Male Sprague-Dawley rats (n = 212, Japan SLC, Shizuoka, Japan) weighing 200-300 g were used in this study. The animals were maintained in a temperature-controlled room (23°C) with a 12/12 hours light-dark cycle. Food and water were freely available. This study was approved by the Animal Experimentation Committee at the Nihon University. All surgery and animal care were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines for Institutional Animal Care, and the guidelines of the International Association for the Study of Pain .
Under anesthesia with an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg; Schering Plough, Whitehouse Station, NJ), the rats were placed on a warm mat (37°C). An incision was made in the floor of the oral cavity beneath the tongue at the left side of lingual frenulum. The lingual nerve was exposed through the floor of the oral cavity and then crushed with an arterial clamp (30 g; Natsume, Tokyo, Japan) for 30 seconds, and then the incision was sutured after LNC. Sham-rats were operated with same procedure without performing the nerve crush.
Head-withdrawal reflex threshold measurement
The thresholds for the head-withdrawal reflex to mechanical and heat stimulation of the lateral edge of tongue (3 mm posterior from tip of tongue) were measured, before and on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25 and 31 after the LNC or sham operation, under light anesthesia with 2% isoflurane (Mylan, Canonsburg, PA) in oxygen. Bipolar enamel-coated stainless steels wire electrodes (Narishige, Tokyo, Japan) were placed in the splenius capitis muscle for electromyogram (EMG) recording of the reflex response (interelectrode distance: 5-6 mm) .
Mechanical stimulation (0-130 g, 10 g/sec, cut off: 130 g) was applied to the lateral edge of the tongue ipsilateral to the LNC or sham operation by using forceps with flat tips (4 mm2 square; Panlab s.l., Barcelona, Spain) in lightly anesthetized rats (n = 7 in each group). The tongue was pinched at dorsum linguae and hypoglottis (Additional file 4: Figure S4A). The stimulus velocity was manually controlled consecutively from 0 g to threshold values at a speed of 10 g/s. The threshold intensity for evoking EMG activity by mechanical stimulation of the tongue was defined as the mechanical head-withdrawal reflex threshold.
Heat stimulation (35-60°C, 1°C/sec, cut off: 60°C) was also applied to the lateral edge of the tongue ipsilateral to the LNC or sham operation by using a contact heat probe (9 mm2 square; Intercross, Tokyo, Japan) in lightly anesthetized rats (n = 7 in each group). The threshold temperature for evoking EMG activity by heat stimulation to the tongue was defined as the heat head-withdrawal reflex threshold. The mechanical or heat stimulation was applied 3 times with 5 minute intervals, and the mean value of the head-withdrawal reflex thresholds were determined.
The baseline of the head-withdrawal reflex thresholds to mechanical and heat stimulation were measured in naïve rats before the LNC or sham operation.
GFAP, P2Y12R, P2Y1R, P2Y13R and NeuN immunohistochemistries
On days 3, 9, 15 and 21 after the LNC or sham operation, rats were transcardially perfused with saline followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under 2% isoflurane in oxygen and sodium pentobarbital anesthesia (50 mg/kg, i.p., n = 5 in each group). LNC- and sham-rats received 2,2-dimethyl-propionic acid 3 -(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395; 18.0 ng in 0.5 μl/day; Sigma-Aldrich) into TG for 3 successive days, and the naïve rats received 2-(Methylthio) adenosine5'-diphosphate trisodium salt hydrate (2-MeSADP; 10.0 nmol in 0.5 μl/day; Sigma-Aldrich) into TG for 3 successive days, and rats were transcardially perfused using a same fixative as described above (n = 5 in each group).
TGs in the ipsilateral side to LNC or sham operation, and left TGs in naïve rats were dissected out after perfusion and immersed in the same fixative for 4 h at 4°C, and then kept in 0.01 M phosphate-buffered saline (PBS) containing 20% sucrose (w/v) for 12 h for cryoprotection. The specimens were then embedded in Tissue Tek (Sakura Finetek, Torrance, CA) and stored until cryosectioning at -20°C. Ten μm TG sections were cut in the horizontal plane along the long axis. Every 15th section was thaw-mounted on MAS-GP micro slide glass (Matunami, Osaka, Japan) and dried overnight at room temperature. Four sections were chosen from each TG in each rat. These sections were processed for GFAP, P2Y12R, P2Y1R, P2Y13R and NeuN immunohistochemistries.
Sections were incubated with mouse anti-GFAP monoclonal antibody (Millipore, Billerica, MA) after dilution at a concentration of 1:800 in 0.01 M PBS containing 4% normal goat serum (NGS) and 0.3% Triton X-100 (Sigma-Aldrich) overnight at 4°C. After rinsing with 0.01 M PBS, sections were incubated in Alexa Fluor 568 anti-mouse IgG (1:200 in 0.01 M PBS; Invitrogen, Paisley, U.K.) for 2 h at room temperature. After rinsing with 0.01 M PBS, sections were coverslipped in mounting medium (Thermo Fisher Scientific, Fremont, CA) and examined under a fluorescence microscope and analyzed using a BZ-9000 system (Keyence, Osaka, Japan). No specific labeling was observed in the absence of primary antibody. The number and cell size of somata of TG neurons encircled with GFAP-IR cells over 2/3 of perimeters of somata of TG neurons were defined as TG neurons encircled with GFAP-IR cells (SensivMeasure; Mitani, Fukui, Japan). The number of TG neurons encircled with GFAP-IR cells was counted in each rat (n = 5 in each group) and the relative number of them was calculated by the following formula: 100 × number of neurons encircled with GFAP-IR cells/total number of neurons.
P2Y12R, P2Y1R, P2Y13R, GFAP and NeuN immunohistochemical analyses were conducted in naïve, sham- and LNC-rats to locate P2Y12R, P2Y1R and P2Y13R in TG (n = 5 in each group). Sections were incubated with rabbit anti-P2Y12R polyclonal antibody (1:200; Anaspec, Fremont, CA), rabbit anti-P2Y1R polyclonal antibody (1:300; Alomone labs, Jerusalem, Israel), rabbit anti-P2Y13R polyclonal antibody (1:300; Chemicon, Temicula, CA), mouse anti-GFAP monoclonal antibody (1:800; Millipore) and/or mouse anti-NeuN monoclonal antibody (1:1000; Chemicon) in 0.01 M PBS containing 4% NGS and 0.3% Triton X-100 (Sigma-Aldrich) overnight at 4°C. After rinsing with 0.01 M PBS, sections were incubated in Alexa Fluor 488 anti-rabbit IgG (1:200 in 0.01 M PBS; Invitrogen) and Alexa Fluor 568 anti-mouse IgG (1:200 in 0.01 M PBS; Invitrogen) for 2 h at room temperature. After rinsing with 0.01 M PBS, sections were coverslipped in mounting medium (Thermo Fisher Scientific) and examined under a fluorescence microscope.
The numbers of TG neurons encircled with P2Y1R-IR or P2Y13R-IR cells and P2Y1R-IR or P2Y13R-IR TG neurons were counted in naïve, sham- and LNC-rats. The relative numbers of them were calculated by the following formula: 100 × number of neurons encircled with P2Y1R-IR cells/total number of neurons; 100 × number of P2Y1R-IR or P2Y13R-IR TG neurons/total number of neurons.
MRS2395 and 2-MeSADP administration into TG
Rats were anesthetized with 2% isoflurane in oxygen and sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic apparatus. The skull was exposed and a small hole (diameter; 1 mm) was drilled above the location of the bifurcation between V1/V2 branch regions and V3 branch region of TG. The guide cannula was extended into the hole 9 mm below the skull surface into TG ipsilateral to the LNC or sham operation (2.8 mm anterior from lambda and 2.7 mm lateral to the midline) and was fixed to the skull with three stainless-steel screws and dental resin. The tip of the trocar was located just below the surface of V1/V2 branch region near the border between V3 and V1/V2 branches to inject drugs into TG. To define the position of the tip of the cannula, multiunit activities by mechanical stimulation of the V1/V2 face area were recorded by using the trocar as electrode. After completion of the surgery, penicillin G potassium (20,000 units; Meiji Seika, Tokyo, Japan) was injected intramuscularly to prevent infection. The rats were allowed to recover for 7 days before experiments were performed [44, 50].
Rats were lightly anesthetized with 2% isoflurane in oxygen, a 31-gauge injection needle (Heraeus Kulzer Japan, Osaka, Japan) was inserted into TG through the guide cannula (positioned as described above and illustrated in Additional file 4: Figure S4B) after the trocar was removed. In advance, we confirmed the diffusion of dye into V3 branch region following 0.5 μl dye injection through the guide cannula. The injection needle was connected to the 10 μl Hamilton syringe to deliver 0.5 μl or 1.0 μl of drugs over a 30 seconds period. LNC- or sham-rats were administered vehicle (0.5 μl; dimethyl sulfoxide (25%) and polyethylene glycol 300 (75%)) or MRS2395 (0.18, 1.8, 9.0 and 18.0 ng in 0.5 μl/day; Sigma-Aldrich) dissolved in vehicle (n = 6 in each group) , and naïve rats were administrated saline (0.5 μl), 2-MeSADP (0.1, 1.0 and 10.0 nmol in 0.5 μl/day; Sigma-Aldrich) dissolved in saline, vehicle (1.0 μl; dimethyl sulfoxide (25%) and polyethylene glycol 300 (75%)) or 2-MeSADP (10.0 nmol in 0.5 μl/day) mixed with MRS2395 (18.0 ng in 0.5 μl/day) once a day into TG for 3 successive days (day 0 through day 2) (n = 6 in each group). Head-withdrawal reflex thresholds were then measured under light anesthesia with 2% isoflurane in oxygen.
Data were expressed as means ± SEM. Statistical analyses were performed by Student's t-test, one-way analysis of variance (ANOVA) followed by Newman-keuls tests, or two-way repeated-measures ANOVA followed by Bonferroni's multiple comparison tests where appropriate. A value of p < 0.05 was considered as significant.