Adult male Sprague Dawley rats (200 to 250 g) were used in this study (Japan SLC, Japan). Rats were exposed to a 12 h light/dark cycle and kept in a temperature-controlled room (23 ± 2°C) with food and water ad libitum. This study was approved by the Animal Experimentation Committee at Nihon University, and all experimental procedures were performed according to the ethical guidelines of the International Association for the Study of Pain
. All possible efforts were made to minimize the number of animals used and their suffering.
The major drugs used in the current study were PD98059 (EMD Biosciences, La Jolla, CA, USA), a well-known MAPK kinase (MEK1/2) inhibitor
, 2-methyl-6-(phenylethynyl)-pyridine (MPEP, Sigma Aldrich, St. Louis, MO, USA), a selective mGluR5 antagonist
, (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG, Tocris, Bristol, UK), a selective mGluR5 agonist
 and CFA (an inflammatory agent, Sigma Aldrich). PD98059 and MPEP were initially dissolved in 100% dimethyl sulfoxide (DMSO) as stock solutions (10 μg/μL) for frozen aliquots, and then further diluted to 0.1 μg/μL in 10% DMSO (in 0.9% saline) for i.t. administration (see below). A solution of 10% DMSO served as the vehicle control. CHPG was diluted to 4.8 mM in 0.9% saline for i.t. administration. For preparation of CFA solution, the original drug was suspended in an oil/saline (1:1) emulsion and stored at 4°C for subsequent use.
Induction and verification of inflammation in the tongue
Under anesthesia from an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Schering Plough, Whitehouse Station, NJ, USA), 5 μL of CFA was submucosally injected into the left side of the anterior dorsolateral two-thirds of the tongue with a 30-gauge needle attaching a Hamilton syringe (1.0 mL, Hamilton, Reno, NV, USA). The same amount of isotonic saline was injected as the vehicle control. Notably, during each injection, the location of the needle was best limited in the superficial skin layer of the tongue without entering the tongue muscle. After the injection, a small cotton swab was placed on the injection site for 1 to 2 min to prevent any leakage. Animals were closely monitored for evidence of distress or pain, and weight gain following the injections.
To verify the occurrence of tongue inflammation, rats were perfused transcardially with 250 mL 0.9% isotonic saline followed by 500 mL ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) on days 8 and 15 after CFA injection. The tongues were removed and immersed in the same fixative for 4 h at 4°C. After post-fixation, tongue tissues were embedded in Tissue Tek (Sakura Finetechnical, Tokyo, Japan), cut in the horizontal plane along the long axis of the tongue on a cryostat at a thickness of 10 μm, stained with hematoxylin and eosin (HE), and evaluated microscopically.
To examine inflammatory extravasation at each specified time point, Evans Blue solution (50 mg/kg, 10 mg/mL in saline) was intravenously injected through the femoral vein at 4 to 5 min before perfusion. Then rats were perfused through the aorta with normal saline. Photographs of tongue sections were taken, and Evans Blue stained regions were observed under the microscope.
Rats were lightly anesthetized with 2% isoflurane (Mylan, Morgantown, WV, USA) mixed with oxygen, and the depth of anesthesia was assured as previously described
. According to our previous reports
[11, 20, 41, 42], a pair of bipolar enamel-coated silver-wire electrodes was inserted into the splenius capitis muscle for electromyographic (EMG) recording (inter-electrode distance, 5 to 6 mm). The EMG activity was amplified, filtered, digitized, and integrated by the Spike 2 software (CED 1401, Cambridge Electronic Design, Cambridge, UK).
For measurement of mechanical head withdrawal threshold (MHWT), an electronic von Frey anesthesiometer (Bioseb, Chaville, France) was used to apply graded mechanical pinch stimuli to CFA- or saline-injected tongue. The MHWT was defined as the lowest pressure (g) required to elicit a robust bursting activity in neck EMG recording accompanied by a clear head withdrawal response. The cutoff mechanical stimulus intensity was 130 g. For assessment of heat head withdrawal threshold (HHWT), heat stimulus was applied using a contact thermal probe (25 mm2, adapted temperature: 35°C, Intercross, Tokyo, Japan) to the tongue. The probe temperature increased 0.3°C per second during the assessment period. The HHWT was defined as the minimum temperature sufficient to elicit a drastic head escape and sudden appearance of a bursting EMG activity. The cutoff temperature was set at 60°C.
Both MHWT and HHWT were measured 1 day before and on days 1, 3, 5, 8, 11, and 15 after saline or CFA injection. Three measurements (at 5-min intervals) were performed and averaged at each time point for each animal. All behavioral tests were conducted under blind conditions.
Tissue preparation and pERK immunohistochemistry
On days 3, 8, and 15 after saline or CFA injection into the tongue and in naive rats, noxious mechanical stimulation was applied to the tongue using an arterial clip (intensity, 120 g; duration, 30s; interval, 30s; total, 10 min) with the rats under sodium pentobarbital anesthesia (50 mg/kg, i.p.). On the basis of our previous results that the number of pERK-IR cells peaked at 5 min after capsaicin injection into the tongue
, rats were perfused transcardially with 250 mL isotonic saline followed by 500 mL cold 4% paraformaldehyde in 0.1 M PB (pH 7.4) at 5 min after noxious stimulation. Furthermore, naive and CFA-injected rats were perfused transcardially in the absence of noxious mechanical stimulation. The medulla and upper cervical spinal cord were removed and placed in the same fixative overnight at 4°C. These tissues were transferred to 20% sucrose in 0.01 M phosphate-buffered saline (PBS) for several days for cryoprotection. Thirty micrometer thick sections of the medulla and upper cervical spinal cord were cut with a freezing microtome at -20°C, and every fourth section was collected in 0.01 M PBS. Free-floating sections were rinsed in 0.01 M PBS, blocked in 3% normal goat serum (NGS) for 1 h at room temperature (RT), and then incubated with rabbit anti-pERK (Thr202/Tyr204) antibody (1:1,000, Cell Signaling Technology, Beverly, MA, USA) in 3% NGS with 0.75% Triton X-100 for 72 h at 4°C. After rinsing, sections were incubated with biotinylated goat anti-rabbit antibody (1:600, Vector Laboratories, Burlingame, CA, USA) for 2 h at RT. Following rinses in 0.01 M PBS, these sections were reacted with peroxidase-conjugated avidin-biotin complex (ABC, 1:50, Vector Laboratories) for 1 h at RT. They were washed in 0.05 M Tris buffer (TB) and incubated with 0.035% 3,3’-diaminobenzidine-tetra hydrochloride (DAB, Tokyo Chemical Industry, Tokyo, Japan) in 0.05 M TB containing 0.2% nickel ammonium sulfate and 0.05% hydrogen peroxide for 3 to 4 min. Sections were finally rinsed in 0.01 M PBS, mounted on gelatin-coated slides, air-dried, dehydrated in ethanol, cleared in xylene, and coverslipped. All specific staining was abolished by omission of the primary antibody in the process.
The pERK-IR cells were counted under a light microscope with an attached camera lucida drawing tube (Neurolucida 2000, MicroBrightField, Colchester, UT, USA). The sections were then grouped into 720 μm segments rostrocaudally with reference to the obex. In order to analyze rostrocaudal distribution of pERK-IR cells, the number of pERK-IR cells from three sections at each level in Vc and C1-C2 was counted, and then averaged from five rats in each group. The cells showing more intense staining than the average background were considered positive for pERK immunoreactivity. The whole counting process was performed by an investigator blind to the experimental treatments.
Three rats received noxious mechanical stimulation of the tongue by an arterial clip (intensity, 120 g; duration, 30s; interval, 30s; total, 10 min) on day 8 after CFA injection into the tongue. Five min after the stimulation, rats were perfused with 250 mL isotonic saline followed by 500 mL cold 4% paraformaldehyde in 0.1 M PB (pH 7.4). After cryoprotection in 20% sucrose, 30 μm thick sections were cut as described previously and processed for double immunofluorescence labeling between pERK or mGluR5 and the neuronal label, NeuN, or the astroglial label, glial fibrillary acidic protein (GFAP). We also performed double-immunostaining between pERK and mGluR5 antibodies. Free-floating tissue sections were rinsed in 0.01 M PBS, blocked in 3% NGS for 1 h and incubated with rabbit anti-pERK antibody (1:300, Cell Signaling Technology) or goat anti-mGluR5 antibody (1:50, Santa Cruz, CA, USA) and mouse anti-NeuN antibody (1:1,000, Chemicon, MA, USA), or mouse anti-GFAP antibody (1:1000, Dako, Tokyo, Japan) for 72 h at 4°C. Similarly, free-floating tissue sections were incubated with rabbit anti-pERK antibody and goat anti-mGluR5 antibody (Merck Millipore, Billerica, MA, USA). After rinsing in 0.01 M PBS, the sections were incubated with the secondary antibodies (anti-rabbit or anti-goat Alexa Fluor 488 and anti-rabbit or anti-mouse Alexa Fluor 568, 1:1,000, Invitrogen, NY, USA) where appropriate for 2 h at RT in a dark room. Then, the sections were rinsed in 0.01 M PBS, mounted on slides, and coverslipped with PermaFluor (Sigma Aldrich, St. Louis, MO, USA). The immunofluorescence images were taken with a confocal laser scanning microscope (LSM 510 V3.2, Carl Zeiss, Tokyo, Japan).
On day 8 after CFA or saline injection into the tongue, the rat was anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused with saline. Medulla containing Vc and C1-C2 was taken out and homogenized in 100 μL of ice-cold lysis buffer (137 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 1 g/mL leupeptin, and 0.5 mM sodium vanadate) using a tube pestle (Thermo Fisher Scientific, Waltham, MA, USA). Sample was centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was collected to new tubes and protein concentration of the sample was determined with a protein assay kit (Bio-Rad, CA, USA). Protein sample was heat-denatured in Laemmli sample buffer solution (Bio-Rad). Sample (30 μg) was subjected to electrophoresis for protein separation on 10% SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (Trans-Blot turb Transfer pack, Bio-Rad) by using Trans-Blot Turbo (Bio-Rad). Following rinsing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with 3% bovine serum albumin (BSA, Bovogen, Essendon, Australia). The membrane was incubated overnight at 4°C with anti-pERK antibody (1:1,000, Cell Signaling) or anti-mGluR5 antibody (1:20,000, Merck Millipore) diluted in TBST containing 5% BSA. Each protein binding was visualized using a horseradish peroxidase-conjugated donkey anti-rabbit antibody (Cell signaling) and Western Lightning ELC Pro (Perikin Elmer, Waltham, MA, USA). Band intensity was quantified using a ChemiDoc MP system (Bio-Rad) and normalized to β-actin immunoreactivity on blots reprobed with anti-β-actin antibody (1:200, Santa Cruz) after removing protein binding using a stripping reagent (Thermo Fisher Scientific).
Effects of PD98059, MPEP and CHPG on nocifensive behavior and ERK phosphorylation
Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus. After a midline skin incision, an opening was made in the caudal part of the skull with a dental drill to insert intrathecally a soft polyethylene tube (PE45, ID, 0.58 mm; OD, 0.96 mm; Natsume, Tokyo, Japan)
. The tube was connected to a mini-osmotic pump (Alzet model 2001, Alzet, Cupertino, CA, USA; total volume, 200 μL) filled with the drug and embedded subcutaneously in the dorsal portion of the body. Thus, PD98059 or MPEP was intrathecally applied for 7 days (1.0 μL/h). The dosage and duration of the two drugs (0.1 μg/μL) for pump infusion were chosen primarily based on previous reports
[11, 20, 23, 39]. After completion of the pump embedding, 5 μL of CFA solution was injected into the anterior dorsolateral two-thirds of the rat tongue, and the behavioral testing was performed on days 1, 3, 5, 8, 11, and 15 for PD98059 treatment or on day 8 for MPEP treatment after CFA injection. CHPG was also intrathecally applied for 7 days (0.5 μL/h) in naive rats and behavioral testing was performed on day 8.
On day 8, CFA-treated rats with continuous PD98059 or MPEP administration received noxious mechanical stimulation to the tongue. Five min after the stimulation, the rats were perfused, and the pERK immunohistochemistry was performed. All subsequent staining steps and counting analysis were the same as described above. All behavioral and immunohistochemical experiments were conducted on animals without any obvious neurological deficits. Also, the Alzet pump was removed at the end of each experiment, and the amount of the drug remaining in the pump was checked. If there was still residual drug in the pump, the data from that rat were excluded from the final analysis.
All results are presented as mean ± SEM. Statistical analyses were performed by Student’s t test, one-way ANOVA followed by Dunnett’s multiple-comparison tests, or two-way repeated-measures ANOVA followed by Bonferroni’s multiple-comparison tests where appropriate. A value of P<0.05 was considered statistically significant.