Introduction
Migraine is a neurobiologic disorder that affects about 27 million women and 10 million men in the US [
1]. Migraine attacks manifest themselves from childhood (usually 8–12 yrs.) to old age, with a decline among women during the postmenopausal years. Migraine is a unilateral throbbing headache that lasts 4–72 hours; it is idiopathic, episodic and recurrent [
2]. Although the causes of migraine are unknown, it is generally thought that the pain originates from chemical activation of sensory nerves that supply intracranial blood vessels and the meninges [
3]. However, the long-term consequences of repeated intermittent attacks of acute migraine on brain function, whatever the origin of the syndrome is, are not well defined.
Two major unanswered questions in the field of migraine relate to (1)
Is there an underlying basis for the increased sensitivity to various stimuli of the migraine brain during [
4,
5]
and even between [
6‐
8]
acute attacks? and (2)
What is the underlying basis for the recent evidence suggesting that migraine, may predispose to significant functional [
9,
10]
and structural changes [
11‐
15]
in the brain? One mechanism by which both of these changes may take place is through alterations in neurochemical systems in the brain that are augmented by the repeated acute attacks. Such changes may eventually drive the process on the evolution from acute migraine to chronic daily headaches [
16] and also the resistance to drug therapy in the chronic daily headache group [
17]. By using magnetic resonance spectroscopy, chemical changes in the brain can be measured in patients. Here we have begun to explore this issue by trying to define these changes during the interictal period in acute intermittent migraine patients for reasons discussed below. A definition of such chemical changes would provide a target for potential interictal therapies that may decrease the severity and/or frequency of migraine and provide a basis for evaluating changes that may take place in the transition to chronic migraine.
A number of recent reports suggest alterations in the interictal migraine brain based on changes in cerebral blood flow [
18‐
20] as well as changes in interictal cognitive function in migraineurs with aura [
21]. A wealth of evidence, including measurements demonstrating changes in physiological (i.e., evoked potentials) measures [
22‐
24]), strongly supports the hypothesis of central neuronal hyperexcitability as playing a key role in the pathogenesis of migraine [
25]. One potential mechanism for neuronal excitability includes an abnormality of the pre-synaptic release of excitatory amino acid neurotransmitters. Although increased platelet [
26,
27] and plasma [
27,
28] levels of neuroexcitatory amino acids including aspartate (Asp), glutamate (Glu), Gln and glycine (Gly) have been reported in migraine patients compared to healthy control subjects [
29], these changes are not always good measures or indicators of changes of synaptic glutamate in the brain. In addition, cerebrospinal fluid (CSF) Gln, Gly and taurine (Tau) concentrations are elevated in migraineurs [
30] suggesting glutamatergic systems are likely to be altered in the migraine brain. Indeed, given that glutamate is the main excitatory transmitter in the brain excess or under production of glutamate through injury or disease can have pathophysiological effects. The glutamate hypothesis for migraine has been discussed by Ramadan [
31] and reviewed recently by Vikelis and Mitsokostkas [
32]. Increased synaptic concentrations of excitatory amino acid neurotransmitters may lead to excessive activity at the N-methyl D-aspartate (NMDA) Glu receptor subtype, which may amplify and reinforce pain transmission in migraine and other types of headache. Indeed, low-affinity NMDA receptor (NMDAr) antagonists, such as memantine, have previously been shown to reduce frequency of migraine and tension-type headaches [
33].A neuroimaging method capable of assessing potential glutamatergic imbalances in the migraine brain
in vivo might provide key insights into the true nature of the neurochemical impairment and to monitor its modulation following pharmacotherapy.
1H-MRS is a potential candidate for investigating glutamate systems
in vivo although its application to migraine is relatively sparse in the literature. Functional
1H-MRS studies have focused predominantly on changes in the visual cortex [
34‐
36]. Other
1H-MRS studies have evaluated metabolite ratios in cluster headache in the hypothalamus and show that N-acetyl aspartate (NAA) to creatine (Cr) ratio is lower in patients with cluster headache vs. chronic migraine or controls [
37]. In addition, single-voxel
1H-MRS studies have investigated potential cerebellar metabolite alterations demonstrating significantly decreased choline (Cho) levels in migraine patients compared to healthy controls [
38]. A similar study detected decreased cerebellar NAA and Glu concentration and increased myo-inositol (mI) levels in familial hemiplegic migraine patients [
39]. More recently, a 3.0 T
1H-MRS study reported differences in thalamic metabolite ratios in migraine patients compared to healthy controls [
40].
Most of these earlier studies employed conventional
1H-MRS methodology at a low static magnetic field strength of 1.5 T and none reported changes in multiple brain regions. For the present study we evaluated brain chemistry using medium field 4.0 T
1H-MRS in two regions, the ACC and the insula. In addition, we employed a variant of single-voxel 2D
J-resolved
1H-MRS method in an attempt to further enhance spectral resolution and sensitivity, and to provide access to the quantification of an increased number of metabolites [
41‐
43]. The brain regions were selected as an initial focus on evaluating brain metabolites in migraine patients for a number of reasons. The ACC is involved in a number of behaviors [
44‐
46] and is usually implicated in most pain studies [
47,
48] including migraine [
49] and related cognitive components [
50]. With respect to the latter it is considered to be involved in reinforcement history [
44] that may be relevant in repeated episodes of migraine. The region has been proposed as a model for understanding components of central sensitization of pain [
51]. As for the insula, the region is involved in both pain [
52] and emotional processing [
53], including the unpleasantness of pain [
54]. Given the nature of the regions in sensory and emotional processing, we hypothesized that differences in glutamatergic metabolism would be observed when comparing spectral data from patients and healthy controls.
Discussion
Here we report novel differences in
1H-MRS defined levels of metabolites in the ACC and insula measured in the interictal period of migraine patients. Although conventional descriptive statistics yielded no differences on the analysis of the spectra, a LDA demonstrated significant differences between migraine subjects and age-gender matched controls. This type of analysis allows for the determination of discrimination of two or more groups (e.g., migraine vs. healthy) based on Cr-normalized levels of specific metabolites. This analysis separated out a relationship between NAAG and Gln within the ACC and insula during their interictal period. NAAG is the most abundant peptide neurotransmitter in the mammalian CNS [
63] being synthesized exclusively in neurons from NAA and Glu by NAAG synthetase. In addition to its role as a neurotransmitter, NAAG is a source of Glu [
64] and like NAA is thought to play a role as a major osmolyte in the vertebrate brain [
65,
66]. Glutamine on the other hand is synthesized exclusively in glial cells from Glu and ammonia by the enzyme glutamine synthetase. Subsequently, Gln is released back into the extracellular space, shuttled back into neurons and converted to Glu by glutaminase. The Glu that is regenerated may then go on to play a direct role in excitatory neurotransmission, packed and stored in vesicles or incorporated into NAAG. An intriguing observation in the present study is the LDA-detected classification of migraine patients and control subjects for two different brain regions based on NAAG and Gln, which are closely linked by this excitatory neurotransmitter system. Interestingly, the ACC and insula LDA plots show oppositely signed gradients, an observation that might be explained by (i) the significant tissue type differences within ACC and insula voxels detected by image segmentation and (ii) the known uneven distribution of Gln and NAAG throughout the brain and within brain tissue type [
67]. The measured changes in these excitatory amino acid neurotransmitters (NAAG) and related species (Gln) provide some insights into altered central nervous system (CNS) mechanisms in migraine and may contribute to abnormal CNS processing including changes during the migraine state (e.g., process of central sensitization [
68,
69], progressing from acute episodic to chronic/daily migraine [
70] or abnormalities during the interictal period [
71‐
74]. We did not detect direct differences in Glu levels between controls and migraine patients although preferential storage of excess synaptic Glu in the form of Gln and/or NAAG might explain comparable Glu levels within the two cohorts. Note that a previous
1H-MRS study showed decreased cerebellar Glu levels in migraine patients compared to healthy controls [
39] yet similar cortical
1H-MRS findings have not been reported to date.
A growing body of preclinical and clinical data supports the notion of aminergic dysfunction in migraine headache including alterations in both the glutamatergic and glutaminergic systems [
31,
32]. For example, NMDA receptor antagonists inhibit cortical spreading depression in the rat brain [
75]. Cerebrospinal fluid [
76] and plasma [
27] Glu and Gln levels are increased in chronic migraine patients, although no such data is available for episodic migraine (i.e. our population). It has been postulated that increased brain Glu leads to cortical hyperexcitability typical of migraine [
77,
78] and potential pharmacological targets for migraine therapy include the ionotropic (NMDA, AMPA and kainate) and metabotropic glutamate receptor antagonists [
79]. The use of a tridimensional personality questionnaire in migraine and tension-type headache clinical sub-populations has shown that glutaminergic dysfunction might also be a specific feature associated with migraine headache [
80]. The development of novel pharmaceutics that can modulate the glutaminergic system and block central and peripheral sensitization might be efficacious for treating migraine. It is also worth noting that, although little is known in the literature for a potential role of NAAG in migraine, there may be a potential role for NAAG antagonists (via mGluR3 receptor blockade) for the therapy of migraine.
A number of reports indicate that modulation of the glutamatergic system in the ACC takes place following pharmacological or sensory manipulation. Alterations in ACC neurons may be dependent on prior events that change or modulate neuronal activity. For example, drugs may decrease levels of glutamate in the ACC [
81] and excitatory synapses into the ACC are in part NMDA mediated changes in this region [
82]. In addition, amputation of a hind paw digit in rats results in a loss of activity-dependent long-term depression in the ACC [
83] and potentiation of sensory responses [
84]. NMDA receptors in the ACC mediates pain-related aversion [
85]. Thus, in migraine patients either as a result of intermittent pain or medications, ACC glutamatergic impairment would account for an increase in activation in this region. In data from another report we observe increased sensitivity in the descending modulatory systems in the brainstem in interictal migraine patients vs. controls [
86]. In functional imaging studies of pain, activation in the insula is observed and it has been suggested that the region has important contributions to both pain and emotional processing [
87,
88]. However,
1H-MRS detected changes in this region in the interictal period have not been reported.
For the present study, we chose to use a 3D localized variant of
J-resolved
1H-MRS, a method that has been shown to enhance spectral resolution at several field strengths including 1.5 T [
42,
43], 3.0 T [
89] and 4.0 T [
90]. Increased spectral resolution is achieved as
J-coupled metabolite resonances are effectively spread over a 2D surface whereas uncoupled peaks remain along F
1 = 0 Hz. Glutamine contains a single methine (CH; 3.75 ppm) and two methylene (CH
2; 2.1 and 2.4 ppm) groups and each proton resonance is split owing to
J-coupling effects [
91]. It follows that, for 2D
J-resolved
1H-MRS data, glutamine shows multiple proton resonances across the 2D surface. In combination with LCModel fitting and GAMMA-simulated basis sets, we use information from the whole 2D datasets and this approach further improves multiple-metabolite quantification of 2D
1H-MRS data. Recently we applied these methods
in vivo and demonstrated their utility for reliably measuring brain glutamate and glutamine levels [
59]. NAAG, a dipeptide composed of NAA and Glu joined by a peptide bond, also benefits from the 2D
1H-MRS approach. The major resonance of NAAG is its CH
3 resonance at 2.04 ppm that appears as a shoulder on the dominating NAA CH
3 2.0 ppm peak. In conventional
1H-MR spectra, this chemical shift region is further complicated by underlying
J-coupled resonances of Gln, Glu and GABA, and a major advantage of 2D
J-resolved data is the fact these
J- coupled metabolite resonances are shifted away from the F
1 = 0 Hz axis. This yields a cleaner chemical shift region that is essentially comprised of NAA and NAAG CH
3 singlet peaks, both of which are more reliably fitted by the described LCmodel template and fitting procedures.
It is certainly true that MRS studies are limited by the relatively low SNR of the spectra and some studies of chronic pain patients have noted larger between group differences. However, the methods that were employed in the present study were designed to allow the detection and quantitation of a larger number of lower concentration metabolites. This has to make observations that would not have been possible using more standard methods. In addition the interictal migraine group may differ from the chronic pain state in that it produces prolonged and continuous brain changes that manifest in profound structural [
15] and functional changes [
92,
93].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AP implemented MRS acquisition and data analysis methods, participated in organization of study design, carried out MRS acquisitions and drafted most parts of the manuscript. LB helped with overall study design and oversaw data analysis procedures. GP performed statistical data analyses. ST was entirely responsible for subject screening and enrollment. EJ provided and oversaw 2D MRS data analysis methods. RH was involved in the overall study design and oversaw data analysis procedures. PR was helped with the original study design and oversaw MRS data acquisition and analysis procedures. RB contributed to direction of the study and oversaw data analysis and interpretation. DB contributed to conceptual framework and overall direction of the study including overseeing data analysis, interpretation and manuscript preparation. All authors have read and approved the final manuscript.