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The Use of Proton MR Spectroscopy in Epilepsy: A Methodological Review

Magnetic Resonance Spectroscopy (MRS) has at least two major roles in the evaluation of epileptic patients. First, MRS can help to understand the interaction between seizures and metabolic function. Thus, MRS is particularly interesting for basic science studies of seizures and epilepsy. Second, MRS can explain the nature of seizure control and/or provide localization information by measuring metabolic changes. So MRS can be used as a powerful complementary technique to structural MRI for diagnosis and assessment of response to therapy, and measurement of disease progression. The aim of this paper is to review the methodological aspects of H-MRS publications between 1994 to 2016, which utilized H-MRS in lateralizing the epileptogenic zone in lesional and non-lesional Temporal Lobe Epilepsy (TLE), Extra-Temporal Lobe Epilepsy (ETLE), and generalized epileptic patients to help the spectroscopist, magnetic resonance imaging technologists, and radiologists to improve the overall diagnostic sensitivity in epileptic patients.


Introduction
According to the World Health Organization (WHO), more than 50 million individuals are living with epilepsy, of whom nearly 80% are in middle-and low-income countries and do not get the proper treatment. Approximately 2.4 million individuals are diagnosed with epilepsy annually, and about 0.2% of all deaths in the world occur among this group of patients every year [1].
The first suggested treatment line for patients with epilepsy is Antiepileptic Drugs (AEDs). Although most patients with generalized seizures and a considerable number of patients with partial epilepsy respond well to antiepileptic drugs, 20-30% of patients in the focal epilepsy category are resistant to the current medications [2]. Surgical resection of seizure foci is frequently one of the most effective treatments for this group of patients. Defining the exact resection margin is crucial in the surgical treatment of epilepsy to remove the epileptogenic zone as much as possible.
Pre-surgical planning for epilepsy surgery includes an extensive medical history, physical exam, Electroencephalogram (EEG) findings, and neuroimaging information. Structural and functional neuroimaging methods, including Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) have a key role in evaluating and selecting patients for surgery. However, advancements in imaging techniques such as Diffusion Tensor Imaging (DTI), MRI morphometry, Magnetic Resonance Spectroscopy (MRS), EEG-fMRI (functional MRI), Magnetoencephalography (MEG)/Magnetic Source Imaging (MSI), and EEG/ESI (Electronic Source Imaging) can improve surgical decision making noticeably [3].
Cellular and biochemical changes occur before structural damage can be detectable by conventional imaging. In addition, there are cases where conventional MRI cannot demonstrate structural changes. Methods such as MRS that discover biochemical changes in the tissue are perfectly suited to assessing such pathological changes even in the absence of morphological alterations. Therefore, MRS can be used as a powerful complementary technique to structural MRI for diagnosis, response to therapy and measurement of disease progression.
Proton Magnetic Resonance Spectroscopy ( 1 H-MRS) identifies hydrogen atoms and uses the same principles of physics as MRI to obtain the biochemical information of tissue metabolites by detecting their resonance frequencies, referred to as chemical shift. MRS can measure concentrations of different metabolites in various structures and different pathological situations of the brain to generate sensible and reliable results.
MRS has at least two major roles in the evaluation of epileptic patients. First, MRS can help to understand the interaction between seizures and metabolic function through the study of the relationship between the clinical characteristics of seizures and 1 H-MRS alternations. Therefore, MRS is particularly interesting for basic science studies of seizures and epilepsy [4,5]. Second, MRS can explain the nature of seizure control and/or provide localization information by measuring metabolic changes [6,7]. On the other hand, no study has reviewed the literature based on methodology and its contribution to 1 H-MRS results in epilepsy although there is significant methodological variability among the relevant studies. Therefore, this paper is designed in such a way that it can provide an overview of the findings of 99 1 H-MRS studies on patients with epilepsy based on various methodological and technical aspects, including the strength of the magnetic field, MRS sequence, and absolute concentration versus metabolite ratios.

Literature Review
We obtained 250 papers through the electronic databases of Medline/PubMed and ScienceDirect from 1994 to 2016 (Table 1, 2, 3). The search terms were epilepsy, Temporal Lobe Epilepsy (TLE), generalized epilepsy, Juvenile Myoclonic Epilepsy (JME), hippocampus, Frontal Lobe Epilepsy (FLE), Hippocampal Sclerosis (HS), and correlation, which were also combined with proton magnetic resonance spectroscopy, proton MR spectroscopy, 1 H MRS, neuroimaging, and EEG. We excluded studies which included children or had already been published in pediatric journals and included studies on patients with HS lesions on MR imaging. Two reviewers studied the titles and abstracts of the studies and excluded some papers because of a lack of necessary criteria for patients or methodology and studied 107 papers based on methodological aspects. A summary of the details of all included studies was tabulated into a spreadsheet. Then, these studies were classified into three groups (generalized epilepsy, TLE, ETLE researches) and seven methodological factors were extracted from them.

MRS in Epilepsy
The most popular metabolites studied in epileptic patients include N-Acetyl Aspartate (NAA, 2.01 p.p.m.), Creatine (Cr, 3.0 p.p.m.), and Choline (Cho, 3.2 p.p.m.). NAA is considered as a neuronal and axonal integrity marker; Cr and Cho are found in neurons and glia. On the other hand, Cr is a key component of Phosphocreatine (PCr) and it can be considered a normalization factor for the bioenergetics parameter [8]. On the other hand, 1 H-MRS is a non-invasive and sensitive method to help detecting subtle variations for Extra-Temporal Lobe Epilepsy (ETLE) patients even in the absence of structural alterations [10,11]. Moreover, it has been shown that 1 H-MRS has successfully identified processes of epileptogenesis in Juvenile Myoclonic Epilepsy (JME), which was characterized by a reduction of NAA levels in the thalami and prefrontal cortex of this group of patients [14]. 1 H-MRS was able to reveal elevated levels of Glutamate (Glu) and Glutamine (Gln) (Glu+ Gln= Glx) and Gamma-Aminobutyric Acid (GABA) in the frontal and occipital lobes of IGE patients [15, 16].

General Considerations
There are only a few factors which affect the information content of the proton spectra of the brain, including the type of the pulse sequence, TE, and field strength. Despite the use of 1 H-MRS as a non-invasive method for investigating brain neurochemistry for understanding psychiatric and neurological diseases, a low Signal-to-Noise Ratio (SNR) has limited its use in clinical investigations. On the other hand, due to an approximately linear increase in the noise voltage with frequency, it is expected that SNR increases linearly with B0. In recent years, thanks to the increased field strength of scanners and improved acquisition techniques and analysis approaches, 1 H-MRS has gained the support and interest of the MR and clinical neuroscience communities [8]. At 1.5 T field strength with long TE, signals from Cr, Cho, and NAA are easily detectable in a normal brain where other chemical compounds such as Alanine and Lactate (Lac) may be observable due to some pathological processes. At short TE, chemical compounds such as Glutamate (Glu), Glutamine (Gln), GABA, Myo-Inositol (mI), lipids and macromolecules can be observed. After increasing the field strength to 3, 4, and 7 T, spectral resolution increases and more components can be recognized with high confidence [8].
Currently, multi-voxel MRS (Chemical Shift Imaging (CSI) or MRSI) and Single-Voxel Spectroscopy (SVS) are two methods of obtaining the spatially localized biochemical information in epilepsy. SVS is a simple and rapid image acquisition technique (2-4 min), but has some limitations such as the voxel position. The SVS voxel should contain the lesion correctly during patient examination. In other words, it should be carefully positioned on the tissue. MRSI can help overcome this limitation by changing the grid to fit precisely over the desired lesion any time during data acquisition. MRSI is important in obtaining spatial information on small structures like amygdala or hippocampus, and SVS is preferred when comparing a pathological zone with a contralateral region. Therefore, whether to choose SVS or MRSI depends heavily on the aim of the study (See [8] for more details).
Both types of localization techniques can use various types of pulse sequences. Point Resolved Surface coil Spectroscopy (PRESS) and Stimulated Emission Acquisition Mode (STEAM) are the two most common pulse sequences that the single-voxel technique uses. PRESS excitation is the most common method for two or three-dimensional MRSI. For more information about PRESS, see [17], and for a detailed comparison between STEAM and PRESS, see [18].
Two parameters, TE and TR are effective on the 1 H-MRS results and should be determined based on the desired metabolite. MR spectra at short TE (based on our knowledge from 1 H-MRS studies in epilepsy "20-72 ms") involve broad macromolecule resonances signals that decay completely after 60-80 ms because of their This journal is © Tehran University of Medical Sciences short T2 relaxation time. However, spectra obtained at short TE values are richer in information because MR spectra obtained using short TE provide more metabolite signals, including Glx or mI (increased Glx levels may reflect seizure activity). The quantification of long TE (107-272 ms) acquisitions is less sensitive to measurement and tissue conditions compared to signals measured at short TE values. SNR and then quantification shall be improved by using a long TR (>3 seconds, about 3000-6000 ms), whereas examination time increases. The typical in-vivo TR value for clinical MRS is in the range of 1-3 sec. Nevertheless, it is necessary to use long TR by keeping TE as short as possible, so that T1 and T2 relaxation do not affect signal intensities. Therefore, choosing between short and long TE along with appropriate TR depends on the aim of the study; for example, if only NAA, Cho, and Cr need to be quantified, 1 H-MRS must be performed at long TE to prevent the effects of macromolecular signals on the quantification procedure [8].

Methodological Review of Hippocampal 1 H-MRS Studies in TLE
There are methodological differences in the hippocampal 1 H-MRS in TLE studies published to date. Our investigation of 78 papers showed that they had all been carried out with different MR scanners (e.g. Philips, General Electric, Siemens) running at a field strength of 1.5 Tesla (T), except for 18 of them: four studies used 2 T equipment [4, 19- One of the most important issues in hippocampal 1 H-MRS studies in TLE patients is the size of investigated voxels, ranging from 1 cm 3 to 6 cm 3 . Because of the hippocampal structure in which the middle part and the tail end are considerably thinner than the edge lengths, the voxel size often contains extra tissue beyond the hippocampus, which can lead to partial volume effects.
Chang et al. obtained spectra from hippocampal areas bilaterally with VOIs of 2.25 cm 3 and 6 cm 3 in healthy and patients groups. They found that the overall lateralization ability of MRS was 72% and the correct lateralization rate was 82% for the VOI of 2.25 cm 3 (1.5×1.5×1 cm 3 ) and 62% for the VOI of 6 cm 3 [37]. Moreover, tissue heterogeneity in the selected VOIs is also another important factor which has been addressed in several studies. The differences in the concentration of  Table 1 shows a comparison of methodological aspects for hippocampus in TLE patients. Based on our current knowledge, the two most popular voxel sizes are 2×2×2 cm 3 and 1.5×1.5×1.5 cm 3 , and the most popular sequence is PRESS with long TE but as discussed above, the choice between short and long TE along with appropriate TR depends on the aim of the study.

Methodological Review of Extrahippocampal 1 H-MRS Studies in TLE
We studied 16 papers in this area. In some studies, authors have examined a set of brain structures and regions that are anatomically and functionally associated with the epileptogenic zone (hippocampus) in TLE patients. This network includes the thalamus, temporal pole, basal ganglia, and insula. There are no significant differences in the reported methodology of 1

Methodological Review of 1 H-MRS Studies in Extra-Temporal Lobe Epilepsy
In all patients in such studies (13 papers), no structural lesions were found on the MRI scans, but based on EEG the diagnosis was lateralized and localized in the parietal lobe or the frontal lobe and in two studies the occipital lobe (

Methodological Review of 1 H-MRS Studies in Generalized Epilepsy
All studies (13 papers) in this section examined subjects with IGE, including nine studies which examined JME patients [12, 14, Table 3 shows a comparison of methodological aspects in generalized epileptic patients. According to current knowledge, the most popular sequence for these studies is PRESS. The most common metabolite ratio in patients with ETLE is NAA/Cr (Table 2 # 1-6, 9-13). Other metabolite ratios in such patients are: Cho/Cr (Table 2 # 1, 5, 12, 13), NAA/Cho (Table 2 # 1, 6, 12, 13), NAA/Cr+Cho (Table 2 # 2, 12, 13), tNAA/Cr, Glx/mI and Glx/Cr (Table 2 # 7,8), and Lac/Cr (Table 2 # 12). Reduced NAA/Cr or NAA/Cho ratios have been found in the seizure focus in FLE patients, which may help to lateralize the epileptogenic zone in MRI-negative FLE patients. Metabolic abnormalities in 1 H-MRS have also been identified in areas away from the seizure focus where there is no evidence of a lesion on MRI, and it is not clear whether this reflects widespread pathology without an MRI-evident lesion or a functional consequence of a seizure focus. tNAA, Glx, Cho, Cr, mI, GABA+, Lac concentrations in ETLE patients has been reported by R. J. Simister and his colleagues (Table 2 # 7,8). In this study, IGE patients showed increased Glx and GABA+.

3.2.The Relationship between EEG Finding and 1 H-MRS Alterations
The results regarding correlations between the distribution of metabolic alterations and interracial EEG spikes showed a moderate level of concordance in the lateralization of TLE and ETLE patients [10,44,52,53,86,97,98]. T. Hammen et al. found that tNAA values and the degree of Interictal Epileptiform Discharges (IEDs) in EEG (p=0.04) have a negative correlation. However, there was a positive correlation between Cr and the duration of the seizure (p=0.01), which represents the relationship between the duration of the seizure and increased levels of Cr. The results of this study showed that the degree of tNAA reduction is probably indicative of neuronal dysfunction [86]. H. Aydin et al. also suggested the MRSI sensitivity index for lateralization in the left and right hippocampi, which were 100% and 92% respectively according to EEG findings [98].

3.3.The Relationship between Cognitive Performance and 1 H-MRS Alterations
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The Relationship between MR Volumetry and 1 H-MRS Alterations
MR volumetry demonstrates volume loss in the hippocampus, which is an MRI abnormality in TLE patients with HS. Six studies investigated the relation between 1 H-MRS neuro-metabolites and MR volumetry in TLE patients with or without HS [9, 10, 32, 103-105]. E. Duzel et al. examined 22 patients with MR abnormality and normal MR. They found that hippocampal volumetry results did not correlate with the Apparent Diffusion Coefficient (ADC) value, NAA/(Cho+Cr), and the duration of the disease in any of the patients (with or without HS) or controls. There was a negative correlation between the NAA/(Cho+Cr) ratio and ADCs in the ipsilateral hippocampus (based on EEG recordings) and MR-positive patients (r= -0.45, p=0.05 and r= -0.94, p=0.001, respectively). In contrast, these two measurements did not have a significant correlation in patients without HS. According to the results, they suggested that these three techniques show the complementary aspects of hippocampal pathology [103]. G. Helms et al. examined 43 IGE patients and 38 healthy controls. They used the quantitative single voxel proton MR spectroscopy, thalamic volumetry, and Voxel Based Morphometry (VBM), to measure concentrations of Glx and NAA in the thalamus and occipital cortex, and fractions of thalamic grey and white matter. They found that quantitative 1 H-MRS and VBM provide further information for the involvement of the thalamus in the generation of seizures in IGE patients. The mean thalamic and cerebral volume was reduced in patients compared with controls (p=0.0001, p=0.0003, respectively) [106].

3.5.The Relationship between SPECT and PET results and 1 H-MRS Alterations
As mentioned above, currently, MRI, SPECT, and PET are the most important non-invasive methods for the detection of structural and functional abnormalities in TLE and ETLE patients.

The Relationship between Clinical Characteristics of Epilepsy and 1 H-MRS Alterations
Some studies have examined the relationship between 1 H-MRS results and seizure frequency [4,109]. In a study conducted by P. A. Garcia et al. there was a negative correlation between NAA concentration and seizure frequency in FLE and TLE patients (r=-0.72, p<0.02 and r=-0.60, p<0.06, respectively). However, the correlation between NAA/Cr ratio, FLE, TLE and the duration of epilepsy was poor (r=0.10, p<0.41 and r=0.18, p<0.34, respectively) [5].
Taking everything into consideration, there is methodological heterogeneity in applied methods (including CSI vs. SVS, MRS sequences, and their parameters) and the quantification methods. Nevertheless, increasing the field strength, improving RF pulse design methods, localization sequences, and postprocessing and quantification procedures, especially at short TEs, can improve the overall diagnostic sensitivity and provide a valuable implication for epilepsy diagnosis in clinics. Mohammadi et al. suggested an interesting approach for the quantification of MRS data. They employed a dimension reduction technique for the spectral fitting of the MR spectroscopy data with short TE (=30 ms) and showed that the sensitivity and specificity of this modality in TLE patients are 60% and 82%, respectively. Overall, H. Aydin et al. suggested that proton MR spectroscopy imaging can be used for the lateralization of an epileptic focus in mTLE patients with 96% sensitivity (sensitivity in the right hippocampi was 92%) and 50% specificity.