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Resveratrol Alleviates Temporomandibular Joint Inflammatory Pain by Recovering Disturbed Gut Microbiota
Resveratrol Alleviates Temporomandibular Joint Inflammatory Pain by Recovering Disturbed Gut Microbiota
Resveratrol Alleviates Temporomandibular Joint Inflammatory Pain by Recovering Disturbed Gut Microbiota
Author manuscript
Brain Behav Immun. Author manuscript; available in PMC 2022 September 05.
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cCenter for Craniofacial Research and Diagnosis, Texas A&M University College of Dentistry,
Dallas, TX, USA
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
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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
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Keywords
Inflammatory joint pain; Gut microbiota; Temporomandibular disorders; Short-chain fatty acids;
Microglial activation; Tumor necrosis factor alpha
*
Corresponding author at: 3302 Gaston Ave., Dallas, TX 75246, USA. ftao81@tamu.edu (F. Tao).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbi.2020.01.016.
Ma et al. Page 2
1. Introduction
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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.
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
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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.
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
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Ma et al. Page 3
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.
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.
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.
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
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Ma et al. Page 4
defined as a sharp withdrawal of the head upon stimulation. The head withdrawal threshold
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was calculated as the force at which the positive response occurred in three of five stimuli.
The SYBR Green was used as a fluorescent dye. Relative quantification was analyzed with
the 2−ΔΔCT method.
Brain Behav Immun. Author manuscript; available in PMC 2022 September 05.
Ma et al. Page 5
perfusion system. The whole brains were post-fixed in 4% PFA solution for 4 h, and then
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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).
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
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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.
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
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Ma et al. Page 6
microglia as the cell body size was measured according to previous studies (Adeluyi et al.,
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3. Results
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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
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i.p. injection of RSV robustly recovered the SCFA-producing gut bacteria compared to the
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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
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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).
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).
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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
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Ma et al. Page 8
percentage of TNFα- and Iba1-co-labeled cells to total Iba1-positive microglia in the “CFA
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+ 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).
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).
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
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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
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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
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have shown that hyperexcitability of primary afferent neurons (Takeda et al., 2006; Takeda et
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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
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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
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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.
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closely linked to their function (Crews and Vetreno, 2016; Fernández-Arjona et al., 2017).
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FMT has recently been employed as an effective therapeutic approach for different
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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.
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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
Refer to Web version on PubMed Central for supplementary material.
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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.).
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Fig. 1.
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
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Fig. 2.
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.
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Fig. 3.
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.
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Fig. 4.
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).
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Fig. 5.
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
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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.)
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Fig. 6.
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
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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.
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Fig. 7.
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.
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(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.
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