Introduction

The cerebral metabolic rate of oxygen (CMRO₂) is an important metric for the evaluation of neurovascular and neurometabolic coupling. The degree of coupling between brain activity, CMRO₂, and other metabolic fluxes remains a matter of debate^1,2. In the rat, literature reported CMRO₂ increases of 15%³ to 200%² during bilateral forepaw activation. To revisit these early experiments, and possibly reconcile conflicting findings, more studies using non-invasive preclinical imaging tools with high spatial resolution are required. The current gold standard for CMRO₂ measurements is PET using ¹⁵O₂ as tracer. However, this technique suffers from an inherently low spatial resolution, precluding regional CMRO₂ mapping in rodents. As an alternative, in vivo MRI detection of ¹⁷O-labeling of brain water during inhalation of ¹⁷O₂ gas enables direct CMRO₂ measurements without any theoretical spatial resolution limits, provided that ¹⁷O MR detection sensitivity is sufficient (fig.1). In this work, we adapted our ¹⁷O zero echo time (ZTE) MRI protocol, previously developed for mice⁴, to measure CMRO₂ during neuronal activation in the rat primary somatosensory cortex at 11.7 T. To improve our measurements sensitivity, we summed data acquired over two consecutive cycles of ¹⁷O₂ inhalation-washout, and we show that a functional increase in CMRO₂ can be detected with good spatial resolution.

Methods

Animals&setup. All data were acquired in female Wistar rats (2-10 months, 240-330 g) under medetomidine anesthesia (0.6 mg.kg^-1.h^-1 intra-venous infusion) using a 11.7 T Bruker system and a ¹H volume coil (72 mm). Either a ¹⁷O custom-built Tx/Rx surface coil (10 mm) or a Bruker ¹H receive surface coil were placed on top of the head for CMRO₂ and BOLD fMRI acquisitions, respectively. Dual forelimb/hindlimb (HL/FL) stimulation was chosen to maximize the cortex activation volume (AM-system, 1.5 mA/limb; 50 µs pulse; 5 Hz) (fig.2). CMRO₂ measurements. Our protocol consisted of two consecutive ¹⁷O₂ inhalation-washout experiments. Series of ¹⁷O ZTE-MRI (TR=1.3 ms, voxel size=1.5 mm³, time resolution=18 s) were acquired before (5 to 10 min), during (4 min) and after (15 min) inhalation of ¹⁷O₂ gas (Nukem Isotopes, 70% enrichment) delivered at 100 mL.min^-1. Electrostimulation of the right (n=3) or the left (n=3) HL/FL paws was started 2 min before inhalation. ¹H-BOLD fMRI. In 4 rats out of 6, BOLD fMRI data were subsequently collected during the same electrical stimulation paradigm to locate the activated area (single-shot EPI, TR/TE: 1000/15 ms, 250 µm² in-plane, 750 µm slice thickness). In addition, to demonstrate the stability of neuronal activation over the duration of the experiment, BOLD fMRI was acquired in one rat during a 30 min stimulation (fig.2). Data analysis and CMRO₂ quantification. The two series of complex ZTE data acquired in each animal were summed and Hamming filter was applied. A 3-phase model⁵ was fitted to the data voxel-wise to map CMRO₂, K_G, and K_L rates (fig.3). K_G and K_L represent the rates of ¹⁷O labelled water gain and loss in the voxel, respectively, and are by definition proportional to cerebral blood flow. In addition, because the blood circulation time (Tc) can greatly affect CMRO₂ determination and depends on anesthesia, we measured Tc in a separate group of 5 rats in our conditions of anesthesia. Using confocal laser endo-microscopy, we detected the first and second passages of a bolus of fluorescent dextran (FITC) (fig.4). We found Tc=9.2±1.0 s and used this value for quantification. BOLD data were pre-processed with Gaussian spatial filtering. After block normalization and averaging, activated voxels were selected by thresholding (>contralateral signal+2*SD). All statistical group comparisons were computed with a simple paired t-test and statistical significance was set to α=0.05.

Results

BOLD signal was increased by up to 3% over a 28.5±3.35 mm³ volume in S1HL/FL, confirming neuronal activation in response to electrical stimulation. This large activated volume allowed the selection of 3 voxels per hemisphere on average on CMRO₂ maps. Our results (fig.3) showed that CMRO₂ was significantly higher (+7±5%, p=0.016) in the activated S1HL/FL (2.74±0.37 µmol.g^-1.min^-1) compared to the contralateral side (2.55±0.31 µmol.g^-1.min^-1). The changes in K_G and K_L with activation were even greater. K_G was 19±11% higher in the activated S1HL/FL (0.44±0.08 min^-1) compared to the contralateral side (0.37±0.04 min^-1, p=0.018), and K_L was 10±6% higher in the activated S1HL/FL (0.32±0.05 min^-1) compared to the contralateral side (0.29±0.03 min^-1, p=0.014).

Discussion

With this work, we demonstrate for the first time the feasibility of functional ¹⁷O-MRI based CMRO₂ measurements in the activated rat brain. Such experiments had previously only been carried out in cats, where a 30% increase was found in the visual cortex⁶. Here, combining dual HL/FL electrostimulation to maximize the activated cortex volume, and our strategy of summing two consecutive data sets improved our detection sensitivity. We found a 7% CMRO₂ increase and an average 15% increase in flow-related parameters (K_G and K_L). These results are in line with previous reports of a decoupling between the vascular response and the oxygen metabolism response to activation in the brain, and consistent with a strong BOLD effect.

Acknowledgements

The 11.7 T scanner and part of this project were funded by ‘NeurATRIS ANR-11-INBS-0011’, of the French Investissements d’Avenir Program run by the Agence Nationale pour la Recherche, and ‘ANR-21-CE44-0023’ collaborative research grant to CB.

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