Animals
Male adult C57BL/6 J mice (21–34 g) used in the experiments were obtained from SPF Biotechnology Co., Ltd. (Beijing, China). The mice were kept in individual cages with a room temperature of 23 ± 2 °C, the humidity of 50 ± 10%, a 12 h light-dark cycle, food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee at the Chinese PLA General Hospital and followed Regulations for The Administration of Affairs Concerning Experimental Animals. After a week of adaption, the animals were randomly assigned to experimental groups.
Surgical procedure
A cannula was implanted to the skull of each mouse for IS or normal saline (NS) infusion with reference to other research [13, 14]. Briefly, a mouse was anesthetized intraperitoneally (i.p.) with 1% pentobarbital sodium (7.5 ml/Kg) and placed onto a stereotaxic frame. The animal temperature was maintained during surgery using a constant temperature heating blanket. After hair removal and disinfection of the surgical area, a 5- to 8-mm-long midline incision was made to expose bregma. A burr hole, 1.5 mm caudal to bregma, 1.5 mm lateral to the midline, adjacent to the superior sagittal sinus, was slowly drilled to expose the dura mater, taking care not to pierce the dura. A small screw (M 1.0 mm, L 2.0 mm) was invertedly affixed to the site, approximately 3 mm contralateral to the midline, by using biomedical glue. A guide cannula (O.D. 0.41 mm, I.D. 0.25 mm, length 0.3 mm, RWD, China) filled with a dummy cannula (O.D. 0.20 mm, length 0.4 mm) was implanted to the drilled hole and affixed to the skull by applying dental cement to cover the foundation of the cannula and the screw. A microsyringe (10 μL, Hamilton), an injection cannula (O.D. 0.21 mm, I.D. 0.11 mm), and a length of polyethylene tube were assembled into a drug delivery system. It was used to deliver IS or NS to the dura mater. After surgery, the mouse was returned to its cage when it was fully awake and recovered for at least 7 days before the next procedure. Analgesics were not used during the recovering period due to the concern of any interference to the pain pattern of the animal model.
Recurrent IS or NS infusion
56 mice were separated into 7 groups (n = 8) at random: (1) IS-20 group (IS infusion, sacrificed on 20 d), (2) IS-22 group (IS infusion, sacrificed on 22 d), (3) IS-26 group (IS infusion, sacrificed on 26 d), (4) IS-33 group (IS infusion, sacrificed on 33 d), (5) IS-40 group (IS infusion, sacrificed on 40 d), (6) IS-47 group (IS infusion, sacrificed on 47 d), and (7) control (CON) group (NS infusion, sacrificed on 47 d). IS contains 1 mM histamine, 1 mM serotonin, 1 mM bradykinin, and 0.1 mM prostaglandin E2 in NS. All procedures took place between 8:00 and 18:00. On infusion days, after the dummy cannula was removed from the guide cannula, mice in IS groups were received 5 μL IS infusion by the drug delivery system through the guide cannula. Mice in the CON group received the same dose of NS instead. The infusion was operated for a total of 10 times on alternate days (days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). It needs to be noted that the mice in the CON group only followed the same timeline of the IS-47 group and were sacrificed on 47 d (Fig. 1)
Cutaneous allodynia behavior
Cutaneous allodynia behavior of mice was evaluated with mechanical thresholds in the periorbital region and the hind paw plantar, as described previously [15, 16]. The animals were habituated in a plexiglass box with a mesh grid bottom for 15–20 min before measurement. Mechanical thresholds were measured by applying von Frey filaments (vFF) perpendicularly to the periorbital region and the hind paw plantar, with the filaments bending for approximately three seconds or until a positive response is evoked. The vFF is calibrated by the manufacturer. The force range of the vFF applied to the periorbital region and the hind paw plantar is 0.008–0.4 g (0.008, 0.02, 0.04, 0.07, 0.16, 0.4 g) and 0.07–4 g (0.07, 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0 g), respectively. The experimenter applied the filaments with sequential increasing order to determine the mechanical thresholds. After a positive response is evoked, the next filament of less force is applied. And if it is a negative response, the next filament of higher force will be applied. The positive response is considered as the animal quickly retracts its head or rubs its face in the measurement of mechanical thresholds in the periorbital region. In the measurement of mechanical thresholds in the hind paw plantar, the positive response is considered as the animal licks, retracts, or shakes its hind paw. The mechanical thresholds are defined as the force which would firstly evoke a positive response three times in five or more trials. Only the mice in the IS-47 group (n = 8) and CON group (n = 8) were performed the measurement. The mechanical thresholds were measured before each IS/NS infusion (days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). On days 20, 22, 26, 33, 40, and 47, we also take mechanical thresholds tests at the same time of day as before (8:00–18:00). The mechanical thresholds measured on day 1 were regarded as a baseline. The experimenter was blinded to the grouping during the tests.
Sample collection
The serum and the TNC tissue of each animal were collected. On the sample collection day (8:00–18:00), the animals in each group were sacrificed under deep anesthesia with 1% pentobarbital sodium. Four mice of each group were taken blood samples from the vein near the eye into Eppendorf (EP) tubes. After 20-min undisturbed incubation and 10-min centrifugation (3000 rpm, 4 °C), the serum was aliquoted and stored at − 80 °C for subsequent analysis. The brains were separated from the skull quickly on ice after transcardial phosphate-buffered solution (PBS) perfusion. Four brains of each group were embedded with optimum cutting temperature compound (OCT, Tissue-Tek), snap-frozen in liquid nitrogen, and then stored at − 80 °C for the next sectioning procedure. The TNC tissue of the other four brains in each group was immediately isolated, snap-frozen in liquid nitrogen, and stored at − 80 °C for the following procedures. The integrity of the dura mater was visually inspected.
Immunofluorescent staining
The brains (n = 4/group) embedded with OCT were manufactured into 10-μm-thick frozen sections of TNC tissue in a cryostat microtome (Leica 1950 M). After fixation in ice-cold acetone, the sections were blocked by blocking buffer (10% normal goat serum, 0.5% Triton X-100, dissolved in 0.1 M PBS) for 1 h at room temperature (RT). Then, the sections were incubated with primary antibody (rabbit anti-GFAP antibody, 1:100, ab207165, Abcam) diluted in primary antibody dilution buffer (P0262, Beyotime) overnight at 4 °C. After cleaning with 0.1 M PBS three times, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, 1:1000, ab150077, Abcam) diluted in secondary antibody dilution buffer (P0265, Beyotime) for 1 h at RT. Then, the sections were rinsed in 0.1 M PBS three times and mounted with an antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, P0131, Beyotime). Magnified Images (× 20 objective) were captured under a fluorescence microscope (BX43, Olympus) using the cellSens standard software (version 1.18, Olympus). In negative-control sections, PBS was used instead of primary antibody, and there were no positive signals. The immunofluorescence area fraction was measured using ImageJ software (version 1.52p, National Institutes of Health).
Western blot assay
We examined the protein levels of GFAP through a western blot assay. The TNC tissue (n = 4 in each group) were homogenized by an electric homogenizer in radioimmunoprecipitation (RIPA) lysis buffer (P0013B, Beyotime) supplemented with phenylmethylsulphonyl fluoride (PMSF, ST506, Beyotime) and protease phosphatase inhibitor mixtures (P1045, Beyotime) for 2 h at 4 °C. The homogenate was centrifuged for 20 min (16,000 rpm, 4 °C). The supernatant was collected as a whole-cell protein extract and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (P0010, Beyotime). Equal amounts of protein were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, electrophoresed, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat milk for 2 h at 37 °C and incubated with rabbit anti-GFAP antibody (1:5000, ab207165, Abcam) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (1:2000, ab8245, Abcam) overnight at 4 °C. The membranes were washed with tris-buffered saline Tween-20 buffer (TBST) three times and incubated with horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (H + L) (1:1000, A0208, Beyotime) for 1 h at 37 °C. The immunoblots were probed with western blot detection kits (BeyoECL Plus, P0018S, Beyotime, China) and visualized with an imaging system (Tanon-5200, China). GAPDH was used as a loading control.
RNAscope in situ hybridization (ISH) assay
We examined the transcriptional levels of GFAP through an RNAscope 2.5 HD Duplex Reagent Kit (322430, Advanced Cell Diagnostics, ACD). 10-μm-thick frozen sections of the TNC tissue (n = 4 in each group) were fixed with 4% paraformaldehyde and dehydrated with 50%, 70%, and 100% concentrations of ethanol step by step. The detection of GFAP and Iba1 in the TNC tissue sections was performed following the manufacturer’s protocols using GFAP probe (313211, ACD) and Iba1 probe (319141-C2, ACD). We also used the positive control probe (PPIB-C1/POLR2A-C2, 321651, ACD) and 2-plex negative control probe (320751, ACD) as the positive and negative control, respectively. The images (× 40 objective) were captured under a light microscope (BX43, Olympus) using the cellSens standard software (version 1.18, Olympus). The numbers of blue (GFAP) and red (Iba1) dots and total cell numbers were calculated using ImageJ software. The mRNA level was presented as dots number per cell.
Quantification detection of cytokines
We quantitatively detected 13 cytokines (including CCL2, CCL5, CCL7, CCL12, CXCL1, CXCL13, IFN-γ, TNF-α, M-CSF, IL-1β, IL-6, IL-10, IL-17A) in serum and TNC tissue extraction (n = 4 in each group) using Luminex Multiplex Immunoassays (R&D systems) according to the manufacture’s protocols. In brief, serum and the TNC tissue extraction were added to a 96-well microplate and mixed with a microparticle cocktail that was already coated with analyte-specific capture antibodies. After 2-h incubation at RT, the biotin-antibody cocktail was added to form an antibody-antigen sandwich in each bead. Subsequently, streptavidin-phycoerythrin was added to bind the biotinylated detection antibodies. Beads were read on a Luminex 100/200 system (Bio-Rad).
Statistical analysis
Data were expressed as the mean ± standard error of mean (SEM). SPSS 25.0 was used for statistical analysis. Statistical differences between two groups were analyzed using the independent-sample t test. Multiple comparisons were statistically analyzed by one-way analysis of variance (ANOVA) and Dunnett’s t test post hoc analysis. P < 0.05 was considered statistically significant.