Background

A series of metabolic processes are associated with neuronal activation, such as increases in blood flow, oxygen and glucose consumption, and oxidative phosphorylation. These processes are essential to meet increased energy needs for the required brain function. Pyruvate dehydrogenase (PDH), a mitochondrial enzyme that regulates pyruvate flux into the mitochondria and the subsequent tricarboxylic acid (TCA) cycle, is the main pathway for oxidative phosphorylation. Blood oxygen level-dependent (BOLD) fMRI detects neuronal activation, based on changes in magnetic susceptibility due to deoxygenation of hemoglobin, and has been the dominant methodology for studying brain activation. However, BOLD fMRI depends on signal changes that are several steps away from neuronal activation and thus, is susceptible to other physiological changes and artifacts, which can result in diluted signal sensitivity. In addition, the temporal resolution is limited due to the slow hemodynamic response. The advent of dissolution dynamic nuclear polarization (DNP) provides an opportunity to assess oxidative phosphorylation as the production of [¹³C]bicarbonate from administered hyperpolarized (HP) [1-¹³C]pyruvate is a direct biomarker of PDH activity. Recent translation of the technique in human brains demonstrated that [¹³C]bicarbonate is primarily produced in cerebral cortex (1-3), with the absence of pyruvate carboxylase-specific products suggesting that bicarbonate is primarily produced from neurons (3). We hypothesized that HP [¹³C]bicarbonate production in the brain reflects the metabolic changes associated with neuronal activation (Figure 1). In this study, we report bicarbonate production in response to visual stimulation in the visual cortex of healthy adults.

Methods

The imaging protocol was approved by the local Institutional Review Board (IRB#: STU-2018-0013). All studies were performed using a clinical SPINlab^TM polarizer (GE Healthcare) and a 3T MRI scanner (GE Healthcare, 750w Discovery). Three healthy volunteers (age: 22 – 45, one male and two female) were recruited. Each participant underwent a ¹H/¹³C-integrated MR protocol (~1hr), which includes ¹H BOLD fMRI, ¹H MPRAGE, and two injections of HP [1-¹³C]pyruvate (pyruvate concentration = 250 mM, dose = 0.1 mmol/kg body weight, injection rate = 5mL/s) with a 30-min interval between injections (Figure 2A). During the first 45 min, a dual-frequency RF head coil (Clinical MR Solutions, LLC), composed of ¹H quadrature Tx/Rx, ¹³C quadrature Tx and 8-channel ¹³C receive array, was used (3,4). The first HP pyruvate injection occurred after a fast GRE localizer and 2D T₂-weighted FLAIR scan. ¹³C spectra were acquired from a brain slice including the visual cortex using a time-resolved ¹³C MRS sequence (TR = 4 s, spectral width = 10,000 Hz, spectral points = 4096, scan time = 4 min, slice thickness = 3 cm). The center frequencies were set on the [1- ¹³C]pyruvate resonance. A spectral-spatial RF pulse that excites bicarbonate with 90°, lactate with 10°, and pyruvate with 5° (Figure 2C) to fully sample bicarbonate signals within each timepoint without perturbing its precursors. During the MRS acquisition, subjects were asked to focus on an alternating black screen with a cross in the center (20 s) and circular checkerboard stimuli (4 Hz, 20 s) (Figure 2B). The second HP injection was performed without the visual activation. Between the two HP pyruvate scans, a 2D ¹H BOLD fMRI with the visual stimulation and 3D ¹H MPRAGE images were acquired. The BOLD fMRI and MPRAGE scans were repeated using a 24-ch ¹H head coil (GE Healthcare). All clinical fluid paths were prepared in a sterile environment and the HP pyruvate solutions passed a quality control analysis prior to the injection, as previously described (3). ¹³C data was reconstructed in absorption mode with pyruvate, lactate, and bicarbonate quantified by integrating the peaks in each time point to display their time courses. The BOLD fMRI data were analyzed using Statistical Parametric Mapping software (SPM12; Functional Imaging Laboratory, University College London) and the resultant brain activation map (p < 0.05) overlaid on the MPRAGE images.

Results and Discussion

Due to the proximity to the visual cortex, ¹³C data from channels #2 and #3 of the ¹³C receive arrays were selectively combined and analyzed (Figure 3A). Participants #1 (23 y.o., male) and #3 (22 y.o., female) showed markedly increased bicarbonate production relative to the pyruvate in response to the stimuli whereas participant #2 (45 y.o., female) showed no change in bicarbonate production. Overall, bicarbonate signal intensity relative to pyruvate peak (Bic/Pyr) in time-averaged (0 – 90 s) ¹³C spectra were 22.1 ± 24.2 % higher with visual stimulation than without stimulation (Figure 3B). Increased bicarbonate production was detected during the first stimulation (20 – 40 s), probably due to the high signal sensitivity (Figure 3C). BOLD fMRI confirmed the regional activation of BOLD signal in the visual cortex (Figure 4).

Conclusion

In this study, we observed increased PDH activity of the human visual cortex in response to visual stimuli. We plan to expand the study to a larger number of subjects and develop analytical methods.

Acknowledgements

Personnel Support: We appreciate the clinical research team and the supporting staffs of the Advanced Imaging Research Center at UT Southwestern for recruiting and imaging the volunteers – Jeannie Baxter, R.N. and Kelley Derner, R.N.
Funding: National Institutes of Health of the United States (R01 NS107409, P41 EB015908, S10 OD018468, S10 RR029119); The Welch Foundation (I-2009-20190330).

References

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