Animals
Adult male Sprague–Dawley rats weighing between 200 and 250 g were procured from the Laboratory Animal Center 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 ± two °C), ad libitum food, and water. After one week acclimatized, animals were randomly assigned to different groups. The investigators were blinded to the group allocation during the animal experiments. 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[21].
Status epilepticus induction and seizure quantification
The induction of status epilepticus using pilocarpine was performed as previously described[23]. Experimental rats were injected with lithium chloride (125 mg/kg, i.p. Sigma-Aldrich, St. Louis, MO). 18 to 24 hours later, atropine sulfate (1mg/kg, i.p. Beijing reagent) was administered to antagonize the peripheral cholinergic effect. Thirty minutes later, SE was induced by injecting pilocarpine hydrochloride (350mg/kg, i.p.; Sigma). Pilocarpine hydrochloride was given repeatedly (10mg/kg, i.p.) every 30 minutes until the rats developed seizures. Sham rats received the same treatment with lithium chloride and atropine sulfate, but an equivalent amount of normal saline replaced pilocarpine.
Seizure behavior 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, (5) rearing and falling[29]. Only rats progressing to stages four or more were selected.
Inflammatory Soup dural Injections and nociceptive behavioral tests
The experimental procedures were carried out as previously reported in our laboratory[13]. 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 expose the dura carefully. 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 IS (1 mM bradykinin, serotonin, and histamine and 0.1 mM prostaglandin E2 or two mM bradykinin, serotonin, and histamine and 0.2 mM prostaglandin E2; pH 5.5) was delivered from the annular tubes to stimulate the dural once a day for four days.
The nociceptive behavior was assessed by head-scratching numbers, and the facial mechanical sensitivity threshold of a von-Frey filament was measured by the Dixon "up-and-down" method[2]. Testing began with the 1gram (g) filament on the face and increased weight until a rat quickly retracted its head. Mechanical thresholds were detected after the last IS dural infusion. The head-scratching numbers were recorded for one hour after the fourth IS administration. All investigators were blinded to experimental conditions.
Drug administration
AZD8797 administration was carried out as described previously[7]. 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 (1mg/kg) or an equal amount of PBS once a day for four days before IS dural administration.
Rat Fractalkine (PeproTech, USA) was dissolved in 0.9% NaCl to yield a final concentration of 1ug/ul; bilateral intracerebral injection of fractalkine was performed stereotactically to TNC at coordinates of 14.08 mm, lateral 2.75 mm, and ventral 8.65 mm relative to the bregma in the rat. A total of 2.5 ul was injected into each site using a 5 µL glass syringe with a fixed needle[1]. At the same time, the sham groups received 2.5 ul of 0.9% saline at each site.
Cell Cultures and drug treatments
BV2 mouse microglial cell line ICLCATL03001 (kindly provided by the emergency department Laboratory, Renmin Hospital of Wuhan University) were 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 cell cultures were activated by LPS (1µg/ml, MCE, USA) for 90 minutes[19]. To examine the influence of FKN on microglia and the role of microglial cell release and synthesis of BDNF, we also give 0.1mg/ml FKN (PeproTech, USA) to the cell cultures for 90 minutes according to the morphological changes of BV2 cells.
HT22 mouse hippocampal neuronal cells were purchased from Wuhan Procell Century Technology. They were cultured in DMEM medium, supplemented with 10% FBS, 100 U/ml penicillin, and 100U/ml streptomycin at 37°C in a humidified environment containing 5% CO2.
Neuron-BV2 microglia transwell coculture
Neuron-BV2 microglia transwell cultures were used to investigate the role of neuron-mediated FKN on microglia. BV2 microglial cells were plated into 6-well dishes (106 cells/well). HT22 mouse hippocampal neurons were plated (5x105 cells/well) in the transwell chambers (0.4um, Cell Biolabs, Inc.), and these inserts were placed on top of the wells containing the microglia culture. After 24 h of coculture, the morphologic change of microglia was observed. Microglia on the lower membrane were collected to test related protein content.
Western blot
The 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. Total protein concentration was determined using a BCA Protein Assay Kit (Beyotime). An equal amount of protein ranging from 15 to 30 µg total protein was separated by SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore). The following antibodies were used: rabbit anti-CX3CR1 antibody (1:3000, Abcam), goat anti-CX3CL1 antibody (1:200, Abcam), rabbit anti-BDNF antibody (1:3000, Abcam), rabbit anti-GAPDH antibody (1:3000, Service), goat anti-iba1 (1:500, Woko). Protein bands were visualized using a chemiluminescence system (ChemiDocTM XRS+, BioRad). The protein expressions were semi-quantitatively analyzed with ImageJ software.
Immunohistochemistry and Immunofluorescence
Rats were anesthetized and transcardially perfused with 4% paraformaldehyde. 20-µm-thick coronal sections were cut through the midbrain at the thalamus or caudal medulla level and the upper cervical spinal cord at the sp5c level. Microwave heating performed deparaffinization, gradient alcohol dehydration, and antigen repair on paraffin sections were deparaffinized and rehydrated. Then, 3% H2O2 was used to reduce nonspecific background staining caused by endogenous peroxidase. After 20 minutes of incubation with 2% Triton X-100 and 1 hour of blocking with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4°C with rabbit-anti-cx3cr1 (1:500, Abcam), goat-anti-cx3cl1 (1:100, R & D), goat-anti-iba1 (1:500, Woko), and mouse-anti-Neun (1:200, Abcam, England). For the Immunohistochemistry (IHC) experiment, the signal was developed following the manufacturer's instructions for the High-Efficiency IHC Detection System Kit (sharp, Wuhan China). For the Immunofluorescence experiment, the sections were incubated with the corresponding florescent-labeled secondary antibodies (FITC or CY3-labeled donkey anti-goat IgG, FITC-labeled donkey anti-rabbit IgG, CY3-labeled donkey anti-mouse) and examined under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).
Immunocytochemistry staining
Microglia cells were fixed in 4% paraformaldehyde and 0.1% Triton X-100 for 15 min. The cells were washed with PBS, blocked with 3% BSA for 30 min, and incubated with goat-anti-iba1 (1:500, Woko) and rabbit-anti-BDNF (1:400, Abcam) antibodies overnight at 4℃. After washing with PBS and incubation with Alexa Fluor 594-conjugated anti-goat and Alexa Fluor 488-conjugated anti-rabbit for two hours at room temperature in the dark, the sections were washed with PBS followed by nuclear staining with DAPI. After a brief rinse in PBS, the sections were then examined under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).
Sholl analysis
The complexity of microglia was analyzed by setting a group of sequential concentric circles covering the cells in the ImageJ software. The innermost circle (35µm radius of activated microglia and 21µm radius for resting microglia) is located right in the nucleus. The other circles were arranged following a step length of 10µm. The diameter of the outermost circles is adaptive according to each cell. Then, the number of intersections was counted at individual concentric circles.
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 were presented as the mean ± SEM. The results were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001, ****p<0.0001.