Activation of microglial GLP-1R in the trigeminal nucleus caudalis suppresses central sensitization of chronic migraine after recurrent nitroglycerin stimulation

Male C57BL/6 mice weighing 18–20 g were kindly provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Animal experiments were conducted in accordance with Chongqing Medical University Animal Ethics Committee and were approved by the National Institutes of Health guidelines on animal care. Mice were housed with a 12 h light/dark cycle, at a stable temperature of 23 ± 2 °C and humidity of 50 ± 10 %, and were given food and water ad libitum. Every effort was made to minimize the number of mice used in the experiments and their suffering.

Nitroglycerin (NTG) (5.0 mg/ml, Henan Reagent, China) was freshly diluted to a final concentration of 1 mg/ml with saline, containing 6 % propylene glycol and 6 % alcohol. The intraperitoneal (i.p.) delivery of the diluted NTG was performed as described previously, that is, injected at a dose of 10 mg/kg every other day for 9 d (i.e., days 1, 3, 5, 7, and 9). Mice in the sham group were injected with an equivalent volume of saline, 6 % propylene glycol, and 6 % alcohol.

To measure the effect of GLP-1R in CM, a selective GLP-1R agonist (liraglutide) and an antagonist (exendin(9–39)) were applied in the experiment. Liraglutide (800 µg/kg; Selleck, TX, USA) and exendin(9–39) (50 µg/kg; MedChemExpress/MCE, USA), diluted with sterile saline, were administered i.p. for 16 consecutive days; that is, these drugs were administered 1 week before NTG injection. The time point for injections was also selected before NTG administration. To examine the role of PI3K/Akt in CM, the PI3K/Ak antagonist LY294002 (20 mg/kg; Selleck, TX, USA), diluted with sterile saline, was injected i.p. five times every other day prior to NTG injections. The dose and delivery method of these drugs were based on previous research [30‐33] and our experience.

After a week of adaptation, the animals were randomly assigned to different experimental groups, as shown in Table 1 in which the experimental sets and sample size were delineated. During the whole experiment, the body weight and blood glucose of mice were measured 3 times, respectively, before drug administration, 1 week and 16 d after delivering liraglutide or exendin(9–39). The time point was selected at 9:00 prior to NTG injection. Glucose measurement was performed by a blood glucose meter (Sinocare, Changsha, China) using blood collected from the tail vein of mice.

BV-2 cells were provided by Procell Life Science & Technology (Wuhan, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY, USA) supplemented with 10 % foetal bovine serum (FBS) (Gibco, NY, USA) and were maintained in a humidified incubator at 37 °C and 5 % CO₂.

Cells were incubated with serum-free media for 24 h prior to each experiment and seeded at 1 × 10⁶ cells/dish in 100 mm culture dishes for western blotting and 3 × 10⁴ cells/well in 24-well- culture plates for immunofluorescence. To study the role of GLP-1R in microglial inflammation, BV-2 cells were preincubated with the selective GLP-1R agonist liraglutide (100 nM, Selleck, TX, USA) and the antagonist exendin(9–39) (200 nM, MCE, USA) for 24 h, followed by LPS (1 µg/ml, Sigma–Aldrich, MO, USA) stimulation for 24 h. The doses of these drugs used with cells were based on previous data [34‐36].

Mice were acclimated to the testing chambers for 3 successive days before the behavioural tests. All experiments were performed in a low-light room between 9:00 and 15:00 at time points before and 2 h after NTG injection. The experimenter was blinded to the dug condition and the behavioural data analysis.

Both head-specific (periorbital) and hindpaw hyperalgesia were measured to reveal the hypersensitivity of the CM animal model. Mechanical responses were detected by a set of von Frey filaments (bending force ranging from 0.008 to 2 g, Aesthesio, USA) according to the up and down method [37, 38]. The first filament was chosed as 0.4 g. For periorbital testing, mice were placed in a paper cup to which they had previously been habituated, and then the periorbital region, positioned caudal to the eyes and near the midline, was stimulated with a von Frey filament. A positive response was considered quick retraction of the head or scratching of the face with the ipsilateral forepaw. In the absence of a response, a heavier filament (up) was applied, and in the presence of a response, a lighter filament (down) was applied. The duration of each stimulus was 3 s with an interval of at least 1 min. This process was followed for a maximum of 4 filaments after the first response. For hindpaw sensitivity, mice were habituated to acrylic cages 30 min before the test, and then the central area of the plantar surface of the hindpaw was stimulated with von Frey filaments. Positive responses were defined as lifting, shaking or licking of the paw. Testing was performed according to the up-down method described above.

To analyze the protein expression of GLP-1, GLP-1R, PI3K and p-Akt on different days after NTG intermittent injection, TNC samples were collected 24 h after the first (NTG 1d), the second (NTG 3d), the third (NTG 5d) and the last (NTG 9d) NTG injections. To examine c-fos expression, TNC tissues were collected 2 h after the last NTG injection, while for other protein targets, TNC was isolated and collected 24 h after the last NTG administration. The sampling time was shown in Table 1. Tissues were homogenized in cold RIPA lysis buffer (Beyotime, Shanghai, China) containing phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China) at 4 °C for 1 h. The total amount of protein was determined using a BCA protein assay kit (Beyotime, Shanghai, China). The protein samples (40 µg) were separated by 10 % SDS-polyacrylamide gel (Beyotime, Shanghai, China) electrophoresis and transferred to PVDF membranes. Following transfer, the membranes were blocked in TBST containing 5 % skim milk for 2 h at room temperature (RT). Then, the blots were incubated with the following antibodies overnight at 4 °C: mouse anti-GLP-1R (1:200, Santa Cruz, CA, USA), mouse anti-GLP-1 (1:100, Santa Cruz, CA, USA), mouse anti-CGRP (1:100, Santa Cruz, CA, USA), mouse anti-c-fos (1:200, Santa Cruz, CA, USA), rabbit anti-Iba-1 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-PI3K (1:1000, Proteintech, China), rabbit anti-Akt (1:1000, Proteintech, China), rabbit anti-phospho-Akt (1:2000, Cell Signalling, Boston, MA, USA), rabbit anti-IL-1β (1:800, Wanleibio, China), rabbit anti-TNF-α (1:800, Wanleibio, China), and mouse anti-β-actin (1:1000, Beyotime, China). After the membranes had been washed with TBST, the secondary antibodies (goat anti-rabbit, 1:1000; goat anti-mouse, 1:1000; Beyotime, China) were applied and incubated for 1 h at RT. After the secondary antibody reaction, the bands were visualized with enhanced chemiluminescence, and the positive pixel area was detected by an image analysis system (Fusion, Germany). The band sensitivities were normalized against the corresponding β-actin loading controls. The western blotting test was repeated six times for each target, and consistent results were obtained.

To investigate the expression level of various proteins in BV-2 cells, we collected cells after LPS incubation. Cells were lysed in cold RIPA buffer containing PMSF for 30 min at 4 °C, and the total protein concentration was determined using a BCA protein assay kit. The following procedures were performed according to the method described above. Each analysis was repeated in three independent experiments.

To stained c-fos, TNC tissues were collected 2 h after the last NTG injection, while for staining other targets, TNC was collected 24 h after the last NTG administration. Mice were deeply anaesthetized and perfused transcardially with 30 ml of PBS (pH 7.4) followed by 30 ml of cold 4 % paraformaldehyde (PFA) in PBS. The whole brain and cervical spinal cord (C1-C2) were collected and postfixed with 4 % PFA overnight at 4 °C.The medullary segment including the TNC between + 1 and − 3 mm from the obex was removed and transferred to 30 % sucrose for 48 h. After being frozen at − 80 °C, samples were cut into 10-µm-thick sections on a cryostat (Thermo). Sample sections were blocked in 5 % goat serum with 0.3 % Triton X-100 for 1 h at RT and then incubated overnight at 4 °C with the following primary antibodies: mouse anti-GLP-1R (1:50, Santa Cruz, CA, USA), mouse anti-GLP-1(1:50, Santa Cruz, CA, USA), rabbit anti-Iba1 (1:500, Wako Chemicals, Tokyo, Japan), rabbit anti-GFAP (1:200, Cell Signalling, Boston, MA, USA), rabbit anti-NeuN (1:100, Proteintech, China), mouse anti-CGRP (1:100, Santa Cruz, CA, USA), and mouse anti-c-fos (1:100, Santa Cruz, CA, USA). The sections were then incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor Cy3 and Alexa Fluor 488, 1:500, Beyotime, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) at RT for 10 min. The sections were visualized and photographed with a confocal microscope (TCS Sp8, Leica). Quantification of the fluorescent signal intensity was performed by image analysis software (Image-Pro Plus 6.2, Media Cybernetics).

To quantify the immunoreactivity of CGRP and the numbers of c-fos- and iba-1- positive cells in the TNC, 6 sections per mouse from 4 mice were selected randomly for each group, and 6–8 visual fields per section were captured. Images centred on the superficial layer of the TNC were captured under a ×100 or × 200 objective, and the immunoreactive cells in this region were counted with Image-Pro Plus.