Adult male mice C57BL/6 mice (8–12 weeks old; 25-30gr; Taconic, Denmark) and Thy1-GFP-M transgenic mice (8–12 weeks old; 25-30gr; the Jackson Laboratory) were housed with free access to food and water for a minimum of seven days prior to surgery.
Central fluid percussion model (cFPI) of diffuse traumatic brain injury
The surgical procedure for central fluid percussion injury (C57BL/6 mice cFPI, n = 53; Thy1-GFP-M transgenic mice cFPI, n = 26) has been described in detail previously . Briefly, the mouse was induced in a ventilated Plexiglas chamber with 4% isoflurane in air, and then moved to the stereotaxic frame and anaesthesia was delivered through a nosecone (isoflurane 1.2% and N2O/O2 70/30%). Local anaesthesia (bupivacaine, AstraZeneca, Stockholm, Sweden) was injected under the scalp on the top of the head, the skin cut open to reveal the skull. A 3.0 mm diameter craniotomy was made carefully over the midline, keeping the dura mater and the superior sagittal sinus intact. Then, a plastic cap was attached to the skull over the craniotomy using tissue adhesive and secured using dental cement. The cap was filled with isotonic saline at room temperature. The mouse was moved to the fluid percussion device (VCU Biomedical Engineering Facility, Richmond, VA) and connected to the spout of the device. In order to induce a diffuse TBI, the fluid percussion pendulum was released to create a pressure wave subsequently transmitted into the cranial cavity. The injury-induced apnea and immediate post-injury seizures were recorded. Post-injury apnea, observed in all cFPI-injured mice, was 25 ± 15 s (range 10–40 s). Twenty cFPI animals died at time of impact resulting in injury related mortality rate of approximately 17%. Sham-injured animals (C57BL/6 mice Sham, n = 34; Thy1-GFP-M transgenic mice Sham, n = 9) were subjected to anaesthesia and surgery, but the pendulum was not released. The cap was removed following surgery, the bone flap replaced, and the skin sutured using resorbable sutures. The animals were put in a heated cage until they recovered from the anaesthesia and subsequently returned to the home cage.
All animal experiments in the study were randomized and performed blindly by the researchers in areas of the study such as behavior, vDISCO, and MRI at both our and at international research centers. For immunohistochemistry (IHC) analyses, the number of included both C57BL/6 and Thy1-GFP-M mice per group was n = 3 for Sham, and n = 4 for cFPI at all time. For Western blotting analyses the number of included animals (C57BL/6 mice) per group was n = 5 for Sham, n = 8 for cFPI. For vDISCO analyses the number of included Thy1-GFP-M mice per group n = 2 for Sham, n = 11 (total) for cFPI [n = 3 (2dpi), n = 4 (7dpi), n = 4 (30dpi)]. For MRI studies the number of included animals per group was n = 4 for Sham, n = 6 for cFPI. For Beam walking assessment the number of included animals per group was n = 5 for naïve, non-injured animals, n = 5 for Sham, n = 5 for cFPI.
vDISCO whole-mount immunolabelling of brains
To visualize whole-brain neuronal connectivity, brains of cFPI and sham-injured animals were stained according to the nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO) whole-mount immunolabeling method . First, the post-fixed brains were incubated in in 4.5 ml of permeabilization solution (1.5% goat serum, 0.5% Triton X-100, 0.5 mM Methylbeta-cyclodextrin (Sigma, 332,615), 0.2% trans-1-Acetyl-4-hydroxy-L-proline (Sigma, 441,562), 0.05% sodium azide (Sigma, 71290) in 0.1 M PBS) for 2 days at 37 °C with gentle shaking. Subsequently, the brains were incubated in 4.5 ml of the permeabilization solution and Atto647N-conjugated anti-GFP nanobooster (Chromotek, gba647n-100) (1:700) for 14 days at 37 °C with gentle shaking. Then, the brains were washed for 2 h 3 times and once overnight with the washing solution (1.5% goat serum, 0.5% Triton X-100, 0.05% of sodium azide in 0.1 M PBS) at room temperature and finally for 2 h 4 times with 0.1 M PBS at room temperature. The immunolabelled brains were cleared with the 3DISCO clearing method . They were first incubated in 4.5 ml of the different gradients of tetrahydrofuran (THF; Sigma, 186,562) diluted in distilled water as follow: 50 Vol% THF, 70 Vol% THF, 80 Vol% THF, 100 Vol% THF and overnight 100 Vol% THF at room temperature with gentle shaking. After dehydration, the samples were incubated for 1 h in dichloromethane, and finally in benzyl alcohol + benzyl benzoate (BABB), (1:2, Sigma, 24122 and W213802) until transparent. During all the clearing steps, the tubes were wrapped with aluminum foil to protect the samples from light.
Immunofluorescence staining and confocal imaging
Coronal cerebellar Sections (40 μm) were washed three times in PBS, then blocked for one hour in PBS-TX supplemented with 3% NDS (Sigma G9023). Sections were incubated overnight at 4 °C with primary antibodies (see below) diluted in blocking solution. After incubation with primary antibodies, sections were washed three times in PBS-TX then incubated with corresponding secondary antibodies (Life Technologies and Jackson) (1:500 dilution in blocking solution for two hours at RT). Three final washes in PBS were conducted before sections were mounted on slides and images digitally captured using a Zeiss LSM 780 Microscope. Figures were composed using Photoshop CS5 software. Primary antibodies were: MBP (1: 4000, Abcam, Cat No: ab40390), CNPase (1: 500, Cat No: ab6319), SMI-32 (1:1000, Biolegend, Cat No: 801601). Secondary staining was conducted using species-specific fluorophore-conjugated antibodies (Streptavidin Alexa 488, Molecular Probes; Cy3 or Cy5, Jackson).
Light-sheet microscopy imaging
In the following procedure, whole mouse brain image stacks were acquired using a II ultramicroscope (LaVision BioTec) with an axial resolution of 4 μm and the following filter sets: ex 470/40 nm, ex 640/40 nm. The optic transparent whole mouse brains were acquired individually for High-magnification tile scanning with 4 × objectives (Olympus XLFLUOR 4× corrected/0.28 NA [WD = 10 mm] and PLAN 12x/0.53 NA [WD = 10 mm], LaVision BioTec MI) coupled to an Olympus rotary zoom unit (U-TVCAC) set at 1×. The tile scans were acquired with a 20% overlap and the width of the light-sheet was reduced to achieve maximum illumination in the field of view. The acquired raw images TIFF were processed with Fiji's stitching plugin.
The cerebellum samples were homogenized by sonication in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM β-Glycerolphosphate, 1 mM Sodium orthovanadate (Na3VO4), 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete™ Protease Inhibitor Cocktail (Sigma-Aldrich) and centrifuged at 14.000 RPM for 20 min at 4 °C. The supernatant was collected and stored at − 80 °C. Protein samples were boiled for 5 min in 2 × Laemelli buffer supplemented with 10% 2-mercaptethanol. Then 10 µg of the samples were subjected to protein separation on Mini-Protean® TGX™ casted gels (Bio-Rad, Hercules, USA); proteins were transferred onto PVDF membranes using a Trans-blot® Turbo™ (Bio-Rad, Hercules, USA) system. Membranes were blocked in TBS (20 mM Tris, 136 mM NaCl, pH 7.6) supplemented with 0.1% Tween 20 and 5% nonfat dry milk before incubation with primary antibodies SMI-32 (1:5000, Biolegend, Cat No. 801601), and Vglut 2 (1:500 Cat No: ab216463) overnight at 4 °C. Signals were boosted by binding of horseradish peroxidase (HRP)-linked secondary antibodies (anti rabbit 1:25,000 and anti-mouse 1:10,000, Sigma-Aldrich, Cat No: A0545) to the primary antibodies. Membranes were stripped and reprobed for β-actin (1:75,000, Sigma-Aldrich, Cat No: A3854). Membranes were exposed on a ChemiDoc™ MP system (BioRad) using a chemiluminescence kit (Merck Millipore, Billerica, MA, USA) that reacted with the HRP-linked secondary antibodies. Densitometry analyses were conducted using ImageJ software (Billerica, MA, USA) and protein levels were calculated as percentage of β-actin expression.
Data were acquired on a 9.4 T Agilent magnet (Agilent, Santa Clara, USA) equipped with Bruker BioSpec AVIII electronics operating with 7.0.0 (PV7) a BGA 12S HP gradient system (Bruker, Ettlingen, Germany) with a maximum gradient strength of 670 mT/m and a rise time of 130 µs. The measurements were performed with a transmit-receive quadrature cryo coil from Bruker.
High-resolution T2-weighted images were obtained with a 2D RARE sequence. The following parameters were used: TE 44 ms, RARE factor 10, TR 3.4 s, resolution 50 × 50 mm2, FOV 18 × 15 mm2 and slice thickness 0.5 mm. 32 slices were acquired axially with 6 averages in 10 m 12 s.
Diffusion tensor imaging
Diffusion tensor imaging was performed with 2D EPI readout with TE 20 ms, TR 6 s and a spectral bandwidth of 250 kHz. An isotropic resolution was used with in plane resolution of 170 × 170 um2, FOV 11 × 8 mm2 and slice thickness 0.17 mm with 80 slices. 64 diffusion directions were used with 5 reference images with no diffusion gradient. The B value was 2500 mm2 s−1 and the total acquisition time 34 m 30 s with 5 averages.
Diffusion data preprocessing involved four steps: manual brain extraction, head motion and eddy currents correction via the eddy method provided by FSL , images registration and group template construction. The b-zero volumes (b = 0 s/mm2) of each imaging session were longitudinally registered using the ANTs rigid scheme then a template for each temporal (1 and 7 dpi) and experimental (TBI, sham) condition was constructed . Generalized q-sampling imaging  (GQI) was used to calculate the orientational distribution of the density of water diffusion, then quantitative anisotropy (QA) estimated for each within-voxel fiber orientation. Template transforms were then applied to the QA metric maps and longitudinally compared using differential tractography  (threshold difference ≥ 30%). Seeding regions were separately placed at the cerebellum and whole brain, while the number of tracts resulting from decreased QA were analysed via false discovery rate (FDR).
Motor coordination assessment
The beam walk test was performed as previously described . The mouse was placed on a wooden beam (5 cm × 4 mm and 80 cm length) with the home cage placed at the end of the beam. The mouse traversed the beam to reach the home cage. Before being subjected to TBI or sham injury the mice were trained to cross the beam until a stable baseline performance was obtained. The performance of mice was recorded with a video camera, and the total number of steps and faults for each paw counted by a trained and blinded observer. The beam walk test was performed between 9 and 11 am.
Graphs and statistical analysis were made with GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Kruskal–Wallis one-way ANOVA with Dunn’s post-test was used following analyses of normality of data distribution by using Shapiro–Wilk normality test. Statistical analyses for Western blot results were performed with Student’s t test between sham-injured and cFPI group for each time point. All data are expressed as the mean ± S.E.M. Significance was set at P < 0.05.