Male Sprague–Dawley (SD) rats weighting 200–250 g were used. All rats were housed under a 12 h light/dark cycle at 22 ± 2 °C, with free access to food and water. They were habituated in experimental environment for one week before each experimental procedure. All experiments were approved by the Committee on Animal Use for Research and Education of the Laboratory Animals Center, Renmin Hospital of Wuhan University (Wuhan, PR China), and followed the recommendations of the International Association for the Study of Pain in Conscious Animals. The number and suffering of animals were minimized as much as possible.
The experimental procedures were carried on according to a previous study. Briefly, a midline incision was performed under pentobarbital anesthesia (60 mg/kg; i.p). A guide cannula was implanted in a previously drilled cranial window adjacent to the superior sagittal sinus, to delivery IS or PBS to the dura mater in all rats. We take special care to prevent infection and to minimize the effect of inflammation.
Repetitive dural infusions.
Rats were divided into two groups randomly: the control (CON) group and the migraine model (IS) group. The migraine models were built via repeated IS (30 ul, including 2 mM histamine, 2 mM serotonin, 2 mM bradykinin, and 0.2 mM prostaglandin E2 in PBS) dural infusions for at least 4 days. Rats in the control group received the same dose of PBS.
A total of 0.8 ml anti-IL-18 antibody (100 ng/µl; R & D Systems) was first administered intraperitoneally 1.5 h prior to the each dural IS infusion [IS + Anti-IL-18(i.p) group]. Then, the intracerebroventricular injection of 5 ul anti-IL-18 antibody was conducted as described previously. Briefly, anti-IL-18 antibody (100 ng/µl; R & D Systems) was injected into the lateral ventricle (-1.0 mm rear from the Bregma and + 1.5 mm lateral, 4.0 mm from the skull plane) 24 h after the 4th IS infusion[IS + Anti-IL-18(i.c.v) group], under adequate anesthesia. TAK (3 mg/kg, Millipore, Bedford, MA), a specific TLR4 antagonist, was administered intraperitoneally 1.5 h prior to the dural IS infusion based on the previous study (TAK group). The 30 µl of IL-18 (5 µg; R & D Systems) was delivered to the dura mater as described above (IL-18 group). TAK was diluted in PBS. The anti-IL-18 antibody and IL-18 were both dissolved in PBS. Rat in the control group received the equivalent volume of PBS. The dosages of above drugs were determined according to previous studies. Random grouping was used.
The headache behavior of rats was evaluated by a nociception threshold and face rubbing. A von Frey filament (vFF) was used when measuring the facial mechanical withdraw threshold, as described previously. The nociceptive threshold was taken as the least force applied to make rats quickly retract its head at least three of five stimulus. Face rubbing was a nociceptive behavior when rats received IS. In process testing, the experimenter was blinded to the grouping.
Quantitative real-time polymerase chain reaction (q-PCR)
Total RNA was extracted from rat medullary dorsal horn using Trizol reagent (Invitrogen) as previously described. A NanoDrop ND1000 Spectrometer (NanoDrop Technologies) was used to measure the RNA concentration. Then, 1 µg of total RNA was reverse-transcribed into cDNA using PrimerScript RT Reagent Kit (Takara, Kyoto, Japan) according to manufacturer's protocols. Real-time PCR amplification was conducted using SYBR Premix Ex Taq™ II (Takara, Kyoto, Japan) according to manufacturer's protocols. The following primers were used: 5’-GACAAAAGAAACCCGCCTG-3’ (IL-18 forward); 5’- ACATCCTTCCATCCTTC-ACAG-3’ (IL-18 reverse); 5’-TGGAGTCTACTGGCGTCTT-3’ (GAPDH forward); 5’-TGTCATATTTCTCGTGGTTCA-3’ (GAPDH reverse). Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), a housekeeping gene, was used to normalize expression. The relative mRNA level was represented as fold change of control by 2−△△Ct method.
After deep anesthesia, rats were perfused transcardially with 9% saline followed by 4% formaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4). After the perfusion, the rat brain tissues were removed and postfixed in 4% formaldehyde. Medullary dorsal horn sections (paraffin-embedded, 5 µm) were manufactured. Then, the sections were placed in xylene for dewaxing, and followed by decreasing grades of alcohol for rehydrating. Sections were blocked by 5% BSA for 60 min at RT, and then incubated in primary antibody diluted in 1% BSA overnight at 4 °C. The dilution concentration of the primary antibody was applied as follows: IL-18 (1:500; Abcam), IL-18R (1:100; Abcam), Iba1 (1:600; Abcam), GFAP (1:500; Gene Tex), p-p38 (1:50; Santa Cruz). After cleaning with PBS, the sections were incubated with respective secondary antibodies. Anti-mouse (488 nm, green), anti-rabbit (594 nm, red), anti-rabbit (Cy3, red), anti-goat (FITC, green) and anti-goat (CY3, red) antibodies were used. DAPI (servicebio) was used to stain nuclei. Images were captured under a 20 × or 40 × objective with an ECLIPSE Ti-U microscope using the NLS-ElementsBR.3.0software (Nikon, Melville, NY). The immunofluorescence intensity was measured using ImageJ software (Version1.52, National Institutes of Health).
Western blot analysis
Tissue from medullary dorsal horn homogenized in lysis buffer (consisting of RIPA, cocktail PMSF and phosphorylase inhibitor). Then, protein concentrations were measured using a BCA Protein Assay Kit (Beyotime). After heated in loading buffer for 10 min at 100℃, equal amounts of protein were loaded and separated on 10% SDS PAGE gels (Bio-Rad), and subsequently transferred to PVDF membranes (Millipore). After blocked with 5% nonfat milk for 90 min at RT, the membrane was in incubated in primary antibody overnight at 4 °C. The dilution concentration of the primary antibody was applied as follows: IL-18 (1:1000; Abcam), IL-18R (1:50; Abcam), GFAP (1:2500; Gene Tex), p65(1:3000, Abcam), p-p65 (1:50; Santa Cruz) and NAPDH (1:1500, servicebio). The membrane then was washed three times in TBST and incubated with respective secondary antibodies for 60 min at RT. The antibody-reactive bands were visualized using Odyssey CLx Image Studio 3. The gray-scale value of straps was quantified using Image J software (Version1.52, National Institutes of Health). All Western blot analysis were conducted at least three times, and consistent results were obtained.
All data are presented as the mean ± SEM. GraphPad Prism 8 (GraphPad Software, San Diego, CA) were used to perform statistical analysis and to generate the graphs. T-test was used to evaluate differences between two independent groups. P < 0.05 was considered significant.