5-HT3/7 and GABA_B receptors mediate inhibition of trigeminal nociception by dietary supplementation of grape seed extract

Abstract

Background:

Temporomandibular joint disorder is a prevalent orofacial pain condition involving sensitization and activation of trigeminal nociceptive neurons. Dietary supplementation with a proanthocyanin-enriched grape seed extract (GSE) was found to inhibit trigeminal nociception in a chronic TMD model. In this study, the cellular mechanisms by which GSE inhibits sustained trigeminal nociception in male and female Sprague Dawley rats were investigated.

Methods:

Some animals were supplemented with 0.5% GSE dissolved in their water one week prior to neck muscle inflammation induced by injection of complete Freund’s adjuvant into the trapezius. To investigate the mechanism of GSE, some animals were injected intracisternally with antagonists of 5-HT3, 5-HT7, GABAA, or GABAB, receptor prior to jaw opening.

Results:

In males and females, trapezius inflammation prior to jaw opening resulted in sustained mechanical hypersensitivity of trigeminal nociceptors that was significantly inhibited by GSE. Further, GSE beginning 14 days post jaw opening also inhibited trigeminal nociception. Intracisternal injection of antagonists of the 5-HT3/7 and GABAB, but not GABAA receptors reduced the anti-nocifensive effect of GSE in both sexes. Neuronal expression of GABAB protein and mRNA in the spinal cord and trigeminal ganglion were detected.

Conclusions:

The inhibitory effect of GSE is mediated via activation of 5-HT3/7 receptors and GABAB to enhance central descending inhibitory pain pathways and suppress ongoing trigeminal nociception. Further, our findings support the use of GSE as a dietary supplement in the management of pain associated with TMD and other orofacial pain conditions involving central sensitization and dysfunction of descending pain modulation.

Graphical Abstract

Introduction

Temporomandibular disorders (TMD) are prevalent orofacial pain conditions that have a significant impact on the quality of life since they affect the ability to speak and eat [1]. TMD is a chronic disease that affects 5–12% of the adult population and is associated with pain in the temporomandibular joint (TMJ) and muscles associated with mastication [2]. Individuals with TMD commonly experience increased sensitivity to other painful stimuli and TMD pathology is frequently comorbid with migraine and other types of headache, and as such is considered a risk factor [3]. Given the prevalence, significant morbidity, and major social and economic ramifications of TMD, there is a critical need for improved abortive and preventative measures for TMD. The primary reason that TMD patients seek treatment is chronic pain, mediated via activation of trigeminal ganglion nerves that provide sensory innervation to the TMJ and associated muscles and surrounding tissues. In response to injury to the TMJ, trigeminal nociceptive neurons relay information to the trigeminal ganglion and spinal trigeminal nucleus (STN) leading to peripheral and central sensitization of the trigeminal system [4].

TMD has a complex etiology involving multiple psychological and physiological risk factors that promote development of chronic TMD [5]. Commonly reported risk factors include prolonged jaw opening and neck muscle tenderness. In a preclinical model of TMD, prolonged jaw opening, which can occur during yawning, molar extractions, root canal procedures, or orthodontic treatment, was shown to cause a prolonged increase in trigeminal sensitivity to mechanical stimulation that resolved 14 days post jaw opening in male animals [6]. More recently, combining the risk factors of neck muscle inflammation prior to prolonged jaw opening resulted in a sustained level of trigeminal nociception that persisted longer in females than males [7]. Chronic pain associated with TMD can persist for years and current treatments are inadequate since they provide limited pain relief, function effectively in only a portion of patients, and are frequently associated with adverse side effects [8]. However, the use of nutritional supplements may offer a safe and effective method for preventing and treating chronic neurological and inflammatory diseases. In support of this notion, heightened trigeminal sensitivity to mechanical stimulation was significantly inhibited by daily dietary inclusion of a 0.5% proanthocyanin-enriched grape seed extract (GSE) in the drinking water prior to neck muscle inflammation [7]. However, the mechanism by which GSE suppresses the development of a sensitized trigeminal system is not well understood.

Similar to other chronic pain conditions, a decrease in descending inhibitory pain modulation is implicated in the pathology of chronic TMD by promoting a persistent state of central sensitization [9]. This regulatory pathway, which is stimulated in response to opioids and endorphins, involves activation of serotonin (5-HT) and gamma aminobutyric acid (GABA) receptors [10]. Activation of 5-HT3 and 5-HT7 receptors expressed on inhibitory interneurons leads to release of the inhibitory neurotransmitter GABA within the medullary horn. Stimulation of GABA_A and GABA_B receptors present on second-order neurons inhibits ascending pain signaling to the thalamus while activation of GABA receptors expressed on processes from primary trigeminal afferents projecting into the STN inhibits release of excitatory neurotransmitters and neuropeptides. The goal of this study was to test the hypothesis that the anti-nociceptive effect of GSE shown in a preclinical TMD model involves activation of serotonergic and GABAergic receptors to restore and/or enhance function of the descending inhibitory pain modulation pathway.

Materials and methods

Animals

Adult Sprague Dawley male and female rats (200–300 g) were purchased from a central management breeding colony (Springfield, MO) and allowed to acclimate for 1 week to facility conditions prior to use. Animals were housed individually in clean, standard plastic rat cages with unrestricted access to food and water with 12 h/ light dark cycles. All protocols were approved by Missouri State University’s Institutional Animal Care and Use Committee and conducted in compliance with all established guidelines in the Animal Welfare Act, National Institutes of Health, and ARRIVE Guidelines. Concerted efforts were made to minimize suffering, as well as the number of animals used in this study. A power of analysis was conducted using G*Power, with a power of 0.8 and an effect size of 0.25, and a minimum sample size of 7 animals per group was suggested to detect significant differences between groups.

Sensitization and activation of trigeminal nociceptive neurons

The experimental design for the chronic TMD model was based on a prior study [7] involving muscle sensitization in conjunction with prolonged jaw opening (Figure 1). Animals were anesthetized with 3% isoflurane in oxygen and received a total of 100 μL of complete Freund’s adjuvant (CFA, Sigma-Aldrich; 1:1 in 0.9% sterile saline) administered as 5 injections of 10 μL each per side into the upper trapezius muscles. Animals recovered in their cages and were monitored for normal behaviors for a total of 8 days prior to prolonged jaw opening, which was performed as described previously [6]. Briefly, a mouth retractor was positioned around the bottom and top incisors and the retractor arms separated until near maximal opening was achieved without subluxation and then maintained for 20 min under 2% isoflurane.

Figure 1.

Timeline outlining experimental paradigm. Animals received daily GSE supplementation starting on day 0 for one week prior to muscle injections on day 7. Jaw opening was performed 8 days following muscle injections (D15). Inhibitors were administered immediately prior to jaw opening. Behavioral von Frey assessments were conducted on day 0, 7, and 15 prior to jaw opening and 2 h,1 d (D16) and 7 days (D22) after jaw opening.

Nociceptive testing

Mechanical nocifensive thresholds were determined in response to a series of calibrated von Frey filaments (26, 60, 100, 180 g) as previously described [7]. Nocifensive withdrawal reactions were verified by two scientists blinded to experimental conditions. Filaments were applied 5 times over each masseter of each animal and averaged to obtain the mean number of reactions out of 5 tests. Data from the 100 g filament were chosen for analysis, since testing with that filament produced average nocifensive responses below 1 reaction out of 5 tests in naïve animals, but robust responses in sensitized animals. Sensitivity levels were established basally and at times designated by asterisks in the timelines (see Figures 1 and 5).

Figure 5.

Sustained trigeminal nociception in sensitized animals was significantly suppressed in response to daily supplementation with GSE initiated 14 days after prolonged jaw opening. The experimental timeline is shown in panel A. Behavioral von Frey assessments were conducted on days indicated by an asterisk. The average nocifensive head withdrawal responses ± SEM of 5 applications to each side are reported for male animals (panel B). Animals were subjected to prolonged jaw opening 8 days after injection of CFA into upper trapezius (M + J, D8). Some animals received daily GSE in lieu of water starting at 14 days post prolonged jaw opening (M + J+GSE, D22) and supplementation was continued for the remainder of the study (D36). * = P < 0.05 when compared to Naïve nociceptive levels at that timepoint. # = P < 0.05 compared to M + J levels.

Dietary supplementation of GSE

Following basal nociceptive testing, some animals had their drinking water replaced with a 0.5% solid solution of MegaNatural^®-BP GSE (Healthy Origins, Pittsburgh, PA) dissolved in water [7]. Animals consumed approximately 800 ml/kg/week of liquid, around 4000 mg GSE per kg rat weight per week, or approximately 570 mg GSE/kg/day. Some animals received GSE starting 1 week prior to induction of neck muscle inflammation, while other animals received daily GSE starting 14 days after jaw opening (study day 22) and continuing for the duration of the study. All other groups consumed tap water throughout the study. No significant differences in body weight, food or liquid consumption were observed between groups at any timepoint.

Intracisternal injection of 5-HT and GABA receptor inhibitors

Animals were lightly anesthetized using 3% isoflurane prior to intracisternal injections of antagonists to 5-HT3, 5-HT7, GABA_A, and GABA_B, receptors (purchased from Tocris) [11]. Ondansetron hydrochloride (5-HT3 inhibitor) and SB 269970 (5-HT7 inhibitor) were dissolved in 0.9% sterile saline to a final concentration of 100 nM. In addition, a mixture of 100 nM ondansetron and 100 nM SB 269970 was prepared in sterile saline. Bicuculline (GABA_A inhibitor) was dissolved in DMSO, then diluted to 20 μM in sterile 0.9% saline, while saclofen (GABA_B inhibitor) was prepared as a 1.5 mg/mL (6 mM) solution in 0.9% sterile saline. Some animals received sterile 0.9% saline alone as a vehicle control. All treatments were administered via injection of 20 μl between the occipital bone and C1 vertebrae immediately prior to jaw opening. Following injections and jaw opening, animals recovered in their cages and were monitored for normal motor function, prior to being tested for nociception 2 h later.

Immunohistochemistry and qPCR

Upper spinal cord tissue containing the STN and the entire trigeminal ganglion were removed from naïve animals and animals that had received GSE supplementation for one week. Briefly, animals were euthanized via CO₂ asphyxiation and decapitation, and tissues obtained through cranial dissection. Tissues designated for immunohistochemical studies were placed in 4% paraformaldehyde overnight at 4°C, and tissues for PCR analysis were snap frozen immediately in liquid nitrogen.

Following sucrose fixation, samples were mounted in Tissue-Tek ^® Optimal Cutting Temperature mounting media (Sakura^® Finetek). Fourteen μm cross sections of the upper spinal cord and longitudinal sections of trigeminal ganglion were attached to Superfrost ^® Plus Microscope slides (Thermo Fischer Scientific) and stored at −20°C.

Immunostaining procedures and analysis were performed essentially as described in prior studies [12]. Slides containing one tissue section from each experimental condition were incubated for 20 min in PBS containing 5% normal donkey serum and 0.1% Triton. A monoclonal antibody against GABA_B Receptor 1 (ab55051, Abcam) was diluted 1:500 in 5% donkey serum in PBS and incubated for 3 h at room temperature. Alexa Fluor 647 donkey anti-mouse secondary antibody (Jackson Laboratories; 1:200 dilution in PBS) was then incubated with tissues for 1 h at room temperature. Tissues were mounted in DAPI-containing Vectashield medium (Vector Laboratories) and a single 3 × 3 tiled 200X image of the dorsal medullary horn region and a 2 × 2 tiled 200X image of the V3 region of the ganglion was captured on a Zeiss fluorescent microscope. Zen 2 software (Carl Zeiss) was utilized to balance the background of each image, followed by densitometric analysis of gray scale images using ImageJ software. For spinal cord tissues, pixel densities in 10 non-overlapping, rectangular regions of interest (ROI) encompassing laminas I-III were measured and averaged. Additionally, background measurements acquired from acellular areas were averaged and subtracted from the ROI values. Relative average means of fluorescent intensities were determined for each condition and data reported as average fold change ± SEM relative to the average mean for naive animals, which was set equal to one.

For trigeminal ganglia, pixel densities of GABA_B receptor staining were measured for the whole image and normalized to the intensity of nuclear staining with DAPI. Relative average means were determined for each condition and data reported as average fold change ± SEM relative to the average mean for naive animals, which was set equal to one.

For qPCR analysis, spinal cord tissue and trigeminal ganglia collected from naïve animals or animals supplemented for one week with GSE were snap frozen in liquid nitrogen and stored at −80 C. Total RNA was isolated using TRIzol™ (Invitrogen), following the manufacturer’s protocol, resuspended in molecular biology grade water, and the concentration determined using a Nanodrop™ 2000c Spectrophotometer. Approximately 400 ng of RNA per sample was reverse transcribed to cDNA in a 20 μl reaction using a SuperScript IV VILO kit, according to the manufacturer’s protocol (Invitrogen). To detect changes in gene expression, qPCR was performed using a StepOne Plus Real-Time PCR System, TaqMan™ Fast Advanced Master Mix, and TaqMan™ Gene Expression Assay primers (Applied Biosystems™, assay ID’s Rn01775763_g1 (Gapdh), and Rn00578911_m1 (Gabbr1)) as recommended by the manufacturer. Results are presented as average cycle number and as ΔCt normalized to the level of glyceraldehyde dehydrogenase (GAPDH) gene.

Statistical analysis

All data were initially evaluated for normality using a Shapiro–Wilk test. Behavioral data were found to be non-normal (P < 0.05), so non-parametric statistical tests were applied. To determine if nociception was different across all groups, a Kruskal Wallis test was performed. Upon reaching a significant result, a Mann–Whitney U test with a Wilcoxon’s W post-hoc test was performed to determine if there were pairwise differences in nociception between groups at each evaluated time point. Immunohistochemical and qPCR data were normally distributed, and differences between naïve tissues and tissues from animals receiving GSE were compared using an Independent Samples T-Test. Statistical analysis was performed using SPSS Statistical Software 24 (IBM), and changes were considered significant if P < 0.05.

Results

Pretreatment with GSE and neck muscle inflammation do not alter basal trigeminal nociception

Initially, the effect of GSE pretreatment and neck muscle inflammation on the level of trigeminal nociception in response to mechanical stimulation was determined. The average number of nocifensive head withdrawals was less than one response out of 5 von Frey applications at the basal time point for all experimental conditions in both males (Figure 2(A)) and females (Figure 2(B)). There was no significant change in the nocifensive response at the pre-muscle inflammation timepoint in naive animals, in animals to be injected with CFA in the trapezius (M + J) or in animals receiving GSE (GSE + M+J) when compared to basal levels. Similarly, no significant changes in nociception were observed in any of the experimental groups 8 days post neck muscle injection (on study day 15, immediately before jaw opening) in either males or females. These results provide evidence that daily supplementation of GSE or neck muscle inflammation mediated by CFA does not cause a change in the basal level of trigeminal sensitivity.

Figure 2.

GSE supplementation inhibits trigeminal nociception mediated by prolonged jaw opening in sensitized animals. The average nocifensive head withdrawal responses ± SEM of 5 applications to each side to mechanical stimuli are reported for male (A) and female animals (B). Animals were subjected to prolonged jaw opening 8 days after injection of CFA into upper trapezius (M + J, D15). Some animals received daily GSE in lieu of water for 1 week prior to trapezius injections (D7) and supplementation was continued for remainder of study (M + J+GSE, D22). * = P < 0.05 when compared to Naïve nociceptive levels at that timepoint. # = P < 0.05 compared to M + J levels.

Dietary GSE supplementation inhibits trigeminal nociception mediated by neck muscle inflammation and prolonged jaw opening

The average number of nocifensive responses was significantly elevated over naïve levels 2 h after jaw opening in sensitized animals that had received neck muscle injections (M + J) in both males (P < 0.001, Figure 2(A)) and females (P < 0.001, Figure 2(B)). This nocifensive response was ameliorated by dietary GSE supplementation prior to jaw opening. The level of trigeminal sensitivity to mechanical stimulation was significantly repressed by GSE to near basal levels at two hours, 1 d, and seven days post jaw opening in males (P < 0.01, for each time) and also in females (P < 0.001, P <0.01, P < 0.01, respectively).

Intracisternal injection of 5-HT antagonists blocks inhibitory effect of GSE

To investigate whether GSE’s anti-nociception effect involves the descending serotonergic pathway, antagonists to 5-HT3 (ondansetron), 5-HT7 (SB-269970), or a mixture of both 5-HT antagonists were administered intrathecally just prior to jaw opening (Figure 3). Administration of ondansetron into the upper spinal cord did not inhibit GSE’s analgesic effects in males (P = 0.281) or females (P = 0.440, data not shown). Similarly, administration of SB-269970 did not have a significant effect on GSE’s inhibitory effect on trigeminal nociception in males (P = 0.587) or females (P = 0.562, data not shown). However, co-injection of both 5-HT receptor antagonists (GSE + M+J + MIX) prior to jaw opening inhibited the anti-nociceptive effect of GSE at 2 h post-jaw opening in females (P < 0.001), which also trended towards, but did not reach, statistical significance in males (P = 0.059) when compared to animals injected intrathecally with the vehicle control saline (GSE + M+J + SAL). This effect was no longer significant in females by one day post-jaw opening (P = 0.173), but now was significant in males at that time (P < 0.001). No significant effects were observed with the antagonist mixture’s administration in males (P = 0.888) or females (P = 1.00) 7 days post jaw opening (study day 22). To demonstrate that the effect of the antagonist mixture was mediated by an inhibition of GSE, rather than activation caused by injection of the antagonists, some sensitized animals receiving dietary supplementation with GSE were injected with the 5-HT3/7 antagonist mixture in the absence of jaw-opening. In those animals, no elevation in nociception levels was observed over naïve levels 2 h later (data not shown).

Figure 3.

The anti-nociceptive effect of GSE involves 5-HT3 and 5-HT7 receptors. The average nocifensive head withdrawal response is reported. Some animals were injected with complete Freund’s adjuvant (CFA) 8 days prior to prolonged jaw opening (M + J, D15) and nociceptive responses determined at 2 h (15 + 2), and day 1 (D16) and day 7 (D22) post jaw opening. Additionally, some animals received GSE beginning one week prior to receiving CFA injections. The GSE + M+J animals received intrathecal injection of saline (SAL) or a mixture of the antagonists ondansetron and SB 269970 (MIX) and nociception measured at the same timepoints. * = P < 0.05 when compared to Naive levels for that condition, # = P < 0.05 compared to M + J, while + = P < 0.05 compared to GSE + M+J + SAL.

Central administration of GABAB antagonist suppresses GSE inhibition of trigeminal sensitization

To elucidate whether GSE might be acting through alteration of GABAergic pathways centrally, animals received antagonists against GABA_A (bicuculline) and GABA_B (saclofen). Blocking GABA_A signaling via bicuculline injection (GSE + M+J + BIC) did not affect the ability of GSE to reduce nociceptive responses in male (P = 0.587) or female rats (P = 0.573) at two hours post jaw opening (Figure 4). However, inhibition of GABA_B activity with saclofen (GSE + M+J + SAC) was sufficient to significantly negate GSE’s efficacy in reduction of nociceptive responses two hours after jaw opening in males (P < 0.05) and females (P < 0.001) when compared to animals injected with saline (GSE + M+J + SAL). Although still inhibitory, these effects were no longer statistically significant from GSE + M +J + SAL the following day in male (P = 0.074) or female (P = 0.408) animals. To ensure that saclofen was not causing trigeminal activation itself, some animals that received GSE supplementation and trapezius injection of CFA to promote sensitization were injected with saclofen but did not receive jaw opening. These animals showed no increase in trigeminal nociception to mechanical stimulation (data not shown).

Figure 4.

GSE inhibition of trigeminal nociception involves primarily GABA_B receptors. The average nocifensive head withdrawal response is reported. Some animals were injected with complete Freund’s adjuvant 8 days prior to prolonged jaw opening (M + J, D15) and nociceptive responses were determined at 2 h (15 + 2), and day 1 (D16) and day 7 (D22) post jaw opening. Additionally, some animals received GSE beginning one week prior to receiving CFA injections. Some of the GSE + M+J animals were injected intrathecally with saline (SAL), bicuculline (BIC), or saclofen (SAC) just prior to jaw opening and nociception measured. * = P < 0.05 when compared to Naive levels for each condition, # = P < 0.05 compared to M + J, while + = P < 0.05 compared to GSE + M +J + SAL.

Daily GSE supplementation post jaw opening significantly inhibits sustained trigeminal nociception

Daily supplementation of GSE 14 days after prolonged jaw opening significantly suppressed the average number of nocifensive responses to naive levels in latent sensitized male animals on day 29, which corresponds to 7 days of dietary GSE (P < 0.001, Figure 5). The level of nociception remained near naive levels in GSE-supplemented animals 14 days post GSE inclusion (day 36) and was no longer significantly different from sensitized animals subjected to jaw opening (M + J).

GABA_B expression in upper spinal cord and trigeminal ganglion

To determine whether supplementation with GSE altered the mRNA expression of the GABA_B receptor (Gabrr1), qPCR was performed on RNA isolated from tissues of the upper spinal cord containing the STN and from trigeminal ganglia. The mRNA expression of Gabbr1 in both spinal cord and ganglion, as measured by the average C_t value normalized to the level of GAPDH, was similar between naive animals and animals that received daily GSE supplementation for one week (Table 1). Similarly, GSE did not significantly change the average intensity of GABA_B1 receptor immunostaining or alter the cellular distribution of GABA_B1 receptors within the spinal cord (Figure 6) or trigeminal ganglia (Figure 7). GABA_B1 expression was observed in the outer lamina of the medullary horn in neuronal and glial cells in naive animals in a pattern similar to animals supplemented with GSE. Within the trigeminal ganglion, GABA_B1 receptors were primarily expressed in neuronal cells within the V3 region of the trigeminal ganglion and no difference in staining intensity was observed between naive and GSE treated animals.

Table 1.

GSE does not alter GABA_B subunit 1 receptor mRNA expression in upper spinal cord tissues or trigeminal ganglion compared to levels in naive, untreated animals.

Tissue	Naïve		GSE-Treated
Gene	Avg. Ct value	ΔCt	Avg. Ct value	ΔCt
Spinal Cord
Gabbr1	22.4 ± 0.23	4.63	22.5 ± 0.16	4.61
Spinal Cord
Gapdh	17.8 ± 0.44	N/A	17.8 ± 0.09	N/A
Ganglion
Gabbr1	22.4 ± 1.07	4.37	23.3 ± 2.47	4.75
Ganglion
Gapdh	18.0 ± 0.56	N/A	18.52 ± 1.36	N/A

Figure 6.

GSE does not change the expression of GABA_B receptors in the medullary horn. Tissues from the upper spinal cored obtained from Naive animals and animals that received GSE for 1 week were costained with the nuclear dye DAPI and an antibody to the GABA_B1 receptor (merged). Representative images from Naive (left panels) and GSE (right panels) animals are shown. GABA_B1 staining was readily detected in both neurons and glia in the outer lamina of the medullary horn. Scale bar = 50 μm. The average intensity of GABA_B1 immunostaining was not significantly different between Naive and GSE-treated animals.

Figure 7.

The expression of GABA_B receptors in the trigeminal ganglion is not changed by GSE supplementation. Representative images from the V3 region of the trigeminal ganglia obtained from Naive (upper panels) and GSE (lower panels) animals were costained with the nuclear dye DAPI and an antibody to the GABA_B1 receptor (merged). GABA_B staining was detected primarily in the neuronal cell body. Scale bar = 50 μm. The average intensity of GABA_B immunostaining in the ganglion was similar in Naive and GSE-treated animals.

Discussion

A major finding from our study is that daily dietary supplementation with GSE inhibited trigeminal nociception in a preclinical model of chronic TMD via a mechanism involving activation of the serotonergic and GABAergic descending pain modulation pathway. The GSE used in this study and our prior studies [7, 12] was MegaNatural^®-BP GSE, a highly purified extract from Vitis vinifera seeds containing >90% polyphenols as gallic acid equivalents. The findings from this study are in agreement with our prior studies in which dietary inclusion of 0.5% GSE in the drinking water was sufficient to inhibit trigeminal nociception [7] and mediate changes in the expression of proteins implicated in peripheral and central sensitization [12]. In this study inclusion of GSE in the diet for one week prior to muscle injections or beginning 14 days post jaw opening significantly inhibited trigeminal nociception, which supports the notion that GSE could function as a preventative or abortive TMD treatment.

TMD is a common orofacial pain condition that is more prevalent in women than men and is associated with pain in the temporomandibular joint and muscles of mastication [13]. In our model of TMD, exposure of animals to the two reported TMD risks factors of neck muscle inflammation and prolonged jaw opening [14] promotes development of a sensitized trigeminal system characterized by enhanced nociception in response to mechanical stimulation in both males and females. Currently, there are no specific FDA approved treatments for TMD and there are few non-pharmacological therapeutic options for managing TMD pain. Results from our studies provide evidence that dietary supplementation with GSE may lower the risk of developing chronic TMD in sensitized individuals caused by prolonged jaw opening, which can occur during routine dental or orthodontic procedures [5]. Although a gender difference was not seen in this study, this finding was likely due to the shorter duration (7 days post jaw opening) since in a prior study using this model, females exhibited elevated nociceptive levels 28 days post jaw opening while males were no longer significantly elevated [7].

Chronic TMD pathology is thought to involve sustained peripheral and central sensitization of trigeminal nociceptive neurons and dysfunction of the descending inhibitory pain modulation pathway [4]. Findings from our study support the notion that the anti-nociceptive effect of GSE is mediated at least in part via stimulation of the serotonergic pathway, which is known to be positively modulated by endorphins and opioids to suppress pain signaling [15]. Co-administration of ondansetron and SB 269970, antagonists to the 5-HT3 and 5-HT7 receptors respectively, just prior to jaw opening in sensitized animals suppressed the inhibitory effect of GSE on trigeminal nociception in male and female animals. This finding is in agreement with results from our study that demonstrated the inhibitory effect of non-invasive vagus nerve stimulation on trigeminal nociception in a model of episodic migraine involved stimulation of 5-HT3 and 5-HT7 receptors [11]. Activation of these receptors on inhibitory interneurons causes the release of the inhibitory neurotransmitters GABA and glycine to decrease signaling of the ascending pain pathway via excitation of primary and secondary trigeminal nociceptive neurons. Dietary GSE supplementation was previously shown to increase the basal expression of the glutamate transporter GLAST in astrocytes in the STN [12]. GLAST functions to remove the excitatory neurotransmitter glutamate from the extracellular space and therefore plays an important role in modulating the excitability state of nociceptive neurons. In the same study, GSE was found to decrease the basal level of the neuropeptide calcitonin gene-related peptide (CGRP), which is implicated in TMD pathology and known to mediate peripheral and central sensitization by modulating glutamate receptors [16]. Taken together, these findings provide evidence that the modulatory effect of GSE on trigeminal nociception involves enhancement of the serotonergic descending inhibitory pain pathway and suppression of glutaminergic excitatory signaling pathways.

In our model of chronic TMD, the inhibitory effect of GSE was found to be mediated primarily via activation of GABA_B receptors since intrathecal injection of the GABA_B antagonist saclofen suppressed GSE-mediated inhibition of trigeminal nociception. Interestingly, administration of the GABA_A antagonist bicuculline prior to stimulation of trigeminal nociception by jaw opening did not significantly reduce the effect of GSE. This finding differs from the results observed in response to non-invasive vagus nerve stimulation (nVNS) in which central administration of bicuculline prevented trigeminal nociception in a model of episodic migraine, thus implicating activation of GABA_A receptors [11]. While activation of GABA_A receptors is known to involve opening of chloride channels to cause hyperpolarization, activation of GABA_B receptors is coupled to the G_i/G_o pathway and modulation of potassium and calcium ion channels to repress neuronal excitability and inhibit secretion [17]. Thus, it appears that GSE functions similarly to nVNS to inhibit trigeminal nociception via activation of 5-HT3 and 5-HT7 receptors, but the treatments mediate their downstream effects via stimulation of different GABA receptors. Based on the efficacy and safety results from multiple clinical trials, nVNS has been cleared by the FDA as a therapeutic option for the treatment and prevention of cluster headache and migraine [18–20]. Since GSE functions via a similar mechanism as nVNS, it is likely that daily GSE supplementation would be beneficial not only as a neuroprotective therapeutic for reducing the risk of chronic TMD but also for cluster headache and migraine.

To determine if the inhibitory effect of GSE might involve modulation of GABA_B receptor expression in the spinal cord or trigeminal ganglion, mRNA levels and protein levels were compared between naive animals and animals receiving daily GSE for one week. Results from qPCR analysis for the GABA_B subunit 1 (GABAB1) receptor demonstrated that GSE supplementation did not cause a significant change in the mRNA for this subunit in upper spinal cord tissue containing the STN or trigeminal ganglion. GABAB receptors are heteromers of the two subunits GABAB1 and GABAB2, which are both required for G-protein signaling since GABAB1 is responsible for binding GABA while GABAB2 couples to G_i protein activation [21]. GABAB1 immunoreactivity was readily detectable in neurons and glial cells in laminas I-III in the medullary horn and was primarily localized to the cell bodies of neuronal cells in the trigeminal ganglion. In agreement with the mRNA results, no significant change in the average immunostaining intensity or pattern for GABAB1 was observed in the spinal cord tissue or V3 region of the trigeminal ganglion. Thus, it does not appear that GSE is mediating its primary effect by modulating the level or pattern of GABA_B receptor expression. We can only speculate that GSE may be functioning as a positive allosteric modulator of the GABA_B receptor to enhance its ability to decrease neuronal excitability [22]. The demonstration that the anti-nociceptive effect of GSE involves activation of GABA_B receptors provides the first evidence to our knowledge of a nutritional supplement enhancing the activity of the GABAergic receptor known to be the target of the allosteric modulator drug baclofen [23]. The anti-nociceptive effects of baclofen, which clinically has been used to treat pain conditions including TMD [24], are likely mediated via inhibition of adenylate cyclase activity, activation of the G-protein gated inward rectifying potassium channel (GIRK), and/or voltage-gated calcium channels [25]. However, the utilization of baclofen systemically at higher doses is limited due to its negative effects including mental confusion and drowsiness [23]. Based on our findings, GSE supplementation may be beneficial alone or used as an adjunctive therapy with lower doses of baclofen to treat chronic pain conditions such as TMD by altering the basal excitability state of trigeminal nociceptive neurons without the negative side effects.

Conclusions

In summary, daily supplementation of 0.5% GSE inhibited trigeminal nociception in a preclinical model of chronic TMD via activation of 5-HT3, 5-HT7, and GABA_B receptors (Figure 8). Thus, GSE may offer a safe and effective alternative or adjunctive therapeutic to opioids and baclofen for reducing the risk of chronic TMD, and likely other chronic orofacial pain conditions whose pathology involves persistent central sensitization and dysfunction of the descending inhibitory pain modulation pathway. Since dietary GSE did not cause a change in the expression of GABA_B receptors in the medullary horn or trigeminal ganglion, we speculate that the inhibitory effect of GSE may be mediated in part by allosteric modulation of GABA_B receptors to suppress trigeminal pain signaling. Future studies will be directed at understanding the cellular mechanisms by which GSE-mediated activation of GABA_B receptors functions to inhibit sensitization and nociception of the trigeminal pathway.

Figure 8.

Schematic representation of GSE suppression of trigeminal nociception in a preclinical model of chronic TMD. Briefly, neck muscle inflammation mediated latent sensitization of trigeminal neurons such that prolonged jaw opening promoted a state of prolonged mechanical nociception. Daily supplementation with GSE in the animals drinking water either prior to or 14 days post jaw opening suppressed trigeminal nociception. The inhibitory effect of GSE involved stimulation of 5-HT3/7 and GABA_B receptors. Based on our findings, GSE appears to function in a neuroprotective capacity via enhancing the descending inhibitory pain modulation pathway.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.