Task activation results in regional ¹³C-lactate signal increase in the human brain

Abstract

Hyperpolarized-¹³C magnetic resonance imaging (HP-¹³C MRI) was used to image changes in ¹³C-lactate signal during a visual stimulus condition in comparison to an eyes-closed control condition. Whole-brain ¹³C-pyruvate, ¹³C-lactate and ¹³C-bicarbonate production was imaged in healthy volunteers (N = 6, ages 24–33) for the two conditions using two separate hyperpolarized ¹³C-pyruvate injections. BOLD-fMRI scans were used to delineate regions of functional activation. ¹³C-metabolite signal was normalized by ¹³C-metabolite signal from the brainstem and the percentage change in ¹³C-metabolite signal conditions was calculated. A one-way Wilcoxon signed-rank test showed a significant increase in ¹³C-lactate in regions of activation when compared to the remainder of the brain ( $p=0.02$ ). No significant increase was observed in ¹³C-pyruvate signal ( $p=0.11$ ) or ¹³C-bicarbonate signal ( $p=0.95$ ). The results show an increase in ¹³C-lactate production in activated regions that is measurable with HP-¹³C MRI.

Introduction

Hyperpolarized-¹³C magnetic resonance imaging (HP-¹³C MRI) is a minimally invasive technique that enables imaging of exogenously administered ¹³C-labelled metabolites and their downstream conversion products in vivo. The most commonly imaged metabolites are ¹³C-pyruvate and its downstream products ¹³C-lactate and ¹³C-bicarbonate. The production of ¹³C-lactate signal indicates the reduction of ¹³C-pyruvate to ¹³C-lactate in the cytosol, wheras ¹³C-bicarbonate production indicates the decarboxylation of ¹³C-pyruvate into acetyl-CoA on the mitochondrial membrane. HP-¹³C MRI has previously been used to image ¹³C-metabolite production in the human brain in healthy and disease states.^1–6

Previous results showed concordant regional patterns of ¹³C-pyruvate, ¹³C-lactate and ¹³C-bicarbonate across individuals suggesting a highly regulated process leading to the pattern. ⁴ At the same time, increased lactate concentration in the brain has been measured using proton spectroscopy and a visual stimulus.^7–9 However, the effect of a visual stimulus, and the concomitant increase in lactate pool size, on the ¹³C-metabolite pattern in the brain has not been documented previously. We hypothesized that the ¹³C-metabolite distribution detected with HP-¹³C MRI would be modulated in response to task activation.

In this study whole-brain HP-¹³C MRI was used to assess regional changes in ¹³C-lactate signal during a visual task stimulus. Blood-oxygen level dependent fMRI (BOLD-fMRI) images were acquired to delineate regions of BOLD activation in each participant. We hypothesized that the regions of activation defined by BOLD-fMRI would have higher ¹³C-lactate signal under stimulus conditions relative to control conditions.

Materials and methods

Written informed consent was obtained from six study participants (3 male, 3 female) and procedures were performed under a protocol approved by the Research Ethics Board of Sunnybrook Health Sciences Centre and by Health Canada under a Clinical Trial Application in compliance with the ICH-GCP and Declaration of Helsinki. The participants ranged in age from 24 to 33 (mean age of 26) and only those deemed cognitively healthy via the Montreal Cognitive Assessment ¹⁰ were scanned.

Several hours prior to scanning, two doses of ¹³C-pyruvic acid were prepared using a 1.47 g sample of [1-¹³C]pyruvic acid (Sigma Aldrich, St. Louis, MO) for each dose and polarized in a SPINLab polarizer system (GE Healthcare, Waukesha, WI) for a minimum of two hours. Immediately prior to scanning, a 20 gauge intravenous catheter was placed into the forearm of each participant before positioning them in the scanner bore.

Images were acquired using a GE MR750 3.0 T MRI scanner (GE Healthcare, Waukesha, WI) with a ¹³C birdcage head coil built in-house for HP-¹³C imaging. Volumetric images of ¹³C-lactate, ¹³C-bicarbonate, and ¹³C-pyruvate were acquired using a 3D echo-planar imaging (EPI) sequence. ¹¹ This sequence, applied extensively in previous brain imaging studies,^4–6,12 uses frequency-selective RF pulses to separately excite individual metabolites.^13,14 This “metabolite specific” imaging approach has the advantage that each RF excitation results in signal from a single metabolite (e.g. ¹³C-lactate), which can be imaged using conventional fast imaging methods such as the echo-planar encoding used here. The sequence of events during the imaging process are: first, a whole-brain 3D image of ¹³C-lactate is acquired with 15 mm isotropic spatial resolution in 1.2 seconds. Then, the RF pulse excitation frequency is changed to the frequency of ¹³C-bicarbonate and a 3D image of ${\hspace{0.17em}}^{13}$ C-bicarbonate is acquired, followed by ¹³C-pyruvate. This allows full brain coverage for all 3 ¹³C-metabolites within a 5 s block, which was repeated 12 times for a net 60 s imaging time. The net flip angle was 80° for ¹³C-lactate and ¹³C-bicarbonate and 11° for ¹³C-pyruvate. This metabolite-specific excitation and imaging approach has the disadvantage that no spectrum is acquired, just images of the pre-chosen resonances.

Each participant underwent two HP-¹³C scans, consisting of a task (¹³C-task) and control (¹³C-control) scan. The visual task consisted of an 8 Hz flashing checkerboard stimulus initiated 5 s before the ¹³C task scan. For the ¹³C-control scan, the participant was asked to close their eyes (and a blank screen with a plus sign was projected onto the screen). To control for any effect of scan ordering, three of the participants had the control scan first, whereas the other three had the control scan second. All stimuli and visual prompts were delivered via a projector and projector screen setup. The projector was placed inside the MRI console room facing into the MRI room, where a projector screen was located. Prior to imaging, the projector was lined up with the projector screen and we verified with the participant in the scanner that they were able to view a test image. There was a half-hour wait period between ¹³C-task and ¹³C-control scans.

During the half-hour wait period, the ¹³C head coil was interchanged with an 8-channel ¹H head coil (Invivo Inc, Pewaukee, WI) to perform anatomical T1-weighted and BOLD-fMRI scans. Anatomical T1-weighted images were acquired with an axial RF-spoiled gradient echo sequence (FOV $25.6\times 25.6\hspace{0.17em}\mathrm{cm}{\hspace{0.17em}}^2$ , 1 mm isotropic resolution, repetition time (TR) = 7.6 ms, echo time (TE) = 2.9 ms, flip angle 11°). BOLD-fMRI data were acquired using a single-shot EPI acquisition with 3.5 mm isotropic resolution, TR = 2000 ms, TE = 29 ms, 40° flip angle, $64\times 64$ matrix size and 38 3.5 mm slices with 0 mm gap. The task block duration was the same as the HP ${}^{13}$ C scan duration (one minute) and the total fMRI scan duration was 5 minutes, including 3 task blocks interleaved with 2 control blocks. The timing of the scan protocol is shown in Figure 1. To minimize head motion between ${}^{13}$ C and ${}^1$ H scans, foam was placed between the head rest and participants’ heads and participants were instructed to keep their head still and move only their eyes during the experiment (i.e. blinking, opening, and closing).

Figure 1.

Image acquisition workflow for the visual stimulus scans. Two sets of ¹³C-images were acquired with a thirty minute wait period between acquisitions to allow for washout of residual ¹³C-metabolites. During this wait period, anatomical images and BOLD-fMRI images were acquired.

The ${}^{13}$ C-metabolite images were summed across time to calculate the total ${}^{13}$ C-metabolite signal during the 1 minute acquisition window. All subsequent analysis was done using these summed-across-time images. The control and task ${}^{13}$ C-lactate images were registered (rigid six-body) to the anatomical T1-weighted images using FMRIB’s Linear Image Registration Tool from the FMRIB software library, ¹⁵ and the resulting transformation matrix was applied to the control and task ${}^{13}$ C-pyruvate and ${}^{13}$ C-bicarbonate images respectively. Finally, post-registered control and task ${}^{13}$ C-lactate and unprocessed BOLD-fMRI images were overlaid with the corresponding anatomical T1 images and visually inspected for misalignment. Additionally, the ${}^{13}$ C-lactate control and ${}^{13}$ C-task images were normalized by dividing by the brainstem signal for each participant/condition and then averaged across participants to create group-average ${}^{13}$ C-control and ${}^{13}$ C-task images.

The T1-weighted anatomical images were parcellated into 132 brain regions according to the BrainCOLOR labelling protocol ¹⁶ using an automated workflow. ¹⁷ These parcellation maps were then used to compute regional ${}^{13}$ C-pyruvate, ${}^{13}$ C-lactate, and ${}^{13}$ C-bicarbonate signals for each participant using the mri segstats function. ¹⁸ Regions with volumes below the voxel volume of 3.4 mL were excluded, resulting in 90 parcellated regions for each participant.

BOLD-fMRI pre- and post-processing was done using the Analysis of Functional Images (AFNI) toolbox,^19,20 and included removal of the first two TRs of each acquisition, registration across time to mitigate motion effects, and blurring with a Gaussian function (4 mm full-width half maximum). Voxelwise linear regression coefficients were fit using the stimulus block design as an independent variable, input as a repeating boxcar function with box length one minute. T-statistics were calculated for each positive regression coefficient using an alpha of 0.001. Cluster correction was used to control for multiple comparisons, with voxels in face-to-face contact comprising each cluster. ²¹

The BOLD-fMRI t-statistic map was used to define an an activation volume for each participant, with the remainder of the brain (including the white matter and ventricles) defined as the non-activation volume.

Each regional ¹³C-metabolite signal, as well as the signal from the activation volume and non-activation volume, were normalized by the ¹³C-metabolite signal from the brainstem region from the particular participant (referred to as ‘brainstem normalization’ below). This was done to normalize for any global signal scaling between scans, such as those caused by differing ¹³C-pyruvate polarization levels, while maintaining the inter-regional signal pattern.

The percentage change in ¹³C-metabolite signal between task and control conditions was calculated as follows:

$$\Delta \mathrm{\%}=\frac{S_{\mathit{task}}-S_{\mathit{control}}}{S_{\mathit{control}}}\times 100\mathrm{\%}$$

where $S_{\mathit{task}}$ is the brainstem-normalized ${}^{13}$ C-metabolite signal from a particular region/participant in the task condition and $S_{\mathit{control}}$ is the signal from the same metabolite/region/participant in the control condition (also brainstem normalized). To account for the paired data from each participant without a requirement for data normality, a one-way Wilcoxon signed-rank test was used to test for a difference in the $\Delta \%$ between the activation and non-activation volumes. The one-way Wilcoxon signed-rank test was also used to test for a difference between $S_{\mathit{task}}$ and $S_{\mathit{control}}$ in each individual region.

The thresholded t-statistic maps from the BOLD-fMRI scans were also used to designate parcellated brain regions as ‘activation regions’. The remaining parcellated brain regions were categorized as ‘non-activation regions’.

Finally, the strength of BOLD-fMRI activation (t-statistic) was tested for correlation with the task-induced change in ${}^{13}$ C-metabolite signal. An average t-statistic was calculated within the activation volume for each volunteer, as well as the $\Delta \%\mathrm{ }{}^{13}$ C-metabolite signal inside the same activation volume. A test for correlation was then done using the Spearman correlation test.

Results

BOLD-fMRI activation was consistently achieved in the visual cortex over the course of the 5 minute block design in all six participants. This confirmed that the stimulus block length was sufficient for inducing activation during the HP ${}^{13}$ C scans within the one minute acquisition time. Example t-statistic maps registered to the MNI152 atlas ²² are shown in Figure 2. Group average ${}^{13}$ C-task and control images are shown in Figure 3. No misalignments between registered ¹³C-metabolite images, t-statistic maps, or anatomical images were found upon visual inspection. The $\Delta \%$ for the ${}^{13}$ C-metabolites in the activation and non-activation volumes are plotted in Figure 4(a).

Figure 2.

Example BOLD fMRI t-statistic map for the visual stimulus registered to the MNI152 atlas. The inset is an example time series curve from a voxel with significant activation (yellow) and the stimulus regressor used in the general linear model (red).

Figure 3.

Group average ¹³C-lactate images overlaid on the MNI152 atlas for the control (left) and task (right) conditions. Increased ¹³C-lactate signal can be observed in the occipital lobe, predominantly in the left hemisphere. A sagittal slice (top) and coronal slice (bottom) are shown with the same color scale used for ¹³C-lactate signal. The A, P, R and L markers denote anterior, posterior, right and left.

Figure 4.

(a) The percentage change between task and control ¹³C-scans for the non-activated volume (left) and activated volume (right). The lines connect the data points from the same individual, the box centre line indicates the mean and whiskers at the 25th and 75th percentile and (b) the percentage change between task and control ¹³C-scans for each BrainCOLOR region. The BOLD-fMRI t-statistic maps were used to designate ‘activation volume regions’ and ‘non-activation volume regions’. An elevated $\Delta \%$ in ${\hspace{0.17em}}^{13}$ C-lactate is visible in the activation volume regions.

The one-way Wilcoxon signed-rank test showed a significant difference in $\Delta \%$ for ${}^{13}$ C-lactate signal between the activation and non-activation volumes ( $p=0.02$ , V = 21), indicating a task-induced increase in ${}^{13}$ C-lactate production in the activation volume relative to the rest of the brain. The Wilcoxon signed-rank tests for $\Delta \%{}^{13}$ C-bicarbonate and $\Delta \%\mathrm{ }{\hspace{0.17em}}^{13}$ C-pyruvate showed no significant difference between the activation and non-activation volumes ( $p=0.95$ , V = 3 and $p=0.11$ , V = 17 respectively). The magnitude of the V-statistic indicates the difference between groups. None of the Wilcoxon signed-rank tests on the individual regions showed a signficant difference between $S_{\mathit{control}}$ and $S_{\mathit{task}}$ for any of the ${}^{13}$ C-metabolites (after controlling for multiple testing), which was expected given the small sample size.

The parcellated regions within the activation volume for all six individuals were: the bilateral calcarine cortex, cuneus, inferior occipital gyrus, lingual gyrus, middle occipital gyrus, occipital pole, occipital fusiform gyrus, and superior occipital gyrus. These regions are highlighted in Figure 4(b), with the $\Delta \%\mathrm{ }{}^{13}$ C-lactate signal visibly higher in these regions in comparison to the non-activation volume regions.

The correlation between the degree of BOLD activation (mean t-statistic) and the percentage change in ¹³C-lactate within the activation volume is plotted in Figure 5. The correlation between ¹³C-lactate and the mean t-statistic within the activation volume was the strongest, at 0.77, though it did not reach significance (p = 0.051). All other correlations were not significant.

Figure 5.

Correlation between the level of BOLD activation (t-statistic) within the activation volume and the percentage ¹³C-lactate signal change between the task and control conditions.

Discussion

In this study hyperpolarized ${}^{13}$ C-MRI was used to measure the metabolic conversion of exogenously administered ${}^{13}$ C-pyruvate into ${}^{13}$ C-lactate and ¹³C-bicarbonate. Increased ${}^{13}$ C-lactate signal in visual cortex regions was observed during a visual stimulus, consistent with prior studies using proton spectroscopy.^8,23,24 These results demonstrate that task activation causes an increase in regional ${}^{13}$ C-lactate signal measurable with HP-¹³C MRI. There was also a strong correlation between the percent change in ¹³C-lactate and t-statistic within the activation volume, suggesting a monotonic relationship between the BOLD response and the production of ¹³C-lactate. Elevated ¹³C-lactate tended to occur in areas that showed BOLD activation consistently across participants. The activation volume for each participant was defined using the BOLD signal and a significant difference in ¹³C-lactate signal between the activation and non-activation non-activation volumes was found ( $p=0.02$ , V = 21). ${\hspace{0.17em}}^{13}$ C-metabolite signals were normalized by the signal from the brainstem, allowing for comparison of relative differences between conditions but not absolute quantification.

The increase in ¹³C-lactate signal may include ¹³C-lactate in the venous blood, in addition to parenchymal signal. However, there is evidence from the rodent brain that ${}^{13}$ C-lactate is largely parenchymal. ²⁵ Cerebral arteriovenous differences in lactate measured during stimulation have shown increased venous lactate consistent with local production, as opposed to arterial transport of lactate to regions of high energy demand.^26,27 Moreover glutamate, the most abundant excitatory neurotransmitter in the brain, has consistently been observed to increase under stimulus conditions when measured through fMRS. ²⁸ Glutamate is cleared from the intracellular space by astrocytes through a process that is bioenergetically supported by the non-oxidative metabolism of pyruvate into lactate. ²⁹ However, lactate can also be used to meet transient energy needs in neurons, ³⁰ and neuronal stimulus directly induces neuronal lactate production. ³¹

No significant differences between task and control conditions were detected for the ${}^{13}$ C-pyruvate signal, although an increasing trend can be observed in Figure 4(a). Increased cerebral blood volume due to functional activation would be expected to increase regional ${}^{13}$ C-pyruvate signal, especially as ${}^{13}$ C-pyruvate signal is largely in the vascular compartment. ²⁵ However, cerebral blood volume increases only slightly during functional activation, ³² so it is possible that the effect size was too small to discern a significant change in ${}^{13}$ C-pyruvate signal with the small sample size.

Surprisingly, a task-induced increase in ${}^{13}$ C-bicarbonate signal was not detected. A recent study by Zaidi et al. using HP-¹³C MR spectroscopy found increased ¹³C-bicarbonate signal in the occipital lobe during functional activation, attributed to increased pyruvate oxidation. ³³ The discrepancy between the two studies may be because ${}^{13}$ C-bicarbonate production is partly derived from oxidative metabolism of ${}^{13}$ C-lactate, as observed in prior studies. ³⁴ Due to the high effective flip angle used at the ${}^{13}$ C-lactate frequency in our study, ${}^{13}$ C-bicarbonate signal resulting from ¹³C-lactate would be suppressed. In the study by Zaidi et al. a reduced flip angle was used for the ${}^{13}$ C-lactate frequency, so ${}^{13}$ C-bicarbonate signal would not be suppressed. ³³ This phenomenon will be explored in future studies.

Conclusions

HP ${}^{13}$ C MRI was used to assess functional activation in a cohort of healthy volunteers. Increased ${}^{13}$ C-lactate signal in visual cortex regions, defined by BOLD-fMRI activation, was observed following a visual stimulus. Thus HP ${}^{13}$ C MRI may be useful for studying stimulus-induced changes in brain lactate metabolism, a distinct pathway implicated in both neuron and astrocyte bioenergetics. Future work will explore different data acquisition strategies to better understand the underlying physiological origins of the observed ¹³C-lactate signal.