Considerations and recommendations from the ISMRM diffusion study group for preclinical diffusion MRI: Part 2-Ex vivo imaging: Added value and acquisition

Abstract

The value of preclinical diffusion MRI (dMRI) is substantial. While dMRI enables in vivo non-invasive characterization of tissue, ex vivo dMRI is increasingly being used to probe tissue microstructure and brain connectivity. Ex vivo dMRI has several experimental advantages including higher SNR and spatial resolution compared to in vivo studies, and enabling more advanced diffusion contrasts for improved microstructure and connectivity characterization. Another major advantage of ex vivo dMRI is the direct comparison with histological data, as a crucial methodological validation. However, there are a number of considerations that must be made when performing ex vivo experiments. The steps from tissue preparation, image acquisition and processing, and interpretation of results are complex, with many decisions that not only differ dramatically from in vivo imaging of small animals, but ultimately affect what questions can be answered using the data. This work represents "Part 2" of a three-part series of recommendations and considerations for preclinical dMRI. We describe best practices for dMRI of ex vivo tissue, with a focus on the value that ex vivo imaging adds to the field of dMRI and considerations in ex vivo image acquisition. We first give general considerations and foundational knowledge that must be considered when designing experiments. We briefly describe differences in specimens and models and discuss why some may be more or less appropriate for different studies. We then give guidelines for ex vivo protocols, including tissue fixation, sample preparation, and MR scanning. In each section, we attempt to provide guidelines and recommendations, but also highlight areas for which no guidelines exist (and why), and where future work should lie. An overarching goal herein is to enhance the rigor and reproducibility of ex vivo dMRI acquisitions and analyses, and thereby advance biomedical knowledge.

Keywords: acquisition; best practices; diffusion MRI; diffusion tensor; ex vivo; microstructure; open science; preclinical; processing; tractography.

FIGURE 1

Four areas in which preclinical brain imaging adds value to the field of dMRI. It enables: (i) correlation with histology on the same subject/sample, (ii) the acquisition of richer datasets than on clinical systems thanks to more advanced hardware and longer scan times available, (iii) the study of tissue changes with disease and treatment in a more controlled setting, and (iv) comparative anatomy between species. Figures reused and adapted from (left to right): (i),, , (ii),, , (iii),, (iv).

FIGURE 2

Considerations in the diffusion process. When performing studies on ex vivo tissue, one must consider effects of (i) chemical fixation (changes in geometry, volume fractions, relaxation rates, permeability, diffusion coefficients), (ii) changes in diffusivity (which can be approximally two to five times reduced from in vivo depending on experimental conditions), and (iii) temporal instabilities over long scan times (causing temporal drift or image artifacts). Figures reused and adapted from (i), (ii),, (iii).,

FIGURE 3

Ex vivo imaging of mouse models facilitates high resolution, high SNR, dense sampling of q‐t space. Here, a fixed mouse brain was imaged on a 16.4T Bruker Eon Ascend scanner equipped with a 10‐mm birdcage coil and gradients capable of producing up to 3 T/m in all directions. Images show mean diffusivity (MD), mean kurtosis (MK), and directionally encoded color (DEC) fractional anisotropy (FA) maps. All animal studies were approved by the competent institutional and national authorities and performed according to European Directive 2010/63. Images kindly provided by Andrada Ianus and Noam Shemesh.

FIGURE 4

Primate models have been used to validate tractography estimates of structural connectivity. Ex vivo imaging offers the ability to investigate and compare the anatomical accuracy of high quality and high resolution dMRI datasets against histological tracers, the gold‐standard for elucidating brain tractography. Figure adapted from and, based on ex vivo macaque data acquired by shows tracer trajectory (left), directionally encoded color map (middle), and tractography streamlines (right).

FIGURE 5

Ex vivo imaging of the human brain facilitates high resolution and high SNR dMRI (left), which offers exceptional tractography, mapping and creation of templates for small structures, and investigation of gray matter laminar structures (right). Images adapted from.

FIGURE 6

For high‐quality ex vivo diffusion MRI, decisions regarding hardware, fixation, preparation, MR scanning, and tissue storage must be carefully considered. Hardware: Utilize the smallest coil that fits the sample under investigation, to maximize SNR. Fixation: For ex vivo tissue to be a good model of in vivo, the post‐mortem interval to fixation must be as short as possible. Preparation: Washing out fixative and soaking tissue in a solution of Gadolinium‐based contrast agent decreases primarily water‐protons T₁ which in turn allows for a favorable trade‐off between SNR maximization and TR reduction (i.e., reduced acquisition time), while a robust physical setup eliminates motion during scanning. MR Scanning: A multi‐shot 3D diffusion‐weighted spin echo EPI or multi‐shot 3D diffusion‐weighted RARE/FSE sequence. While not typically used in vivo due to motion sensitivity and long scan time, these sequences combine advantages of high SNR, minimal distortion, and reasonable scan time ex vivo. Storage: Fixed tissue can be stored for many months to several years if stored in fixative or phosphate buffered solution (often with 1% paraformaldehyde [PFA]) at 5°C.

FIGURE 7

Examples of ex vivo samples prepared for dMRI acquisitions. Sample holders may be syringes or test tubes/falcon tubes, custom‐made or 3D printed holders with ventilation valves, or simply placement within a plastic bag robustly secured to a platform. Photos courtesy of Daniel Colvin, Kurt Schilling, Luisa Ciobanu, Stijn Michielse, Francesco Grussu, Raquel Perez‐Lopez, Ileana Jelescu, Tim Dyrby.

FIGURE 8

Plots showing how SNR efficiency varies with T₁. Curves are based on the SNR‐efficiency equation given in Section 4.5.2 based on a spin echo sequence. The optimal TR is ˜1.25 times the sample T₁, although there is a wide range of near‐maximum efficiency. Similar optimization can be performed for TE, and diffusion weightings (see Other considerations in Section 4.5.3 q‐t coverage, for examples).

FIGURE 9

Approach to optimizing ex vivo diffusion protocols. Relaxometry (top) and diffusion (middle) can be measured as a function of contrast concentration, or fixative solution, for both white and gray matter tissue types, and SNR efficiency can be optimized (bottom) by manipulating sequence parameters and gadolinium contrast agent concentration. Images are adapted and modified from (top) and (bottom).