Introduction

With increased interests in the hippocampus as a potential regulator of the hypothalamic-pituitary-adrenocortical (HPA) axis, the final common pathway in the stress response, differentiate glucose metabolism between dorsal hippocampus and hypothalamus in the very same subject may lay foundation for investigating relevant evidence to the underlying mechanism how the hippocampal regulation of HPA sensitivity.
Although ¹³C Magnetic Resonance Spectroscopy (MRS) has shown to be a very promising tool to study brain metabolism in vivo. For instance, indirect carbon spectroscopy (¹H-[¹³C] MRS) is a technique that allows measuring ¹³C-labelled metabolites with higher sensitivity. Metabolic fluxes including TCA cycle in mouse hypothalamus were assessable using ¹H-[¹³C] MRS upon infusion of [1,6-¹³C₂] glucose at 14.1T. However, at high magnetic field, challenges are substantially amplified for multivoxel MRS in murine brains. For instance, first- and second-order shims become essential to improve local field homogeneity. Given recent studies showing feasibility of acquire ¹H MRS from two distinct brain regions in the very same subject using dynamic shim updates (DSU) ,¹ thus we aimed to demonstrate the feasibility of acquiring in vivo high-field ¹H-[¹³C] MR spectra² concurrently from the mouse hippocampus and hypothalamus at 14.1T on both shims without any supplementary hardware.

Methods

Animals
Male C57BL/6 mice (n=6, 8-12 weeks, 25±2g) were scanned under isoflurane anesthesia (1-2%) in a horizontal 14.1T/26cm Varian magnet (Agilent Inc., USA). A homemade ¹H surface coil in quadrature combined with a linear ¹³C coil was designed specific for ¹H-[¹³C] MRS. The bilateral dorsal hippocampi (2×6×1.5 mm³) and hypothalamus (2×2.7×2.2 mm³⁾ were localized using a set of T₂-weighted FSE images. Field inhomogeneity was adjusted using FASTMAP to reach a water linewidth <25Hz.³ On the target VOI, localized indirect ¹H-[¹³C] detection was applied using the full signal intensity BISEP-SPECIAL sequence (TE/TR=2.8/4000ms, 2) together with OVS and water suppression. Glucose metabolism of both dorsal hippocampus and hypothalamic was evaluated upon infusion of an intial bolus of 99% enriched and followed by 70% enriched 20% (w/v) [1,6-¹³C₂] glucose up to 4 hours. MR methods All experiments were performed in a horizontal 14.1T scanner, equipped with a 400mT/m gradient (200msec) interfaced to a DirectDrive (vnmrj, Agilent Inc.). 2nd order shim coils with maximum strengths of Z2 = 5.3 × 10−2mT/cm2, YZ = 1.2 × 10−1mT/cm2, XZ = 1.2 × 10−1mT/cm2, XY = 4.5 × 10−2mT/cm2 and X2Y2 = 4.2 × 10−2mT/cm2. A home-made quadrature surface coil (two geometrically decoupled 12mm-diameter loops) resonating 600MHz was used for radio-frequency transceiver. Anatomical MR images were acquired using fast-spin-echo images (TEeff/TE=50/4000ms, nt=8). The optimized spectral parameters, including excitation, water suppression, out volume suppression and the number of averages per scan (e.g. 16 averages per scan), for cortex and hippocampus were stored separately.
Dynamic Shim Update (DSU)
With the corresponding shims and the optimized spectral parameters, dynamic shim update (DSU) acquisition scheme (Figure 1) was applied on mouse hippocampus and hypothalamus, in which glutamatergic neurons are abundant in hippocampus. Data processing The unsuppressed water signals acquired from the same VOIs were used for quantification. Spectra were frequency corrected and summed (5.5min of hippocampus and 11.0min for hypothalamus) for LCModel quantification. Non-edited ¹H MR spectra contain 1H resonances coupled to both ¹²C and ¹³C and thus can be quantified with a standard basis set for neurochemical profiles of mouse hippocampus.2,3 The ¹³C-editing spectra were quantified using another simulated basis set as previously.2,3 The fraction of isotope enrichment (FE) in lactate (Lac, LacC₃), glutamate (Glu, GluC₄), glutamine (Gln, GlnC₄), the sum of Glu and Gln (Glx, GlxC₃) and γ-aminobutyric acid (GABA, GABAC₂ and GABAC₃) was obtained.

Results and Discussion

DSU both first- and second-order shims without any extra hardware is feasible for dual-voxel ¹H-[¹³C] spectroscopy at 14.1T, as shown in Figure 1. With the DSU, dynamic quality spectra were obtained in an interleaved fashion from two brain regions (Figure 2, 3&4). Sufficient FEs of labeled substrates can be reliably measured (Figure 5).Without any modeling, we showed that DSU of interleaved high-field spectral acquisition is feasible on ¹³C MRS. This study shows for the first time an in vivo comparison of ¹³C MRS of glucose in both hippocampus and hypothalamus of the very same mouse using indirect ¹H-[¹³C] MRS upon [1,6-C₂] ¹³C-glucose infusion.

Acknowledgements

This work was supported by the CIBM of the UNIL, UNIGE, HUG; CHUV, EPFL and Leenaards and Louis-Jeantet Foundations.