Investigation of NAA and NAAG dynamics underlying visual stimulation using MEGA-PRESS in a functional MRS experiment

doi:10.1016/j.mri.2015.10.038

Magnetic Resonance Imaging

Volume 34, Issue 3, April 2016, Pages 239-245

https://doi.org/10.1016/j.mri.2015.10.038 Get rights and content

Abstract

N-acetyl-aspartate (NAA) is responsible for the majority of the most prominent peak in ¹H-MR spectra, and has been used as diagnostic marker for several pathologies. However, ~ 10% of this peak can be attributed to N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide whose release may be triggered by intense neuronal activation. Separate measurement of NAA and NAAG using MRS is difficult due to large superposition of their spectra. Specifically, in functional MRS (fMRS) experiments, most work has evaluated the sum NAA + NAAG, which does not appear to change during experiments. The aim of this work was to design and perform an fMRS experiment using visual stimulation and a spectral editing sequence, MEGA-PRESS, to further evaluate the individual dynamics of NAA and NAAG during brain activation. The functional paradigm used consisted of three blocks, starting with a rest (baseline) block of 320 s, followed by a stimulus block (640 s) and a rest block (640 s). Twenty healthy subjects participated in this study. On average, subjects followed a pattern of NAA decrease and NAAG increase during stimulation, with a tendency to return to basal levels at the end of the paradigm, with a peak NAA decrease of –(21 ± 19)% and a peak NAAG increase of (64 ± 62)% (Wilcoxon test, p < 0.05). These results may relate to: 1) the only known NAAG synthesis pathway is from NAA and glutamate; 2) a relationship between NAAG and the BOLD response.

Introduction

N-acetyl-aspartate (NAA) is one of the most highly concentrated molecules in the central nervous system (CNS) [1]. Due to its prominent signal in MR spectra, NAA has been used as a diagnostic marker for many neuropathologies, including Canavan disease, ischemia, stroke, Alzheimer disease, epilepsy, brain tumors, multiple sclerosis, traumatic brain injury and schizophrenia. Most of these pathologies, with the exception of Canavan disease, have a decreased NAA peak in the MR spectrum. At first this was interpreted as irreversible neural loss. However, there is currently evidence that decreased NAA concentration may also be associated with mitochondrial dysfunction, which in some cases may be reversible. In the particular case of patients with Canavan disease, which is a demyelinating pathology [2], NAA levels in the CNS are increased due to the absence of the enzyme aspartoacylase (ASPA), which is responsible for breaking down NAA [1]. This has suggested that high NAA levels in the CNS may also have harmful effects.

N-acetyl-aspartyl-glutamate (NAAG) is the most highly concentrated dipeptide in the brain [1], and largely localized to neurons. Although NAA is responsible for the largest contribution to the MRS peak located at 2 ppm, NAAG can contribute 10 to 20% of the signal that is often attributed only to NAA [3], [4]. Decreases of total NAA associated with neurological disease either involve a joint decrease of NAA and NAAG levels, or may underestimate the decrease of NAA levels, in the case where the NAAG levels increase or remain constant [1]. As mentioned, measurement of the separate contributions of NAA and NAAG using MRS is difficult due to the large superposition of their spectra. To separate these contributions, post-processing methods such as LCModel [5] have been used [3], [6], [7], however the efficacy of such an approach is variable [8]. Recently Edden et al. used a MEGA-PRESS pulse sequence to separate the contribution of these metabolites in a standard (not dynamic) MRS experiment [9]. On the other hand, in a recent functional MRS (fMRS) experiment with visual stimulation performed by our group, we found a 20% decrease of the NAA signal and a 200% increase of the NAAG signal with stimulus, while their sum remained constant [10]. Few studies have reported similar results [11], [12], which have been contested by others [13]. These results were obtained using LCModel, which is likely to be impacted by linewidth changes associated with metabolite BOLD, so in the present work we sought to design and perform an fMRS experiment using the MEGA-PRESS sequence, to further evaluate the individual dynamics of NAA and NAAG underlying brain activation, and contribute to the elucidation of their functions in the nervous system.

Section snippets

Subjects

Twenty healthy subjects (mean age 27 ± 6, range 20–40 years, 8 women) participated in this study. The project was approved by the local ethics committee. Informed consent was obtained from all individual participants included in the study.

FMRS experiment

The fMRS paradigm consisted of a rest (baseline) block (320 s, 20 spectra), followed by a stimulus block (640 s, 40 spectra) and a rest block (640 s, 40 spectra), with a total duration of 1600 s (around 27 min, totaling 100 spectra) (Fig. 1). The visual stimulus was

Results

Frequency shifts found during frequency correction of the spectra were within typical drift levels, being at most 12.3 Hz for NAA spectra and 18.4 Hz for NAAG spectra, still below the bandwidth of the MEGA-PRESS inversion pulse (which was 40 Hz). Due to poor quality (edited peaks were not visible), NAA data from one subject (subject 10), and NAAG data from two subjects (subjects 4 and 6) were discarded. The signal-to-noise ratio (SNR) was evaluated for all nine spectra corresponding to one of the

Discussion

The aim of the present work was to evaluate the individual dynamics of NAA and NAAG underlying brain activation, and to contribute to the elucidation of their functions in the nervous system.

Although the function of NAA in the nervous system is not yet well known, it has been hypothesized that it acts as a molecular pump, removing excess water created by neuronal energy metabolism out to the extracellular environment [11], [15]. However, no protein acting to transport NAA and water out of

Acknowledgements

We thank Elvis Lira da Silva (UNICAMP, Brazil) for programming the paradigms in the E-prime software. This work was supported by São Paulo Research Foundation (FAPESP – Brazil), grants 2005/56578-4, 2009/10046-2, 2011/01106-1, 2013/07559-3. This project applied tools developed under NIH grants P41 EB015909 and R01 EB016089.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and

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In-vivo magnetic resonance spectroscopy (MRS) of the brain can potentially bridge between cellular neuroscience and in vivo imaging of physical properties of tissue water by measuring the concentration of endogenous metabolites, particularly those associated with neurotransmission, energy metabolism and oxidative stress defense. Major neurometabolites quantifiable by MRS include the neuronal marker N-acetyl aspartate (NAA) (Landim et al., 2016), as well as glutamate (Glu) (Cheng et al., 2021), the principal excitatory neurotransmitter. Glutamine (Gln) is an MRS-detectable precursor for Glu, though often MRS studies performed at 3T report Glx, the combination of Glu+Gln signals.
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102 healthy volunteers, with approximately equal numbers of male and female participants in each decade of age from the 20s, 30s, 40s, 50s, and 60s, were recruited with IRB approval. MR spectroscopic data were acquired on a 3T MRI scanner. Metabolite spectra were acquired using PRESS localization (TE=30 ms; 96 transients) in the centrum semiovale (CSO) and posterior cingulate cortex (PCC). Water-suppressed spectra were modeled using the Osprey algorithm, employing a basis set of 18 simulated metabolite basis functions and a cohort-mean measured macromolecular spectrum. Pearson correlations were conducted to assess relationships between metabolite concentrations and age for each voxel; Spearman correlations were conducted where metabolite distributions were non-normal. Paired t-tests were run to determine whether metabolite concentrations differed between the PCC and CSO. Finally, robust linear regressions were conducted to assess both age and sex as predictors of metabolite concentrations in the PCC and CSO and separately, to assess age, signal-noise ratio, and full width half maximum (FWHM) linewidth as predictors of metabolite concentrations.
Data from four voxels were excluded (2 ethanol; 2 unacceptably large lipid signal). Statistically-significant age*metabolite Pearson correlations were observed for tCho (r(98)=0.33, p<0.001), tCr (r(98)=0.60, p<0.001), and mI (r(98)=0.32, p=0.001) in the CSO and for NAAG (r(98)=0.26, p=0.008), tCho(r(98)=0.33, p<0.001), tCr (r(98)=0.39, p<0.001), and Gln (r(98)=0.21, p=0.034) in the PCC. Spearman correlations for non-normal variables revealed a statistically significant correlation between sI and age in the CSO (r(86)=0.26, p=0.013). No significant correlations were seen between age and tNAA, NAA, Glx, Glu, GSH, PE, Lac, or Asp in either region (all p>0.20). Age associations for tCho, tCr, mI and sI in the CSO and for NAAG, tCho, and tCr in the PCC remained when controlling for sex in robust regressions. CSO NAAG and Asp, as well as PCC tNAA, sI, and Lac were higher in women; PCC Gln was higher in men. When including an age*sex interaction term in robust regression models, a significant age*sex interaction was seen for tCho (F(1,96)=11.53, p=0.001) and GSH (F(1,96)=7.15, p=0.009) in the CSO and tCho (F(1,96)=9.17, p=0.003), tCr (F(1,96)=9.59, p=0.003), mI (F(1,96)=6.48, p=0.012), and Lac (F(1,78)=6.50, p=0.016) in the PCC. In all significant interactions, metabolite levels increased with age in females, but not males. There was a significant positive correlation between linewidth and age. Age relationships with tCho, tCr, and mI in the CSO and tCho, tCr, mI, and sI in the PCC were significant after controlling for linewidth and FWHM in robust regressions.
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(1) NAA facilitates functional capacity and activity through its close relationship with mitochondrial energy metabolism (Baslow et al., 2007; Clark, 1998). NAA also changes dynamically with neurostimulation, while sedative drugs reduce tNAA, Glx, and Cho (Baslow et al., 2016; Castellano et al., 2012; Gomez et al., 2012; Landim et al., 2016; Zhang et al., 2009). In this view tNAA-related facilitation of functional activity would increase one's responsiveness to low-intensity cues, thereby facilitating immersive emotion and positive agency (Fig. 5).
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Fig. 4 plots the time dynamics of Glx, Glu, tNAA=NAA+NAAG, tCho=GPC+PCh, tCr=Cr+PCr, GSH and GABA for the short and long-cycled designs, with the shaded regions representing CRLBs. Changes in the major singlets (tCho, tCr, tNAA) are rarely reported in the literature (but see Landim et al., 2016), an observation corroborated by our own data (see Fig. 1c), and are included as controls. Subject-level analysis revealed similar dynamics in Glx and Glu as observed for the group-level analysis, with no statistically significant changes for any other metabolite.
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Original contributionInvestigation of NAA and NAAG dynamics underlying visual stimulation using MEGA-PRESS in a functional MRS experiment

Abstract

Introduction

Section snippets

Subjects

FMRS experiment

Results

Discussion

Acknowledgements

Brain Lang

Magn Reson Mat Phys Biol Med

Neuroimage

N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology

Prog Neurobiol

N-acetylaspartate and N-acetylaspartylglutamate: neurobiology and clinical significance

Neurology

Differential distribution of NAA and NAAG in human brain as determined by quantitative localized proton MRS

NMR Biomed

Estimation of metabolite concentrations from localized in vivo proton NMR spectra

Magn Reson Med

MR spectroscopic evidence for glial increase but not for neuro-axonal damage in MS normal-appearing white matter

Magn Reson Med

Physiologic variability of single-voxel proton MR spectroscopic measurements at 3 T

Am J Neuroradiol

In vivo differentiation of N-acetyl aspartyl glutamate from N-acetyl aspartate at 3 Tesla

Magn Reson Med

NAA and NAAG variation in neuronal activation during visual stimulation

Braz J Med Biol Res

Dynamic relationship between neurostimulation and N-acetylaspartate metabolism in the human visual cortex: evidence that NAA functions as a molecular water pump during visual stimulation

J Mol Neurosci

Original contribution
Investigation of NAA and NAAG dynamics underlying visual stimulation using MEGA-PRESS in a functional MRS experiment