Bu çalışma Ulusal Sağlık Enstitüleri (Hibe 1UM1HL112856) tarafından desteklenmiştir.

Tüm prosedürler ve protokoller Chicago Illinois Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Burada anlatılan deneyler akut hayatta olmayan deneyler ve göz merhemi hiçbir kullanımı vardı. Cerrahi aletler dihidro 2 O içinde% 70 etanol ile yıkanmış, zaman steril koşullar bakımı sadece oluşur Deneyin sonunda sıçanlar kurban edilmesi IV, ketamin / ksilazin doz aşımı ile meydana geldi. Araçların ve Kimya 1. Hazırlık <ol><li> PBS içinde 0.05 M, 5-HT&#39;nin HCI stok çözelti hazırlayın. Daha sonra 0.203 mM&#39;lik nihai 5-HT konsantrasyonu PBS stoğu seyreltin. </li><li> 20 ug / ul bir konsantrasyonda susam yağı içinde dronabinol seyreltin. </li><li> (PE) -50 boru (ID 0.58 mm, OD 0.965 mm) polietilen 20 cm uzunluğunda kesin. Hortumun bir ucunda, makas ile konik ucu kesilmiş ve diğer kare sonunda, 23 gr iğneyi. 1 ml bu 23 g iğne iletişimeşırınga ve 50 u / ml heparin 0.3 ml ile doldurmak. </li><li> 4-0 örgü ipek iplik dört adet kesilmiş ve yan ayarlanır. </li><li> Dih 2 O.,% 70 etanol ile 2 kavisli Graefe forseps ve 3 mikro cerrahi makas kelepçeler sterilize </li></ol> Femoral Ven 2. Kateterizasyon Kateterizasyon Protokol Jespersen ark modifiye edilir. 21 <ol><li> (10 mg / kg ila 100 mg / kg) intraperitoneal (ip), ketamin / ksilazin ile Sprague-Davvley sıçan anestezisi. Sıçan ayak Pinch ve anestezi uygun bir seviyede onaylamak için herhangi bir hareket gözlemlemek. Sol uyluk ventral ve boyun ventral Tıraş. Yerel Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından Gerekirse, kuruluğunu önlemek için göz merhemi uygulayın. </li><li> Bir cerrahi gemide bir yatar pozisyonda Güvenli sıçan. Sol arka uyluk cilt kesip cerrahi makas kullanın. </li><li> Forseps kullanarak, davranış künt dissectioyüzeysel kas n femoral ven ortaya çıkarmak. Femoral ven yaklaşık 2 konuları femoral arter ven ayrı ve yerleştirmek için forseps kullanın. </li><li> Pull-up ven kan akışını durdurmak için kavisli Graefe forseps kullanın. 22 G şırınga iğnesi kullanarak, femoral ven delinme ve daha sonra damar içine PE-50 boru kesik ucunu. </li><li> PE-50 boru şırınga pistonu geri çekilmesiyle doğru takılı olup olmadığını kontrol edin. Kan PE-50 boruları giren görülmelidir. 2 konuları kullanarak ven ve PE-50 tüp yaklaşık 2 düğümler. </li></ol> 3. IV İnfüzyon yoluyla Apnea 5-HT-kaynaklı <ol><li> Solunum ölçmek için sıçan etrafında bir piezoelektrik gerilme ölçer yerleştirin. </li><li> Bilgisayarda amplifikatör yazılımı aracılığıyla elektronik amplifikatörü düzeyi ayarlarını değiştirme: yükseltmek (100x) ve bant geçiren filtre (1-10 Hz) suşu ölçer elde edilen solunum sinyalleri, at &amp; # &quot;highpas filtre&quot; ayarlamak için8220;. Toplam Gain &quot;için&quot; 10 Hz, &quot;ve girerek amplifikasyon set&quot; 10 İlk Kazanç &quot;için&quot; &quot;ve&quot; 100 &quot;&quot; at &quot;ac @ 1 HZ&quot; ve &quot;Düşük Geçişli Filtre. </li><li> Bilgisayarınızdaki kayıt yazılımı aracılığıyla &quot;Analog Giriş&quot; açarak örnekleme hızı ayarlarını değiştirin. &quot;1.000 Hz&quot; ve en &quot;örnek&quot; bir analog-dijital dönüştürücü kullanarak (500 Hz örnekleme oranı) dijital ortama aktarmak için &quot;1&quot; (etkili örnekleme hızı 500 Hz) tarafından her kayıt noktası &quot;atlamak&quot;, ve sonra sinyal kayıt kayıt yazılımı kullanarak. </li><li> Kateterinden 1 ml şırınga çıkarın ve katetere 5-HT&#39;nin 0.203 mM ile doldurulmuş bir 500 ul hassas cam şırınga yerleştirin. </li><li> Bir infüzyon pompası içine 500 ul hassas cam şırınga yerleştirin. 63 ml / saatlik bir hızda 350 ul / 5-HT çözeltisi kg başına 12.5 mg / kg infüze. Bir görülür sıçan birden infüzyonu gerçekleştirin ve gözlemlemek apne,bilgisayar monitörü kaydedilen solunum sinyalleri (≥ 2.5 sn) nefes duraklama sa. </li><li> Boyun cerrahisi ile devam etmeden önce, desen nefes izlemek ve sıçan ayak tutam ağrı refleks kontrol edin. Solunum düzensiz veya ayak tutam bir ağrı refleks varsa, IP ketamin / ksilazin yönetmek durumunda (100 mg / kg: 5 mg / kg) ve daha sonra anestezi uygun bir seviyede yeniden onaylayın. </li></ol> 4. Boyun Cerrahisi nodoz ganglionlar Açığa <ol><li> Bir cerrahi gemide bir yatar pozisyonda Güvenli sıçan. Boyun cerrahi makas kullanarak bir orta hat boyuna kesim olun. </li><li> (Cerrahi sitenin açık bu kas tutmak için 2 mikro kelepçeler kullanarak) platizma kası üzerinde künt diseksiyon kullanarak, sternohyoideus ve omohyoideus kasları ortaya. Internal karotid arter ve dallarının biri, pterigopalatin arteri açığa bu kasları ayırın. </li><li> O th boyunca sonra internal karotis arter boyunca çalışır ve gibi vagus siniri gözlemleyin<html> e pterigopalatin arter. Glossofaringeal sinir (kranial sinir IX) ve spinal aksesuar sinir (kranial sinir XI) ile birlikte vagus siniri ve pterigopalatin arter, kafatasının dibinde arka yırtılmış foramen girmek nasıl gözlemleyin. </li><li> Nodoz ganglion o arka yırtılmış foramen girmeden hemen önce vagus sinirinin bir şişlik olarak görüntülenir dikkat edin. Ayrıca farenks ve laringeal sinir dalları nodoz ganglion, sırasıyla, anterior ve posterior yönlerini dökülmek dikkat edin. 5 </li><li> Graefe forseps kullanarak, arterlerden vagus siniri ayırmak ve vagus sinirinin çevresine bir iplik parçası yerleştirin. Vagus sinirinin üzerinde hafif gerginlik uygulamak, ve enjekte ederken daha az direnç sağlamak için herhangi bir bağ dokusu nodoz ganglion temizlemek için iş parçacığı üzerinde bir kelepçe yerleştirin. </li><li> Herhangi bir hasar nodoz ganglion yapıldığını onaylamak için 3. bölümde belirtildiği gibi 5-HT-kaynaklı apnesi tekrarlayın. </li></ol>dronabinol ve itle &quot;&gt; 5. Intranodose Ganglionlar Enjeksiyon <ol><li> Susam yağı dronabinol 5 ul ucu kesik bir 35 ° bir ısmarlama 28 gr yarım inç şırınga iğnesi ile yapıştırılmış bir 10 ul gaz geçirmez hassas cam şırınga doldurun. </li><li> Vagus sinirinin üzerinde hafif gerginlik uygulamak, ve bu kadar delinme değil nodoz ganglion özen delinme parçacığı üzerinde bir mikro kelepçe yerleştirin. Şırınga tüm içeriğini enjekte yavaş yavaş şırınga (≥ 60 sn) bastırın. Şırınga içeriğinin bazı ganglion sızıntı unutmayın. </li><li> 3. bölümde belirtildiği gibi tekrarlayın apne 5-HT-kaynaklı. </li></ol>

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The authors declare that they have no competing financial interests.

nodoz ganglionlar içine nörokimyasalların başarılı enjeksiyon için kritik adımlar şunlardır: 1) belirlenmesi ve boğumlu ganglionlar kapalı bağ dokusu temizleme; 2) Enjeksiyon öncesinde nodoz ganglionlar bütünlüğünü teyit; 3) ve ince enjekte küçük bir iğne kullanarak, ama tamamen, boğumlu ganglionlar ile delme değil. vagus siniri boyun ve karın birçok organ innervates ve bu merkezi sinir sistemi kalp hızı, kan basıncı, bronkopulmoner tahriş, ve gastrointestina...

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.

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	<tbody>
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			<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.

Şekil 1, dronabinol ve intranodose gangliyon enjeksiyon öncesi ve sonrası apne uyarılması için 5-HT infüzyonu uygulanan sıçanlar örnek solunum kaydı temsil eder. 5-HT 5-HT bradikardi, hipotansiyon, apne ve Bezold-Jarisch refleks katkıda nodoz ganglionlar 3 reseptörlerini harekete geçirir. Dronabinol olan 11,17-19 Intranodose ganglion enjeksiyonları inhibitör CB reseptörleri aktive ya da allosterically 5-HT modüle (Şekil 1, üst panel) öncesinde ...

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.

Sprague-Dawley Sıçan nodoz Ganglionlar Enjeksiyon Bir Yöntem

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

18.5K Görüntüleme.  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.