Peripherally increased artemin is a key regulator of TRPA1/V1 expression in primary afferent neurons

All animal experiments conformed to the regulations of the Hyogo College of Medicine Committee on Animal Research and were performed in accordance with the guidelines of the National Institutes of Health on animal care. Male Sprague Dawley rats, aged 4 weeks for the DRG primary culture neurons and calcium imaging study, and those weighing 200–250 g for the other studies, were used. Plantar injection was performed under ether anesthesia, and surgical procedures were performed under sodium pentobarbital (50 mg/kg, i.p.) anesthesia. Additional doses of the anesthetics were given as needed. 50% Complete Freund’s adjuvant saline (CFA, 100 μl) was injected into the plantar surface of the left hind paw. Recombinant mouse artemin (R & D systems, 20 μg/ml, 100 μl) was injected into the plantar surface. DMSO was used as a vehicle control for the models using p38 inhibitor with the DRG primary culture neurons. L5 spinal nerve ligation was performed with some modifications to the original SPNL model [37]. After anesthetizing the rats, the hair of the lower back was shaved and the skin was sterilized with 0.5% chlorhexidine. Using sterilized operating instruments, the left L5 spinal nerve was isolated and tightly ligated with 3–0 silk thread (L5 SPNL). Thereafter, the ligated spinal nerve was cut on the distal side to the ligated part. The right side was not subjected to any surgery. The wound was washed with distilled saline and sutured with 3–0 silk thread.

Rats were perfused transcardially with 1% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) followed by 4% paraformaldehyde in 0.1 M PB. The bilateral L4-5 DRG neurons and sciatic nerve (1 cm length) 2 cm distal to the ligation were removed, and postfixed in the same fixative at 4°C overnight, followed by immersion in 20% sucrose in 0.1 m phosphate buffer at 4°C for 2 d. Thereafter, they were rapidly frozen in powdered dry ice, and cut on a cryostat at a thickness of 8 μm. For plantar skin samples, rats were sacrificed by decapitation under deep ether anesthesia. The plantar skin surface was removed, rapidly frozen, and cut on a cryostat at a thickness of 5 μm. Sections were thaw-mounted onto MAS-coated glass slides (Matsunami, Osaka, Japan). The details of the dual ISHH procedure have been described in our previous study [6]. For autoradiography, the sections were coated with NTB emulsion (Eastman Kodak, Rochester, NY) diluted 3:2 with distilled water at 45°C and exposed for 1–2 weeks in light-tight boxes at 4°C. After development in D-19 (Eastman Kodak) and fixation in 24% sodium thiosulfate, the sections were stained with hematoxylin-eosin, dehydrated in a graded ethanol series, cleared in xylene, and coverslipped [6].

For the co-expression study of the two different mRNAs on DRG neurons in naive rats, we used ³⁵S-labeled RNA probes for TRPV1 (GenBank accession number AF029310, nucleotides 149–505), TRPA1 (GenBank accession number AY496961, nucleotides 302–788), and DIG-labeled probes for TrkA (GenBank accession number X05137, nucleotides 560–979) and GFRα3 (GenBank accession number AF184920, nucleotides 164–604) in the same sections.

The protocol for immunohistochemistry was based on the published ABC (Vectastain Elite ABC kit, Vector, CA, USA) method [38]. The DRG sections on the MAS-coated glass were preincubated in TBS containing 10% normal goat serum (NGS) for 1 hour, then incubated in primary antibody for p-p38 (1:500, Cell Signaling Technology) in the same solution for 48 hours at 4°C. The sections were washed in TBS and then incubated in biotinylated anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA) in TBS containing 5% NGS for 2 hours at 4°C, followed by incubation in avidin-biotin–peroxidase complex (Vectastain Elite ABC kit, Vector, CA, USA) for 1 hour at room temperature. The horseradish peroxidase reaction was developed in 0.1 M Tris-buffered saline, pH 7.4, containing 0.05% 3,39-diaminobenzidine tetrahydrochloride (Sigma, Steinheim, Germany), and 0.01% hydrogen peroxidase. Sections were then washed in TBS, mounted on slides, dried, and cover-slipped.

The statistical analyses of ISHH have been previously described in detail [38-40]. At least 3,000 neuronal profiles from four rats were quantified for each antisense probe in the single ISHH study. Measurements of the density of silver grains over randomly selected tissue profiles were performed using a computerized image analysis system (NIH Image, version 1.61), in which only neuronal profiles that contained nuclei were used for quantification. At a magnification of 200x and with brightfield illumination, upper and lower thresholds of gray level density were set such that only silver grains were accurately discriminated from the background in the outlined cell or tissue profile and were read by the computer, pixel by pixel. Subsequently, the area of discriminated pixels was measured and divided by the area of the outlined neuronal profile, giving a grain density (%) for each cell. To reduce the risk of biased sampling of the data because of varying emulsion thickness, we used a signal-to-noise (S/N) ratio for each cell in each tissue. The S/N ratio of an individual neuron and its cross-sectioned area, which was computed from the outlined profile, was plotted. Based on this scattergram, neurons with a grain density 5-fold that of the background level or higher (5 S/N ratio) were considered positively labeled for TRPA1, TRPV1 and GFRα3 mRNAs. To distinguish cell size-specific changes, we characterized the DRG neurons as small (<600 μm²), medium (600-1200 μm²) and large (>1200 μm²), according to their cross-sectional area. Since a stereological approach was not used in this study, quantification of the data may represent a biased estimate of the actual numbers of neurons. The number of positively labeled DRG neurons was divided by the number of neuronal profiles counted in each DRG. Data are expressed throughout as mean ± SEM (%). The level of statistically significant difference was taken to be a probability of less than 5% (p < 0.05) [40].

For purification of total RNA from these samples, a PureLink^® RNA Micro Kit (Invitrogen, CA, USA) was used. RNA extraction was quantified by measurement of optical density at 260 nm. Samples of 3 μg of total RNA were mixed with 25 μl RT mixture to a final concentration 200 units of M-MLV reverse transcriptase (Promega, Madison, WI), 20 units of RNase inhibitor (Promega), 0.8 μM dNTP, and 1 μg oligo dT primer in 1 x reaction buffer (pH 7.5; Promega). RT was carried out at 37°C for 60 minutes, following inactivation at 70°C for 10 minutes. The forward and reverse primers specific for rat NGF, artemin, TRPV1, TRPA1, GFRα3 and GAPDH were designed as shown in Table 1. The PCR reaction was performed in 10 μl of Taq PCR master mix kit (QIAGEN, CA, USA) and cDNA with a pair of 10-pmol primers for each gene on a DNA Thermal Cycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). The PCR conditions for temperature cycles were different for each gene. The resulting PCR products were electrophoresed through a 1.25% agarose gel containing ethidium bromide and visualized with UV illumination. Each pair of forward and reverse primers presented a single band with the expected size.

Adult male Sprague–Dawley rats (200-250 g) were used for the behavioral analyses. Room temperature and lightning were kept stable for all testing. Rats were sufficiently habituated in the environment and tested for basal pain hypersensitivity. Thereafter, rats were administered 100 μl of artemin or PBS into the left hindpaw. One day after the administration, mechanical and thermal behavioral tests were started with a Dynamic Plantar Aesthesiometer (Ugo Basile, Italy) and the Hargreaves radiant heat apparatus, respectively. The Dynamic Plantar Aesthesiometer was used as the automated von Frey hair system. The threshold was taken as the force applied to the plantar surface at which the rats withdrew from it. For the 5-consecutive-day injection model, testing was performed just before injection. The first value for each test was removed from the data, and the averages from the subsequent three values were taken as the thermal and mechanical thresholds.

Ratiometric calcium imaging was performed using an Olympus fluorescence microscope equipped with a variable filter wheel (Sutter Instruments, Novato, CA, USA) and a cooled digital CCD camera (Hamamatsu, Shizuoka, Japan). Dual images (340 and 380 nm excitation, 510 nm emission) were collected and pseudo-color ratiometric images were monitored every 2 seconds during the experiment using an Axon Imaging Workbench 5.0 (INDEC BioSystems, Inc, Mountain View, CA, USA) [41]. The DRG neurons were removed from 4-week Sprague–Dawley rats. After dissociation of the DRG neurons, the cell suspension was placed onto coverslips coated with poly-L-lysine and cultured for 18 to 24 hours with the same method as above (see DRG primary culture neurons in the Methods section). They were loaded with 4μM Fura-2 acetoxymethyl ester (Nacalai Tesque, Kyoto, Japan) for 40 minutes at 37°C. After the incubation, the coverslips were placed in a chamber connected to a gravity flow system to deliver various chemicals. One microscopic field on the coverslip, which contained 20 to 30 neurons, was randomly selected and measured. The data were collected from a total of 20 fields in each group (n = 4 each group). The system was loaded with a standard bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES and 10 mM glucose pH7.4 (adjusted with NaOH). A standard bath solution was first applied to the cells for 20 seconds, followed by KCl (50 mM) for 20 seconds, and the cells were then washed with the standard bath solution for 5 minutes. For the TRPV1 study, capsaicin (200nM) was applied for 20 seconds and it was washed out again with the standard bath solution for 3 minutes. For the TRPA1 study, AITC (100 μM) was applied for 20 seconds and it was washed out for 3 minutes. Thereafter, capsaicin (1 μM) was perfused for 20 seconds. The number of agonist-responding neurons relative to KCl-responding neurons in each selected microscopic field was calculated and summed.