dc-bTBI causes abnormalities in vestibulomotor performance.

<p>A–D: performance on beam walk (A), beam balance (B), accelerating RotaRod (C) and spontaneous rearing (D) over the course of 1–14 days; n = 10 sham-injury, 10dc-bTBI; day 0 is baseline before dc-bTBI;*, <i>p</i>< 0.05; **, <i>p</i><0.01for comparison between sham and dc-bTBI rats.</p

MRI data of a representative dc-bTBI rat at different time points from baseline to 28 days post injury.

<p>(a) T2w images, FA and MK maps from DKI for a coronal slice. Arrow indicated soft tissue contusion from the injury, which were apparent at Day1 on T2w images but has fully resolved by Day7. (b) In vivo proton MRS acquired in the hippocampus. alanine (Ala), asparttate (Asp), g-aminobutyric acid (GABA), glucose (Glc), glutamine (Gln), glutamate (Glu), glutathione (GSH), myo-inositol (Ins), lactate (Lac), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), taurine (Tau), total creatine (tCr), choline compounds (tCho), glutamate/glutamine complex (Glx), and macromolecules (MM).</p

Changes in Diffusion Kurtosis Imaging and Magnetic Resonance Spectroscopy in a Direct Cranial Blast Traumatic Brain Injury (dc-bTBI) Model

<div><p>Explosive blast-related injuries are one of the hallmark injuries of veterans returning from recent wars, but the effects of a blast overpressure on the brain are poorly understood. In this study, we used <i>in vivo</i> diffusion kurtosis imaging (DKI) and proton magnetic resonance spectroscopy (MRS) to investigate tissue microstructure and metabolic changes in a novel, direct cranial blast traumatic brain injury (dc-bTBI) rat model. Imaging was performed on rats before injury and 1, 7, 14 and 28 days after blast exposure (~517 kPa peak overpressure to the dorsum of the head). No brain parenchyma abnormalities were visible on conventional T2-weighted MRI, but microstructural and metabolic changes were observed with DKI and proton MRS, respectively. Increased mean kurtosis, which peaked at 21 days post injury, was observed in the hippocampus and the internal capsule. Concomitant increases in myo-Inositol (Ins) and Taurine (Tau) were also observed in the hippocampus, while early changes at 1 day in the Glutamine (Gln) were observed in the internal capsule, all indicating glial abnormality in these regions. Neurofunctional testing on a separate but similarly treated group of rats showed early disturbances in vestibulomotor functions (days 1–14), which were associated with imaging changes in the internal capsule. Delayed impairments in spatial memory and in rapid learning, as assessed by Morris Water Maze paradigms (days 14–19), were associated with delayed changes in the hippocampus. Significant microglial activation and neurodegeneration were observed at 28 days in the hippocampus. Overall, our findings indicate delayed neurofunctional and pathological abnormalities following dc-bTBI that are silent on conventional T2-weighted imaging, but are detectable using DKI and proton MRS.</p></div

Placement of DKI ROIs (white contours) on coronal FA maps for bi-lateral hippocampus (1), internal capsule (2), and cerebellum (3).

<p>Placement of DKI ROIs (white contours) on coronal FA maps for bi-lateral hippocampus (1), internal capsule (2), and cerebellum (3).</p

Placement of the MRS voxels (white contours) on (a) cerebellum, (b) hippocampus, and (c) bi-lateral internal capsule on representative MR images of a rat.

<p>Placement of the MRS voxels (white contours) on (a) cerebellum, (b) hippocampus, and (c) bi-lateral internal capsule on representative MR images of a rat.</p

Histochemical findings in the hippocampus in dc-bTBIvs.

<p>sham-injured rats at 28 days.A,B: Iba1 immunolabeling (green) for microglia in a sham-injured and a dc-bTBI rat,respectively. C, D: Fluoro-Jade C (green) for neurodegeneration in a sham-injured and a dc-bTBI rat, respectively. E,F: Quantification of Iba1 immunolabeling and Fluoro-Jade C staining in sham-injured and dc-bTBI rats; in A–D, DAPI (blue) nuclear labeling is also shown; n = 5 per group; **, <i>p</i><0.01for comparison between sham and dc-bTBI rats.</p

Changes of DKI and MRS parameters in the sham and dc-bTBI rats in the hippocampus (a) and internal capsule (b) from baseline to 28 days post injury.

<p>Temporal data in each rat were normalized to its baseline (pre-injury) value. Error bar shows standard error. Comparisons were made between sham and dc-bTBI rats.</p

The effect of cerebral microbleeds on myelination.

<p><b>A–D</b>: Representative images (A–C), with quantification (D), of LFB staining (A) and MBP immunolabeling (B,C) at P52 in naïve pups with vaginal delivery (CTR-VD), pups following prenatal pro-angiogenic stimuli with vaginal delivery (PS-VD), and in pups following prenatal pro-angiogenic stimuli with abdominal delivery (PS-AD); arrows in (A) point to clumped myelinated fibers; arrows in (C) point to poorly myelinated fibers above corpus callosum; rectangles show ROI’s that were quantified; 7 rats/group; **, <i>p</i><0.01 comparing CTR-VD and PS-VD; §§, <i>p</i><0.01 comparing PS-VD and PS-AD; bars, 1 mm (A), 500 μm (B), 250 μm (C). <b>E</b>: Immunoblot (<i>left</i>), with quantification (<i>right</i>), of all bands of MBP at P52 in CTR-VD, PS-VD, and PS-AD rats; HSC70 used as loading control; 3 rats per group.</p

The effect of cerebral microbleeds on neurological function.

<p><b>A</b>: Performance on the righting reflex and on the negative geotaxis test on P3–14 in naïve pups with vaginal delivery (CTR-VD), pups following prenatal pro-angiogenic stimuli with vaginal delivery (PS-VD), and pups following prenatal pro-angiogenic stimuli with abdominal delivery (PS-AD). <b>B</b>: Performance on the open field test at P24, the elevated plus maze at P31, and on thigmotaxis at P35, in CTR-VD pups, PS-VD pups, and PS-AD pups. <b>C</b>: Spontaneous rearing at P31, performance on the beam balance test at P31, and grip strength at P31 in CTR-VD pups, PS-VD pups, and PS-AD pups. <b>D</b>: Incremental spatial learning on P35–39, performance on the memory probe at P40, and on the rapid learning test at P42 in CTR-VD pups, PS-VD pups, and PS-AD pups. For all panels, 19–25 pups/group; * and **, <i>p</i><0.05 and 0.01, respectively, comparing CTR-VD and PS-VD; §§, <i>p</i>< 0.01comparing PS-VD and PS-AD.</p

IUI+mLPS increases proteolytic activity and decreases collagen IV immunoreactivity.

<p><b>A–C</b>: <i>In situ</i> zymography (A,B), with quantification (C), of coronal sections from naïve control (CTR) and 24 hours after dual prenatal pro-angiogenic stimuli (PS) of IUI+mLPS, shown at low (A) and at high (B) magnification; the subventricular zone is shown in (B); nuclei stained with DAPI (blue); scale bars, 1 mm (A), 25 μm (B); 3 pups per group; *, <i>p</i><0.05; **, <i>p</i><0.01. <b>D,E</b>: Images of vessels identified by immunolabeling for RECA (red), that show proteolytic activity on <i>in situ</i> zymography (green); merged images are shown on the right; nuclei stained with DAPI (blue); scale bars, 50 μm. <b>F</b>: Immunolabeling for collagen IV (<i>left</i>), with quantification (<i>right</i>), on P0 in naïve controls (CTR), after IUI alone, after mLPS alone, and after the dual pro-angiogenic stimuli of IUI+mLPS (PS), in all cases after vaginal delivery, in coronal brain sections; 5 pups per group; tissues from the IUI alone group were from a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171163#pone.0171163.ref020" target="_blank">20</a>].</p