Experimental model and subject details
Animals. Male mice were use throughout (25–30 g, 5–8 weeks). The following strains of mice were used C57BL/6J mice (Charles River, RRID:IMSR_JAX:000664); wild-type (Trpa1+/+) and TRPA1-deficient (Trpa1−/−; B6129P-Trpa1tm1Kykw/J; RRID:IMSR_JAX:006401, Jackson Laboratory) mice53; wild-type (Trpv1+/+) and TRPV1-deficient (Trpv1−/−; B6129X1-Trpv1tm1Jul/J, RRID:IMSR_JAX:003770, Jackson Laboratory) mice. Genetically modified mice were maintained as heterozygotes on a C57BL/6J background.
To generate mice in which the Trpa1 and Ramp1 genes were conditionally silenced in Schwann cells/oligodendrocytes, homozygous 129S-Trpa1tm2Kykw/J (floxed TRPA1, Trpa1fl/fl, RRID:IMSR_JAX:008649 Jackson Laboratory) and C57BL/6N-Ramp1< tm1c(EUCOMM)Wtsi>/H (floxed Ramp1, Ramp1fl/fl Stock No: EM:07401, MRC HARWELL Mary Lion Center)54 were crossed with hemizygous B6.Cg-Tg(Plp1-CreERT)3Pop/J mice (Plp1-CreERT, RRID:IMSR_JAX:005975 Jackson Laboratory), expressing a tamoxifen-inducible Cre in myelinating cells (Plp1, proteolipid protein myelin 1)28. The progeny (Plp1-CreERT;Trpa1fl/fl and Ramp1-CreERT;Trpa1fl/fl) was genotyped by PCR for Trpa1, Ramp1 and Plp1-CreERT. Mice negative for Plp1-CreERT (Plp1-CreERT−;Trpa1fl/fl and Plp1-CreERT−;Ramp1fl/fl) were used as control. Both positive and negative mice to CreERT and homozygous for floxed Trpa1 (Plp1-CreERT;Trpa1fl/fl and Plp1-CreERT−;Trpa1fl/fl, respectively) and floxed Ramp1 (Plp1-CreERT;Ramp1fl/fl and Plp1-CreERT−;Ramp1fl/fl) mice were treated with 4-hydroxytamoxifen (4-OHT) by subcutaneous periorbital (p.orb.) injection (0.02 mg/10 µl in corn oil once a day for 3 consecutive days). Some Plp1-CreERT;Ramp1fl/fl and Plp1-CreERT−;Ramp1fl/fl mice were treated with intraperitoneal (i.p.) 4-OHT (1 mg/100 µl in corn oil, once a day for 3 consecutive days).
Treatments resulted in Cre-mediated ablation of Trpa1 and Ramp1 in PLP-expressing Schwann cells/oligodendrocytes. To selectively delete the Trpa1 and Ramp1 gene in primary sensory neurons, Trpa1fl/fl and Ramp1fl/fl mice were crossed with hemizygous Advillin-Cre mice (Adv-Cre)28, 55, 56. Both positive and negative mice to CreERT and homozygous for floxed Trpa1 (Adv-Cre+;Trpa1fl/fl and Adv-Cre−;Trpa1fl/fl, respectively) and floxed Ramp1 (Adv-Cre+;Ramp1fl/fl and Adv-Cre−;Ramp1fl/fl) were used.
The group size of n = 8 animals for behavioral experiments was determined by sample size estimation using G*Power (v3.1)57 to detect size effect in a post-hoc test with type 1 and 2 error rates of 5 and 20%, respectively. Mice were allocated to vehicle or treatment groups using a randomization procedure (http://www.randomizer.org/). Investigators were blinded to the identities (genetic background) and treatments, which were revealed only after data collection. No animals were excluded from experiments.
All behavioral experiments were in accordance with European Union (EU) guidelines for animal care procedures and the Italian legislation (DLgs 26/2014) application of the EU Directive 2010/63/EU. Study was approved by the Italian Ministry of Health (research permits #383/2019-PR and #765/2019-PR). The behavioral studies followed the animal research reporting in vivo experiment (ARRIVE) guidelines58. Mice were housed in a temperature- and humidity-controlled vivarium (12 hr dark/light cycle, free access to food and water, 5 animals per cage). At least 1 hr before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. All the procedures were conducted following the current guidelines for laboratory animal care and the ethical guidelines for investigations of experimental pain in conscious animals set by the International Association for the Study of Pain59. Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO2 plus 10–50% O2.
Cell lines. Primary cultures of human Schwann cells (HSCs, #1700, ScienCell Research Laboratories) were grown and maintained in Schwann cell medium (#1701, ScienCell Research Laboratories) at 37°C in 5% CO2 and 95% O2. Cells were passaged at 90% confluency and discarded after 12 passages. HEK293T (#CRL-3216™, American Type Culture Collection) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with heat inactivated fetal bovine serum (FBS, 10%), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C in 5% CO2 and 95% O2. The mouse Schwann cell line (IMS32 cells, #PMC-SWN-IMS32-COS, Cosmo Bio USA) was grown and maintained in Schwann cell medium (#PMC-SWN-MM-COS, Cosmo Bio USA) at 37°C in 5% CO2 and 95% O260, 61. All cells were used when received without further authentication.
Pharmacological reagents. Supplementary Tables 1 and 2 provide doses, routes of administration and concentrations and of all pharmacological reagents.
Behavioral experiments
Treatment protocol. Subcutaneous injections were made in the periorbital area 2-3 mm from the external eyelid corner17. Briefly, the mouse was lifted by the base of the tail and placed on a solid surface with one hand and the tail was pulled back. Then, it was quickly and firmly picked up by the scruff of the neck with the thumb and index finger of the other hand. Injection was made rapidly by a single operator with minimal animal restraint. Mice received unilateral (right side) injections (10 μl/site) of CGRP (1.5 nmol in 0.9% NaCl), SP (3.5 nmol in 0.9% NaCl), capsaicin (10, 50, 100 pmol in 0.1% dimethyl sulfoxide, DMSO), or vehicles (control). Mice received bilateral injections (10 µl/site, right side same site as stimulus, left side symmetrical to right side) of antagonists and inhibitors. CGRP (1.5 nmol in 0.9% NaCl) or vehicle was also administered by intraplantar (i.pl., 20 μl/site) or systemic (0.1 mg/kg, i.p.) injection. GTN was administered at 10 mg/kg, i.p. injection.
Acute nociception. Immediately after the p.orb. injection, mice were placed inside a plexiglass chamber and spontaneous nociception was assessed for 10 min by measuring the time (s) that the animal spent rubbing the injected area of the face with its paws17, 62.
Periorbital mechanical allodynia. PMA was assessed using the up-down paradigm63, 64 in the same mice in which acute nociceptive responses were monitored. Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds17. One day before the first behavioral observation, mice were habituated to the apparatus. PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum)65 before (basal threshold) and after (0.5, 1, 2, 4, 6, 8 hr) treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. Mechanical stimuli were applied homolaterally outside the periorbital area at a distance of 6-8 mm from the site where stimuli were injected. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. Absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e. lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined.
Paw mechanical allodynia. Paw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm63, 64. Mice were acclimatized (1 hr) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6 and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated.
Primary culture of mouse Schwann cells. Mouse Schwann cells (MSC) were isolated from sciatic nerves of C57BL/6J, Trpa1+/+ and Trpa1-/-, Plp1-CreERT+;Ramp1fl/fl and Plp1-CreERT-;Ramp1fl/fl mice28, 66. The epineurium was removed, and nerve explants were divided into 1 mm segments and dissociated enzymatically using collagenase (0.05%) and hyaluronidase (0.1%) in Hank's Balanced Salt Solution (HBSS, 2 hr, 37 °C). Cells were collected by centrifugation (800 rpm, 10 min, room temperature) and the pellet was resuspended and cultured in DMEM containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 μM). Three days later, cytosine arabinoside (Ara-C, 10 mM) was added to remove fibroblasts. Cells were cultured at 37 °C in 5% CO2 and 95% O2. The culture medium was replaced every 3 days and cells were used after 15 days of culture.
qRT-PCR. Detailed method and the sets of primers for human and mouse cells are reported in Supplementary information and Table S3.
Calcium imaging. HSCs, MSCs and IMS32 cells were plated on poly-L-lysine-coated (8.3 μM) 35 mm glass coverslips and maintained at 37 °C in 5% CO2 and 95% O2 for 24 hr. Cells were loaded (40 min) with Fura-2 AM-ester (5 μM) added to the buffer solution (37 °C) containing (in mM) 2 CaCl2; 5.4 KCl; 0.4 MgSO4; 135 NaCl; 10 D-glucose; 10 HEPES and bovine serum albumin (BSA, 0.1%) at pH 7.4. Cells were exposed to CGRP (0.01-10 μM) or vehicle (0.9% NaCl) and the Ca2+ response was monitored for approximately 40 min. In another set of experiments, IMS32 cells were exposed to AITC (30 μM) or vehicle (0.03% DMSO). The Ca2+ response to CGRP was monitored in the presence of CGRP8-37 (100 nM), olcegepant (100 nM), SQ22536 (100 μM), L-NAME (10 μM), A967079 (50 μM), PBN (50 μM), H89 (1 μM), ML171 (1 μM) or vehicle (0.1 % DMSO), and also in the presence of DIPMA-MK-3207 (1-1000 nM) and MK-3207 free drug (0.01-1000 μM) or DIPMA-empty. Some experiments used Ca2+-free buffer solution containing EDTA (1 mM). Results were expressed as percent increase in ratio340/380 over baseline normalized to the maximum effect induced by ionomycin (5 μM) added at the end of each experiment.
Protein extraction and western immunoblot assay. Detailed method is reported in Supplementary information.
In-cell ELISA assay. HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 105 cells/well) and maintained at 37 °C in 5% CO2 and 95% O2 for 24 hr. HSCs and IMS32 cells were exposed to CGRP (1 and 10 μM, respectively) or its vehicle (phosphate buffered saline, PBS) for 5, 10, 15, 30 and 60 min, at 37 °C, then washed with DMEM pH 2.5 and fixed in 4% paraformaldehyde for 30 min. Cells were then washed with TBST (0.05%) and blocked with donkey serum (5%) for 4 hr at room temperature and incubated overnight 4 °C with eNOSpS1177 (#ab184154, rabbit polyclonal, 1:100, Abcam). Cells were then washed and incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase (HRPO, 1:2000, Bethyl Laboratories Inc.) for 2 hr at room temperature. Cells were then washed and stained using SIGMAFAST OPD for 30 min protected from light. After the incubation period, the absorbance was measured at 450 nm. Change in NOS3 phosphorylation was calculated as a percentage of the signal in vehicle-treated cells.
cAMP ELISA assay. cAMP level was determined by the CatchPoint™ cyclic-AMP fluorescent assay kit (#R8088, Molecular Device) according to the manufacturer’s protocol. Briefly, HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 105 cells/well) and maintained in 5% CO2 and 95% O2 (24 hr, 37 °C). The cultured medium was replaced with HBSS added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM) or vehicle (0.1% DMSO in HBSS) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively), forskolin (1 μM, positive control) or their vehicles (HBSS) and maintained for 40 min at room temperature protected from light. Signal was detected 60 min after exposure to the stimuli. cAMP level was calculated using cAMP standards and expressed as nmol/1.
Nitric oxide assay. Nitric oxide was determined by using the fluorometric-orange assay kit (#ab219932, Abcam) according to the manufacturer’s protocol. HSCs or IMS32 cells were plated 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 105 cells/well) and maintained in 5% CO2 and 95% O2 (24 hr, 37 °C). The cultured medium was replaced with Hanks' balanced salt solution (HBSS) added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM), A967079 (50 μM), PS2 or Dy4 (both 30 µM), PS2 inact or Dy4 inact (30 µM) or vehicle (0.1% DMSO in HBSS) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively), diethylamine NONOate (1 mM, positive control) or their vehicles (HBSS) and maintained for 40 min at room temperature protected from light. Signal was detected 60 min after exposure to the stimuli. Change in nitric oxide level was calculated as percentage of the vehicle.
H2O2 assay. H2O2 was determined by using the Amplex Red assay (#A12222, Thermo Fisher Scientific). Detailed method is reported in Supplementary information.
Immunofluorescence. Anesthetized mice were transcardially perfused with PBS and 4% paraformaldehyde. Trigeminal and sciatic nerves were removed, postfixed for 24 hr, and paraffin-embedded. Human and mouse formalin fixed paraffin embedded (FFPE) sections (5 μm) were incubated with primary antibodies: TRPA1 (#ab58844, rabbit polyclonal, 1:400, Abcam), S100 (#ab14849, mouse monoclonal [4B3], 1:300, Abcam), CLR (#NLS6731, rabbit polyclonal, 1:30, Novus Biologicals), RAMP1 (#ab241335, rabbit polyclonal, 1:200, Abcam), diluted in fresh blocking solution (PBS, pH 7.4, 2.5% normal goat serum, [NGS]). Sections were then incubated with the fluorescent polyclonal secondary antibodies Alexa Fluor 488 and 594 (#A32731, #A32727 1:600; Invitrogen), and coverslipped using mounting medium with DAPI (Abcam). The Pearson correlation (Rcoloc) value for RAMP1 and S100 in the colocalization studies were calculated using the colocalization Plugin of the ImageJ software (ImageJ 1.32J, National Institutes of Health). The use of FFPE sections of human abdominal cutaneous tissues was approved by the Local Ethics Committee of the Florence University Hospital (Area Vasta Toscana Centro) (18271_bio/2020), according to the Helsinki Declaration, and informed consent was obtained.
cAMP cADDIS assay. HSCs were plated on poly-D-lysine-coated 96-well black wall clear bottom plates (Corning Life Sciences) (25 × 103 cells/well) and incubated in 5% CO2 and 95% O2 for 4-6 hr. HSCs were transduced with the baculovirus mediated Green Upward cADDIS cAMP reporter (25 µl/well, Montana Molecular) following manufacturer’s instructions, and cells were incubated in 5% CO2 and 95% O2 (48 hr, 37 °C). HSCs were washed twice in HBSS plus HEPES (10 mM) pH 7.4. Cells were incubated in HBSS/HEPES with the CLR/RAMP1 antagonists olcegepant (100 pM-100 µM) or vehicle (control) for 30 min. Plates were mounted in a FlexStation3 plate reader (Molecular Devices) and fluorescence (485-500 excitation, 515-530 emission with cutoff at 510) was monitored. Baseline was measured for 1 min, and cells were stimulated with human CGRPa (100 pM-10 µM) or forskolin (10 µM, positive control). For single cell imaging, HSCs were plated on poly-D-lysine-coated 35 mm glass bottom dishes (MatTek, Ashland) (40 × 103 cells/dish) and incubated in 5% CO2 and 95% O2 (overnight, 37 °C). HSCs cells were transduced with Green Upward cADDIS and incubated for 48 hr. HSCs were washed twice in HBSS/HBS and mounted on a Leica DMI8 microscope (Wetzlar, Germany). Fluorescence (470/40 excitation, 527/30 emission) was measured every 5 s. Baseline was measured for 30 s, and HSCs were challenged with human CGRPa (100 nM). Images were analyzed with ImageJ (NIH). To inhibit endocytosis, cells were incubated in HBSS containing 0.45 M sucrose or normal HBSS (control) for 30 min at 37 °C before cAMP assays.
CAMYEL BRET cAMP assay. HEK293 cells stably expressing the CAMYEL BRET sensor (~2x106) were seeded into 90 mm Petri dish (Corning™, USA) in DMEM/FBS/Geneticin and incubated in 5% CO2 and 95% O2 (24 hr, 37 °C). Prior to the transfection, the medium was changed to fresh DMEM/FBS/Geneticin and rat CLR/RAMP1 was transfected (2.5 µg CLR/RAMP1 DNA/dish) using JetPEI (Polyplus Transfection, France) at a 1:6 ratio. After 24 hr, cells were plated in poly-L-lysine coated black 96 well CulturPlate (Perkin Elmer, USA) and incubated in 5% CO2 and 95% O2 (24 hr, 37 °C). BRET was assessed using a LUMIstar (BMG LABTECH, Germany). On the day of the assay, cells were equilibrated in HBSS for 30 min, supplemented with 12 mM HEPES at 37 °C in CO2-free incubator. DIPMA-MK-3207 or free MK-3207 was incubated for 25 min, followed by the addition of coelentrazine-h (50 µM) for 5 min. Baseline was then measured for 5 min, followed by stimulation with CGRP (100 nM, ~EC50), vehicle (HBSS) or forskolin (1 µM, positive control), and further measurements for 5 min. Buffer was then replaced by HBSS with coelentrazine-h (50 µM) and measurements were resumed for further 25 min.
Synthesis of TAMRA-CGRP and endocytosis of TAMRA-CGRP and Cy5-DIPMA nanoparticles. Detailed method is reported in supplementary information.
EbBRET assays of G protein and βARR recruitment to the plasma membrane and endosomes cDNAs. Detailed method is reported in supplementary information.
Synthesis and Characterization of Nanoparticles. Detailed method is reported in supplementary information.
Statistical analysis. Results are expressed as mean ± standard error of the mean (SEM). For multiple comparisons, a one-way analysis of variance (ANOVA) followed by the post-hoc Bonferroni’s test or Dunnett’s test was used. Two groups were compared using Student’s t-test. For behavioral experiments with repeated measures, the two-way mixed model ANOVA followed by the post-hoc Bonferroni’s test was used. Statistical analyses were performed on raw data using Graph Pad Prism 8 (GraphPad Software Inc.). IC50 values and confidence intervals were determined from non-linear regression models using Graph Pad Prism 8 (GraphPad Software Inc.). P values less than 0.05 (P<0.05) were considered significant. Statistical tests used and the sample size for each analysis are listed in the Fig. legend.
Data Availability. Materials and data generated from this study are available upon request from Pierangelo Geppetti ([email protected]) or Nigel W. Bunnett ([email protected]).