Hyperpolarized ¹³C metabolic imaging detects long-lasting metabolic alterations following mild repetitive traumatic brain injury

Joint Public Review:

Summary:

The authors present a metabolic imaging study of pyruvate metabolism in a mouse model of repetitive traumatic brain injury in the chronic recovery stage. They measure pyruvate metabolism with hyperpolarised 13C magnetic resonance spectroscopic imaging. This is acquired alongside semi-quantitative MR imaging metrics, a behavioural measure, and postmortem measures of relevant enzyme activity and expression of metabolic transporter proteins. They find that the MRSI-measured cortical lactate/pyruvate ratio (and signal from pyruvate and lactate independently) can differentiate the rTBI group from the sham group. They additionally find that postmortem, cortical pyruvate dehydrogenase activity is a statistically significant discriminator. All other metrics (MRI and enzyme/transporter measures) are not significantly different between groups. Finally, using a machine learning approach, the authors investigate the predictive power of combinations of all measures.

Strengths:

The primary strength of this work is the likely robustness of the primary finding - that hyperpolarised 13C lactate/pyruvate metabolite ratios are perturbed in this chronic rTBI model compared to the sham control.

Weaknesses:

Focal alterations in blood-brain-barrier permeability may affect the primary lactate/pyruvate measures. Whilst 13C urea measures perfusion, urea remains purely extracellular; whilst in the metabolism of the healthy brain, pyruvate must be transported through two levels of monocarboxylate transporters (MCTs) - in the endothelium surrounding the capillary bed and then into the parenchyma. By mechanically disrupting the brain, tight junctions in the BBB may be disrupted, therefore increasing the flux of pyruvate across the BBB and increasing pyruvate availability. In this case, lac/pyr would be a poor measure of metabolism as "delivery" has changed. While the authors assess perfusion using HP urea, it is unclear whether or how this metric would change in the presence of BBB disruption in relatively large and well-vascularised voxels.

The finding that "HP [1-13C]pyruvate levels were 1.05 fold higher" indicates that delivery of pyruvate might be increased. It is unclear if normalisation to the combined amplitude of lactate and pyruvate is fair in the case that the volume fraction in the voxel might have increased. Ideally, the authors would estimate polarisation separately as a normalisation.

No estimate of uncertainty is provided for the primary metabolic measures. Note that the lactate-pyruvate ratio is not normally distributed (see doi: 10.1002/mrm.26615), and this should be accounted for when carrying out statistical tests.

All metabolic maps are shown masked to the brain and interpolated to the structural MRI resolution (around 20 times). Nor is there any characterisation of the spectroscopic imaging's voxel volume, including the effect of the point spread function. It is, therefore, hard to have confidence in any spatial effects or potential partial volume effects from the tissue surrounding the brain.

The t2-weighted and SWI MRI measures used in this work are not quantitative. Normalisation in each case is carried out without regard to any spatially variable transmit and receive coil sensitivities (B1{plus minus}), which vary per subject. This adds intersubject variance, which could mask any effect between groups. No quality metrics (SNR or uncertainty estimates) are given for the MRI metrics.

Spectroscopic imaging was conducted 16 s after injection. Given the high heart rate of a mouse, measures of perfusion (using urea) could , therefore, be considered in a steady state, lowering sensitivity to any changes in perfusion or metabolite delivery. Furthermore, it is unclear how any changes in BBB permeability would manifest with the relatively low spatial resolution of MRSI. Would signal always be dominated by vascular compartments?

There is no apparent attempt to understand if an immune response occurs at this chronic time point. Macrophages are glycolytic and could affect the pyruvate measurement. Furthermore, is there any evidence for cellular changes in this model, namely density, cell type fraction, or microstructure? Are there any expected changes in glucose uptake?

There is no information or references provided for the accuracy or precision of the postmortem assays or their correlation with in vivo processes. What is the effect of cell density changes after injury on the assay kits?

The proposed interpretation of T1 as a measure of oxidative stress would seem to ignore the many confounding interpretations of T1.

Aims and impact:

In summary, the authors broadly achieve one aim, which is to find that HP 13C measured lac/pyruvate is a biomarker for the chronic effects of rTBI in a mouse model. As the authors themselves highlight in the discussion, the interpretation of this finding is tricky alongside their post-mortem assay results. The MR imaging in this work seems inconclusive, given the potential for inter-subject variance in the normalisation method.

The work, therefore, continues to highlight that HP 13C MRSI is a highly promising avenue of investigation to identify, characterise, and understand traumatic brain injury. It suggests that HP 13C MRSI is more promising in this sense than some standard MRI contrasts. The work currently fails to convincingly interpret the HP 13C MR results in conjunction with the other metrics.