Paravision 5.1 imaging software

By measuring tissue integrity at the micron level, ex vivo DTI allows quantification of microstructural injury (Aung et al., 2013 (link)). Observed patterns of abnormalities vary with type of insult (Sierra et al., 2015 (link)), and recovery intervals following injury (Mac Donald et al., 2007 (link)). To assess long-term abnormalities in CA3 hippocampal microstructure, rats at P35–40 were deeply anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde. Brains were then removed and after post-fixation, embedded in 2% agarose containing 3 mM sodium azide for ex vivo magnetic resonance imaging (MRI). MRI was performed on a Bruker 4.7-T BioSpec 47/40 Ultra-Shielded Refrigerated nuclear system equipped with a 72 mm I.D. quadrature RF coil and a small-bore (12 cm I.D.) gradient set with a maximum gradient strength of 50 Gauss/cm. MR protocols consisted of echo-planar diffusion tensor imaging (EP-DTI) sequences. Images of 12 contiguous coronal 1 mm slices were obtained with a field-of-view (FOV) of 3.00 cm, a TR of 3000 ms, TE of 40 ms, and b-value 2000 mm²/s with 30 gradient directions. CA3 was analyzed using Bruker’s Paravision 5.1 imaging software. Diffusion-weighted images and fractional anisotropy (FA) maps were generated. Axial (λ1) and Radial [(λ2 + λ3)/2] diffusivity eigenvectors were also measured by observers blinded to the injury status.

Ex vivo MRI using diffusion sequences was performed on a Bruker BioSpec 7T 70/30 Ultra Shield Refrigerated (USR) nuclear MRI system, consistent with prior published methods (Robinson et al., 2016 (link), 2017b (link); Yellowhair et al., 2018 (link)). Briefly, P21 rats were deeply anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde. Brains were removed from the skull and post-fixed in 4% paraformaldehyde for 1 week and embedded in 2% agarose containing 3 mM sodium azide for immediate ex vivo MR imaging. Echo-planar diffusion tensor imaging (EP-DTI) of twenty contiguous coronal 1 mm slices were obtained with a FOV (field-of-view) of 3.00 cm and an MTX of 256. Brain regions of interest (ROI) in major white matter tracts (corpus callosum and external capsule) and gray matter (hippocampus and thalamus), were traced by an observer blinded to experimental conditions and analyzed using Bruker’s ParaVision 5.1 imaging software. Fractional anisotropy (FA), axial (λ1) and radial [(λ2+λ3)/2] diffusivity eigenvectors were measured and calculated. For bilateral neuroanatomical ROIs, metrics were acquired on each side and averaged per ROI. Directionally encoded diffusion color maps and color-coded FA maps were created.

Pulsed-field gradient NMR (PFG-NMR) was used to observe mat morphology and determine intra-mat porosity and diffusion coefficients. The techniques used were similar to those of Renslow et al. (2013) (link). The details of the NMR imaging techniques and full method descriptions are provided in the Supplementary Information. Briefly, the NMR imaging experiments were conducted at 500.40 MHz for proton (1H) detection using a 89-mm-wide bore 11.7-T magnet with a Bruker Avance III digital NMR spectrometer and ParaVision 5.1 imaging software (Bruker Instruments, Billerica, MA, USA). Each mat sample was placed in a 15-mm NMR tube on a support bed of 2% agar gel. Experiments performed included 2D magnetic resonance imaging (mic_flash), diffusion tensor imaging for determining diffusion coefficients (DtiStandard; Renslow et al., 2013 (link)), and chemical shift selective imaging for generating porosity measurements.