Computational protocol: In Vivo Multimodal Magnetic Resonance Imaging Changes After N-Methyl-d-Aspartate-Triggered Spasms in Infant Rats

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Protocol publication

[…] Animals were maintained under anesthesia with 1% isoflurane in a 1:2 mixture of O2:N2O with monitoring of their respiratory rate, electrocardiogram, and rectal temperature. GluCEST MR imaging was performed using a 7.0 T/160-mm small-animal imaging system (Bruker Pharmascan, Ettlingen, Germany) with a single-channel surface coil. Images were obtained using a 9.4 T/160-mm bore animal MRI system (Agilent Technologies, Santa Clara, CA, USA) for 1H-MRS and diffusion tensor imaging. Radiofrequency excitation and signal detection were accomplished with a 72-mm quadrature volume coil and a two-channel phased-array coil, respectively. Axial slices corresponding to coronal images in the neuroanatomic axis were collected from the cervical spinal cord to the olfactory bulb.GluCEST images were acquired from an axial slice (1-mm thick) that included the hippocampal region. GluCEST images were acquired using T2-weighted imaging (rapid acquisition with relaxation enhancement [RARE]) with a frequency selective saturation preparation pulse comprised a Gaussian pulse with a total duration of 1,000 ms (irradiation offset of 500.0 Hz and interpulse delay of 10 µs) at a B1 peak of 5.6 µT. Z-spectra were obtained from −5.0 ppm to +5.0 ppm with intervals of 0.33 ppm (total, 31 images, Figure S1 in Supplementary Material). The sequence parameters were as follows: repetition time/echo time (TR/TE) = 4,200/36.4 ms, field of view = 30 mm × 30 mm, slice thickness = 1 mm, matrix size = 96 × 96, RARE factor = 16, echo spacing = 6.066 ms, and average = 1. To confirm the linear GluCEST effect, a phantom consisting of test tubes with different concentrations of glutamate (pH 7.0) images at 7 T was also done (Figure S2 in Supplementary Material).To measure the GluCEST value (%), each region of interest (cortex, hippocampus, striatum) was drawn manually on the T2-weighted anatomical MR images without a frequency selective saturation preparation pulse, and the regions of interest were overlaid on the GluCEST maps. GluCEST contrast is measured as the asymmetry between an image obtained with saturation at the resonant frequency of exchangeable amine protons (+3 ppm downfield from water for glutamate) and an image with saturation equidistant upfield from water (–3 ppm), according to the following equation: GluCEST(%)=S−3.0 ppm−S+3.0 ppmS−3.0 ppm*100 where S−3.0ppm and S+3.0ppm are the magnetizations obtained with saturation at a specified offset from the water resonance of 4.7 ppm.The B0/B1 maps on the same slices were acquired for B0 and B1 correction. The B0 map was calculated by linearly fitting the accumulated phase per pixel following phase unwrapping against the echo time differences from gradient echo images collected at TEs of =1.9 and 2.6 ms. B1 maps were calculated by using the double-angle method (flip angles 30° and 60°) and the linear correction for B1 was calculated as the ratio of the actual B1 to the expected value.1H-MRI/MRS images were obtained at the assigned times as follows (Figure ): (1) 1 day after the last cluster of spasms (P16), (2) about 1 week after the spasms (P23), and (3) about 2 weeks after the spasms (P30). The MR spectra were acquired through a signal voxel (from bregma to −3.0 mm in a coronal section, 3 mm × 2 mm × 1.5 mm) in the cingulate cortex using a point-resolved spectroscopy (PRESS) sequence for 128 acquisitions with TR/TE = 5,000/13.4 ms. For quantification, unsuppressed water signals were also acquired from the same voxel (average = 8). All the MR spectra were processed with the linear combination analysis method (LC Model ver. 6.0, Los Angeles, CA, USA) to calculate the metabolite concentrations from a fit to the experimental spectrum, based on a simulated basis set. The following brain metabolites were included in the metabolite basis set: alanine (Ala), aspartate (Asp), creatine (Cr), γ-aminobutyric acid (GABA), glucose (Glc), glutamate (Glu), glutamine (Gln), glycerophosphorylcholine (GPC), phosphorylcholine (PCh), myo-inositol (mIns), lactate (Lac), phosphocreatine (PCr), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), taurine (Tau), macromolecules (MMs), and lipids (Lip). The water-suppressed time domain data were analyzed between 0.2 and 4.0 ppm, without further T1 or T2 correction. Absolute metabolite concentrations (mmol/kg wet weight) were calculated using the unsuppressed water signal as an internal reference (assuming 80% brain water content) (). The in vivo proton spectra were judged to have an acceptable value if the standard deviation of the fit for the metabolite was less than 20% (Cramer–Rao lower bounds). MR diffusion tensor images were acquired using a four-shot DT-echo planar imaging sequence (TR = 3.7 s, TE = 20 ms, B0 = 1,000 s/mm2) with a 10-ms interval (Δ) between the application of diffusion gradient pulses, a 4-ms diffusion gradient duration (δ), a gradient amplitude (G) of 46.52 mT/m, and the Jones 30 gradient scheme.Postprocessing analysis was performed using Diffusion Toolkit software ( The cingulate cortex of each rat was selected and the fractional anisotropy (FA) and mean diffusivity (MD) were calculated from the diffusion tensor parametric maps. Subsequently, repeated measures ANOVA and t-tests were conducted to test for the treatment effect of the different diffusion parameters. […]

Pipeline specifications

Software tools DTK, TrackVis
Applications Magnetic resonance imaging, Diffusion magnetic resonance imaging analysis
Organisms Homo sapiens, Rattus norvegicus
Diseases Epilepsy, Lymphoma, Non-Hodgkin, Spasms, Infantile
Chemicals Betamethasone, Choline, Creatine, gamma-Aminobutyric Acid, Taurine, N-Methylaspartate, Glutamic Acid