This study was supported by National Institutes of Health (Grant 1UM1HL112856).

All procedures and protocols were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. Experiments described here are acute non-survival experiments, and there was no use of eye ointment. Maintenance of sterile conditions only occurs when surgical instruments are washed with 70% ethanol in DiH2O. Sacrifice of rats at the end of the experiment occurred via overdose of IV ketamine/xylazine.
1. Preparation of Instruments and Chemicals
<ol>
	<li>Prepare stock solution of 0.05 M of 5-HT HCl in PBS. Then dilute stock with PBS to a final 5-HT concentration of 0.203 mM.</li>
	<li>Dilute dronabinol in sesame oil at a concentration of 20 &#181;g/&#181;l.</li>
	<li>Cut 20 cm length of polyethylene (PE)-50 tubing (I.D. 0.58 mm, O.D. 0.965 mm). At one end of the tubing, cut a bevel tip using scissors, and at the other square end, insert a 23 g needle. Connect this 23 g needle to a 1 ml syringe, and fill it up with 0.3 ml of 50 u/ml heparin.</li>
	<li>Cut four pieces of 4-0 braided silk thread and set to the side.</li>
	<li>Sterilize surgical scissors, 2 curved Graefe forceps, and 3 micro clamps with 70% ethanol in diH2O.</li>
</ol>
2. Catheterization of Femoral Vein
Protocol for catheterization is modified from Jespersen et al.21
<ol>
	<li>Anesthetize a Sprague-Dawley rat with intraperitoneal (IP) ketamine/xylazine (100 mg/kg:10 mg/kg). Pinch the toe of the rat and observe any movement to confirm a proper level of anesthesia. Shave the ventral aspect of the left thigh, and the ventral aspect of the neck. If necessary by the local Institutional Animal Care and Use Committee, apply eye ointment to prevent dryness.</li>
	<li>Secure rat in a supine position on a surgical board. Use surgical scissors to cut away skin in the left hind thigh.</li>
	<li>Using forceps, conduct blunt dissection of the superficial muscle to expose the femoral vein. Use the forceps to separate the vein from the femoral artery, and place 2 threads around the femoral vein.</li>
	<li>Use the curved Graefe forceps to pull-up the vein to stop blood flow. Using a 22 G syringe needle, puncture the femoral vein and then insert the beveled end of the PE-50 tubing into the vein.</li>
	<li>Check if the PE-50 tubing was inserted correctly by retracting the plunger of the syringe. Blood should be seen entering the PE-50 tubing. Tie 2 knots around the vein and PE-50 tubing using the 2 threads.</li>
</ol>
3. 5-HT-induced Apnea via IV Infusion
<ol>
	<li>Place a piezoelectric strain gauge around the rat to measure respiration.</li>
	<li>Change settings for amplification level of the electronic amplifier via amplifier software on the computer: to amplify (100x) and band-pass filter (1-10 Hz) the respiratory signals obtained from the strain gauge, set &#8220;highpas filter&#8221; at &#8220;AC @ 1 HZ&#8221; and &#8220;Lowpass Filter&#8221; at &#8220;10 Hz,&#8221; and set the amplification by inputting &#8220;10&#8221; for the &#8220;Initial Gain&#8221; and &#8220;100&#8221; for &#8220;Total Gain.&#8221;.</li>
	<li>Change settings for sampling rate by opening &#8220;Analog Input&#8221; via recording software on the computer. To digitize (500 Hz sampling rate) using an analog-to-digital converter, &#8220;sample&#8221; at &#8220;1,000 Hz&#8221; and &#8220;skip&#8221; every other recording point by &#8220;1&#8221; (effective sampling rate 500 Hz), and then record the signal using recording software.</li>
	<li>Remove the 1 ml syringe from the catheter and insert a 500 &#181;l precision glass syringe filled with 0.203 mM of 5-HT into the catheter.</li>
	<li>Place the 500 &#181;l precision glass syringe into an infusion pump. Infuse 12.5 &#181;g/kg per 350 &#181;l/kg of 5-HT solution at a rate of 63 ml/hr. Perform multiple infusions and observe apnea in rat, which is seen as a pause in breathing (&#8805; 2.5 sec) in the recorded respiratory signals on the computer monitor.</li>
	<li>Before proceeding with neck surgery, monitor breathing pattern and check for pain reflex from toe pinch in the rat. If breathing is irregular, or if there is a pain reflex from toe pinch, administer IP ketamine/xylazine (100 mg/kg:5 mg/kg) and then re-confirm a proper level of anesthesia.</li>
</ol>
4. Neck Surgery to Expose Nodose Ganglia
<ol>
	<li>Secure rat in a supine position on a surgical board. Make a midline longitudinal cut using surgical scissors at the neck.</li>
	<li>Using blunt dissection on the platysma muscle (using 2 micro clamps to keep this muscle clear of the surgical site), expose the sternohyoideus and omohyoideus muscles. Separate these muscles to expose the internal carotid artery and one of its branches, the pterygopalatine artery.</li>
	<li>Observe the vagus nerve as it runs along the internal carotid artery and then along the pterygopalatine artery. Observe how the vagus nerve and pterygopalatine artery, along with the glossopharyngeal nerve (cranial nerve IX) and the spinal accessory nerve (cranial nerve XI), enter the posterior lacerated foramen at the base of the skull.</li>
	<li>Notice that the nodose ganglion is displayed as a swelling of the vagus nerve right before it enters the posterior lacerated foramen. Also notice that the pharyngeal and laryngeal nerve branches come off the anterior and posterior aspects, respectively, of the nodose ganglion.5</li>
	<li>Using the Graefe forceps, separate the vagus nerve from the arteries, and place a piece of thread around the vagus nerve. Place a clamp on the thread to apply slight tension on the vagus nerve, and clean the nodose ganglion of any connective tissue to provide less resistance when injecting.</li>
	<li>Repeat 5-HT-induced apnea as stated in section 3 to confirm that no damage was done to the nodose ganglia.</li>
</ol>
5. Intranodose Ganglia Injection of Dronabinol
<ol>
	<li>Fill a 10 &#181;l gastight precision glass syringe affixed with a custom-made 28 g half-inch syringe needle with a 35&#176; beveled tip with 5 &#181;l of dronabinol in sesame oil.</li>
	<li>Place a micro clamp on the thread to apply slight tension on the vagus nerve, and puncture the nodose ganglion taking care not to puncture through it. Depress the syringe slowly (&#8805; 60 sec) to inject the entire contents of the syringe. Note that some of the syringe contents will leak out of the ganglion.</li>
	<li>Repeat 5-HT-induced apnea as stated in section 3.</li>
</ol>

<ol><li>Zhuo, H., Ichikawa, H., Helke, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurochemistry+of+the+nodose+ganglion%5BTitle%5D)+AND+%22Prog.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Pharmacology+of+vagal+afferent+influences+on+disordered+breathing+during+sleep%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">Pharmacology of vagal afferent influences on disordered breathing during sleep.</a> Respir. Physiol. Neurobiol. 164 (1-2), 197-203 (2008).</li>
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<li>Greene, E. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreene%2C+EC+%22Anatomy+of+the+rat%22">Anatomy of the rat</a>. , Hafner. (1963).</li>
<li>Li, Y., Wu, X., Zhou, S., Owyang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-affinity+CCK-A+receptors+are+coexpressed+with+leptin+receptors+in+rat+nodose+ganglia%3A+implications+for+leptin+as+a+regulator+of+short-term+satiety%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Gastrointest.+Liver+Physiol%22%5BJournal%5D)">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/pubmed?term=((Vagal+afferent+innervation+of+the+lower+esophageal+sphincter%5BTitle%5D)+AND+%22Auton.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Sensory+vagal+innervation+of+the+rat+esophagus+and+cardia%3A+a+light+and+electron+microscopic+anterograde+tracing+study%5BTitle%5D)+AND+%22J.+Auton.+Nerv.+Syst%22%5BJournal%5D)">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/pubmed?term=((Live-cell+imaging+methods+for+the+study+of+vagal+afferents+within+the+nucleus+of+the+solitary+tract%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Presynaptic+melanocortin-4+receptors+on+vagal+afferent+fibers+modulate+the+excitability+of+rat+nucleus+tractus+solitarius+neurons%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Bezold-Jarisch+reflex+is+inhibited+by+excitotoxin-induced+destruction+of+vagal+primary+afferent+neurons%5BTitle%5D)+AND+%22Eur.+J.+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Excitotoxin-induced+degeneration+of+rat+vagal+afferent+neurons%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Denervation+of+vagal+cardiopulmonary+receptors+by+injection+of+kainic+acid+into+the+nodose+ganglia+in+dogs%5BTitle%5D)+AND+%22Auton.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Selective+inhibition+of+vagal+afferent+nerve+pathways+regulating+cough+using+Nav+1.7+shRNA+silencing+in+guinea+pig+nodose+ganglia%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Regul.+Integr.+Comp.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Transgene+expression+and+effective+gene+silencing+in+vagal+afferent+neurons+in+vivo+using+recombinant+adeno-associated+virus+vectors%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Activation+of+opioid+mu-receptors%2C+but+not+delta-+or+kappa-receptors%2C+switches+pulmonary+C-fiber-mediated+rapid+shallow+breathing+into+an+apnea+in+anesthetized+rats%29%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Intranodose+ganglion+injections+of+dronabinol+attenuate+serotonin-induced+apnea+in+Sprague-Dawley+rat%5BTitle%5D)+AND+%22Respir.+Physiol.+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Pharmacological+characterization+of+5-hydroxytryptamine-induced+apnea+in+the+rat%5BTitle%5D)+AND+%22J.+Pharmacol.+Exp.+Ther%22%5BJournal%5D)">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/pubmed?term=((5HT2+and+5HT3+receptors%27+contribution+to+modeling+of+post-serotonin+respiratory+pattern+in+cats%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">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/pubmed?term=((Cannabinoid+agonists+inhibit+the+activation+of+5-HT3+receptors+in+rat+nodose+ganglion+neurons%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">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/pubmed?term=((Femoral+arterial+and+venous+catheterization+for+blood+sampling%2C+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Direct+inhibition+by+cannabinoids+of+human+5-HT3A+receptors%3A+probable+involvement+of+an+allosteric+modulatory+site%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">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>
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</ol>

The authors declare that they have no competing financial interests.

The critical steps for successful injection of neurochemicals into the nodose ganglia are: 1) identifying and cleaning the connective tissue off the nodose ganglia; 2) confirming the integrity of the nodose ganglia before injection; 3) and using a small gauge needle to delicately inject into, but not completely puncturing through, the nodose ganglia.
The vagus nerve innervates many organs in the neck and abdomen, and relays important information such as heart rate, blood pressure, bronchopulmo...

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.

Figure 1 represents sample breathing recording in rats that had infusion of 5-HT to induce apnea before and after intranodose ganglia injections of dronabinol. 5-HT activates 5-HT3 receptors on the nodose ganglia that contribute to the Bezold-Jarisch reflex of bradycardia, hypotension, and apnea.11,17-19 Intranodose ganglia injections of dronabinol activate inhibitory CB receptors, or allosterically modulates 5-HT3 receptors that inhibit the 5-HT-induced excitation of the...

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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.

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

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18.7K 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.

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

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

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.

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

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.

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.

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

Lumbar Puncture Delivery of AAV9 in Juvenile Rats for CNS Gene Therapy

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

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.

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

Lumbar Intrathecal Injection of Gene Therapy Vectors for Central Nervous System Targeting in Mice and Rats

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

A lumbar intrathecal injection represents a translationally relevant route of administration for delivering gene therapy to the central nervous system. This comprehensive standardized protocol for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats aims to guide researchers in adopting this technique for preclinical gene therapy studies.

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This ...

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

Medicine

Modeling Sensory Axon Regeneration in a Rat Using Dorsal Root Ganglion Injection and Dorsal Root Crush Injury

Begin the surgery by locating the prominent C2 and T2 spinous processes over the skin. Then use a number 10 scalpel, to make a skin incision between the C2 and T2 spinous processes. Once the skin is opened, a white fibrous tissue midline should be visible on the first layer of muscle, which has a jelly-like texture.Make a similar sized incision on the first layer of muscle along the white midline. Do not go beyond the prominent T2 spinous process, as there is a major blood vessel located near there. Retract the first layer of muscle using two retractors, one placed rostrally and one caudally.The second layer of muscle with a striated appearance should now be visible. Locate the midline of the second layer of muscle, where two longitudinal muscles can be observed, connected by a thin membranous tissue. Then use a pair of micro scissors to dissect the membranous tissue and separate the two longitudinal muscles.Adjust the retractors to expose the third layer of thin muzzle covering the spine. The spinous processes can be felt by lightly touching with a pair of forceps over the third layer of muscle. Use micro scissors to make a small incision on the third layer of muscle. And gently scrape off the muscle from the bone using a curette or scalpel in a sideways manner to clearly expose the vertebrae.To expose the left C5 to C8 DRGs, use a pair of fine rongeurs to perform a left hemilaminectomy on the C4 to T1 vertebrae by carefully removing part of the lamina and pedicle. Once enough of the DRG has been exposed for injection, prepare the syringe by placing the virus filled microliter syringe fitted with a custom made 33-gauge blunt needle, onto the stereotaxic syringe holder. Next, use a 30-gauge beveled needle to make a small superficial opening on each of the targeted DRG to assist with the insertion of the injection needle. Gently adjust the stereotaxic coordinates, to insert the 33-gauge needle into the center of the DRG.Ideally, the needle should be placed at the very center of the DRG to ensure uniform diffusion of the injected solution throughout the DRG.Once the needle is inserted, use the infusion syringe pump to inject 1 microliter of the virus into each DRG. During the injection, the DRG will slowly change color if the virus solution contains a colored dye. Do not over insert the needle, as this may cause fluid to leak out from the ventral side of the DRG.Should leakage occur during the injection, adjust the position of the needle immediately. Three minutes after the end of the injection, withdraw the injection needle. To perform a concurrent C5 to C8 dorsal root crush injury, crush each root three times for 10 seconds using a pair of fine tipped forceps. Completely oppose the ends of the forceps. A white line in the tissue should appear at the crush site.

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

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