Sprague-Dawley (SD) rats were obtained from the Experimental Animal Center of Nantong University. Animal experiments were conducted based on Institutional Animal Care Guidelines of Nantong University and approved ethically by the Administration Committee of Experimental Animals, Jiangsu, China.
Schwann cell culture and treatment
Primary Schwann cells were collected and cultured as previously described . Briefly, cells isolated from neonatal SD rat sciatic nerves were purified with anti-Thy1.1 antibody (Sigma, St. Louis, MO, USA) and rabbit complement (Invitrogen, Carlsbad, CA, USA) and then cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco), 1% penicillin and streptomycin (Invitrogen), 2 μM forskolin (Sigma), and 10 ng/ml HRG (R&D Systems Inc., Minneapolis, MN, USA). For Btc knockdown, cultured primary Schwann cells were transfected with siRNA segments targeting Btc (siRNA sequences: siRNA-1: TCTTCGGAAACATCGCAAA, siRNA-2: CAAGCATTACTGCATCCAT, and siRNA-3: GAAACCAATGGCTCTCTTT) or a non-targeting negative control (random sequence, RiboBio, Guangzhou, Guangdong, China) for 48 hours using Lipofectamine RNAiMAX transfection reagent (Invitrogen). For Btc protein exposure, Schwann cells were pre-treated with 10 ng/ml Btc recombinant protein (100-50-20, PeproTech, Rocky Hill, NJ, USA) dissolved in 0.1% BSA or 0.1% BSA control for 24 hours.
Cultured Schwann cells fixed with 4% paraformaldehyde and exposed to primary antibodies rabbit anti-Btc (1:100, PA5-76664, Invitrogen) and mouse anti-S100 (1:100, ab4066, Abcam, Cambridge, MA, USA) followed by reaction with secondary antibodies Alexa Fluor 488-conjugated anti-rabbit (1:400, ab150077, Abcam) and Cy3-conjugated anti-Mouse (1:400, SA00009-1, Proteintech). Cell nuclei were stained with DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Images were captured using Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH, Jena, Germany).
Real-time RT-PCR was performed using RNAs isolated from cultured Schwann cells and SYBR Green Premix Ex Taq (TaKaRa) on a StepOne Real-time PCR System (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression of Btc was measured using the 2-ΔΔCt method. Primer sequences were: Btc (forward) 5’-TCTCCAGTGCGTGGTGG-3’ and (reverse) 5’-CGAGAGAAGTGGGTTTTCGATT-3’ and GAPDH (forward) 5’- ACAGCAACAGGGTGGTGGAC-3’ and (reverse) 5’- TTTGAGGGTGCAGCGAACTT-3’.
A total of 6 × 104 Schwann cells transfected with Btc siRNA or siRNA control, respectively, were cultured in DMEM for additionally 24 hours. Schwann cell culture supernatants were collected and passed through a 0.22 μm filter (Millipore, Billerica, MA, USA). The amount of secreted Btc protein was determined with a Btc ELISA Kit (ARG81876, Arigo biolaboratories, Hsinchu, Taiwan, China) according to manufacturer’s instructions using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Burlington, VT, USA).
Transwell migration assay
Schwann cells suspended in DMEM at a density of 4 × 104 cells/ml were transferred to the upper chamber of a Transwell with 8 μm pores (Costar, Cambridge, MA, USA). The bottom chamber of the Transwell was filled with cell culture medium with 10% FBS to drive cell migration. The upper surface of the upper chamber was cleaned after 24 hours and the bottom surface of the upper chamber was stained with 0.1% crystal violet. Stained crystal violet was dissolved with 33% acetic acid to measure absorbance using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek). Images were captured using a DMI 3000B inverted microscope (Leica Microsystems, Bensheim, Germany).
Wound healing assay
Schwann cells suspended in cell culture medium at a density of 2 × 104 cells/ml were transferred to a mold chamber with a 1 mm wide insert. Insert was removed after cell reaching > 95% confluent to allow cell migration. Remaining cleaned areas were captured using a DMI 3000B inverted microscope (Leica Microsystems) 9 hours later. Three cleaned areas in each image were randomly selected to calculate relative cleaned areas using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA).
Briefly, dorsal root ganglia were obtained from neonatal SD rats, cut into pieces, and digested with collagenase I. Isolated cells were re-suspended in 15% BSA and subjected to centrifuge to collect pellets. Collected primary neurons were cultured in Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco), 2 mM L-glutamine (ThermoFisher Scientific, Waltham, MA, USA), and 1% penicillin and streptomycin (Invitrogen) in a humidified 5% CO2 incubator 37°C.
Neurons were co-cultured with Schwann cells transfected with Btc siRNA or siRNA control using Transwell co-culture assay as previous described . Briefly, Schwann cells were seeded onto the upper chamber and neurons were seeded onto a PLL–coated glass slide placed on the bottom chamber. After 24-hour co-culture, neurons were fixed with 4% paraformaldehyde and immunostained with primary antibody mouse anti-neurofilament-200kD (NF-H; 1:200, N2912, Sigma) followed by secondary antibody Cy3-conjugated Goat anti-Mouse IgG (1:400, SA00009-1, Proteintech). Cell nuclei were stained with DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Images were captured using Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH). Axon length was determined using Image J (National Institutes of Health, Bethesda, MA, USA).
In vivo experiments
Adult male SD rats weighting 180-220g were subjected sciatic nerve crush and Btc recombinant protein injection to evaluate the in vivo effects of Btc. After anaesthetization, sciatic nerves of SD rats were exposed and crushed with a forcep. After sciatic nerve crush injury, 100 ng Btc recombinant proteins dissolved in 0.1% BSA was diluted in 5 µl saline. Btc or equal volume of saline was injected into the epineurium at the crush site by using a microsyringe. Rat sciatic nerve segments were collected at 4 days after crush injury, fixed with 4% paraformaldehyde, cut with a cryostat (Leica Microsystems, Bensheim, Germany) into 12 µm thick nerve sections, immunostained with primary antibodies rabbit anti-SCG10 (1:500, NBP1-49461, Novus Biologicals, Littleton, CO, USA) and mouse anti-neurofilament-200kD (NF-H; 1:200, N2912, Sigma) overnight at 4°C, and exposed to secondary antibodies Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:400, ab150113, Abcam, Cambridge, MA, USA) and Alexa Fluor 555-conjugated anti-rabbit secondary antibody (1:400, ab150078, Abcam) for 2 hours at room temperature. Images were captured using Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH).
Another group of SD rat were subjected sciatic nerve transection to generate a 6 mm nerve gap. A silicone tube filled with 100 ng Btc recombinant protein dissolved in 0.1% BSA and 8 µl saline or equal volume of saline was implanted to bridge the nerve gap. Rat sciatic nerve segments collected at 10 days after transection were immunostained with primary antibodies mouse anti-NF-H and rabbit anti-S100β (1:200, S2644, Sigma) and secondary antibodies Alexa Fluor 488-conjugated anti-mouse secondary antibody and Alexa Fluor 555-conjugated anti-rabbit secondary antibody. Cell nucleus was stained with Hoechst 33342 (ab228551, Abcam). Images were captured using Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH). Axon elongation length and Schwann cell migration distance were calculated using Zeiss Microscopy Software ZEN 2.3.
Summarized outcomes were demonstrated as means ± SEM from 3-4 paired experiments. Student’s t-test or multiple t-tests was used for comparisons. A p-value < 0.05 was considered as significantly different.