Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain.

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Functional connectivity of hypothalamus in chronic migraine with medication overuse.

OBJECTIVE: To investigate the functional connectivity of the hypothalamus in chronic migraine compared to interictal episodic migraine in order to improve our understanding of migraine chronification. METHODS: Using task-free fMRI and ROI-to-ROI analysis, we compared anterior hypothalamus intrinsic connectivity with the spinal trigeminal nucleus in patients with chronic migraine (n = 25) to age- and sex-matched patients with episodic migraine in the interictal phase (n = 22). We also conducted a seed-to-voxel analysis with anterior hypothalamus as a seed. RESULTS: All patients with chronic migraine had medication overuse. We found a significant connectivity (T = 2.08, p = 0.024) between anterior hypothalamus and spinal trigeminal nucleus in the chronic group, whereas these two regions were not connected in the episodic group. The strength of connectivity was not correlated with pain intensity (rho: 0.09, p = 0.655). In the seed-to-voxel analysis, three regions were more connected with the anterior hypothalamus in the chronic group: The spinal trigeminal nuclei (MNI coordinate x = 2, y = -44, z = -62), the right dorsal anterior insula (MNI coordinate x = 10, y = 10, z = 18), and the right caudate (MNI coordinate x = 12, y = 28, z = 6). However, these correlations were no longer significant after whole brain FWE correction. CONCLUSION: An increased functional connectivity between the anterior hypothalamus and the spinal trigeminal nucleus, as previously reported in preictal episodic migraine, was demonstrated in chronic migraine with medication overuse. This finding confirms a major role of the anterior hypothalamus in migraine and suggests that chronic migraineurs are locked in the preictal phase.

Visual stimulation leads to activation of the nociceptive trigeminal nucleus in chronic migraine.

OBJECTIVE: The visual system has often been described to be sensitized in migraineurs, with light being perceived as aversive or even painful. One possible explanation for this altered perception is crosslinks between the visual and the trigeminonociceptive system. Visual stimulation in chronic migraineurs on the level of the brainstem might lead to enhanced activity within the spinal trigeminal nucleus (sTN) as the main site of trigeminal pain processing within this area. METHODS: Eighteen episodic migraineurs (EM), 17 chronic migraineurs (CM), and 19 healthy controls (HC) underwent one session of high-resolution brainstem imaging during which a rotating checkerboard was presented repeatedly as a visual stimulus. Data were analyzed using SPM12 and MATLAB with the classic first-level-second-level approach of SPM. Analyses of variance were used for group comparisons. RESULTS: CM showed enhanced activation within the sTN as compared to HC. In addition, we observed enhanced activity within the right superior colliculus in CM as compared to HC. When comparing all migraineurs with headaches during scanning with all migraineurs without headaches during scanning and HC, we also found the sTN to be more strongly activated during headaches. CONCLUSION: Our data provide evidence for the existence of visual-nociceptive integration on brainstem level in chronic migraineurs.

Trigeminal long-term potentiation as a cellular substrate for migraine.

Most previous studies suggest that the subnucleus caudalis (Vc) of spinal trigeminal nucleus (Vsp) plays a key role in the generation and maintenance of migraine, a type of primary headache, by participating in the trigeminovascular system. Furthermore, the excitability of the Vc with the stimulation of the peripheral nociceptive fibers innervating the intracranial vessels or dura matter is regarded as a main cellular substrate for migraine. Here, a revised hypothesis is introduced, reinforcing the previous hypothesis and complementing it. This hypothesis suggests that, besides the Vc, much broader areas of the trigeminal sensory nuclei (Vsn), i.e., the principal sensory nucleus (Vp), the oralis nucleus (Vo), and the interpolaris nucleus (Vi), contribute to process and integrate pain signals generated in the head. In addition, the plasticity of synaptic transmission between nuclei or subnuclei in the Vsn, in particular, the Vsp, can be a cellular model for migraine, in the same way as the hippocampal synaptic plasticity is a model for learning and memory. This hypothesis will contribute to the discovery of new therapeutic tools for patients with migraine.

Blockade of 5-HT3 receptors with granisetron does not affect trigeminothalamic nociceptive transmission in rats: Implication for migraine.

One way to expand the existing range of anti-migraine drugs seems to be the search for pharmacological agents with anti-cephalalgic properties among medicines approved for clinical indications other than migraine. Numerous experimental and clinical data imply that selective serotonin 5-HT3 receptor antagonists can be considered as potential anti-migraine agents. Therefore, the objective of our work was to examine the impact of selective 5-HT3 receptor blockade with granisetron on migraine-related nociceptive transmission within the spinal trigeminal nucleus (STN) and the ventroposteromedial nucleus of the thalamus (VPM). Using an electrophysiological model of trigemino-durovascular nociception in anaesthetised male Wistar rats, we evaluated the effects of intravenous administration of granisetron on ongoing firing and dural electrical stimulation-evoked responses of the spinal trigeminal and thalamic cells. Granisetron did not substantially affect responses of the STN and VPM neurons to electrical stimulation of the dura mater as well as did not cause steady changes in ongoing firing of the spinal trigeminal cells. The results obtained argue against the use of 5-HT3 receptor antagonists for treating migraine. These data also lead to the conclusion that in the absence of sustained sensitisation of neurons along the trigemino-thalamo-cortical pathway the role of 5-HT3 receptor-dependent mechanisms in serotonergic modulation of trigeminovascular nociceptive transmission can hardly be considered crucial.

[The effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis in rats].

Objective: To investigate the effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis (Vc). Methods: The animals were randomly divided into the control group, 1-, 3-, 5-, 7-, 9-, 11-and 14-day stress groups, with 10 rats in each group. The body weight increase and behavior tests were used to testify the animal model. The mechanical sensitivity of masseter of the rat before and after the stress was measured with Von Frey filaments. Histological examinations were used to evaluate the expression of neuronal c-fos and astrocytic glial fibrillary acidic protein (GFAP). Results: Restraint stress resulted in remarkable mechanical allodynia in the masseter muscle. The head withdrawal threshold was significantly lower in the 7-, 9-, 11-and 14-day stress groups ([0.071±0.011], [0.059±0.020], [0.052±0.011], [0.033±0.011] N) than that in the control group ([0.120±0.025] N) (P<0.05). Compared to the control group, the rats in the 1-day stress group showed a significant increase of c-fos in neurons of the Vc and then declined to normal level after 1 week gradually. The GFAP expression in astrocytes of the Vc was significantly increased in the 7-, 9-, 11-and 14-day stress groups (4.3±1.0, 4.5±0.6, 4.6±0.5, 4.8±1.3) compared with the control group (2.0±0.8) (P<0.05). Conclusions: Chronic restraint stress could lower the threshold of mechanical allodynia in the masseter muscle and activate the neurons and astrocytes in Vc. The activation of neurons and astrocytes plays an important role in the masseter hyperalgesia induced by restraint stress in rats.

Inhibitory effect of high-frequency greater occipital nerve electrical stimulation on trigeminovascular nociceptive processing in rats.

Electrical stimulation of the greater occipital nerve (GON) has recently shown promise as an effective non-pharmacological prophylactic therapy for drug-resistant chronic primary headaches, but the neurobiological mechanisms underlying its anticephalgic action are not elucidated. Considering that the spinal trigeminal nucleus (STN) is a key segmental structure playing a prominent role in pathophysiology of headaches, in the present study we evaluated the effects of GON electrical stimulation on ongoing and evoked firing of the dura-sensitive STN neurons. The experiments were carried out on urethane/chloralose-anesthetized, paralyzed and artificially ventilated male Wistar rats. Extracellular recordings were made from 11 neurons within the caudal part of the STN that received convergent input from the ipsilateral facial cutaneous receptive fields, dura mater and GON. In each experiment, five various combinations of the GON stimulation frequency (50, 75, 100 Hz) and intensity (1, 3, 6 V) were tested successively in 10 min interval. At all parameter sets, preconditioning GON stimulation (250 ms train of pulses applied before each recording) produced suppression of both the ongoing activity of the STN neurons and their responses to electrical stimulation of the dura mater. The inhibitory effect depended mostly on the GON stimulation intensity, being maximally pronounced when a stimulus of 6 V was applied. Thus, the GON stimulation-induced inhibition of trigeminovascular nociceptive processing at the level of STN has been demonstrated for the first time. The data obtained can contribute to a deeper understanding of neurophysiological mechanisms underlying the therapeutic efficacy of GON stimulation in primary headaches.

The migraine generator revisited: continuous scanning of the migraine cycle over 30 days and three spontaneous attacks.

Functional imaging using positron emission tomography and later functional magnetic resonance imaging revealed a particular brainstem area that is believed to be specifically activated in migraine during, but not outside of the attack, and consequently has been coined the 'migraine generator'. However, the pathophysiological concept behind this term is not undisputed and typical migraine premonitory symptoms such as fatigue and yawning, but also a typical association of attacks to circadian and menstrual cycles, all make the hypothalamus a possible regulating region of migraine attacks. Neuroimaging studies investigating native human migraine attacks however are scarce and for methodological but also clinical reasons there are currently no studies investigating the last 24 h before headache onset. Here we report a migraine patient who had magnetic resonance imaging every day for 30 days, always in the morning, to cover, using functional imaging, a whole month and three complete, untreated migraine attacks. We found that hypothalamic activity as a response to trigeminal nociceptive stimulation is altered during the 24 h prior to pain onset, i.e. increases towards the next migraine attack. More importantly, the hypothalamus shows altered functional coupling with the spinal trigeminal nuclei and the region of the migraine generator, i.e. the dorsal rostral pons during the preictal day and the pain phase of native human migraine attacks. These data suggest that although the brainstem is highly linked to the migraine biology, the real driver of attacks might be the functional changes in hypothalamo-brainstem connectivity.

Trigeminal neuroplasticity underlies allodynia in a preclinical model of mild closed head traumatic brain injury (cTBI).

Post-traumatic headache (PTH) following TBI is a common and often persisting pain disability. PTH is often associated with a multimodal central pain sensitization on the skin surface described as allodynia. However, the particular neurobiology underlying cTBI-induced pain disorders are not known. These studies were performed to assess trigeminal sensory sensitization and to determine if sensitization measured behaviorally correlated with detectable changes in portions of the trigeminal sensory system (TSS), particularly trigeminal nucleus, thalamus, and sensory cortex. Thermal stimulation is particularly well suited to evaluate sensitization and was used in these studies. Recent advances in the use of reward/conflict paradigms permit use of operant measures of behavior, versus reflex-driven response behaviors, for thermal sensitization studies. Thus, to quantitate facial thermal sensitization (allodynia) in the setting of acute TBI, the current study utilized an operant orofacial pain reward/conflict testing paradigm to assess facial thermal sensitivity in uninjured control animals compared with those two weeks after cTBI in a rodent model. Significant reductions in facial contact/lick behaviors were observed in the TBI animals using either cool or warm challenge temperatures compared with behaviors in the normal animals. These facial thermal sensitizations correlated with detectable changes in multiple levels of the TSS. The immunohistochemical (IHC) studies revealed significant alterations in the expression of the serotonin (5-HT), neurokinin 1 receptor (NK1R), norepinephrine (NE), and gamma-aminobutyric acid (GABA) in the caudal trigeminal nucleus, thalamic VPL/VPM nucleus, and sensory cortex of the orofacial pain pathways. There was a strong correlation between increased expression of certain IHC markers and increased behavioral markers for facial sensitization. The authors conclude that TBI-induced changes observed in the TSS are consistent with the expression of generalized facial allodynia following cTBI. To our knowledge, this is the first report of orofacial sensitization correlated with changes in selected neuromodulators/neurotransmitters in the TSS following experimental mild TBI.

Prolonged Jaw Opening Promotes Nociception and Enhanced Cytokine Expression.

AIMS: To test the hypothesis that prolonged jaw opening, as can occur during routine dental procedures, increases nociceptive sensitivity of the masseter muscle and increases cytokine expression. METHODS: Sprague-Dawley rats were used to investigate behavioral and cellular changes in response to prolonged jaw opening. A surgical retractor was placed around the maxillary and mandibular incisors, and the jaw was held at near maximal opening for 20 minutes. Head-withdrawal responses to mechanical stimuli applied to the facial skin overlying the left and right masseter muscles were determined following jaw opening. Cytokine levels in the upper cervical spinal cord containing the caudal part of the spinal trigeminal nucleus were evaluated using protein antibody microarrays (n = 3). Statistical analysis was performed using a nonparametric Mann-Whitney U test. RESULTS: Prolonged jaw opening significantly increased nocifensive head withdrawal to mechanical stimuli at 2 hours, and days 3 and 7 postinduction (P < .05). The increase in nociceptive response resolved after 14 days. Sustained jaw opening also stimulated differential cytokine expression in the trigeminal ganglion and upper cervical spinal cord that persisted 14 days postprocedure (P < .05). CONCLUSION: These findings provide evidence that near maximal jaw opening can lead to activation and prolonged sensitization of trigeminal neurons that results in nociceptive behavior evoked by stimulation of the masseter muscle, a physiologic event often associated with temporomandibular disorders (TMD). Results from this study may provide a plausible explanation for why some patients develop TMD after routine dental procedures that involve prolonged jaw opening.

Effect of carbamazepine and gabapentin on excitability in the trigeminal subnucleus caudalis of neonatal rats using a voltage-sensitive dye imaging technique.

BACKGROUND: The antiepileptic drugs carbamazepine and gabapentin are effective in treating neuropathic pain and trigeminal neuralgia. In the present study, to analyze the effects of carbamazepine and gabapentin on neuronal excitation in the spinal trigeminal subnucleus caudalis (Sp5c) in the medulla oblongata, we recorded temporal changes in nociceptive afferent activity in the Sp5c of trigeminal nerve-attached brainstem slices of neonatal rats using a voltage-sensitive dye imaging technique. RESULTS: Electrical stimulation of the trigeminal nerve rootlet evoked changes in the fluorescence intensity of dye in the Sp5c. The optical signals were composed of two phases, a fast component with a sharp peak followed by a long-lasting component with a period of more than 500 ms. This evoked excitation was not influenced by administration of carbamazepine (10, 100 and 1,000 µM) or gabapentin (1 and 10 µM), but was increased by administration of 100 µM gabapentin. This evoked excitation was increased further in low Mg(2+) (0.8 mM) conditions, and this effect of low Mg(2+) concentration was antagonized by 30 µM DL-2-amino-5-phosphonopentanoic acid (AP5), a N-methyl-D-aspartate (NMDA) receptor blocker. The increased excitation in low Mg(2+) conditions was also antagonized by carbamazepine (1,000 µM) and gabapentin (100 µM). CONCLUSION: Carbamazepine and gabapentin did not decrease electrically evoked excitation in the Sp5c in control conditions. Further excitation in low Mg(2+) conditions was antagonized by the NMDA receptor blocker AP5. Carbamazepine and gabapentin had similar effects to AP5 on evoked excitation in the Sp5c in low Mg(2+) conditions. Thus, we concluded that carbamazepine and gabapentin may act by blocking NMDA receptors in the Sp5c, which contributes to its anti-hypersensitivity in neuropathic pain.

Involvement of trigeminal astrocyte activation in masseter hyperalgesia under stress.

It is commonly accepted that psychological stress contributes to the development of temporomandibular joint disorders, in which chronic orofacial pain is the main symptom. However, the central mechanism underlying the development of these disorders has remained unclear. The current study was performed to determine the involvement of the glia in the trigeminal spinal subnucleus caudalis in stress-induced increases in masseter muscle hyperalgesia in rats. After being subjected to chronic restraint stress, the animals showed decreased body weight gain, behavioral changes and marked masseter allodynia. We also found that astrocytes, but not microglia, in the trigeminal subnucleus caudalis (Vc) were dramatically activated. A further analysis was undertaken to investigate the contribution of the glia; we intrathecally injected l-α-aminoadipate (astrocyte-specific inhibitor) and/or minocycline (microglia-specific inhibitor) into the stressed rats. Our results showed that l-α-aminoadipate (LAA), but not minocycline, could significantly attenuate the mechanical masseter allodynia and behavioral changes induced by restraint stress. In addition, the expression of interleukin-1ß (IL-1ß) and phosphorylated N-methyl-d-aspartic acid receptor 1 (p-NR1) in the Vc was significantly increased after chronic restraint stress, whereas LAA dramatically inhibited the overexpression of IL-1ß and p-NR1. Taken together, these results suggest that activated astrocytes in the Vc may be one of the most important factors in the pathophysiology of masseter hyperalgesia induced by restraint stress and the following overexpression of IL-1ß and excessive NMDAR phosphorylation may ultimately contribute to masseter hyperalgesia. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for the treatment of orofacial pain induced by stress.

Effect of carbamazepine and gabapentin on excitability in the trigeminal subnucleus caudalis of neonatal rats using a voltage-sensitive dye imaging technique

BACKGROUND: The antiepileptic drugs carbamazepine and gabapentin are effective in treating neuropathic pain and trigeminal neuralgia. In the present study, to analyze the effects of carbamazepine and gabapentin on neuronal excitation in the spinal trigeminal subnucleus caudalis (Sp5c) in the medulla oblongata, we recorded temporal changes in nociceptive afferent activity in the Sp5c of trigeminal nerve-attached brainstem slices of neonatal rats using a voltage-sensitive dye imaging technique. RESULTS: Electrical stimulation of the trigeminal nerve rootlet evoked changes in the fluorescence intensity of dye in the Sp5c. The optical signals were composed of two phases, a fast component with a sharp peak followed by a long-lasting component with a period of more than 500 ms. This evoked excitation was not influenced by administration of carbamazepine (10, 100 and 1,000 µM) or gabapentin (1 and 10 µM), but was increased by administration of 100 µM gabapentin. This evoked excitation was increased further in low Mg²+ (0.8 mM) conditions, and this effect of low Mg²+ concentration was antagonized by 30 µM DL-2-amino-5-phosphonopentanoic acid (AP5), a N-methyl-D-as-partate (NMDA) receptor blocker. The increased excitation in low Mg²+ conditions was also antagonized by carbamazepine (1,000 µM) and gabapentin (100 µM). CONCLUSION: Carbamazepine and gabapentin did not decrease electrically evoked excitation in the Sp5c in control conditions. Further excitation in low Mg²+ conditions was antagonized by the NMDA receptor blocker AP5. Carbamazepine and gabapentin had similar effects to AP5 on evoked excitation in the Sp5c in low Mg²+ conditions. Thus, we concluded that carbamazepine and gabapentin may act by blocking NMDA receptors in the Sp5c, which contributes to its anti-hypersensitivity in neuropathic pain.

Estrogen status and psychophysical stress modify temporomandibular joint input to medullary dorsal horn neurons in a lamina-specific manner in female rats.

Estrogen status and psychological stress contribute to the expression of several chronic pain conditions including temporomandibular muscle and joint disorders (TMJD). Sensory neurons that supply the temporomandibular joint (TMJ) region terminate in laminae I and V of the spinal trigeminal nucleus (Vc/C1-2 region); however, little is known about lamina-specificity and environmental influences on the encoding properties of TMJ brainstem neurons. To test the hypothesis that Vc/C1-2 neurons integrate both interoceptive and exteroceptive signals relevant for TMJ nociception, we recorded TMJ-evoked activity in superficial and deep laminae of ovariectomized rats under high and low estradiol (E2) and stress conditions. Rats received daily injections of low (LE) or high (HE) dose E2 and were subjected to forced swim (FS) or sham swim conditioning for 3days. The results revealed marked lamina-specificity in that HE rats displayed enhanced TMJ-evoked activity in superficial, but not deep, laminae independent of stress conditioning. By contrast, FS conditioned rats displayed increased background firing and TMJ-evoked activity of neurons in deep, but not superficial, laminae independent of E2 status. FS also enhanced TMJ-evoked masseter muscle activity and suggested the importance of deep dorsal horn neurons in mediating evoked jaw muscle activity. In conclusion, E2 status and psychophysical stress play a significant role in modifying the encoding properties of TMJ-responsive medullary dorsal horn neurons with a marked lamina-specificity.

CGRP in the trigeminovascular system: a role for CGRP, adrenomedullin and amylin receptors?

UNLABELLED: The neuropeptide calcitonin gene-related peptide (CGRP) is reported to play an important role in migraine. It is expressed throughout the trigeminovascular system. Antagonists targeting the CGRP receptor have been developed and have shown efficacy in clinical trials for migraine. However, no CGRP antagonist is yet approved for treating this condition. The molecular composition of the CGRP receptor is unusual because it comprises two subunits; one is a GPCR, the calcitonin receptor-like receptor (CLR). This associates with receptor activity-modifying protein (RAMP) 1 to yield a functional receptor for CGRP. However, RAMP1 also associates with the calcitonin receptor, creating a receptor for the related peptide amylin but this also has high affinity for CGRP. Other combinations of CLR or the calcitonin receptor with RAMPs can also generate receptors that are responsive to CGRP. CGRP potentially modulates an array of signal transduction pathways downstream of activation of these receptors, in a cell type-dependent manner. The physiological significance of these signalling processes remains unclear but may be a potential avenue for refining drug design. This complexity has prompted us to review the signalling and expression of CGRP and related receptors in the trigeminovascular system. This reveals that more than one CGRP responsive receptor may be expressed in key parts of this system and that further work is required to determine their contribution to CGRP physiology and pathophysiology. LINKED ARTICLES: This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7.

Spinal trigeminal neurons demonstrate an increase in responses to dural electrical stimulation in the orofacial formalin test.

Primary headaches are often associated with pain in the maxillofacial region commonly classified under the term "orofacial pain" (OFP). In turn, long-lasting OFP can trigger and perpetuate headache as an independent entity, which is able to persist after the resolution of the main disorder. A close association between OFP and headache complicates their cause and effect definition and leads to misdiagnosis. The precise mechanisms underlying this phenomenon are poorly understood, partly because of the deficiency of research-related findings. We combined the animal models of OFP and headache-the orofacial formalin test and the model of trigeminovascular nociception-to investigate the neurophysiological mechanisms underlying their comorbidity. In anesthetized rats, the ongoing activity of single convergent neurons in the spinal trigeminal nucleus was recorded in parallel to their responses to the electrical stimulation of the dura mater before and after the injection of formalin into their cutaneous receptive fields. Subcutaneous formalin resulted not only in the biphasic increase in the ongoing activity, but also in an enhancement of neuronal responses to dural electrical stimulation, which had similar time profile. These results demonstrated that under tonic pain in the orofacial region a nociceptive signaling from the dura mater to convergent trigeminal neurons is significantly enhanced apparently because of the development of central sensitization; this may contribute to the comorbidity of OFP and headache.

A trigeminoreticular pathway: implications in pain.

Neurons in the caudalmost ventrolateral medulla (cmVLM) respond to noxious stimulation. We previously have shown most efferent projections from this locus project to areas implicated either in the processing or modulation of pain. Here we show the cmVLM of the rat receives projections from superficial laminae of the medullary dorsal horn (MDH) and has neurons activated with capsaicin injections into the temporalis muscle. Injections of either biotinylated dextran amine (BDA) into the MDH or fluorogold (FG)/fluorescent microbeads into the cmVLM showed projections from lamina I and II of the MDH to the cmVLM. Morphometric analysis showed the retrogradely-labeled neurons were small (area 88.7 µm(2)±3.4) and mostly fusiform in shape. Injections (20-50 µl) of 0.5% capsaicin into the temporalis muscle and subsequent immunohistochemistry for c-Fos showed nuclei labeled in the dorsomedial trigeminocervical complex (TCC), the cmVLM, the lateral medulla, and the internal lateral subnucleus of the parabrachial complex (PBil). Additional labeling with c-Fos was seen in the subnucleus interpolaris of the spinal trigeminal nucleus, the rostral ventrolateral medulla, the superior salivatory nucleus, the rostral ventromedial medulla, and the A1, A5, A7 and subcoeruleus catecholamine areas. Injections of FG into the PBil produced robust label in the lateral medulla and cmVLM while injections of BDA into the lateral medulla showed projections to the PBil. Immunohistochemical experiments to antibodies against substance P, the substance P receptor (NK1), calcitonin gene regulating peptide, leucine enkephalin, VRL1 (TPRV2) receptors and neuropeptide Y showed that these peptides/receptors densely stained the cmVLM. We suggest the MDH- cmVLM projection is important for pain from head and neck areas. We offer a potential new pathway for regulating deep pain via the neurons of the TCC, the cmVLM, the lateral medulla, and the PBil and propose these areas compose a trigeminoreticular pathway, possibly the trigeminal homologue of the spinoreticulothalamic pathway.

[Neuropeptide effects on the trigeminal system: pathophysiology and clinical significance for migraine]. / Neuropeptidwirkungen im trigeminalen System: Pathophysiologie und klinische Bedeutung bei der Migräne.

Neuropeptides, such as calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) are considered important mediators in primary headaches. Increased concentrations of CGRP have been found in jugular venous plasma during attacks of migraine and, concomitant with VIP elevation, during cluster headache. Substance P and CGRP are produced from subsets of trigeminal afferents whereas VIP derives from parasympathetic efferents. Release of these neuropeptides in the meninges causes arterial vasodilatation, mast cell degranulation and plasma extravasation in animal experiments. Particularly CGRP seems to be important, as receptor antagonists have recently been shown to have a therapeutic effect on migraine. Animal models have confirmed the role of CGRP in meningeal nociception. The activity of spinal trigeminal neurons is a sensitive integrative measure of trigeminal activity and CGRP released from central terminals of trigeminal afferents in the spinal trigeminal nucleus has been shown to facilitate nociceptive transmission, most likely by a presynaptic action. The proposed CGRP functions are supported by the distribution of CGRP receptor components localized in the rat cranial dura mater, the trigeminal ganglion and the spinal trigeminal nucleus. The currently available data indicate multiple sites of CGRP action in trigeminal nociception and the pathogenesis of migraine but central CGRP receptors are probably the essential targets in the treatment of migraine using CGRP receptor antagonists.

Altered responses of MeCP2-deficient mouse brain stem to severe hypoxia.

Rett syndrome (RTT) patients suffer from respiratory arrhythmias with frequent apneas causing intermittent hypoxia. In a RTT mouse model (methyl-CpG-binding protein 2-deficient mice; Mecp2(-/y)) we recently discovered an enhanced hippocampal susceptibility to hypoxia and hypoxia-induced spreading depression (HSD). In the present study we investigated whether this also applies to infant Mecp2(-/y) brain stem, which could become life-threatening due to failure of cardiorespiratory control. HSD most reliably occurred in the nucleus of the solitary tract (NTS) and the spinal trigeminal nucleus (Sp5). HSD susceptibility of the Mecp2(-/y) NTS and Sp5 was increased on 8 mM K(+)-mediated conditioning. 5-HT(1A) receptor stimulation with 8-hydroxy-2-(di-propylamino)tetralin (8-OH-DPAT) postponed HSD by up to 40%, mediating genotype-independent protection. The deleterious impact of HSD on in vitro respiration became obvious in rhythmically active slices, where HSD propagation into the pre-Bötzinger complex (pre-BötC) immediately arrested the respiratory rhythm. Compared with wild-type, the Mecp2(-/y) pre-BötC was invaded less frequently by HSD, but if so, HSD occurred earlier. On reoxygenation, in vitro rhythms reappeared with increased frequency, which was less pronounced in Mecp2(-/y) slices. 8-OH-DPAT increased respiratory frequency but failed to postpone HSD in the pre-BötC. Repetitive hypoxia facilitated posthypoxic recovery only if HSD occurred. In 57% of Mecp2(-/y) slices, however, HSD spared the pre-BötC. Although this occasionally promoted residual hypoxic respiratory activity ("gasping"), it also prolonged the posthypoxic recovery, and thus the absence of central inspiratory drive, which in vivo would lengthen respiratory arrest. In view of the breathing disorders in RTTs, the increased hypoxia susceptibility of MeCP2-deficient brain stem potentially contributes to life-threatening disturbances of cardiorespiratory control.

Long lasting pain hypersensitivity following ligation of the tendon of the masseter muscle in rats: a model of myogenic orofacial pain.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.