An Optimised Multishot EPI Protocol for Ex Vivo Mouse Brain Diffusion MRI With Gd-DOTA Contrast Enhancement

Diffusion-weighted magnetic resonance imaging (dMRI) in small animal models is often limited by long acquisition times, low signal-to-noise ratio (SNR) and magnetic susceptibility artefacts. We developed and optimised a 3D spin-echo multishot EPI (SE-msEPI) protocol combined with Gd-DOTA administration to enable efficient, high-quality ex vivo diffusion imaging of the mouse brain. Systematic testing demonstrated that 12 EPI shots provided the optimal trade-off between artefact reduction, SNR and acquisition duration. Gd-DOTA incubation at 3 mM for 7 days allowed the TR to be reduced from 1500 to 400 ms without compromising diffusion metrics, yielding more than threefold SNR increase. This optimisation enabled whole-brain acquisitions at 100-μm isotropic resolution with 60 directions (b = 3000 s/mm²) in ~11 h, a fourfold acceleration compared to standard long-TR protocols. Fractional anisotropy values in Gd-DOTA-enhanced acquisitions closely matched those of the long-TR reference, while low-SNR short-TR baseline and postwashout acquisitions exhibited systematic FA underestimation. Residual error analysis and tractography performance metrics further demonstrated the improved image quality and anatomically plausible reconstruction of white matter pathways under Gd-DOTA-enhanced conditions. Beyond these technical gains, the optimised protocol provides a reproducible framework for high-throughput ex vivo diffusion MRI, enabling connectomics and microstructural studies in mouse models with greater efficiency, increased reuse of tissue sample and reduced animal use.