Male C57BL/6J mice (eight weeks, 25–30 g) were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. IL-17a mutant mouse were supplied by Jackson Laboratory (No. 016879, USA). Five mice were housed in every plastic cage with food and water available ad libitum. The artificial light was provided in a 12:12 h (light/dark) cycle from 7 AM and room temperature was kept at 22 ± 1℃. All animals were sacrificed by an overdose of urethane (25%, 2.5 g/kg, i.p.) after the experiments. These experiment protocols were approved by the Fudan University Animal Care and Use Committee, and were conducted in accordance with the policies issued by the International Association for the Study of Pain (IASP).
2.2. Spared nerve injury (SNI)
All mice were anesthetized with 2% isoflurane and the incision was made at left mid-thigh level as described by Bourquin AF et al. . Two of the three branches of the sciatic nerve (the tibial and common peroneal nerve) were ligated and transected together, while the sural nerve is left intact. After a tight ligation of both nerves was performed, a 1–2 mm section of the two nerves was removed. Muscle and skin were closed in two distinct layers with silk 6 − 0 suture. The sham procedure was consisted of the same surgery without ligation and transection of the nerves.
2.3. von Frey test
A series of von Frey filaments (0.16, 0.4, 0.6, 1.0, 1.4, 2.0 g, Stoelting, USA) were used on ipsilateral hind paws to assess the mechanical sensitivity as described previously . Mice were habituated to the testing environment for 2 h every day at 2–3 days before the behavioral test. They were confined under the 8 cm × 8 cm × 4 cm plexiglass boxes placed on an elevated iron wires floor with 2 mm grids. In ascending order of force, an appropriate von Frey filament was applied on the plantar surface of the hind paw. Different filament was applied 2 s x 5 times between every 15 s. The mechanical threshold was defined as paw withdrawal threshold (PWT), which was the lowest force produced 3 withdraw responses out of 5 applications.
2.4. Allodynia Score
As described by Cheng et al. , dynamic mechanical hypersensitivity was tested by light stroking from heel to toe (velocity: about 2 cm/s) along the external lateral side of the injured hind paw using a trimmed blunt paintbrush (the width of tip: 1 mm, the length of tip: 6 mm). The allodynia scores were graded according to the following 4-point scale: 0, a very fast movement, lifting the stimulated paw for less than 1 s; 1, sustained lifting (more than 2 s) toward the body or a single gentle flinching of the stimulated paw; 2, one strong lateral paw lift, above the level of the body or a startle-like jump; 3, multiple flinching responses or licking of the affected paw. We repeated the stimulation three times at 30 s intervals and obtained an average score for each mouse.
2.5. Drug administration
Mice were briefly anesthetized with 1–2% isoflurane. For lumbar puncture injection, rabbit anti IL-17 antibody (Sigma, PRS4887, USA), normal rabbit IgG (R&D, AB-105-C, USA) or IL-17 recombination (R&D, 7956-ML/CF, USA) was delivered to the cerebral spinal fluid with a 31-gauge needle between the L4 and L5 vertebrae. No more than 10 µl of drug was administered over a period of 5 min. Sterile 0.01 M PBS was used as the vehicle control.
Under anesthesia with urethane (25%, 1.5 g/kg, i.p.), the L4-L6 spinal cord was removed after transcardial perfusion with normal saline and 4% paraformaldehyde in 0.1 M PB. Thereafter, the tissue was post-fixed for 2–4 h at 4°C followed by dehydration in gradient sucrose (10%-20%-30%) for 24–48 h at 4°C. Transverse spinal cord sections were 35 µm made by a cryostat (model 1900, Leica, Germany). After blocked by 10% normal donkey serum with 0.3% Triton X-100 for 2 h at 4°C, the sections were incubated with the primary antibodies of rabbit anti-IL-17 (1:50, Sigma-Aldrich, PRS4887, USA), rabbit anti-IL-17 RA (1:50, Santa Cruz, SC-30175, USA), goat anti-Iba-1 (1:500, Abcam, ab5076, UK), mouse anti-GFAP (1:2000, Sigma-Aldrich, G6171, USA), or mouse anti-NeuN (1:2000, Millipore, MAB377, USA) overnight at 4°C. Then the sections were labeled with secondary antibodies of donkey anti-goat Alexa Flour 546 (1:200, Invitrogen, A-11055, USA), donkey anti-rabbit Alexa Flour 488 (1:200, Invitrogen, A-10040, USA) or donkey anti-mouse Alexa Flour 546 (1:200, Invitrogen, A-10036, USA) for 2 h at room temperature in the dark. The images were captured by a confocal laser-scanning microscope (FV1000; Olympus, Tokyo, Japan).
2.7. Recording of spinal LTP in vivo
According to the previous study , mice were anesthetized with urethane (1.5 g/kg, i.p.). For exposing the lumbar enlargement, a laminectomy was performed at vertebrae T13 to L1. A PE-5 tube was inserted into the gap of L4 and L5 vertebrae and extended to the cavum subarachnoidale space near the lumbar enlargement. Approximately 2 µl sterile normal saline (NS) was filled in the catheter, and the drugs or vehicle (10 µl) were applied in 1 min followed by 2 µl NS. The left sciatic nerve was separated free for bipolar electrical stimuli. A feedback-controlled heating blanket was used for keeping the colorectal temperature at 37 ± 0.5°C.
The field potentials were recorded with glass microelectrodes (3–5 MΩ impedance) in 100–300 µm from the surface of the L4-L5 spinal cord segments. A single rectangular pulse (2 x C-fiber threshold, 0.5 ms, 60 s interval) was applied on the ipsilateral sciatic nerve. Followed the stable recordings more than 30 min, spinal LTP was induced by the conditioning tetanic stimulation (4 x C-fiber threshold, 0.5 ms, 100 Hz, 4 trains of 1 s duration, 10 s interval) delivered to the sciatic nerve. As a control, the sham group was not applied with conditioning tetanic stimulation. The signals were amplified by a microelectrode AC amplifier (A-M System, USA), and converted into a digital signal by CED systems (A/D converter Micro 1401 mkⅡ, UK).
The responses of 10 consecutive test stimuli calculated off-line by Spike 2 version 6 were averaged and normalized to mean amplitudes of C-fiber evoked field potentials in the first 30-min.
All data were expressed as mean ± SEM and tested using Student’s t test, one-way or two-way ANOVA followed by post hoc Student-Newmann-Keuls test. The criterion for statistical significance was P < 0.05.