Universal dynamic fitting of magnetic resonance spectroscopy.

PURPOSE: Dynamic (2D) MRS is a collection of techniques where acquisitions of spectra are repeated under varying experimental or physiological conditions. Dynamic MRS comprises a rich set of contrasts, including diffusion-weighted, relaxation-weighted, functional, edited, or hyperpolarized spectroscopy, leading to quantitative insights into multiple physiological or microstructural processes. Conventional approaches to dynamic MRS analysis ignore the shared information between spectra, and instead proceed by independently fitting noisy individual spectra before modeling temporal changes in the parameters. Here, we propose a universal dynamic MRS toolbox which allows simultaneous fitting of dynamic spectra of arbitrary type. METHODS: A simple user-interface allows information to be shared and precisely modeled across spectra to make inferences on both spectral and dynamic processes. We demonstrate and thoroughly evaluate our approach in three types of dynamic MRS techniques. Simulations of functional and edited MRS are used to demonstrate the advantages of dynamic fitting. RESULTS: Analysis of synthetic functional 1H-MRS data shows a marked decrease in parameter uncertainty as predicted by prior work. Analysis with our tool replicates the results of two previously published studies using the original in vivo functional and diffusion-weighted data. Finally, joint spectral fitting with diffusion orientation models is demonstrated in synthetic data. CONCLUSION: A toolbox for generalized and universal fitting of dynamic, interrelated MR spectra has been released and validated. The toolbox is shared as a fully open-source software with comprehensive documentation, example data, and tutorials.

Diffusion-weighted MR spectroscopy: Consensus, recommendations, and resources from acquisition to modeling.

Brain cell structure and function reflect neurodevelopment, plasticity, and aging; and changes can help flag pathological processes such as neurodegeneration and neuroinflammation. Accurate and quantitative methods to noninvasively disentangle cellular structural features are needed and are a substantial focus of brain research. Diffusion-weighted MRS (dMRS) gives access to diffusion properties of endogenous intracellular brain metabolites that are preferentially located inside specific brain cell populations. Despite its great potential, dMRS remains a challenging technique on all levels: from the data acquisition to the analysis, quantification, modeling, and interpretation of results. These challenges were the motivation behind the organization of the Lorentz Center workshop on "Best Practices & Tools for Diffusion MR Spectroscopy" held in Leiden, the Netherlands, in September 2021. During the workshop, the dMRS community established a set of recommendations to execute robust dMRS studies. This paper provides a description of the steps needed for acquiring, processing, fitting, and modeling dMRS data, and provides links to useful resources.

PDZD8 Disruption Causes Cognitive Impairment in Humans, Mice, and Fruit Flies.

BACKGROUND: The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability. METHODS: Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene's function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing. RESULTS: Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum-anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits. CONCLUSIONS: These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.

High temporal resolution functional magnetic resonance spectroscopy in the mouse upon visual stimulation.

Functional magnetic resonance spectroscopy (fMRS) quantifies metabolic variations upon presentation of a stimulus and can therefore provide complementary information compared to activity inferred from functional magnetic resonance imaging (fMRI). Improving the temporal resolution of fMRS can be beneficial to clinical applications where detailed information on metabolism can assist the characterization of brain function in healthy and sick populations as well as for neuroscience applications where information on the nature of the underlying activity could be potentially gained. Furthermore, fMRS with higher temporal resolution could benefit basic studies on animal models of disease and for investigating brain function in general. However, to date, fMRS has been limited to sustained periods of activation which risk adaptation and other undesirable effects. Here, we performed fMRS experiments in the mouse with high temporal resolution (12 s), and show the feasibility of such an approach for reliably quantifying metabolic variations upon activation. We detected metabolic variations in the superior colliculus of mice subjected to visual stimulation delivered in a block paradigm at 9.4 T. A robust modulation of glutamate is observed on the average time course, on the difference spectra and on the concentration distributions during active and recovery periods. A general linear model is used for the statistical analysis, and for exploring the nature of the modulation. Changes in NAAG, PCr and Cr levels were also detected. A control experiment with no stimulation reveals potential metabolic signal "drifts" that are not correlated with the functional activity, which should be taken into account when analyzing fMRS data in general. Our findings are promising for future applications of fMRS.

Inflammation-driven glial alterations in the cuprizone mouse model probed with diffusion-weighted magnetic resonance spectroscopy at 11.7 T.

Inflammation of brain tissue is a complex response of the immune system to the presence of toxic compounds or to cell injury, leading to a cascade of pathological processes that include glial cell activation. Noninvasive MRI markers of glial reactivity would be very useful for in vivo detection and monitoring of inflammation processes in the brain, as well as for evaluating the efficacy of personalized treatments. Due to their specific location in glial cells, myo-inositol (mIns) and choline compounds (tCho) seem to be the best candidates for probing glial-specific intra-cellular compartments. However, their concentrations quantified using conventional proton MRS are not specific for inflammation. In contrast, it has been recently suggested that mIns intra-cellular diffusion, measured using diffusion-weighted MRS (DW-MRS) in a mouse model of reactive astrocytes, could be a specific marker of astrocytic hypertrophy. In order to evaluate the specificity of both mIns and tCho diffusion to inflammation-driven glial alterations, we performed DW-MRS in a volume of interest containing the corpus callosum and surrounding tissue of cuprizone-fed mice after 6 weeks of intoxication, and evaluated the extent of astrocytic and microglial alterations using immunohistochemistry. Both mIns and tCho apparent diffusion coefficients were significantly elevated in cuprizone-fed mice compared with control mice, and histologic evaluation confirmed the presence of severe inflammation. Additionally, mIns and tCho diffusion showed, respectively, strong and moderate correlations with histological measures of astrocytic and microglial area fractions, confirming DW-MRS as a promising tool for specific detection of glial changes under pathological conditions.

Short echo time relaxation-enhanced MR spectroscopy reveals broad downfield resonances.

PURPOSE: Most MR spectroscopy (MRS) pulse sequences rely on broadband excitation with water saturation and typically focus on upfield signals. By contrast, the downfield spectrum, which contains many potentially useful resonances, is typically not targeted because conventional water-suppressed techniques indirectly saturate the labile protons through exchange. Relaxation-enhanced MRS (RE-MRS) uses frequency-selective excitation while actively avoiding bulk water perturbation, thereby enabling high-quality downfield spectroscopy. However, RE-MRS typically requires very long (typically >40 ms) echo times (TEs) due to its localization module, which inevitably decreases sensitivity and filters shorter T2 components. Here, we overcome this limitation by combining RE-MRS and image selected in vivo spectroscopy (ISIS) localization, abbreviated iRE-MRS, which in turn allows very short TEs (5 ms using our hardware). METHODS: Experiments were performed in vitro for validation as well as and in in vivo rat brains at 9.4T. RESULTS: The new iRE-MRS methodology was validated in phantoms where good performance was noted. When the downfield spectrum was investigated at short TEs in in vivo rat brains, iRE-MRS provided very high sensitivity; the ensuing downfield spectra encompassed numerous broad peaks, as well as a broad baseline. All downfield spectral peaks were highly attenuated by increasing TEs as well as by applying water saturation, although to different extent. The signal ratios also varied between TEs, suggesting that exchange rates are different among the downfield signals. CONCLUSIONS: Short-TE iRE 1 H downfield MRS opens new directions in the investigation of in vivo downfield metabolites and their role on healthy and disease processes.

Diffusion-weighted magnetic resonance spectroscopy enables cell-specific monitoring of astrocyte reactivity in vivo.

Reactive astrocytes exhibit hypertrophic morphology and altered metabolism. Deciphering astrocytic status would be of great importance to understand their role and dysregulation in pathologies, but most analytical methods remain highly invasive or destructive. The diffusion of brain metabolites, as non-invasively measured using diffusion-weighted magnetic resonance spectroscopy (DW-MRS) in vivo, depends on the structure of their micro-environment. Here we perform advanced DW-MRS in a mouse model of reactive astrocytes to determine how cellular compartments confining metabolite diffusion are changing. This reveals myo-inositol as a specific intra-astrocytic marker whose diffusion closely reflects astrocytic morphology, enabling non-invasive detection of astrocyte hypertrophy (subsequently confirmed by confocal microscopy ex vivo). Furthermore, we measure massive variations of lactate diffusion properties, suggesting that intracellular lactate is predominantly astrocytic under control conditions, but predominantly neuronal in case of astrocyte reactivity. This indicates massive remodeling of lactate metabolism, as lactate compartmentation is tightly linked to the astrocyte-to-neuron lactate shuttle mechanism.

Brain Metabolite Diffusion from Ultra-Short to Ultra-Long Time Scales: What Do We Learn, Where Should We Go?

In vivo diffusion-weighted MR spectroscopy (DW-MRS) allows measuring diffusion properties of brain metabolites. Unlike water, most metabolites are confined within cells. Hence, their diffusion is expected to purely reflect intracellular properties, opening unique possibilities to use metabolites as specific probes to explore cellular organization and structure. However, interpretation and modeling of DW-MRS, and more generally of intracellular diffusion, remains difficult. In this perspective paper, we will focus on the study of the time-dependency of brain metabolite apparent diffusion coefficient (ADC). We will see how measuring ADC over several orders of magnitude of diffusion times, from less than 1 ms to more than 1 s, allows clarifying our understanding of brain metabolite diffusion, by firmly establishing that metabolites are neither massively transported by active mechanisms nor massively confined in subcellular compartments or cell bodies. Metabolites appear to be instead diffusing in long fibers typical of neurons and glial cells such as astrocytes. Furthermore, we will evoke modeling of ADC time-dependency to evaluate the effect of, and possibly quantify, some structural parameters at various spatial scales, departing from a simple model of hollow cylinders and introducing additional complexity, either short-ranged (such as dendritic spines) or long-ranged (such as cellular fibers ramification). Finally, we will discuss the experimental feasibility and expected benefits of extending the range of diffusion times toward even shorter and longer values.

Can we detect the effect of spines and leaflets on the diffusion of brain intracellular metabolites?

Prior models used to clarify which aspects of tissue microstructure mostly affect intracellular diffusion and corresponding diffusion-weighted magnetic resonance (DW-MR) signal have focused on relatively simple geometrical descriptions of the cellular microenvironment (spheres, randomly oriented cylinders, etc…), neglecting finer morphological details which may have an important role. Some types of neurons present high density of spines; and astrocytes and macroglial cells processes present leaflets, which may all impact the diffusion process. Here, we use Monte-Carlo simulations of many particles diffusing in cylindrical compartments with secondary structures mimicking spines and leaflets of neuronal and glial cell fibers, to investigate to what extent the diffusion-weighted signal of intracellular molecules is sensitive to spines/leaflets density and length. In order to study the specificity of DW-MR signal to these kinds of secondary structures, beading-like geometry is simulated as "control" deviation from smooth cylinder too. Results suggest that: a) the estimated intracellular tortuosity increases as spines/leaflets density or length (beading amplitude) increase; b) the tortuosity limit is reached for diffusion time td>200 ms for metabolites and td>70 ms for water molecules, suggesting that the effects of these finer morphological details are negligible at td longer than these threshold values; c) fiber diameter is overestimated, while intracellular diffusivity is underestimated, when simple geometrical models based on hollow smooth cylinders are used; d) apparent surface-to-volume, S/V, ratio estimated by linear fit of high frequency OG data appears to be an excellent estimation of the actual S/V ratio, even in the presence of secondary structures, and it increases as spines and leaflets density or length increase (while decreasing as beadings amplitude increases). Comparison between numerical simulations and multimodal metabolites DW-MRS experiments in vivo in mouse brain shows that these fine structures may affect the DW-MRS signal and the derived diffusion metrics consistently with their expected density and geometrical features. This work suggests that finer structures of cell morphology have non-negligible effects on intracellular molecules' diffusion that may be measured by using multimodal DW-MRS approaches, stimulating future developments and applications.

Metabolite diffusion up to very high b in the mouse brain in vivo: Revisiting the potential correlation between relaxation and diffusion properties.

PURPOSE: To assess the potential correlation between metabolites diffusion and relaxation in the mouse brain, which is of importance for interpreting and modeling metabolite diffusion based on pure geometry, irrespective of relaxation properties (multicompartmental relaxation or surface relaxivity). METHODS: A new diffusion-weighted magnetic resonance spectroscopy sequence is introduced, dubbed "STE-LASER," which presents several nice properties, in particular the absence of cross-terms with selection gradients and a very clean localization. Metabolite diffusion is then measured in a large voxel in the mouse brain at 11.7 Tesla using a cryoprobe, resulting in excellent signal-to-noise ratio, up to very high b-values under different echo time, mixing time, and diffusion time combinations. RESULTS: Our results suggest that the correlation between relaxation and diffusion properties is extremely small or even nonexistent for metabolites in the mouse brain. CONCLUSION: The present work strongly supports the interpretation and modeling of metabolite diffusion primarily based on geometry, irrespective of relaxation properties, at least under current experimental conditions. Magn Reson Med 77:1390-1398, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Modeling diffusion of intracellular metabolites in the mouse brain up to very high diffusion-weighting: Diffusion in long fibers (almost) accounts for non-monoexponential attenuation.

PURPOSE: To investigate how intracellular metabolites diffusion measured in vivo up to very high q/b in the mouse brain can be explained in terms of simple geometries. METHODS: 10 mice were scanned using our new STE-LASER sequence, at 11.7 Tesla (T), up to qmax = 1 µm-1 at diffusion time td = 63.2 ms, corresponding to bmax = 60 ms/µm². We model cell fibers as randomly oriented cylinders, with radius a and intracellular diffusivity Dintracyl, and fit experimental data as a function of q to estimate Dintracyl and a. RESULTS: Randomly oriented cylinders account well for measured attenuation, giving fiber radii and Dintracyl in the expected ranges (0.5-1.5 µm and 0.30-0.45 µm2/ms, respectively). The only exception is N-acetyl-aspartate (NAA) (extracted a∼0), which we show to be compatible with a small fraction of the NAA pool being confined in highly restricted compartments (with short T2). CONCLUSION: The non-monoexponential signal attenuation of intracellular metabolites in the mouse brain can be described by diffusion in long and thin cylinders, yielding realistic Dintra and fiber diameters. However, this simple model may require small "corrections" for NAA, in the form of a small fraction of the NAA signal originating from a highly restricted compartment. Magn Reson Med, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Probing metabolite diffusion at ultra-short time scales in the mouse brain using optimized oscillating gradients and "short"-echo-time diffusion-weighted MRS.

Measuring diffusion at ultra-short time scales may yield information about short-range intracellular structure and cytosol viscosity. However, reaching such time scales usually requires oscillating gradients, which in turn imply long echo times TE . Here we propose a new kind of stretched oscillating gradient that allows us to increase diffusion-weighting b while preserving spectral and temporal properties of the gradient modulation. We used these optimized gradients to measure metabolite diffusion in the mouse brain down to effective diffusion times of 1 ms while keeping TE relatively short (60 ms). At such TE , a significant macromolecule signal could still be observed and used as an internal reference of approximately null diffusivity, which proved critical to discard datasets corrupted by some motion artifact. The methods introduced here may be useful to improve the accuracy and precision of metabolite apparent diffusion coefficient measurements with oscillating gradients.

New paradigm to assess brain cell morphology by diffusion-weighted MR spectroscopy in vivo.

The brain is one of the most complex organs, and tools are lacking to assess its cellular morphology in vivo. Here we combine original diffusion-weighted magnetic resonance (MR) spectroscopy acquisition and novel modeling strategies to explore the possibility of quantifying brain cell morphology noninvasively. First, the diffusion of cell-specific metabolites is measured at ultra-long diffusion times in the rodent and primate brain in vivo to observe how cell long-range morphology constrains metabolite diffusion. Massive simulations of particles diffusing in synthetic cells parameterized by morphometric statistics are then iterated to fit experimental data. This method yields synthetic cells (tentatively neurons and astrocytes) that exhibit striking qualitative and quantitative similarities with histology (e.g., using Sholl analysis). With our approach, we measure major interspecies difference regarding astrocytes, whereas dendritic organization appears better conserved throughout species. This work suggests that the time dependence of metabolite diffusion coefficient allows distinguishing and quantitatively characterizing brain cell morphologies noninvasively.