Animals
Adult male Sprague–Dawley (SD) rats weighing 200–250 g were procured from the Laboratory Animal Centre of Renmin Hospital, Wuhan University. All rats were housed under specific pathogen-free (SPF) laboratory conditions with a 12-h light/dark cycle (lights on at 8:00 A.M., humidity 60 ± 5%, temperature 22 ± 2 °C) and ad libitum access to food and water. The protocol was reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration, including the “3R” concept [26].
Status epilepticus induction and seizure quantification
The induction of status epilepticus using pilocarpine was performed as previously described [27]. Experimental rats were injected with lithium chloride (127 mg/kg, i.p., Sigma–Aldrich, St. Louis, MO), and 18 to 24 h later, atropine sulfate (1 mg/kg, i.p., Double-Crane pharmaceutical Co., Ltd) was administered to antagonize the peripheral cholinergic effect. Thirty minutes later, SE was induced by injecting pilocarpine hydrochloride (35 mg/kg, i.p., Sigma). Seizure behaviour was scored by the modified Racine scale with the following stages: (0) no abnormality, (1) mouth and facial movements, (2) head nodding, (3) forelimb clonus, (4) rearing, and (5) rearing and falling [28]. Pilocarpine hydrochloride was administered (10 mg/kg, i.p.) every 30 min until the rats developed seizures at Stage 4 of the Racine scale. Only rats that progressed to Stage 4 or greater were selected. The maximum dose for pilocarpine was 60 mg/kg. Diazepam (10 mg/kg, i.p., Sinopharm) was used to terminate seizures after a one-hour status epilepticus. Sham rats received the same treatment with lithium chloride and atropine sulfate, but an equivalent amount of phosphate buffered saline (PBS) was used instead of pilocarpine.
Dural injection of inflammatory soup and nociceptive behavioural tests
The experimental procedures were carried out as previously reported [25]. After the skull was exposed, a 1 mm hole in the middle of the superior sagittal sinus (between bregma and lambda) was made with a precooled dental drill (DH-0 Pin Vise, Plastics One) to carefully expose the dura. A 0.5 mm guide cannula (22 GA, #C313G, Plastics One) was inserted into the hole and sealed into place with a mixture of dental cement powder and superglue. A dummy cannula (#C313DC, Plastics One Inc.) was inserted to ensure the patency of the guide cannula. The skin incision was closed with a silk suture. After one day of recovery from the surgery, 30 μl of IS (2 mM bradykinin, 2 mM serotonin, 2 mM histamine, and 0.2 mM prostaglandin E2) was administered via the annular tubes to stimulate the dura at a rate of 4 μl/minute once per day for four days according to previously described methods [16, 29]. Sham-operated rats underwent the same surgical procedure, but an equal amount of PBS was used instead of IS.
Nociceptive behaviour was assessed by counting the number of head-scratching actions and measuring the periorbital region mechanical sensitivity threshold. The head-scratching counts were recorded for one hour immediately after the last IS administration to assess nociceptive behaviours. Only rats scratching the face above the eye, which is innervated by the first division of the trigeminal nerve, were counted [30]. To assess the acute pain threshold, mechanical thresholds were measured by Von-Frey filaments after the last IS dural infusion. Testing began with the 1-gram filament on the face. If the rat showed a head-back reaction, the Von-Frey filament was changed to a smaller wire. If there was no reaction, the wire size was increased until the rat quickly retracted its head [31]. The pain threshold is determined by the first change in response. The evaluators were blinded to all experimental conditions.
Drug administration
AZD8797 (an antagonist of CX3CR1) administration was carried out as described previously [32]. AZD8797 (HY-13848, MCE, USA) was dissolved in a DMSO solution to yield a final concentration of 2 mg/ml according to the instructions. Rats received either an intraperitoneal injection of AZD8797 (1 mg/kg) or an equal amount of PBS once per day for four days before dural IS administration. Rat fractalkine (PeproTech, USA) was dissolved in 0.9% NaCl to yield a final concentration of 1 μg/μl; bilateral intracerebral injection of fractalkine was performed stereotactically to the sp5c at the following coordinates: anteroposterior, − 14.08 mm; lateromedial, ± 2.75 mm; dorsoventral, − 8.65 mm relative to the bregma in the rat. A total of 2.5 μl was injected into each site using a 5 μl glass syringe with a fixed needle [33]. Rats in the sham groups received 2.5 μl of PBS at each site. A schematic representation of the intracerebral injection is given in Supplementary Fig. 1.
Experimental design and animal groupings
After one week of acclimatization, a total of 80 rats were randomly divided into ten groups of eight rats each according to experimental needs.
Experiment 1
Experiment 1 was designed to assess the effect of seizures on migraine and the expression of the FKN/CX3CR1 axis in SE and migraine rats. The rats were randomly divided into four groups: sham group, migraine group, SE group, and comorbidity group. The sham group received PBS. Rats in the migraine group underwent IS infusion. LiCl-Pilo was used in the SE group. Repeated IS injection into the dura mater of SE model rats was performed once per day for four consecutive days to establish the comorbidity group (Fig. 1A). After evaluating the behaviour of each group, 3 ~ 4 rats (randomly selected) from each group were immediately used for perform western blotting (WB), and other rats were perfused and used to make paraffin sections for immunohistochemistry (IHC) or immunofluorescence (IF) analysis. In addition, the colocalization of CX3CR1 with microglia and FKN with neurons was evaluated using double immunofluorescence staining in the sham group, migraine group, and comorbidity group.
Experiment 2
Experiment 2 was performed to investigate whether the effect of seizures on migraine was mediated by FKN/CX3CR1. AZD8797 was administered to rats in the comorbidity group. The groups included the sham-PBS group, the comorbidity-PBS group, and the comorbidity-AZD group (Fig. 5A). After evaluating the behaviour of each group, we collected sp5c tissues for WB to examine the antagonistic effect of AZD8797 on FKN/CX3CR1 (n = 3 ~ 4/group). The protein levels of proBDNF/BDNF were also measured to examine the potential molecular pathway associated with the FKN/CX3CR1 axis. Moreover, IF staining was performed to examine changes in microglia (n = 3 ~ 4/group).
Experiment 3
To further validate the effect of FKN/CX3CR1 on facilitating migraine-related behaviours and the specific mechanisms, FKN was infused into rats with migraine. There were three groups: the PBS-sham group, the PBS-migraine group, and the FKN-migraine group (Fig. 6A). Behavioural assessment, WB (n = 3 ~ 4/group), and IF staining (n = 3 ~ 4/group) were conducted in this experiment.
The observers were blinded to group allocations during the animal experiments.
Cell cultures and drug treatments
The BV2 mouse microglial cell line ICLCATL03001 (kindly provided by the Emergency Department Laboratory, Renmin Hospital of Wuhan University) was cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 incubator at 37 °C. BV2 cells were activated with lipopolysaccharide (LPS, 1 μg/ml, MCE, USA) for 90 min [34]. To examine the effects of FKN on microglia and the role of microglial release and synthesis of BDNF, we administered 0.1 mg/ml FKN (PeproTech, USA) to the cell cultures for 90 min according to the morphological changes in BV2 cells.
HT22 mouse hippocampal neurons were purchased from Wuhan Procell Century Technology. The cells were cultured in DMEM, supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin at 37 °C in a humidified environment containing 5% CO2.
Neuron-BV2 microglia transwell coculture
Neuron-BV2 microglial transwell cultures were used to investigate the effect of neuron-mediated FKN on microglia. BV2 microglial cells were plated in 6-well dishes (106 cells/well). HT22 mouse hippocampal neurons were plated (5 × 105 cells/well) in transwell chambers (0.4 µm, Cell Biolabs, Inc.), and these inserts were placed on top of the wells containing microglia. After 24 h of coculture, morphological changes in microglia were observed. Microglia on the lower membrane were collected, and the related protein levels were measured.
Western blotting
Rat brain tissue or cell samples were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail as previously described. Lysates were centrifuged and collected. Equal amounts of total protein ranging from 15 to 30 μg total protein were separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The following antibodies were used: rabbit anti-cfos (1:1000, Abcam), rabbit anti-CX3CR1 (1:3000, Abcam), goat anti-FKN (1:200, Abcam), rabbit anti-BDNF (1:3000, Abcam), rabbit anti-GAPDH (1:3000, Servicebio), and goat anti-iba1 (1:500, Woko). Blots were developed with an HRP-labelled goat anti-rabbit antibody (1:5000, Servicebio) and an HRP-labelled donkey anti-goat secondary antibody (1:5000, Servicebio). Protein bands were visualized using a chemiluminescence system (ChemiDocTM XRS + , BioRad). Protein expression was semiquantitatively analysed with Image J software.
Immunostaining
Paraffin-embedded serial sections and cells were fixed with paraformaldehyde and treated with 3% H2O2 for 10 min. Then, the sections were washed 3 times in PBS solution and blocked in 3% bovine serum albumin (BSA) and 0.3% Triton X-100 for 1 h. The sections were incubated overnight with rabbit anti-cfos (1:500, Abcam), rabbit anti-CX3CR1 (1:500, Abcam), goat anti-FKN (1:100, R&D), goat anti-iba1 (1:500, Woko), mouse anti-Neun (1:200, Abcam), and rabbit anti-BDNF (1:400, Abcam) at 4 ℃. For IHC, after being incubated with HRP-labelled goat anti-rabbit secondary antibodies (1:200, Servicebio) and HRP-labelled donkey anti-goat secondary antibodies (1: 200, Servicebio) for 1 h, the signal was developed according to the manufacturer's instructions for the High-Efficiency IHC Detection System Kit (Sharp, Wuhan China). For IF and immunocytochemistry (ICC) experiment, primary antibodies were detected with an appropriate fluorescent secondary antibody. Fluorescent secondary antibodies included FITC-labelled donkey anti-rabbit IgG (1:200, Servicebio), FITC- or Cy3-labelled donkey anti-goat IgG (1:200, Servicebio), Cy3-labeled donkey anti-mouse IgG (1:200, Servicebio). The slices were washed three times in PBS, covered with a glass cover with mounting solution and examined under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).
Sholl analysis
The complexity of microglia was analysed by setting a group of sequential concentric circles covering the cells with Image J software. The innermost circle (7 μm radius of activated microglia and 4 μm radius of resting microglia) was located in the nucleus. The other circles were arranged following a step length of 2 μm. The diameter of the outermost circles was adaptive according to each cell. Then, the number of intersections of individual concentric circles was counted.
Statistical analysis
Statistical analyses were performed with SPSS version 22.0 and GraphPad Prism 8.0. Unpaired Student's t tests were used to compare the means of two groups. One-way ANOVA followed by Bonferroni post-hoc tests was used for comparisons among three or more groups. All data are presented as the mean ± SEM. The results were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.
