Neuroimmune interactions in the development and chronification of migraine headache.

Migraine is highly prevalent and debilitating. The persistent headaches in this condition are thought to arise from the activation and sensitization of the trigeminovascular pathway. Both clinical and animal model studies have suggested that neuroimmune interactions contribute to the pathophysiology of migraine headache. In this review, we first summarize the findings from human studies implicating the dysregulation of the immune system in migraine, including genetic analyses, measurement of circulatory factors, and neuroimaging data. We next discuss recent advances from rodent studies aimed at elucidating the neuroimmune interactions that manifest at various levels of the trigeminovascular pathway and lead to the recruitment of innate and adaptive immune cells as well as immunocompetent glial cells. These cells reciprocally modulate neuronal activity via multiple pro- and anti-inflammatory mediators, thereby regulating peripheral and central sensitization. Throughout the discussions, we highlight the potential clinical and translational implications of the findings.

Low-Dose Interleukin-2 Reverses Traumatic Brain Injury-Induced Cognitive Deficit and Pain in a Murine Model.

OBJECTIVE: Despite the high prevalence, mild traumatic brain injury (mTBI)-induced chronic headache and cognitive deficits are poorly understood and lack effective treatments. Low-dose interleukin-2 (LD-IL-2) treatment soon after mTBI or overexpressing IL-2 in brain astrocytes prior to injury protects mice from developing post-traumatic headache (PTH)-related behaviors and cognitive decline. The present study addresses a clinically relevant knowledge gap: whether LD-IL-2 treatment long after the initial injury is still effective for chronic PTH and cognitive deficits. METHODS: mTBI was induced by a noninvasive closed-head weight drop method. LD-IL-2 was administered 4-6 weeks post-mTBI to assess its effects on chronic PTH-related facial mechanical hypersensitivity as well as mTBI-induced impairment in novel object recognition and object location tests. Endogenous regulatory T (Treg) cells were depleted to investigate the mechanism of action of LD-IL-2. RESULTS: Delayed LD-IL-2 treatment abolished chronic PTH-related behaviors. It also completely reversed mTBI-induced cognitive impairment in both male and female mice. Treg cell depletion not only prolonged PTH-related behaviors but also abolished the effects of LD-IL-2. Interestingly, LD-IL-2 treatment significantly increased the number of Treg cells in dura but not in brain tissues. INTERPRETATION: These results suggest that the beneficial effects of LD-IL-2 treatment are mediated through the expansion of meningeal Treg cells. Collectively, our study identifies Treg as a cellular target and LD-IL-2 as a promising therapy for both chronic PTH and mTBI-induced cognitive impairment for both males and females, with a wide therapeutic time window and the potential of reducing polypharmacy in mTBI treatment. ANN NEUROL 2024;96:508-525.

Nanoparticle Polymeric Micellar Paclitaxel Versus Paclitaxel for Patients with Advanced Gastric Cancer.

BACKGROUND: Nanoparticle polymeric micellar paclitaxel (NPMP) is a novel Cremophor EL (CrEL)-free nanoparticle micellar formulation of paclitaxel. This study evaluated the efficacy and toxicity of NPMP in the treatment of patients with advanced gastric cancer (AGC). METHODS: Patients with histologically confirmed AGC in Jiangsu Cancer Hospital were retrospectively collected and divided into two groups. Patients in group A received NPMP at a total dose of 360 mg/m2 each cycle, and patients in group B were given paclitaxel at a dose of 210 mg/m2 each cycle. In addition, all patients received 5-fluorouracil at a dose of 0.75 g/m2 on days 1-4 and leucovorin at a dose of 200 mg/m2 on days 1-4 for at least 2 cycles. RESULTS: From January 2021 to May 2023, 63 patients (32 in group A and 31 in group B) could be evaluated for treatment response. A marked disparity in the overall response was observed between groups A and B, indicating statistical significance. The overall response rate was 31% in group A (10/32) and 10% in group B (3/31) (P = 0.034). Disease control rate was 91% in group A (29/32) and 81% in group B (25/31) (P = 0.440). No statistically significant difference in adverse reactions was observed between the two groups. However, the incidence of anemia, leucopenia, nausea, vomiting, diarrhea, liver dysfunction, and allergy in group A was notably lower than that in group B. CONCLUSIONS: NPMP combined chemotherapy offers a new, active, and safe treatment for patients with AGC.

A Cataract-Causing Mutation in the TRPM3 Cation Channel Disrupts Calcium Dynamics in the Lens.

TRPM3 belongs to the melastatin sub-family of transient receptor potential (TRPM) cation channels and has been shown to function as a steroid-activated, heat-sensitive calcium ion (Ca2+) channel. A missense substitution (p.I65M) in the TRPM3 gene of humans (TRPM3) and mice (Trpm3) has been shown to underlie an inherited form of early-onset, progressive cataract. Here, we model the pathogenetic effects of this cataract-causing mutation using 'knock-in' mutant mice and human cell lines. Trpm3 and its intron-hosted micro-RNA gene (Mir204) were strongly co-expressed in the lens epithelium and other non-pigmented and pigmented ocular epithelia. Homozygous Trpm3-mutant lenses displayed elevated cytosolic Ca2+ levels and an imbalance of sodium (Na+) and potassium (K+) ions coupled with increased water content. Homozygous TRPM3-mutant human lens epithelial (HLE-B3) cell lines and Trpm3-mutant lenses exhibited increased levels of phosphorylated mitogen-activated protein kinase 1/extracellular signal-regulated kinase 2 (MAPK1/ERK2/p42) and MAPK3/ERK1/p44. Mutant TRPM3-M65 channels displayed an increased sensitivity to external Ca2+ concentration and an altered dose response to pregnenolone sulfate (PS) activation. Trpm3-mutant lenses shared the downregulation of genes involved in insulin/peptide secretion and the upregulation of genes involved in Ca2+ dynamics. By contrast, Trpm3-deficient lenses did not replicate the pathophysiological changes observed in Trpm3-mutant lenses. Collectively, our data suggest that a cataract-causing substitution in the TRPM3 cation channel elicits a deleterious gain-of-function rather than a loss-of-function mechanism in the lens.

Optimization of green deep eutectic solvent (DES) extraction of Chenopodium quinoa Willd. husks saponins by response surface methodology and their antioxidant activities.

Quinoa saponins have outstanding activity, and there are an increasing number of extraction methods, but there are few research programs on green preparation technology. The extraction conditions of quinoa saponins with deep eutectic solvents (DESs) were optimized by single-factor experiments combined with response surface methodology. The antioxidant capacity of saponins extracted by DESs and traditional methods was evaluated by the DPPH clearance rate, iron ion chelation rate and potassium ferricyanide reducing power. The results show that the optimal DES is choline chloride: 1,2-propylene glycol (1 : 1), and its water content is 40%. The optimal extraction conditions were as follows: the solid-to-solvent ratio was 0.05 g mL-1, the extraction time was 89 min, and the extraction temperature was 75 °C. Under these conditions, the extraction of quinoa saponins by DES was more effective than the traditional extraction methods. The saponins extracted by DES and traditional methods were analyzed by UPLC-MS, and five main saponins were identified. Quantitative analysis by HPLC-UV showed that Q1 (m/z = 971) and Q2 (m/z = 809) had higher contents of saponins. In vitro antioxidant experiments showed that all DES saponin extracts showed good antioxidant capacity. This study provides new insight into the development and utilization of quinoa saponins.

Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone.

Transfer of the zp3a gene results in changes in egg adhesiveness and buoyancy in transgenic zebrafish.

Reproductive strategies and spawning habits play key roles in the evolution of endemic East Asian cyprinids. However, the molecular mechanisms underlying the regulation of spawning habits are not well understood. We recently identified zona pellucida (Zp) as the top differentially expressed protein between East Asian cyprinids that produce adhesive and semi-buoyant eggs, suggesting that Zp protein may play important roles in the regulation of egg type. In this work, we generated transgenic zebrafish in which oocyte-specific expression of zp genes from rare minnow ( Gobiocypris rarus), an East Asian cyprinid laying adhesive eggs, was driven by a zebrafish zp3.2 gene promoter. We found that the transgenic eggs obtained partial adhesiveness and exhibited alteration in hydration and buoyancy. Abnormal metabolism of vitellogenin (VTG) may contribute to enhanced hydration and/or buoyancy. Our work shows that expression of the exogenous zp3a gene from an adhesive-egg producing fish is sufficient to induce changes in both egg adhesiveness and buoyancy in zebrafish, emphasizing the important role of zp genes in the regulation of spawning habits. Our results thus provide new insights into how endemic East Asian cyprinids may have adapted to the Yangtze river-lake system via changes in spawning habits.

Low-dose interleukin-2 reverses chronic migraine-related sensitizations through peripheral interleukin-10 and transforming growth factor beta-1 signaling.

Low-dose interleukin-2 (LD-IL-2) treatment has been shown to effectively reverse chronic migraine-related behaviors and the sensitization of trigeminal ganglion (TG) neurons through expansion and activation of peripheral regulatory T cells (Tregs) in mice. In this study, we investigated the molecular mechanisms underlying the effects of LD-IL-2 and Treg cells. LD-IL-2 treatment increases the production of cytokines interleukin-10 (IL-10) and transforming growth factor beta-1 (TGFß1) in T cells, especially Treg cells, suggesting that they may mediate the therapeutic effect of LD-IL-2. Indeed, neutralizing antibodies against either IL-10 or TGFß completely blocked the effects of LD-IL-2 on the facial mechanical hypersensitivity as well as the sensitization of TG neurons resulting from repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration in mice, indicating that LD-IL-2 and Treg cells engage both peripheral IL-10 and TGFß signaling pathways to reverse chronic-migraine related sensitizations. In an in vitro assay, incubation of TG culture with exogenous IL-10 or TGFß1 fully reversed NTG-induced sensitization of TG neurons, suggesting that the IL-10 and TGFß1 signaling in TG neurons contribute to LD-IL-2's therapeutic effects. Collectively, these results not only elucidate the molecular mechanisms through which LD-IL-2 and Treg cells reverse chronic-migraine related sensitizations, but also suggest that the IL-10 and TGFß1 signaling pathways in TG neurons are potential targets for chronic migraine therapy.

Low-Dose Interleukin-2 and Regulatory T Cell Treatments Attenuate Punctate and Dynamic Mechanical Allodynia in a Mouse Model of Sciatic Nerve Injury.

PURPOSE: Nerve injury-induced mechanical hyper-sensitivity, in particular stroking-induced dynamic allodynia, is highly debilitating and difficult to treat. Previous studies indicate that the immunosuppressive regulatory T (Treg) cells modulate the magnitude of punctate mechanical allodynia resulting from sciatic nerve injury. However, whether enhancing Treg-mediated suppression attenuates dynamic allodynia is not known. In the present study, we addressed this knowledge gap by treating mice with low-dose interleukin-2 (ld-IL2) injections or adoptive transfer of Treg cells. METHODS: Female Swiss Webster mice received daily injections of ld-IL2 (1 µg/mouse, intraperitoneally) either before or after unilateral spared nerve injury (SNI). Male C57BL/6J mice received adoptive transfer of 1 x 106 Treg cells 3 weeks post-SNI. The responses to punctate and dynamic mechanical stimuli on the hindpaw were monitored before and up to 4-6 weeks post-SNI. We also compared the distribution of Treg cells and CD3+ total T cells after SNI and/or ld-IL2 treatment. RESULTS: Ld-IL2 pretreatment in female Swiss Webster mice completely blocked the development of SNI-induced dynamic mechanical allodynia and reduced the magnitude of punctate allodynia. Delayed ld-IL2 treatment in female mice significantly attenuated the morphine-resistant punctate and dynamic allodynia at 3-5 weeks post-SNI. Adoptive transfer of Treg cells to male C57BL/6J mice 3 weeks post-SNI effectively reversed the persistent punctate and dynamic allodynia, supporting that the effect of ld-IL2 is mediated through endogenous Treg cells, and is likely independent of mouse strain and sex. Neither ld-IL2 treatment nor Treg transfer affected the basal responses to punctate or brush stimuli. Ld-IL2 significantly increased the frequency of Treg cells among total CD3+ T cells in the injured sciatic nerves but not in the uninjured nerves or the dorsal root ganglia, suggesting the injured nerve as ld-IL2's site of action. CONCLUSION: Collectively, results from the present study supports Treg as a cellular target and ld-IL2 as a potential therapeutic option for nerve injury-induced persistent punctate and dynamic mechanical allodynia.

Electric Field Stimulation for the Functional Assessment of Isolated Dorsal Root Ganglion Neuron Excitability.

Genetically encoded calcium indicators have proven useful for characterizing dorsal root ganglion neuron excitability in vivo. Challenges persist in achieving high spatial-temporal resolutions in vivo, however, due to deep tissue imaging and motion artifacts that may be limiting technical factors in obtaining measurements. Here we report an ex vivo imaging method, using a peripheral neuron-specific Advillin-GCaMP mouse line and electric field stimulation of dorsal root ganglion tissues, to assess the sensitivity of neurons en bloc. The described method rapidly characterizes Ca2+ activity in hundreds of dorsal root ganglion neurons (221 ± 64 per dorsal root ganglion) with minimal perturbation to the in situ soma environment. We further validate the method for use as a drug screening platform with the voltage-gated sodium channel inhibitor, tetrodotoxin. Drug treatment led to decreased evoked Ca2+ activity; half-maximal response voltage (EV50) increased from 13.4 V in untreated tissues to 21.2, 23.3, 51.5 (p < 0.05), and 60.6 V (p < 0.05) at 0.01, 0.1, 1, and 10 µM doses, respectively. This technique may help improve an understanding of neural signaling while retaining tissue structural organization and serves as a tool for the rapid ex vivo recording and assessment of neural activity.

Increase in trigeminal ganglion neurons that respond to both calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide in mouse models of chronic migraine and posttraumatic headache.

A large body of animal and human studies indicates that blocking peripheral calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling pathways may prevent migraine episodes and reduce headache frequency. To investigate whether recurring migraine episodes alter the strength of CGRP and PACAP signaling in trigeminal ganglion (TG) neurons, we compared the number of TG neurons that respond to CGRP and to PACAP (CGRP-R and PACAP-R, respectively) under normal and chronic migraine-like conditions. In a mouse model of chronic migraine, repeated nitroglycerin (NTG) administration significantly increased the number of CGRP-R and PACAP-R neurons in TG but not dorsal root ganglia. In TG neurons that express endogenous αCGRP, repeated NTG led to a 7-fold increase in the number of neurons that respond to both CGRP and PACAP (CGRP-R&PACAP-R). Most of these neurons were unmyelinated C-fiber nociceptors. This suggests that a larger fraction of CGRP signaling in TG nociceptors may be mediated through the autocrine mechanism, and the release of endogenous αCGRP can be enhanced by both CGRP and PACAP signaling pathways under chronic migraine condition. The number of CGRP-R&PACAP-R TG neurons was also increased in a mouse model of posttraumatic headache (PTH). Interestingly, low-dose interleukin-2 treatment, which completely reverses chronic migraine-related and PTH-related behaviors in mouse models, also blocked the increase in both CGRP-R and PACAP-R TG neurons. Together, these results suggest that inhibition of both CGRP and PACAP signaling in TG neurons may be more effective in treating chronic migraine and PTH than targeting individual signaling pathways.

Low-dose interleukin-2 reverses behavioral sensitization in multiple mouse models of headache disorders.

Headache disorders are highly prevalent and debilitating, with limited treatment options. Previous studies indicate that many proinflammatory immune cells contribute to headache pathophysiology. Given the well-recognized role of regulatory T (Treg) cells in maintaining immune homeostasis, we hypothesized that enhancing Treg function may be effective to treat multiple headache disorders. In a mouse model of chronic migraine, we observed that repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration doubled the number of CD3 T cells in the trigeminal ganglia without altering the number of Treg cells, suggesting a deficiency in Treg-mediated immune homeostasis. We treated mice with low-dose interleukin-2 (ld-IL2) to preferentially expand and activate endogenous Treg cells. This not only prevented the development of NTG-induced persistent sensitization but also completely reversed the established facial skin hypersensitivity resulting from repeated NTG administration. The effect of ld-IL2 was independent of mouse sex and/or strain. Importantly, ld-IL2 treatment did not alter basal nociceptive responses, and repeated usage did not induce tolerance. The therapeutic effect of ld-IL2 was abolished by Treg depletion and was recapitulated by Treg adoptive transfer. Furthermore, treating mice with ld-IL2 1 to 7 days after mild traumatic brain injury effectively prevented as well as reversed the development of behaviors related to acute and chronic post-traumatic headache. In a model of medication overuse headache, Ld-IL2 completely reversed the cutaneous hypersensitivity induced by repeated administration of sumatriptan. Collectively, this study identifies ld-IL2 as a promising prophylactic for multiple headache disorders with a mechanism distinct from the existing treatment options.

MicroRNA-498 promotes proliferation, migration, and invasion of prostate cancer cells and decreases radiation sensitivity by targeting PTEN.

Prostate cancer (PCa) remains the secondary highest cause of cancer-related death in the United States in men. It has been reported that microRNAs can serve as key regulators in tumor development and progression in various cancers including PCa. In this study, we examined the effect of miR-498 on proliferation, radiosensitivity, invasion, and migration of PCa cells. The proliferation of LNCaP and DU-145 PCa cells with altered expression of miR-498 was evaluated by MTT assay. The invasion and migration of LNCaP and DU-145 PCa cells were assess by matrigel invasion assay and transwell migration assay. The protein expression level in PCa cells was examined by western blot. The function of miR-498 on radiation-induced apoptosis in LNCaP and DU-145 PCa cells was detected by Caspase-Glo3/7 assay. Forced expression of miR-498 improved the proliferation, invasion and migration in PCa cells. Furthermore, miR-498 decreased the sensitivity of PCa cells after ionizing radiation treatment. MiR-498 reduced the radiation-induced apoptosis in PCa cells by regulation of BAX and Bcl-2 expression. Meanwhile, miR-498 altered the expression of E-cadherin, N-cadherin, snail, and Vimentin in both LNCaP and DU-145 PCa cells and regulated epithelial to mesenchymal transition (EMT). Further study showed that aberrant expression of miR-498 changed the expression levels of phosphatase and tensin homolog and p-AKT in LNCaP and DU-145 PCa cells. In a summary, miR-498 displayed important roles in tumor development and progression in PCa cells, and might act as a potential prognostic biomarker and predict radiotherapy response in PCa.

TRESK K+ Channel Activity Regulates Trigeminal Nociception and Headache.

Although TWIK-related spinal cord K+ (TRESK) channel is expressed in all primary afferent neurons in trigeminal ganglia (TG) and dorsal root ganglia (DRG), whether TRESK activity regulates trigeminal pain processing is still not established. Dominant-negative TRESK mutations are associated with migraine but not with other types of pain in humans, suggesting that genetic TRESK dysfunction preferentially affects the generation of trigeminal pain, especially headache. Using TRESK global knock-out mice as a model system, we found that loss of TRESK in all TG neurons selectively increased the intrinsic excitability of small-diameter nociceptors, especially those that do not bind to isolectin B4 (IB4-). Similarly, loss of TRESK resulted in hyper-excitation of the small IB4- dural afferent neurons but not those that bind to IB4 (IB4+). Compared with wild-type littermates, both male and female TRESK knock-out mice exhibited more robust trigeminal nociceptive behaviors, including headache-related behaviors, whereas their body and visceral pain responses were normal. Interestingly, neither the total persistent outward current nor the intrinsic excitability was altered in adult TRESK knock-out DRG neurons, which may explain why genetic TRESK dysfunction is not associated with body and/or visceral pain in humans. We reveal for the first time that, among all primary afferent neurons, TG nociceptors are the most vulnerable to the genetic loss of TRESK. Our findings indicate that endogenous TRESK activity regulates trigeminal nociception, likely through controlling the intrinsic excitability of TG nociceptors. Importantly, we provide evidence that genetic loss of TRESK significantly increases the likelihood of developing headache.

Deficits in Burrowing Behaviors Are Associated With Mouse Models of Neuropathic but Not Inflammatory Pain or Migraine.

Burrowing, or the removal of material from an enclosed tube, is emerging as a prominent means of testing changes in a voluntary behavior in rodent models of various pain states. Here, we report no significant differences between male and female mice in terms of burrowing performance, in a substantially shorter time frame than previous reports. We found that the color of the burrow tube affects the variability of burrowing performance when tested in a lit room, suggesting that light aversion is at least a partial driver of this behavior. Spared nerve injury (SNI; as a model of neuropathy) impairs burrowing performance and correlates with enhanced mechanical sensitivity as assessed by von Frey filaments, as well as being pharmacologically reversed by an analgesic, gabapentin. Loss of the SNI-induced burrowing deficit was observed with daily testing post-surgery, but not when the testing interval was increased to 5 days, suggesting a confounding effect of daily repeat testing in this paradigm. Intraplantar complete Freund's adjuvant (as a model of inflammatory pain) and systemic nitroglycerin (as a model of migraine-like symptoms) administration did not induce any burrowing deficit, indicating that assessment of burrowing behavior may not be universally suitable for the detection of behavioral changes across all rodent pain models.

Quantitative Analysis of Mouse Dural Afferent Neurons Expressing TRPM8, VGLUT3, and NF200.

OBJECTIVE: To quantify the abundance of dural afferent neurons expressing transient receptor potential channel melastatin 8 (TRPM8), vesicular glutamate transporter 3 (VGLUT3), and neurofilament 200 (NF200) in adult mice. BACKGROUND: With the increasing use of mice as a model system to study headache mechanisms, it is important to understand the composition of dural afferent neurons in mice. In a previous study, we have measured the abundance of mouse dural afferent neurons that express neuropeptide calcitonin gene-related peptide as well as two TRP channels TRPV1 and TRPA1, respectively. Here, we conducted quantitative analysis of three other dural afferent subpopulations in adult mice. METHODS: We used the fluorescent tracer Fluoro-Gold to retrogradely label dural afferent neurons in adult mice expressing enhanced green fluorescent protein in discrete subpopulations of trigeminal ganglion (TG) neurons. Mechanoreceptors with myelinated fibers were identified by NF200 immunoreactivity. We also conducted Ca2+ -imaging experiments to test the overlap between TRPM8 and VGLUT3 expression in mouse primary afferent neurons (PANs). RESULTS: The abundance of TRPM8-expressing neurons in dural afferent neurons was significantly lower than that in total TG neurons. The percentages of dural afferent neurons expressing VGLUT3 and NF200 were comparable to those of total TG neurons, respectively. TRPM8 agonist menthol evoked Ca2+ influx in less than 7% VGLUT3-expressing PANs in adult mice. CONCLUSIONS: TG neurons expressing TRPM8, VGLUT3, and NF200 all innervate adult mouse dura. TRPM8 and VGLUT3 are expressed in distinct subpopulations of PANs in adult mice. These results provide an anatomical basis to investigate headache mechanisms in mouse models.

TRPM8 and Migraine.

Migraine is among the most common diseases on earth and one of the most disabling, the latter due in large part to poor treatment efficacy. Development of new therapeutics is dependent on the identification of mechanisms contributing to migraine and discovery of targets for new drugs. Numerous genome-wide association studies (GWAS) have implicated the transient receptor-potential M8 (TRPM8) channel in migraine. This channel is predominantly expressed on peripheral sensory neurons and is known as the sensor for cold temperature in cutaneous tissue but is also expressed on deep visceral afferents where cold is not likely a stimulus. Consequently, a number of alternative endogenous agonists have been proposed. Apart from its role in cold sensation, TRPM8 also contributes to cold allodynia after nerve injury or inflammation, and it is necessary for cooling/menthol-based analgesia. How it might contribute to migraine is less clear. The purpose of this review is to discuss the anatomical and physiological mechanisms by which meningeal TRPM8 may play a role in migraine as well as the potential of TRPM8 as a therapeutic target. TRPM8 is expressed on sensory afferents innervating the meninges, and these neurons are subject to developmental changes that may influence their contribution to migraine. As in viscera, meningeal TRPM8 channels are unlikely to be activated by temperature fluctuations and their endogenous ligands remain unknown. Preclinical migraine studies show that activation of meningeal TRPM8 by exogenous agonists can both cause and alleviate headache behaviors, depending on whether other meningeal afferents concurrently receive noxious stimuli. This is reminiscent of the fact that cold can trigger migraine in humans but menthol can also alleviate headache. We propose that both TRPM8 agonists and antagonists may be potential therapeutics, depending on how migraine is triggered in individual patients. In this regard, TRPM8 may be a novel target for personalized medicine in migraine treatment.

Characterization of a mouse model of headache.

Migraine and other primary headache disorders affect a large population and cause debilitating pain. Establishing animal models that display behavioral correlates of long-lasting and ongoing headache, the most common and disabling symptom of migraine, is vital for the elucidation of disease mechanisms and identification of drug targets. We have developed a mouse model of headache, using dural application of capsaicin along with a mixture of inflammatory mediators (IScap) to simulate the induction of a headache episode. This elicited intermittent head-directed wiping and scratching as well as the phosphorylation of c-Jun N-terminal kinase in trigeminal ganglion neurons. Interestingly, dural application of IScap preferentially induced FOS protein expression in the excitatory but not inhibitory cervical/medullary dorsal horn neurons. The duration of IScap-induced behavior and the number of FOS-positive neurons correlated positively in individual mice; both were reduced to the control level by the pretreatment of antimigraine drug sumatriptan. Dural application of CGRP(8-37), the calcitonin gene-related peptide (CGRP) receptor antagonist, also effectively blocked IScap-induced behavior, which suggests that the release of endogenous CGRP in the dura is necessary for IScap-induced nociception. These data suggest that dural IScap-induced nocifensive behavior in mice may be mechanistically related to the ongoing headache in humans. In addition, dural application of IScap increased resting time in female mice. Taken together, we present the first detailed study using dural application of IScap in mice. This headache model can be applied to genetically modified mice to facilitate research on the mechanisms and therapeutic targets for migraine headache.

Function and postnatal changes of dural afferent fibers expressing TRPM8 channels.

BACKGROUND: Genome-wide association studies have identified TRPM8 (transient receptor potential melastatin 8) as one of the susceptibility genes for common migraine. Here, we investigated the postnatal changes of TRPM8-expressing dural afferent fibers as well as the function of dural TRPM8 channels in mice. RESULTS: First, we quantified the density and the number of axonal branches of TRPM8-expressing fibers in the dura of mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from one TRPM8 allele between postnatal day 2 (P2) to adulthood. The number of axonal branches on individual dural EGFP-positive fibers was decreased by 30% between P2 and P11. The density of dural EGFP-positive fibers was subsequently reduced by 50% between P16 and P21. Conversely, the density and the number of branches of axons expressing calcitonin gene-related peptide remained stable in postnatal mouse dura. The density of TRPM8-expressing fibers innervating the mouse cornea epithelium was significantly increased from P2 to adulthood. Next, we tested the function of dural TRPM8 channels in adult mice and found that TRPM8 agonist menthol effectively inhibited the nocifensive behavior evoked by dural application of inflammatory mediators. CONCLUSIONS: Our results indicate that the TRPM8-expressing dural afferent fibers undergo cell- and target tissue-specific axonal pruning during postnatal development. Activation of dural TRPM8 channels decreases meningeal irritation-evoked nocifensive behavior in adult mice. This provides a framework to further explore the role of postnatal changes of TRPM8-expressing dural afferents in the pathophysiology of pediatric and adult migraine.

[Design, synthesis and evaluation of 5-aminobenzimidazolone derivatives as acetylcholinesterase inhibitors].

The target compounds were prepared from 5-aminobenzimidazolone by two steps reaction, and their AChE inhibitory activities were measured by Ellman method in vitro. The AChE inhibitory activity of compound 4d is the best of them, and its IC50 value is equal to 7.2 µmol·L(-1), which is better than that of rivastigmine; moreover the 4d had no inhibitory activities to BuChE. Therefore, the inhibitory activities of 5-aminobenzimidazolone derivatives to acetylcholinesterase are worth further researching.