Poking palpebral conjunctiva evoked upper-eyelid retraction during ophthalmic surgery. Iatrogenic eyelid ptosis occurred if eyelid branch of lachrymal nerve was sectioned. Mesencephalic trigeminal nucleus (Vme) neurons were labeled when tracer injected into lachrymal nerve innervating eyelid Mueller\'s muscle. Masseter afferent Vme neurons projecting to oculomotor nucleus (III) was observed in toad and rat, which helps amphibians to stare prey when they open mouth widely to prey. We hypothesized single Vme neurons may have peripheral collaterals to both eyelid and masseter muscles. WGA-594 was injected into upper eyelid, and WGA-488 was simultaneously delivered into ipsilateral masseter muscle in the same rat. Then, double labeled Vme neurons were found under both conventional and confocal microscope. Meanwhile, contact of WGA-594 positive eyelid afferent Vme neurons with WGA-488 labeled masseter afferent ones were observed sometimes. Combined with our previous observation of oculomotor projection Vme neurons, we thought WGA-594/488 double labeled Vme cells, at least some of them, are oculomotor projecting ones. Contact between eyelid and masseter afferent Vme neurons are supposed to be electrotonically coupled, based on a line of previous studies. If exogenous or genetic factors make these Vme neurons misinterpret masseter input as eyelid afferent signals, these Vme neurons might feedforward massages to eyelid retractor motoneurons in the III. Besides, oculomotor projecting Vme neurons might be co-fired by adjacent masseter afferent Vme neurons through electrotonic coupling once the masseter muscle is activated. In these cases, Marcus Gunn Syndrome might occur. This finding leads to a new hypothesis for the Syndrome.

BACKGROUND: Prior clinical studies suggest a shared mechanism between vestibular symptoms and migraine headache. However, the specific neuroanatomical substrate connecting vestibular symptoms with migraine remains to be largely unknown. Thus, the aim of this study was to further investigate the mechanisms that whether and how trigeminovestibular neurons produce effects on neuronal activation in vestibular nucleus (VN).
METHODS: A chronic-NTG rat model was established by recurrent intermittent administration of nitroglycerin (NTG). Pain- and vestibular-related behaviors were assessed. To selectively inhibit the glutamatergic neurons and trigeminal nucleus caudalis (TNC) to VN projection neurons, the AAVs encoding engineered Gi-coupled hM4D receptor were administered in the TNC or VN area.
RESULTS: We identify a glutamatergic projection from TNC to VN that mediates vestibular dysfunction in a chronic-NTG rat model. Inhibition of the GlutamateTNC neurons alleviates vestibular dysfunction in the chronic-NTG rat. Calcitonin gene-related peptide (CGRP)-expressing neurons in the VN received glutamatergic projections from TNC neurons. Silencing the glutamatergic TNC-VN projection neurons attenuates vestibular dysfunction in the chronic-NTG rat.
CONCLUSIONS: Together, we reveal a modulatory role of glutamatergic TNC-VN projection neurons in vestibular dysfunction of migraine.

OBJECTIVE: Chronic migraine (CM) is a common neurological disorder with complex pathogenesis. Evidence suggests that pituitary adenylate cyclase-activating peptide (PACAP) induces migraine-like attacks and may be potential a new target for migraine treatment, but the therapeutic results of targeting PACAP and its receptors are not uniform. Therefore, the aim of this study was to investigate the regulatory effect of PACAP type I receptor (PAC1R) antagonist, PACAP6-38, on nitroglycerin (NTG)-induced central sensitization in a CM model.
METHODS: Sprague-Dawley (SD) rats received repeated injections of NTG to construct a CM model. Mechanical and thermal thresholds were measured using Von Frey filaments and hot plate tests. C-Fos expression was measured by western blotting and immunofluorescence staining to assess the central sensitization. PACAP6-38 was intracerebrally injected into the trigeminal nucleus caudalis (TNC), and then the changes in c-Fos, the synaptic-associated proteins, phospho-ERK1/2 (p-ERK1/2), phosphorylation of cyclic adenosine monophosphate response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) were detected. Transmission electron microscopy (TEM) and Golgi-Cox staining were used to observe the ultrastructure of synapses and dendritic structures of TNC neurons.
RESULTS: The results showed that PACAP and PAC1R expression were significantly raised in the TNC after repeated NTG injections. Additionally, PACAP6-38 treatment alleviated nociceptive sensitization, inhibited NTG-induced overexpression of c-Fos and synaptic-associated proteins in the TNC of CM rat, restored aberrant synaptic structures. Furthermore, the expression of ERK/CREB/BDNF pathway was depressed by PACAP6-38.
CONCLUSIONS: Our results demonstrated that abnormal synaptic structure in the TNC of CM, which could be reversed by inhibition of PAC1R via down-regulating the ERK/CREB/BDNF signaling pathway. PACAP6-38 improves NTG-induced central sensitization by regulating synaptic plasticity in the TNC of CM rat, which may provide new insights into the treatments targeting PACAP/PAC1R in migraine.

BACKGROUND: The study was designed to explore the correlation of the asymmetric regulation between periaqueductal gray (PAG) and bilateral trigeminal nucleus caudalis (TNC) in migraine rats through studying the changes of metabolites in pain regulatory pathway of acute migraine attack.
METHODS: Thirty male Sprague-Dawley (SD) rats were randomly divided into three groups: blank, control, model groups. Then, blank group was intraperitoneally injected with ultrapure water, while control group injected with saline and model group injected with Glyceryl Trinitrate (GTN). Two hours later, PAG and bilateral TNC were removed respectively, and metabolite concentrations of PAG, Left-TNC, Right-TNC were obtained. Lastly, the differences of metabolite among three brain tissues were compared.
RESULTS: The relative concentrations of rNAA, rGlu, rGln, rTau, rMI in PAG or bilateral TNC had interaction effects between groups and sites. The concentration of rLac of three brain tissues increased in migraine rats, however, the rLac of LTNC and RTNC increased more than that of PAG. Besides, the concentrations of rNAA and rGln increased in RTNC, while rGABA decreased in RTNC.
CONCLUSIONS: There is correlation between PAG, LTNC and RTNC in regulation of pain during acute migraine attack, and the regulation of LTNC and RTNC on pain is asymmetric.

Stressful stimuli can activate the hypothalamic-pituitary-adrenal (HPA) axis. Clinically, it has been widely reported that stressful events are often accompanied by teeth clenching and bruxism, while mastication (chewing) can promote coping with stress. Trigeminal motoneurons in the trigeminal motor nucleus supplying the chewing muscles receive direct inputs from interneurons within the peritrigeminal premotor area (Peri5). Previous studies found that the paraventricular hypothalamic nucleus (PVH) participates in trigeminal activities during stressful events. However, the neural pathway by which the stress-induced oral movements alleviate stress is largely unknown. We hypothesized that paraventricular-trigeminal circuits might be associated with the stress-induced chewing movements and anxiety levels. First, we observed the stress-coping effect of wood gnawing on stress-induced anxiety, with less anxiety-like behaviors seen in the open field test and elevated plus maze, as well as decreased corticosterone and blood glucose levels, in response to stress in mice. We then found that excitotoxic lesions of PVH reduced the effect of gnawing on stress, reflected in more anxiety-like behaviors; this emphasizes the importance of the PVH in stress responses. Anterograde, retrograde, transsynaptic, and nontranssynaptic tracing through central and peripheral injections confirmed monosynaptic projections from PVH to Peri5. We discovered that PVH receives proprioceptive sensory inputs from the jaw muscle and periodontal ligaments, as well as provides motor outputs via the mesencephalic trigeminal nucleus (Me5) and Peri5. Next, pathway-specific functional manipulation by chemogenetic inhibition was conducted to further explore the role of PVH-Peri5 monosynaptic projections. Remarkably, PVH-Peri5 inhibition decreased gnawing but did not necessarily reduce stress-induced anxiety. Moreover, neuropeptide B (NPB) was expressed in Peri5-projecting PVH neurons, indicating that NPB signaling may mediate the effects of PVH-Peri5. In conclusion, our data revealed a PVH-Peri5 circuit that plays a role in the stress response via its associations with oromotor movements and relative anxiety-like behaviors.

BACKGROUND: Astrocytic activation might play a significant role in the central sensitization of chronic migraine (CM). However, the temporal characteristics of the astrocytic activation in the trigeminal nucleus caudalis (TNC) and the molecular mechanism under the process remain not fully understood. Therefore, this study aims to investigate the duration and levels change of astrocytic activation and to explore the correlation between astrocytic activation and the levels change of cytokines release.
METHODS: We used a mice model induced by recurrent dural infusion of inflammatory soup (IS). The variation with time of IS-induced mechanical thresholds in the periorbital and hind paw plantar regions were evaluated using the von Frey filaments test. We detected the expression profile of glial fibrillary acidic protein (GFAP) in the TNC through immunofluorescence staining and western blot assay. We also investigated the variation with time of the transcriptional levels of GFAP and ionized calcium binding adapter molecule 1 (Iba1) through RNAscope in situ hybridization analysis. Then, we detected the variation with time of cytokines levels in the TNC tissue extraction and serum, including c-c motif chemokine ligand 2 (CCL2), c-c motif chemokine ligand 5 (CCL5), c-c motif chemokine ligand 7 (CCL7), c-c motif chemokine ligand 12 (CCL12), c-x-c motif chemokine ligand 1 (CXCL1), c-x-c motif chemokine ligand 13 (CXCL13), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), macrophage colony-stimulating factor (M-CSF), interleukin 1beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17A (IL-17A).
RESULTS: Recurrent IS infusion resulted in cutaneous allodynia in both the periorbital region and hind paw plantar, ranging from 5 d (after the second IS infusion) to 47 d (28 d after the last infusion) and 5 d to 26 d (7 d after the last infusion), respectively. The protein levels of GFAP and messenger ribonucleic acid (mRNA) levels of GFAP and Iba1 significantly increased and sustained from 20 d to 47 d (1 d to 28 d after the last infusion), which was associated with the temporal characteristics of astrocytic activation in the TNC. The CCL7 levels in the TNC decreased from 20 d to 47 d. But the CCL7 levels in serum only decreased on 20 d (1 d after the last infusion). The CCL12 levels in the TNC decreased on 22 d (3 d after the last infusion) and 33 d (14 d after the last infusion). In serum, the CCL12 levels only decreased on 22 d. The IL-10 levels in the TNC increased on 20 d.
CONCLUSIONS: Our results indicate that the astrocytic activation generated and sustained in the IS-induced mice model from 1 d to 28 d after the last infusion and may contribute to the pathology through modulating CCL7, CCL12, and IL-10 release.

Occlusal trauma (OT), by causing periodontal tissue damage, can activate and enhance the activity of the peripheral and central nervous system (CNS) neuropeptides. The brain-derived neurotrophic factor (BDNF) gene is activity-dependent and exhibits marked alterations, characterized by protection against injury and repair. Our results show the possible molecular mechanism through which noxious environmental stimuli induce alterations in BDNF activity in the local periodontal tissue, the primary sensory neurons-Vc, and the hippocampus, suggesting systemic impairment. BDNF serves a more positive and enduring trauma protection and repair function in Vc compared to that in local dental tissue.

BACKGROUND: Central sensitization is considered a critical pathogenic mechanism of chronic migraine (CM). Activation of microglia in the trigeminal nucleus caudalis (TNC) contributes to this progression. Microglial glucagon-like peptide-1 receptor (GLP-1R) activation can alleviate pain; however, whether it is involved in the mechanism of CM has not been determined. Thus, this study aims to investigate the precise role of GLP-1R in the central sensitization of CM.
METHODS: Repeated nitroglycerin injection-treated mice were used as a CM animal model in the experiment. To identify the distribution and cell localization of GLP-1R in the TNC, we performed immunofluorescence staining. Changes in the expression of GLP-1R, Iba-1, PI3K and p-Akt in the TNC were examined by western blotting. To confirm the effect of GLP-1R and PI3K/Akt in CM, a GLP-1R selective agonist (liraglutide) and antagonist (exendin(9-39)) and a PI3K selective antagonist (LY294002) were administered. Mechanical hypersensitivity was measured through von Frey filaments. To investigate the role of GLP-1R in central sensitization, calcitonin gene-related peptide (CGRP) and c-fos were determined using western blotting and immunofluorescence. To determine the changes in microglial activation, IL-1β and TNF-α were examined by western blotting, and the number and morphology of microglia were measured by immunofluorescence. We also confirmed the effect of GLP-1R on microglial activation in lipopolysaccharide-treated BV-2 microglia.
RESULTS: The protein expression of GLP-1R was increased in the TNC after nitroglycerin injection. GLP-1R was colocalized with microglia and astrocytes in the TNC and was fully expressed in BV-2 microglia. The GLP-1R agonist liraglutide alleviated basal allodynia and suppressed the upregulation of CGRP, c-fos and PI3K/p-Akt in the TNC. Similarly, the PI3K inhibitor LY294002 prevented nitroglycerin-induced hyperalgesia. In addition, activating GLP-1R reduced Iba-1, IL-1β and TNF-α release and inhibited TNC microglial number and morphological changes (process retraction) following nitroglycerin administration. In vitro, the protein levels of IL-1β and TNF-α in lipopolysaccharide-stimulated BV-2 microglia were also decreased by liraglutide.
CONCLUSIONS: These findings suggest that microglial GLP-1R activation in the TNC may suppress the central sensitization of CM by regulating TNC microglial activation via the PI3K/Akt pathway.

Although the mechanism of chronic migraine is still unclear, more and more studies have shown that mitochondrial dysfunction plays a possible role in migraine pathophysiology. Silent information regulator 1 (SIRT1) plays a vital role in mitochondrial dysfunction in many diseases. However, there is no research on the role of SIRT1 in mitochondrial dysfunction of chronic migraine. The aim of this study was to explore the role of SIRT1 in mitochondrial dysfunction in chronic migraine. A rat model was established through repeated dural infusions of inflammatory soup for 7 days to simulate chronic migraine attacks. Cutaneous hyperalgesia caused by the repeated infusions of inflammatory soup was detected using the von Frey test. Then, we detected SIRT1 expression in the trigeminal nucleus caudalis. To explore the effect of SIRT1 on mitochondrial dysfunction in chronic migraine rats, we examined whether SRT1720, an activator of SIRT1, altered mitochondrial dysfunction in chronic migraine rats. Repeated infusions of inflammatory soup resulted in cutaneous hyperalgesia accompanied by downregulation of SIRT1. SRT1720 significantly alleviated the cutaneous hyperalgesia induced by repeated infusions of inflammatory soup. Furthermore, activation of SIRT1 markedly increased the expression of peroxisome proliferator-activated receptor gamma-coactivator 1-alpha, transcription factor A, nuclear respiratory factor 1 and nuclear respiratory factor 2 mitochondrial DNA and increased the ATP content and mitochondrial membrane potential. Our results indicate that SIRT1 may have an effect on mitochondrial dysfunction in chronic migraine rats. Activation of SIRT1 has a protective effect on mitochondrial function in chronic migraine rats.

The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain.
In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry.
In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05).
Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.