All animal experiments were performed in accordance with the National Institutes of Health guidelines on animal care, and were approved by the Institutional Animal Care Committee. All efforts were made to minimize the number of animals used and animal suffering. Adult male Sprague-Dawley rats (n=30) weighing 290-310 grams (Samtako Bio, Osan, Korea) were randomly and blindly allocated into three groups. In group 1 (sham, n=8), laminectomy was performed. In group 2 (SCI-VPA, n=11), the animals received a dose of 200 mg/kg of VPA (Sigma-Aldrich, St. Louis, MO, USA). In group 3 (SCI-saline, n=11), animals received 1.0 mL of the saline vehicle solution. Rats were anesthetized intraperitoneally with a mixture of xylazine (10 mg/kg) and ketamine (60 mg/kg). After laminectomy at T9, the extradural plane between the dura and adjacent vertebrae was carefully dissected. A modified aneurysm clip with a closing force of 30 grams (Aesculap, Tuttlingen, Germany) was held in an applicator in the open position. The clip was rapidly released from the applicator and applied vertically onto the exposed spinal cord for a 2-minute compression. For the sham controls the same surgical procedure was followed, but clip compression was not applied. After surgery, the muscle, fascia, and skin were sutured using a 4-0 silk suture. Rectal temperature was maintained at 37.0±0.5℃ by a thermostatically-regulated heating pad during surgery, and during recovery, animals were placed overnight in a temperature and humidity controlled chamber. To reduce post-surgery isolation-induced stress, rats were housed in pairs at an ambient temperature of 22-25℃ in an alternating 12-hour light/dark cycle. Bladders were manually emptied twice daily until spontaneous voiding occurred. At three days after surgery, we checked functional deficit using the open locomotor rating scale by Basso, Beattie, and Bresnahan (the BBB score)3). All rats showed 4 or 5 BBB score, indicating proper cord damage in SCI model. A dose of 200 mg/kg of VPA or normal saline as a vehicle control was intraperitoneally injected twice daily at 12 hours intervals for 7 days. The total daily VPA dose of 400 mg/kg/day was similar to doses used in previous studies9,34). To evaluate histological changes, the animals were sacrificed and the spinal cords were collected 1 week after SCI. For immunohistochemistry analysis, samples were prepared from the sham group (n=5), SCI-saline group (n=7), and SCI-VPA group (n=7). Samples for western blotting analysis were prepared from rats in the three groups (group 1=3 rats, group 2, 3=4 rats).

Rats were deeply anesthetized by an intraperitoneal injection of ketamine and were perfused intracardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH=7.4). The thoracic spinal cord was excised, postfixed for 24 hours, and maintained overnight in 30% sucrose in 0.1 M PB. Spinal cord tissues were sectioned at a thickness of 30 µm on a cryostat, and sections were floated on the surface of 0.1 M PB. A 5 and 6 mm section rostral to the center of injury was selected. To detect nestin (marker for neural stem cell), spinal cord sections were blocked with 4% normal serum in 0.5% Triton X-100 for 1 hour at room temperature and incubated overnight at 4℃ with a 1 : 2000 dilution of mouse monoclonal anti-nestin (R&D Systems Inc., Minneapolis, MN, USA), and rinsed for 3×10 min in 0.1 M PB. Sections were then incubated in 0.1 M PB containing 4% normal serum and 0.5% Triton X-100 for 2 hours at 25℃ on a shaker, and then in primary antiserum in 0.1 M PB containing 4% normal serum and 0.5% Triton X-100 for 12 hours at 25℃. After rinsing (3×10 min) in 0.1 M PB, sections were incubated in a 1 : 200 dilution of biotinylated anti-mouse IgG (Sigma, St. Louis, MO, USA) in 0.1 M PB containing 4% normal serum and 0.5% Triton X-100 at 25℃ for 2 hours. The sections were then incubated in a 1 : 50 dilution of avidin-biotinylated horseradish peroxidase (Vector Laboratory) in 0.1 M PB for 2 hours and rinsed (3×10 min) in 0.25 M Tris. Finally, staining was visualized by reaction with 3, 3'-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide in 0.25 M Tris for 3-10 min using a DAB reagent set (Kierkegaard&Perry, Gaithersburg, MD, USA). All sections were rinsed in 0.1 M PB and mounted on Superfrost Plus slides (Fisher, Pittsburgh, PA, USA) and dried overnight at 37℃. The mounted sections were dehydrated with alcohol, cleared with xylene, and cover slipped with Permount mounting medium (Fisher). The labeled cells were identified and counted with separation of antibody at three tissues in each different animal. The labeled tissues were photographed using a Zeiss Axiopan microscope with high power DIC optics (Carl Zeiss Meditec Incorporation, Jena, Germany). The images were viewed on a computer monitor using a Zeiss Plan-Apochromat 40x objective (Carl Zeiss) and photographs of the central canal region and ventral side of white matter of left and right sides were taken with a Zeiss AxioCam HRc digital camera (Carl Zeiss). Enumeration of immune-positive cells used a Labworks, version 4.5, computer-assisted image analyzer (UVP, Upland, CA, USA).

Rats in three groups were decapitated rapidly under anesthesia. The thoracic spinal cord was rapidly dissected and then immediately frozen in liquid nitrogen. Frozen tissue was mixed with RIPA buffer (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany) and immediately homogenized. The homogenate was centrifuged at 13000 rpm for 30 min at 4℃ and the supernatants were determined using the BCA protein assay (Sigma, St. Louis, MO, USA). Proteins were separated by 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After incubation in a blocking solution of 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membrane was incubated with a 1 : 1000 dilution of mouse monoclonal anti-SOX2 (cell signaling) and mouse monoclonal anti-β-actin (Sigma-Aldrich), and overnight at 4℃, and then with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The proteins were detected with chemiluminescence reagents. Immune-positive bands used an image J, version 1.46r, computer-assisted image analyzer (National Institutes of Health, USA).

All statistical comparisons were computed using SPSS 17.0 (SPSS, Inc., an IBM Company, Chicago, IL, USA). Data are expressed as mean±standard error of the mean. Repeated measure ANOVA was used to compare groups. Null hypotheses of no difference were rejected if p-values were less than 0.05.

We analyzed the nestin immunoreactivity from two different sites of the spinal cord based on methods from Sibuya et al.32). One site is the white matter of ventral side and the other is the ependyma of central canal in a cross-section of spinal cord. We used ANOVA analysis for confirmation of density areas of nestin.

In the white matter of ventral side, nestin immunoreactivity was almost undetectable in the sham group (Fig. 1A, D). SCI groups showed remarkable nestin immunoreactivity compared with sham group. In the SCI-saline group, nestin immunoreactivity extended in arboroid processes from the pial surface toward the spinal cord center (Fig. 1B, E). In the SCI-VPA group, nestin immunoreactivity was much stronger than in SCI-saline group (Fig. 1C, F). Density areas of nestin expression in the white matter of ventral side differed significantly between the SCI and sham group (Fig. 2). The SCI group showed significantly large density area compared with sham group and within SCI groups, the SCI-VPA group had a much larger density area of nestin than the SCI-saline group.

In the ependyma of the central canal, nestin immunoreactivity also was strongest in the SCI-VPA group (Fig. 3C). In sham group, nestin immunoreactivity was observed in some cells (Fig. 3A). SCI groups had significant nestin immunoreactivity compared to the sham group and extended in processes from the whole ependymal area (Fig. 3B, C). The density areas of nestin expression in the ependyma of the central canal were similar to the white matter of ventral side. The SCI groups had significantly large density areas of nestin compared to the sham group, and SCI-VPA group had a much larger density area of nestin than SCI-saline group (Fig. 4).

To sum up, VPA increased expression of nestin, a marker for NSPCs, in the white matter of the ventral side and ependyma of the central canal.

SOX2 was detectable in SCI groups and undetectable in sham group. Besides, in SCI groups, VPA significantly increased the SOX2 positive band than in the SCI-saline group (Fig. 5). This difference was confirmed by ANOVA analysis. SOX2 protein levels were highest in SCI-VPA group, followed by SCI-saline (Fig. 6).