Animals and Groups
Male Sprague Dawley (SD) rats were used in this study. Rats were housed in standard cages at a constant temperature under a 12 hours light/dark cycle, with free access to water and food. The rats weighed 200 ~ 220g at the time of surgery. This study was approved by Institutional Animal Care and Use Committee, Sun Yat-Sen University (Approval NO. SYSU-IACUC-2022-000798). An observer who was blinded to the groups of animals performed the behavioral tests.
The rats were randomly divided into Sham group (sham operated rats treated with saline), CCI group (chronic constrictive injury rats treated with saline) and BoNT/A group (CCI rats treated with BoNT/A). In order to further study the regulatory effect of BoNT/A on Wnt pathway, rats treated with BoNT/A were randomly divided into DMSO group (CCI rats were treated with DMSO after administration of BoNT/A) and Wnt agonist group (CCI rats were treated with Wnt agonist after administration of BoNT/A).
Surgery
CCI surgery was performed according to the method described in the literature [13]. Briefly, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg). After making a skin incision along the lateral surface of the biceps femoris, blunt forceps were inserted into the muscle belly to split the biceps femoris muscle and expose the left sciatic nerve. Next, three ligatures (3 − 0 chromic gut suture) were loosely tied around the nerve at 1-mm spacing. The sciatic nerves in the Sham group were only exposed without ligation.
Drugs Administration
The BoNT/A (Lanzhou Biotechnique Development Co., LTD) was dissolved in normal saline (100U/5ml). Perineural injection of BoNT/A (15 U/kg) or an equivalent volume of normal saline was administrated under the guidance of ultrasound 4 days after surgery. The Wnt agonist BML-284 (AbMole, America) was dissolved in DMSO accordance with the instructions. BML-284 (40 uM in 20ul) or an equivalent volume of DMSO was injected intrathecally at the L5 and L6 intervertebral space in rats by a direct transcutaneous intrathecal injection method as described in the literature [14] at days 5, 8 and 11 after CCI. The drug doses were chosen based on previous research [15].
Ultrasound Guided Perineural Injection
Perineural injection was performed 4 days after surgery under the high-resolution musculoskeletal ultrasound device (Ultrasound System, M-Turbo, Sonosite, America; transducer, L25x/13 − 6 MHz, Sonosite, America). Rats were positioned on their right side under anesthesia. The hind portion was shaved and gel was placed, then the ultrasound transducer probe was placed on the lateral side of hind limb at the mid-thigh, close to the skin incisions for CCI surgery. Small adjustments were usually required to provide optimal image quality. Following this technique, the sciatic nerve was identified as an oval hypoechoic structure underneath the gluteal fascia (Fig. 1. A). Once the nerve was identified, moving the probe as close to the distal knot as possible, then insert a 29G needle in an in-plane approach (Fig. 1. B). When the tip of the needle was considered as close as possible to the nerve without touching it, BoNT/A was injected to separate the nerve from surrounding soft tissues. The ultrasound-guided injection technique referred to the literature [16] and our previous study [17].
Behavioral Studies
pMWT was assessed with the up-down method and a 50% paw withdrawal threshold was determined according to the method described in the literature [13]. A set of von-Frey hair (Aesthesio, USA) of different forces was applied perpendicularly to the ipsilateral hind paws until bent. A positive response was recorded when the rats withdrew, flicked, or licked their paw. A stronger force was used in case of no response and a weaker force was used in case of a positive response. Every hind paw was tested alternately at 5 min intervals. The largest force used was 15 g to reduce animal suffering. At least 4 valid readings were obtained after the first positive response.
A BL-200 radiant heat apparatus (Chengdu Taimeng Technology & Market Corporation, China) was applied to assess pTWL according to the method described in the literature [13]. Rats were placed in a clear plastic chamber with a glass floor, and a radiant heat source beneath the floor was aimed at the plantar surface of the ipsilateral hind paw. The time rats needed to withdraw from the heat stimulus was automatically recorded as withdrawal latency. The intensity of the light source was adjusted to produce a withdrawal latency of 12–15 seconds (s) in the sham group, and the maximum cutoff time was set at 20 s to reduce animal suffering.
The baseline threshold of mechanical allodynia and thermal hyperalgesia of rats were tested before surgery, and no differences was observed among the rats.
C-fiber Evoked Field Potentials Recording
C-fiber evoked field potentials in the spinal dorsal horn were recorded, according to the method described in the literature [18]. Briefly, C-fiber evoked field potentials in response to electrical stimulation of the sciatic nerve were recorded in the spinal dorsal horn (L4 and L5 segments) with a glass microelectrode, which was driven by an electronically controlled microstepping motor (Narishige Scientific Instrument Laboratory, Japan). The amplitudes of C-fiber evoked field potentials were recorded and analyzed by the LTP program. Test stimuli delivered to the sciatic nerve were single square pulses (0.5 ms duration, in 1-min intervals). The intensity of stimulation was 1V、2.5V、5V、7.5V、10V、12.5V、15V、20V、25V, respectively. The distance from the stimulation site at the sciatic nerve to recording site in the lumbar spinal dorsal horn was about 10 cm.
Tissue Harvesting
12 days after surgery, rats were deeply anesthetized with pentobarbital sodium (50mg/kg) by intraperitoneal injection, after which, rats were perfused with 0.9% saline. Ipsilateral sciatic nerves and spinal cord were collected for immunofluorescence staining or western blotting analyses. The samples for western blotting were snap frozen in liquid nitrogen immediately after collection and stored at -80 C. The samples for immunofluorescence staining were fixed in 4% paraformaldehyde at 4℃ for 12 hours.
Immunofluorescence
Samples were dehydrated using graded sucrose (20% and 30% sucrose in 0.1 M PBS) at 4℃ for 3–4 days. Transverse sections of the lumbar spinal cord and the sciatic nerve were cut at 14 mm using a cryostat (Leica, Germany). Sections were blocked in immunol staining blocking buffer (Beyotime Biotechnology, China) at room temperature for 1 h, then washed in 0.01 M PBS. The sections of sciatic nerve were stained with primary antibodies against S100 (1:200, Abcam, UK) and NF200 (1:200, Abcam, UK), while the sections of spinal cord were stained with primary antibodies against β-catenin (1:200, Abcam, UK), SNAP-25 (1:100, Thermo Fisher Scientific, MA) and Iba-1 (1:200, Wako, Germany) for 12–14 h at 4℃, followed by incubation with the corresponding secondary antibodies (1:500, Cell Signaling Technology, USA). Images were captured using a fluorescence microscope. (Nikon, Japan).
Western Blot Analysis
Western blot analysis was performed as literature described [13]. Tissue samples (injured sciatic nerve or lumbar spinal dorsal horn) were lysed in RIPA buffer containing phosphatase and protease inhibitors, then centrifuged at 12000 g for 30 min at 4℃. After centrifugation, the supernatant solution was collected, and the protein concentrations were determined by a BCA assay kit (Invitrogen, USA). The proteins were subsequently boiled and equal concentrations of total protein were loaded on 12.5% SDS-PAGE gels, separated by electrophoresis and transferred to PVDF membranes (Millipore, USA). The membranes were then incubated with the appropriate primary antibodies against MBP (1:1000, Affinity Biosciences, USA), LC3-I/II (1:2000, Proteintech, China), p62 (1:1000, CST, USA), NLRP3 (1:1000, Affinity Biosciences, USA), ASC (1:1000, Proteintech, USA), Caspase-1 (1:1000, Proteintech, USA), IL-18 (1:1000, Affinity Biosciences, USA), pNR2B (1:1000, Millipore, USA), Wnt 3a (1:1000, Abcam, USA), β-catenin (1:1000, Abcam, USA), c-Myc (1:1000, Affinity Biosciences, USA) and β-tublin (1:1000, CST, USA). The membranes were then washed with TBST buffer and incubated with HRP-conjugated anti-rabbit secondary antibodies. Protein bands were visualized with enhanced chemiluminescence Western blot detection reagents (Millipore, USA). Individual protein bands were quantified by densitometry using ImageJ software.
Data analysis
Data are presented as the mean ± SD. Statistical analysis of all data was conducted with SPSS 17.0. The results from the behavioral tests and C-fiber evoked field potentials recordings were analyzed with repeated-measure analysis of variance (ANOVA). The comparisons of multiple groups were analyzed by one-way ANOVA with post hoc least significant difference (LSD) test. Student’s t test was used for comparisons between two groups. A two-tailed p < 0.05 was regarded as statistically significant.