Macrophages in trigeminal ganglion contribute to ectopic mechanical hypersensitivity following inferior alveolar nerve injury in rats

We used male Sprague-Dawley rats (n = 101, Japan SLC, Shizuoka, Japan) weighing 170–260 g. All rats were housed individually in transparent polycarbonate cages (length 48 cm; width 26.5 cm; height 21 cm) with paper shavings as bedding. The room temperature was controlled (23 °C) with a 12 h light/dark cycle (lights on at 7:00, off at 19:00), and animals were allowed ad libitum access to food and water. All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the International Association for the Study of Pain [14]. This study was approved by the Animal Experimentation Committee at Nihon University (AP16D010).

IANX was performed as described previously under deep anesthesia with intraperitoneal (i.p.) 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) [15]. Briefly, a small incision was made in the skin of the left cheek, and the masseter muscle was dissected to expose the mandibular bone surface. The surface of the mandible was scraped using a low-speed dental drill bur to expose the inferior alveolar nerve. The exposed inferior alveolar nerve was gently pulled, transected, and replaced into the mandibular canal. In the control group, the skin incision, muscle dissection, and bone scraping without IANX were performed (sham operation). The dissected muscle and skin incision were sutured with 6-0 silk.

Rats were trained daily for 5 to 7 days to protrude their snout from a cage that had a small fenestration and to withdraw their snout freely following application of mechanical stimuli to the whisker pad skin by von Frey filaments (Touch-Test Sensory Evaluator, North Coast Medical, Morgan Hill, CA) as described previously [4]. After stabilizing the mechanical head-withdrawal threshold (MHWT), IANX or sham operation was performed and followed by behavioral testing under the same conditions every other day for 15 days. The MHWT was determined as the lowest intensity that evoked head-withdrawal responses to five stimuli (duration 1 s). The behavioral testing was conducted under blinded conditions.

Under deep anesthesia with i.p. butorphanol (2.5 mg/kg), medetomidine (0.375 mg/kg), and midazolam (2.0 mg/kg), a head of rat was placed in a stereotaxic instrument for guidance [4, 16]. The skull was exposed and a small hole (1 mm in diameter) was drilled above the location of the TG (2.8 mm anterior from the posterior fontanelle, 2.7 mm lateral to the sagittal suture) ipsilateral to the location of IANX or sham operation. A guide cannula was extended in the TG (9 mm below the skull surface) and was anchored to the skull bone with dental cement and three stainless-steel screws. To confirm the correct positioning of the tip of the cannula, a trocar was inserted into the cannula as an electrode and multi-unit activities induced by mechanical stimulation of the whisker pad skin were conformed. After implantation of the cannula, rats were allowed to recover for 5 days before performing the IANX or sham operation. Under light anesthesia with 2% isoflurane in oxygen, liposomal clodronate Clophosome-A (LCCA, 6 μl, 7 mg/mL, product code: F70101CA, FormuMax Scientific, Sunnyvale, CA, USA) or plain control liposomes for LCCA (Cont-LCCA, 6 μl, 20 mM, product code: F70101-A, FormuMax Scientific) were administered into the TG through the guide cannula prior to operation and on days 1 to 4 following IANX or sham operation. LCCA has been shown to be effective in successfully depleting proliferated and resident macrophages [17]. Etanercept (1 μg; Pfizer, New York, NY, USA) was dissolved in physiological saline or vehicle alone (physiological saline) and administered (6 μl) into the TG through the guide cannula under light anesthesia with 2% isoflurane in oxygen prior to operation and on days 1 to 4 following IANX or sham operation. The LCCA and etanercept administration were performed immediately after the measurement of the MHWT of whisker pad skin. The MHWT of the whisker pad skin was measured before operation and every other day for 5 days after IANX or sham operation.

Moreover, rats were allowed to recover after implantation of the cannula for 5 days. Under light anesthesia with 2% isoflurane in oxygen, 2 μl of 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine methanesulfonate (DiI, Thermo Fisher Scientific, Waltham, MA) was administered into the TG through the guide cannula. On day 3 after the administration, rats were deeply anesthetized with the above-described solution and transcardially perfused with saline followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After the perfusion, TGs were removed and immersed in a fixative containing 4% paraformaldehyde for more than 4 h at 4 °C then transferred to 0.01 M phosphate-buffered saline (PBS) for 12 h for cryoprotection. TG from naive rat was also processed in a similar manner. Next, the tissues were embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan) and were cut in 12-μm-thick sections in a horizontal plane along the long axis.

Under deep anesthesia with i.p. butorphanol (2.5 mg/kg), medetomidine (0.375 mg/kg), and midazolam (2.0 mg/kg), a retrograde labeling tracer, FluoroGold (7 μl dissolved in saline; FG; 4% hydroxystilbamidine; Fluorochrome, Denver, CO, USA), was injected into the skin of the left whisker pad ipsilateral to IANX using a 30-gauge needle before performing the IANX or sham operation. On day 3, rats were deeply anesthetized with the above-described solution and transcardially perfused with saline followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After the perfusion, TGs were removed and immersed in the same fixative for more than 4 h at 4 °C then transferred to 0.01 M PBS containing 20% sucrose for 12 h for cryoprotection. Next, the tissues were embedded in Tissue-Tek and stored at − 20 °C until cryosectioning. Then, the ganglion tissues were cut in 12-μm-thick sections in a horizontal plane along the long axis. Every 10th section (five sections per rat) was thaw-mounted on a MAS-GP slide glass (Matsunami, Osaka, Japan) and dried overnight at room temperature. The five sections were used for immunohistological analysis. After rinsing with 0.01 M PBS three times for 5 min each, sections were incubated in mouse anti-Iba1 polyclonal antibody (1:200; Abcam, Cambridge, MA, USA) and rabbit anti-TNFα monoclonal antibody (1:200; Abcam) or rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; cat.No. ab7260, Abcam) or rabbit anti-F4/80 monoclonal antibody (1:500; cat.No. ab111101, Abcam), or anti-tumor necrosis factor receptor-1 (TNFR1) monoclonal antibody (1:50; Santa Cruz, Santa Cruz, CA, USA), diluted in 0.01 M PBS containing 4% normal donkey serum (Merck, Darmstadt, Germany) in 0.3% Triton X-100 (Merck) overnight at 4 °C. After rinsing with 0.01 M PBS, the sections were incubated in Alexa Fluor 568 anti-rabbit IgG (1:200; Thermo Fisher Scientific) and/or Alexa Fluor 488 anti-mouse IgG (1:200; Thermo Fisher Scientific) in 0.01 M PBS for 2 h at room temperature. After rinsing with 0.01 M PBS, sections were coverslipped in PermaFluor (Thermo Fisher Scientific). The sections were observed using a BZ-9000 system (Keyence, Osaka, Japan). Iba1-IR and TNFα-IR cells in the TGs of the ophthalmic-maxillary division (V1-V2) and mandibular division (V3) were analyzed. The mean relative area (density) of Iba1-IR or TNFα-IR cells were calculated by using a computer-assisted imaging analysis system (ImageJ, NIH, Bethesda, MD, USA). The mean relative area (density) of F4/80- and Iba1-IR area was calculated by using a computer-assisted imaging analysis system (BZ-X analyzer, Keyence, Osaka, Japan). All immunohistochemical measurements were conducted under blinded conditions, performed by observers who were unaware of which treatment the animals received. Using the same conditions, no specific labeling was observed in the absence of primary antibody. Measurement of the area occupied by the immuno-products was made in a region selected identically from V1/V2 and V3 in the TG.

On day 3 following IANX or sham operation, the rats were perfused through the aorta with physiological saline under deep anesthesia as described above. Immediately, the TG was removed and homogenized in 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; 0.05 mM sodium vanadate). The homogenate was centrifuged at 15,000 rpm for 10 min at 4 °C, and the protein concentration of the resulting supernatants was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). The supernatants were heat-denatured in Laemmli sample buffer solution (Bio-Rad), and 30 μg of each protein sample was subjected to SDS-PAGE using a 10% acrylamide gel. The samples were transferred to a polyvinylidene difluoride membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) using Trans-Blot Turbo (Bio-Rad). The membrane was rinsed with Tris-buffered saline mixed with 0.1% Tween 20 (TBST) and incubated in 3% bovine serum albumin (BSA; Bovogen, Essendon, Australia). The membrane was then incubated with rabbit anti-TNFα polyclonal antibody (1:200; diluted in TBST and 3% BSA, Abcam) or rabbit anti-Iba1 monoclonal antibody (1:1000; diluted in TBST and 3% BSA, cat.No. ab178847, Abcam) overnight at 4 °C. Next, it was incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (Cell Signaling, Danvers, MA, USA) for 2 h at room temperature. Antibody binding was detected using Western Lightning ELC Pro (PerkinElmer, Waltham, MA, USA). Band intensity was analyzed using a ChemiDoc MP system (Bio-Rad) and normalized to β-actin on blots re-probed with the anti-β-actin antibody (1:200; Santa Cruz) after removing bound protein using a stripping reagent (Thermo Scientific).

Data were expressed as mean ± standard error. Statistical analyses were performed by Student’s t test, one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test, and two-way ANOVA with repeated measures followed by Bonferroni’s multiple-comparison test where appropriate. A value of p < 0.05 was defined as statistically significant.

The MHWT of the whisker pad skin ipsilateral to IANX was significantly decreased on day 1 following IANX, and this decrease persisted until day 15 when compared with sham-operated animals (Fig. 1a). On day 3 following IANX, Iba1-IR cells were observed in V1/V2 FG-labeled neurons, as well as in V3, in which FG-labeled neurons were absent (Fig. 1b, c). The relative area of Iba1-IR cells in V1/V2 and V3 in TG ipsilateral to IANX was significantly increased on day 3 following IANX compared to that of the sham-operated group (Fig. 1d). The Iba1 protein level was increased in TG ipsilateral to IANX on day 3 following IANX compared to sham-operated animals (Fig. 1e).

Primarily, it was confirmed that drug administration into the TG through the guide cannula was accurate (Fig. 2a). Daily administration of LCCA into the TG prevented the development and progress of mechanical allodynia in the whisker pad skin ipsilateral to IANX from days 1 to 5 following IANX (Fig. 2b). On day 3 after IANX, Iba1-IR cells in V1/V2, in which FG-labeled neurons were abundant, were observed in the LCCA- and Cont-LCCA-treated groups (Fig. 2c). Moreover, the relative area of Iba1-IR cells in the LCCA-treated group was significantly lower compared to the group treated with Cont-LCCA (Fig. 2d). Before intra-TG LCCA and Cont-LCCA administration, it was confirmed that the guide cannula implantation did not change the mechanical sensitivity (data not shown).

TNFα was expressed in activated Iba1-IR cells presented with morphological characteristics such as the bigger cell bodies and thicker ramifications and in activated GFAP-IR cells in V1/V2 on day 3 in the IANX group rather than sham group (Fig. 3a). F4/80 immunoreactivity was observed in the Iba1-IR cells in V1/V2 on day 3 in IANX or sham groups, and the relative F4/80-IR area in the Iba1-IR area was decreased in V1/V2 ipsilateral to IANX compared to that of the sham-operated group (Fig. 3b). The relative area of TNFα immuno-products increased in V1/V2 ipsilateral to IANX compared to that of the sham-operated group (Fig. 3c, d). The TNFα protein level was also increased in TG ipsilateral to IANX on day 3 following IANX compared to naive and sham-operated animals (Fig. 3e). Daily LCCA administration significantly reduced the relative area of TNFα immuno-products in V1/V2 in TG on day 3 following IANX compared to the Cont-LCCA-treated IANX group or sham group (Fig. 4a, b).

Daily administration of etanercept into the ipsilateral TG prevented mechanical hypersensitivity in whisker pad skin ipsilateral to IANX from days 1 to 5 (Fig. 5a). Immunostaining was performed to examine TNFR1 expression in the TG on day 3 following IANX. TNFR1 expression was observed in FG-labeled neurons, and the mean percentage of FG-labeled TNFR1-IR neurons in TG ipsilateral to IANX was significantly increased compared to the sham-operated group (Fig. 5b).