Experimental animals were purchased from the Animal Experimental Center of Nantong University, Jiangsu, China. Experimental procedures were conducted in accordance with Institutional Animal Care guidelines of Nantong University and were ethically approved by Administration Committee of Experimental Animals, Jiangsu, China.
Schwann cell isolation, culture, and transfection
Primary Schwann cells were collected from sciatic nerve stumps of neonatal 1-day-old Sprague-Dawley (SD) rats, purified, and cultured as previous described . Cultured Schwann cells were transfected with 20 nM let-7 mimic, 100 nM let-7 inhibitor, and corresponding mimic or inhibitor control (RiboBio, Guangzhou, Guangdong, China) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA).
Total RNAs were isolated from cultured Schwann cells and reversely transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and stem-loop RT primers (Ribobio). Quantitative RT-PCR was performed using QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany) on an Applied Biosystems Stepone real-time PCR System (Applied Biosystems, Foster City, CA). Relative expression levels of let-7 were determined by the ΔΔCt method.
EdU cell proliferation assay
Schwann cells were resuspended, seeded onto 96-well plates at a volume of 100 µL and a density of 2×105 cells/ml, and transfected with let-7 mimic, let-7 inhibitor, or the corresponding controls for 24 hours. 100 µM EdU was added to the cell culture medium and cells were cultured for additional 12 hours. After the fixation with 4% paraformaldehyde, the proliferation rate of Schwann cells was measured with Cell-Light EdU DNA Cell Proliferation Kit (RiboBio). Images were captured with a DMR fluorescence microscope (Leica Microsystems, Bensheim, Germany).
Transwell-based cell migration assay
Schwann cells transfected with let-7 mimic, let-7 inhibitor, or the corresponding controls were resuspended in DMEM medium and seeded onto the upper chamber of a 6.5 mm transwell with 8 µm pores (Corning, Tewksbury, MA) at a volume of 100 µL and a density of 3×105 cells/ml. The bottom chamber of transwell was filled with 500 µL culture medium. After 24 hours incubation, Schwann cells left in the upper surface of the upper chamber were cleaned with a cotton swab while Schwann cells migrated to the bottom surface were stained with 0.1% crystal violet. Images were captured with a Leica DMI3000 B (Leica Microsystems). Migrated cells were dissolved in crystal violet with 33% acetic acid and measured the intensity of the absorbance of crystal violet staining using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Burlington, VT, USA).
Adult, male SD rats was anesthetized and injected with 100 nmol let-7a antagomir (RiboBio) dissolved in 1 ml saline through caudal vein injection. At 5 days after injection, rats were sacrificed and their hearts, livers, spleens, lungs, and kidneys were collected and fixed in 4% PFA. Hetamotylin-eosin staining was performed for histopathological examinations. At 5 days and 4 weeks after injection, rats were subjected to blood sample test. Blood cellular and electrolyte parameters, biochemical parameters, and immunological parameters were measured.
Flow cytometry analysis
Rats were subjected to sciatic nerve crush and 5 nmol let-7a antagomir was injected in each rat at the injury site immediately after nerve injury. Flow cytometry was conducted to measure the proportions of let-7a antagomir positive cells at 1 and 4 days after nerve injury. Rat sciatic nerve stumps were harvested, trypsinized, fixed, and incubated with primary antibodies rabbit anti-S100β antibody (1:100, Abcam, Cambridge, MA), rabbit anti-P4HB antibody (1:100, Abcam), mouse anti-CD68 antibody (1:200, Abcam). Cells were then stained with Alexa Fluor 488 donkey anti-rabbit IgG and subjected to flow cytometry analysis (BD Bioscience, San Jose, CA). The positive ratio of anti-let-7a containing cells was calculated with the following formula: Positive ratio=UR/(UR+LR) ×100%, where UR represented upper right and LR represented left right.
Animal surgery and application of let-7 antagomir
5 nmol let-7a antagomir was dissolved in 20 µL DEPC-treated water and mixed with 10 µL hydrogel (Beaver for Life Sciences, Suzhou, Jiangsu, China). The mixture of let-7a antagomir and hydrogel was injected into chitosan conduit to construct a let-7a antagomir. Adult, male SD rats (Anti-let-7a group) was subjected to 7 mm sciatic nerve transection and their nerve gaps were bridged with let-7a antagomir. Rats in the control group were bridged with chitosan scaffolds containing 5 nmol let-7a antagomir non-targeting control, 20 µL DEPC-treated water, and 10 µL hydrogel.
Immunohistochemistry and immunofluorescence staining
At 4 or 8 weeks after surgery, rat sciatic nerve tissues were mounted onto microscope slides, fixed in 4% PFA, and blocked with 5% goat serum. Sections were incubated with primary antibodies rabbit anti-S100β antibody, rabbit anti-P4HB antibody, mouse anti-CD68 antibody, mouse anti-NF-200 (1:100; Sigma) and secondary antibodies goat anti-rabbit or anti-mouse 488 (1:500; Proteintech, Rosemont, IL) and cy3 (1:200, Proteintech) for axon morphological examination. Sections were stained with α-Bungarotoxin (1:500; Sigma) for motor endplate observation. Immunohistochemistrical images were taken under a fluorescence microscopy (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany).
Compound muscle action potential (CMAP) recording
At 8 weeks after surgery, rats in the control group and the Anti-let-7 group were used for CMAP recording by using a Keypoint 2 portable electromyography (Dantec, Denmark). Recording electrodes were inserted into the mid-belly of gastrocnemius and stimulating electrodes were inserted into the proximal and distal sciatic nerve stumps. An electric stimulus of 5 mV was delivered to evoke CMAP responses. CMAP amplitudes both at the proximal nerve stump and the distal nerve stumps were recorded.
CatWalk gait analysis
At 8 weeks after surgery, rats in the control group and the Anti-let-7a group were used for CatWalk gait analysis. The CatWalk XT system (Noldus Information Technology, the Netherlands) with a high-speed camera that detects digital images at a high-speed rate was used to determine the intensity of rat paws as previously described . Sciatic function index (SFI) was calculated with the following formula: SFI=-38.3[(EPL-NPL)/NPL] +109.5[(ETS-NTS)/NTS] +13.3[(EIT-NIT)/NIT]-8.8, where EPL represented injured experimental site, NPL represented uninjured normal site, ETS represented toe spread, NTS represented the normal toe spread and NIT represented intermediate toe spread. A SFI value of -100 indicated loss of nerve function while a SFI value of 0 indicated normal nerve function.
Muscle weight measurement and Masson trichrome staining
At 4 and 8 weeks after surgery, the anterior tibial muscles and gastrocnemius muscles of rats in each group were collected to determine muscle wet weight ratio. Muscles on both the injured site and the contralateral uninjured site were weighed. The wet weight ratio was calculated by dividing the wet weight of muscle on the injured site to muscle on the contralateral uninjured site. The belly of anterior tibial muscle was collected, paraffin embedded, and stained with Masson trichrome. Muscle fibers were stained in red while collagen fibers were stained in blue.
Student’s t-test and ANOVA by Dunnett’s post hoc test were used to compare the statistical differences among groups. Statistical analysis and histograms were conducted with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA). A p-value < 0.05 was considered significant.