Mice

All procedures were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis and were conducted in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals. To avoid social isolation stress, all mice were group housed (2–5 per cage, same sex) in the animal facility of Washington University in St. Louis on a 12-hour light-dark cycle with constant temperature (23–24 °C), humidity (45–50%), and food and water ad libitum. All experiments were performed during the light phase (9 am – 4 pm). Adult mice (8–14 weeks old, both male and female) were used in the experiments. A total of 90 male and 170 female mice of various genotypes were used in this study. Estrous cycle analysis was not performed in female mice. In experiments using only wild-type mice, C57BL/6J or CD-1 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or Charles River (O’Fallon, MO), respectively.

CCL2 and CCR2 global knockout mice

CCL2 and CCR2 global knockout (KO) breeders on C57BL/6J background were purchased from the Jackson Laboratory (strains 004434 and 027619) and were crossed with wild-type C57BL/6J mice to generate heterozygous (HZ) breeders. Wild-type and KO mice were generated by crossing heterozygous breeders. CCR2 KO mice express enhanced green fluorescent protein (EGFP) from Ccr2 alleles, abolishing endogenous CCR2 expression [15].

CGRPα knockout mice

Wild-type and CGRPα global KO mice on C57BL/6J background were generated by crossing HZ breeders purchased from the Mutant Mouse Resource & Research Center (MMRRC_036773_UNC, Chapel Hill, NC).

DEREG transgenic mice

DEREG (Depletion of regulatory T cell) breeders (strain 32050) on C57BL/6J background were purchased from the Jackson Laboratory. The DEREG mice contain a transgenic allele that expresses the diphtheria toxin receptor-EGFP (DTR-EGFP) fusion protein under the control of the genomic sequences that regulate the expression of endogenous FOXP3 [16]. DEREG breeders were crossed with C57BL/6J mice to generate HZ for experiments. We also crossed DEREG and CCL2 KO mice to generate DEREG_CCL2KO that express EGFP signal in Treg cells on CCL2 KO background.

Genotypes of genetically engineered mice were determined by polymerase chain reaction (PCR) of tail DNA as described previously [15,16,17,18].

Mouse model of medication overuse headache

Mice were extensively handled by the experimenters for 2 weeks and were well-habituated to the test room and the test apparatus before baseline measurement. The experimenters were blinded to the genotype and the treatments mice received during data collection and analysis. To model MOH, mice received intraperitoneal (i.p.) injections of sumatriptan succinate (0.6 mg/kg, Fresenius Kabi, Lake Zurich, IL [12]), once every day for 12 days. The withdrawal thresholds to mechanical stimuli on facial skin were measured at various time points.

One to three days before testing, mice were lightly anesthetized with isofluorane. The hair on the mouse forehead (above and between the eyes) was shaved and trimmed with a Multi-Groom Ultra Precise Beard Styler trimmer Groomer (Philips, PHMG1100/16, Cambridge MA). On the test day, the experimenters gently held the mouse on the palm with minimal restraint and applied the calibrated von Frey filament perpendicularly to the shaved skin, causing the filament to bend for 5 s. A positive response was determined by the following criteria as in previous studies: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking [12]. The up-down paradigm was used to determine the 50% withdrawal threshold [19].

In some experiments, after the facial mechanical threshold returned to baseline level, mice received 2 daily injections of nitroglycerin (NTG, 0.1 mg/kg, i.p.). Facial mechanical thresholds were measured 1 day later to assess repeated sumatriptan-induced hyperalgesic priming.

Drug treatments

On the days that the mouse behaviors were tested, all drugs were injected after the completion of the behavioral assays. Sumatriptan succinate (Fresenius Kabi, Melrose Park, IL) was freshly diluted from a stock solution (12 mg/ml, stored at 4oC) with saline before each injection (0.6 mg/kg, i.p.). The NTG stock solution (10% in propylene glycol, SDM27, Copperhead Chemical, Tamaqua, PA) was stored in airtight glass vials at 4oC and was freshly diluted with saline before each injection. Mice received 2 daily i.p. injections of NTG (0.1 mg/kg, in saline with 0.01% propylene glycol) to assess hyperalgesic priming.

To neutralize the endogenous CCL2, mice received i.p. injections of the anti-mouse CCL2 antibody (CCL2 ab, 200 µg/mouse, BE0185; BioXcell, West Lebanon, NH [10]) every 4–7 days. The control group received isotype-matched control immunoglobulin (IgG, 200 µg/mouse, BE0091; BioXcell) in parallel.

Recombinant mouse IL-2 (carrier-free, Biolegend, San Diego, CA) was freshly diluted from the stock (1 mg/ml aliquots at -80oC) every day. Mice received daily i.p. injections of saline or IL-2 (1 µg in 100 µl saline) for 6–12 days.

To deplete endogenous Treg cells, DEREG mice received i.p. injections of 0.5 µg diphtheria toxin (Sigma) in 100 µl saline for two consecutive days every 6 days [20, 21].

RNA extraction, reverse transcription and quantitative polymerase chain reaction

Female CD-1 mice received i.p. saline or sumatriptan (0.6 mg/kg) injections once every days for 4 or 12 times. Mice were euthanized 1 day after the last sumatriptan administration. Dura and TG tissues were quickly removed, stored in RNAprotect Tissue reagent (Qiagen, Valencia, CA) and then homogenized using a Polytron homogenizer (Qiagen). Total RNA was isolated using a RNeasy mini Kit (Qiagen) and treated with DNase. Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) based on the manufacturer’s protocols.

The quantitative PCR (qPCR) reactions were performed with samples in triplicate on an ABI 7500 fast real-time PCR system using the Taqman Gene Expression Master Mix (Applied Biosystems) [10]. The primer Taqman probe for mouse CCL2 and CCR2 cDNAs (Mm00441242_m1 and Mm99999051_gH, Applied Biosystems) were used. The real-time qPCR reactions underwent 50 cycles; cycling conditions for these genes were as follows: 2 min (min) at 95 °C for denaturing, 3 s at 95 °C for annealing, and 30 s at 60 °C for extension. Duplicate samples without cDNA (no-template control) for each gene showed no contaminating DNA. Relative CCL2 and CCR2 mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm 99999915_g1, Applied Biosystems) and quantified by use of the comparative CT (ΔΔCT) method for calculating relative quantitation of gene expression.

Primary culture of mouse trigeminal ganglion neurons and ratiometric Ca2+ imaging

Ratiometric Ca2+ imaging of TG neurons was performed as described in a previous study [22]. Briefly, after 12 daily injections of saline or sumatriptan, TG tissues were collected and were treated with 2.5 mg/ml collagenase IV for 10 min followed by 2.5 mg/ml trypsin at 37oC for 15 min. Cells were dissociated by triturating with fire-polished glass pipettes, resuspended in MEM-based culture medium containing 5% fetal bovine serum, 25 ng/ml nerve growth factor (R&D, Minneapolis, MN) and 10 ng/ml glial cell-derived neurotrophic factor (R&D), and seeded on Matrigel-coated coverslips. Ca2+ imaging was performed 2 days later. Each experiment contained neurons from at least 2 batches of culture.

Coverslips containing cultured TG neurons were incubated with HBSS/HEPES solution containing 2.5 µM fura-2 AM and 10% Pluronic F-68 (both from Molecular Probes, Eugene, OR) at 37oC for 45 min to load the ratiometric Ca2+ indicator. De-esterification of the dye was carried out by washing the coverslips 3 times with HBSS/HEPES solution and incubating the coverslips in HBSS/HEPES solution in the dark for an additional 15 min at 37oC. Neurons were used for Ca2+ imaging experiments within 1 h after Fura-2 loading.

Coverslips with fura-2 loaded neurons were placed in a flow chamber mounted on a Nikon TE2000S inverted epifluorescent microscope and were perfused with room temperature (RT) Tyrode’s solution (1 ml/min) containing (in mM): 130 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 25 Hepes, 30 glucose, pH 7.3–7.4 with NaOH, and 310 mosmol/kgH2O. Healthy neurons were chosen based on the differential interference contrast images. Fura-2 was alternately excited by 340 and 380 nm light (Sutter Lambda LS, Sutter Instrument, Navato, CA) and the emission was detected at 510 ± 20 nm by a UV-transmitting 20x objective (N.A. 0.75) and a prime BSI back illuminated sCMOS camera (Photometrics, Tuscon, AZ). The frame capture period was 50 milliseconds at 1.5 s interval. Metamorph software (Molecular Devices, San Jose, CA) was used for controlling and synchronizing the devices as well as image acquisition and analysis. After a 2–3 min baseline measurement in Tyrode’s solution, neurons were perfused with 50 nM PACAP1 − 38 (pituitary adenylate cyclase–activating polypeptide 1–38, Tocris, Ellisville, MO) for 1 min followed by washing with Tyrode’s for 4 min. Subsequently, the coverslip was incubated with 3 µM human CGRPα (Tocris) for 1 min followed by washing with Tyrode’s for 4 min. CGRP and PACAP were freshly diluted from the stock (aliquots at -80oC) in Tyrode’s solution every day.

Regions of interest (ROIs) encompassing individual neurons were defined a priori. The ratio of fluorescence excited by 340 nm divided by fluorescence excited by 380 nm (R340/380) was determined on a pixel-by-pixel basis and was averaged for each ROI. An additional background area was recorded in each field for off-line subtraction of background fluorescence. Peak responses were determined by calculating the relative increase in R340/380 above baseline (F0, the average R340/380 during the 2–3 min baseline measurement). A ΔF/F0 > 20% was set as the threshold for a response.

Blood collection, antibody staining and flow cytometry

Mice used for flow cytometry analysis were not used in behavioral tests. Female DEREG and DEREG_CCL2KO mice received 5 daily injections of LD-IL-2. About 100 µl blood was collected from each mouse by submandibular bleeding. Cell suspensions were stained with the antibodies (all from Biolegend) that recognize mouse CD3ε (clone 145-2C11), CD4 (clone GK1.5 and RM4-5), and CD25 (clone PC61) for 20 min. The frequency of individual cell subpopulations was determined via flow cytometric analysis. Data were collected with FACScan (Becton Dickinson, Franklin Lakes, NJ) and analyzed with the CellQuest Pro (Becton Dickinson) and Rainbow X Alias (Cytek, Freemont, CA) software.

Tissue preparation, immunohistochemistry, and image analysis

Immunohistochemistry approach was used to quantify Treg cells in the dura because the distribution of Treg cells is highly uneven in different regions of the dura. Immunohistochemistry analysis allows us to focus on Treg cells in dura areas adjacent to the middle meningeal artery (MMA). This increases the sensitivity of detecting the effect of CCL2-CCR2 signaling on Treg cell infiltration from peripheral blood to tissues. Moreover, dural afferent neurons innervating the MMA region have been implicated in headache pathophysiology by many studies, Treg cells in this region likely contribute to the neuroimmune interactions that regulate dural afferent activities and headache susceptibility. We also used immunohistochemistry to quantify Treg cells in TG. This allowed us to easily distinguish Treg cells localized within the TG tissues from those that are localized in the membrane surrounding the TG tissues.

Immunohistochemistry and image analysis were performed as previously described [10, 12], with experimenters blinded to the treatments mice received. Briefly, mice were euthanized with i.p. injection of barbiturate (200 mg/kg) and were transcardially perfused with warm 0.1 M phosphate buffered saline (PBS, pH 7.2) followed by cold 4% formaldehyde in 0.1 M phosphate buffer (PB, pH 7.2) for fixation. TG tissues were collected and sectioned at 15 μm in the transverse plane on a cryostat, collected on Superfrost Plus glass slides in sequence and stored at -20 °C.

One in every 3 TG sections were processed for each immunohistochemistry experiment. The sections were dried at RT, washed three times in 0.01 M PBS, and incubated in blocking buffer consisting 0.01 M PBS, 10% normal goat serum (NGS), and 0.3% triton X-100 for 1 h at RT. Sections from DEREG and DEREG_CCL2KO mice were then incubated with the chicken anti-EGFP antibody (1:1000 dilution in blocking buffer, AVES Lab, Davis, CA) in a humidity chamber at 4 °C overnight. After 6 washes (5 min each) in washing buffer containing 0.01 M PBS with 1% NGS and 0.3% triton, sections were incubated with blocking buffer for 1 h, followed by the incubation with AlexaFluor 488-conjugated secondary antibody (1:1000 dilution in blocking buffer, Invitrogen, Waltham, MA) at RT for 1 h. After washing off the antibody, sections were rinsed with PBS, cover-slipped using Fluoromount-G Slide Mounting Medium (Electron Microscopy, Hatfield, PA), sealed with nail polish, and stored at 4 °C. Sections from CCR2 HZ mice were incubated overnight with AlexaFluor 594-conjugated anti-mouse CD25 antibody (1:50, clone PC61, Biolegend) in a humidity chamber at 4 °C.

Immunofluorescence was observed through a 40x objective on a Nikon TE2000S inverted epifluorescence microscope. To quantify Treg cells in TG, all CD25+ cells in individual sections from CCR2 HZ mice and all EGFP+ cells in individual sections from DEREG_WT and DEREG_CCL2KO mice were counted, and the number was multiplied by 3 to obtain the total number of cells per TG in each mouse. Cells that were localized in the membranes surrounding the TG tissues were not included in the quantification.

Dura was carefully dissected from the skull using forceps after 4 h fixation, washed 3 times in 0.01 M PBS and stained as whole-mount in a 48-well plate. The blocking and washing buffers used for dura staining contained 0.1% triton. Treg cells in the area adjacent to the MMA were quantified.

Statistical analysis

For behavioral experiments, power analysis was conducted to estimate sample size with > 80% power to reach a significance level of 0.05. For immunohistochemistry and flow cytometry experiments, sample sizes were estimated based on our prior experience.

All data were reported as mean ± standard error of the mean. The Shapiro–Wilk test was used to check data normality. Statistical significance within or between experimental groups was assessed by two-tailed t test, analysis of variance (ANOVA, one-way or two-way, with or without repeated-measures [RM]) with post hoc Bonferroni test or Student-Newman-Keuls test, where appropriate, using Origin (OriginLab Corporation, Northampton, MA) or Statistica (StatSoft Inc, Tulsa, OK). Differences with p < 0.05 were considered statistically significant. The statistical analysis for individual experiments was described in figure legends or in Table 1.

Table 1 Statistical analysis in individual experiments