Male MRL/MPJ and C57BL/6 J mice aged 8–12 weeks at surgery were used in this study. Mice were originally purchased from Jackson Laboratory and multiplied in the animal facility of Zhejiang University. They were housed in ventilated cages in a clean room with controlled humidity (45- 65%) and temperature (22–24℃) on a standard 12-h light–dark cycle (lights on at 8 am). Regular chew and clean water were available ad libitum. All experiments were in accordance with guidelines of The International Association for the Study of Pain  and were approved by the Zhejiang University Animal Experimentation Committee. Mice were assigned into different groups randomly. Efforts were made to minimize the animal use and suffering.
Partial sciatic nerve ligation (PSL)
Mice were anesthetized by isoflurane inhalation (4% for induction and 2% for maintenance). PSL surgery was done as reported . Briefly, the left sciatic nerve was exposed and under the surgical microscope, an 8/0 silk suture was inserted into the dorsum of the nerve trunk with a 3/8 curved, reversed-cutting needle and was tightly ligated so that the dorsal 1/3–1/2 of the nerve thickness was trapped in the ligature. The sciatic nerve in the sham group was exposed but left intact. The wound was closed in layers.
Partial infraorbital nerve transection (p-IONX)
The p-IONX surgery was performed as previously described . In brief, under isoflurane anesthesia, the mouth of the mouse was opened by pulling the lower and upper fore teeth with a rubber thread. Under the surgical microscope, a 2–2.5 mm incision was made from the gingival mucosa of the first molar on the left side to expose the deep branches of the ION. Approximately 1 mm of the nerve fibers was excised with a pair of microsurgical scissors and an absorbent gelatin sponge was placed on the wound. The nerve branches of mice in the sham group were exposed but left uninjured.
All behavioral tests were conducted during the light phase (from 9 am to 5 pm). Mice were habituated in the testing room for at least 30 min before the test. Examiners were blinded to the groups of mice. The testing room was sound-proof and the illumination intensity was maintained at 80 Lux with temperature at 24–26 °C. After each trial, the mice were put back into their home cages and the experimental apparatus was cleaned with 75% alcohol to eliminate the odor that may affect animal behavior. Anxio-depressive behaviors and evoked pain-like behaviors were assessed on different days after surgery (postoperatively, PO), while the latter were also tested 2 days before surgery (baseline, BL).
Elevated plus maze (EPM) test
The apparatus used for the EPM test was a cross-shaped device consisting of an intermediate platform region (5 × 0.5 cm2) and two pairs of open arms and closed arms (25 × 5 cm2) connected thereto. The closed arms are surrounded by high walls (16 cm), whereas the open arms have slight walls (0.5 cm). The entire maze was elevated to a height of 50 cm. The mouse was first placed in the intermediate region at the same position with its head toward open arms, and its behavior in the device was then recorded by a camera for 5 min and the time spent by the animal in each arm was analyzed by ANY-maze .
Light dark box (LDB) test
The apparatus for LDB test was a rectangular box comprising three connected chambers: a light open chamber with white walls (~ 100 Lux, 15 × 20 × 25 cm3), a dark covered chamber with black walls (~ 5 Lux, 30 × 20 × 25 cm3) and an intermediate small chamber (5 × 20 × 25 cm3). The apparatus was placed on the floor. For every trial, the mouse was placed at the same position in the light box. Animal behavior in the apparatus was then recorded for 5 min by a digital camera and the time the animal spent in each box was analyzed by ANY-maze .
Open field test (OFT)
The OFT was performed in an open rectangular box with a bottom edge length of 50 cm and a height of 60 cm. The box was placed directly on the floor. After placing the mouse in the intermediate region of the box, animal behavior was recorded by a camera for 5 min. The bottom of the box was divided into the central (25 × 25 cm2) and the rest peripheral region by ANY-maze. The time spent by the animal in each region as well as the moving distance in the box were calculated .
Forced swimming test (FST)
A glass cylinder with a height of 25 cm and a diameter of 10 cm filled with water at 25 °C in a depth of 10 cm was used for FST. The whole test was fulfilled in two days. The mice were placed in the water for 15 min on the first day and 6 min on the next day. The animal behavior was recorded with a camera. After the time had elapsed, the animals were removed from the cylinder and dried before being returned to the home cage. The immobility (or floating) time within the last 4 min on the second day was analyzed by ANY-maze .
Tail suspension test (TST)
The apparatus for the TST was a steel frame with a suspended chain on the middle bar. A black plastic plate was placed behind the chain to provide optical contrast. At the beginning of the test, the mouse was fixed to the chain with a tape at 1.5 cm to the distal end of the tail and was suspended 15 cm above the ground. The camera was then turned on to record the animal behavior for 6 min. Immobility was defined as no physical struggles and the total immobility time from the second to last minute was calculated manually with a stopwatch .
Evoked pain-like behavior test
To test pain-like behaviors evoked by noxious thermal stimulation of the left vibrissal pad, the mouse was placed in a small cage made with metal mesh ceiling and wood bottom (10 × 5 × 5 cm3). The laser with a pulse width of 150 ms generated by an infrared diode laser machine (LYPE, China) was shot at the left vibrissal pad with the guidance of a red aiming beam. Evoked pain-like behaviors included scratching the vibrissal pad, shaking head or turning around the body. The laser intensity (A) was started from 15 A and incremented by 1 A. Each intensity was tested 3 times with an interval of at least 5 min. The threshold was defined as the intensity that induced pain-like behaviors at least 2 times out of 3 trails . To test the pain-like behaviors evoked by stimulation of the hind paw, the mouse was placed in a plastic cylinder (height of 9 cm and diameter of 8 cm) with metal mesh bottom. Mechanical stimulation was applied to the plantar surface of the left hind paw by a set of von Frey hairs numbered 1–9 with bending force 0.008, 0.02, 0.03, 0.07, 0.16, 0.4, 0.6, 1.0 and 1.4 g, respectively . The test was started from hair No.5 (0.16 g) and progressed according to an up-down method. Each test constituted a constant number of five stimuli with an interval of at least 5 min. Each stimulus lasted 2 s. A sharp withdrawal or an immediate flinch of the hind paw indicated a positive response. The final number of von Frey hair was determined by adding 0.5 to the number of the fifth test if it evoked responses or reducing 0.5 if it did not. The paw withdrawal threshold (PWT) to mechanical stimulation was calculated by the equation: PWT force = 10(x*F+B) (F is the final number of von Frey hair, x = 0.240, B = − 2.00). Noxious thermal stimulation was supplied by a laser pulse with a wave width of 200 ms that was shot at the plantar surface of the left hind paw. The laser intensity was increased by 1A step and each intensity was tried 3 times with an interval of at least 5 min. The PWT was defined as the intensity that evoked withdrawal responses at least 2 times out of 3 trails.
Conditioned place preference (CPP) test
The CPP test was performed in a standard three-box apparatus consisting of two large boxes with the same size (45 × 40 × 35 cm3) and a middle connecting channel (40 × 9 × 35 cm3). According to previous study , the test was divided into consecutive three phases, i.e. habituation or preconditioning, conditioning and testing. During the habituation phase for two days, the mice were placed in the apparatus for 30 min each day and were allowed to move freely with access to all three boxes. On the second day, the movements of each animal in the first 15 min were recorded and analyzed with ANY-maze to verify the absence of preference for each box. Animal spending > 80% or < 20% of the total time in any box were excluded from further testing. A four-day conditioning experiment was then performed. The mice receiving ds-HMGB1 (HMGBiotech Srl, Italy), the pro-inflammatory from of HMGB1 or anti-HMBG1 monoclonal antibody (mAb), the mAb against HMGB1 we developed to neutralize the secreted HMGB1 , were placed into the box on one side for 30 min without access to other boxes on the third and the fifth days, while those receiving vehicle were placed into the other side on the fourth and the sixth days for 30 min. On the seventh day, the mice were placed in the middle box with free access to other boxes and their movements were recorded for 15 min. The time that the animal spent in each box and the percentage occupancy (preference) and shifts in occupancy for one side were analyzed by ANY-maze and compared with that on the second day.
Mice were perfused intracardially with ice-cold 0.9% saline after anesthetized with intraperitoneal (i.p.) injection of pentobarbital (100 mg/kg). The medial prefrontal cortex (mPFC), basolateral amygdala (BLA) and medulla oblongata were removed quickly and frozen in -80 °C fridge. For western blot analysis, frozen tissues were homogenized and lysed in homogenization buffer on ice. Proteins in nucleus and cytoplasm are separated by nuclear and cytoplasmic protein extraction kit (Beyotime, China). Protein concentrations were determined by a bicinchoninic acid assay kit. Protein samples (80 μg) were separated by SDS-PAGE gel electrophoresis and electro-transferred onto a nitrocellulose membrane. After blocking with 5% fat-free milk, the membranes were then incubated with rat anti-HMGB1 mAb (1:1000) and mouse anti-β-tubulin (1:1000; Boster, China) or histone3 (1:1000; CST, USA) polyclonal antibody at 4 °C overnight followed by secondary antibodies conjugated with HRP against either rat or mouse IgG (1:5000; Cell Signaling Technology, USA) for 2 h. Images were captured and quantified by Quantity-One software (Bio-Rad, USA). The ratios between HMGB1 and β-tubulin were calculated and then normalized to the values measured in the control group.
Mice were perfused intracardially with ice-cold saline followed by phosphate-buffered 4% paraformaldehyde (pH 7.4) after anesthetized with pentobarbital. The brain was removed and post-fixed overnight in the same fixative and dehydrated in 30% sucrose for 48 h at 4 °C. Coronal brain sections were cut at 20 μm by a cryostat (NX50, Thermo, USA). For immunohistochemical staining, the sections containing the mPFC, BLA, ventral hippocampus (vHPC) and parabrachial nucleus (PBN), four nuclei conventionally implicated in anxiety, were incubated in 0.1% Triton X-100 for 15 min and in 5% donkey serum for 2 h firstly, and then were incubated with rabbit or rat anti-mouse primary antibodies against S100b (1:200, Abcam, UK) /NeuN (1:500, Millipore, USA) /Iba1 (1:500, Wako, Japan) or HMGB1 (1:500) overnight at 4 °C, then with anti-rabbit IgG-Alexa Fluor 488 or anti-rat IgG-Alexa Fluor 594 (1:2000, Invitrogen, USA) for 2 h at room temperature (RT). After repeated washing, the sections were then covered with glass coverslips and fluorescent images were captured by a fluorescence microscope (BX51, Olympus, Japan). The analysis of fluorescence intensity and cell counting were performed by Image J software (NIH, USA). The cytoplasmic translocation of HMGB1 was defined when the intracellular area of HMGB1 immunofluorescence was greater than that of DAPI.
To implant the canula for intracerebral injection, the mouse was placed on a heating pad and mounted on a stereotaxic apparatus (Stoelting, USA) under anesthesia with sodium pentobarbital (50 mg/kg, i.p.). The skull was exposed and small craniotomies were made over the bilateral mPFC or BLA for guide cannula (0.30 mm in out diameter, RWD Life Science, China) implantation. The coordinates relative to bregma were as follows according to the Paxinos and Franklin (2001) atlas: mPFC (AP: 1.80 mm, ML: ± 0.20 mm, DV: 2.50 mm), BLA (AP: -1.45 mm, ML: ± 2.30 mm, DV: 4.30 mm). The cannulas were held in place by dental cement and the sites of cannula placement were confirmed by histochemistry at the end of all experiments. Ds-HMGB1 (1 μg), anti-HMGB1 mAb (2 μg) or control Ig G (2 μg), all in 1 μl, was infused in 2 min by an injection pump (World Precision Instruments, USA) through a needle that was connected to a 1 μL Hamilton syringe and fit for the cannula. After infusion, the needle was left in place for additional 5 min before slowly withdrawn. For systemic administration injection, anti-HMGB1 mAb (1 mg/kg) or gabapentin (10 or 20 mg/kg) was injected intraperitoneally. Behavioral tests were carried out 1 h after agent administration. All the doses of administered agents were determined by pilot studies.
Mice were anesthetized with sodium pentobarbital and mounted in a stereotaxic apparatus. A craniotomy was performed unilaterally and a glass micropipette was introduced into the mPFC (AP: 1.80 mm; ML: 0.20 mm; DV: 2.50 mm) for infusing virus targeting glutamatergic neurons (pAAV-CaMKIIα-eArch3.0-eYFP) in a volume of 100 nl at 0.05 μl/min. The pipette was not removed until 10 min after infusion to allow diffusion of the virus. Animals were kept for 3 weeks to allow the maximal in vivo viral expression before implantation of chronic fiber optic cannula (core diameter 200 μm, 0.22 NA, Newdoon, China) into the mPFC. Behavioral test was carried out another week later. Persistent laser stimulation at 594 nm (5 mW, direct current) was applied through an optogenetic patch cord (Newdoon, China) to inhibit neurons throughout behavioral tests. Mice receiving virus that were connected with the optic fiber but without illumination served as the control for those receiving both virus and illumination. The effective transduction of virus was confirmed by colocalization of eYFP and CaMKIIα, a marker of glutamatergic neurons and by reduction of firing in putative glutamatergic neurons upon laser stimulation.
In vivo single-unit recordings
Under anesthesia with 20% urethane (1.4 g/kg, i.p.; Sigma-Aldrich, USA), a small craniotomy was performed over the mPFC on D7 PO. The microelectrodes that consisted of 8 channels of wires (25 μm; AM-Systems, USA) with impedances of 1–2 MΩ were lowered into the mPFC by micromanipulator as previously described . Signals were acquired by a multichannel acquisition system (Blackrock Microsystems, USA) at a sampling rate of 30 kHz and high- and low-pass at 250 Hz and 7.5 kHz, respectively, and analyzed by Offline Sorter (Plexon, USA) and NeuroExplorer 4.0 (NEX, USA). Putative glutamatergic neurons were identified according to their wide spike waveform (full width at half maximum ≥ 0.30 ms) and sharp autocorrelation [35, 36].
Patch clamp electrophysiology
Preparation of brain slices
The mouse was anesthetized deeply with isoflurane on day 7 (D7) PO and the brain was quickly removed to icy cold artificial cerebral spinal fluid (ACSF) oxygenated with 95% O2 and 5% CO2. The brain slices in 300 mm containing the mPFC were obtained by a vibratome (VT1000, Leica Instruments, Germany). The slices were then incubated for at least 30 min at 33 °C and another 1 h at RT in oxygenated ACSF. Then the slices were transferred to a recording chamber and were continuously perfused with oxygenated ASCF at a rate of 3–4 ml/min before electrophysiological recordings at RT. For action potential recordings, the ACSF contained (in mM): 120 NaCl, 11 Dextrose, 2.5 KCl, 1.28 MgSO4, 3.3 CaCl2, 1 NaH2PO4, and 14.3 NaHCO3, with pH at 7.4 and osmolarity at 310.5 mOsm. For spontaneous post-synaptic current recordings, a low divalent ion ACSF containing (in mM): 125 NaCl, 3.5 KCl, 1.25 NaH2PO4, 0.5 MgCl2, 26 NaHCO3, 25 Dextrose, and 1 CaCl2, with pH at 7.4 and osmolarity at 310.5 mOsm was used.
Whole-cell patch-clamp recordings
Pyramidal neurons of the mPFC slices (coordinates: AP 1.70 mm, ML ± 0.20 mm or ± 0.20 mm, DV 2.10 mm) were visualized and recorded under an infrared differential interference contrast video microscopy mounted on an upright microscope (FN1, Nikon, Japan) equipped with a 340/0.80 water-immersion objective and a charge-coupled device camera (Clara-E, Andor Technology, UK). To record action potentials, pipettes with resistance of 5–10 MΩ and outer diameter of 1.5 mm were filled with a K+-based recording solution containing (in mM): 140 K-gluconate, 5 NaCl, 0.2 EGTA, 2 Mg-ATP and 10 HEPES. Stepped currents (0–100 pA, 5 pA per step) were injected into neurons to elicit action potentials. To record spontaneous excitatory and inhibitory post-synaptic currents (sEPSCs and sIPSCs), cesium-based recording solution containing (in mM): 100 CsCH3SO3, 20 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Tris-GTP, 7 Tris2-Phosphocreatine, and 3 QX-314) was used. The holding potential was -60 mV and + 10 mV for recording sEPSCs and sIPSCs, respectively. The signals were amplified by the amplifier (EPC10, HEKA Instruments, Germany), and digitized at 10 kHz. The lowpass filter was set at 2.8 kHz. If the series resistance changed more than 20% during the recordings, the neuron was immediately abandoned. Data were further and analyzed with MiniAnalysis Program (Synatosoft Inc, USA) and Clampfit 10.7 software (Molecular Devices, USA) to provide spreadsheets for the generation of cumulative probability plots. The amplitude and interevent interval of post-synaptic currents were collected. The ratio of charge transfer of sEPSCs (∣CsEPSC∣) and sIPSCs (CsIPSC) was defined as EI ratio.
All data are expressed as the mean ± SEM. The required sample sizes were estimated based on our experience and Power analysis was used to justify the sample size. Statistical analysis was conducted by GraphPad Prism 8.0 (GraphPad Software, USA). Shapiro–Wilk test was used to assess whether the data followed a normal distribution. If the data was normally distributed, two-tailed paired or unpaired Student’s t test was used for comparison between two groups; if not, Mann–Whitney test was used instead. One-way analysis of variance (ANOVA) with Dunnett or Tukey post hoc test or Kruskal–Wallis test with Dunn’s post hoc was used for comparison of more than two groups with one factor. When comparing thresholds to thermal or mechanical stimulation among groups, two-way ANOVA with Bonferroni post hoc multiple comparisons test was used. The significance level was set at P < 0.05.