Animals

All animal experiments were approved by the Animal Ethics Committee of the Second Hospital of Lanzhou University and were conducted in accordance with the standards of the Institutional Animal Care and Use Committee [18]. Approximately 8-week-old female C57BL/6 mice weighing 18–22 g were used in the study. All mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute. The mice were housed in the laboratory of Lanzhou University under controlled conditions of 45–60% humidity, 20–25 °C temperature, and a 12-hour light-dark cycle. All efforts were taken to minimize animal suffering and reduce the number of animals used. Mice were randomly assigned to different experimental groups, and the sample sizes of each group were shown in Table 1. Before starting all experiments, mice were given 1 week to adapt to the experimental environment.

Table 1 The experimental groups and sample size of mice per groupCM mouse model

We established a CM mouse model using repeated intermittent intraperitoneal injections (i.p.) of NTG, which is a classic model that has been accepted by numerous studies [19, 20]. A stock solution of 5 mg/ml NTG (Beijing Yimin, China) was diluted to a concentration of 1 mg/ml in 0.9% saline. The diluted NTG was freshly prepared before each experiment and stored protected from light. After the nociceptive threshold tests, the mice were administered (i.p.) with 10 mg/kg of NTG or an equal volume of vehicle (VEH), 0.9% saline, in the morning every second day for 9 days (5 injections total).

Behavioral tests

All behavioral tests were conducted in a quiet environment between 8:00 AM and 3:00 PM. The investigator was blinded to the experimental groups and was not involved in the analysis of behavioral data. Before testing, mice were habituated to the testing environment for 2 days, with each adaptation session lasting at least 30 minutes. On days 1, 3, 5, 7, and 9, mechanical nociceptive thresholds and thermal nociceptive thresholds of the hind paw were assessed in mice before NTG/VEH injection (basal response) and 2 hours after NTG/VEH injection (post-treatment response). Two hours after the NTG/VEH injection on day 9, following the establishment of the CM model, mechanical nociceptive thresholds around the periorbital were evaluated (Figs. 1A, 3A). Von Frey monofilaments ranging from 0.008 g to 2 g were applied perpendicularly to the hind paw or periorbital region using the up-down method to evaluate the mechanical nociceptive threshold, following the previously described protocol [21, 22]. If the mice exhibited a positive/negative response, the next lighter/heavier filament was selected (up-and-down method). After the mice made a positive response, an additional four filaments were tested using the same method. Each measurement was tested for approximately 60 seconds. When assessing the mechanical threshold of the hind paw, the mice were separately placed in a transparent plexiglass chamber (L*W*H: 10*7*7 cm) on a wire grid floor, and the filaments were applied to the central area of the plantar surface, avoiding contact with the fat pad of the paw. A positive response was defined as a rapid withdrawal or shaking of the hind paw upon filament stimulation. When assessing the mechanical thresholds in the periorbital region, the lower half of the mouse’s body was placed in a cylindrical restraint device (inner diameter 25 mm), and the mice were controlled by the tail while allowing free movement of the head and forelimbs. After a 5–10 minutes adaptation period in the restraint device, when the mice were calm, the filaments were applied to the periorbital region (middle and upper areas of the orbits on both sides). A positive response was defined as a quick withdrawal of the head, head shaking, or scratching of the face with the ipsilateral forelimb. Finally, the mechanical nociceptive threshold was calculated using online [21], “(https://bioapps.shinyapps.io/von_frey_app/)”. For the measurement of thermal nociceptive thresholds, the mice were placed in a testing apparatus with a bottom surface heated to 55 degrees Celsius (diameter 30 cm) and surrounded by transparent plexiglass (YLS-6B Intelligent Hot Plate Instrument, Jinan, China). The latency for the mouse to first lick its hind paw was recorded as the thermal nociceptive threshold. The maximum cutoff time was set at 30 seconds to prevent thermal injury to the mouse’s paw.

Fig. 1

Repeated NTG administration causes hyperalgesia in female mice. A. The schematic diagram illustrates the establishment of the CM mouse model and behavioral assessments through repeated NTG administration. B. In the NTG mice, the mechanical thresholds in the hind paw were significantly decreased compared to the VEH mice both before and after NTG administration. C. The thermal thresholds in the hind paw of NTG mice were significantly decreased compared to the VEH mice both before and after NTG administration. D. On the 9th day, after NTG administration, the mechanical threshold in the periorbital region of NTG mice showed a significant decrease compared to the VEH mice. Two-way ANOVA with the Tukey post hoc tests, * p < 0.05, **p < 0.01, n = 10 /group, Abbreviations: NTG, nitroglycerin; CM, chronic migraine; VEH, vehicle

Environmental enrichment

EE was established by placing the mice in large cages (480*350*210 mm), equipped with a house, a running wheel, and 4–5 toys: swing, bridge, tunnel, seesaw, rolling ball, tube and so on. Five mice were housed in each large cage, and toys were changed every 2 weeks. In contrast, the control mice were housed in a standard environment (SD) (290*178*160 mm), with 5 mice per cage. A total of 30 adult female mice were randomly divided into two groups: the EE group (15 mice) and the SD group (15 mice). These mice were raised in EE or SD for 2 months and then subjected to CM modeling. During the modeling period, they continued to be raised in their original cages until finally sacrificed under anesthesia.

Immunofluorescence staining

On day 9, at 12 hours after administering NTG/VEH, four or five mice from each group were randomly selected for immunofluorescence staining. Mice were deeply anesthetized by intraperitoneal injection of 1% pentobarbital sodium and perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) through the heart. After sufficient perfusion, the mice brains were immediately dissected and immersed in 4% PFA overnight. Then, the brains were dehydrated in 15% and 30% sucrose solutions sequentially until they sank completely. The brains were embedded in Tissue-Tek O.C.T. Compound (Sakura 4583) and sectioned using a cryostat microtome (Leica, CM1950) to obtain 25 μm thick sections. The sections were stored in a frozen protective solution at minus 30 degrees Celsius. For immunostaining, the brain sections were selected and rinsed with 0.01 M phosphate-buffered saline (PBS) and blocked with 10% goat serum in PBS at 37 °C for 1 hour. The brain sections were incubated at 4 °C overnight with the following primary antibodies: rabbit c-Fos antibody (1:1000; ab222699 Abcam), mouse CGRP antibody (1:100; sc-57,053 Santa Cruz), rabbit Iba1 antibody (1:200; ab178847 Abcam), rabbit GFAP antibody (1:800; GTX108711 GeneTex) and rabbit VGluT1 antibody (1:1000; ab227805 Abcam) in antibody dilution buffer (0.01 M PBS, 1% goat serum, 3% Triton X-100). The sections were then rinsed with 0.01 M PBS and incubated with goat anti-rabbit Alexa Fluor 594 (1:200; Abbkine) or goat anti-mouse Alexa Fluor 594 (1:200; Abbkine) at 37 °C for 1 h in the dark. After washing with PBS, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Finally, the sections were mounted with 50% glycerol for imaging. Images were acquired with an inverted microscope and were taken under identical exposures and conditions. For quantification of positive staining, three adjacent sections were acquired from each mouse. The average percentage of the positively stained area was calculated using ImageJ software to reflect the degree of positive immunoreactivity.

Western blot analysis

On day 9, at 12 hours after administering NTG/VEH, the mice were deeply anesthetized with 1% pentobarbital sodium, and the brain TNC tissues of the medulla oblongata were quickly harvested on ice, frozen in liquid nitrogen, and stored at − 80 °C. The TNC tissues were sonicated in RIPA lysis buffer, added with 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 20 mM NaF, and 2 mM protease inhibitor cocktail, and quantified using the Bradford method. Proteins were separated using 8%–12% SDS-PAGE gels and were transferred to the PVDF membrane (Millipore). The PVDF membranes were blocked with 5% milk for 2 hours at room temperature, and were incubated overnight at 4 °C with the following primary antibodies: anti-c-Fos (1:1000; ab222699 Abcam), anti-CGRP (1:200; sc-57,053 Santa Cruz), anti-VGluT1 antibody (1:1000; ab227805 Abcam) and anti-GAPDH (1:5000; YM3445 ImmunoWay). Then, the membranes were washed in TBST and incubated with goat anti-rabbit or anti-mouse secondary antibodies (1:1000; Boster) for 1 hour at room temperature. The bands were visualized with Chemiluminescent HRP substrate (Millipore) on Azure Biosystem C500 and quantified using ImageJ.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from TNC tissues using RNAiso Plus reagent (TaKaRa, China) and reversely transcribed into cDNA by using the PrimeScript RT Master Mix kit (TaKaRa, China). The relative expression level of the target and reference genes was determined by real-time fluorescence quantitative PCR (Thermo, PikoReal 96, USA) using the SYBR Premix Ex Taq II kit (TaKaRa, China). The reaction procedure was as follows: 30 seconds at 95 °C, followed by 40 cycles of 15 seconds at 95 °C, 30 seconds at 60 °C, then melting curve (65 ~ 95 °C, every 0.5 °C temperature rise, collect fluorescence signal once). The primer sequences are shown in Table 2.

Table 2 The primer sequences for qRT-PCRRNA-seq data processing

RNA-seq was performed from Shanghai Applied Protein Technology (Shanghai, China) with the following specific parameters. Total RNA was extracted from brain TNC tissues using TRIzol® Reagent (Magen) according to the manufacturer’s instructions. The quality of RNA samples was assessed by measuring the A260/A280 absorbance ratio using a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RNA integrity was determined using an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Only high-quality samples were selected for library construction. Paired-end libraries were prepared using the ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s protocol. Library quality was assessed using the Agilent Bioanalyzer 4150 system. The libraries were sequenced on an MGISEQ-T7 platform, generating 150 bp paired-end reads. The obtained data were subjected to bioinformatics analysis. The clean reads were aligned to the reference genome using HISAT2 software “(http://daehwankimlab.github.io/hisat2/)” with the orientation mode to obtain mapped reads. Feature counts “(http://subread.sourceforge.net/)” was used to count the reads mapped to each gene. The fragments per kilobase million (FPKM) value of each gene was calculated based on the gene length and the number of reads mapped to that gene. Differential expression genes (DEGs) analysis was performed using DESeq2 “(http://bioconductor.org/packages/release/bioc/html/DESeq2.html)”. Genes with a |log2FC| > 1 and an adjusted p-value (Padj) < 0.05 were considered significantly differentially expressed. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted using the clusterProfiler R software package. A significance threshold of p < 0.05 was used to determine significantly enriched GO terms or KEGG pathways. Venn diagrams were generated using online “(https://www.visual-paradigm.com/cn/)”.

Statistical analysis

All statistical analyses were performed with PRISM 8 software and SPSS 22.0 (IBM Statistics, IBM Corporation, New York, NY, USA). Data are shown as the mean ± SEM. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon’s rank sum tests. Parametric comparisons were done by two-sided unpaired Student’s t tests or two-way ANOVA and post-hoc Tukey test. All statistical tests were two-sided and a p-value of < 0.05 was considered statistically significant.