Naloxone Reversed Orofacial Analgesia Induced by Treadmill Exercise in a Rat Model of Parkinson's Disease: Exploring Opioid Receptors Involvement

Naloxone Reversed Orofacial Analgesia Induced by Treadmill Exercise in a Rat Model of Parkinson's Disease: Exploring Opioid Receptors Involvement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Naloxone Reversed Orofacial Analgesia Induced by Treadmill Exercise in a Rat Model of Parkinson's Disease: Exploring Opioid Receptors Involvement Karina Henrique Binda, Marucia Chacur, Daniel Oliveira Martins This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9452205/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Exercise-induced analgesia has been consistently associated with the activation of endogenous opioid pathways; however, the specific contribution of opioid receptor signaling to the reduction of orofacial pain in Parkinsonian conditions remains incompletely understood. Therefore, this study investigated whether pharmacological blockade of opioid receptors reverses the antinociceptive effect induced by exercise in a rat model of Parkinson’s disease (PD). Hemiparkinsonian rats were generated by unilateral 6-OHDA injection and subsequently submitted to a treadmill exercise protocol. To determine the involvement of endogenous opioids in exercise-induced analgesia, animals received the non-selective opioid receptor antagonist naloxone (1 mg/kg, i.p.), which exhibits high affinity for µ-opioid receptors, on day 28 after lesion induction, following six exercise sessions. Mechanical nociceptive thresholds in the orofacial region were assessed using the von Frey test at 15 and 30 minutes after naloxone administration. In addition, protein expression of µ- and δ-opioid receptors and the endogenous opioid peptide enkephalin was quantified in the trigeminal ganglion (TG) using Western blot analysis. We observed that the 6-OHDA + SED group showed a decrease in the mechanical threshold, which was reversed in the 6-OHDA + EX group. The antinociceptive effects of EX on orofacial pain were significantly reduced by naloxone, as showed in the behavioral test. Additionally, western blot analysis showed that the PD decreased the expression mu, delta and enkephalin in the TG and EX increased this expression. The current study shows that inhibiting opioid receptors, diminishes the facial antinociceptive effects of EX, indicating the involvement of opioid receptor in the analgesic effect of EX. Parkinson’s disease Trigeminal pain Treadmill Exercise Rats 6-OHDA Opioid System Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background According to the World Health Organization, oral health is a critical measure of human quality of life, physical health, and well-being (Bawaskar & Bawaskar, 2020). While improvements in dental care, including enhanced understanding and new treatment strategies (Lobbezoo & Aarab, 2021), have occurred, serious oral health complications may still emerge as people age, often due to a reduction in motor function. An illustrative example of a condition linked to this decline is Parkinson's Disease (PD) (Verhoeff et al., 2023). Pain experienced by PD patients might be an intrinsic feature of the disorder or it could stem from separate causes. To date, there is limited understanding regarding orofacial pain and related dysfunction in individuals affected by PD. While our previous study has reported the benefits of EX for orofacial pain relief in a hemi-parkinsonian rat model, the underlying mechanisms remain unclear. The effect of EX on opioid response remains controversial, but some studies have already assessed this link (Arida et al., 2015; Nelson et al., 2024). Opioid analgesics are the standard therapeutic agents for the management of moderate to severe pain and alleviate pain predominantly by stimulating the mu-opioid receptors in the central nervous system (Alorfi, 2023). Opioid receptors are found in the central nervous system (CNS), the dorsal root and trigeminal ganglia (TG) (Machelska & Celik, 2020). The classical opioid receptors are sensitive to the antagonist naloxone and their endogenous agonists are opioid peptides, such as β-endorphin, enkephalins (Met-, Leu-enkephalin), and dynorphins (dynorphin A, B, α-neoendorphin) (Ananthan, 2006; Hughes et al., 1975; Li et al., 1976). Naloxone is an antagonist with a high binding affinity for the μ-opioid receptor (Kanemasa et al., 2020), and μ-opioid receptor is a well-established target for analgesia (Jamison & Mao, 2015). Given that different exercise modalities affect opioid signaling in the brain, the role of opioid expression in exercise-induced pain relief has been evaluated in hemi-parkinsonian rats. A hemi-parkinsonian rat model produced by 6-hydroxydopamine (6-OHDA) injection into the nigrostriatal pathway (Binda et al., 2020, 2023; Garcia et al., 2017) was used to investigate behavioral and western blot responses to PD induces pain in the orofacial region. We recently demonstrated that exercise-induced analgesia in 6-OHDA animals relies on the endogenous cannabinoid system (Binda et al., 2023). This study, therefore, sought to determine if endogenous opioids represent an additional neurochemical mechanism responsible for reducing the orofacial pain associated with 6-OHDA-induced PD. Given the potential involvement of this system in the nociceptive changes seen in PD patients, we specifically investigated the hypothesis that exercise can restore the altered expression of opioid receptors resulting from the 6-OHDA lesion in the nigrostriatal pathway and, consequently, modulate the orofacial nociceptive response. Methods Animals Twenty-three male Wistar rats with body weight between 250 - 300g (5 per group for western blotting analysis and 3 for behavioral test with naloxone) were used. Male Wistar rats (Rattus norvegicus) from the Animal Resource of the Institute of Biomedical Sciences (São Paulo, Brazil) were maintained in a temperature and humidity-controlled animal facility, five rats per cage, under a 12-hour dark/light cycle. All tests occurred in the light cycle time between 9:00 a.m. and 12:00 a.m. The Institutional Animal Care Committee approved the study of the Institute of Biomedical Sciences, University of São Paulo (protocol number 4860310118). All efforts were made to reduce the number of animals used and their suffering (Zimmermann, 1983), and this study is reported following the ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines). The animals were taken to the experimental environment and kept for 1 h in the room for 3 days before the beginning of behavioral tests to minimize stress. The rats were randomly divided into 2 groups: (1) saline control animals which were injected with the vehicle into the right striatum (SAL); and (2) 6-OHDA animals, which were injected with 6-OHDA into the right striatum. Fifteen days after stereotactic surgery, half of the animals were submitted to treadmill exercise (EX), and half remained sedentary (SED), so the experimental groups were: (1) SAL + SED (n=5); (2) SAL + EX (n=5); (3) 6-OHDA + SED (n=5); (4) 6-OHDA + EX (n=5). Rats were randomly assigned to the experimental groups and all data were collected by researchers blinded to the experimental group and the pharmacological treatment. Furthermore, an extra treatment group was established to assess the impact of intrathecal naloxone administration (n = 3) throughout the exercise protocol. Opioid Receptor antagonist Naloxone hydrochloride (as an opioid antagonist (Abolghasemi et al., 2025) was dissolved into physiological saline (1 mg/Kg). The drug was administered intrathecally (i.t.) in a volume of 50 µl. Animals were anesthetized by isoflurane inhalation, and a 29-gauge needle was introduced through the shaved skin into the L5-L6 intervertebral space (Milligan et al., 2003; Rocha et al., 2020; Rosa et al., 2017a). The correct positioning of the needle was assured by a tail-flick reaction. After that, the drug was administered and the needle was carefully removed (Mestre et al., 1994). Mechanical hyperalgesia was measured on day 28 PO, after six sessions of physical exercise, 15 and 30 min after injection of the antagonist. Stereotaxic Surgery The experiment was performed as described previously (Binda et al., 2020, 2023). Animals were anesthetized with isoflurane mixed with oxygen (5% induction, 2% maintenance, 0.8 Liters/minute). The animals were placed in a stereotaxic frame and in a stereotaxic apparatus (Kopf Instruments, Germany). After craniotomy, rats were injected unilaterally in the right striatum (CPu) with 1.5 μl of a drug containing 9 μg of neurotoxin 6-OHDA hydrochloride (H4381, Sigma) diluted in 0.3% ascorbic acid in saline at two different brain coordinates: (1) L: 2.7 mm; AP: -0.5mm; V: 4.5 mm; (2) L: 2.7 mm; AP: +0,5 mm; V: 5 mm (Paxinos, 2013). The final concentration of the neurotoxin was 18 micrograms; sham-lesioned rats received only the vehicle at the same coordinates. After infusion, the syringe needle was left in the infused region for 5 min to avoid reflux of the solution. The incision was then sutured. Pain medication (ketoprofen – 1 mg/kg, s.c.) was administered before and 24 h after surgery to reduce discomfort. If animals still showed discomfort after 48 h, an extra dose of analgesic was given (Binda et al., 2020, 2023). Their regular diet was supplemented with a dietary supplement (Ensure, Abbott, SP, BRA) once a day for 3 consecutive days to ensure full recovery of the animals after the nigrostriatal injury. Rout of Drug Administration Four weeks after the 6-OHDA injection and after six treadmill exercise sessions, a subset of animals was briefly anesthetized with 2% isoflurane using a mask. They received intrathecal administration (i.t. in a volume of 50 µl) of naloxone, a non-specific opioid receptor antagonist. Naloxone was dissolved into physiological saline (1 mg/Kg). Naloxone was administered intrathecally (i.t.) in a volume of 50 µl. Animals were anesthetized by isoflurane inhalation and a 29-gauge needle was introduced through the shaved skin into the L5-L6 intervertebral space (Milligan et al., 2003; Rocha et al., 2020; Rosa et al., 2017b). The correct positioning of the needle was assured by a tail-flick reaction. After that, the drug was administered and the needle was carefully removed (Mestre et al., 1994). Following a complete recovery from inhalation anesthesia (about 5 min), the facial mechanical sensitivity of each animal was assessed by Von Frey filaments at two timepoints: 15 and 30 min after injection of the antagonist. Von Frey test The region below the eye and caudal to the whisker pad, a region innervated by the second branch of the trigeminal ganglion (Leiser & Moxon, 2006) was tested using traditional von Frey filaments (Stoelting, USA), according of the method previously described (Binda et al., 2023; Martins et al., 2024). The measurements were conducted one day prior to injection of 6-OHDA, fourteen days post-injection and at intervals of 21, 28, 35, 42 and 49 (final measurement) days PO, always after three sessions of EX. All groups in the experiment included 5 animals/group, except the trial with naloxone which consisted of 3 animals/group. Treadmill Training Program Treadmill exercise is an established exercise training model in which we can control the exercise intensity and duration. The treadmill exercise program was carried out as previously described (Binda et al., 2020, 2023). Rats were exercised 3 days a week every other day on a treadmill (KT 3000, IMBRAMED) during the light cycle to decrease stress (Holmes et al., 2004). The animals were habituated to the motorized treadmill by running for 15 min on two consecutive days (6.7 meters/minute). Rats that refused to run were excluded and transferred to another study protocol for our group. Following the injection of 6-OHDA or saline, animals were randomized to a sedentary or exercise-trained group. Rats were then divided into four sub-groups: (1) sedentary saline (SAL SED); (2) saline exercised (SAL + EX); (3) 6-OHDA sedentary (6-OHDA SED), and (4) 6-OHDA exercised (6-OHDA + EX). The animals were then subjected to a light treadmill exercise protocol for 40 minutes, at a speed of 10 meters/minute, running approximately 400 meters/day (Binda et al., 2020, 2023). SAL SED animals, which were considered control groups, and 6-OHDA SED animals were placed in cages near the treadmill to expose them to the same environmental conditions as exercised animals. Experimental Design Experiment I: Evaluation of 6-OHDA Microinjection on Orofacial Pain-Related Behaviour The first phase of the study focused on evaluating how unilateral microinjection pf 6-OHDA into striatum influenced orofacial pain-related behavior and the impact of EX on behavioral responses (Fig. 1). In this phase, the vehicle group was administered 2 µl of saline solution directly into the right striatum (SAL), whereas the 6-OHDA group received 1,5 µl of a solution containing 9 µg of neurotoxin 6-OHDA (H4381, Sigma) diluted in 0,3% ascorbic acid in saline in the right striatum, in two different striatum coordinates: (1) L: -2.7 mm; AP: - 0,5 mm; V: - 4,5 mm; (2) L: - 2,7 mm; AP: + 0,5 mm; and V: - 5 mm (Paxinos, 2013). Them, they were randomly divided into 4 groups: (1) vehicle injected into the right striatum (SAL), (2) 6-OHDA injected into the right striatum (6-OHDA). 15 days after surgery, half of the animals were submitted to forced treadmill exercise (EX), and half of them remain sedentary (SED). So, the experimental group design of the first phase were: (1) SAL + SED; (2) SAL + EX; (3) 6-OHDA + SED; (4) 6-OHDA + EX (n = 5). Figure 1 . Experimental design. Animals were subjected to the DP induction model (6-OHDA injection) or control (saline). After 14 days of the surgical procedure, the animals were evaluated in the mechanical nociceptive test (measure 1 before treatment). In the 15th after of the unilateral 6-OHDA induction model (or Saline), they were submitted to 3 sessions of exercise per week during 5 weeks. After each exercise sessions, the animals were evaluated in the mechanical nociceptive test. After one day, the animals were euthanized for fresh tissue collection (for western blotting analyses). The trigeminal ganglion was collected for evaluation. PO - Postoperative period. Experiment II: Investigating the impact of intratecal administration of naloxone on behavioral orofacial pain-related behavior The second phase of the research analyzed the role of mu-opioid receptor blockade by the intrathecally (i.t.) administration of a volume of 50 ul of naloxone hydrochloride (dissolved into saline (1 mg/Kg). In this phase, the animals administered naloxone (n = 3) on day 28 post-6-OHDA injection, after six sessions of EX; and at 15 and 30 minutes after naloxone injection, we employed the von Frey test to asses pain behavior (Fig. 2). Experiment III: Evaluate the expression of mu, kappa opioid receptors by Western blot analysis In the third phase and concluding phase of the research to determine the participation of mu-opioid, delta-opioid receptors and enkephalin in the antinociceptive effect of EX. To study mu and delta expression, protein quantification was performed in the TG (FIG. 3A, 3B, and 3C). Protein quantification Upon the completion of the experimental procedures, the rats were anesthetized with isoflurane (5% induction, Cristália LTDA, Sao Paulo, Bra) and euthanized by decapitation. The next step involved the extraction of TG, which was subsequently homogenized in extraction buffer containing 100 mM Tris, pH 7.4, 10 mM EDTA, 2 mM PMSF, and 10 μg/ml aprotinin. They were then homogenized using an ultrasonic processor (Sonics & Materials, Newtown, PA). The homogenates were centrifuged at 13,000 rpm at 4 °C for 20 min, and the protein concentration of the supernatant was determined using the Bradford protein assay (Bio-Rad, Melville, NY) (Bradford, 1976). Samples containing 60 μg of protein were loaded on an acrylamide gradient gel (Miller et al., 2016) and transferred by electrophoresis to nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo Transfer System during the 30-min protocol. After transfer, the membranes were treated for 2 h at room temperature with a blocking solution containing 5% powdered milk, washed, and incubated overnight at 4 °C with an anti-Mu Opioid Receptor (rabbit polyclonal antibody, Abcam Cat# ab17934, RRID: AB_2283186), anti-Opioid receptor delta (rabbit polyclonal antibody, Millipore Cat# AB1560, RRID: AB_90778), anti-enkephalin (mouse monoclonal antibody, Millipore Cat# MAB350, RRID: AB_11213781), diluted 1:1000. The membranes were then washed and incubated for 2 h at room temperature with peroxidase-conjugated anti-rabbit and anti-mouse (GE Healthcare) secondary antibody, diluted 1:5000. β-actin (mouse monoclonal antibody, Sigma Cat# A-5316, RRID: AB_476743) was used as an internal control (1: 10000; Sigma). The specifically bound antibody was visualized using a chemiluminescence kit (Amersham Biosciences). The blot was analyzed densitometrically using the NIH-Scion Image 4.0.2, quantified by optical densitometry of the bands (Scion Corporation, USA), and corrected by the optical density for β-actin. In contrast, samples from control animals were used as the standard for normalization of the results (assuming 100% for sedentary animals). Statistics GraphPad Prism, version 9.00 (Graph-Pad Software Inc., San Diego, CA), was used to conduct the statistical analysis. For behavioral data comparing groups (treatment x time), we used the two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Western blot data were normalized (by defining the naive group as 100% for mu, delta, and enkephalin) and analyzed using the one-way ANOVA (nonparametric) followed by the Tukey's post hoc test. P < 0.05 was considered statistically significant, the data satisfied a normal distribution, and all values were expressed as the means ± SE and adjusted p values for multiple comparisons (Snedecor et al., 1946). Results Experiment I: Evaluation of 6-OHDA Microinjection and Exercise on Orofacial Pain-Related Behaviour We have previously shown that injection of 6-OHDA, a model of PD disease, causes facial hypersensitivity which started 14 days following the injection and remained until the last evaluation, and exercise improved this behaviour. In the pain-related behaviour, statistical analysis indicated that the animals who received unilateral injection of 6-OHDA, showed a significant decrease in the nociceptive threshold after 14 days compared with the baseline measurement (6-OHDA + SED and 6-OHDA + EX p ≤ 0.001) and compared with the control groups, in the baseline (SAL + SED and SAL + EX). After 21 days the pain response in the group exercised (3 sessions) returned to the baseline measurements and remained similar to the response of the control groups until the last assessment. While the group of animals that received unilateral injection of 6-OHDA and remain sedentary maintained a low nociceptive threshold until the last measurement. This response was like the ipsilateral whisker pad to injection side (Fig. 2 ). Experiment II: Investigating the impact of intratecal administration of naloxone on behavioral orofacial pain-related behavior As described above, the injection of 6-OHDA, caused facial hypersensitivity which started 14 days following the injection and remained until the last evaluation and exercise improved this behaviour to day 21 (after 3 sessions) until day 49 (last time evaluated). In this phase a new group of animals (N = 3) that received unilateral injection of 6-OHDA and that accomplished six sessions of EX, 28 days after 6-OHDA injection, received the intrathecally (i.t.) administration of a volume of 50 ul of naloxone dissolved into saline (1 mg/Kg). The nociceptive threshold was measured after 15 and 30 min of naloxone injection. After 15 min naloxone no showed effects in nociceptive threshold compared with baseline and 28 days measure (last measure before naloxone injection). After 30 min von Frey test values revealed that naloxone significantly reduced the nociceptive threshold compared to the baseline and 28 days measure (Fig. 3 ), diminished the antinociceptive effects induced by EX. After this, the animals were returned for the cages and followed proposed protocol until the end of evaluations (Fig. 3 ). Experiment III: Evaluate the expression of mu and delta opioid receptors and enkephalin by Western blot analysis The findings described above showed that 6-OHDA injection caused facial hypersensitivity, that an exercise protocol could improve this behavior, and that naloxone administration can reverse the analgesia induced by exercise. To provide information regarding the expression of opioid receptors mediating the effects of exercise, the expression of mu and kappa opioid receptors and enkephalin was evaluated within the trigeminal system using Western blot analysis. Western blot Analysis of Delta opioid in the trigeminal ganglion The TG from each of the four groups were obtained to analyze the expression patterns of DOR. Analysis revealed that there were no significant differences in the average MSD of DOR-expressing in control groups (SAL + SED vs. SAL + EX) and 6-OHDA + SED group compared with controls (F (4,12) = 9.11, p = 0,0249; Fig. 3 ). Notably, there was a significant difference between the 6-OHDA + SED vs. 6-OHDA + EX group (p = 0,0003), and SAL + SED vs. 6-OHDA + EX (p = 0,0051). Western blot Analysis of MOR in the trigeminal ganglion The expression of MOR in the TG tissues of saline groups (sedentary and exercise) did not show any significant difference (p > 0.05). The expression of MOR in the TG of PD group decreased in comparison with the control’s groups (SAL + SED and SAL + EX), although the differences were not statistically significant among the groups (p > 0.05). The MOR expression in the TG of 6-OHDA + EX group show a significant difference after exercise training compared with SAL + SED and 6-OHDA + SED (*p = 0,0365 and ***p = 0,0006, respectively) (Fig. 4 ). Western blot Analysis of ENK in the trigeminal ganglion We further examined whether exercise affected the expression of enkephalin in the TG. The expression of enkephalin in saline exercise group was unchanged in comparison to saline sedentary group, but the expression of enkephalin increased in 6-OHDA + SED group, although the difference was not statistically significant in comparison with the control’s groups (SAL + SED and SAL + EX) and was significantly upregulated after exercise training compared with the others groups (SAL + SED, SAL + EX and 6-OHDA + SED (**p = 0,0133; *** p = 0,0019 and *p = 0,0443, respectively) (Fig. 6 ). Discussion Although there are a lot of clinical and experimental works using exercise to pain, the analgesic effect remains incomplete and unsatisfactory for both the patients and the clinicians, making it challenging to identify and propose specific treatment. In the context of orofacial pain in PD patients’ knowledge is even more scarce. In a previous work we showed the involvement of cannabinoid receptors in orofacial analgesia induce by exercise (Binda et al., 2023 ). The aim of this study was to investigate the effect of intrathecal injection of naloxone on mechanical allodynia pain behavior under 6-OHDA model of PD condition during the protocol of treadmill exercise and whether opioids receptors are involved in this effect. Opioids are routinely prescribed to manage moderate to severe pain. They are effective against both spontaneous pain and mechanical allodynia (Grenald et al., 2017 ; Zubieta et al., 2001 ). Naloxone, a competitive opioid receptor antagonist, is widely used in experimental pain research to evaluate the role of the endogenous opioid system in various nociceptive states (Benedetti et al., 2023 ; Lee et al., 2021 ). In experimental models of pain, naloxone has been shown to produce paradoxical effects. For instance, high doses of naloxone typically induce hyperalgesia (increased pain sensitivity) and mechanical allodynia (Springborg et al., 2016 ), and low doses of naloxone have been reported to enhance morphine-induced analgesia and even reduce hyperalgesia in rat models of neuropathic and inflammatory pain (Lewis et al., 2012 ; Yang et al., 2013 ). Consistent with our previous findings, behavioral analysis revealed that hemiparkinsonian rats presented a significant reduction in withdrawal thresholds in the von Frey test, confirming that the PD model induced orofacial mechanical allodynia. Notably, the exercise protocol progressively restored withdrawal thresholds to baseline levels after three exercise sessions, indicating a robust antinociceptive effect. However, on postoperative day 28, following six exercise sessions, intrathecal administration of naloxone abolished the exercise-induced analgesia, demonstrating the involvement of opioid receptor signaling in this response. These findings are further supported by Western blot analysis, which showed that exercise increased the expression of enkephalin as well as µ- and δ-opioid receptors in the trigeminal ganglion compared with the PD sedentary group. Considering the inhibitory action of naloxone on opioid receptors (Benyamin et al., 2008 ; Trescot et al., 2008 ), these results collectively indicate that activation of the endogenous opioid system plays a central role in mediating exercise-induced orofacial antinociception. Widely recognized for its extensive health benefits, exercise enhances the opioid system by boosting endogenous opioids like endorphins and supporting in pain modulation (Belle et al., 2026 ). The analgesia induced by exercise appears to be mediated in part by the activation of endogenous opioid systems (enkephalins, endorphins and dynorphins), which act on mor, dor and kappa opioid-receptors (Da Silva Santos & Galdino, 2018 ; Goldfarb et al., 2024 ). Our previous studies using this same model and protocol have demonstrated the involvement of cannabinoids in mediating exercise-induced orofacial analgesia in hemiparkinsonian rats (Binda et al., 2023 ). Research demonstrates a reciprocal relationship between the endogenous opioid and cannabinoid systems, where each system significantly contributes to the antinociceptive effects of the other (Bushlin et al., 2010 ; Cichewicz, 2004 ; Wilson-Poe et al., 2013 ). It is known that opioids and cannabinoids exhibit overlapping neuroanatomical distribution and comparable functional neurobiological properties (Navarro et al., 2001 ). The present findings indicate that exercise-induced analgesia depends on a highly regulated opioid response, requiring endogenous opioids to achieve and maintain sufficient concentrations to reduce pain. Taken together with our previous results, opioids and cannabinoids, were likely to act synergistically to reduce the orofacial pain in hemiparkinsonian rats. Thus, in a murine model of PD induced by the administration of 1.5 µl of a drug containing 9 µg of 6-OHDA which produces, allodynia to mechanical stimuli to orofacial region than 40 days we have demonstrated that: 1) hemiparkinsonian rat have a reduced head withdrawal threshold compared to control group, 2) EX group have restored the threshold compared to DP rats and 3) the intrathecal injection of naloxone during the exercise protocol vanish analgesia induced by EX and this antinociceptive effects produced by EX may be involved with the differential expression of enkephalin and µ- and δ-opioid receptor during DP progression. Some limitations warrant consideration: the lack of an opioid receptor-specific antagonist group, the absence of female group, the latter of which prevents the evaluation of sex differences in exercise-induced analgesia. Conclusions Our findings demonstrate that exercise-induced orofacial analgesia in hemiparkinsonian rats is mediated by activation of the endogenous opioid system, as evidenced by increased expression of enkephalin and µ- and δ-opioid receptors in the trigeminal ganglion. These neurochemical adaptations appear to contribute to the modulation of trigeminal nociceptive transmission, supporting endogenous opioid signaling as a key mechanism underlying exercise-induced antinociception in Parkinsonian conditions. Declarations Ethics approval and consent to participate All experimental procedures carried out in this study have been approved by the Institutional Animal Care and Use Committee of the University of São Paulo (protocol number 4860310118) and were in compliance with the guidelines for animal care and use set forth by that committee. Consent for publication “Not applicable” Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests None of the authors have any potential or actual conflicts of interest to declare. Funding Martins, D.O., was the recipient of a FAPESP Postdoctoral scholarship (2015/24256-0). Binda K.H., was the recipient of a FAPESP Master scholarship (2017/26821-1). Chacur M., was the recipient of a FAPESP grant number (2017/05218-5, 2021/02897-4). The funding agencies play no role in the design of the study, data collection, analysis, interpretation of the data, or in writing the manuscript. Authors' contributions All authors made substantial contributions to the following aspects of this research: initial conception (Binda K.H., Chacur M., Martins D.O.); design (Binda K.H., Chacur M., Martins D.O.); provision of resources (Chacur M., Martins D.O.); collection of data (Binda K.H., Martins D.O.); analysis and interpretation of data (Binda K.H., Chacur M., Martins D.O.); writing the first draft of the paper or important intellectual content (Binda K.H., Chacur M., Martins D.O.); revision of the paper (Martins D.O.). All authors read and approved the final manuscript. Acknowledgements We thank Adilson da Silva Alves for technical assistance. This work was supported by FAPESP, Hospital Sírio-Libanês and CNPq; Martins D.O., was the recipient of a FAPESP Postdoctoral scholarship (Grant number: 2015/24256-0); Binda, K.H., was the recipient of a Master’s scholarship (Grant number: 2017/26821-1). Chacur, M., supported by FAPESP (Grant number: 2017/05218-5). 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G., Chacur M (2020) Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats. Mol Cell Neurosci 105:103497. https://doi.org/10.1016/j.mcn.2020.103497 Rosa AS, Freitas MF, Rocha IRC, Chacur M (2017a) Gabapentin decreases microglial cells and reverses bilateral hyperalgesia and allodynia in rats with chronic myositis. Eur J Pharmacol 799:111–117. https://doi.org/10.1016/j.ejphar.2017.02.012 Rosa AS, Freitas MF, Rocha IR, Chacur M (2017b) Gabapentin decreases microglial cells and reverses bilateral hyperalgesia and allodynia in rats with chronic myositis. Eur J Pharmacol 799:111–117. https://doi.org/10.1016/j.ejphar.2017.02.012 Snedecor GW, Sokal RR, Rohlf FJ (1946) Statistical methods Biometry. Owa State University Springborg AD, Jensen EK, Taylor BK, Werner MU (2016) Effects of target-controlled infusion of high-dose naloxone on pain and hyperalgesia in a human thermal injury model: A study protocol A randomized, double-blind, placebo-controlled, crossover trial with an enriched design. Medicine 95(46):e5336. https://doi.org/10.1097/MD.0000000000005336 Trescot AM, Datta S, Lee M, Hansen H (2008) Opioid pharmacology. Pain Physician 11(2 Suppl):133–153 Verhoeff MC, Eikenboom D, Koutris M, De Vries R, Berendse HW, Van Dijk KD, Lobbezoo F (2023) Parkinson’s disease and oral health: A systematic review. Arch Oral Biol 151:105712. https://doi.org/10.1016/j.archoralbio.2023.105712 Wilson-Poe AR, Pocius E, Herschbach M, Morgan MM (2013) The periaqueductal gray contributes to bidirectional enhancement of antinociception between morphine and cannabinoids. Pharmacol Biochem Behav 103(3):444–449. https://doi.org/10.1016/j.pbb.2012.10.002 Yang C-P, Cherng C-H, Wu C-T, Huang H-Y, Tao P-L, Lee S-O, Wong C-S (2013) Intrathecal Ultra-Low Dose Naloxone Enhances the Antihyperalgesic Effects of Morphine and Attenuates Tumor Necrosis Factor-α and Tumor Necrosis Factor-α Receptor 1 Expression in the Dorsal Horn of Rats with Partial Sciatic Nerve Transection. Anesth Analgesia 117(6):1493–1502. https://doi.org/10.1213/ANE.0000000000000020 Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16(2):109–110. https://doi.org/10.1016/0304-3959(83)90201-4 Zubieta J-K, Smith YR, Bueller JA, Xu Y, Kilbourn MR, Jewett DM, Meyer CR, Koeppe RA, Stohler CS (2001) Regional Mu Opioid Receptor Regulation of Sensory and Affective Dimensions of Pain. Science 293(5528):311–315. https://doi.org/10.1126/science.1060952 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9452205","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625166298,"identity":"45aafa2f-324c-4a3c-a1cf-22b564979ff5","order_by":0,"name":"Karina Henrique Binda","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Karina","middleName":"Henrique","lastName":"Binda","suffix":""},{"id":625166302,"identity":"2e1a1f81-5745-4dea-b950-2a2b8f18568a","order_by":1,"name":"Marucia Chacur","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Marucia","middleName":"","lastName":"Chacur","suffix":""},{"id":625166304,"identity":"de08c3d7-0396-49df-8022-8679238fb92f","order_by":2,"name":"Daniel Oliveira Martins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYDCCAyBUAGfbJDCwQzgJ+LUYgFjMIHZaApAmrIUBpgUIDhPWwnf87MEDHwwYovlnnz94uODP+Tz5ZgbGxxW/GPLMG7BrkTyTl3BwhgFD7oxzyQyHZ7bdLjY4zMBseLaPoVjmAHYtBgdyDA7zALU0nGFmOMzbcDtxAzMDm2RjD0PiDBwOMzj/xuDwH6CW+SAtPH/OJc5vJqTlBtAWoPdzN4C1sB1IbDgM1NLwA7cWyRtvDA72GEjkbjzDbHCYty05ccNhxmbDxgaJYglcIXY+x/jDjwqb3HlnGB9/5vljlzi/vfngw4Y/Nnm4tEABijRjAwNjGwENWMAfknWMglEwCkbB8AUAqRphiZERSU8AAAAASUVORK5CYII=","orcid":"","institution":"Hospital Sírio-Libanês","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"Oliveira","lastName":"Martins","suffix":""}],"badges":[],"createdAt":"2026-04-17 19:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9452205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9452205/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107304102,"identity":"460c05d6-e586-480b-9983-898dd6825346","added_by":"auto","created_at":"2026-04-20 08:07:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design.\u003c/strong\u003e Animals were subjected to the DP induction model (6-OHDA injection) or control (saline). After 14 days of the surgical procedure, the animals were evaluated in the mechanical nociceptive test (measure 1 before treatment). In the 15th after of the unilateral 6-OHDA induction model (or Saline), they were submitted to 3 sessions of exercise per week during 5 weeks. After each exercise sessions, the animals were evaluated in the mechanical nociceptive test. After one day, the animals were euthanized for fresh tissue collection (for western blotting analyses). The trigeminal ganglion was collected for evaluation. PO - Postoperative period.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/00c9549b426679bc97e343f5.png"},{"id":107304107,"identity":"2f97656c-2a60-45de-95bc-ebe3849ea776","added_by":"auto","created_at":"2026-04-20 08:07:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118258,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of the facial mechanical sensitivity threshold – Von Frey test. *p \u0026lt; 0.001 comparison between 6-OHDA + EX and 6-OHDA + SED groups. #p \u0026lt; 0.001 comparison between baseline and postoperative days measurements for the other groups (n = 5 per group). SAL + SED: saline-injected animals that remained sedentary; SAL + EX: saline-injected animals that performed exercise; 6-OHDA + SED: 6-OHDA-injected animals that remained sedentary; 6-OHDA + EX: 6-OHDA-injected animals that performed exercise.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/31d8258baa08538af51d1162.png"},{"id":107484545,"identity":"57b3ba61-18f3-48bf-b346-0f15ea8ed992","added_by":"auto","created_at":"2026-04-22 02:32:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98306,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of the facial mechanical sensitivity threshold before and after naloxone antagonist administration. **p \u0026lt; 0.05 and ***p \u0026lt; 0.05 in comparison to baseline; # p \u0026lt; 0.05 and $p \u0026lt; 0.01 in comparison to nociceptive threshold at day 28 PO before injection of the antagonist (n = 3). 6-OHDA + EX: 6-OHDA-injected animals that performed exercise.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/5390dcfe6a04f959db5ce15d.png"},{"id":107485335,"identity":"821a3a14-f691-49a5-8282-527c978de361","added_by":"auto","created_at":"2026-04-22 02:34:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72218,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of delta opioid receptor in the trigeminal ganglion. Data from all groups were normalized to the SAL + SED group. The effect of treadmill exercise on DOR expression is shown. Data are reported as mean ± SEM (n = 5 animals per group). *p = 0,0051compared to the control groups (SAL + SED) and *** p = 0,0003 compared to the 6-OHDA group. SAL + SED: saline-injected animals that remained sedentary; SAL + EX: saline-injected animals that performed exercise; 6-OHDA + SED: 6-OHDA-injected animals that remained sedentary; 6-OHDA + EX: 6-OHDA-injected animals that performed exercise\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/f93d05621a9416821d113340.png"},{"id":107304103,"identity":"7572a80d-b088-4f6f-87b0-fce25c80ac03","added_by":"auto","created_at":"2026-04-20 08:07:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71446,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of MOR in the trigeminal ganglion. Data from all groups were normalized to the SAL + SED group. The effect of treadmill exercise on MOR expression is shown. Data are reported as mean ± SEM (n = 5 animals per group). *p = 0,0365 compared to the control group (SAL + SED) and *** p = 0,0006 compared to the 6-OHDA + SED group. SAL + SED: saline-injected animals that remained sedentary; SAL + EX: saline-injected animals that performed exercise; 6-OHDA + SED: 6-OHDA-injected animals that remained sedentary; 6-OHDA + EX: 6-OHDA-injected animals that performed exercise.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/add71b9c03436b3a51f5edd0.png"},{"id":107485912,"identity":"82f9186b-eacc-4e2a-8592-5794e1ffff2c","added_by":"auto","created_at":"2026-04-22 02:36:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70064,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of enkephalin in the trigeminal ganglion. Data from all groups were normalized to the SAL + SED group. The effect of treadmill exercise on enkephalin expression is shown. Data are reported as mean ± SEM (n = 5 animals per group). **p = 0,0133 and ***p=0,0019 compared to the control’s groups (SAL + SED and Sal + EX) and * p = 0,0443 compared to the 6-OHDA + SED group. SAL + SED: saline-injected animals that remained sedentary; SAL + EX: saline-injected animals that performed exercise; 6-OHDA + SED: 6-OHDA-injected animals that remained sedentary; 6-OHDA + EX: 6-OHDA-injected animals that performed exercise.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/56c21f61e215f5dfe98e30d5.png"},{"id":107487807,"identity":"73f86c1b-f9a2-4f50-ad1d-03bd9358adff","added_by":"auto","created_at":"2026-04-22 02:42:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1048153,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9452205/v1/aed3c060-51e6-4b00-b414-1bf0918cffde.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNaloxone Reversed Orofacial Analgesia Induced by Treadmill Exercise in a Rat Model of Parkinson's Disease: Exploring Opioid Receptors Involvement\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAccording to the World Health Organization, oral health is a critical measure of human quality of life, physical health, and well-being\u0026nbsp;(Bawaskar \u0026amp; Bawaskar, 2020). While improvements in dental care, including enhanced understanding and new treatment strategies\u0026nbsp;(Lobbezoo \u0026amp; Aarab, 2021), have occurred, serious oral health complications may still emerge as people age, often due to a reduction in motor function. An illustrative example of a condition linked to this decline is Parkinson\u0026apos;s Disease (PD)\u0026nbsp;(Verhoeff et al., 2023). Pain experienced by PD patients might be an intrinsic feature of the disorder or it could stem from separate causes. To date, there is limited understanding regarding orofacial pain and related dysfunction in individuals affected by PD.\u003c/p\u003e\n\u003cp\u003eWhile our previous study has reported the benefits of EX for orofacial pain relief in a hemi-parkinsonian rat model, the underlying mechanisms remain unclear. The effect of EX on opioid response remains controversial, but some studies have already assessed this link\u0026nbsp;(Arida et al., 2015; Nelson et al., 2024). Opioid analgesics are the standard therapeutic agents for the management of moderate to severe pain and alleviate pain predominantly by stimulating the mu-opioid receptors in the central nervous system\u0026nbsp;(Alorfi, 2023). Opioid receptors are found in the central nervous system (CNS), the dorsal root and trigeminal ganglia (TG)\u0026nbsp;(Machelska \u0026amp; Celik, 2020). The classical opioid receptors are sensitive to the antagonist naloxone and their endogenous agonists are opioid peptides, such as \u0026beta;-endorphin, enkephalins (Met-, Leu-enkephalin), and dynorphins (dynorphin A, B, \u0026alpha;-neoendorphin)\u0026nbsp;(Ananthan, 2006; Hughes et al., 1975; Li et al., 1976). Naloxone is an antagonist with a high binding affinity for the \u0026mu;-opioid receptor\u0026nbsp;(Kanemasa et al., 2020), and \u0026mu;-opioid receptor is a well-established target for analgesia\u0026nbsp;(Jamison \u0026amp; Mao, 2015).\u003c/p\u003e\n\u003cp\u003eGiven that different exercise modalities affect opioid signaling in the brain, the role of opioid expression in exercise-induced pain relief has been evaluated in hemi-parkinsonian rats. A hemi-parkinsonian rat model produced by 6-hydroxydopamine (6-OHDA) injection into the nigrostriatal pathway (Binda et al., 2020, 2023; Garcia et al., 2017) was used to investigate behavioral and western blot responses to PD induces pain in the orofacial region. We recently demonstrated that exercise-induced analgesia in 6-OHDA animals relies on the endogenous cannabinoid system (Binda et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study, therefore, sought to determine if endogenous opioids represent an additional neurochemical mechanism responsible for reducing the orofacial pain associated with 6-OHDA-induced PD. Given the potential involvement of this system in the nociceptive changes seen in PD patients, we specifically investigated the hypothesis that exercise can restore the altered expression of opioid receptors resulting from the 6-OHDA lesion in the nigrostriatal pathway and, consequently, modulate the orofacial nociceptive response.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-three male Wistar rats with body weight between 250 - 300g (5 per group for western blotting analysis and 3 for behavioral test with naloxone) were used. Male Wistar rats (Rattus norvegicus) from the Animal Resource of the Institute of Biomedical Sciences (S\u0026atilde;o Paulo, Brazil) were maintained in a temperature and humidity-controlled animal facility, five rats per cage, under a 12-hour dark/light cycle. All tests occurred in the light cycle time between 9:00 a.m. and 12:00 a.m. The Institutional Animal Care Committee approved the study of the Institute of Biomedical Sciences, University of S\u0026atilde;o Paulo (protocol number 4860310118). All efforts were made to reduce the number of animals used and their suffering (Zimmermann, 1983), and this study is reported following the ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines). The animals were taken to the experimental environment and kept for 1 h in the room for 3 days before the beginning of behavioral tests to minimize stress. The rats were randomly divided into 2 groups: (1) saline control animals which were injected with the vehicle into the right striatum (SAL); and (2) 6-OHDA animals, which were injected with 6-OHDA into the right striatum. Fifteen days after stereotactic surgery, half of the animals were submitted to treadmill exercise (EX), and half remained sedentary (SED), so the experimental groups were: (1) SAL + SED (n=5); (2) SAL + EX (n=5); (3) 6-OHDA + SED (n=5); (4) 6-OHDA + EX (n=5). Rats were randomly assigned to the experimental groups and all data were collected by researchers blinded to the experimental group and the pharmacological treatment. Furthermore, an extra treatment group was established to assess the impact of intrathecal naloxone administration (n = 3) throughout the exercise protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpioid Receptor antagonist\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaloxone hydrochloride (as an opioid antagonist (Abolghasemi et al., 2025) was dissolved into physiological saline (1 mg/Kg). The drug was administered intrathecally (i.t.) in a volume of 50 \u0026micro;l. Animals were anesthetized by isoflurane inhalation, and a 29-gauge needle was introduced through the shaved skin into the L5-L6 intervertebral space (Milligan et al., 2003; Rocha et al., 2020; Rosa et al., 2017a). The correct positioning of the needle was assured by a tail-flick reaction. After that, the drug was administered and the needle was carefully removed (Mestre et al., 1994). Mechanical hyperalgesia was measured on day 28 PO, after six sessions of physical exercise, 15 and 30 min after injection of the antagonist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic Surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was performed as described previously (Binda et al., 2020, 2023). Animals were anesthetized with isoflurane mixed with oxygen (5% induction, 2% maintenance, 0.8 Liters/minute). The animals were placed in a stereotaxic frame and in a stereotaxic apparatus (Kopf Instruments, Germany). After craniotomy, rats were injected unilaterally in the right striatum (CPu) with 1.5 \u0026mu;l of a drug containing 9 \u0026mu;g of neurotoxin 6-OHDA hydrochloride (H4381, Sigma) diluted in 0.3% ascorbic acid in saline at two different brain coordinates: (1) L: 2.7 mm; AP: -0.5mm; V: 4.5 mm; (2) L: 2.7 mm; AP: +0,5 mm; V: 5 mm (Paxinos, 2013). The final concentration of the neurotoxin was 18 micrograms; sham-lesioned rats received only the vehicle at the same coordinates. After infusion, the syringe needle was left in the infused region for 5 min to avoid reflux of the solution. The incision was then sutured. Pain medication (ketoprofen \u0026ndash; 1 mg/kg, s.c.) was administered before and 24 h after surgery to reduce discomfort. If animals still showed discomfort after 48 h, an extra dose of analgesic was given (Binda et al., 2020, 2023). Their regular diet was supplemented with a dietary supplement (Ensure, Abbott, SP, BRA) once a day for 3 consecutive days to ensure full recovery of the animals after the nigrostriatal injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRout of Drug Administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour weeks after the 6-OHDA injection and after six treadmill exercise sessions, a subset of animals was briefly anesthetized with 2% isoflurane using a mask. They received intrathecal administration (i.t. in a volume of 50 \u0026micro;l) of naloxone, a non-specific opioid receptor antagonist. Naloxone was dissolved into physiological saline (1 mg/Kg). Naloxone was administered intrathecally (i.t.) in a volume of 50 \u0026micro;l. Animals were anesthetized by isoflurane inhalation and a 29-gauge needle was introduced through the shaved skin into the L5-L6 intervertebral space (Milligan et al., 2003; Rocha et al., 2020; Rosa et al., 2017b). The correct positioning of the needle was assured by a tail-flick reaction. After that, the drug was administered and the needle was carefully removed (Mestre et al., 1994). Following a complete recovery from inhalation anesthesia (about 5 min), the facial mechanical sensitivity of each animal was assessed by Von Frey filaments at two timepoints: 15 and 30 min after injection of the antagonist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVon Frey test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe region below the eye and caudal to the whisker pad, a region innervated by the second branch of the trigeminal ganglion (Leiser \u0026amp; Moxon, 2006) was tested using traditional von Frey filaments (Stoelting, USA), according of the method previously described (Binda et al., 2023; Martins et al., 2024). The measurements were conducted one day prior to injection of 6-OHDA, fourteen days post-injection and at intervals of 21, 28, 35, 42 and 49 (final measurement) days PO, always after three sessions of EX. All groups in the experiment included 5 animals/group, except the trial with naloxone which consisted of 3 animals/group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreadmill Training Program\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreadmill exercise is an established exercise training model in which we can control the exercise intensity and duration. The treadmill exercise program was carried out as previously described (Binda et al., 2020, 2023). Rats were exercised 3 days a week every other day on a treadmill (KT 3000, IMBRAMED) during the light cycle to decrease stress (Holmes et al., 2004). The animals were habituated to the motorized treadmill by running for 15 min on two consecutive days (6.7 meters/minute). Rats that refused to run were excluded and transferred to another study protocol for our group. Following the injection of 6-OHDA or saline, animals were randomized to a sedentary or exercise-trained group. Rats were then divided into four sub-groups: (1) sedentary saline (SAL SED); (2) saline exercised (SAL + EX); (3) 6-OHDA sedentary (6-OHDA SED), and (4) 6-OHDA exercised (6-OHDA + EX). The animals were then subjected to a light treadmill exercise protocol for 40 minutes, at a speed of 10 meters/minute, running approximately 400 meters/day (Binda et al., 2020, 2023). SAL SED animals, which were considered control groups, and 6-OHDA SED animals were placed in cages near the treadmill to expose them to the same environmental conditions as exercised animals.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment I: Evaluation of 6-OHDA Microinjection on Orofacial Pain-Related Behaviour\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first phase of the study focused on evaluating how unilateral microinjection pf 6-OHDA into striatum influenced orofacial pain-related behavior and the impact of EX on behavioral responses (Fig. 1). In this phase, the vehicle group was administered 2 \u0026micro;l of saline solution directly into the right striatum (SAL), whereas the 6-OHDA group received 1,5 \u0026micro;l of a solution containing 9 \u0026micro;g of neurotoxin 6-OHDA (H4381, Sigma) diluted in 0,3% ascorbic acid in saline in the right striatum, in two different striatum coordinates: (1) L: -2.7 mm; AP: - 0,5 mm; V: - 4,5 mm; (2) L: - 2,7 mm; AP: + 0,5 mm; and V: - 5 mm (Paxinos, 2013). Them, they were randomly divided into 4 groups: (1) vehicle injected into the right striatum (SAL), (2) 6-OHDA injected into the right striatum (6-OHDA). 15 days after surgery, half of the animals were submitted to forced treadmill exercise (EX), and half of them remain sedentary (SED). So, the experimental group design of the first phase were: (1) SAL + SED; (2) SAL + EX; (3) 6-OHDA + SED; (4) 6-OHDA + EX (n = 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 1\u003cstrong\u003e. Experimental design.\u003c/strong\u003e Animals were subjected to the DP induction model (6-OHDA injection) or control (saline). After 14 days of the surgical procedure, the animals were evaluated in the mechanical nociceptive test (measure 1 before treatment). In the 15th after of the unilateral 6-OHDA induction model (or Saline), they were submitted to 3 sessions of exercise per week during 5 weeks. After each exercise sessions, the animals were evaluated in the mechanical nociceptive test. After one day, the animals were euthanized for fresh tissue collection (for western blotting analyses). The trigeminal ganglion was collected for evaluation. PO - Postoperative period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment II: Investigating the impact of intratecal administration of naloxone on behavioral orofacial pain-related behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe second phase of the research analyzed the role of mu-opioid receptor blockade by the intrathecally (i.t.) administration of a volume of 50 ul of naloxone hydrochloride (dissolved into saline (1 mg/Kg). In this phase, the animals administered naloxone (n = 3) \u0026nbsp; on day 28 post-6-OHDA injection, after six sessions of EX; and at 15 and 30 minutes after naloxone injection, we employed the von Frey test to asses pain behavior (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment III: Evaluate the expression of mu, kappa opioid receptors by Western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the third phase and concluding phase of the research to determine the participation of\u0026nbsp;mu-opioid, delta-opioid\u0026nbsp;receptors and enkephalin in the antinociceptive effect of EX. To study mu and delta expression, protein quantification was performed in the TG (FIG. 3A, 3B, and 3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon the completion of the experimental procedures, the rats were anesthetized with isoflurane (5% induction, Crist\u0026aacute;lia LTDA, Sao Paulo, Bra) and euthanized by decapitation. The next step involved the extraction of TG, which was subsequently homogenized in extraction buffer containing 100 mM Tris, pH 7.4, 10 mM EDTA, 2 mM PMSF, and 10 \u0026mu;g/ml aprotinin. They were then homogenized using an ultrasonic processor (Sonics \u0026amp; Materials, Newtown, PA).\u003c/p\u003e\n\u003cp\u003eThe homogenates were centrifuged at 13,000 rpm at 4 \u0026deg;C for 20 min, and the protein concentration of the supernatant was determined using the Bradford protein assay (Bio-Rad, Melville, NY)\u0026nbsp;(Bradford, 1976). Samples containing 60 \u0026mu;g of protein were loaded on an acrylamide gradient gel\u0026nbsp;(Miller et al., 2016)\u0026nbsp;and transferred by electrophoresis to nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo Transfer System during the 30-min protocol. After transfer, the membranes were treated for 2 h at room temperature with a blocking solution containing 5% powdered milk, washed, and incubated overnight at 4 \u0026deg;C with an anti-Mu Opioid Receptor (rabbit polyclonal antibody, Abcam Cat# ab17934, RRID: AB_2283186), anti-Opioid receptor delta (rabbit polyclonal antibody, Millipore Cat# AB1560, RRID: AB_90778), anti-enkephalin (mouse monoclonal antibody, Millipore Cat# MAB350, RRID: AB_11213781), diluted 1:1000. The membranes were then washed and incubated for 2 h at room temperature with peroxidase-conjugated anti-rabbit and anti-mouse (GE Healthcare) secondary antibody, diluted 1:5000. \u0026beta;-actin\u0026nbsp;(mouse monoclonal antibody, Sigma Cat# A-5316, RRID: AB_476743) was used as an internal control (1: 10000; Sigma). The specifically bound antibody was visualized using a chemiluminescence kit (Amersham Biosciences). The blot was analyzed densitometrically using the NIH-Scion Image 4.0.2, quantified by optical densitometry of the bands (Scion Corporation, USA), and corrected by the optical density for \u0026beta;-actin. In contrast, samples from control animals were used as the standard for normalization of the results (assuming 100% for sedentary animals).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism, version 9.00 (Graph-Pad Software Inc., San Diego, CA), was used to conduct the statistical analysis. For behavioral data comparing groups (treatment x time), we used the two-way analysis of variance (ANOVA) followed by Bonferroni\u0026apos;s post hoc test. Western blot data were normalized (by defining the naive group as 100% for mu, delta, and enkephalin) and analyzed using the one-way ANOVA (nonparametric) followed by the Tukey\u0026apos;s post hoc test. P \u0026lt; 0.05 was considered statistically significant, the data satisfied a normal distribution, and all values were expressed as the means \u0026plusmn; SE and adjusted p values for multiple comparisons\u0026nbsp;(Snedecor et al., 1946).\u003c/p\u003e\n"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eExperiment I: Evaluation of 6-OHDA Microinjection and Exercise on Orofacial Pain-Related Behaviour\u003c/h2\u003e \u003cp\u003eWe have previously shown that injection of 6-OHDA, a model of PD disease, causes facial hypersensitivity which started 14 days following the injection and remained until the last evaluation, and exercise improved this behaviour. In the pain-related behaviour, statistical analysis indicated that the animals who received unilateral injection of 6-OHDA, showed a significant decrease in the nociceptive threshold after 14 days compared with the baseline measurement (6-OHDA\u0026thinsp;+\u0026thinsp;SED and 6-OHDA\u0026thinsp;+\u0026thinsp;EX p\u0026thinsp;\u0026le;\u0026thinsp;0.001) and compared with the control groups, in the baseline (SAL\u0026thinsp;+\u0026thinsp;SED and SAL\u0026thinsp;+\u0026thinsp;EX). After 21 days the pain response in the group exercised (3 sessions) returned to the baseline measurements and remained similar to the response of the control groups until the last assessment. While the group of animals that received unilateral injection of 6-OHDA and remain sedentary maintained a low nociceptive threshold until the last measurement. This response was like the ipsilateral whisker pad to injection side (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperiment II: Investigating the impact of intratecal administration of naloxone on behavioral orofacial pain-related behavior\u003c/h2\u003e \u003cp\u003eAs described above, the injection of 6-OHDA, caused facial hypersensitivity which started 14 days following the injection and remained until the last evaluation and exercise improved this behaviour to day 21 (after 3 sessions) until day 49 (last time evaluated). In this phase a new group of animals (N\u0026thinsp;=\u0026thinsp;3) that received unilateral injection of 6-OHDA and that accomplished six sessions of EX, 28 days after 6-OHDA injection, received the intrathecally (i.t.) administration of a volume of 50 ul of naloxone dissolved into saline (1 mg/Kg). The nociceptive threshold was measured after 15 and 30 min of naloxone injection. After 15 min naloxone no showed effects in nociceptive threshold compared with baseline and 28 days measure (last measure before naloxone injection). After 30 min von Frey test values revealed that naloxone significantly reduced the nociceptive threshold compared to the baseline and 28 days measure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), diminished the antinociceptive effects induced by EX. After this, the animals were returned for the cages and followed proposed protocol until the end of evaluations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment III: Evaluate the expression of mu and delta opioid receptors and enkephalin by Western blot analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe findings described above showed that 6-OHDA injection caused facial hypersensitivity, that an exercise protocol could improve this behavior, and that naloxone administration can reverse the analgesia induced by exercise. To provide information regarding the expression of opioid receptors mediating the effects of exercise, the expression of mu and kappa opioid receptors and enkephalin was evaluated within the trigeminal system using Western blot analysis.\u003c/p\u003e \u003cp\u003eWestern blot Analysis of Delta opioid in the trigeminal ganglion\u003c/p\u003e \u003cp\u003eThe TG from each of the four groups were obtained to analyze the expression patterns of DOR. Analysis revealed that there were no significant differences in the average MSD of DOR-expressing in control groups (SAL\u0026thinsp;+\u0026thinsp;SED vs. SAL\u0026thinsp;+\u0026thinsp;EX) and 6-OHDA\u0026thinsp;+\u0026thinsp;SED group compared with controls (F (4,12)\u0026thinsp;=\u0026thinsp;9.11, p\u0026thinsp;=\u0026thinsp;0,0249; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, there was a significant difference between the 6-OHDA\u0026thinsp;+\u0026thinsp;SED vs. 6-OHDA\u0026thinsp;+\u0026thinsp;EX group (p\u0026thinsp;=\u0026thinsp;0,0003), and SAL\u0026thinsp;+\u0026thinsp;SED vs. 6-OHDA\u0026thinsp;+\u0026thinsp;EX (p\u0026thinsp;=\u0026thinsp;0,0051).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern blot Analysis of MOR in the trigeminal ganglion\u003c/p\u003e \u003cp\u003eThe expression of MOR in the TG tissues of saline groups (sedentary and exercise) did not show any significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The expression of MOR in the TG of PD group decreased in comparison with the control\u0026rsquo;s groups (SAL\u0026thinsp;+\u0026thinsp;SED and SAL\u0026thinsp;+\u0026thinsp;EX), although the differences were not statistically significant among the groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The MOR expression in the TG of 6-OHDA\u0026thinsp;+\u0026thinsp;EX group show a significant difference after exercise training compared with SAL\u0026thinsp;+\u0026thinsp;SED and 6-OHDA\u0026thinsp;+\u0026thinsp;SED (*p\u0026thinsp;=\u0026thinsp;0,0365 and ***p\u0026thinsp;=\u0026thinsp;0,0006, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern blot Analysis of ENK in the trigeminal ganglion\u003c/p\u003e \u003cp\u003eWe further examined whether exercise affected the expression of enkephalin in the TG. The expression of enkephalin in saline exercise group was unchanged in comparison to saline sedentary group, but the expression of enkephalin increased in 6-OHDA\u0026thinsp;+\u0026thinsp;SED group, although the difference was not statistically significant in comparison with the control\u0026rsquo;s groups (SAL\u0026thinsp;+\u0026thinsp;SED and SAL\u0026thinsp;+\u0026thinsp;EX) and was significantly upregulated after exercise training compared with the others groups (SAL\u0026thinsp;+\u0026thinsp;SED, SAL\u0026thinsp;+\u0026thinsp;EX and 6-OHDA\u0026thinsp;+\u0026thinsp;SED (**p\u0026thinsp;=\u0026thinsp;0,0133; *** p\u0026thinsp;=\u0026thinsp;0,0019 and *p\u0026thinsp;=\u0026thinsp;0,0443, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough there are a lot of clinical and experimental works using exercise to pain, the analgesic effect remains incomplete and unsatisfactory for both the patients and the clinicians, making it challenging to identify and propose specific treatment. In the context of orofacial pain in PD patients\u0026rsquo; knowledge is even more scarce. In a previous work we showed the involvement of cannabinoid receptors in orofacial analgesia induce by exercise (Binda et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The aim of this study was to investigate the effect of intrathecal injection of naloxone on mechanical allodynia pain behavior under 6-OHDA model of PD condition during the protocol of treadmill exercise and whether opioids receptors are involved in this effect.\u003c/p\u003e \u003cp\u003eOpioids are routinely prescribed to manage moderate to severe pain. They are effective against both spontaneous pain and mechanical allodynia (Grenald et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zubieta et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Naloxone, a competitive opioid receptor antagonist, is widely used in experimental pain research to evaluate the role of the endogenous opioid system in various nociceptive states (Benedetti et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In experimental models of pain, naloxone has been shown to produce paradoxical effects. For instance, high doses of naloxone typically induce hyperalgesia (increased pain sensitivity) and mechanical allodynia (Springborg et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and low doses of naloxone have been reported to enhance morphine-induced analgesia and even reduce hyperalgesia in rat models of neuropathic and inflammatory pain (Lewis et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsistent with our previous findings, behavioral analysis revealed that hemiparkinsonian rats presented a significant reduction in withdrawal thresholds in the von Frey test, confirming that the PD model induced orofacial mechanical allodynia. Notably, the exercise protocol progressively restored withdrawal thresholds to baseline levels after three exercise sessions, indicating a robust antinociceptive effect. However, on postoperative day 28, following six exercise sessions, intrathecal administration of naloxone abolished the exercise-induced analgesia, demonstrating the involvement of opioid receptor signaling in this response. These findings are further supported by Western blot analysis, which showed that exercise increased the expression of enkephalin as well as \u0026micro;- and δ-opioid receptors in the trigeminal ganglion compared with the PD sedentary group. Considering the inhibitory action of naloxone on opioid receptors (Benyamin et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Trescot et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), these results collectively indicate that activation of the endogenous opioid system plays a central role in mediating exercise-induced orofacial antinociception.\u003c/p\u003e \u003cp\u003eWidely recognized for its extensive health benefits, exercise enhances the opioid system by boosting endogenous opioids like endorphins and supporting in pain modulation (Belle et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). The analgesia induced by exercise appears to be mediated in part by the activation of endogenous opioid systems (enkephalins, endorphins and dynorphins), which act on mor, dor and kappa opioid-receptors (Da Silva Santos \u0026amp; Galdino, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Goldfarb et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur previous studies using this same model and protocol have demonstrated the involvement of cannabinoids in mediating exercise-induced orofacial analgesia in hemiparkinsonian rats (Binda et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research demonstrates a reciprocal relationship between the endogenous opioid and cannabinoid systems, where each system significantly contributes to the antinociceptive effects of the other (Bushlin et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cichewicz, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wilson-Poe et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It is known that opioids and cannabinoids exhibit overlapping neuroanatomical distribution and comparable functional neurobiological properties (Navarro et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The present findings indicate that exercise-induced analgesia depends on a highly regulated opioid response, requiring endogenous opioids to achieve and maintain sufficient concentrations to reduce pain. Taken together with our previous results, opioids and cannabinoids, were likely to act synergistically to reduce the orofacial pain in hemiparkinsonian rats.\u003c/p\u003e \u003cp\u003eThus, in a murine model of PD induced by the administration of 1.5 \u0026micro;l of a drug containing 9 \u0026micro;g of 6-OHDA which produces, allodynia to mechanical stimuli to orofacial region than 40 days we have demonstrated that: 1) hemiparkinsonian rat have a reduced head withdrawal threshold compared to control group, 2) EX group have restored the threshold compared to DP rats and 3) the intrathecal injection of naloxone during the exercise protocol vanish analgesia induced by EX and this antinociceptive effects produced by EX may be involved with the differential expression of enkephalin and \u0026micro;- and δ-opioid receptor during DP progression.\u003c/p\u003e \u003cp\u003eSome limitations warrant consideration: the lack of an opioid receptor-specific antagonist group, the absence of female group, the latter of which prevents the evaluation of sex differences in exercise-induced analgesia.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings demonstrate that exercise-induced orofacial analgesia in hemiparkinsonian rats is mediated by activation of the endogenous opioid system, as evidenced by increased expression of enkephalin and \u0026micro;- and δ-opioid receptors in the trigeminal ganglion. These neurochemical adaptations appear to contribute to the modulation of trigeminal nociceptive transmission, supporting endogenous opioid signaling as a key mechanism underlying exercise-induced antinociception in Parkinsonian conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures carried out in this study have been approved by the Institutional Animal Care and Use Committee of the University of S\u0026atilde;o Paulo (protocol number 4860310118) and were in compliance with the guidelines for animal care and use set forth by that committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Not applicable\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors have any potential or actual conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMartins, D.O., was the recipient of a FAPESP Postdoctoral scholarship (2015/24256-0). Binda K.H., was the recipient of a FAPESP Master scholarship (2017/26821-1). Chacur M., was the recipient of a FAPESP grant number (2017/05218-5, 2021/02897-4). The funding agencies play no role in the design of the study, data collection, analysis, interpretation of the data, or in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors made substantial contributions to the following aspects of this research: initial conception (Binda K.H., Chacur M., Martins D.O.); design (Binda K.H., Chacur M., Martins D.O.); provision of resources (Chacur M., Martins D.O.); collection of data (Binda K.H., Martins D.O.); analysis and interpretation of data (Binda K.H., Chacur M., Martins D.O.); writing the first draft of the paper or important intellectual content (Binda K.H., Chacur M., Martins D.O.); revision of the paper (Martins D.O.). All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Adilson da Silva Alves for technical assistance. This work was supported by FAPESP, Hospital S\u0026iacute;rio-Liban\u0026ecirc;s and CNPq; Martins D.O., was the recipient of a FAPESP Postdoctoral scholarship (Grant number: 2015/24256-0); Binda, K.H., was the recipient of a Master\u0026rsquo;s scholarship (Grant number: 2017/26821-1). Chacur, M., supported by FAPESP (Grant number: 2017/05218-5).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbolghasemi H, Shahani P, Mozafari R, Barikrow N, Yekta BG, Haghparast A (2025) The dopaminergic and opioidergic interactions in the nucleus accumbens in the suppression of pain affect: Exploring their impact on formalin-induced pain in rats. 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Science 293(5528):311\u0026ndash;315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1060952\u003c/span\u003e\u003cspan address=\"10.1126/science.1060952\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, Trigeminal pain, Treadmill Exercise, Rats, 6-OHDA, Opioid System","lastPublishedDoi":"10.21203/rs.3.rs-9452205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9452205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExercise-induced analgesia has been consistently associated with the activation of endogenous opioid pathways; however, the specific contribution of opioid receptor signaling to the reduction of orofacial pain in Parkinsonian conditions remains incompletely understood. Therefore, this study investigated whether pharmacological blockade of opioid receptors reverses the antinociceptive effect induced by exercise in a rat model of Parkinson\u0026rsquo;s disease (PD). Hemiparkinsonian rats were generated by unilateral 6-OHDA injection and subsequently submitted to a treadmill exercise protocol. To determine the involvement of endogenous opioids in exercise-induced analgesia, animals received the non-selective opioid receptor antagonist naloxone (1 mg/kg, i.p.), which exhibits high affinity for \u0026micro;-opioid receptors, on day 28 after lesion induction, following six exercise sessions. Mechanical nociceptive thresholds in the orofacial region were assessed using the von Frey test at 15 and 30 minutes after naloxone administration. In addition, protein expression of \u0026micro;- and δ-opioid receptors and the endogenous opioid peptide enkephalin was quantified in the trigeminal ganglion (TG) using Western blot analysis. We observed that the 6-OHDA\u0026thinsp;+\u0026thinsp;SED group showed a decrease in the mechanical threshold, which was reversed in the 6-OHDA\u0026thinsp;+\u0026thinsp;EX group. The antinociceptive effects of EX on orofacial pain were significantly reduced by naloxone, as showed in the behavioral test. Additionally, western blot analysis showed that the PD decreased the expression mu, delta and enkephalin in the TG and EX increased this expression. The current study shows that inhibiting opioid receptors, diminishes the facial antinociceptive effects of EX, indicating the involvement of opioid receptor in the analgesic effect of EX.\u003c/p\u003e","manuscriptTitle":"Naloxone Reversed Orofacial Analgesia Induced by Treadmill Exercise in a Rat Model of Parkinson's Disease: Exploring Opioid Receptors Involvement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 08:07:22","doi":"10.21203/rs.3.rs-9452205/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24fa26ab-acb0-4adc-996a-86bf667a0de4","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T08:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 08:07:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9452205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9452205","identity":"rs-9452205","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}