Animals
Male C57BL/6 mice weighing 20-30g were used in this study. All animals were obtained from the Experiment Animal Center of Chongqing Medical University (Chongqing, China). Mice were kept in the standard experimental environment at 22±2℃ and 50±5% relative humidity with an alternating 12/12-h light/dark cycle. Food and water were provided ad libitum. Before the experiment, all the animals were acclimatized to the environment for at least one week and then be randomly assigned to different experimental groups on the basis of a random number sequence generated by Excel Software. The detailed procedure is illustrated as follows: 1) Mice fulfilling the experimental criteria were selected and numbered. 2) Animal numbers were input into an Excel table, and a random sequence was generated using the ”rand” function in Excel software; 3) The animal numbers were rearranged according to the ascending order of random sequence; 4) Based on the new order, experimental animals were assigned to receive different treatments, with 6 mice in each group. Since the model induces pain in animals, the number of mice used was the minimum necessary to achieve sufficient statistical power. Fig. 1B shows the detail information about experimental group and sample size of mice for each experiment. The experimental protocol was approved by the Animal Care and Use Committee at Chongqing Medical University in China. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Establishment of the chronic migraine model
The CM model was established as the previously described[29]. Nitroglycerin (NTG) (Beijing Regent, China) was prepared from a stock solution of 5.0 mg/ml NTG dissolved in 30% alcohol, 30% propylene glycol, and water. Before injection, NTG was freshly diluted to 1mg/ml with 0.9% saline. Solution containing 0.9% saline, 6% propylene glycol, and 6% alcohol was used as a vehicle control. The animals intraperitoneally (i.p.) administered of 10mg/kg of NTG or an equal volume of vehicle every second day for 9 days (five times in total) (Fig. 1A). All experimental animals were subjected to the behavioral tests described in detail below before and 2 hours after NTG injection (Fig. 1A).
Drug administration
To explore the level of autophagic flux in CM, 60 mg/kg chloroquine (CQ, MedChemExpress/ MCE, American) was intraperitoneally injected once a day for 9 consecutive days, before NTG treatment and after baseline threshold measurement (Fig. 1A). To investigate the role of autophagy in CM, the autophagy inducer, rapamycin (RAPA, Selleck, TX, USA), was delivered in the same manner at a concentration of 1 mg/kg (Fig. 1A). The P2X7R selective antagonist, Brilliant Blue G (BBG, Sigma-Aldrich, Hungary), was used to figure out the function of P2X7R in CM. BBG was intraperitoneally administered at a dose of 50 mg/kg every other day for 9 days, in an identical manner to NTG treatment (Fig. 1A). CQ and BBG were diluted in 0.9% saline. The RAPA was dissolved in 2% DMSO, 30% PEG 300 and 5% Tween 80. An equivalent volume of solvent corresponding to each drug was used as a vehicle control. The drug dosage and methods of delivery were determined by literature data and preliminary experiments[16, 30, 31]. All drug solutions were freshly prepared on the day of use.
Behavioral assessment
In the clinic, central sensitization is manifested as cutaneous allodynia and expansion of the pain area, including the craniofacial and non-craniofacial region[32]. Therefore, in the animal model, we measured both the periorbital and hind paw withdrawal threshold to mechanical stimulation, as performed in the previous references. The plantar thermal sensitivity was also evaluated, since chronic NTG delivery induces thermal hyperalgesia[33]. All the behavioral assessments were performed between 9:00 am to 15:00 pm in a quiet environment. Mice underwent a three-day training period before the experiment. All the behavioral tests including mechanical withdrawal threshold and thermal withdrawal latency were performed in the same sets of animals. At the end of the experiment, six mice with successful modeling and significant behavioral tests were selected for further biochemical and morphological analyses. The behavioral testing was performed by the same investigators, who were blinded to the treatment groups.
Measurement of the mechanical withdrawal threshold
The mechanical withdrawal threshold of the hind paw and periorbit was assessed every other day before and 2 hours after NTG injection (Fig. 1A). We used Von Frey filaments with the up-down method to determine the withdrawal threshold to mechanical stimulation as previously described[7]. Briefly, a series of Von Frey filaments (range from 0.01 g to 2 g) were applied to the hind paw or periorbit, with an initial stimulation strength of 0.4 g. If there was no response to the stimulation, the filament strength was increased; Otherwise, the filament strength was decreased until there was a positive reaction. Each filament was held for 5 seconds at the testing site with an interval of 1 minute. The threshold was recorded as the lowest force evoking a positive response and averaged from three repetitive measurements.
For the hind paw test, the mice were separately placed in a suspended acrylic chamber covered with a wire mesh floor. The animals were acclimated to the new environment for at least 30 minutes. The Von Frey filaments were applied perpendicularly to the central area of the hind paw surface. Brisk withdrawal, shaking, lifting or licking of the testing paw were considered positive responses. For the periorbital assessment, the mice were placed individually in a 4 oz. paper cup, allowing only the head to poke out. The head and fore paw could move freely, but body could not turn in the cup. The periorbital region included the area from caudal of eyes to approximately the midline. Vocalization, quick retraction of head from the stimulation or scratching of face with the ipsilateral fore paw were considered positive responses.
Measurement of thermal withdrawal latency
The thermal withdrawal latency of the hind paw was assessed every other day before and 2 hours after NTG injection (Fig. 1A). We used a plantar test apparatus (Techman PL-200, Chengdu, China) with an intensity adjustable radiant heat to assess the thermal sensitivity. Briefly, mice were placed separately in a transparent chamber with a temperature controlled glass floor. After 30 minutes of acclimatization, the radiant thermal stimulus was delivered to the central part of the hind paw through the glass. The stimulus was shut off once the hind paw moved and the thermal withdrawal latencies were recorded automatically. The radiant heat intensity was calibrated to produce a basal withdrawal latency of 8-10 s in the control group. The cut-off time was 20 s to prevent tissue damage[33]. The radiant heat was delivered three times to each hind paw with an interval of 5 minutes. The thermal withdrawal latencies were defined as the average of three recordings.
Western blot analysis
Mice were sacrificed under anesthesia with 10% chloral hydrate. The TNC was collected immediately and stored at -80℃. Tissues were homogenized in cold RIPA lysis buffer (Byotime, Shanghai, China) containing a protease inhibitor, phenylmethylsulphonyl fluoride (PMSF, Byotime, Shanghai, China ) at 4 ℃ for 1 h. The lysate was centrifuged at 12000 xg for 15 minutes in 4 ℃, and then the protein concentration of the supernatant was determined with a BCA protein assay kit (Beyotime, Shanghai, China). The protein was denatured by heating at 100 °C for 5 minutes and stored at -80 ℃. Equal amounts of tissue protein (40 ug) were separated on 10% or 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels (Beyotime, Shanghai, China), and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in Tris-buffered saline with Tween-20 (TBST buffer) containing 5% nonfat milk for 2 h at room temperature and incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-P2X7R, rabbit anti-CGRP, mouse anti-c-fos, rabbit anti-LC3, rabbit anti-SQSTM1/p62, rabbit anti-beclin1, rat anti-NLRP3, rabbit anti-IL-1β, rabbit anti-IL-18 and mouse anti-β-actin. The detailed information for all the antibodies used in this study is provided in table 1. All the primary antibodies were diluted with special diluent (Beyotime, Shanghai, China). The next day, the membranes were washed three times with TBST for 10 minutes each and incubated with corresponding horseradish peroxidase conjugated secondary antibodies, including goat-anti-rabbit, goat-anti-mouse and goat-anti-rat, for 1 h at room temperature. The immunoreacted bands were revealed with an ECL detection kit (Advansta Inc., USA), visualized and analyzed with an imaging system (Fusion, Germany). β-actin was used to normalize the relative expression of the target proteins in different groups. Each western blot was repeated at least six times, and the consistent results were obtained.
Immunofluorescence staining
To detect c-fos, tissues were collected 2 h after the last NTG or vehicle injection, while for other targets, tissues were collected within 12 h. Mice were anesthetized with 10% chloral hydrate and perfused transcardially with 60 ml of cold phosphate-buffered saline (PBS, PH 7.4) followed by 60 ml of 4% cold paraformaldehyde (PFA) in 0.1% PBS (PH 7.4). The brainstem and cervical spinal cord (C1-C2) were harvested and postfixed in 4% PFA for 24 h at 4 ℃. The medullary segment containing the TNC between + 1 and −3 mm from the obex was removed and dehydrated sequentially in 20% and 30% sucrose until the tissue sank. Tissues were embedded, flash frozen with 2-methylbutane (aladding, Shanghai, China) and sliced coronally into 10 um sections with a cryostat (Leica, Japan). After antigen retrieval with sodium citrate (Beyotime, Shanghai, China), the sections were blocked and permeabilized simultaneously with 0.3% Triton X-100 (Beyotime, Shanghai, China) in 5% donkey or goat serum (Boster, Wuhan, China) for 30 minutes at 37 °C. Then, the sections were incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-P2X7R, mouse anti-CGRP, rabbit anti-c-fos, rabbit anti-Iba1, goat anti-Iba1, mouse anti-NeuN and mouse anti-GFAP. The detailed information for all the antibodies is provided in table 1. All the primary antibodies were diluted with blocking solution. After rinsing three times for 15 minutes in PBS, the sections were reacted with corresponding secondary antibodies (conjugated to Alexa Fluor 488, 555 or cy3) for 90 minutes at 37 ℃. Nuclei were stained with 4′,6-diamidino-2-phenylinodole (DAPI) (Beyotime, Shanghai, China) for 10 minutes at 37 ℃. Images were captured with a confocal microscope (LSM800, ZEISS, Germany). Negative control sections were treated with PBS instead of primary antibody and showed no positive signals.
Immunofluorescence imaging data analysis
The TNC area was determined based on the morphology under a light microscope according to the Mouse Brain Atlas[34]. The mean optical density of CGRP was analyzed using ImageJ software (version 1.8.0_112) with a 10x objective. To quantify the number of immunopositive cells of Iba1 and c-fos, the squared images (field of view, FOV, 320 x 320 um2) in the superficial layer of the TNC (Fig. 4F) was taken at 200x magnification. Four-six FOV per section were investigated. ImageJ software (version 1.8.0_112) was used to count the immunoreactive cells. To analyze microglial morphology, Neuron J, an ImageJ plug-in, was used to determine the total and mean length of microglial processes. Each cohort consisted of 4 mice, and 6-8 sections from each mouse were analyzed. The image collection and analysis were performed by an experimenter who was blinded to the treatment groups.
Statistical analysis
GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego,CA, USA) was used for the statistical analysis and graph generation. All the data are presented as the mean ± standard error of the mean (SEM). The Kolmogorov-Smirnov test and Bartlett’s tests were used to analyze the normality and homogeneity of the data, respectively. Differences between two groups were determined by an independent-sample t-test. Comparison among three or more groups were investigated by one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s multiple comparison tests to detect pair-wise between-group differences. Behavioral data were analyzed by two-way ANOVA with the Bonferroni post hoc test. Two-tailed tests were applied in all statistical analyses. P<0.05 was considered to be statistically significant.