Tanguy Duval

In vivo histology of the myelin g-ratio with magnetic resonance imaging

The myelin g-ratio, defined as the ratio between the inner and the outer diameter of the myelin sheath, is a fundamental property of white matter that can be computed from a simple formula relating the myelin volume fraction to the fiber volume fraction or the axon volume fraction. In this paper, a unique combination of magnetization transfer, diffusion imaging and histology is presented, providing a novel method for in vivo magnetic resonance imaging of the axon volume fraction and the myelin g-ratio. Our method was demonstrated in the corpus callosum of one cynomolgus macaque, and applied to obtain full-brain g-ratio maps in one healthy human subject and one multiple sclerosis patient. In the macaque, the g-ratio was relatively constant across the corpus callosum, as measured by both MRI and electron microscopy. In the human subjects, the g-ratio in multiple sclerosis lesions was higher than in normal appearing white matter, which was in turn higher than in healthy white matter. Measuring the g-ratio brings us one step closer to fully characterizing white matter non-invasively, making it possible to perform in vivo histology of the human brain during development, aging, disease and treatment.

The impact of gradient strength on in vivo diffusion MRI estimates of axon diameter

Diffusion magnetic resonance imaging (MRI) methods for axon diameter mapping benefit from higher maximum gradient strengths than are currently available on commercial human scanners. Using a dedicated high-gradient 3T human MRI scanner with a maximum gradient strength of 300 mT/m, we systematically studied the effect of gradient strength on in vivo axon diameter and density estimates in the human corpus callosum. Pulsed gradient spin echo experiments were performed in a single scan session lasting approximately 2h on each of three human subjects. The data were then divided into subsets with maximum gradient strengths of 77, 145, 212, and 293 mT/m and diffusion times encompassing short (16 and 25 ms) and long (60 and 94 ms) diffusion time regimes. A three-compartment model of intra-axonal diffusion, extra-axonal diffusion, and free diffusion in cerebrospinal fluid was fitted to the data using a Markov chain Monte Carlo approach. For the acquisition parameters, model, and fitting routine used in our study, it was found that higher maximum gradient strengths decreased the mean axon diameter estimates by two to three fold and decreased the uncertainty in axon diameter estimates by more than half across the corpus callosum. The exclusive use of longer diffusion times resulted in axon diameter estimates that were up to two times larger than those obtained with shorter diffusion times. Axon diameter and density maps appeared less noisy and showed improved contrast between different regions of the corpus callosum with higher maximum gradient strength. Known differences in axon diameter and density between the genu, body, and splenium of the corpus callosum were preserved and became more reproducible at higher maximum gradient strengths. Our results suggest that an optimal q-space sampling scheme for estimating in vivo axon diameters should incorporate the highest possible gradient strength. The improvement in axon diameter and density estimates that we demonstrate from increasing maximum gradient strength will inform protocol development and encourage the adoption of higher maximum gradient strengths for use in commercial human scanners.

In vivo mapping of human spinal cord microstructure at 300 mT/m

The ability to characterize white matter microstructure non-invasively has important applications for the diagnosis and follow-up of several neurological diseases. There exists a family of diffusion MRI techniques, such as AxCaliber, that provide indices of axon microstructure, such as axon diameter and density. However, to obtain accurate measurements of axons with small diameters (<5μm), these techniques require strong gradients, i.e. an order of magnitude higher than the 40-80mT/m currently available in clinical systems. In this study we acquired AxCaliber diffusion data at a variety of different q-values and diffusion times in the spinal cord of five healthy subjects using a 300mT/m whole body gradient system. Acquisition and processing were optimized using state-of-the-art methods (e.g., 64-channel coil, template-based analysis). Results consistently show an average axon diameter of 4.5+/-1.1μm in the spinal cord white matter. Diameters ranged from 3.0μm (gracilis) to 5.9μm (spinocerebellar tracts). Values were similar across laterality (left-right), but statistically different across spinal cord pathways (p<10(-5)). The observed trends are similar to those observed in animal histology. This study shows, for the first time, in vivo mapping of axon diameter in the spinal cord at 300mT/m, thus creating opportunities for applications in spinal cord diseases.

g-Ratio weighted imaging of the human spinal cord in vivo.

Abstract The fiber g‐ratio is defined as the ratio of the inner to the outer diameter of the myelin sheath. This ratio provides a measure of the myelin thickness that complements axon morphology (diameter and density) for assessment of demyelination in diseases such as multiple sclerosis. Previous work has shown that an aggregate g‐ratio map can be computed using a formula that combines axon and myelin density measured with quantitative MRI. In this work, we computed g‐ratio weighted maps in the cervical spinal cord of nine healthy subjects. We utilized the 300 mT/m gradients from the CONNECTOM scanner to estimate the fraction of restricted water (fr) with high accuracy, using the CHARMED model. Myelin density was estimated using the lipid and macromolecular tissue volume (MTV) method, derived from normalized proton density (PD) mapping. The variability across spinal level, laterality and subject were assessed using a three‐way ANOVA. The average g‐ratio value obtained in the white matter was 0.76+/−0.03, consistent with previous histology work. Coefficients of variation of fr and MTV were respectively 4.3% and 13.7%. fr and myelin density were significantly different across spinal tracts (p=3×10−7 and 0.004 respectively) and were positively correlated in the white matter (r=0.42), suggesting shared microstructural information. The aggregate g‐ratio did not show significant differences across tracts (p=0.6). This study suggests that fr and myelin density can be measured in vivo with high precision and that they can be combined to produce a g‐ratio‐weighted map robust to free water pool contamination from cerebrospinal fluid or veins. Potential applications include the study of early demyelination in multiple sclerosis, and the quantitative assessment of remyelination drugs. Graphical abstract Figure. No caption available.

Quantitative analysis of the myelin g-ratio from electron microscopy images of the macaque corpus callosum

We provide a detailed morphometric analysis of eight transmission electron micrographs (TEMs) obtained from the corpus callosum of one cynomolgus macaque. The raw TEM images are included in the article, along with the distributions of the axon caliber and the myelin g-ratio in each image. The distributions are analyzed to determine the relationship between axon caliber and g-ratio, and compared against the aggregate metrics (myelin volume fraction, fiber volume fraction, and the aggregate g-ratio), as defined in the accompanying research article entitled ‘In vivo histology of the myelin g-ratio with magnetic resonance imaging’ (Stikov et al., NeuroImage, 2015).

Promise and pitfalls of g-ratio estimation with MRI

Abstract The fiber g‐ratio is the ratio of the inner to the outer diameter of the myelin sheath of a myelinated axon. It has a limited dynamic range in healthy white matter, as it is optimized for speed of signal conduction, cellular energetics, and spatial constraints. In vivo imaging of the g‐ratio in health and disease would greatly increase our knowledge of the nervous system and our ability to diagnose, monitor, and treat disease. MRI based g‐ratio imaging was first conceived in 2011, and expanded to be feasible in full brain white matter with preliminary results in 2013. This manuscript reviews the growing g‐ratio imaging literature and speculates on future applications. It details the methodology for imaging the g‐ratio with MRI, and describes the known pitfalls and challenges in doing so. HighlightsThis article reviews the recently developed g‐ratio imaging framework.Confounds in the methodology are detailed.Recent progress and applications are reviewed.

AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis.

Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg.

Axon and Myelin Morphology in Animal and Human Spinal Cord

Characterizing precisely the microstructure of axons, their density, size and myelination is of interest for the neuroscientific community, for example to help maximize the outcome of studies on white matter (WM) pathologies of the spinal cord (SC). The existence of a comprehensive and structured database of axonal measurements in healthy and disease models could help the validation of results obtained by different researchers. The purpose of this article is to provide such a database of healthy SC WM, to discuss the potential sources of variability and to suggest avenues for robust and accurate quantification of axon morphometry based on novel acquisition and processing techniques. The article is organized in three sections. The first section reviews morphometric results across species according to range of densities and counts of myelinated axons, axon diameter and myelin thickness, and characteristics of unmyelinated axons in different regions. The second section discusses the sources of variability across studies, such as age, sex, spinal pathways, spinal levels, statistical power and terminology in regard to tracts and protocols. The third section presents new techniques and perspectives that could benefit histology studies. For example, coherent anti-stokes Raman spectroscopy (CARS) imaging can provide sub-micrometric resolution without the need for fixation and staining, while slide scanners and stitching algorithms can provide full cross-sectional area of SC. In combination with these acquisition techniques, automatic segmentation algorithms for delineating axons and myelin sheath can help provide large-scale statistics on axon morphometry.

AxonPacking: An Open-Source Software to Simulate Arrangements of Axons in White Matter

HIGHLIGHTS AxonPacking: Open-source software for simulating white matter microstructure. Validation on a theoretical disk packing problem. Reproducible and stable for various densities and diameter distributions. Can be used to study interplay between myelin/fiber density and restricted fraction. Quantitative Magnetic Resonance Imaging (MRI) can provide parameters that describe white matter microstructure, such as the fiber volume fraction (FVF), the myelin volume fraction (MVF) or the axon volume fraction (AVF) via the fraction of restricted water (fr). While already being used for clinical application, the complex interplay between these parameters requires thorough validation via simulations. These simulations required a realistic, controlled and adaptable model of the white matter axons with the surrounding myelin sheath. While there already exist useful algorithms to perform this task, none of them combine optimisation of axon packing, presence of myelin sheath and availability as free and open source software. Here, we introduce a novel disk packing algorithm that addresses these issues. The performance of the algorithm is tested in term of reproducibility over 50 runs, resulting density, and stability over iterations. This tool was then used to derive multiple values of FVF and to study the impact of this parameter on fr and MVF in light of the known microstructure based on histology sample. The standard deviation of the axon density over runs was lower than 10−3 and the expected hexagonal packing for monodisperse disks was obtained with a density close to the optimal density (obtained: 0.892, theoretical: 0.907). Using an FVF ranging within [0.58, 0.82] and a mean inter-axon gap ranging within [0.1, 1.1] μm, MVF ranged within [0.32, 0.44] and fr ranged within [0.39, 0.71], which is consistent with the histology. The proposed algorithm is implemented in the open-source software AxonPacking (https://github.com/neuropoly/axonpacking) and can be useful for validating diffusion models as well as for enabling researchers to study the interplay between microstructure parameters when evaluating qMRI methods.

Scan-rescan of axcaliber, macromolecular tissue volume, and g-ratio in the spinal cord: Scan-Rescan of qMRI in the Spinal Cord

Recent MRI techniques have been introduced that can extract microstructural information in the white matter, such as the density or macromolecular content. Translating quantitative MRI to the clinic raises many challenges in terms of acquisition strategy, modeling of the MRI signal, artifact corrections, and metric extraction (template registration and partial volume effects). In this work, we investigated the scan–rescan repeatability of several quantitative MRI techniques in the human spinal cord.