Animals, spared nerve injury surgery and SAFit2 treatment

In all experiments, wild-type mice (male C57Bl/6N mice, age from 8 to 12 weeks at the start of the study) were purchased from commercial breeding companies (Janvier and Charles River). For inducing neuropathic pain in mice, a spared nerve injury (SNI) surgery was performed under anesthesia to establish neuroinflammation. During the surgery, the sciatic nerve was exposed by a blunt dissection on the level of the knee joint. Then, two of the three sciatic nerve branches, common peroneal and tibial branch, were ligated with 6/0 non-sterile silk thread and cut distally from the ligature, leaving the sural nerve branch intact. Contact with the sural nerve branch was avoided to prevent stretching or harming. Afterwards, muscle and skin were closed in two layers [9].

For assessing the impact of SAFit2 on neuropathic pain behavior and neuroinflammation, animals were treated intraperitoneally with either 10 mg/kg SAFit2 or vehicle (PBS supplemented with 5% PEG400, 5% Tween and 0.7% ethanol) two times daily on six consecutive days starting on day five after surgery.

Behavioral experiments

During all behavioral experiments, the experimenter was blinded. The motoric function of all animals was verified via rotarod before treatment. All animals were transferred into respective test cages for at least one hour before the measurement to allow habituation. For the determination of the mechanical withdrawal threshold, a dynamic plantar test was performed, using a Dynamic Plantar Aesthesiometer (Ugo Basile) as described previously. Briefly, a steel rod was pushed against the mid-plantar area of the hind paw with linear ascending force up to 5 g over 10 s and a holding force of 5 g until a withdrawal response occurred. For behavioral experiments a cut-off time of 20 s was set [49].

Tissue isolation

For tissue isolation, mice were euthanized by isoflurane, cardio puncture and cranial dislocation either on day 14 or on day 21 after surgery covering different time points in neuroinflammation. The sciatic nerve, lumbar (L4–L6) dorsal root ganglia (DRGs) and the respective segments of the spinal cord were dissected from injured (ipsilateral) and unimpaired (contralateral) sites, followed by freezing tissue samples in liquid nitrogen for either RNA isolation, Western blot or multiplex assay. For flow cytometry analysis, the tissue was dissected and stored in 500 µl ice-cooled PBS at 4 °C until further processing.

Quantitative real-time PCR

Total RNA was isolated from L4–L6 DRGs and the respective segments of spinal cord using the mirVana miRNA Isolation Kit (Applied Biosystems) according to the manufacturer´s instructions. Afterwards, RNA concentrations were quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and a cDNA synthesis was performed with 200 ng RNA from DRGs and 400 ng RNA from spinal cord. The reverse transcription was performed with the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The quantitative real-time PCR was conducted with QuantStudio™ Design & Analysis Software v 1.4.3 (Thermo Fisher Scientific) in a TaqMan® Gene Expression Assay System (Table 1, Thermo Fisher Scientific) according to the manufacturer´s instructions. The raw data were evaluated using the ΔΔC(T) method, as described previously [29, 47].

Table 1 List of used TaqMan® gene expression assaysMultiplex assay

For performing the ProcartaPlex multiplex immunoassay (Thermo Fisher), proteins were isolated with a manufacturer recommended cell lysis buffer, which was further supplemented with a phosphatase inhibitor cocktail (PhosSTOP, Roche) and a protease inhibitor cocktail (cOmplete, Roche). DRG samples were suspended in 100 µl and spinal cord samples in 200 µl of cell lysis buffer. The spinal cord samples were further processed by a cell grinder. Afterwards, the tissue was homogenized two times per sample using a Sonopuls Sonicator (Bandelin) with the setting 6 × 10%. During sonication, the samples were cooled in an ice bath, preventing proteins from degradation. Finally, the samples were centrifuged with 16,000 × g at 4 °C for 10 min, followed by collecting the supernatant for a protein concentration determination via Bradford.

The ProcartaPlex multiplex immunoassay was performed according to the manufacturer’s recommendations. Briefly, a dark wall 96-well plate was prepared by several washing steps and coating steps with respective magnetic beads. Afterwards, standards were prepared in a serial dilution (1:4, (v/v)) and added to the plate, followed by further washing steps. Lastly, the samples were diluted (1:2, (v/v)) and added to the plate, which was sealed and incubated for 40 min with 500 rpm on an orbital shaker at room temperature, overnight at 4 °C and further 50 min with 500 rpm at room temperature. On the next day, the detection antibody mixture was added after a washing step and the plate was further incubated on an orbital shaker for 30 min at room temperature. Again, the plate was washed and streptavidin phycoerythrin was added to the plate and incubated for 30 min as described above. After the last washing step, the plate was prepared with reading buffer, incubated for 5 min with 500 rpm on an orbital shaker, and measured with the Luminex 200 system (Bio-Rad).

Western blot

For Western blot purposes, the tissue of five animals was pooled and proteins isolated from the respective tissue samples. Therefore, 300 µl of cell lysis buffer, which was described in part 2.5, were added to spinal cord samples and 100 µl to DRG samples. The tissue samples were homogenized as previously described and the protein amount was determined by a Bradford assay.

Afterwards, 30 µg tissue lysate was loaded and separated by SDS-polyacrylamide gel electrophoresis (4% stacking gel, 12% running gel) and transferred on a nitrocellulose membrane with the Trans-Blot®Turbo™ Transfer System (BioRad). For total protein detection, the membrane was blocked with TBST buffer (20 mM Tris, 150 mM NaCl and 0.1% Tween20) containing 5% skimmed milk powder at room temperature for two hours, followed by an incubation overnight with primary antibodies at 4 °C: p65 1:500 (8242S, cell signaling technology), IΚBα 1:500 (4812S, cell signaling technology) and IKKβ 1:250 (2370S, cell signaling technology). For phosphorylated protein detection, the membranes were blocked with TBST buffer containing 5% BSA at room temperature for 2 h, followed by an incubation for at least 48 h with primary antibodies at 4 °C: p-p65 1:500 (3033, cell signaling technology) and p-IΚBα 1:250 (2859S, cell signaling technology). Beta-actin 1:1000 (ab8229, abcam) was used as a loading control. For labeling targets, the fluorescent-labeled secondary antibodies anti-rabbit labeled with IRDye680ED from donkey (Licor), and anti-goat labeled with IRDye800CW from donkey (Licor) were used in 1:5000 dilutions for one hour at room temperature. The antibody detection was performed with an Odyssey CLx device from Licor, followed by a quantification with Image Studio Software.

Flow cytometry analysis

For creating single-cell suspensions, sciatic nerves were sliced with a tissue scissor and spinal cords were briefly pottered with a tissue grinder at first. Afterwards, all tissue samples (DRGs, sciatic nerve and spinal cord) were incubated in 500 µl Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 3 mg/mL collagenase and 1 µl/mL DNase for 30 min at 37 °C. Then, the enzymatic reaction was stopped by further adding 500 µl DMEM supplemented with 10% FCS. To obtain a single-cell suspension, the samples were filtered through a 70-µm cell strainer and centrifuged at 400 g for 5 min. The supernatant was discarded and the cell pellet washed with 500 µl PBS supplemented with 0.5% BSA, followed by a further centrifugation step. Lastly, the cell pellet was suspended in 500 µl PBS supplemented with 0.5% BSA and 1 mM EDTA to prevent cells from aggregating until and during flow cytometry analysis. The flow cytometry analysis was performed essentially as described previously [37]. Single-cell suspensions were blocked with FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS-BSA for 20 min, stained with fluorochrome-conjugated antibodies (Table 2) and analyzed on a FACSymphony A5 flow cytometer (BD Biosciences). The data were analyzed using FlowJo VX (TreeStar). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp-Beads (BD Biosciences) were used for single-color compensation to create multicolor compensation matrices. For gating, fluorescence minus one control were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD Biosciences). For characterization of immune cell subsets, the antibodies in Table 2 were used.

Table 2 List of antibodies used for flow cytometry analysisIsolation and purification of dorsal root ganglia (DRGs)

For methods using sensory neurons in cell culture, DRGs were dissected and transferred into ice-cold HBSS with CaCl2 and MgCl2 (Gibco) directly after dissection. Afterwards, DRGs were treated with a collagenase/dispase solution, 500 U/mL collagenase and 2.5 U/mL dispase diluted in neurobasal medium (Gibco), at 37 °C for 75 min. The collagenase/dispase solution was removed by centrifuging and discarding the supernatant, the sensory neurons were washed twice with neurobasal medium containing 10% FCS, followed by an incubation with 0.05% trypsin (v/v) (Gibco) for another 10 min. The washing steps were repeated, and the cells were mechanically dissociated in neurobasal medium (Gibco) supplemented with L-glutamine (2 mM; Gibco), penicillin (100 U/mL; Gibco), streptomycin (100 µg/mL; Gibco), B-27 (Gibco) and gentamicin (50 µg/mL; Gibco). Afterwards, the cell solution was plated on poly-L-lysine-coated cover slips or 48-well plates. After two hours of incubation, 2 mL of neurobasal medium was added and the cells were incubated overnight at 37 °C. For further investigations, the cultured DRGs were further used and treated as described in part 2.9 and 2.13.

Calcium imaging

For calcium imaging, the cultured sensory neurons were stained with fura-2-AM (Thermo Fisher) for at least 60 min at 37 °C and washed afterwards twice with Ringer´s solution. This was set up freshly with 145 mM NaCl, 1.25 mM CaCl2 × 2H2O, 1 mM MgCl2 × 6 H2O, 5 mM KCl, 10 mM D-glucose and 10 mM HEPES and adjusted to a pH of 7.3. During the experiments, Ringer’s solution was also used for baseline measurements and for washing out agonists and compounds between stimulations. For investigating the effect of SAFit2 on different TRP channels, the sensory neurons were pre-incubated with the respective compound for 2 min and stimulated with respective agonists afterwards: 100 nM capsaicin for 30 s (TRPV1 agonist) and 100 mM allyl isothiocyanate for 45 s (TRPA1 agonist). Control experiments were performed with the respective volume of the corresponding vehicle. All stimulating compounds were dissolved in Ringer’s solution to their final concentration. The measurements were performed using a DMI4000 B Microscope, the compact light source CTR550 HS (Leica) and the ValveBank II system (AutoMate Scientific).

Flexstation method

Cells were grown in 96-well black-walled plates. Human TRPV1 was transiently expressed in HEK-293t cells using jetPEI transfection reagent (Polyplus Transfection). Cells were loaded with Calcium 6 for two hours (Calcium 6 Kit; Molecular devices, San Jose, CA), in an extracellular solution containing 145 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 1.25 mM CaCl2, and 1 mM MgCl2, buffered to pH 7.4 with NaOH. According to the manufacturer's protocol, cells are not washed, but extracellular dye is chemically quenched. Calcium 6 fluorescence excited at 488 nm every 2.5 s served as an index of intracellular calcium. Assays were performed at 25 °C. A volume of 50 µL of each applied solution was pipetted automatically according to a preset protocol into 100 µL of extracellular solution in the wells. Fluorescence change was measured by a Flexstation3 (Molecular devices) and is reported relative to baseline fluorescence.

Transfection of HEK-293 cells

For functional expression of green fluorescent protein (GFP) flanked TRPV1, HEK-293 cells were transfected 16–24 h before the start of patch-clamp experiments using TransfeX™ Transfection Reagent (LGC Standards GmbH) according to the manufacturer’s instructions. HEK-293 cells (German Collection of Microorganisms and cell cultures) were grown at 37 °C in a humidified 95% air/ 5% CO2 incubator in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% v/v heat-inactivated fetal bovine serum, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate and 2 mM L-glutamine (all from Invitrogen). After reaching approximately 80% confluence HEK-293 cells were transfected in a 35-mm petri dish with 1 µg of hTRPV1_GFP (NM_080705, RG217713, OrgiGene) plasmid DNA.

Electrophysiology

On the day of experiment, transfected HEK-293 cells were separated by trypsinization, seeded at low density on coverslips (15 mm in diameter), and then incubated for 2 to 4 h to allow adhering of cells on the glass surface. For patch-clamp experiments, coverslips were transferred to a perfusion chamber filled with bath solution and placed on the stage of an inverted microscope. Transfected cells were identified by the fluorescence of the GFP fused to the C-terminus of TRPV1. Patch-clamp experiments were performed in the whole-cell configuration with an EPC-10 patch-clamp amplifier (HEKA Elektronik) at room temperature (20–25 °C). Patch-pipettes were pulled from borosilicate capillaries (DWK Life Sciences) using a single stage glass microelectrode puller (PP-830) resulting in pipettes with 1.5–3 MOhm resistances. The capillaries were coated with Sigmacote® (Merck KGaA) and baked after pulling at 65 °C for 45 min. The pipette solution contained: 140 mM CsCl, 1.93 mM CaCl2, 2 mM MgCl2, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 2 mM MgATP. pH and osmolarity were adjusted to 7.2 with 1 M CsOH and 320 mOsmol/kg with D-mannitol, respectively. The bath solution contained: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM D-glucose. pH and osmolarity were adjusted to 7.4 with 1 M NaOH and 320 mOsmol/kg with D-mannitol, respectively. Capsaicin (M2028, Sigma Aldrich), SAFit2 and Ruthenium Red (RuR) (557,450, Sigma Aldrich) stock solutions were prepared by dissolving 10 mM, 2.5 mM and 10 mM in DMSO, respectively. Capsaicin, SAFit2 and RuR supplemented bath solutions were prepared directly before the measurements by adding the appropriate volume of stock solutions to the external solution. Bath solution exchange was performed using a gravity flow perfusion system. Whole-cell currents were elicited by repetitively applying 500 ms voltage pulses from − 120 mV to + 80 mV (20 mV increments) from a holding potential of − 40 mV and 500 ms voltage ramps from − 60 mV to + 60 mV from a holding potential of − 60 mV. Currents were recorded with a 10 kHz low-pass filter and sampled with a frequency of 50 kHz. Data were memorized with Patchmaster (HEKA Elektronik) and analyzed with Fitmaster (HEKA Elektronik). To account for the different sizes of the measured cells, whole-cell currents were normalized to the cell membrane capacity.

Calcitonin gene-related peptide assay

For measuring the effect of SAFit2 on calcitonin gene-related peptide (CGRP) secretion, DRGs were isolated as previously described (part 2.8) and stimulated with the respective compound the next day. Therefore, the cells were washed with HBSS and incubated with 250 µl of the respective compound, diluted in HBSS to their final concentration, and 1 µM capsaicin for 15 min at 37 °C. The respective volume of the vehicle was used as a negative control, and 1 µM capsaicin alone as positive control. The CGRP quantification was performed with an ELISA kit from Bertin Bioreagent, which was used according to the manufacturer´s recommendations. The final detection was performed measuring the absorption at 405 nm. The raw data were evaluated using linear regression according to the manufacturer´s recommendation.

Calcineurin assay

The calcineurin activation was measured with a cellular calcineurin phosphatase activity assay kit from abcam, which was used according to the manufacturer’s instructions. The enzyme calcineurin was isolated from HEK-293 cells using 1 mL of TBS buffer containing 20 mM Tris and 150 mM NaCl (adjusted to pH 7.3) for a 90% confluent T75 flask. The cell lysate was further purified according to the kit protocol. To measure the impact of the respective compounds on the activity of calcineurin, the purified cellular extract with calcineurin was supplemented with the respective compound and added to the prepared assay plate. Afterwards, the plate was incubated for 30 min at 30 °C to stimulate the activity of the enzyme for a distinct period of time. Finally, the reaction was terminated by adding the assay reagent to the plate and the absorption was detected after 20 to 30 min at 620 nm. The raw data were evaluated using linear regression according to the manufacturer’s instructions.

Differentiation of bone marrow-derived macrophages (BMDM)

For bone marrow isolation, mice were killed and hind legs were dissected and transferred into ice-cold PBS. The bones were cleaned from muscle tissue, cut open and transferred into 0.5-mL Eppendorf tubes with holes at their peak end. To isolate the bone marrow via centrifugation (1300 rpm, 1 min, RT), the tubes were placed into 1.5-mL Eppendorf tubes, containing 50 µL of BMDM medium (RPMI + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FCS (Sigma-Aldrich), 100 U/mL penicillin + 100 µg/mL streptomycin (Thermo Fisher Scientific) and 20 ng/mL M-CSF (Peprotech). After centrifugation, the bone marrow pellet was resuspended in BMDM medium and distributed equally into a 6-well plate. For macrophage differentiation, cells were incubated overnight at 37 °C, followed by a change of BMDM medium the next day. At day four of differentiation, fresh growth factors were provided by adding the same amount of BMDM medium to the differentiating cells. After 7 days, the cells were fully differentiated and used for further experiments [57].

Human macrophage isolation and purification

For isolating buffy coats from whole bloods samples, 50-mL Leucosep tubes were equilibrated with 15 mL lymphocyte separation medium, which was passed through the membrane by centrifuging at 1000 × g for 5 min. Afterwards, 40 mL whole blood was transferred into a tube, which was then filled up to 50 mL with PBS containing 2 mM EDTA. The samples were centrifuged at 440 × g for 35 min at room temperature without break to enable a clear separation of fractions comprising a plasma fraction at the top, the buffy coat in the middle and an erythrocyte and granulocyte fraction at the bottom. The plasma was discarded and the buffy coat transferred into a fresh 50-mL tube, which was then again filled up to 50 mL with PBS containing 2 mM EDTA for purification. The suspension was again centrifuged at 500 × g at room temperature for 5 min and the purification step was repeated until the supernatant was almost clear. Afterwards, the cell pellet was resuspended in 50 mL RPMI medium supplemented with 1% P/S and the cell solution was plated with 1 mL/ well into 6-well plates. The cells were incubated for one hour at 37 °C, followed by a medium change with 2 mL RPMI supplemented with 1% P/S and 3.3% human plasma for differentiation purpose. The medium was changes every 2–3 days until day seven of differentiation. Monocytes were now fully differentiated into macrophages and can be used for further experiments [43].

Transwell assay

For investigating the migration of macrophages, cultivated macrophages were starved overnight and harvested the next day by incubating them 10 min with 600 µl accutase/ well (Sigma-Aldrich) at 37 °C and using a cell scraper. The reaction was terminated using 1% (w/v) BSA solution for murine macrophages. Afterwards, the macrophages were centrifuged at 1000 rpm for 5 min and the cell pellet was resuspended in 1 mL of the respective starving medium. The cell concentration was determined, using a Neubauer counting chamber and the cell concentration adjusted to 106 cells/mL. Transwell inserts (Greiner) were placed into 24-well plates, which contain 500 µl medium/well with either 10% FCS as chemoattractant for murine cells or 3.3% human plasma for human cells and the respective compound. The cells were seeded by adding 300 µl cell solution per insert. After incubating the setup for two hours at 37 °C, the inserts were rinsed with PBS and cells were fixed with 2% PFA for two minutes, followed by further washing steps and a further fixation and permeabilization step with 100% ice-cooled methanol for 20 min. Lastly, the membranes were stained with DAPI in a final concentration of 0.2 ng/mL in the dark for two minutes and embedded on slides with mounting media for quantification purposes. Five images were taken per membrane with the fluorescence microscope Observer.Z1 (Carl Zeiss) and quantified with ImageJ software.

Cytotoxicity assay with WST-1

Primary murine macrophages were isolated and differentiated as previously described (part 2.15). The cells were harvested the same way as described in part 2.17. Afterwards, the cell concentration was adjusted to 3.6 × 105 cells/ mL and cells were seeded with 1 mL/ well into 24-well plates, followed by an incubation for further 48 h at 37 °C. For analyzing the compound´s cytotoxicity, DMEM medium was supplemented with 10% (v/v) WST-1 and either the compound of interest or the according volume of the dissolvent. The cells were treated with 300 µl of prepared medium for two hours at 37 °C and the absorption wavelength was measured at 450 nm and 600 nm afterwards.

Seahorse bioanalyzer

For measuring the metabolic parameters with the Seahorse bioanalyzer device, primary murine macrophages were isolated and differentiated as previously described (part 2.15). The macrophages were harvested in the same way as described in the part 2.17, seeded (25 × 103 cells/well) into Seahorse 96-well cell culture plates and incubated overnight. On the day of measurement, the media was replaced by XF RPMI medium supplemented with 10 mM glucose and 2 mM glutamine (all from Agilent). The plate was equilibrated for 30 min in a non-CO2 incubator at 37 °C. Metabolic parameters were measured on a Seahorse XFe 96 extracellular flux analyzer (Agilent). To analyze the ATP production rate, 2.5 µM oligomycin and 500 nM rotenone together with antimycin A were sequentially added to the cells. All chemicals were purchased from Cayman chemicals. Data were processed using Wave Desktop (Version 2.6.0.31) and ATP rates were calculated using the Seahorse Analytics online tool (Version from February 2022).

Data analysis and statistics

All data are presented as mean ± SEM. Normal distribution was confirmed using the Shapiro–Wilk test. For in vitro experiments comparing only two groups, an unpaired and heteroskedastic Student’s t test was conducted with Welch’s correction. When comparing more than two groups, a one-way analysis of variance (ANOVA) was used and for the comparison of more than three groups a two-way ANOVA was conducted. For statistical analysis of behavioral experiments, an ANOVA was performed, followed by Bonferroni’s post hoc correction. For all statistical analysis the software GraphPad Prism 9 was used. A p value of < 0.05 was considered statistically significant.