Name: a method of nodose ganglia injection in sprague-dawley rat
Uploaded: 2014-11-25T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

这项研究是由美国国立卫生研究院（批准1UM1HL112856）的支持。

所有的程序和协议批准了伊利诺伊大学芝加哥分校的机构动物护理和使用委员会。这里描述的实验是急性非存活实验中，也没有使用眼软膏。无菌条件下维持，只有当外科手术器械在DIH 2 O洗涤，用70％的乙醇发生大鼠在实验结束时牺牲经由第IV氯胺酮/赛拉嗪的药物过量发生。 1.准备仪器和化学品<ol><li>制备0.05M的5-羟色胺的盐酸在PBS原液。然后稀释股票与PBS到最终5-HT的0.203毫米的浓度。 </li><li>稀屈大麻酚在芝麻油在20微克/微升的浓度。 </li><li>切聚乙烯20厘米长（PE）的-50油管（ID0.58毫米，外径0.965毫米）。在管道的一端，切割用剪刀斜面尖端，而在另一方端，插入一个23克针。此23克针连接到1毫升注射器，并填写了0.3毫升50 U / ml肝素。 </li><li>切成四片4-0编织丝线，并设置到侧。 </li><li>消毒手术剪，2弯曲格雷夫钳，并用70％乙醇3微夹钳在DIH 2 O. </li></ol> 2.导管股静脉协议导管从耶斯佩森等进行修改。21 <ol><li>麻醉一个的Sprague-Dawley大鼠腹膜内（IP）的氯胺酮/赛拉嗪（100毫克/公斤10毫克/公斤）。捏的大鼠的脚趾和观察到任何运动，以确认麻醉的一个适当的水平。剃左大腿的腹侧部分，而颈的腹面。如果由本地机构动物护理和使用委员会必要，应用眼膏，以防止干燥。 </li><li>安全大鼠在手术板仰卧位置。用手术剪剪去皮肤左后肢大腿。 </li><li>使用镊子，行为钝dissection个肤浅的肌肉暴露股静脉。使用镊子到静脉从股动脉中分离出来，并将其放置2个线程围绕股静脉。 </li><li>使用弯曲格雷夫钳拉静脉阻止血液流动。用22克注射器针头，穿刺股静脉，然后插入PE-50管插入静脉的倾斜端。 </li><li>检查是否在PE-50管材是由回缩注射器的柱塞正确插入。血液应被视为进入的PE-50管材。配合周围的静脉和PE-50管2节使用2个线程。 </li></ol> 3.通过静脉输注5-HT引起的呼吸暂停<ol><li>周围放置老鼠的压电应变计来测量呼吸。 </li><li>用于经由放大器软件的电子放大器在计算机上的放大电平改变设置:扩增（100X）和带通滤波器（1-10赫兹）从所述应变仪获得的呼吸信号中，设置"highpas滤波器"在＆＃8220; AC @ 1 HZ"和"低通滤波器"，在"10赫兹"，并通过输入设定放大"10"为"初始增益"和"100"的"总收益"。 </li><li>由通过录音软件的计算机上打开"模拟输入"更改设置采样率。以使用模拟 - 数字转换器，"样品"在"1000赫兹"和数字化（500赫兹的采样速率）"跳过"由"1"（有效抽样率500赫兹）每隔一个的记录点，然后记录该信号使用刻录软件。 </li><li>取出1ml注射器从导管和插入一个500μl的精密玻璃注射器填充有0.203毫5-HT的进入导管。 </li><li>放置500微升精密玻璃注射器插入输液泵。注入12.5微克/千克每350微升/ kg的5-羟色胺的溶液在63毫升/小时的速度。执行多个输液，观察呼吸暂停大鼠，这被看作一个SA暂停在计算机显示器上所记录的呼吸呼吸信号（≥2.5秒）。 </li><li>在继续进行颈部手术，监测呼吸模式，并检查疼痛反射从脚趾捏在大鼠。如呼吸不规则，或如果从脚趾捏痛反射，管理IP氯胺酮/甲苯​​噻嗪（100毫克/公斤:5毫克/千克），然后再确定麻醉的适当水平。 </li></ol> 4.头颈外科杂志揭露节状神经节<ol><li>安全大鼠在手术板仰卧位置。使用手术剪在颈部正中纵向切。 </li><li>在颈阔肌（使用2微夹具保持这一肌肉清晰的手术部位的）使用钝性分离，露出胸骨舌骨肌和omohyoideus肌肉。分开这些肌肉，露出颈内动脉及其分支之一，翼腭动脉。 </li><li>观察的迷走神经，因为它沿内颈动脉运行，然后沿第<html> Ë翼腭动脉。观察一下迷走神经和翼腭动脉，随着舌咽神经（颅神经IX）与副神经（脑神经XI），进入后撕裂孔在颅底。 </li><li>注意，结状神经节被显示为迷走神经在进入后撕裂孔权之前的膨胀。还要注意，咽和喉上神经分支来的结状神经节的断前部和后部方面，分别为5 </li><li>使用格雷夫镊子，从动脉分开的迷走神经，并将其放置一块螺纹围绕迷走神经。放置在线程上的夹具上应用迷走神经轻微张力，和清洁任何结缔组织的结状神经节注入时提供较小的阻力。 </li><li>重复5-HT引起的呼吸暂停在第3节规定，确认无损伤做是为了结状神经节。 </li></ol>书名"&gt; 5。Intranodose神经节注射屈大麻酚<ol><li>补贴有特制28克半英寸的注射器针头斜面尖端5微升屈大麻酚的香油35°10微升气密精密玻璃注射器。 </li><li>放置在线程上的微型钳施加于迷走神经张力轻微，并刺破结状神经节，注意不要刺穿它。压下注射器缓慢（≥60秒）注入注射器的全部内容。注意，某些在注射器内容将漏出的神经节。 </li><li>在第3节规定，重复5-HT引起的呼吸暂停。 </li></ol>

<ol>
	<li>Zhuo, H., Ichikawa, H., Helke, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurochemistry+of+the+nodose+ganglion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+vagal+afferent+influences+on+disordered+breathing+during+sleep.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+acid+suppression:+new+pharmacologic+approaches+for+treatment+of+GERD.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-protein+coupled+receptors+regulating+cough.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the rat. , (1963).</li><li>Li, Y., Wu, X., Zhou, S., Owyang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-affinity+CCK-A+receptors+are+coexpressed+with+leptin+receptors+in+rat+nodose+ganglia:+implications+for+leptin+as+a+regulator+of+short-term+satiety.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagal+afferent+innervation+of+the+lower+esophageal+sphincter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+vagal+innervation+of+the+rat+esophagus+and+cardia:+a+light+and+electron+microscopic+anterograde+tracing+study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+methods+for+the+study+of+vagal+afferents+within+the+nucleus+of+the+solitary+tract.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+melanocortin-4+receptors+on+vagal+afferent+fibers+modulate+the+excitability+of+rat+nucleus+tractus+solitarius+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezold-Jarisch+reflex+is+inhibited+by+excitotoxin-induced+destruction+of+vagal+primary+afferent+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxin-induced+degeneration+of+rat+vagal+afferent+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denervation+of+vagal+cardiopulmonary+receptors+by+injection+of+kainic+acid+into+the+nodose+ganglia+in+dogs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+vagal+afferent+nerve+pathways+regulating+cough+using+Nav+1.7+shRNA+silencing+in+guinea+pig+nodose+ganglia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+expression+and+effective+gene+silencing+in+vagal+afferent+neurons+in+vivo+using+recombinant+adeno-associated+virus+vectors.">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/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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranodose+ganglion+injections+of+dronabinol+attenuate+serotonin-induced+apnea+in+Sprague-Dawley+rat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+5-hydroxytryptamine-induced+apnea+in+the+rat.">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/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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+by+cannabinoids+of+human+5-HT3A+receptors:+probable+involvement+of+an+allosteric+modulatory+site.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation:+from+epilepsy+to+the+cholinergic+anti-inflammatory+pathway.">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.

的关键步骤成功注射神经化学物质的进节状神经节是:1）识别和清洁结缔组织断节状神经节; 2）确认注射前的结状神经节的完整性; 3）和使用小型量规针以微妙注入，但不能完全通过删截，所述结状神经节。 迷走神经支配许多器官在颈部和腹部，并传递重要的信息，如心脏速率，血压，支气管发炎，和胃肠膨胀到中枢神经系统。迷走神经的结状神经节包含受体氨基酸，单胺，...

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.

图1表示在大鼠中有输注5-HT的前后intranodose神经节注射屈大麻酚诱导呼吸暂停呼吸样本记录。 5-HT的激活的5-HT 3受体上的结状神经节，有助于心动过缓，低血压和呼吸暂停的的Bezold-Jarisch反射。11,17-19 Intranodose神经节注射屈大麻酚的激活抑制性的CB受体，或变构调节5-HT的3受体抑制16,17,20,22的迷走神经。对5-HT诱导的激发前（ 图1，上图）和后?...

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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.

结状神经节注射液只SD大鼠的方法

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

18.7K 次观看.  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.

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

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

啮齿动物脑显微注射学习动机行为的分子基板

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

科研

行为学

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.

Laminotomy 对猪腰背根神经节通路和注射的治疗

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

神经科学

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.

腺相关病毒介导的脊髓基因神经元的转基因表达

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.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

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

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

佩尔维奇自主性甘利亚与雄性大鼠和雌性大鼠相关神经的解剖

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

低输入细胞核分离和多路复用小鼠交感神经节条形码抗体进行单核RNA测序

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

医学

用于神经干细胞和少突胶质细胞祖细胞分离的大鼠"脑挤奶"

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

作者聚焦:使用新型脑挤奶方案从活体动物中收集神经干细胞和祖细胞 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

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.

大鼠直接迷走神经注射方案

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

结状神经节注射液只SD大鼠的方法

摘要

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此视频中的章节

啮齿动物脑显微注射学习动机行为的分子基板

Laminotomy 对猪腰背根神经节通路和注射的治疗

腺相关病毒介导的脊髓基因神经元的转基因表达

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

佩尔维奇自主性甘利亚与雄性大鼠和雌性大鼠相关神经的解剖

低输入细胞核分离和多路复用小鼠交感神经节条形码抗体进行单核RNA测序

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

大鼠直接迷走神经注射方案