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 cellspecific 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.cell morphology | noninvasive histology | diffusion-weighted NMR spectroscopy | numerical simulations | metabolites T he brain is one of the most complex organs, and it has defined an inexhaustible field of research over the last centuries. Unfortunately, brain's complexity is paralleled by the difficulty in examining it noninvasively. Some fundamental questions regarding morphological modifications of neurons and astrocytes along brain development, aging, or disease, as well as interspecies differences, can only be investigated postmortem using histology, the current gold standard to study cellular morphology. The development of a noninvasive neuroimaging tool to evaluate and monitor brain cell morphology under normal and pathological conditions in vivo would thus represent a major breakthrough.MRI and magnetic resonance spectroscopy (MRS) techniques have opened new doors for examining brain tissues in vivo at both meso-and macroscales. Diffusion-weighted (DW)-MRI and -MRS, which allow the investigation of the diffusion process of endogenous molecules in biological tissues at these scales (1), have made it clear that cell architecture has a critical influence on molecular displacement (2-5). To quantitatively evaluate the impact of cell structure on measured molecular diffusion, mainly two modeling strategies have been developed. The first approach consists in performing numerical simulations of many particles diffusing in arbitrary geometries (e.g., defined by 3D meshes) mimicking "realistic" cell architectures (6-9). Because these realistic geometries are generally built directly from microscopy data rather than being described and generated by a (small) set of parameters, and because simulations are extremely computationally demanding, this approach does not seem adapted to fit experimental data. The second approach consists in simplifying cell architec...

The non-monoexponential signal attenuation of intracellular metabolites in the mouse brain can be described by diffusion in long and thin cylinders, yielding realistic D 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.

PurposeTo 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).MethodsA 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.ResultsOur results suggest that the correlation between relaxation and diffusion properties is extremely small or even nonexistent for metabolites in the mouse brain.ConclusionThe 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.

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 t>200 ms for metabolites and t>70 ms for water molecules, suggesting that the effects of these finer morphological details are negligible at t 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.

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 intraastrocytic 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.

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 T . 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 T relatively short (60 ms). At such T , 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.

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 1H downfield MRS opens new directions in the investigation of in vivo downfield metabolites and their role on healthy and disease processes.

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.