Measurement of CMRO2 changes by somatosensory stimulation in rat using oxygen-17 at 16.4 T

Introduction The measurement of cerebral metabolic rate of oxygen (CMRO2) via direct NMR detection of the stable oxygen isotope O in water is a promising tool to study neuroenergetics, brain activity and pathology [1]. Due to the low natural abundance of H2O (0.037%) and the fast relaxation rates of the O nucleus, sequences with short acquisition delays and ultra-high field strengths are mandatory to determine CMRO2. In this study, we determined CMRO2 in rat somatosensory cortex during peripheral stimulation using inhalations of enriched O2 gas at 16.4T. Methods A 3-D chemical shift imaging sequence (CSI) with an acquisition delay of 538 μs was used for O MRSI. All acquisitions were performed on a 16.4 Tesla magnet (Magnex/Varian Inc.) with 26 cm bore diameter, maximal gradient-strength 1 T/m (Resonance Research Inc.) interfaced to an Avance III Paravision 5.0 (Bruker BioSpin) console. RF excitation and reception was performed by custom-built hetero-nuclear H/O silver wire surface-coils tuned at ω1H=698 MHz / ω17O=94.63 MHz. Animal preparation: 2 mechanically ventilated male Wistar rats (540g; 300g) anesthetized with i.v. infusion of alpha-chloralose at 50 mg/kg/h were fixated in a stereotaxic frame directly below the O surface coil. Exhaled gases were monitored continuously, arterial blood samples from the tail artery were taken 20 min before in-vivo acquisitions to adjust ventilation, and after the experiment. Body temperature was maintained at rectally measured 37.3±0.1 °C by an electric heat blanket. All procedures and experiments were approved by the local authorities (EUVD 86/609/EEC). Peripheral stimulation: subcutaneous electric stimulation of single forepaw using 300 μs pulse width, 4 mA at frequencies of 1, 3 and 6 Hz. Parameters for H EPI: FOV 1.5×1.5×0.7 cm3, matrix 64×64×7 (voxel-volume: 0.1 μl); TR 2s. fMRI task: 40s/60s ON/OFF stimulation paradigms. Parameters for O MRSI: A weighted 3-D CSI [4] sequence was used with a FOV 2.75×1.25×1.8 cm3; matrix 9×4×4 (voxel-volume: 43.1 μl); 200-μs RF hard pulse with Ernst angle 68°, spectral acquisition 375 points in 3.75 ms [5]; TR 4.92 ms, 6144 FIDs per volume (max. 74 averages in the center of k-space) with an overall duration of 30.2 s per measurement. 109 volumes were acquired while performing 15 min inhalations of enriched O2 gas (Ventilation mixture: 33% O2 in N2O (rat 1) or N2 (rat 2), enrichment fraction 50-70% O2, Nukem GmbH) for each determination of CMRO2 [2,3]. Five consecutive CMRO2 acquisitions with 3 stimulation and 2 baseline blocks were performed on each animal (Fig. 1c). Stimulation blocks consisted of continuous stimulation from shortly before inhalations until ~20 minutes after end of inhalations. Post-mortem high-resolution H2O (for coregistration with H-FLASH): Classic CSI (unweighted k-Space) FOV 2.75×1.61×2.5 cm3; matrix 41×24×25 (voxel-volume: 0.45 μl); TR 12 ms with an acquisition duration of 10 ms with 1000 spectral points; total acquired FIDs 3,148,800 with an overall duration of ~10 hours per volume. Data were reconstructed using MATLAB: Spatial zero-filling by a factor of 2 and line-broadening (120 Hz) were used for enhanced SNR and visualization of muscle vs. cortex in the following coregistrations of H2O and proton modality images (Fig. 1b). Coregistration was performed using FSL [6] and Freesurfer [7] by manual alignments based on FOV and contrast. All voxels selected for CMRO2 difference measurements were masked to be less than 1 cm distant from the coil and to have a significantly higher CMRO2 than the surrounding muscle tissue. Standard fMRI analysis was conducted on H-BOLD using FSL [6]. H-FLASH images at 59 μm in-plane resolution were used for final coregistration of proton BOLD activations and CMRO2 differences obtained from fitting the 3D-CSI. Results Estimated brain CMRO2 from H2O time courses using a three-phase model [2] was calculated as 1.42 and 1.8 μmol/g brain tissue/min. Baseline fluctuations in brain CMRO2 from systemic physiological fluctuations were below expected stimulation dependent ΔCMRO2 effects (Fig.1 a;rat 1: ±4%; rat 2: ±2% SD of baseline CMRO2). Peripheral somatosensory stimulation effects were detected in both H-BOLD and H2OCMRO2 modalities. (CMRO2 change in the stimulated S1: +5.4% in rat 1, +12.1% in rat 2). In one rat also the secondary somatosensoric cortex showed colocalized fMRI/CMRO2 activation (Fig. 1b). In dependence of the frequency of stimulation a highest ΔCMRO2 was observed in primary somatosensoric cortex (Fig. 1c) at a frequency of 1 Hz (+11.5% above baseline CMRO2) and decaying with higher frequencies (3 Hz: +8.8%; 6Hz: +5.1% ΔCMRO2). Established dependence on stimulation frequency [8] was found in H-fMRI of S1 (+1 Hz: 10%; 3Hz: +8%; 6 Hz: +4% BOLD).