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Mechanical hyperalgesia/allodynia induced by injection of IL-1 into the Vi/Vc transition zone of the rat. A. An example of caudal brain stem section stained with cresyl violet for histological verification of the site of microinjection. Arrow indicates the injection site in ventral Vi/Vc. Scale bar = 500 μm. cc, central canal; Py, pyramidal tract; sp5, spinal trigeminal tract; Vi/Vc, trigeminal subnuclei interpolaris/caudalis transition zone. B. Stimulus-response function curves illustrating the intensity-dependent head withdrawal responses to mechanical stimuli. Each curve was established with a series of subthreshold to suprathreshold range of von Frey filament forces and the response frequency is plotted against the stimulus intensity. IL-1 (160 fmol) was injected into the ventral Vi/Vc. The skin site above the masseter muscle was probed. Note that there was a leftward shift of the curve at 30 min after IL-1 injection compared to the pre-IL-1 curve (p<0.01), suggesting the development of mechanical hyperalgesia and allodynia. Best-fit curves were generated by nonlinear regression analysis (GraphPad Prism). C, D. The EF 50 s were derived from the respective stimulus-response frequency function curves and are plotted against time. Note significant decreases in EF 50 s at 30 min-6h after IL-1 injection, indicating IL-1 induced bilateral hyperalgesia/allodynia. +, #: p<0.05; **, ++, ##: p<0.01 (ANOVA with repeated measures and post-hoc test). Dashed lines indicate interruption of the linearity of the time scale.
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Our recent studies indicate that the prototypic proinflammatory cytokine IL-1β is upregulated in astroglial cells in the trigeminal interplolaris/caudalis (Vi/Vc) transition zone, a region of the spinal trigeminal complex involved in trigeminal pain processing, after masseter muscle inflammation. Here we investigated the effect of microinjection of...
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Citations
... Complex signaling cascades, including both peripheral mechanisms (Ahn et al., 2009;Salas et al., 2009;Simonic-Kocijan et al., 2013;Xu et al., 2016) and mechanisms in the CNS, such as central sensitization in the MDH (Chiang et al., 2010;Iwata et al., 2011;Cao et al., 2013a,b;Wang et al., 2014;Chichorro et al., 2017;Sessle, 2021), underlie the establishment and maintenance of chronic orofacial pain. The RVM projections to the MDH are also involved in modulating trigeminal nociceptive transmission and nociceptive behavior (Sessle et al., 1981;Shimizu et al., 2009;Bryan et al., 2012). ...
The imbalanced conditions of pronociceptive ON-cells and antinociceptive OFF-cells in the rostral ventromedial medulla (RVM) alter nociceptive transmission and play an important role in the development of chronic pain. This study aimed to explore the neuroplastic mechanisms of the RVM ON-cells and OFF-cells in a male rat model of experimental occlusal interference (EOI)-induced nociceptive behavior reflecting orofacial hyperalgesia and in modified models involving EOI removal at early and later stages. We recorded the mechanical head withdrawal thresholds (HWTs), orofacial operant behaviors, and the activity of identified RVM ON-cells and OFF-cells in these rats. EOI-induced orofacial hyperalgesia could be relieved by EOI removal around postoperative day 3; this effect could be inhibited by intra-RVM microinjection of the kappa-opioid receptor agonist U-69593. EOI removal around postoperative day 8 did not relieve the orofacial hyperalgesia which could however be reversed by intra-RVM microinjection of the NK-1 receptor antagonist L-733060. The activity of ON-cells and OFF-cells did not change during both the initial 3 and 6 days of EOI. When EOI was removed on postoperative day 3, OFF-cell responses decreased, contributing to the reversal of hyperalgesia. When EOI lasted for 8 days or was removed on postoperative day 8, spontaneous activity and stimulus-evoked responses of ON-cell increased, contributing to the maintained hyperalgesia. In contrast, when the EOI lasted for 14 days, OFF-cell responses decreased, possibly participating in the maintenance of hyperalgesia with persistent EOI. Our results reveal that adaptive changes in the RVM were associated with orofacial pain following EOI placement and removal.SIGNIFICANCE STATEMENTA considerable proportion of patients suffered from chronic orofacial pain throughout life despite the therapies given or removal of potential etiological factors. However, current therapies lack effectiveness due to limited knowledge of the chronicity mechanisms. Using electrophysiological recording, combined with a behavioral test, we found that the prevailing descending facilitation in the rostral ventromedial medulla (RVM) participates in the maintenance of orofacial hyperalgesia following late removal of nociceptive stimuli, while the prevailing descending inhibition from the RVM may contribute to the reversal of orofacial hyperalgesia following early removal of nociceptive stimuli. Thus, variable clinical outcomes of orofacial pain may be associated with descending modulation and an optimal window of time may exist in the management of chronic orofacial pain.
... In addition, a wide variety of rat studies have reported the use of a series of calibrated von Frey filaments, with bending forces ranging from 8 mg to 112 g, to probe the facial site and determine an EF 50 value, which is defined as the von Frey filament force necessary to induce a 50% response frequency. 9,32,33 However, this method may not be optimal for mouse testing because it requires the serial application of more than a dozen filaments to the orofacial region, which can cause an uncomfortable, irritable reaction in mice, leading to potential injuries that could confound the experimental results. In this study, we used an ascending series of filaments, starting with the smallest filament, and terminated the test when the experimenter observed three positive responses out of five tests for a particular filament. ...
Orofacial pain or tenderness is a primary symptom associated with temporomandibular joint (TMJ) disorders (TMDs). To understand the pathological mechanisms underlying TMDs, several mouse models have been developed, including mechanical stimulus-induced TMD and genetic mouse models. However, a lack of feasible approaches for assessing TMD-related nociceptive behaviours in the orofacial region of mice has hindered the in-depth study of TMD-associated mechanisms. This study aimed to explore modifications of three existing methods to analyse nociceptive behaviours using two TMD mouse models: (1) mechanical allodynia was tested using von Frey filaments in the mouse TMJ region by placing mice in specially designed chambers; (2) bite force was measured using the Economical Load and Force (ELF) system; and (3) spontaneous feeding behaviour tests, including eating duration and frequency, were analysed using the Laboratory Animal Behaviour Observation Registration and Analysis System (LABORAS). We successfully assessed changes in nociceptive behaviours in two TMD mouse models, a unilateral anterior crossbite (UAC)-induced TMD mouse model and a β-catenin conditional activation mouse model. We found that the UAC model and β-catenin conditional activation mouse model were significantly associated with signs of increased mechanical allodynia, lower bite force, and decreased spontaneous feeding behaviour, indicating manifestations of TMD. These behavioural changes were consistent with the cartilage degradation phenotype observed in these mouse models. Our studies have shown reliable methods to analyse nociceptive behaviours in mice and may indicate that these methods are valid to assess signs of TMD in mice.
... Recent studies have demonstrated the important role of trigeminal interpolaris/caudalis (Vi/Vc) transition zone and rostral ventromedial medulla (RVM) in processing trigeminal pain arising from masseter muscle inflammation and in the development of bilateral hyperalgesia/allodynia. It has been suggested that contralateral hyperalgesia in the orofacial region is mediated through descending facilitatory mechanisms of the RVM-Vi/Vc circuitry (33)(34)(35). It has also been suggested that inhibition of glia and inflammatory cytokines cascade blockage following inflammation provides pain relief (35). ...
Aim:
To determine the relationship between bilateral allodynia induced by masseter inflammation and P2X3 receptor expression changes in trigeminal ganglia (TRG) and the influence of intramasseteric P2X3 antagonist administration on bilateral masseter allodynia.
Methods:
To induce bilateral allodynia, rats received a unilateral injection of complete Freund's adjuvant (CFA) into the masseter muscle. Bilateral head withdrawal threshold (HWT) was measured 4 days later. Behavioral measurements were followed by bilateral masseter muscle and TRG dissection. Masseter tissue was evaluated histopathologically and TRG tissue was analyzed for P2X3 receptor mRNA expression by using quantitative real-time polymerase chain reaction (PCR) analysis. To assess the P2X3 receptor involvement in nocifensive behavior, two doses (6 and 60 μg/50 μL) of selective P2X3 antagonist A-317491 were administrated into the inflamed masseter muscle 4 days after the CFA injection. Bilateral HWT was measured at 15-, 30-, 60-, and 120-minute time points.
Results:
HWT was bilaterally reduced after the CFA injection (P<0.001). Intramasseteric inflammation was confirmed ipsilaterally to the CFA injection. Quantitative real-time PCR analysis demonstrated enhanced P2X3 expression in TRG ipsilaterally to CFA administration (P<0.01). In comparison with controls, the dose of 6 μg of A-317491 significantly increased bilateral HWT at 15-, 30-, and 60-minute time points after the A-317491 administration (P<0.001), whereas the dose of 60 μg of A-317491 was efficient at all time points ipsilaterally (P=0.004) and at 15-, 30-, and 60-minute time points contralaterally (P<0.001).
Conclusion:
Unilateral masseter inflammation can induce bilateral allodynia in rats. The study provided evidence that P2X3 receptors can functionally influence masseter muscle allodynia and suggested that P2X3 receptors expressed in TRG neurons are involved in masseter inflammatory pain conditions.
... This is consistent with the bilateral effects in ION or inferior alveolar nerve injury models observed in other studies Miyamoto et al., 2011;Cao et al., 2013). The bilateral effects could derive from dysregulation of circulating factors such as pro-inflammatory cytokines (Anderson & Rao, 2001), nerve growth factors (Anderson et al., 1998;Anderson & Rao, 2001), or descending facilitatory mechanisms (Shimizu et al., 2009;Chai et al., 2012) by unilateral nerve injury. Since changes in these bilateral factors might not be specifically correlating with the onset or duration of neuropathic pain states post CCI-ION (Anderson & Rao, 2001), we only used CCI-ION rats with unilateral behavioral hypersensitivity in our studies so that we could focus on TSP4 effects in behavioral hypersensitivity at the injury side. ...
Injury to the trigeminal nerve often results in the development of chronic pain states including tactile allodynia, or hypersensitivity to light touch, in orofacial area, but its underlying mechanisms are poorly understood. Peripheral nerve injury has been shown to cause up-regulation of thrombospondin-4 (TSP4) in dorsal spinal cord that correlates with neuropathic pain development. In this study, we examined whether injury-induced TSP4 is critical in mediating orofacial pain development in a rat model of chronic constriction injury to the infraorbital nerve.
Orofacial sensitivity to mechanical stimulation was examined in a unilateral infraorbital nerve ligation rat model. The levels of TSP4 in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 spinal cord (Vc/C2) from injured rats were examined at time points correlating with the initiation and peak orofacial hypersensitivity. TSP4 antisense and mismatch oligodeoxynucleotides were intrathecally injected into injured rats to see if antisense oligodeoxynucleotide treatment could reverse injury-induced TSP4 up-regulation and orofacial behavioural hypersensitivity.
Our data indicated that trigeminal nerve injury induced TSP4 up-regulation in Vc/C2 at a time point correlated with orofacial tactile allodynia. In addition, intrathecal treatment with TSP4 antisense, but not mismatch, oligodeoxynucleotides blocked both injury-induced TSP4 up-regulation in Vc/C2 and behavioural hypersensitivity.
Our data support that infraorbital nerve injury leads to TSP4 up-regulation in trigeminal spinal complex that contributes to orofacial neuropathic pain states. Blocking this pathway may provide an alternative approach in management of orofacial neuropathic pain states.
... Despite the emphasis of orofacial pain research in the Vc, the subnucleus interpolaris/subnucleus caudalis transition zone (Vi/Vc) of the STN has been more recently shown to be involved in mechanisms of deep tissue trigeminal pain891011. We have shown that complete Freund's adjuvant (CFA)-induced masseter inflammation and intra-Vi/Vc microinjection of the pro-inflammatory cytokine, interleukin-1β (IL-1β), induce contralateral orofacial hyperalgesia in rat [12,13] . However, contralateral hyperalgesia is not seen with intra-Vc IL-1β microinjection. ...
... Similar to the behavioral findings, our previous studies showed that CFA-induced masseter inflammation results in bilateral expression of Fos protein in the Vi/Vc and ipsilateral expression of Fos protein in the Vc [14]. We have also shown that IL-1β-induced contralateral hyperalgesia is attenuated with ibotenic acid lesions in the rostral ventromedial medulla (RVM), a midline site that is critical in descending pain modulation [12]. Our previous studies have shown that bilateral reciprocal connections exist from the Vi/Vc to the RVM [14]. ...
... Our previous studies have shown that CFA injection into the masseter muscle and IL-1β injections into the Vi/Vc transition zone generate bilateral mechanical hyperalgesia as seen by the significant decreases in EF 50 values [12,13] . To test whether activation of IL-1 receptors (IL- 1R) in the Vi/Vc is involved in CFA-induced contralateral hyperalgesia, an IL-1R antagonist (IL-1ra) was injected. ...
Background
Our previous studies have shown that complete Freund’s adjuvant (CFA)-induced masseter inflammation and microinjection of the pro-inflammatory cytokine interleukin-1β (IL-1β) into the subnucleus interpolaris/subnucleus caudalis transition zone of the spinal trigeminal nucleus (Vi/Vc) can induce contralateral orofacial hyperalgesia in rat models. We have also shown that contralateral hyperalgesia is attenuated with a lesion of the rostral ventromedial medulla (RVM), a critical site of descending pain modulation. Here we investigated the involvement of the RVM-Vi/Vc circuitry in mediating contralateral orofacial hyperalgesia after an injection of CFA into the masseter muscle.
Results
Microinjection of the IL-1 receptor antagonist (5 nmol, n=6) into the ipsilateral Vi/Vc attenuated the CFA-induced contralateral hyperalgesia but not the ipsilateral hyperalgesia. Intra-RVM post-treatment injection of the NK1 receptor antagonists, RP67580 (0.5-11.4 nmol) and L-733,060 (0.5-11.4 nmol), attenuated CFA-induced bilateral hyperalgesia and IL-1β induced bilateral hyperalgesia. Serotonin depletion in RVM neurons prior to intra-masseter CFA injection prevented the development of contralateral hyperalgesia 1–3 days after CFA injection. Inhibition of 5-HT3 receptors in the contralateral Vi/Vc with direct microinjection of the select 5-HT3 receptor antagonist, Y-25130 (2.6-12.9 nmol), attenuated CFA-induced contralateral hyperalgesia. Lesions to the ipsilateral Vc prevented the development of ipsilateral hyperalgesia but did not prevent the development of contralateral hyperalgesia.
Conclusions
These results suggest that the development of CFA-induced contralateral orofacial hyperalgesia is mediated through descending facilitatory mechanisms of the RVM-Vi/Vc circuitry.
... When an IL-1 receptor antagonist (IL-1ra) was administered intrathecally via osmotic pumps through a cannula implanted at the level of the obex prior to the induction of masseter inflammation, the behavioral allodynia and hyperalgesia were significantly attenuated (Guo et al., 2007). Direct injection of IL-1beta into the ventral Vi/Vc transition zone produces mechanical allodynia and hyperalgesia (Shimizu et al.,2009a). ...
... These findings show that the effect of IL-1beta on NMDAR phosphorylation is attributable to the post-translational regulation through IL-1R signaling; and that the intermediate pathway involves PLC, PLA2 and subsequent PKC activation and intracellular Ca 2+ release. Direct administration of IL-1beta into the ventral trigeminal transition zone produced orofacial hyperalgesia that lasted for hours (Shimizu et al., 2009a). Pretreatment with glial inhibitors does not block IL-1beta-induced hyperalgesia even with relatively high doses that have been shown to be effective in attenuating hyperalgesia after injury (Wei et al., 2008; Shimizu et al., 2009b). ...
... Behavioral studies confirm the functional significance of the RVM-Vi/Vc connections (Sugiyo et al., 2005; Shimizu et al., 2009a). Unilateral CFA-induced inflammation of the masseter muscle produces mechanical allodynia and hyper-algesia in the orofacial region overlapping the masseter muscle. ...
Previous studies have established the role of the medullary dorsal horn or the subnucleus caudalis of the spinal trigeminal complex, a homolog of the dorsal horn of the spinal cord, in trigeminal pain processing. In addition to the medullary dorsal horn, recent studies have pointed out increased excitability and sensitization of trigeminal interpolaris and caudalis transition zone (Vi/Vc) following deep orofacial injury, involving neuron-glia-cytokine interactions. The Vi/Vc transition zone accesses rostral brain regions that are important for descending pain modulation, and somatovisceral and somatoautonomic processing and plays a unique role in coordinating trigeminal nocifensive responses.
... We used EF 50 values as a measure of mechanical sensitivity. A leftward shift of the S-R curve [9,18,19], resulting in a reduction of EF 50 , occurred after ligation of the TASM. This shift of the curve suggests the presence of mechanical hyperalgesia and allodynia since there was an increase in response to suprathreshold stimuli and a decreased response threshold for nocifensive behavior. ...
... Activation of central pain facilitatory pathways also leads http://www.molecularpain.com/content/6/1/40 Page 11 of 12 to secondary hyperalgesia [9,19]. Assuming a further diminished role of primary afferent input during the later phase of tendon injury, it would be interesting to determine whether persistence of hyperalgesia seen in the present model relies on a transition from peripheral to central mechanisms. ...
A major subgroup of patients with temporomandibular joint (TMJ) disorders have masticatory muscle hypersensitivity. To study myofacial temporomandibular pain, a number of preclinical models have been developed to induce myogenic pain of the masseter muscle, one of the four muscles involved in mastication. The currently used models, however, generate pain that decreases over time and only lasts from hours to weeks and hence are not suitable for studying chronicity of the myogenic pain in TMJ disorders. Here we report a model of constant myogenic orofacial pain that lasts for months.
The model involves unilateral ligation of the tendon of the anterior superficial part of the rat masseter muscle (TASM). The ligation of the TASM was achieved with two chromic gut (4.0) ligatures via an intraoral approach. Nocifensive behavior of the rat was assessed by probing the skin site above the TASM with a series of von Frey filaments. The response frequencies were determined and an EF50 value, defined as the von Frey filament force that produces a 50% response frequency, was derived and used as a measure of mechanical sensitivity. Following TASM ligation, the EF50 of the injured side was significantly reduced and maintained throughout the 8-week observation period, suggesting the presence of mechanical hyperalgesia/allodynia. In sham-operated rats, the EF50 of the injured side was transiently reduced for about a week, likely due to injury produced by the surgery. Somatotopically relevant Fos protein expression was indentified in the subnucleus caudalis of the spinal trigeminal sensory complex. In the same region, persistent upregulation of NMDA receptor NR1 phosphorylation and protein expression and increased expression of glial markers glial fibrillary acidic protein (astroglia) and CD11b (microglia) were found. Morphine (0.4-8 mg/kg, s.c.) and duloxetine (0.4-20 mg/kg, i.p.), a selective serotonin-norepinephrine reuptake inhibitor, produced dose-dependent attenuation of hyperalgesia.
Ligation injury of the TASM in rats led to long-lasting and constant mechanical hypersensitivity of myogenic origin. The model will be particularly useful in studying the chronicity of myogenic pain TMJ disorders. The model can also be adapted to other regions of the body for studying pathology of painful tendinopathy seen in sports injury, muscle overuse, and rheumatoid arthritis.
Peripheral and central sensitizations of the trigeminal nervous system are the main mechanisms to promote the development and maintenance of chronic orofacial pain characterized by allodynia, hyperalgesia, and ectopic pain after trigeminal nerve injury or inflammation. Although the pathomechanisms of chronic orofacial pain are complex and not well known, sufficient clinical and preclinical evidence supports the contribution of the N-methyl-D-aspartate receptors (NMDARs, a subclass of ionotropic glutamate receptors) to the trigeminal nociceptive signal processing pathway under various pathological conditions. NMDARs not only have been implicated as a potential mediator of pain-related neuroplasticity in the peripheral nervous system (PNS) but also mediate excitatory synaptic transmission and synaptic plasticity in the central nervous system (CNS). In this review, we focus on the pivotal roles and mechanisms of NMDARs in the trigeminal nervous system under orofacial neuropathic and inflammatory pain. In particular, we summarize the types, components, and distribution of NMDARs in the trigeminal nervous system. Besides, we discuss the regulatory roles of neuron-nonneuronal cell/neuron-neuron communication mediated by NMDARs in the peripheral mechanisms of chronic orofacial pain following neuropathic injury and inflammation. Furthermore, we review the functional roles and mechanisms of NMDARs in the ascending and descending circuits under orofacial neuropathic and inflammatory pain conditions, which contribute to the central sensitization. These findings are not only relevant to understanding the underlying mechanisms, but also shed new light on the targeted therapy of chronic orofacial pain.
The Chronic Constriction Injury of the Infraorbital Nerve (CCI-ION) is a well-established model to study facial sensory changes related to trigeminal neuropathic pain. CCI-ION induces heat hypersensitivity that resolves within 2-3 weeks and a delayed mechanical hypersensitivity that emerges during the second week post-injury. The role of descending facilitatory pain pathways from the rostro ventromedial medulla (RVM) in mediating the heat and tactile hypersensitivity was examined. CCI-ION induced heat hypersensitivity observed 5days post-surgery was reversed by systemic, but not RVM lidocaine. CCI-ION-induced tactile hypersensitivity observed 15days post-surgery was reversed by systemic lidocaine and attenuated by RVM lidocaine. CCI-ION-induced spontaneous pain was determined using conditioned place preference (CPP) to pain relief at each time-point. At day 5 post-CCI-ION, neither systemic nor RVM lidocaine induced CPP. However, at 15days post-CCI-ION, CPP was observed to the chamber paired with RVM lidocaine, but not systemic lidocaine. These data indicate that CCI-ION induced heat hypersensitivity is not dependent on descending facilitatory pain pathways 5-days post-injury whereas descending facilitatory pain pathways mediate tactile allodynia and spontaneous pain 15days post-CCI-ION. This suggests that CCI-ION induces early peripheral sensitization followed by development of central sensitization that mediates spontaneous pain and contributes to mechanical hypersensitivity.
Recent studies show that peripheral injury activates both neuronal and nonneuronal or glial components of the peripheral and central cellular circuitry. The subsequent neuron-glia interactions contribute to pain hypersensitivity. This review will briefly discuss novel findings that have shed light on the cellular mechanisms of neuron-glia interactions in persistent pain.
Two fundamental questions related to neuron-glia interactions in pain mechanisms have been addressed: what are the signals that lead to central glial activation after injury and how do glial cells affect central nervous system neuronal activity and promote hyperalgesia?
Evidence indicates that central glial activation depends on nerve inputs from the site of injury and release of chemical mediators. Hematogenous immune cells may migrate to/infiltrate the brain and circulating inflammatory mediators may penetrate the blood-brain barrier to participate in central glial responses to injury. Inflammatory cytokines such as interleukin-1beta released from glia may facilitate pain transmission through its coupling to neuronal glutamate receptors. This bidirectional neuron-glia signaling plays a key role in glial activation, cytokine production and the initiation and maintenance of hyperalgesia. Recognition of the contribution of the mutual neuron-glia interactions to central sensitization and hyperalgesia prompts new treatment for chronic pain.
