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Objective: To investigate the influence of chronic masseter hyperalgesia induced by 17β-estradiol (E2) and experimental occlusal interference (EOI) on underlying mechanism in hippocampus of ovariectomized (OVX) rats., Methods: In the study, 32 OVX rats were randomly divided into 4 groups (8 rats/group): The control group was OVX group, and 0 μg/d E2 (vehicle) injection was started 7 d after OVX without EOI; in the experimental group (1) OVX + E2 group, 80 μg/d E2 injection was started 7 d after OVX without EOI; in the experimental group (2) OVX + EOI group, vehicle injection was started 7 d after OVX and EOI was applied 17 d after OVX; in the experimental group (3) OVX + E2 + EOI group, 80 μg/d E2 injection was started 7 d after OVX and EOI was applied 17 d after OVX. Bilateral masseter muscle mechanical withdrawal thresholds were measured before OVX, 7 days after OVX (before E2 injection), 17 days after OVX (10 days after E2 injection and before EOI) and 24 days after OVX (7 days after EOI). Immunofluorescence staining was used to reveal phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2)-positive neurons in CA3 of hippocampus. The protein expression of p-ERK1/2 in hippocampus was detected using Western Blot., Results: Compared with the control group [left side: (135.3±8.5) g, right side: (135.4±10.8) g], bilateral masseter muscle mechanical withdrawal thresholds of OVX+E2 group [left side: (113.3±5.6) g, right side: (112.5 ± 5.6) g] and OVX+EOI group [left side: (93.3±5.4) g, right side: 90.8±5.5) g] were decreased ( P < 0.01). Bilateral masseter muscle mechanical withdrawal thresholds were significantly lower in OVX+E2+EOI group [left side: (81.2±6.2) g, right side: 79.8±7.7) g] than in the control, OVX+E2 and OVX+EOI groups ( P < 0.05). The proportion of p-ERK1/2 positive neurons in the CA3 region of the hippocampus was increased in the control, OVX+E2, OVX+EOI and OVX+E2+EOI groups in turn, and the difference between the groups was statistically significant ( P < 0.05). p-ERK1/2 protein expression was increased in the control, OVX+E2 and OVX+EOI groups in turn, but the difference was not statistically significant ( P >0.05). p-ERK1/2 expression was significantly higher in OVX+E2+EOI group than in the other three groups ( P < 0.05)., Conclusion: High concentration of E2 could exacerbated EOI-induced chronic masseter hyperalgesia in ovariectomized rats, and its central mechanism may be related to the upregulation of the phosphorylation of ERK1/2 in hippocampus.

Acute occlusal interference following improper occlusal alteration in dental practice can induce chronic masticatory muscle pain. The underlying mechanism has not been clarified. Synaptic plasticity in the anterior cingulate cortex (ACC) plays a key role in the chronic pain state. This study investigated the role of synaptic plasticity in the ACC in acute occlusal interference-induced chronic masticatory muscle pain. A rat model of experimental occlusal interference (EOI) was established. In vivo local field potential (LFP) recording was conducted to evaluate the change of synaptic strength and plasticity from the medial thalamus (MT) to the ACC after EOI application. The effects of microdialysis of antagonists of glutamate receptors into the ACC on synaptic transmission from the MT to the ACC were examined. Furthermore, the influence of inhibiting glutamate receptors in the ACC on EOI-induced mechanical hyperalgesia in the masseter muscles of rats was investigated. The amplitude of LFP in the ACC evoked by MT stimulation was significantly potentiated since 14 d of EOI application. Long-term potentiation of LFP in the ACC was reliably induced by theta burst stimulation to the MT in control rats but was occluded in 14-d EOI rats. Microdialysis of AMPA/kainate receptor antagonist CNQX into the ACC attenuated LFP in the ACC evoked by stimulating the MT in control and EOI rats. Administration of NMDA receptor subunit NR2B antagonist Ro 25-6981 into the ACC significantly alleviated the potentiation of MT stimulation-evoked LFP in the ACC of EOI rats without affecting that in control rats. EOI-induced hyperalgesia in the bilateral masseter muscles of rats was dose-dependently relieved after microdialysis of Ro 25-6981 into ACC. These findings provide direct evidence that prolonged acute occlusal interference potentiates synaptic transmission in the ACC, which in turn mediates chronic masticatory muscle pain.

Objective: To investigate the effect of long-term resistance exercise of hindlimb on mechanical hyperalgesia of bilateral masseter muscle in rats with or without occlusal interference., Methods: Six-teen male Sprague-Dawley rats (220-250 g) were randomly divided into four groups: the naive control group, naive exercise group, occlusal interference control group, and occlusal interference exercise group. The rats in occlusal interference groups (occlusal interference control group and occlusal interference exercise group) obtained occlusal interference with 0.4 mm-thick crowns bonded to the right maxillary first molars. The rats in exercise groups (naive exercise group and occlusal interference exercise group) performed squat-type resistance exercises for 30 minutes, once a day, 5 days/week, lasting for 14 weeks. Resistance exercise was recorded every day. Mechanical withdrawal thresholds of bilateral masseter muscle were tested per week by use of modified electronic von-frey anesthesiometer. The rats were weighed per week. After the 14-week exercise, the muscle strength of the hindlimb was tested with a grip strength meter. Muscle (gastrocnemius and soleus) weight of bilateral hindlimb and length of bilateral fibula of the rats were obtained. The muscle-mass/body-mass ratios and muscle-mass/fibula-length ratios were calculated., Results: Between the naive control group and naive exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-4 weeks (P>0.05). During the 5-14 weeks, the mechanical withdrawal thresholds of the rats in the naive exercise group were higher than those in the naive control group (P<0.05). Between the occlusal interference control group and occlusal interference exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-6 weeks (P>0.05). During the 7-14 weeks, the mechanical withdrawal thresholds of rats in the naive exercise group were higher than those in the occlusal interference control group (P<0.05). After the 14week exercise, the body mass of the rats in nonexercise group (the naive control group and occlusal interference control group) were larger than those in exercise group [(462±6) g vs. (418±14) g, P<0.05]. And the muscle strength of hindlimb of the rats in exercise group were bigger than those in non-exercise group [(6.75±0.13) N vs. (5.41±0.15) N, P<0.01]., Conclusion: long-term resistance exercise can increase mechanical withdrawal thresholds of the bilateral masseter muscle in rats with or without masseter muscle mechanical hyperalgesia.

Objective: To investigate the effect of occlusal interference on the energy metabolism of masticatory muscle by studying the changes of adenosine triphosphate (ATP), adenosine diphosphate (ADP), inosine monophosphate (IMP), phosphocreatine, creatine, lactate and pH level in masseter muscles of rats after occlusal interference., Methods: Fifty male Sprague-Dawley rats were randomly assigned into experimental group (n=40) and control group (n=10). In experimental group, 0.4 mm thick metal crown was cemented to the upper right first molar of the rat, and maintained for 3, 7, 10, 14 d separately (n=10 for each time point). No occlusal interference was applied for control group. Bilateral masseter muscles of all the rats were acquired under general anesthesia. The samples of 5 rats in each group were fully homogenized with 0.4 mol/L perchlorate (10 mL/g). The homogenates were centrifuged, filtered and analyzed for ATP, ADP, IMP, phosphocreatine, creatine and lactate content by high performance liquid chromatography. The other samples in each group were mixed with homogenates containing 5 mmol/L sodium iodoacetate (10 mL/g), then homogenized and measured for pH value by pH meter in thermostatic water bathunder 37 degrees centigrade., Results: Compared with control group, ATP content in bilateral masseter of the rats increased 3 d after occlusal interference [right side:(5.36±0.13) μmol/g,left side:(5.77±0.25) μmol/g] (P<0.05), and back to normal on 7, 10 and 14 d. There was an increase in IMP [right side:(0.21±0.03) μmol/g,left side:(0.19±0.03) μmol/g]and creatine content [right side:(24.76±2.94) μmol/g,left side:(27.75±2.23) μmol/g]in bilateral masseter of the rats 7 d after occlusal interference (P<0.05) and no difference was detected on 3, 10, and 14. Phosphocreatine content in bilateral masseter started to decline 7 d after occlusal interference and maintained the low level on 10 and 14 d [right side:(10.70±0.71) μmol/g, (11.57±0.52) μmol/g, (10.74±1.39) μmol/g, left side:(10.05±0.57) μmol/g, (10.75±1.12)μmol/g, (10.61±1.15) μmol/g](P<0.05). No change of ADP, lactate or pH level in bilateral muscles of the rats after occlusal interference was observed (P>0.05)., Conclusion: Occlusal interference influences the content of energy metabolites in masticatory muscle of rats, which may be related to the pathological process of masticatory muscles induced by occlusal interference, such as muscle pain, dysfunction and altered fiber architecture.

Background: Masticatory muscle pain may occur following immediate occlusal alteration by dental treatment. The underlying mechanisms are poorly understood. Transient receptor potential vanilloid-1 (TRPV1) and acid-sensing ion channel-3 (ASIC3) mediate muscle hyperalgesia under various pathologic conditions. We have developed a rat model of experimental occlusal interference (EOI) that consistently induces mechanical hyperalgesia in jaw muscles. Whether TRPV1 and ASIC3 mediate this EOI-induced hyperalgesia is unknown., Methods: Rat model of EOI-induced masseter hyperalgesia was established. Real-time polymerase chain reaction, Western blot and retrograde labelling combined with immunofluorescence were performed to evaluate the modulation of TRPV1 and ASIC3 expression in trigeminal ganglia (TGs) and masseter afferents of rats after EOI. The effects of intramuscular administration of TRPV1 and ASIC3 antagonists on the EOI-induced hyperalgesia in masseter muscle were examined., Results: After EOI, gene expressions and protein levels of TRPV1 and ASIC3 in bilateral TGs were up-regulated. The percentage of ASIC3- (but not TRPV1-) positive neurons in masseter afferents increased after EOI. More small-sized and small to medium-sized masseter afferents expressed TRPV1 and ASIC3 separately following EOI. These changes peaked at day 7 and then returned to original status within 10 days after EOI. Intramuscular administration of the TRPV1 antagonist AMG-9810 partially reversed this mechanical hyperalgesia in masseter muscle. No improvement was exhibited after administration of the ASIC3 antagonist APETx2. Co-injection of AMG-9810 and APETx2 enhanced the effect of AMG-9810 administration alone., Conclusions: Peripheral TRPV1 and ASIC3 contribute to the development of the EOI-induced mechanical hyperalgesia in masseter muscle., (© 2015 European Pain Federation - EFIC®)

Objective: To investigate the peripheral mechanism by studying the histological changes of masseter muscles using HE stains and substance P (SP) and protein gene product 9.5 (PGP9.5) immunohistochemical stains., Methods: Fifteen male Sprague-Dawley were randomly assigned into occlusal interference group (n=12) and control group (n=3). In occlusal interference group, 0.4 mm thick crowns were bonded to the rats' first molar of the maxillary. In the control group, rats were anesthetized and mouths were forced open for about 5 min but restorations were not applied. 1, 5, 10, and 21 d after 0.4 mm occlusal alteration treatment, mechanical pain thresholds of bilateral masseter muscles were quantitatively measured by modified electronic anesthesiometer in control group and occlusal interference group. The rats were euthanized by transcardiac perfusion after deep anesthetization at different time points. The paraffin sections of masseter muscles were made and processed for HE, SP, and PGP9.5 immunohistochemical staining., Results: Decreased head withdrawal threshold to mechanical pressure was detected in masseter muscles on both sides following occlusal interference. Histological stains of masseter muscles presented intact following occlusal interference, and no inflammatory cells were observed in both sides. Intensely stained PGP9.5 was observed at 1 d in occlusal interference groups and maintained until the end of the experiment. SP expression was the most obviously increased at 5 d in both sides and gradually decreased to the level of control., Conclusion: Experimental occlusal interference-induced masticatory muscle pain is associated with peripheral sensitization of nociceptive neurons rather than muscle damage and inflammation.