Adult male Sprague-Dawley rats (3 months old; 150 to 350 g; Harlan, IN, USA) were used. All animals were housed in a temperature-controlled room under a 12:12 light-dark cycle with access to food and water ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 80–23) and under a University of Maryland Baltimore approved Institutional Animal Care and Use Committee protocol.

Inflammation was induced by injecting 50 μl of 50% Complete Freund's Adjuvant (CFA) in isotonic saline (Sigma-Alridch, St. Louis, MO) into the mid-region of the masseter muscle via a 27-gauge needle. Rats were briefly anesthetized with 3% isoflurane for the injection procedure. The characteristics of inflammation following CFA injections in the rat masseter have been described previously (28, 29).

The methods for the ROS assay were described in our previous study (20). Briefly, ROS levels were quantified using a cell-permeant oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen, Carlsbad, CA, USA). H₂DCFDA is de-esterified within the cytoplasm and turns highly fluorescent upon oxidation. H₂DCFDA detects hydrogen peroxide (H₂O₂), peroxyl radicals (ROO•), and peroxynitrite (ONOO−), but it is possible that other biologically relevant ROS, such as superoxide radicals (O2•−) and hydroxyl radicals (OH•), are also involved. Rats were injected with either CFA or the same volume of vehicle into the left masseter muscle. Naïve rats that did not receive either CFA or vehicle treatment served as a control group. The TG ipsilateral to the injected muscle was removed either 1, 4, 7, 14, or 28 days after the injection. TG was quickly removed and washed with phosphate-buffered saline (PBS). Immediately after extraction and dissection, the tissues were minced finely in PBS and were incubated in 96-well plates in 200 μl PBS for 30 min at 37°C. The background fluorescence for each specimen was determined with a fluorimeter (DTX880 Multimode Detector, Beckman Coulter) at 485 nm for excitation and 535 nm for emission. After the background reading, H₂DCFDA was added to each well to a final concentration of 10 μM. The plates were again incubated for 30 min at 37°C, and the fluorescence was re-measured. ROS levels were estimated as the intensity of fluorescence after subtraction of the background fluorescence (Multimode Analysis Software). To minimize experimental variations, samples from naïve, CFA-, and vehicle-treated groups at each time point were analyzed on the same day. The results from CFA- or vehicle-treated group were normalized to the results from naïve rats at each time point. We have previously shown that negative control groups without TG tissues (PBS alone, PBS with H₂DCFDA, PBS with H₂O₂, PBS with H₂O₂ and H₂DCFDA) generated little or no positive signal, which is only approximately 1% or less of signals obtained from TG tissues (20). We have also shown that TG samples with exogenously added H₂O₂ exhibit robust fluorescence signal in the presence of H₂DCFDA (20). These control groups validate that our methods allow reliable detection of ROS in TG tissues by H₂DCFDA.

All microinjections into TG were made through a 26-gauge guide cannula (P1 Technologies, Roanoke, VA, USA) that was surgically implanted over TG (2.5 mm posterior and 1.5 mm lateral to the bregma). Briefly, the scalp was incised 1 cm in length with a sterile scalpel at the midline. A small hole was drilled in the skull using a dental drill with a sterile burr bit (2.35 mm shank, 2.7 × 2.5 mm) to enable implantation of a sterile 26-gauge guide cannula (Roanoke, VA) over TG (2.5 mm posterior and 1.5 mm lateral to the bregma) using stereotaxic coordinates. The cannula was secured to the skull using pharmaceutical grade acrylic cement and two small screws (3 mm) inserted into the parietal bones. The incision edges were cleansed with Betadine and rinsed with 0.9% saline and will be closed with a Wax or Silicone coated braided non-absorbable suture (4-0 or 5-0) material in a simple interrupted pattern. Sutures were removed 7 to 10 days after surgery.

The rats were briefly anesthetized with isoflurane (> 3%–4.5% Induction-chamber) (> 1.5%–3% Maintenance—nosecone) for microinjection procedures. The depth of anesthesia was confirmed by pinching the toe using serrated forceps. All microinjections were made manually using a sterile 30-gauge injection cannula attached to a 1.0 ml syringe (Hamilton, Reno, NV, USA) via PE tubing (0.2 mm ID, 1.75 mm OD). The external end of the cannula was prepped using Betadine solution / scrub and rinsed with alcohol prior to removal of access plug. The substances were infused over a 30 s period and the injection syringe was left in place for an additional 30 s to prevent backflow.

The procedures for primary TG cultures are described previously (37, 38, 39). Both TG from each animal were dissected out and dissociated by sequential digestion with 0.1% collagenase D in DMEM-F12 medium (with L-glutamine) at 37°C for 30 min, followed by additional digestion in a medium containing 0.25% trypsin, 50 μg DNase, and 0.02% EDTA at 37°C for 15 min. After trituration, cells were plated on laminin pre-coated 24-well plates and cultured in a 37°C incubator at 5% CO₂ for 1 to 3 days.

To examine whether ROS are involved in the transcription of the TRPA1 gene in TG neurons, we took two complimentary approaches. First, we treated TG primary culture with H₂O₂ (10 μmol, 1 h). Second, we administered a ROS donor, t-BOOH (2 μmol/10 μl, 2 consecutive days), directly into TG of intact animals without inflammation. Total RNA was extracted from either the dissociated TG cells in culture or dissected TG from intact animals, respectively, using an RNeasy kit (Qiagen Sciences, Germantown, MD) followed by DNase treatment to remove genomic DNA. Reverse transcription was carried out using SuperScript II kit (Invitrogen, Waltham, MA) was used to generate cDNA from 500 ng of RNA along with 2.5 ng of random primer per reaction. Real-time PCR analysis of cDNA (equal to 15 ng of RNA) was performed using Maxima SYBR Green/ROX qPCR Master Mix in an Eppendorf Mastercycler Ep Realplex 2.0 (Fermentas, Forest City, CA, USA). In all our RT-PCR experiments, each sample was analyzed in triplicates, and we routinely added a control with no template as a means of checking for any nucleic acid contamination, and a control with no reverse transcriptase to verify that there was no DNA contamination in the RNA preparation. The no template control also serves to identify any potential formation of primer dimers during the SYBR Green assay. The following primer pairs were used to detect Trpa1 mRNA: forward 5′-TCCTATACTG GAAGCAGCGA-3′, reverse 5′-CTCCTGATTGCCATC GACT-3′, and GAPDH, mRNA, used as a control: forward 5′-TCACCACCAT GGAGAAGGC G-3′, reverse 5′-GCTAAGCAGTTGGTG GTGCA-3′. We obtained the ratios between Trpa1 and GAPDH to calculate the relative abundance of mRNA levels in each sample. Relative quantification of the Trpa1 mRNA was calculated by the comparative CT method (2^−ΔΔCT method) between control and experimental groups.

Total proteins were extracted from the TG of naïve and CFA treated rats. The protein samples were dissolved in RIPA buffer containing protease inhibitor cocktail. The protein concentration of lysates was determined using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Fifty micrograms of protein for each sample were separated on 4%–12% NuPAGE gel with MOPS SDS running buffer and transferred to a PVDF membrane (Bio-rad, Hercules, CA, USA). After blocking for 1 h in 5% milk PBST at room temperature, membranes were probed with primary antibodies for TRPA1 (1:5,000, Millipore #ABN-1009, Burlington, MA) and an internal control protein GAPDH (1:5,000, Calbiochem, San Diego, CA), diluted in blocking solution. The TRPA1 antibody was raised against the N-terminus of rat TRPA1 and detects a 90–98-kDa protein, which disappears in TG lysates probed with TRPA1 antibody pre-incubated with a commercially available peptide used to generate the antibody. We have validated the specificity of this antibody in our previous study (33). Membranes from TG samples were incubated with primary antibodies overnight at 4°C and washed four times with PBST. HRP-conjugated secondary antibodies (anti-rabbit secondary antibody (Cell Signaling, Danvers, MA) and anti- mouse secondary antibody (Millipore, Burlington, MA) were diluted to 1:5,000 in PBST and incubated with membranes for 1 h at room temperature. Bands were visualized using ECL (Western Lightning, PerkinElmer Inc., Waltham, MA, USA) or ECL plus Western blotting detection reagent (Lumigen PS-3, GE Healthcare, Chicago, IL). Protein level for TRPA1 was normalized to that of GAPDH within the same sample.

The time-dependent changes in mechanical hyperalgesia before and after CFA or vehicle were analyzed with a Two-Way analysis of variance (ANOVA) with repeated measures. Data obtained from RT-PCR and Western blot experiments were analyzed with a one-way ANOVA on means or Kruskal–Wallis one-way ANOVA on ranks depending on the outcome of a normality test. Unless otherwise indicated, statistical comparisons of two independent groups were made with either Student's t- test or Mann–Whitney Rank Sum test. Data are presented as mean ± SE and differences were considered significant at p < 0.05. All multiple group comparisons were followed by Bonferroni post hoc test. Power analyses were conducted to determine the minimum sample size required for the behavioral experiments to detect a significant effect with a power of 0.8. We used G*Power Software (Heinrich-Heine, Universität Düsseldorf) to confirm that the sample sizes we used yielded a power greater than 0.85 even with a moderate effect size of 0.5.

We previously demonstrated that an injection of CFA in the rat masseter induces a time-dependent and significant decrease in mechanical thresholds that lasts over 3 weeks (33, 38). We confirmed these results, showing the development of mechanical hypersensitivity following CFA treatment in the masseter. Our data show that there were significant treatment (F = 157.7) and time (F = 89.20) effects, as well as significant interactions between treatment and time (F = 96.31), with a peak decrease in mechanical thresholds during the first 3 days (p < 0.0001 for pre-injection vs. post-CFA values, and p < 0.0001 for CFA vs. vehicle groups). A significant decrease in mechanical threshold was observed until day 14 (p < 0.001 for pre-injection vs. post-CFA values and p < 0.0001 for CFA vs. vehicle groups), which gradually returned to a baseline level by 28 days after CFA treatment (Figure 1A). The vehicle treatment did not alter mechanical thresholds for the entire observation period of 28 days (Figure 1A).

Our previous study demonstrated ROS levels increase within TG 1 day after masseter inflammation by CFA (20). A more comprehensive temporal profile of intraganglionic ROS under inflammatory conditions needs to be determined to firmly establish the relationship between inflammatory hyperalgesia with intraganglionic ROS. The inflammation-induced production of ROS in TG was assessed by comparing the intensity of fluorescence from ipsilateral TG obtained 1, 4, 7, 14, and 28 days following the injection of CFA or vehicle into the masseter muscle to that of naïve rats. Fluorescent signals from the TG of CFA-injected rats were significantly greater than those from vehicle-treated rats on days 1, 4, 7, and 14 after CFA treatment (Figure 1B, p < 0.0005 for days 1 and 4 post CFA vs. vehicle groups, p < 0.05 for day 7 post CFA vs. vehicle groups, and p < 0.005 for day 14 post CFA vs. vehicle groups). On day 28, intraganglionic levels of ROS were no longer significantly different between the two groups (Figure 1B). We have previously shown that the ROS level in TG contralateral to the CFA-injection site was not significantly different from that of naïve rats (20). These results demonstrate that masseter inflammation upregulates ROS production within TG, especially during the period when inflammatory mechanical hyperalgesia is pronounced. We conducted the present study using only male rats since the CFA condition we employed produces comparable levels and duration of masseter hyperalgesia in both male and female rats (38). We assumed that shared mechanisms account for the similar behavioral responses in both sexes under our CFA condition. However, we acknowledge that there may be potential differences in ROS accumulation between males and females that require further investigation.

To examine the functional involvement of intraganglionic ROS in the development of mechanical hyperalgesia, phenyl N-tert-butylnitrone (PBN) was injected directly into TG 30 min prior to CFA treatment in the masseter muscle. There were significant treatment (F = 29.33) and time (F = 390.3) effects, as well as significant interactions between treatment and time (F = 55.98). Preemptive intraganglionic PBN, but not vehicle, treatment significantly attenuated mechanical hyperalgesia one day after CFA treatment (Figure 2A, p < 0.005 for PBN vs. vehicle groups). However, PBN was no longer effective on day 2 (Figure 2A). We then examined whether scavenging of ROS within TG can also attenuate fully developed inflammatory mechanical hyperalgesia. There were significant treatment (F = 21.24) and time (F = 165.0) effects, as well as significant interactions between treatment and time (F = 47.09). PBN administered in TG one day after CFA treatment in the masseter muscle significantly blunted mechanical hyperalgesia (Figure 2B, p < 0.01 for PBN vs. vehicle groups). The PBN effect was transient, lasting only a few hours. Once the effect wears off, hyperalgesia returned as shown by pre-treatment measures at each time point. Interestingly, when the same concentration of PBN was administered in the same animals again on day 3 after CFA treatment, it blocked the CFA-induced mechanical hyperalgesia to a greater extent (Figure 2B, p < 0.0005 for PBN vs. vehicle groups). When PBN was administered a third time on day 7, the mechanical threshold was restored to the pre-CFA baseline sensitivity (Figure 2B). Vehicle administration in TG did not alter mechanical sensitivity at any of the time points (Figure 2B). These data demonstrate that excess production of ROS within TG remote from a peripheral inflammatory site contributes to the development and maintenance of inflammatory hyperalgesia.

We have previously shown that CFA-induced mechanical hypersensitivity and spontaneous muscle pain responses were significantly reversed by post-treatment with AP18 in the muscle (33). We have also shown that TRPA1 is amply expressed in the soma of small to medium size TG neurons (34). In order to examine whether TRPA1 expressed within TG could also functionally contribute to inflammatory hyperalgesia, we administered AP18 directly into TG 1, 3 and 7 days after CFA treatment in the masseter muscle. AP18, but not the vehicle, administration in TG one day after CFA treatment in the masseter muscle significantly attenuated the mechanical hyperalgesia (Figure 3, p < 0.0005 for AP18 vs. vehicle groups). The inflamed muscle, however, became hypersensitive again within 24 h of AP18 treatment (Figure 3). The same concentration of AP18 administered in the same animals both 1 day and 3 days after CFA treatment blocked the CFA-induced mechanical hyperalgesia to a similar extent of single AP18 treatment on day 1 (Figure 3, p < 0.0005 for AP18 vs. vehicle groups). The additional administration of AP18 on day 7 continued to be effective (p < 0.005 for AP18 vs. vehicle groups), but it did not further reduce the mechanical hyperalgesia beyond the extent observed on days 1 and 3.

Since both PBN and AP18 administered directly into TG effectively attenuated inflammatory hyperalgesia, and ROS are known to directly activate TRPA1, we investigated whether exogenous administration of ROS can induce pain-related responses without peripheral inflammation and whether that effect is mediated by TRPA1. Since the effects of H₂O₂ are expected to be short-lived, we evaluated evoked and spontaneous pain responses within an hour of H₂O₂ administration. Initially, we directly administered H₂O₂ into the trigeminal ganglion (TG) with or without AP18 and assessed masseter mechanical hypersensitivity. Direct administration of H₂O₂ into TG (20 μM in 5 μl) resulted in a significant decrease in the mechanical sensitivity of the masseter muscle, indicating the development of profound hyperalgesia (Figure 4A; p < 0.0001 for before vs. after). However, when the same concentration of H₂O₂ was co-administered with AP18, it effectively prevented H₂O₂-induced masseter hyperalgesia (Figure 4B). We also assessed RGS since H₂O₂ administration in TG could produce a widespread spontaneous pain not limited to selective orofacial regions. Animals administered with either AP18 or vehicle did not show any changes in facial expression prior to H₂O₂ administration. However, when H₂O₂ was administered, rats exhibited visible changes in facial expressions, indicated by a high RGS score. The increased RGS score due to H₂O₂ treatment was significantly attenuated by co-administration with AP18 (Figure 4C, p < 0.005 for AP18 vs. vehicle groups).

Since intraganglionic ROS have direct access to transcriptional machineries within the soma, we examined whether ROS are involved in the transcription of the Trpa1 gene in TG. Relative changes in the mRNA levels of the Trpa1 gene was determined from H₂O₂-treated primary TG culture samples (10 μmol, 1 h). H₂O₂ treatment induced a significant increase in TRPA1 (Figure 5A, p < 0.05 for H₂O₂ vs. naive groups). To confirm that ROS build up in TG can induce a transcriptional increase of Trpa1, we administered a ROS donor, t-BOOH, directly into TG of intact animals without inflammation. t-BOOH significantly upregulated the transcript levels of TRPA1 in TG (Figure 5B, p < 0.0001 for t-BOOH vs. naive groups), suggesting that ROS alone is sufficient to induce transcriptional changes of Trpa1 in TG. In our previous study, we showed that CFA-induced masseter inflammation results in a time-dependent increase in the level of Trpa1 mRNA in TG (33). The level of Trpa1 mRNA TG is significantly upregulated on days 1, 3 and 7 compared to the baseline. To examine whether ROS buildup in TG can increase TRPA1 protein expression, we administered PBN or vehicle directly into TG on days 1, 3, and 7 of CFA treatment. Here, we confirmed that TRPA1 protein expression is significantly upregulated on day 7 following CFA treatment in the masseter muscle in vehicle treated rats compared to that of naïve rats (Figure 6). However, TRPA1 expression levels in PBN treated rats were significantly lower compared to vehicle treated rats (Figure 6, p < 0.05 for Saline vs. PBN groups), suggesting that excess intraganglionic ROS under inflammatory conditions induce upregulation of TRPA1 expression in TG.