A total of 120 healthy SD rats were used for this study. These animals were randomly allocated into the following three groups: an EGCG group, an Avulsion (control) group, and Sham group. There were 40 rats in each group. The animals in each group received daily doses of 100 mg/kg EGCG (Hangzhou Gosun Biotechnologies Co., Ltd., China) as described in our previous studies(26) or normal saline i.p. from day 1 to day 7. The animals received treatment immediately following the injuries. Five rats were randomly selected from each group on days 3, 7, 14 and 28 post-surgery for the harvesting of spinal cord samples for Nissl staining, immunohistochemistry and western blot analysis. All the experiments were performed in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments were conducted in accordance with the relevant guidelines and regulations. The experimental studies followed the recommendations in the ARRIVE guideline. All experiments were approved by the Ethics Committee of the Fourth Affiliated Hospital of China Medical University. All of the experimental animals were housed in the same conditions (i.e., a controlled temperature of 22°C on a 12-h light/dark cycle) and food and water were provided to the rats.
The root avulsions of the left brachial plexus were performed according to the procedures described in previous publications(27-31). Briefly, the animals were anesthetized with 350 mg/kg of 10% chloral hydrate via intraperitoneal injections. Following anesthesia, the skin was shaved and cleaned with povidone iodine, and the surgeries were performed under sterile conditions.The left brachial plexus was exposed, and the left cervical C5- T1 nerve roots were isolated under a surgical microscope in the supine position. Extravertebral root avulsion was performed by pulling the spinal nerves out one by one with microhemostatic forceps. The avulsed ventral and dorsal roots, together with the dorsal root ganglia, were cut away from the distal ends of the spinal nerves and confirmed under the microscope. For the sham-operated controls, similar procedures were performed until the left brachial plexus was exposed and identified, but not damage to the nerves was inflicted. The surgical wounds were closed in layers. The animals were allowed to recover, and they were returned to their cages upon awakening.
Perfusion and tissue preparation
At the end of the survival time, the animals were deeply anesthetized with an overdose of 10% chloral hydrate and perfused transcardially with 100 ml of salinefollowed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. After perfusion, the C6- C8 segments were quickly removed and postfixed for 24 hours in the same fixative at 4°C overnight. The C6- C8 segments were defined as the region between the uppermost root of the C6 nerve and the lowermost root of the C8 nerve of the contralateral cord. The fixed segments were dehydrated and embedded in paraffin sections. Serial 10-μm thick sections of the segments were cut transversely. Every fifth section from each animal was used for Nissl staining and immunohistochemisty analysis.
After deparaffinization and rehydration, the sections were stained with warmed 0.5% cresyl violet solution (10 minutes) as described(32). Next, the sections were dehydrated in increasing concentrations of ethanol and cleared via immersion in xylene for 2 min before cover slipping with Permount. Both sides of the motor neurons located in the anterior horn that had maximum diametersof no less than 20 μm and contained a well-defined nucleolus and a soma rich in Nissl bodies, were counted by two investigators who were blinded to the grouping(33). The numbers of surviving motoneurons on the injured side were described quantitatively as the percentage of the surviving motoneurons on the contralateral side in the same section(28,29,33).
An avidin-biotin kit was used for the immunohistochemical staining. Briefly, the sections were deparaffinized and treated with 3% H2O2 for 15 min to block the endogenous peroxidase. The sections were exposed to normal bovine serum for 30 min and then incubated with the primary antibodies overnight at 4°C. These antibodies included rabbit monoclonal anti-p-JNK antibody (diluted 1:50, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-p-c-Jun(Ser73) antibody (diluted 1:50, Cell Signaling Technology, Danvers, MA, USA) and rabbit monoclonal anti-caspase-3 antibody (diluted 1:200, Cell Signaling Technology, Danvers, MA, USA). After washing 3 times with 0.01M PBS (PH 7.4), the sections were incubated with the appropriate secondary antibody (Boster, China) for 15 min at 37°C. The immunolabeling was visualized as brown using diaminobenzidine, and counterstaining was performed with hematoxylin. Sections stained without primary or second antibodies served as negative controls. The motoneurons with yellowish-brown nuclei in the p-JNK or p-c-Jun(ser73) immunohistochemical sections were counted as a positive cells, and motoneuron with yellowish-brown cytoplasm in the caspase-3 immunohistochemical sections were counted as positive cells. The results were counted by two observers (who were blind to the groups) and averaged to obtain the final countfor each section. The numbers of IHC-positive motoneurons in each rat are expressed as the total numbers in 10 serial IHC-stained sections(27,34).
For the western blot assays, the spinal cord (C6-C8) was quickly removed and preserved in liquid nitrogen for further analysis. After homogenization in RIPA buffer, the samples were centrifuged at 12,000 g for 30 min at 4°C. The protein concentrations of soluble materials were determined by the Coomassie G250 binding method. The protein samples were separated on 10% polyacrylamide gels containing 0.1% SDS, followed by transfer to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 2 h and then incubated with primary anti-JNK antibody (dilution 1:000,Santa Cruz Biotechnology, Burlingame, CA, USA), anti-p-JNK antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA), anti-c-Jun antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA), anti-p-c-Jun(Ser73) antibody (dilution 1:000, Cell Signaling Technology, Danvers, MA, USA) or β-actin (dilution 1:2000,Santa Cruz Biotechnology, Burlingame, CA, USA) at 4°C overnight. The appropriate secondary antibodies (goat anti-rabbit or -mouse IgG conjugated with horseradish peroxidase) were subsequently used at room temperature for 1 h. Finally, the EC3 Imaging System (UVP Inc., Upland, CA, USA) was used to quantify the p-JNK, JNK, p-c-Jun, c-Jun and β-actin protein bands. Quantification was performed using an ImageJ software (NIH, Bethesda, MD, USA) on a computer.
The data are presented as the mean ± SD and were analyzed using the SPSS 20.0 software. The statistical analyses were performed with one-way analyses of variance (ANOVA) followed by Student-Newman-Keuls tests. P values less than 0.05 were considered significant. The data quantification and analysis were performed by two independent persons who were blinded to the injury.