We utilized male Sprague-Dawley rats (n = 260, Japan SLC, Shizuoka, Japan) weighing 250–350 g. Rats were housed at a stable temperature (23 °C) with a 12 h light:dark cycle (7:00–19:00:19:00–7:00). Rats were raised under aseptic conditions with free intake to food and water. The study was approved by the Nihon University Animal Experiment Committee (protocol numbers AP17D021 and AP19DEN009-1). The study was conducted according to the National Institutes of Health guidelines on laboratory animal management and use and based the previous pain study reports . The statistical design of these experiments minimized the number of rats used. The study complied with ARRIVE guidelines.
Histochemical analysis of TPE pulp in M1-TPE rats
We performed light inhalant anesthesia using a mixture of isoflurane (2%, Mylan, Canonsburg, PA) and oxygen. Rats were deeply anesthetized by an intraperitoneal (i.p.) injection of butorphanol (2.5 mg/kg, Meiji Seika Pharma, Tokyo, Japan), medetomidine (0.375 mg/kg, Zenoaq, Fukushima, Japan), and midazolam (2.0 mg/kg, Sandoz, Tokyo, Japan) dissolved in saline solution (three-mixed anesthetic). The M1-TPE procedure was performed with reference to a previous report . One day after TPE, rats (n = 3) were deeply anesthetized with the three-mixed anesthetic and transcardially perfused as previously described . The mandibular jaw was removed and decalcified with EDTA for 7 days, 4-mm tooth sections were cut from TPE, and sham teeth were stained with hematoxylin and eosin.
Head withdrawal threshold (HWT) measurements
The HWT was measured under light anesthesia with 2% isoflurane and oxygen for mechanical and heat stimulation of the tongue ipsilateral to TPE (3 mm behind the tip of the tongue). HWT measurements were performed on days 1, 2, 3, 5, and 9 after TPE as previously described . Briefly, the lower jaw was gently pulled with plastic strings to hold the mouth open and then mechanical stimulation (0 to 130 g; 10 g/s; cutoff, 130 g) or heat stimulation (35–60 °C; 1 °C/s; cutoff, 60 °C) was applied to the lateral tongue edge. Flat-tip forceps (4 mm2; Panlab s.l., Barcelona, Spain) or a contact heat probe (9 mm2; Intercross, Tokyo, Japan) were used for mechanical or heat stimulation, respectively. The changes in mean HWT values over time were measured in TPE or sham rats. Baseline HWT levels were measured before TPE or sham treatment.
TG neurons innervating the tongue were visualized using fluorogold (FG, Fluorochrome, Denver, CO) which was injected into the tongue. In rats anesthetized with 2% isoflurane, 10.0 µL of 4% FG dissolved in saline was injected into the tongue ipsilateral to TPE. TPE was performed on the second day after FG injection. The next day (day 1 after TPE), the rats were deeply anesthetized with the three-mixed anesthetic and transcardially perfused as previously described . TG sections were prepared (15-µm-thick), mounted on microscope slides (Matsunami, Tokyo, Japan), and blocked to avoid non-specific binding of antibodies. Sections were then incubated in a solution of rabbit anti-TLR4 (1:1000, ab13556; Abcam, Cambridge, UK), rabbit anti-IL1RI (1:500, SC-689; Santa Cruz Biotechnology, Dallas, TX), mouse anti-IL-1β (1:800, ab8320; Abcam), rabbit anti-IBA1 (1:2000, 019-19741; Wako, Shiga, Japan), mouse anti-IL1RI (1:500, ab40101; Abcam), or guinea-pig anti-TRPV1 (1:500, AB10295; Abcam) polyclonal antibody. The sections were then incubated in the secondary antibody solution of Alexa Fluor 568-conjugated goat anti-rabbit IgG, Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, and/or Alexa Fluor 568-conjugated goat anti-guinea pig IgG (all 1:200 in 0.01 M phosphate-buffered saline; Thermo Fisher Scientific, Waltham, MA). Following the application of mounting medium, TG sections were cover-slipped, and the immunoreactive (IR) cells were observed by fluorescence microscopy using a BZ-9000 system (Keyence, Osaka, Japan). Double- or triple-labeled cells (FG and TLR4; FG and IL-1RI; IBA1 and IL-1β; FG, TLR4, and IL-1RI; FG and TRPV1) were recognized as those exhibiting co-expression of respective markers. Then, we precisely counted the number of cells and performed analysis using a computer-assisted imaging analysis system (BZ-X analyzer, Keyence). Cells displaying the signal over twice the saturation level compared to the mean background signal were identified as positive. FG-labeled IR cells in the V3 branch region of the TG were counted. The equations used to calculate and analyze IR cells in the TG of the third branch region are given in the legends to Figs. 2–4.
Drug injection into the TG
Under anesthesia, a midline skin incision (2-cm-long) was made from head to neck and a small hole (1 mm in diameter) was made in the skull above the TG. A guide cannula was inserted into the brain through the hole to reach the TG; the cannula tip was placed 9 mm below the skull surface. Next, as previously reported, the cannulation procedure was performed on the skull surface . Phosphate-buffered saline (1 µL/day), LPS-RS (0.1 mM, 1 µL/day; InvivoGen, San Diego, CA), LCCA (7 mg/mL, 1 µL/day; F70101CA, FormuMax Scientific, Sunnyvale, CA), or plain control liposomes for LCCA (Cont-LCCA; 20 mM, 1 µL/day; F70101-A, FormuMax Scientific) was injected into the TG once daily for two successive days. Then, we measured HWT and performed immunohistochemistry to investigate each drug’s effect on tongue hypersensitivity following M1-TPE.
Injection of HSP70 or IL-1β into the TG
Rats were anesthetized with the three-mixed anesthetic, then recombinant human HSP70/HSPA1A (1 mL, 1 mg/mL, AP-100-100, R&D Systems, Minneapolis, MN) or recombinant mouse IL-1β protein (1 mL, 1 mg/mL, AB9723, Abcam) was injected into the left TG using a guide cannula on day 2 following FG injection. On day 1 after injection, HWT to mechanical and heat stimulation of the tongue was measured, then rats were transcardially perfused as previously described [13, 21, 22]. Then, we prepared TG sections, mounted them on microscopic slides, and incubated them with rabbit anti-IL1RI or guinea pig anti-TRPV1 polyclonal antibody solutions. Saline was also injected as vehicle control.
Data are presented as the mean ± standard error of the mean (SEM). HWTs between the groups were compared using two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post hoc tests. The percentages of positive cells between the groups were compared using the Student’s t-test. All statistical analyses were conducted with a significance level of α = 0.05 (P < 0.05).