Animals
C57BL/6 mice were purchased from the Guangdong Medical Experimental Animal Center. The mice were housed in temperature-controlled conditions and supplied with free food and water. All animal experiments were approved by the Ethics Committee of Guangzhou University of Chinese Medicine and performed according to the guidelines of the Chinese National Institutes of Health (Guangzhou, China, Certificate No. 44005800012426).
TUDCA preparation
TUDCA used in this study (purity >98% of the total weight) was purchased from Shanghai yuanye Bio-Technology Company Limited (Shanghai, China). TUDCA was dissolved in 0.9% normal saline.
Primary culture of cortical neurons
Cortical neurons were extracted from embryos of pregnant C57BL/6 mice (E16.5) according to protocol [26]. Shortly, the cerebral cortex was separated and cut into approximately 1-mm pieces in precooled Dulbecco's Modified Eagle Medium: F-12 (DMEM/F12 medium, Gibco). Subsequently, the tissues were digested with 200 ug/ml papain (sigma, Beijing, China) for 25min at 37℃. After digestion with papain, the solution was filtered using a 100-um cell strainer (BD Falcon) and then centrifuged at 800rpm for 5min. The cell pellet was resuspended in complete Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS, Gibco). And then the cells were seeded into poly-D-lysine (sigma, Beijing, China) pre-coated 6-well plates, 24-well plates, or 96-well plates, and incubated in 5% CO2 at 37℃. 4 hours later, the cells were refreshed and cultured in Neurobasal medium (Gibco) containing 0.5mM L-glutamine (Gibco) and 2% B27 (B-27™ Supplement, Gibco). Fresh neurobasal medium containing L-glutamine and B27 was used to replace half of the medium every two days until the seventh day. All cells used in the assay were at day 4.
Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, KeyGEN, China) assay according to the protocol of the kit. Cortical neurons were seeded into 96-well plates (2×104 cells per well) precoated with poly-D-Lysine, and cortical neurons were treated with H2O2 with or without TUDCA at the indicated concentrations for 24 hours or 48 hours. Then 10 μL CCK-8 solution was added to the culture per well, and incubated at 37℃ for 2 hours. Finally, the absorbance of the culture medium at 450 nm read on microplate reader (Bio-Rad) was analyzed to determined cell viability.
ROS generation
Intracellular ROS generation were measured using DCFH-DA fluorescent probe (KeyGEN, China). After treatment, cortical neurons were washed with PBS, then 20 µM DCFH-DA fluorescent probe were added into the cultures with serum free medium. Cells were further cultured at 37°C for 1 h according to instructions of the probe. ROS production in cells was determined by DCF fluorescence observed under fluorescence microscopy (Olympus IX73).
LDH, SOD and GSH measurement
The biomedical kits (Jiancheng Institute of Biology, Nanjing, China) were used to measure and normalize the level of LDH, SOD and reduced GSH according to the protocol of the kit.
SCI model and treatment
The model of SCI was induced under sterile conditions and performed according to Allen’s method as previously described [27]. Before surgery, the mice were anesthetized using 1% pentobarbital sodium (50 mg/kg) by intraperitoneal injection (i.p.), and the spinal cord were exposed at the T9-T10 levels by laminectomy. The SCI model was induced using a pneumatic impact device. The force was set at 0.5 m/s, and the duration time was 80 ms. After surgery, bladders were manually voided twice a day until the mice could urinate normally. The mice were randomly divided into sham, SCI and TUDCA groups. The mice in the sham group were only subjected to surgical procedure without SCI. TUDCA at 200 mg/kg dosage was given to the mice by oral route once daily post injury. Meanwhile, equal volume of saline was given orally once daily in the sham group and SCI group.
Functional behavior evaluation
Hindlimb motor function was evaluated using the Basso-Beattie-Bresnahan (BBB) locomotion scale and the footprint test at different timepoints post-injury. The BBB scale ranges from 0 to 21 (0 = complete paralysis to 21 = normal gait) based on hindlimb joints movement and coordination.
Footprint test was performed by dipping the posterior limb of the mice with black dye. Then the mice were encouraged to walk straight to across a narrow path to record the footprints. The footprints were scanned, and the digitized images were used to analyze their gaits.
Tissue preparation
The mice were sacrificed under anesthetized and transcranial perfused with 0.9% normal saline at specific time points. For western blot, the spinal cord tissues around the lesion epicenter (± 0.5cm) were dissected out, then homogenized in RIPA buffer (Beyotime, Jiangsu, China) with 10 µl/ml protease inhibitor cocktail. The tissue lysates were centrifuged at 12,000 rpm for 15 min at 4℃, and then the supernatants were collected. Protein assay kit (BCA, Beyotime, Jiangsu, China) was used to determine the protein concentration.
For immunofluorescence staining and histological assessment, the mice were perfused 4% paraformaldehyde (PFA in 0.1 M PBS, pH 7.4) transcardially under anesthetic after normal saline perfusion. Spinal cord tissues were dissected out and post-fixed with 4% paraformaldehyde overnight. Tissues were dehydrated sequentially with 70%, 80%, 95% and 100% ethanol. Then the processed tisues were embedded in an appropriate orientation. The paraffin-embedded spinal cord was sectioned at 5mm using a microtome, and sections were mounted onto histological glass slides and dried overnight for immunostaining or stored at room temperature until use.
Immunofluorescence Staining on cells and sections
The primary cortical neurons cultured on the coverslips were fixed at designated time points with 4% PFA for at least 2 hours at 4℃. The sections were dewaxed in xylene three times each for 5 minutes and hydrated through a series of alcohol of decreasing concentrations (100% to 95% to 80% to 70% and then tap water twice, each step for 5 minutes). The prepared tissue sections and cells were blocked in PBS with 10% normal horse serum at room temperature for 1 hour, and then incubated at 4℃ overnight in PBS with the following primary antibodies from different species diluted in 10% normal horse serum: microtubule-associated protein 2 (MAP2, 1:200, Boster Biological Engineering Co.), β-tubulin III (Tuj1, 1:200, Millipore), GFAP (1:500, Boster Biological Engineering Co.), GAP43 (1:200, NOVUS), MBP (1:200, NOVUS), and CD68 (1:300, Boster Biological Engineering Co.). Secondary antibodies tagged with different Alexa fluor® fluorochrome (1: 300) were used. The immunostained sections or cells were examined under a fluorescence microscope (Olympus IX73).
TUNEL assay
Apoptotic cells in the sections were identified by TUNEL staining using the TUNEL cell apoptosis detection kit (Yeasen Biotech, Shanghai, China) following the manufacturer’s protocol. The immunofluorescent images were captured under a fluorescence microscope (Olympus IX73). After TUNEL labeling, the numbers of apoptotic cells (TUNEL positive cells) and the total number of cells (DAPI positive cells) on each section were counted.
Statistical analysis
All values are presented as the means ± SD. All data were conducted in GraphPad Prism 8 software (GraphPad Software Inc.). Statistical analysis was performed using one-way ANOVA for more than two groups, or using unpaired Student’s t test to compare two groups. In all the analyses, p<0.05 was considered statistically significant (expressed as *p<0.05 or **p<0.01).