MiR-615 Agomir and Hydrogel Preparation
To obtain higher biostability in animal experiments, miR-615 agomir and its negative control-agomir (NC-agomir) were synthesized through a particular chemical modification process. The miR-615 agomir and its negative control were supported by RiboBio (Guangzhou, China).
Pluronic F-127 (Sigma, Aldrich, USA) was prepared as follows: PF-127 hydrogel powder was mixed with 0.1 M phosphate-buffered saline (PBS, pH=7.6) and a 25% (w/v) suspension was obtained. The mixture was shaken gently at 4℃ overnight to fully dissolve into solution and stored at 4℃ for further use after filtrating with a filter (0.22 µm). All procedures were carried out under aseptic conditions.
Establishment of BPA-reimplantation Rat Model
Seventy-five adult female Sprague-Dawley (SD) rats (age, 8-10 weeks; weight, 180-220g) supplied by the Experimental Animal Center of Southern Medical University (Guangdong, China) were used in this study. All procedures using laboratory animals were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and approved by the Administration Committee of Experimental Animals, Guangdong Province, China.
Before the operation, all rats were anesthetized with 1% pentobarbital sodium (40 mg/kg) intraperitoneally. After skin preparation, an incision along the centerline of animal body in the skin and muscles was prepared and the spinal segments from the 4th cervical (C4) to 2nd thoracic (T2) lamina were exposed. Subsequently, a unilateral dorsal laminectomy of the right C5 to C7 laminae was performed to expose the dorsal root. Microscissors were used to remove the right C5-C7 dorsal roots and the corresponding ventral roots were avulsed using a slender glass hook under the stereomicroscope. The C5 and C7 spinal nerves were cut partly, leaving an obvious gap between the nerve roots and spinal cord. The C6 ventral root was replanted to the avulsed site for regeneration. The Terzis grooming test was performed for the upper extremity on the right side at the first day after surgery. All animals scoring 0 indicated the successful establishment of BPA-reimplantation models
Transplantation of MiR-615 agomir and PF-127 Hydrogel into BPA Rats
In order to determine the role of miR-615 in vivo, the BPA rats were randomly divided into five groups: PBS group (n=15), NC-agomir group (n=15), miR-615 agomir group (n=15), miR-615 agomir + Gel group (n=15) and Gel group (n=15). After establishment of BPA model, each group received respective implants (10 µL PBS, 10 µL NC-agomir, 5 µL miR-615 agomir+5 µL PBS, 5 µL miR-615 agomir+5 µL Gel, 10 µL Gel) in the avulsion site of the C6 ventral root through 10 µL Hamilton syringe. The needle remained in reimplanted site for 2 min and then slowly removed. After transplantation, the wound was disinfected and closed. The animals were place on an electric blanket until emergence from anesthesia. All rats were intraperitoneally administrated with penicillin streptomycin (10,000 U/mL; Thermo Fisher Scientific, Waltham, MA, USA) once a day during the first week post-operation to prevent infection.
Gross Specimen Analysis
At the end of 6-week survival period, rats were perfused after overdose anesthesia and both side of C5-7 cervical spinal cord, biceps and coterminous musculocutaneous nerves were carefully separated. The gross specimen was fixed in 4% paraformaldehyde at 4℃ for further observation.
Hematoxylin and Eosin Staining
Bicep paraffin sections were collected at 6 weeks post-operation to perform hematoxylin and eosin (H&E) staining. The sections were dewaxed in xylene, dehydrated by graded ethanol and rinsed with distilled water. Subsequently, the bicep sections were stained with hematoxylin solution and counterstained with eosin solution for 30–60 seconds after differentiation in 1% acid alcohol for 30 seconds and soak in running water. After rinsing with distilled water for 5 min, the sections were again dehydrated by graded ethanol and cleared by xylene. Finally, the sections were mounted onto coverslips with netural resin. More than six fields of each bicep were randomly photographed using a light microscope (20×, ECLIPSE TS100; Nikon, Tokyo, Japan). The muscle fiber diameter was measured and quantified using ImageJ software. The extent of fibrosis was determined by the ratio of fibroblast nuclei number in ipsilateral to contralateral biceps.
To observe the neurons survival in the C6 spinal segments, cross sections of spinal cord were collected and performed Nissl staining at 6 weeks post-surgery. Sections were washed in PBS three times and 5 minutes each time. After dehydration in graded ethanol, samples were stained in 0.05% toluidine blue for 30 minutes and washed in distilled water three times (1 minutes each time). Subsequently, C6 spinal cord sections were differentiated in 95% alcohol for 10 minutes, dehydration in absolute ethanol (I, II; 1 minute each), cleared in xylene (I, II; 5 minutes each) and mounted with neutral resins.
Western Blot Assay
At one week after surgery, the C6 spinal cord segments were quickly dissected. Protein from the C6 segments were extracted using protein lysis buffer. Equal amounts of total protein were electrophoresed on 10% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membrane (0.2 µm; Millipore) and then blocked with 5% defatted milk in TBS with 0.05% Tween (TBST) for 2 hours at room temperature. The membranes were incubated with rabbit anti-LINGO-1 antibody (1:1000, Sigma, USA), NeuN (1:1000; Millipore, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000; Thermo Fisher, USA) overnight at 4℃. After washing in TBST, three times for 5 minutes each, primary antibodies were detected with secondary antibodies as follows for 2 hours at room temperature: horseradish peroxidase goat anti-mouse/rabbit IgG (1:5000; ABclonal, USA). Blots were then washed as described above and visualized by chemiluminescence (ECL) kit (Pierce, USA). The density of the immunoreactive bands was analyzed using ImageJ software. GAPDH was used as the internal control.
The motor function of the upper extremity was evaluated by Terzis grooming test (TGT) weekly. The entire test process was conducted in a spacious and quiet environment. Using a 50 mL syringe to spray bacterial-free water on the neck of rats to elicit grooming behavior of the bilateral upper extremities. The function of the right upper extremity was assess by the following 0-5 point scale: grade 0, the upper extremity of affected side does not response; grade 1, the elbow of affected side can bent, but the upper extremity of affected side cannot touch nose; grade 2, the upper extremity of affected side can touch nose; grade 3, the elbow of affected side can bent and the forelimb of affected side can touch the site below the eyes; grade 4, the forelimb of affected side can touch the eyes; grade 5, the forelimb of the affected side can touch the ears or back of the ears.
Before operation, all animals were evaluated and scored 5. Twenty-four hours after operation, all animals were reevaluated and exhibited a successful grade 0. From the first week post-operation to the endpoint, the Terzis grooming test was performed weekly and recorded by two observers who were blinded to group assignment. If there exists a disagreement, the test was assessed by the third people.
Fluorogold Retrograde Tracing
To observe the spinal motor neurons, the fluorogold retrograde labeling was performed at two days before the 6-week endpoint. Animals from each group were anesthetized by intraperitoneal injection and preoperative skin preparation was performed. Then, their right musculocutaneous nerve was exposed under stereoscopic microscopy and a total of 0.5 µL fluorogold (Sigma-Aldrich) was slowly injected into using a micropump at 4.0 ± 0.5 mm distal to the avulsed site of the lateral cord. At two days after injection, animals were perfused and C5-C7 spinal cord segments were collected. Spinal cord segments were fixed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for two days. Subsequently, 20 µm frozen sections of the spinal segments were collected. Fluorogold-retrograde labeled motor neurons of C5-7 spinal cord ventral horn in affected side were calculated under fluorescence microscopy (Carl–Zeiss Axioplan 2 imaging E, Baden–Wurttemberg, Germany).
Electron Microscopy Observation
In order to observe the axonal structure of musculocutaneous nerve in each group, rats were perfused with 0.9% normal saline followed by a mixture of 4% PFA and 25% glutaraldehyde (v/v = 9:1) and the right musculocutaneous nerve was dissected and extracted for electron microscopy at 6 weeks post-surgery. The right musculocutaneous nerve was fixed in 4% glutaraldehyde (SPI-CHEM, USA) for 4h at 4℃ and washed three times with 0.1 M sodium cacodylate buffer. Subsequently, the tissues were fixed in 1% osmium tetroxide for at least 1 h and washed three times with distilled water. After that, the tissues underwent gradient ethanol hydration to dehydration and embedded with epoxy resin. The tissues were then heated to 60℃ for 48h and cut into 90 nm ultrathin cross section. Finally, the sections were stained with 2% uranyl acetate and lead citrate, and examined using a transmission electron microscope.
To explore whether the motor functional recovery is related to the electromyography changes of the forelimbs, electrophysiology was performed. Firstly, the right biceps brachii and homolateral musculocutaneous nerve was dissected, then the stimulating electrode was hooked in musculocutaneous nerve, while recording electrodes were inserted into the biceps with the depth of 1-2 mm and distance of 3-9 mm. Electrode stimulation intensity was 0.8-1.2 V and electrical activity was performed at three different motor unit locations in each bicep. Stimulation was delivered by a same electrical stimulator from MedLab Biological Signal Collection System (Meiyi Technology Ltd., Nanjing, China).
To observe the condition of neuronal survival, astrocytes activation, and angiogenesis, C6 spinal segments in BPA animals receiving different treatments were determined using immunohistochemistry staining. The primary antibodies used were: rabbit polyclonal anti-NeuN (neuronal marker; 1:200; Millipore), rabbit polyclonal anti- glial fibrillary acidic protein (GFAP) (astrocyte activation marker; 1:200; Thermo Fisher), mouse polyclonal anti-CD31(angiogenesis marker; 1:200; Millipore). The secondary antibody used was mouse anti-rabbit conjugated to Alexa Fluor 568 or goat anti-rat conjugated to Alexa Fluor488 (1:400, Thermo Fisher Scientific). Briefly, after permeabilization in 0.3% Triton X-100 for 15 minutes, the sections were blocked in 10% natural goat serum for 30minutes. Then, sections were incubated with primary antibodies overnight at 4°C. The next day, sections were incubated with secondary antibody in the dark for two hours at 37°C after washing three times in PBS. After repeated washes, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:5000, Cell Signaling Technology) for 15 minutes. Finally, fluorescence Mounting Medium (Dako, Copenhagen, Denmark) was used to mount tissue onto coverslips.
The data were analyzed using One-way analysis of variance (ANOVA) or Two-way analysis of variance using SPSS (Version 20.0). All data were presented as means ± standard deviation (SD). P < 0.05 was considered statistically significant.