Метод Узловые ганглиев инъекций в Спрэг Dawley крыс
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
Критические шаги для успешного введения нейрохимических минуте нодозного ганглиев являются: 1) выявление и очистки соединительной ткани и выключение нодозного ганглиев; 2) подтверждения целостности нодозного ганглиев перед инъекцией; 3) и с помощью небольшой иглы, чтобы впрыснуть в то?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Это исследование было поддержано Национальным институтом здравоохранения (грант 1UM1HL112856).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| 5-HT HCl | MP Biomedicals | 215376591 | 12.5 µg/kg per 350 µl/kg |
| Dronabinol (Marinol) 10 mg Capsules (80 µg/µl) | AbbVie | NDC 0051-0023-21 | Dilute with sesame oil to 20 µg/µl |
| Sesame Oil | Sigma-Aldrich | S3547 | |
| Intramedic Polyethylene-50 | BD | 427411 | Ordered from VWR (Cat. # 63019-047) |
| Graefe Forceps | Roboz | RS-5138 | Two are needed |
| Johns Hopkins Bulldog Clamp | Roboz | RS-7441 | Three are needed |
| Piezoelectric Strain Gauge | Ambu | 813255-100 | |
| Data Acquisition USB Subsystems | DataWave Technologies | NA | |
| Sciworks Experimenter Software | NA | ||
| CyberAmp | Axon Instruments | NA | |
| Syringe, 500 µL, Model 1750 TLL | Hamilton Company | 81220 | |
| Syringe, 10 µL, Model 1801 RN | 7659-01 | ||
| Needle, 28 gauge, Small Hub RN | 7803-02 | Point Style 4, Angle 35, Length 0.5 in |
Referencias
- Zhuo, H., Ichikawa, H., Helke, C. J. Neurochemistry of the nodose ganglion. Prog. Neurobiol. 52 (2), 79-107 (1997).
- Carley, D. W., Radulovacki, M. Pharmacology of vagal afferent influences on disordered breathing during sleep. Respir. Physiol. Neurobiol. 164 (1-2), 197-203 (2008).
- Kuo, P., Holloway, R. H. Beyond acid suppression: new pharmacologic approaches for treatment of GERD. Curr. Gastroenterol. Rep. 12 (3), 175-180 (2010).
- Maher, S. A., Dubuis, E. D., Belvisi, M. G. G-protein coupled receptors regulating cough. Curr. Opin. Pharmacol. 11 (3), 248-253 (2011).
- Greene, E. C. . Anatomy of the rat. , (1963).
- Li, Y., Wu, X., Zhou, S., Owyang, C. Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety. Am. J. Physiol. Gastrointest. Liver Physiol. 300 (2), 217-227 (1152).
- Powley, T. L., et al. Vagal afferent innervation of the lower esophageal sphincter. Auton. Neurosci. 177 (2), 129-142 (2013).
- Neuhuber, W. L. Sensory vagal innervation of the rat esophagus and cardia: a light and electron microscopic anterograde tracing study. J. Auton. Nerv. Syst. 20, 243-255 (1987).
- Rogers, R. C., Nasse, J. S., Hermann, G. E. Live-cell imaging methods for the study of vagal afferents within the nucleus of the solitary tract. J. Neurosci. Methods. 150 (1), 47-58 (2006).
- Wan, S., et al. Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons. J. Neurosci. 28 (19), 4957-4966 (2008).
- Verberne, A. J., Lewis, S. J., Jarrott, B., Louis, W. J. Bezold-Jarisch reflex is inhibited by excitotoxin-induced destruction of vagal primary afferent neurons. Eur. J. Pharmacol. 139 (3), 365-367 (1987).
- Lewis, S. J., et al. Excitotoxin-induced degeneration of rat vagal afferent neurons. Neurociencias. 34 (2), 331-339 (1990).
- Wallick, D. W., Dunlap, M. E., Stuesse, S. S., Thames, M. D. Denervation of vagal cardiopulmonary receptors by injection of kainic acid into the nodose ganglia in dogs. Auton. Neurosci. 102 (1-2), 85-89 (2002).
- Muroi, Y., et al. Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (11), 1017-1023 (2011).
- Kollarik, M., et al. Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors. J. Physiol. 588 (21), 4303-4315 (2010).
- Zhang, Z., Zhang, C., Zhou, M., Xu, F. 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). Respir. Physiol. Neurobiol. 183 (3), 211-217 (2012).
- Calik, M. W., Radulovacki, M., Carley, D. W. Intranodose ganglion injections of dronabinol attenuate serotonin-induced apnea in Sprague-Dawley rat. Respir. Physiol. Neurobiol. 190, 20-24 (2014).
- Yoshioka, M., Goda, Y., Togashi, H., Matsumoto, M., Saito, H. Pharmacological characterization of 5-hydroxytryptamine-induced apnea in the rat. J. Pharmacol. Exp. Ther. 260 (2), 917-924 (1992).
- Kopczynska, B., Szereda-Przestaszewska, M. 5HT2 and 5HT3 receptors’ contribution to modeling of post-serotonin respiratory pattern in cats. Life Sci. 75 (19), 2281-2290 (2004).
- Fan, P. Cannabinoid agonists inhibit the activation of 5-HT3 receptors in rat nodose ganglion neurons. J. Neurophysiol. 73 (2), 907-910 (1995).
- Jespersen, B., Knupp, L., Northcott, C. A. Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements. J. Vis. Exp. (59), (2012).
- Barann, M., et al. Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site. Br. J. Pharmacol. 137 (5), 589-596 (2002).
- Bonaz, B., Picq, C., Sinniger, V., Mayol, J. F., Clarencon, D. Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway. Neurogastroenterol. Motil. 25 (3), 208-221 (2013).
