## 2 Materials and Methods
### Perfusion, Tissue Processing and Histological Assessment  
Six weeks after SCI, when all six weekly neurotest timepoints were completed, the animals were deeply anesthetized with isoflurane (5%) and transcardially perfused with 50 mL 0.1 M cold phosphate-buffered saline (PBS) followed by 150 mL 4% paraformaldehyde (in 0.1 M PBS). The spinal cords were extracted, cut into a 2 cm piece centered around the visible injury lesion, fixed with 4% paraformaldehyde for 24 h and cryoproteted with 30% sucrose for 48 h. The spinal cord pieces were then embedded in tissue embedding medium (Sakura Finetek Europa B.V., Alphen aan den Rijn, The Netherlands) on dry ice and a cryostat (Leica CM3050S, Leica Biosystems, Nuss Bloch, Germany) was used to cut consecutive cross-sections (every 240 \(\upmu\)m) with a thickness of 30 \(\upmu\)m. The spinal cord cross-sections were labeled, dried and stored at \(-80\)\({}^{\circ}\)C until further processing.  
For histological assessment of the preserved spinal cord tissue, immunofluorescence staining with glial fibrillary acidic protein (GFAP) was performed. To this end, the spinal cord cross-sections were blocked with a blocking solution containing 5% non-fat milk, 1% bovine serum albumin, and 0.3% Triton-X100 in 0.1 M PBS for 1 h. A mouse anti-GFAP (1:1000; Millipore, Burlington, VT, USA) primary antibody, diluted in blocking solution, was then applied overnight. An isotype control with nonspecific immunoglobulin at the same concentration was performed to ensure specificity of the antibody staining (data not shown). Alexa Fluor 568 goat anti-mouse (1:400; Invitrogen, Carlsbad, CA, USA) diluted in blocking solution without Triton-X100 was used as the secondary antibody and applied for 1 h. Nonspecific binding of the secondary antibody was ruled out by incubating sections with only the antibody diluent without adding the primary antibody (data not shown). After removal of the secondary antibody and washing with PBS, the spinal cord cross-sections were coverslipped after applying mounting medium, dried and then subjected to imaging analysis.  
All images were taken with a confocal laser scanning microscope (LSM 700; Carl Zeiss Microscopy GmbH, Oberkochen, Germany) in the \".lsm\" file format and analyzed with the image processing software ImageJ (National Institute of Health, New York City, NY, USA). For the assessment of the preserved spinal cord tissue, only images of the same 11 spinal cord cross-sections with the following distinct distances to the lesion epicenter were used: 0 \(\upmu\)m (epicenter), +/\(-\)240 \(\upmu\)m, +/\(-\)480 \(\upmu\)m, +/\(-\)720 \(\upmu\)m, +/\(-\)960 \(\upmu\)m, and +/\(-\)1200 \(\upmu\)m. ROIs were drawn around the entire spinal cord and, if present, around the posttraumatic cystic cavity with the \"Free-hand\" tool and the \"Measure\" tool was used to output the area of the total spinal cord and the preserved spinal cord tissue (total spinal cord area--cyst area). The percentage of preserved spinal cord tissue (preserved spinal cord tissue area divided by the total spinal cord area) was then calculated for every spinal cord cross-section and averaged per animal.