Questo studio è stato sostenuto dal National Institutes of Health (Grant 1UM1HL112856).

Tutte le procedure e protocolli sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale della University of Illinois a Chicago. Gli esperimenti qui descritti sono esperimenti acuti non di sopravvivenza, e non c&#39;era l&#39;uso di unguento oculare. Mantenimento delle condizioni sterili verifica solo quando strumenti chirurgici vengono lavati con etanolo al 70% in DIH 2 O. Sacrificio di ratti alla fine dell&#39;esperimento verificato tramite sovradosaggio di IV ketamina / xilazina. 1. Preparazione degli strumenti e prodotti chimici <ol><li> Preparare soluzione madre di 0,05 M di 5-HT HCl in PBS. Poi diluire azione con PBS a una concentrazione finale 5-HT di 0,203 mm. </li><li> Diluire dronabinol in olio di sesamo ad una concentrazione di 20 mg / mL. </li><li> Tagliare lungo 20 cm di polietilene (PE) -50 tubo (ID 0,58 millimetri, OD 0,965 millimetri). Ad una estremità del tubo, tagliare una punta conica con forbici, e all&#39;altra estremità quadrata, inserire un ago 23 g. Collegare questo 23 g ago per un 1 mlsiringa e riempirlo con 0,3 ml di 50 U / ml di eparina. </li><li> Tagliate quattro pezzi di filo di seta 4-0 intrecciato e impostato sul lato. </li><li> Sterilizzare forbici chirurgiche, 2 pinze Graefe curve e 3 micro pinze con il 70% di etanolo in diH 2 O. </li></ol> 2. cateterizzazione di vena femorale Protocollo per la cateterizzazione è modificato da Jespersen et al. 21 <ol><li> Anestetizzare un ratto Sprague-Dawley con intraperitoneale (IP) ketamina / xilazina (100 mg / kg: 10 mg / kg). Pizzicare la punta del ratto e osservare qualsiasi movimento per confermare un adeguato livello di anestesia. Radere l&#39;aspetto ventrale della coscia sinistra, e l&#39;aspetto ventrale del collo. Se necessario, dalla cura e l&#39;uso degli animali Comitato Istituzionale locale, applicare una pomata oculare per prevenire la secchezza. </li><li> Rat sicura in posizione supina su un bordo chirurgica. Usare le forbici chirurgiche per tagliare via la pelle nella coscia posteriore sinistra. </li><li> Utilizzando pinze, condotta dissectio smussaton del muscolo superficiali per esporre la vena femorale. Utilizzare le pinze per separare la vena dalla arteria femorale, e mettere 2 filetti intorno alla vena femorale. </li><li> Utilizzare le pinze Graefe curve di pull-up la vena a fermare il flusso di sangue. Usando una G 22 della siringa, forare la vena femorale e poi inserire l&#39;estremità smussata del tubo PE-50 nella vena. </li><li> Controllare se il tubo PE-50 è stato inserito correttamente ritraendo lo stantuffo della siringa. Il sangue deve essere visto entrare il tubo PE-50. Tie 2 nodi intorno alla vena e PE-50 tubo utilizzando le 2 fili. </li></ol> 3. 5-HT-indotta Apnea via IV Infusion <ol><li> Posizionare un estensimetro piezoelettrico intorno al topo per misurare la respirazione. </li><li> Modifica delle impostazioni per livello di amplificazione dell&#39;amplificatore elettronico via software amplificatore computer: per amplificare (100x) e filtro passa-banda (1-10 Hz) i segnali respiratori ottenuti dal estensimetro, set &quot;filtro highpas&quot; at &amp; #8220; AC @ 1 HZ &quot;e&quot; filtro passa-basso &quot;a&quot; 10 Hz &quot;, e impostare l&#39;amplificazione inserendo&quot; 10 &quot;per il&quot; guadagno iniziale &quot;e&quot; 100 &quot;per&quot; Total Gain &quot;.. </li><li> Modificare le impostazioni per frequenza di campionamento con l&#39;apertura di &quot;ingresso analogico&quot; via software di registrazione sul computer. Per digitalizzare (500 Hz frequenza di campionamento) con un convertitore analogico-digitale, &quot;campione&quot; a &quot;1.000 Hz&quot; e &quot;saltare&quot; ogni altro punto di registrazione da &quot;1&quot; (frequenza di campionamento effettiva 500 Hz), e si registra il segnale utilizzando il software di registrazione. </li><li> Rimuovere la siringa 1 ml dal catetere e inserire 500 microlitri siringa di vetro riempita con precisione 0,203 mM di 5-HT nel catetere. </li><li> Posizionare il 500 microlitri siringa di vetro di precisione in una pompa per infusione. Infondere 12,5 mg / kg a 350 ml / kg di soluzione di 5-HT ad una velocità di 63 ml / h. Eseguire infusioni multiple e osservare apnea nel ratto, che si vede undello SA pausa nella respirazione (≥ 2,5 sec) nei segnali respiratori registrati sul monitor del computer. </li><li> Prima di procedere con la chirurgia del collo, monitorare modello di respirazione e controllare il dolore riflesso dal pizzico punta nel ratto. Se la respirazione è irregolare, o se c&#39;è un riflesso dolore pizzico punta, amministrare IP ketamina / xilazina (100 mg / kg: 5 mg / kg) e poi ri-confermare un adeguato livello di anestesia. </li></ol> 4. Neck Surgery per esporre nodose gangli <ol><li> Rat sicura in posizione supina su un bordo chirurgica. Fare un taglio longitudinale mediana con le forbici chirurgiche al collo. </li><li> Utilizzando smussa sul muscolo platisma (utilizzando 2 micro pinze per mantenere questo muscolo chiara del sito chirurgico), esporre i muscoli sternohyoideus e omohyoideus. Separare questi muscoli per esporre l&#39;arteria carotide interna e uno dei suoi rami, l&#39;arteria pterigopalatino. </li><li> Osservare il nervo vago come corre lungo l&#39;arteria carotide interna e poi lungo th<html> e pterygopalatine arteria. Osservare come il nervo vago e arteria pterygopalatine, insieme con il nervo glossofaringeo (IX nervo cranico) e il nervo accessorio spinale (cranico XI nervo), entrano nel forame posteriori lacerati alla base del cranio. </li><li> Si noti che il ganglio nodoso è visualizzato come un rigonfiamento del nervo vago giusto prima che entri i posteriori lacerato forame. Si noti inoltre che i faringei e nervo laringeo rami si staccano gli aspetti anteriore e posteriore, rispettivamente, del ganglio nodose. 5 </li><li> Utilizzando le pinze Graefe, separare il nervo vago dalle arterie, e mettere un pezzo di filo intorno al nervo vago. Posizionare un morsetto sul filo di applicare una leggera tensione sul nervo vago, e pulire il ganglio nodose di qualunque tessuto connettivo di fornire meno resistenza quando l&#39;iniezione. </li><li> Ripetere apnea-5-HT indotta come indicato nella sezione 3 per confermare che nessun danno è stato fatto ai gangli nodose. </li></ol>TITOLO &quot;&gt; 5. Intranodose gangli Iniezione di dronabinol <ol><li> Riempire un 10 microlitri siringa di vetro di precisione a tenuta di gas apposto con 28 g di mezzo pollice ago della siringa su misura con un 35 ° smussato punta con 5 ml di dronabinol in olio di sesamo. </li><li> Inserire un micro pinza sul filo di applicare leggera tensione sul nervo vago, e forare il ganglio nodoso facendo attenzione a non forare attraverso di essa. Premere la siringa lentamente (≥ 60 sec) per iniettare l&#39;intero contenuto della siringa. Si noti che alcuni dei contenuti della siringa si fuoriuscire del ganglio. </li><li> Apnea Ripeti 5-HT-indotta come indicato al punto 3. </li></ol>

<ol><li>Zhuo, H., Ichikawa, H., Helke, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurochemistry+of+the+nodose+ganglion%5BTitle%5D)+AND+%22Prog.+Neurobiol%22%5BJournal%5D)">Neurochemistry of the nodose ganglion.</a> Prog. Neurobiol. 52 (2), 79-107 (1997).</li>
<li>Carley, D. W., Radulovacki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacology+of+vagal+afferent+influences+on+disordered+breathing+during+sleep%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">Pharmacology of vagal afferent influences on disordered breathing during sleep.</a> Respir. Physiol. Neurobiol. 164 (1-2), 197-203 (2008).</li>
<li>Kuo, P., Holloway, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+acid+suppression%3A+new+pharmacologic+approaches+for+treatment+of+GERD%5BTitle%5D)+AND+%22Curr.+Gastroenterol.+Rep%22%5BJournal%5D)">Beyond acid suppression: new pharmacologic approaches for treatment of GERD.</a> Curr. Gastroenterol. Rep. 12 (3), 175-180 (2010).</li>
<li>Maher, S. A., Dubuis, E. D., Belvisi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G-protein+coupled+receptors+regulating+cough%5BTitle%5D)+AND+%22Curr.+Opin.+Pharmacol%22%5BJournal%5D)">G-protein coupled receptors regulating cough.</a> Curr. Opin. Pharmacol. 11 (3), 248-253 (2011).</li>
<li>Greene, E. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreene%2C+EC+%22Anatomy+of+the+rat%22">Anatomy of the rat</a>. , Hafner. (1963).</li>
<li>Li, Y., Wu, X., Zhou, S., Owyang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-affinity+CCK-A+receptors+are+coexpressed+with+leptin+receptors+in+rat+nodose+ganglia%3A+implications+for+leptin+as+a+regulator+of+short-term+satiety%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Gastrointest.+Liver+Physiol%22%5BJournal%5D)">Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300 (2), 217-227 (1152).</li>
<li>Powley, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vagal+afferent+innervation+of+the+lower+esophageal+sphincter%5BTitle%5D)+AND+%22Auton.+Neurosci%22%5BJournal%5D)">Vagal afferent innervation of the lower esophageal sphincter.</a> Auton. Neurosci. 177 (2), 129-142 (2013).</li>
<li>Neuhuber, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+vagal+innervation+of+the+rat+esophagus+and+cardia%3A+a+light+and+electron+microscopic+anterograde+tracing+study%5BTitle%5D)+AND+%22J.+Auton.+Nerv.+Syst%22%5BJournal%5D)">Sensory vagal innervation of the rat esophagus and cardia: a light and electron microscopic anterograde tracing study.</a> J. Auton. Nerv. Syst. 20, 243-255 (1987).</li>
<li>Rogers, R. C., Nasse, J. S., Hermann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live-cell+imaging+methods+for+the+study+of+vagal+afferents+within+the+nucleus+of+the+solitary+tract%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Live-cell imaging methods for the study of vagal afferents within the nucleus of the solitary tract.</a> J. Neurosci. Methods. 150 (1), 47-58 (2006).</li>
<li>Wan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presynaptic+melanocortin-4+receptors+on+vagal+afferent+fibers+modulate+the+excitability+of+rat+nucleus+tractus+solitarius+neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons.</a> J. Neurosci. 28 (19), 4957-4966 (2008).</li>
<li>Verberne, A. J., Lewis, S. J., Jarrott, B., Louis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bezold-Jarisch+reflex+is+inhibited+by+excitotoxin-induced+destruction+of+vagal+primary+afferent+neurons%5BTitle%5D)+AND+%22Eur.+J.+Pharmacol%22%5BJournal%5D)">Bezold-Jarisch reflex is inhibited by excitotoxin-induced destruction of vagal primary afferent neurons.</a> Eur. J. Pharmacol. 139 (3), 365-367 (1987).</li>
<li>Lewis, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitotoxin-induced+degeneration+of+rat+vagal+afferent+neurons%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Excitotoxin-induced degeneration of rat vagal afferent neurons.</a> Neuroscience. 34 (2), 331-339 (1990).</li>
<li>Wallick, D. W., Dunlap, M. E., Stuesse, S. S., Thames, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Denervation+of+vagal+cardiopulmonary+receptors+by+injection+of+kainic+acid+into+the+nodose+ganglia+in+dogs%5BTitle%5D)+AND+%22Auton.+Neurosci%22%5BJournal%5D)">Denervation of vagal cardiopulmonary receptors by injection of kainic acid into the nodose ganglia in dogs.</a> Auton. Neurosci. 102 (1-2), 85-89 (2002).</li>
<li>Muroi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+inhibition+of+vagal+afferent+nerve+pathways+regulating+cough+using+Nav+1.7+shRNA+silencing+in+guinea+pig+nodose+ganglia%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Regul.+Integr.+Comp.+Physiol%22%5BJournal%5D)">Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (11), 1017-1023 (2011).</li>
<li>Kollarik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgene+expression+and+effective+gene+silencing+in+vagal+afferent+neurons+in+vivo+using+recombinant+adeno-associated+virus+vectors%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.</a> J. Physiol. 588 (21), 4303-4315 (2010).</li>
<li>Zhang, Z., Zhang, C., Zhou, M., Xu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+opioid+mu-receptors%2C+but+not+delta-+or+kappa-receptors%2C+switches+pulmonary+C-fiber-mediated+rapid+shallow+breathing+into+an+apnea+in+anesthetized+rats%29%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">Activation of opioid mu-receptors, but not delta- or kappa-receptors, switches pulmonary C-fiber-mediated rapid shallow breathing into an apnea in anesthetized rats).</a> Respir. Physiol. Neurobiol. 183 (3), 211-217 (2012).</li>
<li>Calik, M. W., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intranodose+ganglion+injections+of+dronabinol+attenuate+serotonin-induced+apnea+in+Sprague-Dawley+rat%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">Intranodose ganglion injections of dronabinol attenuate serotonin-induced apnea in Sprague-Dawley rat.</a> Respir. Physiol. Neurobiol. 190, 20-24 (2014).</li>
<li>Yoshioka, M., Goda, Y., Togashi, H., Matsumoto, M., Saito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+characterization+of+5-hydroxytryptamine-induced+apnea+in+the+rat%5BTitle%5D)+AND+%22J.+Pharmacol.+Exp.+Ther%22%5BJournal%5D)">Pharmacological characterization of 5-hydroxytryptamine-induced apnea in the rat.</a> J. Pharmacol. Exp. Ther. 260 (2), 917-924 (1992).</li>
<li>Kopczynska, B., Szereda-Przestaszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((5HT2+and+5HT3+receptors%27+contribution+to+modeling+of+post-serotonin+respiratory+pattern+in+cats%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">5HT2 and 5HT3 receptors' contribution to modeling of post-serotonin respiratory pattern in cats.</a> Life Sci. 75 (19), 2281-2290 (2004).</li>
<li>Fan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cannabinoid+agonists+inhibit+the+activation+of+5-HT3+receptors+in+rat+nodose+ganglion+neurons%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">Cannabinoid agonists inhibit the activation of 5-HT3 receptors in rat nodose ganglion neurons.</a> J. Neurophysiol. 73 (2), 907-910 (1995).</li>
<li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Femoral+arterial+and+venous+catheterization+for+blood+sampling%2C+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> J. Vis. Exp. (59), (2012).</li>
<li>Barann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+inhibition+by+cannabinoids+of+human+5-HT3A+receptors%3A+probable+involvement+of+an+allosteric+modulatory+site%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site.</a> Br. J. Pharmacol. 137 (5), 589-596 (2002).</li>
<li>Bonaz, B., Picq, C., Sinniger, V., Mayol, J. F., Clarencon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vagus+nerve+stimulation%3A+from+epilepsy+to+the+cholinergic+anti-inflammatory+pathway%5BTitle%5D)+AND+%22Neurogastroenterol.+Motil%22%5BJournal%5D)">Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.</a> Neurogastroenterol. Motil. 25 (3), 208-221 (2013).</li>
</ol>

The authors declare that they have no competing financial interests.

Le fasi critiche per l&#39;iniezione di sostanze neurochimiche successo nella gangli nodosi sono: 1) identificare e pulizia del tessuto connettivo off gangli nodosi; 2) confermando l&#39;integrità dei gangli nodosi prima dell&#39;iniezione; 3) e utilizzando un ago piccolo per iniettare delicatamente in, ma non completamente attraverso la puntura, gangli nodoso. Il nervo vago innerva molti organi del collo e dell&#39;addome, e trasmette le informazioni importanti come la frequenza cardiaca, ...

Afferent signaling via the vagus nerve (cranial nerve X) transmits important general visceral information to the central nervous system (CNS) from baro-, chemo-, hepatic osmo-, cardiac, pulmonary, and gastric 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 (NTS) of the medulla. The cell bodies of the pseudounipolar neurons of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority is found in the former. Nodose ganglion cells contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that when activated, can modify afferent vagus nerve activity.1 Numerous innervations of the afferent vagus nerves coupled with the diversity of receptors located on the nodose ganglia illustrate the biological importance of this cranial nerve, and systemic drugs that do not cross into the CNS targeted at receptors on nodose ganglia can be used to treat various diseases, such as sleep apnea, gastroesophageal reflux disease, or chronic cough.2-4
The ease of access to the nodose ganglia lends itself to experimental manipulation by midline longitudinal incision made at the neck. The vagus nerve emerges from the posterior lacerated foramen at the base of the skull, and immediately displays a swelling of the nerve that is the nodose ganglion. The nodose ganglion is easily recognizable due to two nerve branches that arise from it: anteriorly the pharyngeal branch; and posteriorly superior laryngeal branch.5 Previous experimental manipulations of the nodose ganglia involved electrophysiological recordings,6 injections of immunohistochemical or immunofluorescent compounds for nerve tracings,7-10 superfusion or injections of neuroexcitotoxins,11-13 injections of adeno-associated virus to knockdown receptors,14,15 and injections of receptor-specific neurochemicals to change the activity of the vagus nerve.16,17
Systemic injections of neurochemicals are problematic in that systemic treatment affects both peripheral and central nervous systems. Thus, systemic treatment does not isolate the effect of neurochemicals on afferent vagal nerve activity. This protocol describes a method using readily available equipment of intranodose injections in the Sprague-Dawley rat that modulates vagus nerve activity without affecting the central nervous system. Stimulation of serotonin type 3 (5-HT3) receptors on nodose ganglia by intravenous (IV) infusion of serotonin (5-HT) induces the Bezold-Jarisch reflex, a vagal response trifecta of bradycardia, hypotension, and apnea, which can be abolished by supranodose vagatomy.11,17-19 Apnea is easily measured by placing a respiratory transducer around the abdomen of the rat.17,18 Cannabinoids decrease 5-HT-induced current in nodose ganglia cells,20 and intranodose ganglia injections of dronabinol attenuate 5-HT-induced apnea.

<table><tbody><tr><td>5-HT HCl</td><td>MP Biomedicals</td><td>215376591</td><td>12.5 &amp;micro;g/kg per 350 &amp;micro;l/kg</td></tr><tr><td>Dronabinol (Marinol) 10 mg Capsules (80 &amp;micro;g/&amp;micro;l)</td><td>AbbVie</td><td>NDC 0051-0023-21</td><td>Dilute with sesame oil to 20 &amp;micro;g/&amp;micro;l</td></tr><tr><td>Sesame Oil</td><td>Sigma-Aldrich</td><td>S3547</td><td></td></tr><tr><td>Intramedic Polyethylene-50</td><td>BD</td><td>427411</td><td>Ordered from VWR (Cat. # 63019-047)</td></tr><tr><td>Graefe Forceps</td><td>Roboz</td><td>RS-5138</td><td>Two are needed</td></tr><tr><td>Johns Hopkins Bulldog Clamp</td><td>Roboz</td><td>RS-7441</td><td>Three are needed</td></tr><tr><td>Piezoelectric Strain Gauge</td><td>Ambu</td><td>813255-100</td><td></td></tr><tr><td>Data Acquisition USB Subsystems</td><td>DataWave Technologies</td><td></td><td></td></tr><tr><td>Sciworks Experimenter Software</td><td></td><td></td><td></td></tr><tr><td>CyberAmp</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Syringe, 500 &amp;micro;l, Model 1750 TLL</td><td>Hamilton Company</td><td>81220</td><td></td></tr><tr><td>Syringe, 10 &amp;micro;l, Model 1801 RN</td><td>7659-01</td><td></td><td></td></tr><tr><td>Needle, 28 G, Small Hub RN</td><td>7803-02</td><td>Point Style 4, Angle 35, Length 0.5 in</td><td></td></tr></tbody></table>

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.

Figura 1 rappresenta registrazione respirazione campione in ratti che avevano infusione di 5-HT per indurre apnea prima e dopo le iniezioni gangli intranodose di dronabinol. 5-HT attiva 5-HT 3 recettori sulla gangli nodose che contribuiscono al riflesso Bezold-Jarisch di bradicardia, ipotensione, e apnea. 11,17-19 Intranodose iniezioni gangli di dronabinol attivano recettori CB inibitori, o modula allostericamente 5-HT 3 recettori che inibiscono l&#39;eccitazione-5-HT in...

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A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

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 ...

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18.7K Views.  University of Illinois at Chicago. 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.

Video: A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Tecnica mini-invasiva per iniezione in Rat nervo ottico

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

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Neuroscienze

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomia per accesso del ganglio di radice dorsale lombare e l'iniezione nei suini

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Espressione del Transgene di mediata da Virus adeno-associato in neuroni geneticamente definiti del midollo spinale

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Consegna intratecala di Oligonucleotidi Antisense nel Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

Somministrazione di AAV9 con puntura lombare in ratti giovani per la terapia genica del SNC

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Autore in primo piano: Valutazione dell'efficacia della terapia genica intratecale nei ratti giovani

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Somministrazione intratecale di vettori in ratti giovani tramite iniezione nella cisterna lombare

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

Direct Vagus Nerve Injection Protocol For Rats

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

Protocollo di iniezione diretta del nervo vago per i ratti

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly ...

Lumbar Intrathecal Injection of Gene Therapy Vectors for Central Nervous System Targeting in Mice and Rats

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

A lumbar intrathecal injection represents a translationally relevant route of administration for delivering gene therapy to the central nervous system. This comprehensive standardized protocol for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats aims to guide researchers in adopting this technique for preclinical gene therapy studies.

Iniezione intratecale lombare di vettori di terapia genica per il targeting del sistema nervoso centrale in topi e ratti

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This ...

Un metodo di nodose Ganglia iniezione in Sprague-Dawley Rat

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Tecnica mini-invasiva per iniezione in Rat nervo ottico

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

Laminotomia per accesso del ganglio di radice dorsale lombare e l'iniezione nei suini

Espressione del Transgene di mediata da Virus adeno-associato in neuroni geneticamente definiti del midollo spinale

Consegna intratecala di Oligonucleotidi Antisense nel Rat Central Nervous System

Somministrazione intratecale di vettori in ratti giovani tramite iniezione nella cisterna lombare

Somministrazione intratecale di vettori in ratti giovani tramite iniezione nella cisterna lombare

Somministrazione intratecale di vettori in ratti giovani tramite iniezione nella cisterna lombare

Protocollo di iniezione diretta del nervo vago per i ratti

Iniezione intratecale lombare di vettori di terapia genica per il targeting del sistema nervoso centrale in topi e ratti