2.1 Animals and surgery
Animal procedures were performed in strict accordance with protocols approved by the Institutional Animal Care and Use Committee at Jinan University. Adult female Sprague-Dawley rats (weight 220 to 250 g) were randomly classified into SCI and sham-operated groups, housed under conditions of constant temperature (25±1°C) and humidity (55±5%), with a 12 h light-dark cycle. A T10 contusion injury was produced by a trained technician as described in previous studies[17, 19]. Briefly, the rats were anesthetized by intraperitoneal (ip) injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Once rats were confirmed to be unconscious by toe-pinch, a laminectomy was performed at vertebral level T10 on the back of the rat. After immobilizing the spine stereotaxically, a weight-drop injury was induced by using a standardized instrument (New York University Impactor) releasing a weight (10 g, rod diameter of 2 mm) from a height of 12.5 mm on the exposed dura of the spinal cord at thoracic level T10, inflicting a moderate contusion injury. The rats were randomly assigned to either PD168393 or vehicle treatment groups. Immediately following SCI, an osmotic mini pump (Alzet Corp., Palo Alto, CA, USA) placed between the shoulder blades was connected to a 32 gauge catheter and the catheter tip was positioned subdurally on the dorsal side of the spinal cord over the center of the injury through a small hole in the dura mater. In the PD168393-treated group, the osmotic pump was filled with 2 mM PD168393 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 5% DMSO, then the rats were micro-infused immediately after pump placement at a rate of 0.5 μL/h for 14 days[17]. The Control animal were given 5% DMSO. Lastly, the back muscles and skin layers were then sutured and animals were returned to their original warm incubators for recovery. After 14 days, the pumps were removed and then the wound was closed with surgical suture. The Sham-operated animals underwent laminectomy alone. Bladders were expressed twice daily until urinary incontinence disappeared.
2.2 Rat Primary spinal cord mixed glial cells (astrocytes and microglia) culture
Spinal cord mixed glial cells (astrocytes and microglia) were derived from neonatal rat spinal cord as described in our previous study [8, 22]. Briefly, the rat pups at postnatal day 0~2 were sacrificed by cervical dislocation and disinfected with 75% alcohol for about 3~5 min, followed by dissection and separation from the whole spinal column via ophthalmological instruments. The spinal cord was discharged by injecting cold D-Hank’s solution into the spinal column and then the meninges were removed. After that, the extracted spinal cord was minced, digested in PBS (pH = 7.4) containing 0.25% collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37°C, filtered through a 70 μm cell strainer, and centrifuged at 1000 rpm for 5 min. Subsequently, the resulting pellet was re-suspended and cells were plated in culture flasks at a density of 1 × 106 cells/ mL and maintained in culture at 37℃ with 5% CO2 in DMEM supplemented with 10% FBS (Hyclone) and 0.5 mg/mL penicillin/streptomycin. After 48 or 72 h, the medium was changed every 3 days thereafter. After a total of 21 days in culture, the mixed glia were passaged with 0.25% trypsin, re-suspended in astrocytic freezing medium (DMEM, 10% FBS, 10% DMSO) at a concentration of 2~3 × 106 cells/mL, and frozen at a rate of 1°C/min at −80°C using a Nalgene freezing container.
2.3 Rat primary spinal cord astrocytes culture
Spinal cord astrocytes from neonatal rat spinal cord were prepared according to an established protocol[23]. Briefly, pups at postnatal day 0–2 were decapitated following sterilization of the body with 75% ethanol. The spinal cords of rat pups were isolated following painless sacrifice. Meninges were removed and the spinal cord was minced, digested in 0.25% collagenase in PBS (pH 7.4), for 10 to 20 minutes at 37°C and then passed through a 70 μm nylon mesh Digested tissues were centrifuged at 1,000 rpm for 2 minutes, then the supernatant removed carefully, the tissue pellet re-suspended, cells were plated on culture flasks at a density of 1~3× 106 /mL and maintained in DMEM supplemented with 10% FBS (Hyclone) and 0.5 mg/mL penicillin/streptomycin at 37°C with 5% CO2. Cell culture medium was changed 2 days after plating and every 3 days thereafter until astrocytes reached confluence. During week 2 in vitro, non-astroglial cells were removed by mildly shaking. The astrocytes remaining in culture medium were identified by immunostaining with anti-GFAP. Approximately 95% of the cells were immunopositive for GFAP. All experiments were performed on spinal cord astrocytes maintained for two to three weeks in culture.
2.4 Rat primary spinal cord microvascular endothelial cells culture
Primary spinal cord microvascular endothelial cells (MEC) were obtained from rats pups at postnatal day 0~2, and the isolation and culture of MEC was performed according to the protocol previously described[8, 24]. Briefly, spinal cords were isolated following painless sacrifice and then large vessels and meninges were eliminated successively. Then, the dissected spinal cord tissues were finely minced into small pieces using iris scissors in cold DMEM/F-12 and manually homogenized on ice for several rounds in a glass homogenizer. Subsequently, the homogenates were digested in pre-warmed ACCUTASE in a shaker for 30 min at 37°C, centrifuged for 5 min at 2000 rpm. Solid fraction pellets were then re-suspended in 22% bovine serum albumin, followed by centrifugation for 15 min at 4000 rpm. The upper myelin layer was aspirated and washed 3 times with HBSS, followed by digestion with pre-warmed ACCUTASE in a shaker at 37 °C for 30 min. Then, the digested tissues were centrifuged at 2000 rpm for 5 min at 4°C and re-suspensed in MEC culture medium [DMEM/F12, supplemented with 10% FBS, 0.5 mg/mL penicillin/streptomycin and 30 ng/mL endothelial cell growth factor (PeproTech)]. Finally, cells were plated on poly-D-lysine-coated culture flasks at a density of 1 × 104 cells/mL and cultured at 37°C in 5% CO2. The MEC were identified by immunofluorescence labeling with anti-CD31. The rate of MEC (CD31-positive) was greater than 95%.
2.5 BSCB damage model in vitro
A BSCB damage model in vitro was produced as described in a previous study[8]. Briefly, re-suspended rat spinal cord MEC cells were seeded on the upper chambers (inserts) of a 12-well Transwell system (polyester membrane, 12 mm diameter, 0.4 μm pore size, Corning Costar, HighWycombe, UK) at a concentration of approximately 5 × 104 cells/mL and 400 μL/well. Then, the upper chambers with MEC were added onto the bottom chambers of the transwell system with pre-seeded spinal cord mixed glial cells and co-cultured in DMEM/MVGS culture medium for 7~14 days with media changes occurring every 3 days.
Oxygen and glucose deprivation/reoxygenation (OGD/R) treatment was applied to the above-mentioned cells in the Transwell system in vitro to mimic BSCB damage in vivo. After three washes with PBS, the cells were cultured with glucose-free DMEM/F12 in a hypoxia incubator (oxygen concentration ≤1%) equilibrated with 95% N2 and 5% CO2 at 37℃ for 3 h in order to induce hypoxic damage to the in vitro BSCB model. After OGD exposure, cells were returned to normal culture in glucose-containing DMEM/F12 under normoxic conditions for reoxygenation. As a control group, cells were washed with glucose-containing DMEM/F12 three times, and then cultured in glucose-containing DMEM/F12 under normoxic conditions at an oxygen concentration of 20%.
2.6 Cell treatment
2.6.1 Agonists and inhibitors for EGFR and p38 MAPK in AS and MEC
Cells were seeded at 1 × 105 cells/cm2 onto glass coverslips in 24-well culture plates. Agonists for EGFR and p38 MAPK were given following OGD treatment, with final concentrations at 5nM (EGF) (Sigma-Aldrich, St. Louis, MO, USA)or 10mM (Isoprenaline) (MCE). Inhibitors for EGFR and p38 MAPK were given following OGD treatment, with final concentrations at 1 nM (PD168393) (Millipore) or 10μM (SB203580) (Sigma-Aldrich, St. Louis, MO, USA). The solvent served as the control treatment. Supernatants were collected for ELISA, while cells were fixed by methanol (−20°C, 15 min) for staining at various harvesting time points.
2.6.2. Overexpressing of EGFR and p38 MAPK in AS and MECs
Cells were grown to confluence on 100cm2 culture plates in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, 3.7 g/L sodium bicarbonate, 4 mM glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin. Cells were transfected with lentivirus vector (1×109TU/ml) containing the cDNA fragments encoding the full-length rattus EGFR and p38 MAPK protein. The target gene overexpresses lentivirus was purchased from Genechem (Shanghai, China). Cells transfected the empty with expression vector served as controls. Transfection efficiency was monitored by fluorescence microscopy.
2.6.3 EGFR and p38 MAPK siRNA transfection in AS and MECs
A lentiviral EGFR short hairpin RNA (shRNA) was purchased from Genechem (Shanghai, China). A scrambled shRNA was included as a negative control. To knockdown endogenous EGFR, and p38 MAPK, cells were transfected with Infection enhancer fluid HitransG A (Genechem, Shanghai, China) plus shRNA according to the manufacturer recommendations. In general, cell suspensions were prepared as a density of 3~5 × 104 cells/mL with complete medium, 2mL cell suspension was added to each well of a six-well plate (bottom area of each well is 10 cm2). Cells were cultured in a humidified incubator containing 95% air and 5% CO2 at 37℃ for 16~24h, with regular media changes. Two uL shRNA (2×109TU/mL) in the medium containing 40uL infection enhancement solution HitransGA was then added to each well, cultured for 6~8hours, and then replaced with conventional medium. Transfection efficiency was monitored by fluorescence microscopy after transfection for 72 h.
2.7 Immunocytochemistry staining of rat spinal cord sections
Rats in the sham, vehicle, and PD168393-treated injury groups were sacrificed 3 days post-injury for GFAP, CD31, and pEGFR double staining and sacrificed at days seven post-injury for ZO-1, Onccludin and CD31 staining (n=5 for each time points). The tissues were dissected and processed as previously described[17, 19]. Briefly, the rats were transcardially perfused with saline, followed by ice-cold 4% paraformaldehyde. After harvesting the 15mm length spinal cord samples centered on the injury site, the spinal cord segments were embedded in Optimum Cutting Temperature Compound (Sakura, USA), rapidly frozen in nitrogen-cooled isopentane, and stored at -80°C until time of processing. Spinal cord tissue blocks were cut horizontally at a thickness of 10 μm. Sections were mounted on poly-L-lysine-coated glass slides and stored at −20°C until further processed. Sections were then washed in PBS buffer solution and incubated with blocking solution containing 0.3% Triton-x and 5% normal goat serum in PBS at 37℃ for 1 hour. Immunofluorescence sections were treated with respectively with a combination of two primary antibodies: mouse anti-GFAP (1:400; Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-pEGFR (1:300; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-GFAP (1:300; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-CD31 (1:400; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-ZO-1 (1:200; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-CD31 (1:400; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-occludin (1:200; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-CD31 (1:400; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. After PBS washing, the sections for double-labelling immunofluorescence were incubated in a mixture of two secondary antibodies: Cy3-conjugated goat anti-rabbit IgG anti-body (1:300; Jackson ImmunoResearch, West Grove, PA, USA) and FITC-conjugated goat anti-mouse IgG antibody (1:300; Jackson ImmunoResearch, West Grove, PA, USA) or Cy3-conjugated goat anti-mouse IgG antibody (1:300; Jackson ImmunoResearch, West Grove, PA, USA) and FITC-conjugated goat anti-rabbit IgG antibody (1:300; Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature and stained with DAPI. Finally, the slides were washed twice in PBS and cover slips were mounted on slides using antifade medium (Sigma). Sections were observed under an Olympus BX-51 light microscope (Olympus, Tokyo, Japan) or a confocal laser-scanning microscopy (TCS SP8, Leica) and analyzed with Image-Pro Plus analysis software (Media Cybernetics, Inc., Silver Spring, MD, USA).
2.8 Western blotting analysis
Animals in the sham, injury, and PD168393-treated groups were deeply anesthetized and sacrificed at days 3, 7, and14 post-injury (n = 3 in each group for each time point). A 15 mm length spinal cord containing the injury epicenter was quickly removed for protein semiquantitative analysis in each group. For cell culture experiments, cells were harvested at 12 h of reoxygenation following OGD. And the Protein extracts were prepared and Western blotting was performed as described previously[17, 19]. Samples containing an equal amount of total proteins were loaded on SDS-PAGE (10% for EGFR/pEGFR; 12% for other proteins). The electrophoresed proteins were transferred onto nitrocellulose membranes (300 mA, four hours for EGFR/pEGFR; 250 mA, two hours for others). After being blocked by incubation with Tris-buffered salina containing 0.1% Tween 20, 2% BSA, and 5% nonfat dry milk, the membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer as follows:. monoclonal mouse anti-GFAP (1:1,000; Neomarkers, Fremont, CA, USA) for astrocytes, polyclonal rabbit anti-CD31 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for microvascular endothelial cells, polyclonal rabbit anti-pEGFR (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-EGFR (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-ZO-1 (1:200; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-occludin (1:200; Sigma-Aldrich, St. Louis, MO, USA) , rabbit anti-COX-2(1:300 Invitrogen, USA), rabbit anti-iNOS (1:300 Invitrogen, USA), rabbit anti-IL1β(1:300 Invitrogen, USA), rabbit anti-TNFα (1:300 Invitrogen, USA), mouse anti-p65 (1:2000; Abcam, UK), mouse anti-p38 (1:2000; Abcam, UK), rabbit anti-β-Tubulin, mouse anti-Histone3 (1:2000; Abcam, UK), mouse anti-GAPDH(1:2000; Abcam, UK), or rabbit anti-β-actin (1:2,000; Neomarkers, Fremont, CA, USA) as a loading control. After washing the membranes with TBS containing 0.1% Tween 20, the blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:2,000; Neomarkers, Fremont, CA, USA) at room temperature for 1 hour and visualized with an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific Inc., USA). The membranes were scanned at 600 dpi, and digital images were quantitatively analyzed by a Kodak Digital Science 1D system. The optical density (OD) of the blots was semiquantified and expressed as the ratio of OD of the tested proteins to that of loading control (β-actin, β-Tubulin, GAPDH or Histone 3).
2.9 Reverse transcriptase-polymerase chain reaction (RT-PCR)
Rats in sham, injury, and PD168393-treated groups were deeply anesthetized and sacrificed at day 3 post-injury (n = 3 in each group). The 15 mm length spinal cord sample centered on the injury site was quickly separated, immediately frozen with dry ice, and stored at -80 °C until use. Total RNA was extracted from spinal cord segments and the cultured cells using Trizol Reagent (Gibco, USA) according to the manufacturer's protocol. RNA was transcribed into single-stranded DNA using MMLV according to the manufacturer’s instructions (Invitrogen). PCRs for TNF-α, iNOS, COX-2, and IL-1β and GAPDH were performed using Taq PCR Master Mix kit (201443 Qiagen) on a Mastercycler gradient PCR (Eppendorf, Gene Company) using a protocol consisting of 94 °C for 2 min in 1 cycle and denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s in each of 35 cycles for GFAP and 25 cycles for GAPDH, respectively. The primers were as follows: for Cox-2,5’-CCA TGT CAA AAC CGT GGT GAA TG-3’ (sense) and 5’-ATG GGA GTT GGG CAG TCA TCA G-3’ (antisense); iNOS, 5’-CTC CAT GAC TCT CAG CAC AGA G-3’ (sense) and 5’-GCA CCG AAG ATA TCC TCA TGA T-3’ (antisense); IL-1β, 5’-GCA GCT ACC TAT GTC TTG CCC GTG-3’ (sense) and 5’-GTC GTT GCT TGT CTC TCC TTG TA-3’ (antisense); TNF-α, 5’-CCC AGA CCC TCA CAC TCA GAT-3’ (sense) and 5’-TTG TCC CTT GAA GAG AAC CTG-3’ (antisense). The PCR products were electrophoresed through a 1% agarose gel. Then Images were captured and the data processed using a Gene Genius Bio-Imaging system (Syngene) with GeneSnap and GeneTools software. Levels of mRNA were calculated as specific mRNA to GAPDH intensity ratios to determine the relative amount of the specific mRNA.
2.10 Measurement of pro-inflammatory factors by ELISA
The amount of pro-inflammatory factors (TNF-α, iNOS, COX-2, and IL-1β) was detected in the conditioned medium from each group using ELISA immunoassay kits according to a previous study [19]. Briefly, after constructing a standard curve, several dilutions of the test sample were assayed in duplicate for each protein using the appropriate kit. Plates were read using a VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a 450-nm wavelength with a reference wavelength of 570 nm. Regression analysis was then performed on standards and sample concentrations were determined with reference to the linear portion of the standard curve.
2.11 Evaluation of blood-spinal cord barrier disruption in vitro
2.11.1 Measurement of transendothelial electrical resistance (TEER)
Measurement of TEER was performed as previously described [8, 25]. The Transwell system, BSCB in virro, was treated with OGD for 3 h followed by re-oxygenation at 3 h, 6 h, 12 h, 24 h, and 48 h, respectively. TEER values in the OGD/R group and control group were measured by Millcell ERS-2 (Merk-Millipore) at each time point. In order to accurately calculate TEER (Ω×cm2), the electrical resistance across the insert membrane (blank resistance) was subtracted from the readings obtained on inserts with cells (sample resistance). This value was multiplied by the surface area of the insert (0.33 cm2).
2.11.2 Transendothelial FITC-dextran permeability evaluation
The experimental protocol of FITC-dextran permeability evaluation was described in detail in our previous studies [8, 26]. Following OGD/R treatment of the BSCB in vitro, FITC-dextran (1 mg/mL) in serum-free DMEM was added to the inserts of the Transwell system and incubated for 30 min at 37℃. FITC-dextran content accumulated in the bottom chamber of the Transwell system was subsequently quantified by transfer of 100 μL of every sample to a black walled-96 well plate for Envision fluorescence microplate assessment.
2.12 Evaluation of blood-spinal cord barrier disruption in vivo
2.12.1 Measurement of BSCB permeability by Evans blue dye extravasation
Permeability of the BSCB was assessed by Evans blue dye extravasation according to previous report[27] with minor modifications. Rats (n=6 in each group) were given a 2% EB saline solution (2mL/kg) by intravenous injection at day 3 after SCI, and then transcardially perfused with normal saline until colorless, transparent fluid flowed out of the right auricle after 2 h of EB circulation. A 15mm length of spinal cord surrounding the T10 injury site was extracted and homogenized in a 50% trichloroacetic acid solution, and cleared by centrifugation 18,000 G for 20 minutes at 4℃. A standard curve with Evans Blue dye (in formamide) was generated and fluorescence intensity was measured in supernatants (in duplicate) using a spectrophotometer (Thermo Fisher Scientific MultiSkan Go) with an excitation wavelength of 620 nm and an emission wavelength of 680 nm. All measurements were converted to dye concentration per tissue weight(μg/g of tissue).
Half of the rats from all groups (n=5 in each group) received a cardiac perfusion with 4% paraformaldehyde after 2h of EB injection. Then 10 μm spinal cord sections were cut with a cryotome (Thermo Shandon). Exosmotic EB in spinal cord was observed using an Olympus bx-60 fluorescence microscope under 550 nm wavelength green light excitation.
2.12.2 Evaluation of spinal cord edema
The water content of the spinal cord can be used to evaluate of spinal cord edema[28]. Spinal cord samples from lesions were excised at day 3 post-injury and immediately weighed (wet weight). Afterwards, the Samples were incubated in a dryer for 24 h at 80℃ and then weighed again (dry weight). The water content of the spinal cord was measured using the following formula[29]: percentage of water = [1- (dry weight/wet weight)]×100.
2.13 Statistical analysis
Data are presented as mean ± standard deviation (SD). Statistically significant differences between data (defined as P < 0.05) were evaluated by Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. For comparison between groups over time, a multiple-measurement ANOVA was used.