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>
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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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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).">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. 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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 border="0" cellpadding="0" cellspacing="0" width="660">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">5-HT HCl</td>
			<td style="width:155px;">MP Biomedicals</td>
			<td style="width:141px;">215376591</td>
			<td style="width:175px;">12.5 &#181;g/kg per 350 &#181;l/kg</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Dronabinol (Marinol) 10 mg Capsules (80 &#181;g/&#181;l)</td>
			<td style="width:155px;">AbbVie</td>
			<td style="width:141px;">NDC 0051-0023-21</td>
			<td style="width:175px;">Dilute with sesame oil to 20 &#181;g/&#181;l</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sesame Oil</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:141px;">S3547</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Intramedic Polyethylene-50</td>
			<td style="width:155px;">BD</td>
			<td style="width:141px;">427411</td>
			<td style="width:175px;">Ordered from VWR (Cat. # 63019-047)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Graefe Forceps</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-5138</td>
			<td style="width:175px;">Two are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Johns Hopkins Bulldog Clamp</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-7441</td>
			<td style="width:175px;">Three are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Piezoelectric Strain Gauge</td>
			<td style="width:155px;">Ambu</td>
			<td style="width:141px;">813255-100</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Data Acquisition USB Subsystems</td>
			<td rowspan="2" style="width:155px;">DataWave Technologies</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sciworks Experimenter Software</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">CyberAmp</td>
			<td style="width:155px;">Axon Instruments</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 500 &#181;L, Model 1750 TLL</td>
			<td rowspan="3" style="width:155px;">Hamilton Company</td>
			<td style="width:141px;">81220</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 10 &#181;L, Model 1801 RN</td>
			<td style="width:141px;">7659-01</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Needle, 28 gauge, Small Hub RN</td>
			<td style="width:141px;">7803-02</td>
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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...

Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

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 ...

a-method-of-nodose-ganglia-injection-in-sprague-dawley-rat

Research

JoVE Journal

Neuroscience

18.4K 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.