RESUMO
Multicellular organisms have co-evolved with complex consortia of viruses, bacteria, fungi and parasites, collectively referred to as the microbiota1. In mammals, changes in the composition of the microbiota can influence many physiologic processes (including development, metabolism and immune cell function) and are associated with susceptibility to multiple diseases2. Alterations in the microbiota can also modulate host behaviours-such as social activity, stress, and anxiety-related responses-that are linked to diverse neuropsychiatric disorders3. However, the mechanisms by which the microbiota influence neuronal activity and host behaviour remain poorly defined. Here we show that manipulation of the microbiota in antibiotic-treated or germ-free adult mice results in significant deficits in fear extinction learning. Single-nucleus RNA sequencing of the medial prefrontal cortex of the brain revealed significant alterations in gene expression in excitatory neurons, glia and other cell types. Transcranial two-photon imaging showed that deficits in extinction learning after manipulation of the microbiota in adult mice were associated with defective learning-related remodelling of postsynaptic dendritic spines and reduced activity in cue-encoding neurons in the medial prefrontal cortex. In addition, selective re-establishment of the microbiota revealed a limited neonatal developmental window in which microbiota-derived signals can restore normal extinction learning in adulthood. Finally, unbiased metabolomic analysis identified four metabolites that were significantly downregulated in germ-free mice and have been reported to be related to neuropsychiatric disorders in humans and mouse models, suggesting that microbiota-derived compounds may directly affect brain function and behaviour. Together, these data indicate that fear extinction learning requires microbiota-derived signals both during early postnatal neurodevelopment and in adult mice, with implications for our understanding of how diet, infection, and lifestyle influence brain health and subsequent susceptibility to neuropsychiatric disorders.
Assuntos
Extinção Psicológica/fisiologia , Medo/fisiologia , Metabolômica , Microbiota/fisiologia , Neurônios/fisiologia , Animais , Antibacterianos/farmacologia , Transtorno Autístico/metabolismo , Sangue/metabolismo , Cálcio/metabolismo , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/metabolismo , Sinais (Psicologia) , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/patologia , Espinhas Dendríticas/fisiologia , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Fezes/química , Vida Livre de Germes , Indicã/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Microbiota/imunologia , Inibição Neural , Neuroglia/patologia , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/patologia , Fenilpropionatos/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/imunologia , Córtex Pré-Frontal/fisiologia , Esquizofrenia/metabolismo , Transcriptoma , Nervo Vago/fisiologiaRESUMO
BACKGROUND: Choline acetyltransferase (ChAT) is required for the biosynthesis of acetylcholine, the molecular mediator that inhibits cytokine production in the cholinergic anti-inflammatory pathway of the vagus nerve inflammatory reflex. Abundant work has established the biology of cytoplasmic ChAT in neurons, but much less is known about the potential presence and function of ChAT in the extracellular milieu. OBJECTIVES: We evaluated the hypothesis that extracellular ChAT activity responds to inflammation and serves to inhibit cytokine release and attenuate inflammation. METHODS: After developing novel methods for quantification of ChAT activity in plasma, we determined whether ChAT activity changes in response to inflammatory challenges. RESULTS: Active ChAT circulates within the plasma compartment of mice and responds to immunological perturbations. Following the administration of bacterial endotoxin, plasma ChAT activity increases for 12-48 h, a time period that coincides with declining tumor necrosis factor (TNF) levels. Further, a direct activation of the cholinergic anti-inflammatory pathway by vagus nerve stimulation significantly increases plasma ChAT activity, whereas the administration of bioactive recombinant ChAT (r-ChAT) inhibits endotoxin-stimulated TNF production and anti-ChAT antibodies exacerbate endotoxin-induced TNF levels, results of which suggest that ChAT activity regulates endogenous TNF production. Administration of r-ChAT significantly attenuates pro-inflammatory cytokine production and disease activity in the dextran sodium sulfate preclinical model of inflammatory bowel disease. Finally, plasma ChAT levels are also elevated in humans with sepsis, with the highest levels observed in a patient who succumbed to infection. CONCLUSION: As a group, these results support further investigation of ChAT as a counter-regulator of inflammation and potential therapeutic agent.
Assuntos
Acetilcolina , Colina O-Acetiltransferase , Humanos , Colina O-Acetiltransferase/metabolismo , Inflamação , Fator de Necrose Tumoral alfa/metabolismo , Citocinas , EndotoxinasRESUMO
Inflammation, the body's primary defensive response system to injury and infection, is triggered by molecular signatures of microbes and tissue injury. These molecules also stimulate specialized sensory neurons, termed nociceptors. Activation of nociceptors mediates inflammation through antidromic release of neuropeptides into infected or injured tissue, producing neurogenic inflammation. Because HMGB1 is an important inflammatory mediator that is synthesized by neurons, we reasoned nociceptor release of HMGB1 might be a component of the neuroinflammatory response. In support of this possibility, we show here that transgenic nociceptors expressing channelrhodopsin-2 (ChR2) directly release HMGB1 in response to light stimulation. Additionally, HMGB1 expression in neurons was silenced by crossing synapsin-Cre (Syn-Cre) mice with floxed HMGB1 mice (HMGB1f/f). When these mice undergo sciatic nerve injury to activate neurogenic inflammation, they are protected from the development of cutaneous inflammation and allodynia as compared to wild-type controls. Syn-Cre/HMGB1fl/fl mice subjected to experimental collagen antibody-induced arthritis, a disease model in which nociceptor-dependent inflammation plays a significant pathological role, are protected from the development of allodynia and joint inflammation. Thus, nociceptor HMGB1 is required to mediate pain and inflammation during sciatic nerve injury and collagen antibody-induced arthritis.
Assuntos
Proteína HMGB1/metabolismo , Neurônios/fisiologia , Nociceptores/metabolismo , Animais , Anticorpos/imunologia , Artrite/induzido quimicamente , Células Cultivadas , Colágeno/imunologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica , Proteína HMGB1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Neuropatia Ciática/metabolismoRESUMO
BACKGROUND: Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4+ T cells as an intermediate. Activating the CAP ameliorates experimental acute pancreatitis. Galantamine is an acetylcholinesterase inhibitor (AChEI) which amplifies the CAP via modulation of central muscarinic ACh receptors (mAChRs). However, as mAChRs also activate pancreatitis, it is currently unknown whether galantamine would be beneficial in acute pancreatitis. METHODS: The effect of galantamine (1-6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed. RESULTS: Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase+ T cells or α7nAChR, or administration of the nAChR ganglionic blocker mecamylamine or the more selective α7nAChR antagonist methyllycaconitine. CONCLUSION: Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.
Assuntos
Galantamina , Pancreatite , Humanos , Camundongos , Animais , Galantamina/farmacologia , Galantamina/uso terapêutico , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Acetilcolinesterase/metabolismo , Acetilcolinesterase/uso terapêutico , Ceruletídeo/metabolismo , Ceruletídeo/uso terapêutico , Doença Aguda , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Citocinas/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Peso CorporalRESUMO
BACKGROUND: Inflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1ß (IL-1ß) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines. METHODS: To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1ß. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1ß-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant. RESULTS: Here, we describe a previously unrecognized mechanism by which IL-1ß activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis. CONCLUSION: Thus, IL-1ß activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity.
Assuntos
Hipotermia Induzida , Hipotermia , Interleucina-1beta , Canais de Potencial de Receptor Transitório , Animais , Camundongos , Anquirinas/metabolismo , Citocinas/metabolismo , Hipotermia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Fibras Nervosas/metabolismo , Dor/metabolismo , Reflexo , Células Receptoras Sensoriais/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Nervo Vago/metabolismoRESUMO
In the brain, compact clusters of neuron cell bodies, termed nuclei, are essential for maintaining parameters of host physiology within a narrow range optimal for health. Neurons residing in the brainstem dorsal motor nucleus (DMN) project in the vagus nerve to communicate with the lungs, liver, gastrointestinal tract, and other organs. Vagus nerve-mediated reflexes also control immune system responses to infection and injury by inhibiting the production of tumor necrosis factor (TNF) and other cytokines in the spleen, although the function of DMN neurons in regulating TNF release is not known. Here, optogenetics and functional mapping reveal cholinergic neurons in the DMN, which project to the celiac-superior mesenteric ganglia, significantly increase splenic nerve activity and inhibit TNF production. Efferent vagus nerve fibers terminating in the celiac-superior mesenteric ganglia form varicose-like structures surrounding individual nerve cell bodies innervating the spleen. Selective optogenetic activation of DMN cholinergic neurons or electrical activation of the cervical vagus nerve evokes action potentials in the splenic nerve. Pharmacological blockade and surgical transection of the vagus nerve inhibit vagus nerve-evoked splenic nerve responses. These results indicate that cholinergic neurons residing in the brainstem DMN control TNF production, revealing a role for brainstem coordination of immunity.
Assuntos
Endotoxemia/fisiopatologia , Inflamação/patologia , Bulbo/fisiologia , Baço/inervação , Fatores de Necrose Tumoral/metabolismo , Nervo Vago/fisiologia , Potenciais de Ação/imunologia , Animais , Neurônios Colinérgicos/fisiologia , Modelos Animais de Doenças , Endotoxemia/imunologia , Gânglios Simpáticos/fisiologia , Humanos , Inflamação/imunologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Masculino , Bulbo/citologia , Camundongos , Camundongos Transgênicos , Optogenética , Ratos , Transdução de Sinais/imunologia , Baço/metabolismo , Técnicas EstereotáxicasRESUMO
BACKGROUND: Severe COVID-19 is characterized by pro-inflammatory cytokine release syndrome (cytokine storm) which causes high morbidity and mortality. Recent observational and clinical studies suggest famotidine, a histamine 2 receptor (H2R) antagonist widely used to treat gastroesophageal reflux disease, attenuates the clinical course of COVID-19. Because evidence is lacking for a direct antiviral activity of famotidine, a proposed mechanism of action is blocking the effects of histamine released by mast cells. Here we hypothesized that famotidine activates the inflammatory reflex, a brain-integrated vagus nerve mechanism which inhibits inflammation via alpha 7 nicotinic acetylcholine receptor (α7nAChR) signal transduction, to prevent cytokine storm. METHODS: The potential anti-inflammatory effects of famotidine and other H2R antagonists were assessed in mice exposed to lipopolysaccharide (LPS)-induced cytokine storm. As the inflammatory reflex is integrated and can be stimulated in the brain, and H2R antagonists penetrate the blood brain barrier poorly, famotidine was administered by intracerebroventricular (ICV) or intraperitoneal (IP) routes. RESULTS: Famotidine administered IP significantly reduced serum and splenic LPS-stimulated tumor necrosis factor (TNF) and IL-6 concentrations, significantly improving survival. The effects of ICV famotidine were significantly more potent as compared to the peripheral route. Mice lacking mast cells by genetic deletion also responded to famotidine, indicating the anti-inflammatory effects are not mast cell-dependent. Either bilateral sub-diaphragmatic vagotomy or genetic knock-out of α7nAChR abolished the anti-inflammatory effects of famotidine, indicating the inflammatory reflex as famotidine's mechanism of action. While the structurally similar H2R antagonist tiotidine displayed equivalent anti-inflammatory activity, the H2R antagonists cimetidine or ranitidine were ineffective even at very high dosages. CONCLUSIONS: These observations reveal a previously unidentified vagus nerve-dependent anti-inflammatory effect of famotidine in the setting of cytokine storm which is not replicated by high dosages of other H2R antagonists in clinical use. Because famotidine is more potent when administered intrathecally, these findings are also consistent with a primarily central nervous system mechanism of action.
Assuntos
COVID-19 , Famotidina , Animais , Anti-Inflamatórios , Síndrome da Liberação de Citocina , Famotidina/farmacologia , Histamina , Antagonistas dos Receptores H2 da Histamina , Lipopolissacarídeos , Camundongos , Reflexo , Nervo Vago , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker L-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Colina O-Acetiltransferase/farmacologia , Modelos Animais de Doenças , Hipertensão/prevenção & controle , Proteínas Recombinantes/farmacologia , Acetilcolina/metabolismo , Angiotensina II , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Polietilenoglicóis/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
The nervous system maintains physiological homeostasis through reflex pathways that modulate organ function. This process begins when changes in the internal milieu (e.g., blood pressure, temperature, or pH) activate visceral sensory neurons that transmit action potentials along the vagus nerve to the brainstem. IL-1ß and TNF, inflammatory cytokines produced by immune cells during infection and injury, and other inflammatory mediators have been implicated in activating sensory action potentials in the vagus nerve. However, it remains unclear whether neural responses encode cytokine-specific information. Here we develop methods to isolate and decode specific neural signals to discriminate between two different cytokines. Nerve impulses recorded from the vagus nerve of mice exposed to IL-1ß and TNF were sorted into groups based on their shape and amplitude, and their respective firing rates were computed. This revealed sensory neural groups responding specifically to TNF and IL-1ß in a dose-dependent manner. These cytokine-mediated responses were subsequently decoded using a Naive Bayes algorithm that discriminated between no exposure and exposures to IL-1ß and TNF (mean successful identification rate 82.9 ± 17.8%, chance level 33%). Recordings obtained in IL-1 receptor-KO mice were devoid of IL-1ß-related signals but retained their responses to TNF. Genetic ablation of TRPV1 neurons attenuated the vagus neural signals mediated by IL-1ß, and distal lidocaine nerve block attenuated all vagus neural signals recorded. The results obtained in this study using the methodological framework suggest that cytokine-specific information is present in sensory neural signals within the vagus nerve.
Assuntos
Interleucina-1beta/farmacologia , Receptores Tipo I de Interleucina-1/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Nervo Vago/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Teorema de Bayes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Nervo Vago/citologia , Nervo Vago/efeitos dos fármacosRESUMO
BACKGROUND: Chronic alcohol use depletes brain serotonin (5-hydroxytryptamine [5-HT]), yet we previously found more tryptophan hydroxylase 2 (TPH2), the rate-limiting biosynthetic enzyme for 5-HT, in the dorsal raphe nucleus (DRN) of alcoholics. We sought to determine whether the increase in amount of TPH2 enzyme is associated with more TPH2 mRNA gene expression in the DRN of a new cohort of alcoholics and controls. METHODS: TPH2 mRNA and protein were measured by in situ hybridization and immunoautoradiography, respectively, in the DRN and median raphe nucleus (MRN) of age- and sex-matched pairs (n = 16) of alcoholics and nonpsychiatric controls. Alcohol use disorder diagnosis and medical, psychiatric, and family histories were obtained by psychological autopsy. Age and sex were covariates in the analyses. RESULTS: TPH2 mRNA in alcoholics was greater in the DRN and MRN compared to controls (DRN: controls: 3.6 ± 1.6, alcoholics: 4.8 ± 1.8 nCi/mg of tissue, F = 4.106, p = 0.02; MRN: controls: 2.6 ± 1.2, alcoholics: 3.5 ± 1.1 nCi/mg of tissue, F = 3.96, p = 0.024). The difference in TPH2 mRNA was present in all DRN subnuclei (dorsal [DRd]: 135%, interfascicular [DRif]: 139%, ventral [DRv]: 135%, ventrolateral [DRvl]: 136% of control p < 0.05) except the caudal subnucleus. Alcoholics also had more TPH2 protein in the DRN and MRN than controls (DRN: controls: 265 ± 47, alcoholics: 318 ± 47 µCi/g, F = 8.72, p = 0.001; MRN: controls: 250 ± 33, alcoholics: 345 ± 39 µCi/g, F = 7.78, p = 0.001). There is a positive correlation between TPH2 protein and mRNA expression in the DRN (r = 0.815, p < 0.001), suggesting that the higher amount of TPH2 protein is due to an increase in TPH2 gene expression. CONCLUSIONS: These findings suggest that greater TPH2 gene expression is the basis for more TPH2 protein in the DRN and MRN in alcoholics.
Assuntos
Alcoolismo/enzimologia , Alcoolismo/genética , Regulação Enzimológica da Expressão Gênica/genética , Núcleos da Rafe do Mesencéfalo/enzimologia , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Adulto , Alcoólicos , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Triptofano Hidroxilase/biossíntese , Adulto JovemRESUMO
BACKGROUND: Key to the advancement of the field of bioelectronic medicine is the identification of novel pathways of neural regulation of immune function. Sensory neurons (termed nociceptors) recognize harmful stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate host defense and inflammatory responses. However, it is still unclear whether nociceptors participate in regulating primary IgG antibody responses to novel antigens. METHODS: To understand the role of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons in IgG responses, we generated TRPV1-Cre/Rosa-ChannelRhodopsin2 mice for precise optogenetic activation of TRPV1 + neurons and TRPV1-Cre/Lox-diphtheria toxin A mice for targeted ablation of TRPV1-expressing neurons. Antigen-specific antibody responses were longitudinally monitored for 28 days. RESULTS: Here we show that TRPV1 expressing neurons are required to develop an antigen-specific immune response. We demonstrate that selective optogenetic stimulation of TRPV1+ nociceptors during immunization significantly enhances primary IgG antibody responses to novel antigens. Further, mice rendered deficient in TRPV1- expressing nociceptors fail to develop primary IgG antibody responses to keyhole limpet hemocyanin or haptenated antigen. CONCLUSION: This functional and genetic evidence indicates a critical role for nociceptor TRPV1 in antigen-specific primary antibody responses to novel antigens. These results also support consideration of potential therapeutic manipulation of nociceptor pathways using bioelectronic devices to enhance immune responses to foreign antigens.
RESUMO
Vagus nerve stimulation (VNS) is being actively explored as a treatment for multiple conditions as part of bioelectronic medicine research. Reliable and safe VNS in mouse models is a critical need for understanding mechanisms of these. We report on the development and evaluation of a microfabricated cuff electrode (MouseFlex) constructed of polyimide (PI) and with iridium oxide (IrOx) electrodes that is thermoformed to 86 µm ± 12 µm radius to interface the mouse cervical vagus nerve (r ≈ 50 µm). Innovative bench-top methods were used to evaluate the stimulation stability and electrochemical properties of electrodes. Our aggressive stimulation stability (Stim-Stab) test utilized 1 billion pulses at a 1000 Hz with a current density of 6.28 A/cm2 (1.51 mC/cm2/phase) delivering 3023 × 103 C/cm2 to evaluate electrode lifetimes, and all electrodes remained functional. We also investigated the effects of thermoforming on their impedance, charge storage capacity (CSC), and charge injection capacity (CIC). The modest changes in electrochemical properties indicate that the thermoforming process was well tolerated. Thermoformed electrode safety and efficacy were evaluated in-vivo by performing acute VNS in mice and monitoring their heart and respiration rate as biomarkers. Their electrochemical properties were also measured before, during and after VNS. Bradycardia and bradypnea were reliably induced at stimulation currents of 100 to 200 µA, well below the in-vivo CIC of â¼1250 µA (â¼0.5 mC/cm2), supporting their safety and efficacy. The electrode impedance increased and CIC decreased during in-vivo use, but largely reversed these changes in in-vitro testing after enzymatic cleaning, supporting their tolerance for surgical use. STATEMENT OF SIGNIFICANCE: Vagus nerve stimulation (VNS) is a rapidly growing aspect of healthcare and bioelectronic medicine research. Reliable and safe VNS in mice with small diameter (d ≈ 100 µm) nerves has been a challenge due to achieving intimate contact with the nerve, and the stimulation stability of commonly used electrodes. We demonstrate a microfabricated (MouseFlex) cuff electrode constructed of polyimide with IrOx electrodes that is thermoformed to contact the mouse cervical vagus. Bench studies highlight the stimulation stability exceeded 109 pulses at 6.28 A/cm2 and their electrochemical properties were measured before, during, and after bench and nerve stimulation. Nerve stimulation induced bradycardia and bradypnea at currents below the in-vivo charge injection capacity, supporting their safety, efficacy, and tolerance for surgical handling.
Assuntos
Estimulação do Nervo Vago , Camundongos , Animais , Estimulação do Nervo Vago/métodos , Bradicardia , Eletrodos , Nervo Vago/fisiologia , Coração , Estimulação ElétricaRESUMO
Introduction: Inflammation is an inherently self-amplifying process, resulting in progressive tissue damage when unresolved. A brake on this positive feedback system is provided by the nervous system which has evolved to detect inflammatory signals and respond by activating anti-inflammatory processes, including the cholinergic anti-inflammatory pathway mediated by the vagus nerve. Acute pancreatitis, a common and serious condition without effective therapy, develops when acinar cell injury activates intrapancreatic inflammation. Prior study has shown that electrical stimulation of the carotid sheath, which contains the vagus nerve, boosts the endogenous anti-inflammatory response and ameliorates acute pancreatitis, but it remains unknown whether these anti-inflammatory signals originate in the brain. Methods: Here, we used optogenetics to selectively activate efferent vagus nerve fibers originating in the brainstem dorsal motor nucleus of the vagus (DMN) and evaluated the effects on caerulein-induced pancreatitis. Results: Stimulation of the cholinergic neurons in the DMN significantly attenuates the severity of pancreatitis as indicated by reduced serum amylase, pancreatic cytokines, tissue damage, and edema. Either vagotomy or silencing cholinergic nicotinic receptor signaling by pre-administration of the antagonist mecamylamine abolishes the beneficial effects. Discussion: These results provide the first evidence that efferent vagus cholinergic neurons residing in the brainstem DMN can inhibit pancreatic inflammation and implicate the cholinergic anti-inflammatory pathway as a potential therapeutic target for acute pancreatitis.
Assuntos
Pancreatite , Humanos , Pancreatite/tratamento farmacológico , Doença Aguda , Optogenética , Inflamação , Tronco EncefálicoRESUMO
Deficiency of coagulation factor VIII in hemophilia A disrupts clotting and prolongs bleeding. While the current mainstay of therapy is infusion of factor VIII concentrates, inhibitor antibodies often render these ineffective. Because preclinical evidence shows electrical vagus nerve stimulation accelerates clotting to reduce hemorrhage without precipitating systemic thrombosis, we reasoned it might reduce bleeding in hemophilia A. Using two different male murine hemorrhage and thrombosis models, we show vagus nerve stimulation bypasses the factor VIII deficiency of hemophilia A to decrease bleeding and accelerate clotting. Vagus nerve stimulation targets acetylcholine-producing T lymphocytes in spleen and α7 nicotinic acetylcholine receptors (α7nAChR) on platelets to increase calcium uptake and enhance alpha granule release. Splenectomy or genetic deletion of T cells or α7nAChR abolishes vagal control of platelet activation, thrombus formation, and bleeding in male mice. Vagus nerve stimulation warrants clinical study as a therapy for coagulation disorders and surgical or traumatic bleeding.
Assuntos
Hemofilia A , Trombose , Estimulação do Nervo Vago , Camundongos , Masculino , Animais , Hemofilia A/complicações , Hemofilia A/terapia , Receptor Nicotínico de Acetilcolina alfa7/genética , Plaquetas , Hemorragia/terapia , Nervo VagoRESUMO
Background. Severe COVID-19 is characterized by pro-inflammatory cytokine release syndrome (cytokine storm) which causes high morbidity and mortality. Recent observational and clinical studies suggest famotidine, a histamine 2 receptor (H2R) antagonist widely used to treat gastroesophageal reflux disease , attenuates the clinical course of COVID-19. Because evidence is lacking for a direct antiviral activity of famotidine, a proposed mechanism of action is blocking the effects of histamine released by mast cells. Here we hypothesized that famotidine activates the inflammatory reflex, a brain-integrated vagus nerve mechanism which inhibits inflammation via alpha 7 nicotinic acetylcholine receptor ( α7nAChR ) signal transduction, to prevent cytokine storm. Methods. The potential anti-inflammatory effects of famotidine and other H2R antagonists was assessed in mice exposed to lipopolysaccharide (LPS)-induced cytokine storm. As the inflammatory reflex is integrated and can be stimulated in the brain, and H2R antagonists penetrate the blood brain barrier poorly, famotidine was administered by intracerebroventricular (ICV) or intraperitoneal (IP) routes. Results. Famotidine administered IP significantly reduced serum and splenic LPS-stimulated tumor necrosis factor α and interleukin-6 concentrations, significantly improving survival. The effects of ICV famotidine were significantly more potent as compared to the peripheral route. Mice lacking mast cells by genetic deletion also responded to famotidine, indicating the anti-inflammatory effects are not mast cell dependent. Either bilateral sub-diaphragmatic vagotomy or genetic knock-out of α7nAChR abolished the anti-inflammatory effects of famotidine, indicating the inflammatory reflex as famotidine's mechanism of action. While the structurally similar H2R antagonist tiotidine displayed equivalent anti-inflammatory activity, the H2R antagonists cimetidine or ranitidine were ineffective even at very high dosages. Conclusions. These observations reveal a previously unidentified vagus nerve-dependent anti-inflammatory effect of famotidine in the setting of cytokine storm which is not replicated by high dosages of other H2R antagonists in clinical use. Because famotidine is more potent when administered intrathecally, these findings are also consistent with a primarily central nervous system mechanism of action.
RESUMO
BACKGROUND: Brain metabolic alterations and neuroinflammation have been reported in several peripheral inflammatory conditions and present significant potential for targeting with new diagnostic approaches and treatments. However, non-invasive evaluation of these alterations remains a challenge. METHODS: Here, we studied the utility of a micro positron emission tomography (microPET) dual tracer ([11C]PBR28 - for microglial activation and [18F]FDG for energy metabolism) approach to assess brain dysfunction, including neuroinflammation in murine endotoxemia. MicroPET imaging data were subjected to advanced conjunction and individual analyses, followed by post-hoc analysis. RESULTS: There were significant increases in [11C]PBR28 and [18F]FDG uptake in the hippocampus of C57BL/6 J mice 6 h following LPS (2 mg/kg) intraperitoneal (i.p.) administration compared with saline administration. These results confirmed previous postmortem observations. In addition, patterns of significant simultaneous activation were demonstrated in the hippocampus, the thalamus, and the hypothalamus in parallel with other tracer-specific and region-specific alterations. These changes were observed in the presence of robust systemic inflammatory responses manifested by significantly increased serum cytokine levels. CONCLUSIONS: Together, these findings demonstrate the applicability of [11C]PBR28 - [18F]FDG dual tracer microPET imaging for assessing neuroinflammation and brain metabolic alterations in conditions "classically" characterized by peripheral inflammatory and metabolic pathogenesis.
RESUMO
Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.
Assuntos
Diabetes Mellitus Experimental , Glucose , Animais , Diabetes Mellitus Experimental/terapia , Glucose/metabolismo , Homeostase , Hipotálamo/metabolismo , Fígado/metabolismo , Camundongos , Ratos , SuínosRESUMO
Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate "Western" diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.
Assuntos
Metabolismo dos Lipídeos/efeitos da radiação , Fígado/efeitos da radiação , Obesidade/sangue , Obesidade/terapia , Terapia por Ultrassom/métodos , Adipocinas/sangue , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos da radiação , Adiposidade/efeitos da radiação , Animais , Citocinas/sangue , Dieta Hiperlipídica/efeitos adversos , Dieta Ocidental/efeitos adversos , Inflamação/metabolismo , Inflamação/terapia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Transdução de Sinais/efeitos da radiação , Aumento de Peso/efeitos da radiaçãoRESUMO
BACKGROUND: The peripheral nervous system is involved in a multitude of physiological functions. Recording neural signals provides information that can be used by diagnostic bioelectronic medicine devices, closed-loop neuromodulation therapies and other neuroprosthetic applications. The ability to accurately record these signals is challenging, due to the presence of various biological and instrument-related interference sources. NEW METHOD: We developed a common-mode interference rejection algorithm based on an impedance matching approach for bipolar cuff electrodes. Two unipolar channels were recorded from the two electrode contacts of a bipolar cuff. The impedance mismatch was estimated and used to correct one of the two channels. RESULTS: When applied to electrocardiographic (ECG) artifacts collected from three mice using CorTec electrodes, the algorithm reduced the interference to noise ratio (INR) over simple subtraction by 12â¯dB on average. The algorithm also reduced the INR of stimulation artifacts in recordings from three rats collected using flexible electrodes by an additional 2.4â¯dB. In the same experiments evoked electromyographic (EMG) interference was suppressed by 1.3â¯dB. COMPARISON WITH EXISTING METHODS: Simple subtraction is the common approach for reducing common-mode interference in bipolar recordings, however impedance mismatches that exist or emerge compromise its efficiency. CONCLUSIONS: The algorithm significantly reduced the common-mode interference from ECG artifacts, stimulation artifacts, and evoked EMG interference, while retaining neural signals, in two animal models and two recording setups. This approach can be used in a variety of different neurophysiological setups to remove common-mode interference from a variety of sources.