This study demonstrated that ES induced mechanical sensitization in the masseter and temporalis muscles. This sensitization was evidenced by reduced response thresholds to the electronic von Frey test and a concomitant increase in c-Fos expression within the TG and the Sp5C, indicating activation of the trigeminal pain pathway. Furthermore, animals subjected to ES exhibited upregulated expression of both cannabinoid receptors CB₁ and CB₂ throughout this neural pathway. The functional relevance of this upregulation was confirmed pharmacologically, as the administration of CB₁/CB₂ receptor agonists and antagonists significantly modulated the stress-induced nociceptive behavioral responses.

The communication box is an experimental model used to investigate stress induced by auditory, olfactory, and visual exposure to other animals undergoing distress (14,16). This model has been shown to be effective to elicit systemic stress responses, such as increased plasma corticosterone and adrenocorticotropic hormone (ACTH) levels (18,19) and behavior changes in the elevated plus maze apparatus (16). In our study, the ES group animals presented stress signals confirmed by the plus-maze test, as evidenced by a reduced number of entries into the open arms.

Another parameter used to assess stress was the evaluation of the adrenal glands. Stressful situations activate the HPA axis, leading to stimulation of the zona fasciculata and production of glucocorticoid (20). Results showed an increased number of cytoplasmic vacuoles in the zona fasciculata of the adrenal gland cortex in the ES group. These vacuoles may be related to changes in lipid enzymatic activity in the adrenal glands associated with an increased synthesis of corticosterone, a hormone related to stress in rodents (20,21). Interestingly, body weight gain and adrenal gland gross weight were not significantly different between Control and ES groups, which can be related to a short ES induction time.

The model by Rosales et al. (14), which was used in this study, evaluates the impact of ES using the communication box to promote hyperactivation in the masticatory muscles, suggesting a bruxism-like effect. In addition, another study (16) demonstrated changes in the masticatory muscle nociceptive threshold. Accordingly, this work focused on the orofacial region, and nociceptive thresholds in other regions of the body were not assessed. This represents a limitation of this study, as it cannot be determined whether this model of ES selectively affects the masticatory muscles or also influences nociception in other regions.

Stressed animals developed changes in the mechanical threshold of masticatory muscles, showing nociceptive behavior after 8 experimental days and lasting until the last day of the experimental protocol, with a peak in D11. The study of Huang et al. (16) concluded that stressed animals showed a decrease in the masticatory muscle nociceptive threshold, but with a peak on D7, a difference that may be associated with the animal strain used. The authors used Sprague-Dawley rats, which are more sensitive to mechanical stimuli than Wistar rats and other species. The authors also showed that mechanical sensitivity in the masticatory muscles is associated with stress exposure, since the mechanical threshold returns to baseline 14 days after the end of the experimental stress protocol.

The results shown here suggest that emotional stress activates the trigeminal nociceptive pathway, which is corroborated by the increase in c-Fos immunostaining observed in the TG and Sp5C nucleus. The TG contains the cell bodies of primary sensory afferent neurons, while the Sp5C area within the medulla oblongata constitutes the principal site of synaptic relay between afferent neurons and second-order central neurons of the trigeminal nociceptive pathway. This circuitry mediates orofacial pain transmission arising from masticatory muscles, particularly the temporalis and masseter. Other preclinical studies have shown that psychological stress induces masticatory muscle mechanical sensitivity in animal models (16,22); however, these studies did not assess c-Fos labeling in the trigeminal nociceptive pathway. The increased expression of c-Fos, a well-established marker of neuronal activation (23), in these regions corroborates the neuronal activation in the trigeminal pathway (24).

In the present study, the pain in the masticatory muscles that developed from the 8th experimental day onward was exclusively induced by emotional stress. Notably, no hyperalgesic or phlogistic substances were injected into the masticatory muscles. Thus, this model intends to simulate alterations in the brain regions involved in pain processing, stimulating a central sensitization process that is directly related to the pathophysiology of nociplastic pain. Chronic stress is closely related to this type of pain condition (25).

In the same manner, the origin of symptoms in patients with painful TMD may not always be attributed to local anatomical alterations. Psychosocial factors must also be considered, as they are directly related to the neurobiology of pain. Supporting this view, painful TMD are classified as a form of nociplastic pain, as described by Svensson (1) and consistent with the altered nociception criteria proposed by Kosek et al. (2), where alterations in pain processing, such as central sensitization, play a pivotal role in the underlying mechanism.

Extending this perspective to the epidemiological context, TMD has recently been reported to have an incidence of 34% in the world population, and the majority of affected people have pain related to masticatory muscles (OMP), which is considered the main type of non-odontogenic orofacial pain (4,26). In this clinical condition, stress can be a predicting factor. Furthermore, individuals with OMP have high levels of anxiety, depression, and catastrophization, which are factors closely related to stress. Psychological or emotional stress is clearly implicated in the etiology of OMP (27), initially functioning as a systemic adaptive response, but leading to the maintenance of pain in the long term. Additionally, stress increases sympathetic activity and contributes to changes in pain threshold, influencing the descending pain modulatory systems (28).

TMD in women is possibly influenced by sex hormones and biopsychosocial factors (4,26). Sex-related neurobiological differences are known to influence stress-induced analgesia and pain responses, but these differences were not studied in the present work which, to the best of the authors' knowledge, is the first to investigate the relationship between emotional stress, masticatory muscle pain, and the endocannabinoid system in rats. To ensure greater control of experimental variables and improved standardization of the evaluated groups, the present study was conducted using only male animals. This methodological choice represents a limitation of the study and highlights the importance of including females in future investigations.

The descending pain modulatory system operates through several mechanisms, including the opioid and endocannabinoid systems. The ECS comprises endogenous ligands (anandamide and 2-AG), cannabinoid receptors, and enzymes responsible for their synthesis and degradation (9). In this sense, cannabinoid receptors CB₁ and CB₂ are pivotal in the regulation of physiological pain, exerting their effects through the modulation of neuronal glutamate release and interactions with microglial cells, thereby maintaining a fine balance between excitatory and inhibitory neurotransmission (9).

Emerging evidence has proposed that nociplastic pain may be underpinned by an ECS deficiency, a framework that could account for the increased expression of cannabinoid receptors observed in our study within the TG and the Sp5C, both critical nodes for the activation and transmission of nociceptive signal in the masseter and temporalis muscles (29). Such upregulation may constitute a homeostatic response to persistent emotional stress, a well-recognized factor contributing to pain chronification. Additionally, it is known that nociplastic pain, in which central sensitization represents a key feature, involves microglial activation within the context of neuroinflammation, as well as glutamatergic changes among the mechanisms studied. The ECS may influence the development of these mechanisms, highlighting the need for future investigations that differentiate central and peripheral mechanisms (9).

Another example of chronic pain mediated by the trigeminal pathway is chronic migraine, a neurovascular disorder arising from abnormal sensory processing and involving both central and peripheral sensitization mechanisms. Recent studies have suggested that the ECS may represent a promising therapeutic avenue to mitigate the physiological and inflammatory components underlying migraine-related pain (30). Nevertheless, in painful TMD, these therapeutic benefits remain largely unsubstantiated, underscoring the relevance of our findings as an initial step toward elucidating the compensatory upregulation of CB₁ and CB₂ receptors and their modulatory role in orofacial pain. Moreover, the involvement of the ECS in stress processing may further reinforce its contribution to the maintenance of maladaptive mechanisms associated with the transition from acute to chronic pain states (29).

Our study focused on CB₁ and CB₂ receptor modulation, with the absence of direct biochemical assessment of anandamide and 2-AG representing a limitation. Future studies may incorporate endocannabinoid quantification to further elucidate system dynamics in stress-induced orofacial pain, which would help to determine whether the observed upregulation of CB₁ and CB₂ receptors reflects a compensatory response or an endocannabinoid system deficiency.

The data showing the upregulation of CB₁ and CB₂ receptors in trigeminal pain-processing areas, prompted us to investigate whether these receptors participated in the modulation of OMP induced by ES. Thus, cannabinoid receptor antagonists (AM251 and AM630) and non-selective cannabinoid receptor agonist (WIN55,212-2) were administered to investigate if the blockade or activation of CB₁ and CB₂ receptors would modify the painful response. The findings showed that a single administration of WIN55,212-2 inhibited ongoing mechanical sensitivity on masticatory muscles caused by ES, and the analgesic effect remained until the last experimental day. Interestingly, the cannabinoid agonist increased the nociceptive threshold suggesting a hypoalgesic effect in the non-stressed group. Although WIN55,212-2 caused this effect, it does not appear to be a cannabimimetic effect, as other studies showed that the utilized dose (1 mg/kg) did not promote cannabimimetic effects (31,32).

Conversely, the blockade of the cannabinoid receptors, CB₁ and CB₂, by the acute administration of the respective selective antagonists, AM251 and AM630, provoked an increase in the mechanical sensitivity associated with ES, anticipating the nociceptive state to the 4th experimental day of the stress protocol and increased mechanical sensitivity on the 8th day. This suggests that cannabinoid receptor blockade possibly prevented endocannabinoids modulation of the nociceptive threshold.

An additional finding was that the effect of AM251 was longer lasting when compared to AM630, especially in the masseter muscle, suggesting a greater participation of CB₁ in this process. In line with this observation, previous studies indicate a greater participation of CB₁ than CB₂ receptors in pain pathway modulation (8,9). Corroborating these results, CB₁ expression increases in both the TG and the Sp5C, whereas CB₂ expression is upregulated only in the Sp5C nucleus. Other authors have also demonstrated that CB₁ plays a critical role in mediating the effects of 2-AG on the glutamatergic system. In this context, elevated 2-AG levels are thought to exert a protective function by stimulating CB₁ receptors, thereby restricting glutamate release and safeguarding the nervous system from the excitotoxic effects of this neurotransmitter (33). Corroborating these findings, Zoppi et al. (34) demonstrated the regulatory role of the CB₁ receptor in stress-induced excitotoxicity and neuroinflammation. The results obtained by western blotting and PCR show an increase in CB₁ protein expression in agreement with the increase in gene expression of its mRNA. The authors associate this increase of the homeostatic response to the excess glutamate that is normally released upon exposure to stress (35). Another study showed that rodents exposed to chronic unpredictable stress exhibited an increase in the maximum density of the CB₁ receptor binding site in the prefrontal cortex, showing that this receptor is important in the homeostatic response to stress (35).

CB₂ also appears to be differentially modulated depending on the type of pain. In an inflammatory pain model, its expression increases in peripheral sites such as DRG and peripheral nerve endings and, in a neuropathic pain model, it increases in DRG and spinal cord (36). An interesting finding was observed when evaluating cannabinoid receptor labeling in the masseter muscle of animals subjected to emotional stress. A higher density of CB₂ labeling was found in stressed animals, while no changes were detected in CB₁ labeling. Indeed, skeletal muscle expresses both types of receptors, as demonstrated in both humans and rodents (37). To our knowledge, no studies have assessed cannabinoid receptor expression in masticatory muscles. Thus, this would be the first study to evaluate differences in CB₁ and CB₂ receptor density in the masseter muscle of male rats with stress-induced OMP and to integrate emotional stress, OMP, and ECS. These findings highlight a potential nociplastic mechanism underlying stress-related OMP and suggest the ECS as a promising therapeutic target.

The cannabinoid receptor expression has been investigated in other muscles. Dalle and Koppo (38) reported higher CB₂ expression in type II fibers and higher CB₁ expression in type I fibers in the human vastus lateralis skeletal muscle of older men compared to younger. This is particularly interesting because type II fibers have low fatigue resistance and appear to be involved in the pathophysiology of bruxism. Studies suggest that bruxism may shift the composition of the masseter muscle toward a higher proportion of type II fibers (39). This shift could be related to increased mechanical demand on masticatory muscles during bruxism episodes, such as clenching or grinding (40). Thus, the increase in type II fibers might explain the higher CB₂ density in the masseters of stressed animals, given that the communication box ES model not only enhances masseter mechanical sensitivity, but also increases masticatory muscle activity, mimicking bruxism (14). However, further experiments would be needed to confirm this correlation.

Here, we demonstrated that the non-selective agonist WIN55,212-2 effectively inhibited OMP, which may result from its action on CB₁ and CB₂ receptors in the trigeminal pain pathway. However, the fact that the masseter muscle expresses both CB₁ and CB₂ receptors, and that CB₂ expression increases in stressed animals, suggests that WIN55,212-2 might also influence masticatory muscle contraction. This provides additional support for the potential use of cannabinoids in reducing pain and fatigue in patients with painful TMD. Furthermore, there is consistent evidence that the ECS is involved in the modulation of stress, plays a central role in pain control, and participates in the trigeminal pain pathway. These findings highlight a potential nociplastic mechanism underlying stress-related OMP and suggest the ECS as a promising therapeutic target.

In conclusion, the data suggest that ES causes mechanical sensitivity in the temporalis and masseter muscles, mimicking what happens in patients with painful TMD. ES also increases the expression of cannabinoid receptors in the trigeminal pain pathway and in the masseter muscle (Figure 7). The blockade or activation of these receptors, mainly CB₁, modifies the sensitivity in the evaluated muscles, indicating that cannabinoid drugs or the modulation of the ECS may represent a promising therapeutic strategy for the treatment of stress-associated OMP, which is frequently observed in patients with TMD.