Hyperpolarised MRI with Dynamic Nuclear Polarisation overcomes the fundamental thermodynamic limitations of conventional magnetic resonance, and is translating to human studies with several early-phase clinical trials in progress including early reports that demonstrate the utility of the technique to observe lactate production in human brain cancer patients. Owing to the fundamental coupling of metabolism and tissue function, metabolic neuroimaging with hyperpolarised [1-13C]pyruvate has the potential to be revolutionary in numerous neurological disorders (e.g. brain tumour, ischemic stroke, and multiple sclerosis). Through the use of [1-13C]pyruvate and ethyl-[1-13C]pyruvate in naïve brain, a rodent model of metastasis to the brain, or porcine brain subjected to mannitol osmotic shock, we show that pyruvate transport across the blood-brain barrier of anaesthetised animals is rate-limiting. We show through use of a well-characterised rat model of brain metastasis that the appearance of hyperpolarized [1-13C]lactate production corresponds to the point of blood-brain barrier breakdown in the disease. With the more lipophilic ethyl-[1-13C]pyruvate, we observe pyruvate production endogenously throughout the entire brain and lactate production only in the region of disease. In the in vivo porcine brain we show that mannitol shock permeabilises the blood-brain barrier sufficiently for a dramatic 90-fold increase in pyruvate transport and conversion to lactate in the brain, which is otherwise not resolvable. This suggests that earlier reports of whole-brain metabolism in anaesthetised animals may be confounded by partial volume effects and not informative enough for translational studies. Issues relating to pyruvate transport and partial volume effects must therefore be considered in pre-clinical studies investigating neuro-metabolism in anaesthetised animals, and we additionally note that these same techniques may provide a distinct biomarker of blood-brain barrier permeability in future studies.

Abnormal pH is a common feature of malignant tumours and has been associated clinically with sub-optimal outcomes. Amide proton transfer magnetic resonance imaging (APT MRI) holds promise as a means to non-invasively measure tumour pH, yet multiple factors collectively make quantification of tumour pH from APT MRI data challenging. The purpose of this study was to improve our understanding of the biophysical sources of altered APT MRI signals in tumours. Combining in vivo APT MRI measurements with ex vivo histological measurements of protein concentration in a rat model of brain metastasis, we determined that the proportion of APT MRI signal originating from changes in protein concentration was approximately 66%, with the remaining 34% originating from changes in tumour pH. In a mouse model of hypopharyngeal squamous cell carcinoma (FaDu), APT MRI showed that a reduction in tumour hypoxia was associated with a shift in tumour pH. The results of this study extend our understanding of APT MRI data and may enable the use of APT MRI to infer the pH of individual patients' tumours as either a biomarker for therapy stratification or as a measure of therapeutic response in clinical settings.

Cerebral blood flow is an important parameter in many diseases and functional studies that can be accurately measured in humans using arterial spin labelling (ASL) MRI. However, although rat models are frequently used for preclinical studies of both human disease and brain function, rat CBF measurements show poor consistency between studies. This lack of reproducibility is due, partly, to the smaller size and differing head geometry of rats compared to humans, as well as the differing analysis methodologies employed and higher field strengths used for preclinical MRI. To address these issues, we have implemented, optimised and validated a multiphase pseudo-continuous ASL technique, which overcomes many of the limitations of rat CBF measurement. Three rat strains (Wistar, Sprague Dawley and Berlin Druckrey IX) were used, and CBF values validated against gold-standard autoradiography measurements. Label positioning was found to be optimal at 45°, while post-label delay was optimised to 0.55 s. Whole brain CBF measures were 109 ± 22, 111 ± 18 and 100 ± 15 mL/100 g/min by multiphase pCASL, and 108 ± 12, 116 ± 14 and 122 ± 16 mL/100 g/min by autoradiography in Wistar, SD and BDIX cohorts, respectively. Tumour model analysis shows that the developed methods also apply in disease states. Thus, optimised multiphase pCASL provides robust, reproducible and non-invasive measurement of CBF in rats.

The purpose of this study was to develop realistic phantom models of the intracellular environment of metastatic breast tumour and naïve brain, and using these models determine an analysis metric for quantification of CEST MRI data that is sensitive to only labile proton exchange rate and concentration. The ability of the optimal metric to quantify pH differences in the phantoms was also evaluated.Novel phantom models were produced, by adding perchloric acid extracts of either metastatic mouse breast carcinoma cells or healthy mouse brain to bovine serum albumin. The phantom model was validated using 1H NMR spectroscopy, then utilized to determine the sensitivity of CEST MRI to changes in pH, labile proton concentration, T 1 time and T 2 time; six different CEST MRI analysis metrics (MTRasym, APT*, MTRRex, AREX and CESTR* with and without T 1/T 2 compensation) were compared.The new phantom models were highly representative of the in vivo intracellular environment of both tumour and brain tissue. Of the analysis methods compared, CESTR* with T 1 and T 2 time compensation was optimally specific to changes in the CEST effect (i.e. minimal contamination from T 1 or T 2 variation). In phantoms with identical protein concentrations, pH differences between phantoms could be quantified with a mean accuracy of 0.6 pH units.We propose that CESTR* with T 1 and T 2 time compensation is the optimal analysis method for these phantoms. Analysis of CEST MRI data with T 1/T 2 time compensated CESTR* is reproducible between phantoms, and its application in vivo may resolve the intracellular alkalosis associated with breast cancer brain metastases without the need for exogenous contrast agents.

To assess the correlation and differences between common amide proton transfer (APT) quantification methods in the diagnosis of ischemic stroke. Methods: Five APT quantification methods, including asymmetry analysis and its variants as well as two Lorentzian model-based methods, were applied to data acquired from six rats that underwent middle cerebral artery occlusion scanned at 9.4T. Diffusion and perfusion-weighted images, and water relaxation time maps were also acquired to study the relationship of these conventional imaging modalities with the different APT quantification methods. Results: The APT ischemic area estimates had varying sizes (Jaccard index: 0.544 ≤ J ≤ 0.971) and had varying correlations in their distributions (Pearson correlation coefficient: 0.104 ≤ r ≤ 0.995), revealing discrepancies in the quantified ischemic areas. The Lorentzian methods produced the highest contrast-to-noise ratios (CNRs; 1.427 ≤ CNR ≤ 2.002), but generated APT ischemic areas that were comparable in size to the cerebral blood flow (CBF) deficit areas; asymmetry analysis and its variants produced APT ischemic areas that were smaller than the CBF deficit areas but larger than the apparent diffusion coefficient deficit areas, though having lower CNRs (0.561 ≤ CNR ≤ 1.083). Conclusion: There is a need to further investigate the accuracy and correlation of each quantification method with the pathophysiology using a larger scale | 2189 FOO et al.

Purpose: Chemical exchange saturation transfer (CEST) is an MRI technique sensitive to the presence of low-concentration solute protons exchanging with water. However, magnetization transfer (MT) effects also arise when large semisolid molecules interact with water, which biases CEST parameter estimates if quantitative models do not account for macromolecular effects. This study establishes under what conditions this bias is significant and demonstrates how using an appropriate model provides more accurate quantitative CEST measurements. Methods: CEST and MT data were acquired in phantoms containing bovine serum albumin and agarose. Several quantitative CEST and MT models were used with the phantom data to demonstrate how underfitting can influence estimates of the CEST effect. CEST and MT data were acquired in healthy volunteers, and a twopool model was fit in vivo and in vitro, whereas removing increasing amounts of CEST data to show biases in the CEST analysis also corrupts MT parameter estimates. Results: When all significant CEST/MT effects were included, the derived parameter estimates for each CEST/MT pool significantly correlated (P < .05) with bovine serum albumin/agarose concentration; minimal or negative correlations were found with underfitted data. Additionally, a bootstrap analysis demonstrated that significant biases occur in MT parameter estimates (P < .001) when unmodeled CEST data are included in the analysis. Conclusions: These results indicate that current practices of simultaneously fitting both CEST and MT effects in model-based analyses can lead to significant bias in all 1360 | SMITH eT al. | THEORYSimilar to the analytical solution for a two-pool model described in the various papers by Yarnykh et al 36,37 and Zhou et al,4,38 we parameter estimates unless a sufficiently detailed model is utilized. Therefore, care must be taken when quantifying CEST and MT effects in vivo by properly modeling data to minimize these biases. K E Y W O R D S amide, CEST, MT, NOE, qMT, quantitative 1366 | SMITH eT al. F I G U R E 3 Semisolid (PSR), and amide and NOE (MTR*) maps for the variable agarose phantom experiment. The top row illustrates the full, four-pool model estimation, the middle displays the fixed-qMT estimation, and the bottom row shows the same fit assuming the NOE and MT pools are a single pool, similar to Tee et al 34 F I G U R E 4 Semisolid (PSR), and amide and NOE (MTR*) maps for the variable BSA phantom experiment. The top row illustrates the full, four-pool model estimation, the middle displays the fixed-qMT estimation, and the bottom row shows the same fit assuming the NOE and MT pools are a single pool, similar to similar to Tee et al 34

In chemical exchange saturation transfer imaging, saturation effects between −2 to −5 ppm (nuclear Overhauser effects, NOEs) have been shown to exhibit contrast in preclinical stroke models. Our previous work on NOEs in human stroke used an analysis model that combined NOEs and semisolid MT; however their combination might feasibly have reduced sensitivity to changes in NOEs. The aim of this study was to explore the information a 4-pool Bloch-McConnell model provides about the NOE contribution in ischemic stroke, contrasting that with an intentionally approximate 3-pool model. Methods: MRI data from 12 patients presenting with ischemic stroke were retrospectively analyzed, as well as from six animals induced with an ischemic lesion. Two Bloch-McConnell models (4 pools, and a 3-pool approximation)were compared for their ability to distinguish pathological tissue in acute stroke. The association of NOEs with pH was also explored, using pH phantoms that mimic the intracellular environment of naïve mouse brain. Results:The 4-pool measure of NOEs exhibited a different association with tissue outcome compared to 3-pool approximation in the ischemic core and in tissue that underwent delayed infarction. In the ischemic core, the 4-pool measure was elevated in patient white matter (1.20 ± 0.20) and in animals (1.27 ± 0.20). In the naïve brain pH phantoms, significant positive correlation between the NOE and pH was observed. Conclusion:Associations of NOEs with tissue pathology were found using the 4-pool metric that were not observed using the 3-pool approximation. The 4-pool model more adequately captured in vivo changes in NOEs and revealed trends depending on tissue pathology in stroke.

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