Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice.

BACKGROUND: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM. METHODS: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated. RESULTS: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE. CONCLUSION: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

TLR2 Mediates Microglial Activation and Contributes to Central Sensitization in a Recurrent Nitroglycerin-induced Chronic Migraine Model.

Central sensitization is an important pathophysiological mechanism underlying chronic migraine (CM). Previous studies have shown that microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to central sensitization. Toll-like receptor 2 (TLR2) is a receptor expressed on the membrane of microglia and participates in central sensitization in inflammatory and chronic pain; however, its role in CM is unclear. Therefore, this study investigated TLR2 involvement in CM in detail. Mice treated with recurrent nitroglycerin (NTG) were used as a CM model. Hyperalgesia was assessed using a 50% paw mechanical threshold and a 50% periorbital threshold on a Von Frey filament pain meter. Western blotting and immunofluorescence analyses were used to detect the expression of TLR2, microglia, c-fos and CGRP in TNC. The expression of inflammatory factors (IL-6, IL-1ß、 IL-10、TNF-α and IFN-ß1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A selective TLR2 antagonist (C29) was systematically administered to observe its effect on hyperalgesia, microglia activation and the expression of c-fos, CGRP and inflammatory factors. Recurrent administration of NTG resulted in acute and chronic hypersensitivity, accompanied by upregulation of TLR2 expression and microglial activation in TNC. C29 partially inhibited pain hypersensitivity. C29 suppressed microglial activation induced by NTG administration. Inhibition of TLR2 reduced the expression of c-fos and CGRP in TNC after NTG treatment. C29 inhibited the expression of inflammatory mediators in TNC. These data showed that microglial TLR2 plays a critical role in the pathogenesis of CM by regulating microglial activation in TNC.

A c-Fos activation map in nitroglycerin/levcromakalim-induced models of migraine.

BACKGROUND: Chronic migraine is a common and highly disabling disorder. Functional MRI has indicated that abnormal brain region activation is linked with chronic migraine. Drugs targeting the calcitonin gene-related peptide (CGRP) or its receptor have been reported to be efficient for treating chronic migraine. The CGRP signaling was also shared in two types of chronic migraine models (CMMs). However, it remains unclear whether the activation of specific brain regions could contribute to persistent behavioral sensitization, and CGRP receptor antagonists relieve migraine-like pain in CMMs by altering specific brain region activation. Therefore, it's of great interest to investigate brain activation pattern and the effect of olcegepant (a CGRP receptor-specific antagonist) treatment on alleviating hyperalgesia by altering brain activation in two CMMs, and provide a reference for future research on neural circuits. METHODS: Repeated administration of nitroglycerin (NTG) or levcromakalim (LEV) was conducted to stimulate human migraine-like pain and establish two types of CMMs in mice. Mechanical hypersensitivity was evaluated by using the von Frey filament test. Then, we evaluated the activation of different brain regions with c-Fos and NeuN staining. Olcegepant was administered to explore its effect on mechanical hyperalgesia and brain region activation. RESULTS: In two CMMs, acute and basal mechanical hyperalgesia was observed, and olcegepant alleviated mechanical hyperalgesia. In the NTG-induced CMM, the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and the caudal part of the spinal trigeminal nucleus (Sp5c) showed a significant increase of c-Fos expression in the NTG group (p < 0.05), while pre-treatment with olcegepant reduced c-Fos expression compared with NTG group (p < 0.05). No significant difference of c-Fos expression was found in the paraventricular thalamic nucleus (PVT) and ventrolateral periaqueductal gray (vlPAG) between the vehicle control and NTG group (p > 0.05). In the LEV-induced CMM, mPFC, PVT, and Sp5c showed a significant increase of c-Fos expression between vehicle control and LEV group, and olcegepant reduced c-Fos expression (p < 0.05). No significant difference in c-Fos expression was found in vlPAG and ACC (p > 0.05). CONCLUSIONS: Our study demonstrated the activation of mPFC and Sp5c in two CMMs. Olcegepant may alleviate hyperalgesia of the hind paw and periorbital area by attenuating brain activation in CMMs.

Toll-like receptors and their role in neuropathic pain and migraine.

Migraine is a complex neurological disease of unknown etiology involving both genetic and environmental factors. It has previously been reported that persistent pain may be mediated by the immune and inflammatory systems. Toll-like receptors (TLRs) play a significant role in immune and inflammatory responses and are expressed by microglia and astrocytes. One of the fundamental mechanisms of the innate immune system in coordinating inflammatory signal transduction is through TLRs, which protect the host organism by initiating inflammatory signaling cascades in response to tissue damage or stress. TLRs reside at the neuroimmune interface, and accumulating evidence has suggested that the inflammatory consequences of TLR activation on glia (mainly microglia and astrocytes), sensory neurons, and other cell types can influence nociceptive processing and lead to pain. Several studies have shown that TLRs may play a key role in neuropathic pain and migraine etiology by activating the microglia. The pathogenesis of migraine may involve a TLR-mediated crosstalk between neurons and immune cells. Innate responses in the central nervous system (CNS) occur during neuroinflammatory phenomena, including migraine. Antigens found in the environment play a crucial role in the inflammatory response, causing a broad range of diseases, including migraines. These can be recognized by several innate immune cells, including macrophages, microglia, and dendritic cells, and can be activated through TLR signaling. Given the prevalence of migraine and the insufficient efficacy and safety of current treatment options, a deeper understanding of TLRs is expected to provide novel therapies for managing chronic migraine. This review aimed to justify the view that TLRs may be involved in migraine.

Anti-N-methyl-D-aspartate receptor encephalitis: a prospective study focused on cerebrospinal fluid and clinical symptoms.

BACKGROUND AND OBJECTIVE: To analyze the relationship between brain MRI, clinical symptoms, and cerebrospinal fluid (CSF) and to provide a reference for early diagnosis of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. MATERIALS AND METHODS: A total of 62 patients with anti-NMDAR encephalitis in Zhengzhou, China (2016-2018) were observed and registered prospectively. First, we analyzed the characteristics of clinical symptoms. Second, according to the disease duration, patients were divided into two groups, and then we analyzed the CSF features. In addition, they were divided into two groups according to the brain MRI, and then the CSF features were analyzed. Finally, the characteristics of cerebrospinal fluid in patients with seizure as the initial symptom were analyzed. RESULTS: Seizure presents as the initial symptom in 14 patients (22.5%), including 11 males (78.57%). The proportion and concentration of CSF total protein abnormalities in the early stage group were significantly higher than those in the middle and late group (P < 0.05). The total protein concentration in the abnormal brain MRI group was significantly higher than that in the normal MRI group (P < 0.05). CONCLUSION: Anti-NMDAR encephalitis should be suspected, when male patients complain of seizure as an initial symptom and have simultaneously had headaches or fever prior to onset. The sensitivity of anti-NMDAR antibody is higher in CSF than in serum. Total CSF protein is more prone to elevation in the middle and late stages of anti-NMDAR encephalitis. Brain MRI abnormalities with anti-NMDAR encephalitis are related to the total protein concentration of CSF, which may be related to the disease duration.

Botulinum Toxin Type A for refractory trigeminal neuralgia in older patients: a better therapeutic effect.

BACKGROUND: Some studies have indicated that a local injection of Botulinum Toxin Type A (BTX-A) is a promising therapy for trigeminal neuralgia (TN). However, BTX-A treatment is still ineffective for approximately 10-43% of patients. We therefore investigated which factors are associated with the therapeutic effect in BTX-A treatment of medically refractory, classical TN. METHODS: We performed a retrospective cohort study with a total of 104 patients who were receiving BTX-A injection for medically refractory classical TN between August 2013 and October 2016. A VAS score, pain attack frequency per day as well as patients' overall response to treatment and side effects were evaluated in 104 patients with TN who were receiving BTX-A. RESULTS: A total of 87 patients reported successful results; 41 stated that their pain was completely controlled while 46 reported adequate pain relief, totaling 83.7%. Our study suggests that treatment success was higher in patients 50 or older (OR=3.66, 95% CI: 1.231-10.885). Univariate and multivariate analyses demonstrated that the patient's age was independently associated with treatment outcome (OR=1.72, 95% CI: 1.063-2.282), with ≥50 years being a significant predictor of pain relief (P=0.020 and P=0.033, respectively). Seventeen patients (16.3%) reported mild side effects. CONCLUSION: A local injection of BTX-A may be a safe and efficient treatment for classical TN which lasts for several months. BTX-A is a novel strategy which is particularly worth trying for particularly middle-aged and elderly patients who cannot tolerate drug side effects and may be afraid of serious complications from microvascular decompression.

Intranasal Delivery of Botulinum Neurotoxin A Protects against Hippocampal Neuron Death in the Lithium-Pilocarpine Rat Model.

Botulinum neurotoxins (BoNTs) block the release of a series of neurotransmitters, which are pivotal for neuron action. Intrahippocampal administration of BoNTs inhibits glutamate release, protects neurons against cell death, and attenuates epileptic seizures. Compared with intrahippocampal administration, intranasal delivery is less invasive and more practical for chronic drug administration. To assess whether intranasal administration is feasible, we examined the role of botulinum neurotoxin A (BoNT/A) in hippocampal neuronal injury after status epilepticus (SE) induced by pilocarpine. Our data showed BoNT/A could bypass the blood-brain barrier (BBB) and entered the olfactory bulb and hippocampal neurons. In addition, SE could result in up-regulation of pro-apoptotic proteins (Caspase-3, Bax), down-regulation of anti-apoptotic protein Bcl-2 and neuronal death in hippocampus. BoNT/A could suppress the expression of Caspase-3 and Bax, attenuate the decrease of Bcl-2, and inhibit hippocampal neuron death induced by SE. Meanwhile, there was no significant difference in cognitive behavior between the BoNT/A-pretreated rats and normal rats. Thus, we provided a more convenient and less invasive route for taking advantage of BoNT/A in the field of anti-epilepsy.

A case-control study of the determinants for cervicocerebral artery dissection.

BACKGROUND: Cervicocerebral artery dissection (CAD) is a major cause of ischemic stroke in young adults. There are many existing studies on determinants for CAD; however, they are still not totally defined. We conduct the study to further investigate the determinants for CAD based on ischemic stroke patients. METHODS: 81 ischemic stroke patients with CAD were enrolled in the CAD stroke group and 84 ischemic stroke patients without CAD were enrolled in the non-CAD stroke group. Their clinical data, such as age, gender, vascular risk factors, headache and neck pain and clinical laboratory data, were collected to analyze the differences between the two groups. RESULTS: A total of 165 ischemic stroke patients were included. The mean age of CAD stroke group was (51.6 ± 12.4) years, and (55.5 ± 8.1) years in non-CAD stroke group, with a statistically significant difference (P = 0.017). The average level of triglycerides in CAD stroke group was (1.3 ± 0.7) mmol/L, and (1.7 ± 1.1) mmol/L in non-CAD stroke group, with a statistically significant difference (P = 0.012). There were 42.0% (34/81) of headache and neck pain in CAD stroke group and 22.6% (19/84) in non-CAD stroke group, with a statistically significant difference (P = 0.008). The key findings with significant difference were stratified and multivariate logistic regression analysis showed that age < 50 years old (OR 2.98, 95% CI 1.43-6.21, P = 0.004), triglycerides < 1.6 mmol/L (OR 3.51, 95% CI 1.69-7.27, P = 0.001) and headache and neck pain (OR 2.94, 95% CI 1.39-6.20, P = 0.005) showed a positive correlation with CAD. CONCLUSION: In the process of diagnosis and treatment of ischemic stroke, for patients with age < 50 years old, headache and neck pain and triglycerides < 1.6 mmol/L, the cervicocerebral artery dissection should be considered, and vascular imaging examination needs to be performed in time.

Characteristics and relative factors of headache caused by cervicocerebral artery dissection.

OBJECTIVE: To analyze the characteristics and relative factors of headache and neck pain due to cervicocerebral artery dissection (CAD). METHODS: A total of 146 consecutive patients with CAD in Zhengzhou, China (2010-2017) were observed and registered prospectively. There were 60 (60/146) cases who complained of headache and neck pain, and we analyzed the characteristics of pain according to their clinical features. For the 130 (130/146) patients with complete clinical laboratory data, they were divided into two groups according to pain, and the relative factors of pain were analyzed. RESULTS: The headache and neck pain in 60 CAD patients was mostly acute onset (98.3%), 70.6% (12/17) of patients with anterior circulation dissection and 88.4% (38/43) of patients with posterior circulation dissection complained of moderate to severe pain. 41.2% (7/17) of patients with anterior circulation dissection had temporal pain, while 46.5% (20/43) of the patients with posterior circulation dissection had occipital pain. There were 23.5% (4/17) and 32.6% (14/43) of patients with anterior and posterior circulation dissection complained of throbbing pain, respectively, 23.5% (4/17) and 20.9% (9/43) of patients with anterior and posterior circulation dissection complained of pulsating pain. The pain could occur in the ipsilateral (40.0%), bilateral (52.7%), or contralateral (7.3%) sites of the dissection. In the 130 patients, there were 56 cases (43.1%) in the pain group, and 74 cases (56.9%) in the non-pain group. Multivariate logistic regression analysis showed that female gender (OR 4.01, 95% CI 1.63-9.85, P = 0.002), posterior circulation (OR 3.18, 95% CI 1.39-7.28, P = 0.006), history of headache (OR 4.72, 95% CI 1.08-20.52, P = 0.039), and low-density lipoprotein less than 1.8 mmol/L (OR 2.90, 95% CI 1.15-7.34, P = 0.025) were risk factors of the occurrence of the pain related to CAD. CONCLUSION: The headache and neck pain caused by CAD is a moderate to severe pain occurring suddenly. The pain nature may be diverse but mostly like throbbing and pulsating. When the dissected artery is located in the posterior circulation, the pain is mostly in the occipital region, and mostly in the temporal region when the dissected artery is located in the anterior circulation. The pain can occur in ipsilateral, bilateral, or contralateral of the dissection. In addition, several factors might contribute to the occurrence of headache and neck pain.

[Determination of angiotensin converting enzyme inhibitor activity by high performance liquid chromatography].

A high performance liquid chromatographic method to determine angiotensin-converting enzyme inhibitor activity in vitro was established by using N-hippuryl-His-Leu tetrahydrate as the reaction substrate and hippuric acid as the reaction product. The chromatographic conditions were as follows: column, ZORBAX SB-C18 (4.6 mm i. d. x 150 mm, 5 microm); column temperature, 25 degrees C; mobile phase, acetonitrile-distilled water (25:75, v/v, both containing 0.05% (v/v) trifluoroacetic acid and 0.1% (v/v) triethylamine); flow rate, 0.5 mL/min; detection wavelength, 228 nm. An excellent linearity over the range of 0.005-1.000 mmol/L (r = 0.9999) was observed. The detection limit was 0.50 micromol/L. The recoveries of hippuric acid ware 99.48%-105.64%, with a relative standard deviation (RSD) of 2.20% (n = 6). It is a simple, precise and reliable assay method for developing antihypertension drugs.

[Determination of the inhibitory activity of angiotensin-converting enzyme inhibitor captopril by high performance capillary electrophoresis].

AIM: To establish a method for determinate of the inhibitory activity of angiotensin-converting enzyme inhibitor captopril by high performance capillary electrophoresis. METHODS: The characteristic absorptive wavelength of hippuric acid determined by ultraviolet spectrophotometer is 228 nm. The method employed a melted capillary column, 50 mmol.L-1 phosphoric acid (pH 8.3) buffer solution, inject pressure 4.8 kPa, inject time 3 s, separation voltage 20 kV and detection wavelength 228 nm. RESULTS: The reactant and resultant was separated completed within 7 min. IC50 of captopril was 0.019 mumol.L-1. Captopril is a competitive inhibitor, which was proved by enzyme reaction dynamics. CONCLUSION: The method was shown to be accurate, simple and rapid and can be used for determination of the inhibitory activity of captopril.