [8][9][10][11][12]. Quantitative susceptibility mapping (QSM) is a noninvasive MRI technique that measures spatial distribution of magnetic susceptibility using phase images of gradient-recalled echoes [13]. It is validated that susceptibility measured by QSM is linearly correlated with concentration of iron in brain nuclei [14] and thus provides us an effective way to detect iron deposition in the brain.
Among the studies probing into the iron deposition in migraine brain, increased iron deposition level has been described in periaqueductal gray matter (PAG), red nucleus, globus pallidus and putamen in migraine patients compared to controls using MRI [9][10][11][12]. While most of these researches focus iron deposition on the deep brain nuclei and PAG regions, cerebrum has been randomly studied. In our study we aim to investigate cerebral iron deposition in CM using voxel-based QSM.

Subjects
The written informed consents were obtained from all subjects according to the approval of the ethics committee of local institutional review board. Fourteen chronic migraine patients [4 males and 10 females, mean age 41.71 ± 8.87 years] and 28 normal controls [10 males and 18 females, mean age 42.36 ± 11.10 years]were sequentially recruited from Headache Clinic and the staffs of Chinese PLA General Hospital.
All the patients should meet the International Classi cation of Headache Disorders, 3rd Edition (ICHD-III) criteria of chronic migraine without aura [15]. The inclusion criteria was ful l the following: (1) the diagnosis of migraine and CM refers to 1.1 Migraine without aura and 1.3CM in ICHD-III, respectively; (2) without migraine preventive medication in the past 3 months; (3) absence of other subtypes of headache, chronic pain other than headache, severe anxiety or depression, and psychiatric diseases; (4) absence of alcohol, nicotine, or other substance abuse. The exclusion criteria should meet the following: cranium trauma, the cerebrovascular disease, long-standing hypertension, diabetes mellitus, tumor history and brain surgery. All the subjects should have no MRI scan contraindications such as metal clips within the body and claustrophobia. The image processing steps included as following: (1) The e images and phase images were imported into the STI software and then automatically generated magnetic susceptibility images; (2) 3D T1 images ( Fig. 1A) were coregistered with the magnitude images (TE = 4.72 ms) (Fig. 1B), and then generated the warped T1 images (Fig. 1C); (3) The warped T1 images were normalized with the T1 template (provided by the SPM software) (Fig. 1D), and then generated the normalized parameters; (4) The normalized parameters were applied with the quantitative susceptibility images (Fig. 1E), and then generated the normalized susceptibility images (Fig. 1F), and then all the normalized images were smoothed by 8 mm full width at half maximum(FWHM); (5) voxel-based analysis was performed using two-sample t-test between CM patients and NC. Signi cance difference was set at a P value of < 0.001 without false discovery rate (FDR) correction; (6) all the positive clusters were saved as a binary regions of interest (ROI) to extract the susceptibility value.

Statistical analysis
The data with normal distribution was described as mean ± standard deviation. Correlation analysis was performed between susceptibility value of all the positive brain regions and the clinical variables, P value of < 0.05 was considered to indicate a statistically signi cant correlation.

Results
Voxel-based QSM over the whole cerebrum in chronic migraine patients Figure 2 presented that the brain regions with increased susceptibility value located in right supramarginal gyrus, precuneus, dorsolateral superior frontal gyrus, postcentral gyrus, cuneus, insula and left postcentral gyrus (Table 1). There were no brain regions with decreased susceptibility value over the whole cerebrum. Correlation analysis between the susceptibility value of all the positive brain regions and the clinical variables Table 2 presented that there was a signi cant positive correlation between the susceptibility value of all the brain regions with increased iron (0.010 ± 0.004) and VAS score (r = 0.0555, P value = 0.039). The other clinical variables, including MIDAS, diseased duration, headache frequency, HAMA, HAMD and MoCA socre, showed no signi cant correlation with the susceptibility value (P value > 0.05).

Discussion
In this study, we used QSM to analyze 14 CM patients and 28 normal controls, and con rmed elevated iron deposition in several cortical regions in CM patients. There is a positive correlation between susceptibility value of all the positive brain regions and VAS score of CM patients.
To the best of our knowledge, we are the rst to use QSM for evaluation of cerebral iron concentration in migraineurs. QSM may provide an effective as well as noninvasive way of revealing iron deposition in the brain in vivo. Previous studies usually used phase imaging or susceptibility weighted imaging for iron evaluation. These phase methods have high spatial resolution and take less scanning time. On the other hand, these methods may yield to nonlocal eld in uence that caused by calci cations and vessels [16]. Some researches were performed on 1.5T MR scanner and the low eld strength may as well result lower sensitivity to local susceptibility effects caused by iron [17].
Iron is a key nutrient for normal central nervous system development and function, including oxygen transportation, DNA and myelin synthesis, mitochondrial respiration and neurotransmitter [18]. Physically there are high levels of iron in the basal nuclei due to the synthesis and metabolism function in dopamine [19]. Meanwhile iron can generate reactive oxygen species, and both iron and reactive oxygen species are indicated to be important initiators and mediators of cell death [20]. Excessive accumulation of iron has been associated with aging and neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Friedreich's ataxia [18].
Iron deposition in the migraine brain has been studied in the past 20 years. Welch et al [12] revealed increased iron level in PAG in EM and chronic daily headache patients compared with healthy ones, and the iron increment was correlated to the illness duration. In CAMERA-study, Kruit et al [9] evaluated 138 migraineurs and 75 controls and showed higher iron level in putamen, globus pallidus and red nucleus in migraineurs under age of 50, and the migraine history was positively correlated with iron accumulation. Follow-up study of CAMERA nine years later showed no difference in iron level in deep brain nuclei between groups, and the authors assumed that aging might obscure the migraine-induced iron changes [8]. Tepper et al [10] investigated iron deposition in deep brain nuclei and indicated that T2 imaging in globus pallidus can be used to distinguish between EM and CM patients. Dominguez et al [11] revealed elevated iron deposition in red nucleus and PAG in CM patients compared with EM and controls.
Previous studies have focused on PAG as a key center in the generation and regulation of migraine. And these results referring to PAG and deep brain nuclei, iron accumulation indicates sensory dysregulation in migraine. Our ndings revealed elevated iron deposition in the cerebral cortical regions in CM patients compared to normal subjects, which indicated that the impaired iron homeostasis in migraine was not limited to the deep brain nuclei and PAG. The elevated iron deposition areas in this study were in right precuneus, dorsolateral superior frontal gyrus, postcentral gyrus, cuneus, insula and left postcentral gyrus. Among these areas, the precuneus has been indicated to be associated with cognitive modulation of pain sensitivity [21], and the insula is involved in pain experience and expectation [22,23]. Meanwhile the insula and precuneus both take part in pain catastrophizing on mild pain [24]. The postcentral gyrus and supramarginal gyrus are part of the somatosensory associated cortex. The postcentral gyrus consists of the rst somatosensory cortex which is responsible for recognizing the localization and intensity of pain [25]. While the supramarginal gyrus interprets tactile sensory information [26]. A recent functional MRI study focused on the pain matrix network which includes the anterior insula, precuneus, supramarginal gyrus, thalami, and several other regions, showed that the increased connectivity of the pain matrix may play a role in migraine chronicization [27]. Our result of the iron deposition in the cerebral cortex as well added more support of cortical dysfunction in the pathogenesis mechanism of migraine chronicization. Moreover, higher VAS scores were associated with increased iron deposition or accumulation in cerebral cortex in migraine patients, which suggested that the iron accumulation levels in CM are related to the severity of headache attacks. It remains unclear whether iron accumulation in the cortex has a causative role in the development of chronic migraine headache or the consequence of repetitive of migraine attack.
This study was limited by the modest number of CM patients. And we are not able to draw the causative conclusion of the iron deposition in cortex and migraine chronicization.
In the future, more details of connectivity between cerebral cortex, deep brain nuclei and PAG may be performed and follow-up study is as well necessary to indicate the dynamic change of iron deposition in migraine patients with age.

Declarations
Ethics approval and consent to participate The Institutional Review Board of the Chinese PLA General Hospital approved the research protocol, and the procedures conformed to the tenets of the Declaration of Helsinki.

Consent for publication
Not applicable.

Availability of data and material
All the data supporting our ndings is contained within the manuscript.

Figure 1
The ow chart of voxel-based quantitative susceptibility mapping Figure 1 The ow chart of voxel-based quantitative susceptibility mapping The brain regions with increased susceptibility value over the whole cerebrum in chronic migraine patients. Figure 2