Experimental animals and groups
The animal care and use committee of China Medical university approved the experimental procedures (IACUC no. 20190129). Healthy adult Sprague-Dawley (SD) rats weighing 220 to 250 g were purchased from the Laboratory Animal Center of China Medical University (Shenyang, China). The rats were reared at 22 to 24 °C, with 40–60% relative humidity, a 12-h light-dark cycle, and ad libitum access to water and food. The rats (n = 60) were allocated randomly into three groups: A sham group (exposure of the sciatic nerve with no transection and the RF needle was placed but no energy was delivered), the SNI group (exposure of the sciatic nerve with partial transection and the RF needle was placed but no energy was delivered), and the PRF group (the exposure of the sciatic nerve with partial transection and the RF needle was placed and energy treatment was provided).
Behavioral tests
The rats were kept in separate metal mesh-bottomed, transparent cages, and allowed to acclimate for 2 h to test the basal paw withdrawal mechanical threshold (PWMT). The previously described up-down method, using von Frey hairs, was used to assess the PWMT16. The first application comprised the 0.4 g stimulus, which was in the middle of the series. If a positive response (paw licking, shaking, or lifting) occurred within 5 seconds, a thinner filament was applied. In the case of a negative response, a thicker filament was used.
SNI model induction
Rat SNI surgery was executed following a previously described method 17. The rats were anesthetized via intraperitoneal injection of 10% chloral hydrate at 400 mg/kg. Then, an incision was made in the skin of the lateral thigh, followed by blunt dissection of the biceps femoris muscle to reveal the right sciatic nerve together with its three terminal branches (the tibial, common peroneal, and sural nerves). 5-0 silk was used to ligate the tibial and common peroneal nerves tightly. Then, transection of the nerves was performed distal to the ligation, removing a 2–4 mm section of the nerve fiber. Contact or stretching of the intact sural nerve was carefully avoided. The Sham group underwent this procedure without nerve injury. After surgery, the rats were returned to their original cages and environments.
Application of PRF
In the PRF group, a 21G PRF needle was inserted into the right L5 intervertebral foramen and placed in contact with the surrounding non-neural tissues. RF pluses were generated by the RF instrument (PMG-230, Baylis Medical Inc., Montreal, Canada) using the following settings: 2 Hz, 42 °C, and 50 V for 120 seconds. No energy was delivered through the needle in the sham and SNI groups. In a preliminary experiment we found that the peak of PWMT value and CCL2 expression were detected on day 7 after nerve injury; therefore, PRF treatment was administered on day 6.
Specimen collection
The rats were placed individually in a transparent sealed box for euthanasia. Isoflurane was administered into this box at 5 ml/kg and the rats were left into the box to breath in the isoflurane until they died (approximately 1–2 min). We isolated the l4-6 spine cord tissues from the rats, which were placed at -80 °C until western blotting analysis.
Western blotting
The spinal cord of the lumbar (L4-5) ipsilateral to the lesion was collected. Samples were ground in Radioimmunoprecipitation assay (RIPA) on ice. A BCA (bicinchoninic acid) Protein Assay kit (Pierce, Rockford, IL, USA) was used to determine the protein concentration. The samples were subjected to SDS-PAGE and then the separated proteins were electroblotted onto a polyvinylidene fluoride membrane.
5% Skimmed milk was used to block the proteins on the membrane for 2 h at room temperature. The membrane was washed using Tris buffered saline Tween (TBST) three times for 5 min each, before being incubated TBST-diluted primary antibodies at 4 °C overnight. The primary antibodies were anti-Phospho-NF-κB p65(Ser536) antibody (1:1000, 3303; Cell Signaling Technology, Danvers, MA, USA), anti-CCL2 antibody (1:2000, ab25124; Abcam, Cambridge, MA, USA), and anti-β-actin (1:4000, 20536-1-AP, Proteintech, Rosemont, IL, USA). The membranes were then washed in TBST (5 min, three times) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:10000, SA00001-2, Proteintech) for 2 h at room temperature. The membranes were washed again in TBST (10 min, three times), and then the immunoreactive proteins were visualized using electrochemiluminescence (ECL) reagents, and results were analyzed using the Gel Doc XR and ChemiDoc XRS system (Biorad, Hercules, CA, USA). The levels of the immunoreactive proteins were assessed quantitively using the grayscale values.
Statistical analysis
All statistical analyses were accomplished using SPSS 18.0 (IBM Corp., Armonk, NY, USA). All date are shown as the mean ± standard error. Repeated-measures analysis of variance (ANOVA) was used to analyze the PWMT. One way ANOVA was used to analyze the levels of CCL2 and phospho-NF-κB p65. Statistical significance was accepted at P < 0.05 was considered significant.