Ethics Statement

This retrospective study was approved by the institutional ethical review board of the Kanagawa Children’s Medical Center, where all of the clinical data in this study was acquired. Written informed consent was waived by the review board.

Subjects

From October 2009 – November 2012, MRS examinations were performed on a total of 235 neonates, who ranged in PCA from 30 – 43 weeks, due to neurological symptoms such as hypoxic ischemic encephalopathy, a suspicion of organic disease, or as routine evaluations before discharge. The inclusion criteria were as follows: the subjects had to be free of radiological abnormalities, organic illness, and neurological symptoms at their latest radiological and clinical examinations. The MR images were interpreted by an experienced pediatric neuroradiologist and clinical and neurological conditions were evaluated by board-certificated neonatologists at our medical center. As a result, 79 neonates (males 36, females 43), including 60 preterm infants (gestational age (GA): 23 – 36 weeks; studied at PCA of 30 – 43 weeks; postnatal age (PNA): 39.9 ± 28.3 d) and 19 term infants (GA: 37 – 41 weeks; studied at PCA of 38 – 42 weeks; PNA: 7.1 ± 6.9 d) were included. Four of the 79 neonates were examined twice in different weeks, which resulted in a total of 83 sets of neonate MR spectra (males 38, females 45).

Proton MRI and MRS

All proton MRI and MRS scans were performed on a clinical 3T MR system (MAGNETOM Verio; Siemens, Erlangen, Germany) using a standard 12-element head coil (diameter: 25 cm) in the quadrature detection mode. During the examinations, the neonates were wrapped in vacuum-type immobilization bags (CFI Medical Solutions, Fenton, MI, USA) to prevent excessive head movement and protect their hearing. Sedation with thiopental (dose, 2 – 10 mg/kg of body weight) was employed where necessary (27 out of 83 neonates). During the examinations, heart rate and transcutaneous oxygen saturation were monitored continuously with a pulse oximeter (Nonin, Plymouth, MN, USA). For single-voxel MRS, the volume of interests (VOI) were placed in three regions: 1) the basal ganglia (BG) as a representative of the subcortical gray matter, 2) the centrum semiovale (CS) as a representative of the white matter, and 3) the cerebellum (Figure 1). T1-weighted (echo time (TE)/repetition time (TR): 9.4/400 ms) or T2-weighted (TE/TR: 120/5000 ms) MR images that had been obtained for clinical diagnosis were also used for the VOI positioning. The MRS was performed using a point-resolved spectroscopic localization sequence (PRESS) [14], a water presaturation pulse sequence, and the following parameters: TE/TR: 30/5000 ms; number of excitations: 4 to 32; spectral bandwidth: 1200 or 2000Hz; number of points: 1024. The excitation frequency was set to the proton resonance of water (4.7ppm, 123.24 MHz). The volume ranges of the BG, CS, and cerebellum were 2.4-6.7, 2.3-7.3, and 2.3-6.3 mL, respectively. Spectra of the same VOI were also obtained without the water presaturation pulse sequence using number of excitations of 2-4 and used to correct eddy-current-induced phase shifts and to quantify the subjects’ brain metabolite concentrations. It took about five minutes to obtain the pairs of MRS scans (with/without water presaturation) for each region; therefore, the total time required to perform MRS examinations of the three regions was about 15 min.

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Figure 1. Representative LCModel outputs of in vivo proton MR spectra for the basal ganglia (a), centrum semiovale (b), and cerebellum (c) (volume, 4.2-6.5 mL; TE/TR, 30/5000 ms) obtained from a neonate at a postconceptional age of 37 weeks.

Volumes of interest were imposed on the T1-weighted images. In the spectra, the bold lines represent the fitted lines produced by LCModel, and the thin lines are the original spectra.

https://doi.org/10.1371/journal.pone.0082746.g001

The MRS data processing including the signal quantification was performed using LCModel (ver. 6.2-1G, Stephen Provencher Inc., Oakville, Ontario, Canada) [15,16] on a LINUX-based personal computer. To quantify the spectra, the “basis-set” of model spectra for the Siemens 3T PRESS sequence, which is internally defined in the LCModel package, was used as a reference to facilitate the separation of the spectra using linear combinations of individual metabolite spectra. The model spectra consisted of data for 16 reference compounds: alanine, aspartate, creatine (Cr), phosphorylcreatine (PCr), γ-aminobutyrate, glucose, glutamate (Glu), glutamine (Gln), glycerophosphorylcholine (GPC), phosphorylcholine (PCh), myo-inositol (mIns), lactate (Lac), N-acetylaspartate (NAA), N-acetyl-aspartyl-glutamate (NAAG), scyllo-inositol, and taurine. Metabolite concentrations were automatically calculated by comparing the proton peaks of the metabolites with that of unsuppressed water in the same VOI. The reference water concentration was assumed to be 46.9 M in all regions [5,10]. The signal-to-noise ratios (SNR) of the spectra and the percentage standard deviation (%S.D.) values of the metabolites were displayed in the LCModel output files. The SNR was defined as the ratio of the spectrum maximum minus the baseline divided by twice the root mean square residual, and the %S.D. was equal to the Cramér–Rao lower bound and was used as an index of the uncertainty in the metabolite concentration estimates [16]. The reliability of the data obtained in this study was ensured by excluding any data that was associated with an SNR of less than 4 or a %S.D. value of more than 30. These are similar protocol to our previous studies [17-19].

In this study, the concentrations of five metabolites were assessed: tCr, the total concentration of Cr and PCr; tCho, the total concentration of GPC (including other choline-containing compounds [16]) and PCh; tNAA, the total concentration of NAA and NAAG; mIns; and Glx, the total concentration of Glu and Gln complex. In the statistical analysis, Pearson’s correlation coefficients were calculated for the relationships between the concentration of each metabolite and PCA. Then, regression line equations were calculated for the statistically significant relationships. The independent samples t-test was used for group comparisons of metabolite concentrations between preterm infants at term (PT) and full term infants at term (FT). For the comparisons of the metabolites between the sexes, the analysis of covariance (ANCOVA) with covariate of PCA was used. P-values of less than 0.05 were considered to be significant.

Metabolite concentration changes with PCA

tNAA (NAA + NAAG) showed marked increases in its concentration during the neonatal period, as was reported previously [1,2,4-7,9-13]. NAA is produced in neuronal mitochondria and plays a role in myelination as a source of acetyl groups for lipid synthesis. NAA also serves as a precursor for the synthesis of NAAG, which acts as a cotransmitter in combination with several other neurotransmitters [1,9,12,20,21]. It was found that the concentration of NAA increased markedly during the period from 32 - 42 weeks gestation; i.e., prior to the initiation of myelination. Such dynamic NAA increases during this period might reflect the use of acetyl groups for myelin deposition [3-7,9].

tCr acts as a “battery” for replenishing ATP levels [1,5,12,22]. In adults, its concentration usually remains relatively stable during aging as well as in a variety of diseases, and hence, it is sometimes used as an internal reference for other metabolites [22]. However, as has been reported in previous neonatal studies, the concentration of tCr increased markedly with PCA in the present study, which might have been due to the increasing energy demand associated with cerebral maturation [5,6,10,12].

tCho is involved in membrane synthesis in the brain and acts as a precursor for the neurotransmitter acetylcholine, which has many functions in the nervous system [1,12,22,23]. Our study did not detect any correlations between the concentration of tCho and PCA in the BG, CS, or cerebellum, and similar results have been reported previously; i.e., no correlations between these parameters were detected in the gray matter, thalamus, or white matter during the period from 32 - 43 weeks PCA [10] or in the motor cortex region during the period from 33 - 42 weeks PCA [6], and no concentration differences were observed between preterm and term infants in the precentral area [7] or cerebellum [4]. On the other hand, tCho displays higher concentrations during early life than in adulthood, indicating that choline-containing compounds are turned over more rapidly during early human development [1,4-6,10]. Brighina et al. reported that the most prominent reduction in the concentration of tCho occurs after the age of 2, by which point myelination has almost been completed, indicating that tCho is progressively incorporated into the lipids that make up myelin [5,12].

The peak observed at 3.56 ppm, which was considered to represent mIns, is also known to contain glycine [22], and it is sometimes difficult to determine the individual concentrations of these metabolites. However, it is likely that mIns is the main component of this peak [5,10] and so the glycine component was not considered in this study. mIns has many functions, for example, it plays roles in osmotic regulation and cellular nutrition and forms complexes such as inositol-1-phosphateand phosphatidyl inositol [1,5,11,12]. Therefore, it is sometimes difficult to identify which concentration increases/decreases are caused by particular functional changes. In this study, decreases in the concentration of mIns were observed in the BG. Marked reductions in the concentration of mIns have also been reported in previous neonatal studies; however, the cause of these changes remains unclear [1,5,6,9-12]. Kreis et al. [5] suggested that mIns plays a role in surfactant production in neonates. In addition, they found that in some supratentorial regions its concentration decreased rapidly after birth, and these reductions appeared to be associated with PNA rather than GA. However, in our analyses we found that the mIns concentration was more strongly correlated with GA than PNA (data not shown). On the other hand, Roelants-van Rijn et al. [11] did not detect any association between the concentration of mIns and PNA, suggesting that reductions in the brain concentrations of mIns might be due to the decreases in brain water levels that accompany changes in the osmoregulation of the brain. Further studies are needed to clarify the changes in the concentration of mIns that occur during the neonatal period.

A novel finding of the present study was the increase in the concentration of mIns observed in the cerebellum (Figure 2, p < 0.005) with more sample size (n = 23) and higher magnetic field (3T) than previous studies: Hüppi et al. [4] did not observe any difference in the concentration of mIns between preterm and term infants. It was reported that cerebellar volume increased by about four-fold from birth to 12 months, whereas that of the whole brain was about two-fold [24,25]. Thus, the increase in the mIns concentration observed in the cerebellum might be more closely associated with the late-onset of brain development than the changes seen in the supratentorial region.

Glu is an excitatory neurotransmitter, and Gln is involved in Glu detoxification, osmolyte synthesis, and ammonia detoxification [1,12,22,26]. Glx concentration increases have been detected during the neonatal period in previous studies [10,11]. As was suggested for tNAA and tCr, the large increase in the Glx concentration observed during the progression of PCA might have been associated with cerebral maturation.

Regarding the cerebellum, only 24 out of the 83 MR spectra were included in the analysis because the rest had low SNR and/or high %S.D. values, mainly due to the small volume of the cerebellum (about 26.9 cm³ even at full term) [27]. However, since our data plots (Figure 2) showed significant dynamic changes in the concentration of mIns with PCA, despite the small sample size, mIns might be useful for evaluating neonatal brain development.

None of the metabolites displayed concentration differences between the PT and FT (Table 1), which agrees with the findings of Hüppi et al. [6,7]. Therefore, for preterm infants metabolite concentrations might be useful indices of brain development, although other factors such as brain volume also need to be evaluated [28,29].

In this study, the Lac concentration was not analyzed quantitatively (for the reason, see Limitations); however, Lac peaks were frequently observed (Figure 1), as has been reported previously [9,10,12,13,30-32]. It was suggested that Lac might be produced in astrocytes and then taken up by neurons and metabolized into energy, which is known as the Lac shuttle [9,12,26,33,34]. Pellerin et al. [33] reported that the Lac shuttle is much more active in the immature brain, which could explain the higher lactate peaks detected by proton MRS in the immature normal brain.

Limitations