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. Author manuscript; available in PMC: 2022 Sep 5.
Published in final edited form as: Brain Behav Immun. 2020 Jan 27;87:455–464. doi: 10.1016/j.bbi.2020.01.016

Resveratrol alleviates temporomandibular joint inflammatory pain by recovering disturbed gut microbiota

Yajing Ma a,b, Sufang Liu a,b, Hui Shu a, Joshua Crawford a, Ying Xing b, Feng Tao a,c,*
PMCID: PMC9444375  NIHMSID: NIHMS1831114  PMID: 32001342

Abstract

Patients with temporomandibular disorders (TMDs) often experience persistent facial pain. However, the treatment of TMD pain is still inadequate. In recent years, the disturbance of gut microbiota has been shown to play an important role in the pathogenesis of different neurological diseases including chronic pain. In the present study, we investigated the involvement of gut microbiota in the development of temporomandibular joint (TMJ) inflammation. Intra-temporomandibular joint injection of complete Freund’s adjuvant (CFA) was employed to induce TMJ inflammation. Resveratrol (RSV), a natural bioactive compound with anti-inflammatory property, was used to treat the CFA-induced TMJ inflammation. We observed that CFA injection not only induces persistent joint pain, but also causes the reduction of short-chain fatty acids (SCFAs, including acetic acid, propionic acid and butyric acid) in the gut as well as decreases relevant gut bacteria Bacteroidetes and Lachnospiraceae. Interestingly, systemic administration of RSV (i.p.) dose-dependently inhibits CFA-induced TMJ inflammation, reverses CFA-caused reduction of SCFAs and these gut bacteria. Moreover, CFA injection causes blood–brain barrier (BBB) leakage, activates microglia and enhances tumor necrosis factor alpha (TNFα) release in the spinal trigeminal nucleus caudalis (Sp5C). The RSV treatment restores the BBB integrity, inhibits microglial activation and decreases the release of TNFα in the Sp5C. Furthermore, fecal microbiota transplantation with feces from RSV-treated mice significantly diminishes the CFA-induced TMJ inflammation. Taken together, our results suggest that gut microbiome perturbation is critical for the development of TMJ inflammation and that recovering gut microbiome to normal levels could be a new therapeutic approach for treating such pain.

Keywords: Inflammatory joint pain, Gut microbiota, Temporomandibular disorders, Short-chain fatty acids, Microglial activation, Tumor necrosis factor alpha

1. Introduction

Patients with temporomandibular disorders (TMDs) suffer from persistent facial pain, a condition characterized by painful temporomandibular joint (TMJ) sounds and limited or asymmetric mandibular motion, and the pain is often localized in the TMJ and muscles of mastication (Scrivani et al., 2008). Nonsteroidal anti-inflammatory drugs are frequently used to manage inflammatory TMD pain (Dionne et al., 1997; Ta and Dionne, 2004). However, TMD pain management requires different therapeutic strategies due to diverse causes of this disorder (Wieckiewicz et al., 2015). Therefore, it is necessary to identify new targets and develop novel therapies for TMD pain.

Accumulating evidence in preclinical studies indicates that the dysregulated expression of a variety of pronociceptive mediators, such as pro-inflammatory cytokines, released from activated microglia in trigeminal nociceptive system is crucial to the development and maintenance of TMJ inflammation and trigeminal pain (Magni et al., 2018; Villa et al., 2010). Although inhibition of these pronociceptive cytokines and chemokines in animal models has shown its efficiency in the treatment of this type of pain, the analgesic effect is usually shortlasting and not always robust. Thus, targeting the mechanisms that cause the production of pronociceptive cytokines in the trigeminal nociceptive system rather than inhibiting these molecules may be a better therapeutic strategy for treating such pain.

The emerging role of gut microbiota in neurological disorders has recently been demonstrated. Growing evidence suggests that the disturbance of gut microbiota significantly influences microglia maturation and its function (Erny et al., 2015; Erny et al., 2017). Moreover, short-chain fatty acids (SCFAs), including acetic acid, propionic acid and butyric acid, are derived from bacterial fermentation of nondigestible carbohydrates in the gut (Koh et al., 2016). And these SCFAs have important roles in regulating microglia morphology and function. Specifically, Bacteroidetes account for approximately 23% of gut bacteria and members of this phylum mainly produce acetic and propionic acids (den Besten et al., 2013), and Lachnospiraceae mainly produces butyric acid in the gut (Meehan and Beiko, 2014). Thus, the gut microbiome may serve as a vital mediator for TMJ inflammation through the regulation of microglial activation in the trigeminal nociceptive system.

Resveratrol (RSV) is a natural bioactive compound found in various plants, especially in grape skins and red wines (Bastianetto et al., 2015; Yu et al., 2012). Due to its anti-oxidant, anti-inflammatory properties, RSV has been proved to improve pathological and behavioral outcomes in the treatment of different neurological disorders including trigeminal neuropathic pain in rats (Yang et al., 2016) and chronic neuropathic pain in mice (Tao et al., 2016). In addition, a recent study shows that RSV is able to alter gut microbiome in obese mice, which is the vital mechanism underlying its effect on glucose homeostasis (Sung et al., 2017). These studies suggest that RSV may be used to treat TMJ inflammatory pain by restoring normal gut microbiota and thereafter regulating microglial activation and the release of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα).

In the present study, we hypothesized that gut microbiota is a potential target to develop an effective therapy for TMJ inflammatory pain. We showed that RSV significantly inhibits complete Freund’s adjuvant (CFA)-induced TMJ inflammation, reverses CFA-caused reduction of SCFAs and the relevant gut bacteria, restores the integrity of the blood–brain barrier (BBB), inhibits the activation of microglia and decreases the release of TNFα in the spinal trigeminal nucleus caudalis (Sp5C). Furthermore, we found that fecal microbiota transplantation (FMT) with feces from RSV-treated mice significantly diminishes the CFA-induced TMJ inflammatory pain. Together, our results suggest that gut microbiome perturbation is critical for the development of TMJ inflammation and that recovering gut microbiome to normal levels could be a new therapeutic approach for treating TMJ inflammatory pain.

2. Materials and methods

2.1. Animals

Eight-week-old male C57BL/6 mice (The Jackson Laboratory) were used in this study. We chose male mice in this study to avoid the influence of estrogen level alteration on systemic RSV-produced treatment of TMJ inflammation. Animals were housed under standard conditions with 12 h light/dark cycle and allowed access to water and food ad libitum. All behavioral tests were performed by an investigator blinded to the assignment of animal groups, and mice were acclimated with the test environment 30 min per day for three days. All procedures were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Texas A&M University College of Dentistry.

2.2. TMJ inflammation mouse model

CFA (10 μl, 5 mg/ml, Chondrex, Inc.) or saline (10 μl) was injected into the superior TMJ space of mice to induce TMJ inflammation as described in our previous study (Bai et al., 2019). Bilateral injection of CFA was performed to observe the effect of systemic administration of RSV on CFA-induced inflammatory pain in TMJ, SCFA production, SCFA-producing bacteria in the gut, and BBB permeability.

2.3. Drug treatment

Mice received RSV (Sigma, R5010) at 40 mg/kg or 80 mg/kg (i.p.) once a day for consecutive 4 days starting from 1 h post-CFA. Stock solution of RSV was prepared by dissolving it in 100% of dimethyl sulfoxide (DMSO) at concentration of 160 mg/ml (Li et al., 2010) and stored at −20°C until use. Equivalent DMSO was used as a vehicle control.

2.4. Assessment of facial mechanical hypersensitivity

The calibrated von Frey filaments were used to assess facial mechanical hypersensitivity as described in our previous study (Bai et al., 2019). Briefly, mice were placed into 10-cm long Plexiglass cylinder tubes and allowed to poke their heads out and forepaws, but the tube prevented them from turning around. Following acclimation for 30 min, the filament was applied to the skin area innervated by the trigeminal nerve V3 branch. Each filament was applied five times to the V3-innervated skin area for 1–2 s with a 10 s interval, starting from the lowest force of filament and continuing in ascending order. A positive response was defined as a sharp withdrawal of the head upon stimulation. The head withdrawal threshold was calculated as the force at which the positive response occurred in three of five stimuli.

2.5. Scfas measurement

The concentration of SCFAs in the fecal samples was measured by Gas Chromatography–Mass Spectrometer (TSQ 8000 EVO GC–MS, ThermoFisher Scientific) in the Integrated Metabolomics Analysis Core at the Texas A&M University. In brief, each fecal sample (50 mg) was homogenized with 600 μl of cold hydrochloric acid (30 mM) and centrifuged at 13,000 g for 10 min at 4 °C. Next, the supernatant (300 μl) was diluted by 300 μl of cold diethyl ether, and the mixture was vortexed for 10 s and incubated on ice for 5 min and the centrifuged at 13,000 g for 1 min at 4 °C. After layers have separated, upper layer was transferred to autosampler vial and injected into the TSQ 8000 EVO GC–MS (Thermo Scientific) for the measurement of SCFAs.

2.6. Gut microbiome analysis

Fecal samples from individually housed mice with different treatments were collected in sterile eppendorf tubes and immediately stored at −80 °C until use. Fecal microbiome genomic DNA was extracted with QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the manufacturer’s instructions and amplified with the following primers: 1) Bacteroidetes: forward, 5′-GTTTAATTCGATGATACGCGAG-3′; reverse, 5′-TTAASCCGACACCTCACGG-3′ (Yang et al., 2015); 2) Lachnospiraceae: forward, 5′-CGGTACCTGACTAAGAAGC-3′; reverse, 5′-AGTTT(C/T)ATTCTTGCGAACG-3′ (Nava et al., 2011). The microbiome quantification was performed by the CFX96 real-time PCR detection system with optical 96-well plates, and the PCR reactions were designed as follows: 95 °C for 10 min, 40 cycles of 95 °C 30 s and 60 °C 1 min, and then 72 °C 30 s. The SYBR Green was used as a fluorescent dye. Relative quantification was analyzed with the 2−ΔΔCT method.

2.7. Fecal microbiota transplantation (FMT)

We performed FMT as described in our recent work (Tang et al., 2019). 100 mg of fresh feces from mice with RSV or DMSO treatment following CFA injection was collected and resuspended in 2 ml of sterile PBS to prepare fecal slurry. Next, recipient mice were gavaged with 200 μl of the fecal slurry once a day for consecutive 4 days (Suez et al., 2014). The FMT with feces from DMSO-treated mice (FMT1) was used as a control for the FMT with feces from RSV-treated mice (FMT2). Oral gavage of sterile PBS served as a control for FMT procedure.

2.8. Examination of BBB integrity

Evans blue perfusion was performed to examine BBB integrity as described previously (Braniste et al., 2014; del Valle et al., 2008). Briefly, 4 days after CFA or saline injection, the mice were anesthetized with i.p. injection of sodium pentobarbital (50 mg/kg). Next, the thoracic cavity was opened and cardiac perfusion was performed with 50 ml of PBS (pH 7.2) followed by 50 ml of the cocktail containing 1% Evans blue (Sigma–Aldrich) dissolved in 4% paraformaldehyde (PFA) solution using a Masterflex peristaltic pump perfusion system. The whole brains were post-fixed in 4% PFA solution for 4 h, and then cryoprotected in 30% sucrose for 24 h at 4 °C. The brainstems removed from dehydrated whole brains were embedded in OCT medium for sectioning with a cryostat (CM1950, Leica). Coronal brainstem sections (20 μm) were mounted on slides and visualized using fluorescent microscope (DMi8, Leica) with excitation light on 543-nm laser beams. Percentage of Evans blue extravasated area to the whole image area was quantified with NIH Image J software as described previously (Li et al., 2014).

2.9. Immunofluorescence staining

Following perfusion, the whole brains were post-fixed in 4% PFA solution for 24 h, and then cryoprotected in 30% sucrose for 48 h. Next, the brainstems were isolated and cut at 20 μm with a cryostat (CM1950, Leica). Free-floating sections were blocked with 5% donkey serum and 0.3% Triton X-100 in PBS for 1 h followed by incubation with primary antibodies overnight at 4 °C, and then the sections were washed and incubated with corresponding secondary donkey anti-goat Alexa 488 (1:200, Jackson, 125100) or donkey anti-mouse Cy3 (1:200, Jackson, 124774) for 1 h at room temperature. The following primary antibodies were used in this study: goat anti-Iba1 (1:700, Abcam, ab5076), mouse anti-TNFα (1:300, Abcam, ab1793). The specificity of the TNFα antibody was validated using Sp5C tissue from TNFα knockout mice (see Supplemental Fig. 1). Immunofluorescent images were visualized and acquired using a Leica fluorescence microscope (DMi8, Leica). Cell counting was done with NIH ImageJ software. For quantification of the co-expression of TNFα and Iba1 in the Sp5C, three brain sections from each animal were used to count positive cells under the 40X objective lens of the fluorescence microscope. Quantitative analysis of TNFα and Iba1 positive cells in the Sp5C-V3 was performed using the plugin QuantIF of the ImageJ (Handala et al., 2019). Briefly, color images were first converted into 8-bit gray images. After background subtraction, colocalization analysis was done using QuantIF. The TNFα staining image was converted to a TNFα staining mask and the Iba1 staining image was converted to an Iba1 staining mask. A third mask corresponding to the co-immunostained cells was created using the “Image Calculator” command and the “AND” operator. Finally, the total numbers of Iba1-positive cells and co-expression cells were counted using the “Analyze Particles” tool.

2.10. Morphological examination of microglia

The morphometric analysis of microglia was carried out with Iba1-immunostained brainstem sections (3 sections from each mouse). Skeleton images were acquired for quantitative analysis of average endpoints, process length and cell size of microglia in saline- or CFA- treated mice on day 4 post-injection using NIH ImageJ software as described previously (Young and Morrison, 2018). For skeleton analysis, a series of ImageJ plugins were progressively performed for visualization of all microglia processes before the conversion to binary and skeletonized images. The AnalyzeSkeleton (2D/3D) plugin was then applied to all skeletonized images to tag elements of microglia skeletons for collecting data on the number of endpoints and process length. Cell body perimeter was measured in each Iba1-immunopositive cell using the same software. The free-hand selection tool was used to trace the outline of individual microglia cell body, and then cell body perimeter of microglia as the cell body size was measured according to previous studies (Adeluyi et al., 2019; Hinwood et al., 2012; Kongsui et al., 2014).

2.11. Statistical analysis

Data were expressed as the mean ± S.E.M. All statistical analyses were performed using Graph Pad Prism 8.0.1. Behavioral data were normalized as percentage of respective baseline and the data before and after normalization were analyzed by two-way analyses of variance (ANOVA) with repeated measures. The interaction between two variables (RSV treatment and different concentration of RSV) was assessed to detect whether there is a dose-dependent effect of RSV. Other data were analyzed with one-way ANOVA and unpaired t-test. P < 0.05 was considered statistically significant.

3. Results

3.1. RSV dose-dependently inhibits CFA-induced TMJ inflammation

To determine whether RSV can treat TMD-like facial pain, we administered (i.p.) it (40 mg/kg and 80 mg/kg) once a day for consecutive 4 days starting from 1 h after intra-TMJ injection of CFA. The CFA-induced TMJ inflammatory pain was assessed by measuring mechanical hypersensitivity with von Frey filaments. We observed that bilateral injection of CFA robustly decreased head withdrawal threshold in both sides of trigeminal nerve V3 branch-innervated facial skin area (Fig. 1A and B). In the CFA-induced TMJ inflammation model, we found that i.p. injection of RSV dose-dependently increased the CFA-decreased head withdrawal threshold compared to the DMSO treatment (Fig. 1C and D). As a control, RSV alone at high dose (80 mg/kg) had no effect on the head withdrawal threshold in animals not receiving CFA (Fig. 1C and D). These results indicate that systemic administration of RSV dose-dependently inhibits CFA-induced TMJ inflammation.

Fig. 1.

Fig. 1.

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RSV dose-dependently inhibits CFA-induced TMJ inflammation. (A and B) Bilateral injection of CFA robustly decreased head withdrawal threshold in both sides of trigeminal nerve V3 branch-innervated facial skin area. (C and D) i.p. injection of RSV (40 mg/kg and 80 mg/kg) dose-dependently increased the CFA-decreased head withdrawal threshold compared to the DMSO treatment. As a control, RSV alone at high dose (80 mg/kg) had no effect on the head withdrawal threshold in animals not receiving CFA. In (A and B), *P < 0.05, **P < 0.01, P < 0.001 vs. the Saline group at corresponding time points; In (C and D), ++P < 0.01, +++P < 0.001 vs. the “Saline + DMSO” group at corresponding time points, and #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the “CFA + DMSO” group at corresponding time points. (n = 6 per group).

3.2. RSV reverses CFA-caused reduction of SCFAs in the gut

To reveal the involvement of gut SCFAs in the CFA-induced TMJ inflammation, we measured three SCFAs (acetic acid, propionic acid and butyric acid) in the gut on day 4 after CFA injection. The three SCFAs were significantly decreased in the CFA-treated mice compared with the saline control group (Fig. 2A). Interestingly, i.p. injection of RSV completely reversed the CFA-caused reduction of SCFAs in the gut compared to the DMSO-treated mice (Fig. 2B), suggesting that restoration of gut SCFAs may mediate the analgesic effect of RSV on the CFA-induced TMJ inflammation.

Fig. 2.

Fig. 2.

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RSV reverses CFA-caused reduction of SCFAs in the gut on day 4 after CFA injection. (A) The three SCFAs including acetic acid, propionic acid and butyric acid were significantly decreased in the CFA-treated mice compared with the Saline control group (n = 3 per group). (B) i.p. injection of RSV (40 mg/kg) completely reversed the CFA-caused reduction of SCFAs in the gut compared to the “CFA + DMSO” group (n = 5 for the “CFA + DMSO” group; n = 6 for the “CFA + RSV” group). *P < 0.05, **P < 0.01 as indicated in the figure.

3.3. RSV recovers CFA-decreased Bacteroidetes and Lachnospiraceae in the gut

SCFAs in the gut are mainly produced by Bacteroidetes and Lachnospiraceae. And Bacteroidetes produce acetic and propionic acids (den Besten et al., 2013), while Lachnospiraceae produces butyric acid (Meehan and Beiko, 2014). To investigate whether the SCFA-producing gut bacteria contribute to the analgesic effect of RSV, we analyzed the abundance of Bacteroidetes and Lachnospiraceae in the gut. Our data showed that intra-TMJ injection of CFA significantly decreased these gut bacteria on day 4 after CFA injection compared with the saline control group (Fig. 3A and B). More importantly, we found that i.p. injection of RSV robustly recovered the SCFA-producing gut bacteria compared to the DMSO-treated mice (Fig. 3C and D).

Fig. 3.

Fig. 3.

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RSV recovers CFA-decreased Bacteroidetes and Lachnospiraceae in the gut. (A and B) Intra-TMJ injection of CFA significantly decreased the gut bacteria on day 4 after CFA injection. The disturbed bacteria returned to the baseline level on day 12 after CFA injection (n = 3 per group). (C and D) i.p. injection of RSV (40 mg/kg) robustly recovered the disturbed gut bacteria on day 4 post-CFA compared to the “CFA + DMSO” group (n = 4 for the “CFA + DMSO” group; n = 6 for the “CFA + RSV” group). *P < 0.05, **P < 0.01 as indicated in the figure.

3.4. FMT with feces from RSV-treated mice attenuates CFA-induced TMJ inflammatory pain

To further verify that the analgesic effect of RSV is mediated by recovering disturbed gut microbiota, we conducted FMT in mice with unilateral intra-TMJ injection of CFA. The FMT was carried out once a day for consecutive 4 days starting at 1 h after CFA injection. The transplantation (FMT2) of fecal slurry from RSV-treated mice significantly increased head withdrawal threshold in the ipsilateral V3-innervated facial skin area compared with the transplantation (FMT1) of fecal slurry from DMSO-treated mice (Fig. 4A), while the FMT2 had no effect on the head withdrawal threshold in the contralateral V3-innervated facial skin area (Fig. 4B). The ipsilateral head withdrawal thresholds were significantly increased following FMT2 from day 3 (before and after transplantation: 0.35 ± 0.07 g and 0.63 ± 0.08 g) until day 9 (before and after transplantation: 1.00 ± 0.10 g and 1.33 ± 0.07 g) after CFA injection. On the other hand, the head withdrawal thresholds of both ipsilateral and contralateral sides in mice with FMT1 were similar to those in the PBS-treated group (Fig. 4). These results suggest that RSV-produced recovery of disturbed gut microbiota is critical for its analgesic effect on CFA-induced TMJ inflammatory pain. To provide evidence indicating the contribution of SCFAs to the effect of FMT, we measured SCFA concentrations and SCFA-producing bacteria in the gut after FMT. Our data showed that the FMT2 completely reversed CFA-caused reduction of SCFAs and significantly recovered CFA-decreased Bacteroidetes and Lachnospiraceae in the gut compared with the FMT1 group (Supplemental Fig. 2).

Fig. 4.

Fig. 4.

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FMT with feces from RSV-treated mice attenuates CFA-induced TMJ inflammatory pain. The FMT was carried out once a day for consecutive 4 days starting at 1 h after CFA injection. (A) The transplantation (FMT2) of fecal slurry from RSV-treated mice significantly increased head withdrawal threshold in the ipsilateral V3-innervated facial skin area compared with the transplantation (FMT1) of fecal slurry from DMSO-treated mice. (B) The FMT2 had no effect on the head withdrawal threshold in the contralateral V3-innervated facial skin area. Note that the head withdrawal thresholds of both ipsilateral and contralateral sides in mice with FMT1 were similar to those in the PBS-treated group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the “CFA + FMT1” group at corresponding time points (n = 6 per group).

3.5. RSV rescues CFA-caused BBB leakage

To investigate the effects of intra-TMJ injection of CFA and RSV treatment on the BBB permeability, we examined extravasation of Evans blue following its cardiac perfusion. The BBB integrity in the control mice treated with saline (intra-TMJ) and DMSO (i.p.) was normal and no Evans blue signal was shown in the brainstem sections (Fig. 5A). However, CFA injection produced extravasation of Evans blue (Fig. 5B), and RSV treatment completely blocked the CFA-caused BBB leakage (Fig. 5C). The quantification of Evans blue extravasated area indicated that intra-TMJ CFA robustly increased the percentage of Evans blue extravasated area to the whole image area but the treatment with RSV rescued BBB integrity (Fig. 5D). Using FMT, we further showed that the FMT with feces from RSV-treated mice blocked BBB leakage on day 4 after CFA injection (Supplemental Fig. 3).

Fig. 5.

Fig. 5.

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RSV rescues BBB integrity after CFA injection. (A) The BBB integrity in the control mice treated with saline (intra-TMJ) and DMSO (i.p.) was normal and no Evans blue signal was shown in the brainstem sections. (B) CFA injection produced extravasation of Evans blue in the brainstem. (C) RSV treatment (40 mg/kg) completely blocked the CFA-caused BBB leakage. Scale bar, 50 μm. (D) Statistical analysis of percentage of Evans blue extravasated area to the whole image area. ***P < 0.001 as indicated in the figure (n = 3 per group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6. RSV blocks CFA-enhanced microglial activation and expression of TNFα in the Sp5C

To further understand the mechanism underlying the analgesic effect of RSV, we assessed microglial activation and expression of microglial mediator TNFα in the Sp5C on day 4 after intra-TMJ injection of CFA. Using double immunofluorescence staining, we observed that CFA injection markedly increased Iba1-labeled activated microglia and robustly upregulated TNFα expression in the ipsilateral Sp5C (Fig. 6, right column), and that the CFA-upregulated TNFα was primarily expressed in Iba1-positive microglia (Fig. 6, right column). Quantification of the co-expression of TNFα and Iba1 in the Sp5C showed that percentage of TNFα- and Iba1-co-labeled cells to total Iba1-positive microglia in the “CFA + DMSO” group (62%) was significantly increased compared with that in the “Saline + DMSO” group (8%). More importantly, RSV treatment returned the CFA-enhanced microglial activation and TNFα expression to baseline level (Fig. 6, right column). The percentage of the co-labeled cells was markedly decreased in the “CFA + RSV” group (15%). However, both CFA and RSV had no effect on the microglial activation and TNFα expression in the contralateral Sp5C (Fig. 6, left column). To show direct evidence indicating that these effects of RSV are through restoration of disturbed gut microbiome, we examined microglial activation and TNFα expression in the Sp5C after FMT. We observed that the FMT with feces from RSV-treated mice dramatically inhibited microglial activation and TNFα expression in the Sp5C on day 4 after CFA (Supplemental Fig. 4).

Fig. 6.

Fig. 6.

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RSV blocks CFA-enhanced microglial activation and expression of TNFα in the Sp5C on day 4 after CFA injection. Double immunofluorescence staining showed that intra-TMJ injection of CFA markedly increased Iba1-positive microglia and robustly upregulated TNFα expression in the ipsilateral Sp5C (right column). The CFA-upregulated TNFα was co-expressed with Iba1in activated microglia (right column). RSV treatment (40 mg/kg) returned the CFA-enhanced microglial activation and TNFα expression to baseline level (right column). However, both CFA and RSV had no effect on the microglial activation and TNFα expression in the contralateral Sp5C (left column). The immunofluorescence staining experiment was repeated three times to confirm the data shown in this figure. Scale bars, 50 μm for lower magnification and 25 μm for higher magnification.

Next, we analyzed the morphological characteristics of Iba1-labeled activated microglia in the ipsilateral Sp5C. Three parameters (endpoint number, process length, and cell body size) were used to characterize the morphology of activated microglia after different treatments. The fluorescence images in the boxes (Fig. 7A, left side) were magnified and expressed as black-white, skeletonized images (Fig. 7A, right side) for measuring the three parameters. We observed that intra-TMJ injection of CFA significantly decreased endpoints and process length, but dramatically increased cell body size of the activated microglia (Fig. 7B). Strikingly, RSV treatment reversed the CFA-produced morphological changes of microglia in the Sp5C (Fig. 7B).

Fig. 7.

Fig. 7.

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RSV restores CFA-induced morphological changes of microglia in the Sp5C on day 4 after CFA injection. Three parameters (endpoint number, process length, and cell body size) were used to characterize the morphology of activated microglia after different treatments. (A) The fluorescence images in the boxes (left side) were magnified and expressed as black-white images and their skeletonized images (right side) for measuring the three parameters. (B) Note that intra-TMJ injection of CFA significantly decreased endpoints and process length, but dramatically increased cell body size of the activated microglia, and that RSV treatment (40 mg/kg) reversed the CFA-produced morphological changes of microglia in the Sp5C. *P < 0.05, ***P < 0.001 as indicated in this figure (n = 3 per group). Scale bars, 50 μm for lower magnification and 25 μm for higher magnification.

4. Discussion

Accumulating evidence has suggested an important role of gut microbiome in nociceptive response and the pathogenesis of different types of pain (Amaral et al., 2008; Moloney et al., 2016; Tang et al., 2019). Gut microbial dysbiosis is common in people suffering from chronic pain, and restoring gut microbiome composition and its function leads to great amelioration of such pain (Aamodt et al., 2008; Minerbi et al., 2019; Schott et al., 2018). It has been shown that alterations in gut microbiota composition and microbial metabolites are key factors affecting host nociceptive and inflammatory responses mediated by microglia in the central nervous system (CNS) (Erny et al., 2015; Erny et al., 2017; Wang et al., 2018). On the other hand, proinflammatory cytokines, released from activated microglia in trigeminal nociceptive system, are crucial to the development and maintenance of trigeminal pain and TMJ inflammation (Magni et al., 2018). Thus, gut microbiota could regulate microglial activation in the trigeminal nociceptive system and targeting the gut microbiome may provide a promising and effective approach for TMD pain management. In the present study, we observed that gut microbiota disturbance contributes to CFA-induced TMJ inflammation through reducing the production of SCFAs in the gut, impairing BBB integrity and activating microglia in the trigeminal nociceptive system. Interestingly, we found that RSV, a natural bioactive compound, markedly recovers disturbed gut microbiota, restores dysregulated gut SCFAs, BBB permeability and microglia activation, and then alleviates the TMJ inflammation. We noticed that the low dose of RSV (40 Mg/kg) completely reversed SCFA, Bacteroidetes, and Lachnospiraceae reduction, but only slightly attenuated mechanical sensitivity. This discrepancy suggests that gut microbiota disturbance contributes to the CFA-induced TMJ inflammation, but it is not the only mechanism. Previous studies have shown that hyperexcitability of primary afferent neurons (Takeda et al., 2006; Takeda et al., 2005) and activation of glial and immune cells in trigeminal ganglia (Villa et al., 2010) are involved in TMJ inflammation. Thus, different mechanisms including gut microbiota disturbance can be integrated together to underlie the CFA-induced mechanical sensitivity in the TMJ inflammation model. Moreover, our FMT experiment further demonstrates that gut microbiome recovery mediates the analgesic effect of RSV on such pain condition.

SCFAs, including acetic acid, propionic acid and butyric acid, are important mediators for gut bacteria to exert their immune function and inflammatory regulation in both gut and brain (Tedelind et al., 2007; Vinolo et al., 2011). It has been shown that SCFAs inhibit microglia activation in adult mice and control maturation and function of microglia in the CNS (Erny et al., 2015). Fecal SCFAs and SCFA-producing bacteria in human gut microbiome have many positive health effects, such as modulating release of pro-inflammatory mediators via regulating T cells, coelomocytes and neutrophils (Maslowski et al., 2009; Rau et al., 2018; Sun et al., 2018). SCFAs also mediate the regulation of BBB integrity by gut microbiota (Braniste et al., 2014). Previous studies have shown that Bacteroidetes and Lachnospiraceae mainly produce acetic and propionic acids and butyric acid in the gut, respectively (den Besten et al., 2013; Meehan and Beiko, 2014). These gut bacteria have been reported to contribute to pain signaling. For instance, reduction of Bacteroides in vitamin D deficient mice increases spared nerve injury-caused neuropathic pain (Guida et al., 2019). Decreased abundance of Lachnospiraceae is involved in the development of stress-induced visceral hypersensitivity (Zhang et al., 2018). In the present study, we revealed that intra-TMJ injection of CFA reduces the levels of SCFAs and decreases the SCFAs-producing Bacteroides and Lachnospiraceae in the gut. More importantly, we found that RSV treatment counteracts these CFA-produced effects and rescues gut SCFAs and relevant bacteria. Therefore, our results suggest that gut microbiome perturbation is critical for the development of TMJ inflammation and that recovering gut microbiome to normal levels could be a new therapeutic approach for treating such pain.

Currently the mechanism underlying the participation of BBB leakage in the pathogenesis of TMD is unknown. Based on our results in this study, we postulate that CFA-caused BBB leakage may promote peripheral immune cells (such as macrophages) infiltration into brain and the infiltrated immune cells can differentiate to microglia, which can be activated by CFA treatment. The activated microglia will release pro-inflammatory cytokines (such as TNFα) to lead to the development of TMD pain. Further investigation needs to be conducted to demonstrate the role of BBB leakage in TMD.

Microglia activation and neuroinflammation play a critical role in the development and maintenance of inflammatory pain. Gut microbiota remarkably influences microglia function, and alteration in composition and microbial metabolites of gut microbiota plays an important role in microglia-mediated inflammatory response in the CNS (Wang et al., 2018). Our recent study showed that release of TNFα from microglia in the trigeminal nociceptive system is critical for CFA-induced inflammatory TMJ pain in mice (Bai et al., 2019), which is consistent with a previous study indicating that activated microglia in the Sp5C are involved in orofacial hypersensitivity in rats caused by intra-TMJ injection of CFA (Magni et al., 2018). It is well known that morphological features of microglia are closely linked to their function (Crews and Vetreno, 2016; Fernández-Arjona et al., 2017). We observed morphological changes of microglia (microgliosis) after CFA administration. However, it is unclear whether microgliosis directly modulates pain. We also observed that CFA enhances microglial activation and the expression of microglial mediator TNFα in the Sp5C, which contributes to the pathogenesis of chronic pain. Upon activation, resident microglia in the CNS can transform from a resting state to an activated pro-inflammatory phenotype, which is characterized by releasing pro-inflammatory cytokines, such as TNFα, and glial activation in the trigeminal nociceptive system can mediate the development and maintenance of trigeminal pain (Yang et al., 2016). In the present study, we confirmed that intra-TMJ injection of CFA activates microglia and enhances the release of TNFα in the Sp5C. And we further revealed that RSV treatment alleviates CFA-induced TMJ inflammation by inhibiting microglia activation and TNFα production in the Sp5C.

FMT has recently been employed as an effective therapeutic approach for different painful conditions, such as irritable bowel syndrome (Cruz-Aguliar et al., 2018), diabetic neuropathy (Cai et al., 2018), visceral pain (Rea et al., 2017), and opioid-dependent hyperpiesia (Lee et al., 2018). RSV is metabolized by gut microbiome (Bode et al., 2013) and it can affect the composition of gut microbiota (Carrera-Quintanar et al., 2018). It has been reported that FMT from RSV-fed donor mice is sufficient to restore altered gut microbiota in obese mice (Sung et al., 2017). In the present study, we carried out FMT with feces from RSV-treated mice and found that the fecal transplantation returns CFA-caused reduction of SCFAs and SCFA-producing bacteria in the gut to baseline levels, blocks CFA-induced BBB leakage, inhibits CFA-enhanced microglial activation and expression of TNFα in the Sp5C, and significantly diminishes CFA-induced TMJ inflammation pain. These results suggest that gut microbiome recovery is an essential mechanism for RSV-produced pain relief in our model.

5. Conclusions

Our results in this study demonstrated that gut microbiome perturbation is crucial for TMJ inflammation. By restoring disturbed gut microbiome-caused dysregulation of SCFAs in the gut, BBB integrity, and microglia activation and TNFα release in the Sp5C, RSV can alleviate TMJ inflammation. Therefore, targeting gut microbiota could be a promising strategy for developing a new therapy for TMD pain.

Supplementary Material

Supplementary file

Acknowledgments

This work was supported by National Institutes of Health Grants R01 DE022880 (F.T.) and K02 DE023551 (F.T.) as well as Texas A&M University Interdisciplinary Faculty T3 Award (F.T.).

Footnotes

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbi.2020.01.016.

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Supplementary Materials

Supplementary file
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