The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

Tutti gli esperimenti sugli animali sono stati eseguiti secondo le linee guida per la cura e l&#39;uso di animali approvati dal Comitato Etico locale Karolinska Institutet, Stoccolma. Il Comitato Etico locale segue le direttive dell&#39;Unione europea in materia di cura degli animali. NOTA: I principali cambiamenti rispetto al protocollo originale sono il tempo di recupero dopo l&#39;intervento chirurgico (6 ore contro 1 ora) e il livello di LPS iniettati (2 mg / kg contro 15 mg / kg). In caso contrario, le diverse impostazioni relative alla chirurgia stessa non sono stati modificati. 1. preparazione del materiale per la stimolazione <ol><li> Accendere il computer e il sistema di acquisizione dati collegato alla stimolazione elettrodo (Figura 1A). </li><li> Inserire il Riconoscimento programma. </li><li> Preparare una soluzione stock di LPS ad una concentrazione di 5 mg / ml in PBS 1x, aliquota, e conservarlo a -20 ° C. Il giorno dell&#39;esperimento, scongelare un&#39;aliquota e preparare un adequate campione di LPS (0,5 mg / ml) per iniettare circa 100 microlitri nell&#39;animale, in base al peso. </li></ol> 2. Preparare l&#39;animale per anestesia <ol><li> Utilizzare C57BL 6 topi /. Mantenerle in condizioni climatiche controllate con un ciclo luce-12 hr / scuro, alimentarli chow roditori standard e dare loro acqua ad libitum. </li><li> Eseguire un intervento chirurgico su topi che pesano circa 25 g al giorno dell&#39;esperimento. NOTA: Quando reazioni infiammatorie sono indotti, è particolarmente importante effettuare regolari controlli e osservazioni delle reazioni cliniche negli animali. L&#39;eutanasia precoce da CO 2 inalazione è necessaria se la condizione animale non soddisfa i criteri etici. </li><li> Impostare la macchina anestetico. <ol><li> Assicurarsi che il tubo sia collegato correttamente e non sia danneggiato in alcun modo. Assicurarsi che la zona ventilata funziona correttamente. Collegare il filtro chiave per la bottiglia isoflurano e riempire il vaporizer con una quantità sufficiente di isoflurano. </li><li> Aprire l&#39;alimentazione del gas (aria e ossigeno) e fare in modo che le bottiglie contengono abbastanza gas per l&#39;esperimento. Un connettore a tre vie è quindi in grado di inviare il flusso isoflurano alla camera di induzione o la maschera. </li></ol></li><li> Ruotare il connettore a tre vie per la camera di induzione. Scegli un mouse dalla gabbia casa e inserire l&#39;animale nella camera. Regolare il regolatore di flusso di 1,0 L / min di ossigeno e 1,0 L / min di aria. Regolare la concentrazione isoflurano al 4-5%. </li><li> Quando si raggiunge il livello desiderato di anestesia, quando si raggiunge il livello desiderato di anestesia, radere l&#39;area chirurgica e spostare l&#39;animale dalla camera alla maschera. Girare il connettore a tre vie per il flusso maschera. Regolare il regolatore di flusso di 0,25 L / min ossigeno e 0,25 L / min di aria. <ol><li> Regolare la concentrazione isoflurano al 1,5-2,5%. Controllare il livello di anestesia per il controllo del riflesso e la frequenza respiratoria prima di iniziare SEGUIRE IL PROCEDIMENTO chirurgicae. </li></ol></li><li> Fissare le gambe del mouse al banco di lavoro con nastro adesivo. Assicurarsi che il naso dell&#39;animale è ancora accuratamente posizionato nella maschera. </li></ol> 3. Chirurgia e stimolazione del nervo vago <ol><li> Disinfettare l&#39;area chirurgica con il 70% di etanolo. </li><li> Usando un bisturi, incidere con cura la pelle a livello del collo (un&#39;incisione di circa 1 a 1,5 cm). Nota: Nel protocollo, la procedura di chirurgia finisce qui per gli animali sham-operati. Infatti, è stato dimostrato che sfiora il nervo con un attrezzo metallico stimola già in una certa misura. Pertanto, per ottenere un più accurato controllo della chirurgia degli animali quando si utilizza una piccola quantità di LPS, fermare l&#39;operazione in questa fase. </li><li> Con l&#39;aiuto di un microscopio (obiettivo 12,5X), isolare il nervo vago sinistro dalla carotide utilizzando dissettore. In primo luogo individuare il muscolo sternocleidomastoideo rimuovendo pelle e grasso strati, e poi rientrare inordinare di mettere la pinza dietro sia il nervo e l&#39;arteria. Nota: Il passo successivo è molto difficile, come il nervo e arteria sono strettamente aderire l&#39;uno all&#39;altro. Pertanto, è molto facile da tagliare la nave e uccidere l&#39;animale. Tuttavia, ponendo molta attenzione la pinza tra il nervo e l&#39;arteria, che alla fine si separano, ed è possibile isolare il nervo. </li><li> Posizionare l&#39;elettrodo (Figura 1B) sotto il nervo vago. L&#39;elettrodo ad ago è piuttosto lungo, anche se il nervo muove leggermente durante la stimolazione, sarà sempre in contatto con l&#39;elettrodo. </li><li> Eseguire un intraperitoneale (ip) iniezione di LPS (2 mg / kg) con l&#39;aiuto di una siringa (per la lettura su livelli di citochine, cioè per misurare l&#39;effetto down-regulation). </li><li> Attendere 5 minuti prima di iniziare la stimolazione. </li><li> Stimolare il nervo vago per 5 minuti a 5 V e 1 Hz premendo il pulsante di avvio nel programma riconoscere. </li><li> Rispostare l&#39;elettrodo e suturare la ferita dell&#39;animale con filo di sutura chirurgica. </li><li> Spruzzare un No Sting Barriera Film (NSBF) sulla ferita al fine di migliorare la guarigione e per proteggere dalle infezioni. </li></ol> 4. Recupero del Animal <ol><li> Dopo l&#39;intervento chirurgico, spostare l&#39;animale torna alla sua gabbia casa per il risveglio e il recupero. Sotto la luce infrarossa al fine di mantenere la temperatura corporea, assicurarsi di monitorare l&#39;animale fino piena coscienza è stato ripreso. </li><li> Lasciate che l&#39;animale recuperare nella sua gabbia per 6 ore prima del sacrificio per l&#39;analisi. Nota: L&#39;effetto della stimolazione del nervo vago è molto veloce e ha anche dimostrato di essere di lunga durata (fino a 48 ore), in modo che il tempo di recupero può essere impostato dallo sperimentatore secondo le esigenze dello studio. </li></ol> 5. Il sacrificio per ulteriori analisi <ol><li> Posto l&#39;animale in una gabbia collegato a un dispositivo di somministrazione di CO 2. </li><li> Impostare il dispositivo su un 5Ciclo -min di CO 2 inalazione. </li><li> Quando l&#39;eutanasia è fatto, raccogliere gli organi di interesse e direttamente congelarli in ghiaccio secco per ulteriori analisi (ad esempio, la misurazione dei livelli di citochine negli estratti di milza usando un topo TH1 / TH2 9-Plex assay) 3. </li></ol>

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Fin dalla sua scoperta nei primi anni 2000, i meccanismi della PAC sono stati accuratamente studiati. Ora abbiamo una buona immagine del percorso, e in particolare, l&#39;organo bersaglio, la milza, dove NE, le cellule T memoria, Ach, e macrofagi lavorare come una squadra molto efficiente per down-regolare mediatori infiammatori 2. Abbiamo inoltre recentemente pubblicato dati sulla importanza di un sistema prostaglandine funzionale nei topi, in particolare, PGE 2, che è ovviamente un ...

L&#39;immunità innata fornisce immediatamente prima linea di difesa contro le infezioni e malattie in una vasta gamma di organismi. Essa non solo avvia la risposta immunitaria primaria per eliminare la minaccia, ma svolge anche un ruolo fondamentale nell&#39;attivazione e educare l&#39;immunità adattativa che svolge risposte immunitarie secondarie in modo specifico agente patogeno. L&#39;infiammazione è orchestrato da una pletora di citochine e chemochine, che a loro volta hanno la capacità di attrarre altre cellule immunitarie al sito di infezione e di indurre i segni cardinali di infiammazione, come rossore, gonfiore, dolore, perdita di funzione, e la febbre . La durata e l&#39;intensità dell&#39;infiammazione dipendono da diversi fattori, ma risolvere l&#39;infiammazione e ripristinando l&#39;omeostasi è un passo fondamentale per evitare l&#39;insorgenza di malattie infiammatorie croniche. I recenti progressi nel campo delle neuroscienze e immunologia hanno svelato i meccanismi neurali specifiche con immenso potenziale terapeutico per il controllo inflinfiammazione sia nel sistema nervoso centrale e nella periferia. Uno di essi consiste nello colinergica via anti-infiammatori (PAC), noto anche come il riflesso infiammatoria, che è azionato dal sistema nervoso autonomo 4, 5. Attualmente è pensato che mediatori infiammatori attivano nervi sensoriali e inviare segnali relativi allo stato di infiammazione del sistema nervoso centrale. Una risposta riflessa viene quindi attivato attraverso il nervo vago efferente. Un ampio studio sui dettagli anatomici della PAC ha rivelato un modello parasimpatico-simpatico composto da due nervi, il nervo vago e nervo splenico, rispettivamente 6. Nella PAC, il nervo vago colinergica efferente attivato termina nel ganglio celiaco mesenterico, causando l&#39;attivazione del nervo splenico adrenergico da un meccanismo ancora da esplorare. Il nervo splenico, così attivato, si caratterizza per internovate in prossimità di cellule immunitarie nella polpa bianca, zona marginale, e polpa rossa della milza, la principale e organo obbligatorio della PAC 7, 8. Norepinefrina (NE) dalle terminazioni nervose splenici lega ai corrispondenti β 2 adrenergici espressi sui linfociti T splenici. Questo induce il rilascio colina acetil transferasi (ChAT) acetilcolina mediata (ACh), che a sua volta attiva i recettori nicotinici dell&#39;acetilcolina (a7) α7nACh sui macrofagi, limitando in tal modo la produzione di citochine e infiammazione 2. Di conseguenza, è chiaro che il sistema nervoso è in grado di regolare l&#39;infiammazione nei tessuti periferici e di ripristinare omeostasi immunitaria locale. Come il nome del percorso suggerisce, il sistema ACh è di importanza fondamentale per il funzionamento di questo percorso di regolazione neuro-immune. È interessante notare che i meccanismi coinvolti nell&#39;attivazione diPAC sembrano essere differente nella periferia e nel sistema nervoso centrale. Mentre l&#39;importanza dei recettori nicotinici (α7nAChR) nella milza è stata dimostrata in precedenza 9, recettori muscarinici (mAChR) sono obbligatorie per l&#39;attivazione centrale del percorso 10, 11. Più recentemente, la somministrazione periferica di una azione centrale M1 agonista muscarinico notevolmente soppressa fattore di necrosi tumorale siero e milza α (TNFa) durante letale endotossemia murina, un&#39;azione che richiedeva intatto nervo vago e nervo splenico segnalazione 12. Abbiamo inoltre recentemente dimostrato che topi privi prostaglandina E2 (PGE2) non erano in grado di rispondere alla stimolazione del nervo vago e non down-regolano il rilascio indotto da LPS di citochine nel siero e milza 3. Pertanto, la PAC potrebbe anche essere regolata da sistemi diversi dal principale pathw AChAy. Il nervo vago è stata indicata come tale per la sua naturalmente vagare nel corpo, innervano organi principali compreso il fegato, polmone, milza, reni, intestino e 13. Considerando questo grande innervazione e molto potente effetto immunosoppressivo del nervo vago, il potenziale terapeutico della PAC potrebbe coprire un&#39;ampia gamma di condizioni infiammatorie. Il nervo vago può essere elettricamente (o meccanicamente) attivato, con controllo della tensione e frequenza, e contrariamente al trattamento convenzionale, senza farmaci aggiunti al corpo. Le prove sono attualmente in corso in pazienti reumatici, per esempio, per testare il significato clinico di VNS nel trattamento di infiammazioni croniche 14. Complessivamente, la comunicazione e la regolazione di infiammazione neuro-immunitario sono attualmente allo studio, che fornirà un trattamento alternativo alla terapia convenzionale. Pertanto, l&#39;analisi del nervo vago stimulatioEffetto n nei diversi organi innervati, ma anche la caratterizzazione del potenziale azione terapeutica in modelli animali di infiammazione cronica, sarebbe sicuramente dare spunti e di speranza per nuovi potenziali bersagli terapeutici. La metodologia originale sviluppato da Tracey e colleghi 4 non poteva essere trasposto al nostro campo di ricerca a causa di eccesso di stimolazione della risposta infiammatoria (da una dose letale di LPS) e una troppo breve intervallo di tempo tra l&#39;attivazione della PAC e il read-out. Nel presente documento, presenteremo le modifiche apportate al protocollo originale, confrontare i due metodi diversi su livelli di citochine, ed evidenziare un nuovo fronte osservazione sull&#39;organo bersaglio (la milza).

<table><tbody><tr><td>Computer</td><td>Toshiba</td><td>-</td><td>Any computer is actually compatible</td></tr><tr><td>MP-150 data acquisition system</td><td>Biopac Systems</td><td>MP150WSW</td><td></td></tr><tr><td>Acknowledge software</td><td>Biopac Systems</td><td></td><td></td></tr><tr><td>Mice C57Bl/6</td><td>Charles River</td><td></td><td></td></tr><tr><td>Anesthetic machine</td><td>Simtec Engineering</td><td></td><td></td></tr><tr><td>Medical oxygen bottle</td><td>AGA</td><td>107563</td><td></td></tr><tr><td>Medical air bottle&amp;nbsp;</td><td>AGA</td><td>108639</td><td></td></tr><tr><td>Vetflurane (1,000 mg/g)</td><td>Virbac</td><td>137317</td><td></td></tr><tr><td>LPS</td><td>Sigma-Aldrich</td><td>L2630</td><td></td></tr><tr><td>Saline</td><td>Merck Millipore</td><td>1024060080</td><td></td></tr><tr><td>PBS 10x</td><td>Sigma-Aldrich</td><td>P5493</td><td>Diluted 10 times for used concentration</td></tr><tr><td>Syringe (1 ml)</td><td>BD Plastipak</td><td>303172</td><td></td></tr><tr><td>Needles 23 G</td><td>KD-FINE</td><td>900284</td><td>0.6 x 30 mm (blue)</td></tr><tr><td>Microdissecting forceps (curved)</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Dissecting scissors</td><td>Sigma-Aldrich</td><td>Z265969</td><td></td></tr><tr><td>Surgical suture 4-0</td><td>Ethicon</td><td>G667G</td><td></td></tr><tr><td>Euthanasia unit</td><td>Euthanex Smartbox</td><td>EA-32000</td><td></td></tr><tr><td>Cavilon No Sting Barrier Film</td><td>3M Health Care</td><td>3346N</td><td></td></tr><tr><td>TH1/TH2 9-Plex assay, ultrasensitive kit</td><td>MesoScale Discovery</td><td>K15013C-1</td><td></td></tr><tr><td>Stimulating electrode device</td><td>Biopac Systems</td><td>STIMSOC</td><td></td></tr><tr><td>Aesculap Isis shaver</td><td>Agnthos</td><td>GT420</td><td></td></tr><tr><td>R70</td><td></td><td></td><td>Rodent diet from Lantmannen, Stockholm, Sweden</td></tr></tbody></table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Livello di TNFa e interleuchina-1β (IL-1β) dopo aver aumentato il Time Lapse dopo l&#39;intervento e ridurre la dose di LPS Come mostrato in precedenza, utilizzando il protocollo originale, VNS diminuzione dei livelli di TNFa (169,3 ± 24,9 pg / mg in SHAM contro 39,7 ± 10,8 pg / mg in VNS, p &lt;0.001) e IL-1β (360.0 ± 40.21 pg / mg SHAM contro 191,7 ± 27,2 pg / mg in VNS, p &lt;0,01) nella milza dopo l&#39;iniezione intra...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Aumento tempo di recupero e Diminuzione LPS Per studiare il nervo vago meccanismi di stimolazione in risposta infiammatoria limitati

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.8K Views.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Video: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Stimolazione del nervo vago come strumento per indurre plasticità Percorsi rilevanti per l&#39;apprendimento Extinction

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Ricerca

Comportamento

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Immunologia e infezioni

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Un metodo di nodose Ganglia iniezione in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Neuroscienze

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

Iniezioni di Lipopolysaccharide in topi per simulare l'ingresso di prodotti microbici derivati dopo la violazione della barriera intestinale

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus nerve stimulation (taVNS). Unlike cervically-implanted VNS, taVNS is an inexpensive and non-surgical method used to modulate the vagus system. taVNS is appealing as it allows for rapid translation of basic VNS research and serves as a safe, inexpensive, and portable neurostimulation system for the future treatment of central and peripheral disease. The background and rationale for taVNS is described, along with electrical and parametric considerations, proper ear targeting and attachment of stimulation electrodes, individual dosing via determination of perception threshold (PT), and safe administration of taVNS.

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation taVNS: Technique, Targeting, and Considerations

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

Laboratorio amministrazione dell'auricolare del nervo vago stimolazione transcutanea (taVNS): tecnica, Targeting e considerazioni

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Stimolazione del nervo vago come un attrezzo di neurostimolazione Adjunctive nella depressione trattamento-resistente

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Medicina

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Preparazione degli elettrodi di stimolazione nervosa periferica per l'impianto cronico nei ratti

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Misurazione della neuromodulazione mediante stimolazione transcutanea del nervo vago auricolare

Understanding the Effects of Non&#45;Invasive Transauricular Vagus Nerve Stimulation on EEG and HRV

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no mechanistic studies have investigated this technique's neural network and autonomic nervous system effects. This study aims to describe how taVNS can affect EEG metrics, HRV, and pain levels. Healthy subjects were randomly allocated into two groups: the active taVNS group and the sham taVNS group. Electroencephalography (EEG) and Heart Rate Variability (HRV) were recorded at baseline, 30 min, and after 60 min of 30 Hz, 200-250 &#181;s taVNS, or sham stimulation, and the differences between the metrics were calculated. Regarding vagal projections, some studies have demonstrated the role of the vagus nerve in modulating brain activity, the autonomic system, and pain pathways. However, more data is still needed to understand the mechanisms of taVNS on these systems. In this context, this study presents methods to provide data for a deeper discussion about the physiological impacts of this technique, which can help future therapeutic investigations in various conditions.

Autore in primo piano: Esplorare gli effetti della stimolazione transauricolare del nervo vago

This protocol provides information on how to apply transcutaneous auricular vagus nerve stimulation (taVNS) in a clinical trial, including potential biomarkers such as EEG metrics and heart rate variability (HRV) to measure the effect of this treatment on the autonomic nervous system.

Comprendere gli effetti della stimolazione non invasiva del nervo vago transauricolare su EEG e HRV

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no ...

Aumento tempo di recupero e Diminuzione LPS Per studiare il nervo vago meccanismi di stimolazione in risposta infiammatoria limitati

Riepilogo

Esplora Altri Video

Capitoli in questo video

Stimolazione del nervo vago come strumento per indurre plasticità Percorsi rilevanti per l'apprendimento Extinction

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Un metodo di nodose Ganglia iniezione in Sprague-Dawley Rat

Iniezioni di Lipopolysaccharide in topi per simulare l'ingresso di prodotti microbici derivati dopo la violazione della barriera intestinale

Laboratorio amministrazione dell'auricolare del nervo vago stimolazione transcutanea (taVNS): tecnica, Targeting e considerazioni

Stimolazione del nervo vago come un attrezzo di neurostimolazione Adjunctive nella depressione trattamento-resistente

Preparazione degli elettrodi di stimolazione nervosa periferica per l'impianto cronico nei ratti

Comprendere gli effetti della stimolazione non invasiva del nervo vago transauricolare su EEG e HRV

Comprendere gli effetti della stimolazione non invasiva del nervo vago transauricolare su EEG e HRV