Este estudo foi financiado pelo National Institutes of Health (Grant 1UM1HL112856).

Todos os procedimentos e protocolos foram aprovados pelo Comitê de Cuidados e Uso da Universidade de Illinois, em Chicago animal Institucional. As experiências aqui descritas são experiências agudas não-sobrevivência, e não houve uso de pomada. Manutenção de condições estéreis só ocorre quando os instrumentos cirúrgicos são lavados com etanol 70% em DIH 2 O. Sacrifício de ratos no final da experiência ocorreu através de sobredosagem de IV cetamina / xilazina. 1. Preparação de Instrumentos e Produtos Químicos <ol><li> Preparar a solução de estoque de 0,05 M de 5-HT de HCl em PBS. Depois dilui-se estoque com PBS a uma concentração final de 5-HT de 0,203 mM. </li><li> Diluir dronabinol em óleo de sésamo com uma concentração de 20 ug / uL. </li><li> Corte de 20 cm de comprimento de polietileno (PE) -50 tubagem (ID 0,58 milímetros, 0,965 milímetros OD). Em uma extremidade do tubo, corte uma ponta bisel com uma tesoura, e na outra ponta quadrada, inserir uma agulha de 23 g. Ligue este 23 g agulha para um 1 mlseringa, e encha-o com 0,3 ml de 50 u / ml de heparina. </li><li> Corte quatro pedaços de fio de seda 4-0 trançado e definido para o lado. </li><li> Esterilizar tesouras cirúrgicas, 2 fórceps Graefe curvas, e 3 grampos de micro com etanol a 70% em DIH 2 O. </li></ol> 2. O cateterismo de veia femoral Protocolo para o cateterismo é modificado a partir Jespersen et al 21. <ol><li> Anestesiar uma ratazana Sprague-Dawley com intraperitoneal (IP) de cetamina / xilazina (100 mg / kg: 10 mg / kg). Aperte o dedo do pé do rato e observar qualquer movimento para confirmar um nível adequado de anestesia. Raspar o aspecto ventral da coxa esquerda, e do aspecto ventral do pescoço. Se necessário pelo Comitê de Cuidados e Uso de Animais Institucional do local, aplicar pomada para evitar o ressecamento. </li><li> Rat seguro em decúbito dorsal em uma placa cirúrgica. Utilize uma tesoura cirúrgica para cortar a pele na coxa posterior esquerda. </li><li> Utilizando uma pinça, conduta dissectio contuson do músculo superficial para expor a veia femoral. Utilizar as pinças para separar a veia a partir da artéria femoral, e colocar dois segmentos em torno da veia femoral. </li><li> Use a pinça Graefe curvas para puxar-up da veia para parar o fluxo de sangue. Usando uma seringa de agulha G 22, por punção da veia femoral e, em seguida, insira a ponta chanfrada do tubo PE-50 na veia. </li><li> Verificar se o tubo PE-50 foi inserida correctamente por retracção do êmbolo da seringa. O sangue deve ser visto de entrar no tubo de PE-50. Amarre 2 nós ao redor da veia e PE-50 tubos usando as duas threads. </li></ol> 3. 5-HT induzido por Apnea via IV Infusion <ol><li> Coloque um medidor de tensão piezoelétrico em torno do rato para medir a respiração. </li><li> Altere as configurações para o nível de amplificação do amplificador eletrônico via software amplificador no computador: para amplificar (100x) e filtro passa-banda (10/01 Hz), os sinais respiratórios obtidos a partir do medidor de tensão, ajuste &quot;filtro highpas&quot; at &amp; #8220; AC @ 1 HZ &quot;e&quot; Filter Lowpass &quot;at&quot; 10 Hz &quot;, e definir a amplificação através da introdução de&quot; 10 &quot;para o&quot; ganho inicial &quot;e&quot; 100 &quot;para&quot; Total Gain &quot;.. </li><li> Altere as configurações de taxa de amostragem, abrindo &quot;Entrada Analógica&quot; via software de gravação no computador. Para digitalizar (500 taxa de amostragem Hz) usando um conversor analógico-digital, &quot;sample&quot; at &quot;1.000 Hz&quot; e &quot;pular&quot; todos os outros pontos de gravação por &quot;1&quot; (taxa de amostragem eficaz 500 Hz), e depois gravar o sinal usando o software de gravação. </li><li> Remover a seringa de 1 ml a partir do cateter e inserir uma seringa de vidro de 500 mL cheio com precisão de 0,203 mM de 5-HT no interior do cateter. </li><li> Coloque a 500 ul seringa de vidro de precisão em uma bomba de infusão. Infundir 12,5 ug / kg por 350 ul / kg de uma solução de 5-HT a uma taxa de 63 ml / hr. Executar várias infusões e observar apnéia em rato, que é visto umsa pausa na respiração (≥ 2,5 seg) nos sinais respiratórios gravados no monitor do computador. </li><li> Antes de prosseguir com a cirurgia de pescoço, monitorar o padrão respiratório e verificar se há reflexo dor de toe pitada no rato. Se a respiração for irregular, ou se existe um reflexo dor de aperto do dedo do pé, administrar IP cetamina / xilazina (100 mg / kg: 5 mg / kg) e, em seguida, voltar a confirmar a um nível adequado de anestesia. </li></ol> 4. Neck Surgery para expor Nodoso Ganglia <ol><li> Rat seguro em decúbito dorsal em uma placa cirúrgica. Faça um corte longitudinal mediana usando uma tesoura cirúrgica no pescoço. </li><li> Usando dissecção romba no músculo platisma (usando dois micro grampos para manter esse músculo clara do sítio cirúrgico), expor os músculos sternohyoideus e omohyoideus. Separe esses músculos para expor a artéria carótida interna e um de seus ramos, a artéria pterigopalatina. </li><li> Observe o nervo vago, uma vez que corre ao longo da artéria carótida interna e, em seguida, ao longo th<html> e pterygopalatine artéria. Observe como o nervo vago e artéria pterigopalatina, juntamente com o nervo glossofaríngeo (IX nervo craniano) e do nervo acessório (XI nervo craniano), digite o forame lacerados posteriores na base do crânio. </li><li> Observe que o gânglio nodoso é exibido como um inchaço do nervo vago direito antes de entrar no forame posterior dilacerado. Note também que os ramos da faringe e da laringe nervosas saem os aspectos anterior e posterior, respectivamente, do gânglio nodoso. 5 </li><li> Usando a pinça Graefe, separar o nervo vago a partir das artérias, e coloque um pedaço de fio ao redor do nervo vago. Coloque um grampo na linha para aplicar uma leve tensão no nervo vago, e limpar o gânglio nodoso de qualquer tecido conjuntivo para proporcionar menor resistência ao injetar. </li><li> Repita apnéia 5-HT induzido conforme previsto no ponto 3 para confirmar que nenhum dano foi feito para o gânglio nodoso. </li></ol>itle &quot;&gt; 5. Intranodose Ganglia Injeção de Dronabinol <ol><li> Encha a 10 ul precisão gastight seringa de vidro afixado com um 28 g de meia polegada agulha de seringa feito por encomenda com um 35 ° chanfradas ponta com 5 mL de dronabinol em óleo de gergelim. </li><li> Coloque um grampo micro no segmento de aplicar uma leve tensão no nervo vago, e perfurar o gânglio nodoso tomando cuidado para não perfurar através dele. Pressione a seringa lentamente (≥ 60 seg) para injetar todo o conteúdo da seringa. Note-se que alguns dos conteúdos de seringas vai vazar para fora do gânglio. </li><li> Apneia Repita 5-HT-induzida como indicado na seção 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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Ther. 260 (2), 917-924 (1992).</li><li>Kopczynska, B., Szereda-Przestaszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5HT2+and+5HT3+receptors'+contribution+to+modeling+of+post-serotonin+respiratory+pattern+in+cats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+agonists+inhibit+the+activation+of+5-HT3+receptors+in+rat+nodose+ganglion+neurons.">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. 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The authors declare that they have no competing financial interests.

Os passos críticos para o sucesso da injecção de neurotransmissores na gânglios nodosos são: 1) a identificação e a limpeza do tecido conjuntivo fora do gânglios nodosos; 2) confirmando a integridade do gânglio nodoso antes da injecção; 3) e usando uma agulha de calibre pequeno para injectar delicadamente em, mas não completamente, através de punção, os gânglios nodosos. O nervo vago inerva muitos órgãos no pescoço e abdômen, e transmite informações importantes, tais co...

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>
			<td style="width:175px;">Point Style 4, Angle 35, Length 0.5 in</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.

A Figura 1 representa gravação respiração amostra em ratos que tinham infusão de 5-HT para provocar apneia antes e depois da injecção dos gânglios intranodose de dronabinol. 5-HT activa 5-HT3 sobre os gânglios nodosos que contribuem para o reflexo de Bezold-Jarisch de bradicardia, hipotensão, e apneia. 11,17-19 injecções gânglios Intranodose dronabinol de activar os receptores CB inibitórios, ou modula alostericamente 5-HT 3 receptores que inibem a excitaç...

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.

Um método de Nodoso Gânglios Injecção em ratos Sprague-Dawley

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 visualizações.  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.