This study was supported by the National Institute for Nursing Research (K99/R00NR014369).

All methods described were approved by the Institutional Animal Care Committee at the University of Illinois at Chicago.
1. Create Bipolar RSNA Electrodes
<ol>
	<li>To create the electrode, cut two pieces of stainless steel wire each approximately 18 mm long. Cut one piece of polyethylene (PE-50) tubing approximately 15 mm long. Feed both pieces of wire into the tubing, leaving the wire protruding from both ends.</li>
	<li>Remove the insulation from the ends of the wires; trim the wires leaving 2-3 mm of exposed wire. On one end, crimp male pins over the exposed wire. Solder the pins securely to the wire, secure the pins inside a connector strip, and cover the connection with epoxy. 
	NOTE: An alternative approach that avoids soldering is to use quick connect/release alligator clips.</li>
	<li>On the opposite end of the electrode, remove the insulation from the ends of the wires, leaving 2-3 mm of exposed wire. Bend this portion of the wires to create small &#34;V&#34; shaped hooks in the uninsulated wire. 
	NOTE: This is the portion of the electrode that will be in contact with the renal sympathetic nerve. It is important to seal this end to prevent fluids from entering the tubing. Silicone or epoxy effectively can be used.</li>
</ol>
2. Administer Anesthesia and Prepare Surgical Sites
<ol>
	<li>Administer anesthesia to a male Sprague Dawley rat (age 9-11 weeks, weighing 150-400 g). Administer pentobarbital sodium 50 mg/kg via an intraperitoneal (IP) injection. To assess a stable plane of anesthesia during surgery, check toe-pinch reflex every 15 min and re-dose anesthesia as needed. 
	NOTE: Pentobarbital sodium (Nembutal) was used in previous studies to achieve a sustained plane of anesthesia without interfering with the modulation of RSNA4,5,6. This protocol is for non-survival surgery, so there is no recovery/post-operative monitoring period.</li>
	<li>Prepare the surgical site according to institutional animal care guidelines (i.e., shave the rat&#39;s abdomen, back, and head; cleanse the skin with 10% povidone-iodine solution; apply eye lubricant; and place the rat on a heating pad). Maintain the body temperature at 37 &#176;C during the experiments.</li>
</ol>
3. Cannulate the Femoral Vein (for Intravenous Access)
<ol>
	<li>Add heparin to 0.9% sterile saline (to achieve 20 units/mL). Fill a 1 mL syringe with the heparinized saline through a 22G needle. Connect 15 cm of PE-50 tubing to the needle and fill tubing with the solution.</li>
	<li>With the rat lying supine, create a 2 cm horizontal incision through the inguinal area. Using cotton-tipped applicators, dissect the connective tissue to expose the femoral vein and artery. To hold the incision open, either apply single-hook elastic surgical stays secured to the surgical field with silk tape or use small hemostats.</li>
	<li>Use hemostats to bend the tip of a 22G needle at a 90&#176; angle to serve as a catheter introducer10.</li>
	<li>Visualize the vessels under the microscope. Gently separate the vein and artery using curved forceps. Place two 12 cm long 5-0 silk suture beneath the vein (one distal and one proximal); place the suture in the same manner beneath the artery.
	<ol>
		<li>Tie the distal (bottom) suture to occlude the vein; secure the edges of this suture to the surgical field using either silk tape or small hemostats. Gently pull the vein taut but not with so much force that the vessel will tear. Position the vein perpendicular to the surgeon&#39;s dominant hand.</li>
	</ol>
	</li>
	<li>Use a loose overhand half knot in the proximal suture to briefly occlude the vein. With delicate hemostatic forceps, gently clamp this suture to occlude blood flow. Hold the 22G needle with the bent tip in the non-dominant hand; clasp the tubing with forceps using the dominant hand.</li>
	<li>Puncture a small hole in the vein with the catheter introducer (Step 3.3) and insert the PE-50 tubing (pre-filled with heparinized saline) into the vessel; use the bent needle to keep the opening in the vessel open and to assist in positioning the tip of the catheter into the vessel10.
	<ol>
		<li>Release the proximal suture and gently flush 0.2 mL of heparinized saline into the vein; advance the catheter. Check the blood return from the vein to ensure proper placement. Complete the proximal knot and, with distal suture tie, secure the tubing inside the vein.</li>
	</ol>
	</li>
	<li>Use venous access during the experiments for administering supplemental anesthesia or medications and for blood collection. Incorporate a 3-way connector if regular intravenous infusions and blood sampling will be necessary. Check noxious toe-pinch reflex every 15 min to titrate anesthesia.</li>
</ol>
4. Cannulate the Femoral Artery for Mean Arterial Pressure Monitoring
<ol>
	<li>Visualize the artery under the microscope. Similar to the method used for venous cannulation, tie the distal suture (Step 3.4) to occlude the artery; secure the edges of this suture to the surgical field with silk tape and position the artery perpendicular to the surgeon&#39;s dominant hand.</li>
	<li>Arterial access if using a pressure transducer/infusion system
	<ol>
		<li>Connect the pressure transducer/tubing to a 500 mL 0.9% saline bag. Flush the tubing with saline, removing all bubbles, and place the bag inside a pressure inducer bag to pressurize the system.</li>
		<li>As described in Step 3.1, fill a 1 mL syringe with the heparinized saline through a 22G needle and connect 15 cm of PE-50 to the needle (flush tubing with heparinized saline).</li>
		<li>Use a loose half knot in the proximal suture to briefly occlude the artery. Hold the catheter introducer (Step 3.3) in the non-dominant hand; hold the distal tip of the PE-50 with vessel cannulation forceps in the dominant hand. Puncture a hole in the artery with the bent 22G needle and insert the cannula into the vessel.</li>
		<li>Release the proximal suture, gently flush 0.2 mL of heparinized saline into the artery and advance the catheter as far as possible. Check for arterial blood return to ensure proper placement. Complete the proximal knot and, with distal suture tie, secure the tubing inside the artery. Connect the arterial line to the pressure transducer/tubing. 
		NOTE: The distal portion of tubing can be taped to the rat&#39;s hindlimb to secure the arterial line. An alternative approach to vessel cannulation is described by Jespersen et al.;11 their protocol differs by using retractors to spread the incision, glue-rather than suture-to secure the tubing, and the approach does not include the bent needle introducer.</li>
	</ol>
	</li>
	<li>Arterial access if using telemetry
	<ol>
		<li>Inspect the pressure-sensing catheter under high magnification before arterial cannulation. Ensure that the catheter is free of bubbles/debris and has an intact meniscus between the fluid-filled (proximal) and gel-filled (distal) components. Prior to each implantation, refill the gel at the distal tip of the catheter. Turn on the transmitter using a magnet; monitor BP during the surgery to endure perfect placement.</li>
		<li>Use a loose overhand half knot in the proximal suture to briefly occlude the femoral artery. Hold the catheter introducer (Step 3.3) in the non-dominant hand. Hold the tip of the cannula of the telemetry unit with vessel cannulation forceps to avoid displacing gel from the tip.</li>
		<li>Puncture a hole in the artery with the bent 22G needle and insert the cannula into the artery using vessel cannulation forceps to avoid displacing gel from the tip. Advance the cannula as far as possible. Using the proximal and distal suture ties, secure the pressure catheter.</li>
		<li>Tuck the body of the telemetry implant inside the flank adjacent to the incision, and close this incision using 4-0 nylon suture on a cutting needle. Turn the telemetry devices off by magnet at the conclusion of the recording period to conserve battery life.</li>
	</ol>
	</li>
</ol>
5. Position the Rat in the Stereotaxic Surgery Frame to Access the Brain
<ol>
	<li>Gently move the rat into the prone position in the stereotaxic surgery frame.</li>
	<li>Position the rat between the ear bars, and adjust the incisor bar to equalize the height of lambda and bregma. Positioning may depend on the rat strain, weight, and locations of CNS targets.</li>
	<li>Make a 2 cm rostrocaudal scalpel incision through the midline of the scalp. Using cotton-tipped applicators, firmly remove connective tissue from the skull surface. Apply hydrogen peroxide to the skull to assist in visualizing the bregma, lambda, and midline sutures.</li>
	<li>Using an atlas of the rat brain to guide targeting12, drill a burr hole osteotomy, sized for electrode access, through the skull.</li>
</ol>
6. Isolating the Renal Sympathetic Nerves
<ol>
	<li>Connect the wire RSNA electrode (Steps 1.1-1.3) to a 10X pre-amplifier and microelectrode amplifier.</li>
	<li>Isolate the renal nerves through a retroperitoneal incision prior to, or after, the rat is secured in the stereotaxic frame. Position the RSNA electrodes once the rat is in the stereotaxic frame. Make a scalpel incision extending from 4-5 cm below the ribs in the caudal direction, slightly lateral to the spine. Blunt dissect the incision to visualize the paraspinal muscles.</li>
	<li>Use scissors to make a very superficial 1-2 cm rostrocaudal incision where the fat meets the muscle. Using cotton-tipped applicators, spread the fat away from the muscle to visualize the kidney. It is important not to enter the peritoneal space.</li>
	<li>Use retractors to gently separate the kidney from the paraspinal muscles to visualize the renal artery and abdominal aorta. Do not stretch the vessels excessively to avoid damaging the renal nerves. Use a 2&#34; x 2&#34; cotton gauze pad soaked in saline to protect the kidney from injury.</li>
	<li>Under high magnification, identify the renal nerves in the incision pocket. The nerve bundles are most easily visible in the right angle formed by the aorta and the renal artery. The renal nerves closely follow the renal artery from the aorta to the kidneys.</li>
	<li>Select a segment of the nerve bundle that will be placed on the recording electrode. Gently dissect the nerve fibers from the surrounding tissue/vessel using micro-dissecting tweezers.</li>
	<li>Secure the wire RSNA electrode in a holder (e.g., an alligator clip attached to a support stand). Lower the electrode to the level of the nerve segment. Use a nerve hook to gently lift the segment of the renal nerve onto the electrode without stretching the nerve. 
	NOTE: The nerve should rest inside both &#34;V&#34; shaped hooks in the uninsulated wire, parallel to the nerve. The electrode wires must not touch any other tissue, blood, or lymph fluid.</li>
	<li>Fill the incision with mineral oil to prevent the exposed renal sympathetic nerve from becoming dry. Use a grounding clip with one end on the skin of the incision and the other attached to the Faraday cage.</li>
	<li>Direct the signal to the amplifiers using high- and low-pass filtering (10 Hz and 3 kHz). Adjust the gain up to 10 K. Include an audio monitor to assess the bursting pattern of the RSNA. Use sampling rates ranging between 2,000-10,000 Hz4,5,6,7,8. Use an increased sampling rate when a CNS manipulation is hypothesized to cause rapid/brief sympathetic responses.</li>
</ol>
7. Record Data
<ol>
	<li>Assess the quality of the RSNA recording by evoking the baroreflex with a bolus injection of 1 mL of saline or 10 &#181;g/mL phenylephrine (in 0.1 mL) intravenously. As illustrated in Figure 1, the infusion should increase BP and inhibit RSNA. An increase in mean arterial pressure of 60-80 mmHg is sufficient for renal sympathoinhibition4,13,14.</li>
	<li>Adjust the position of the electrodes to improve the signal if necessary. Repositioning is required if the nerve is not in contact with both hooks on the electrode or if any tissue, blood, or lymph fluid is in contact with the wires. 
	NOTE: The need for repositioning is based on the auditory characteristics of the nerve discharges.
	<ol>
		<li>If the bursts of RSNA are not occurring cyclically with the cardiac cycle, and if there is any interference in the recording, then carefully reposition the electrode.</li>
		<li>As respiratory movements can also affect the quality of the RSNA recording, improve the signal by gently moving the electrode into a position where the muscle movements do not disrupt the electrode during breathing.</li>
	</ol>
	</li>
	<li>Once a clear signal is obtained, secure the RSNA electrode in place by withdrawing mineral oil and applying a silica gel to cover the nerve/electrode connection in the incision pocket. Do not move the rat before the gel has set completely.</li>
	<li>Perform CNS manipulation protocols while continuously recording RSNA and mean arterial pressure. If a microinjector/pulser is used for brainstem manipulations, a logic signal from this device can be introduced into the RSNA/BP recordings to document the timing of CNS manipulations.</li>
	<li>When the experiment is complete, determine the noise level by crushing the nerve proximal to the recording electrodes between the silica gel and the spinal muscle. Record at least 30 s of this &#34;zero&#34; value for RSNA4,5,6. As an alternative approach for quantifying noise, administer a short-acting ganglionic blocker such as atropine, hexamethonium, chlorisondamine, or pentolinium tartrate8,15,16,17.</li>
	<li>Carefully remove the RSNA electrode and remove any traces of silica gel from the wire electrodes. Save the electrode for re-use. Turn off the telemetry transmitter and remove it, taking care not to damage the tip of the catheter.</li>
</ol>
8. Euthanasia (Transcardiac Perfusion)
<ol>
	<li>Identify the locations of CNS manipulations by injecting dyes or fluorochromes, creating electrolytic lesions, or by detecting of c-fos expression.</li>
	<li>If brain analyses will require fixation, prepare the rat for transcardiac perfusion. Assess the toe-pinch reflex to ensure that the rat remains deeply anesthetized. Provide supplemental anesthesia if needed. Perform transcardiac perfusion of paraformaldehyde fixative in a fume hood. 
	CAUTION: Avid skin/eye irritant.</li>
	<li>Insert tubing into the perfusion pump and prime with 0.9% normal saline.</li>
	<li>Make a 5-6 cm lateral incision through the skin and abdominal wall immediately beneath the rib cage and open the chest cavity. Carefully separate the liver from the diaphragm. Make a small incision in the diaphragm using curved blunt scissors. Inject 0.1 mL of heparin directly into the left ventricle.</li>
	<li>Pass a perfusion needle into the left ventricle (a stainless steel gavage needle works well for this step) by either puncturing it into the heart or by cutting a small incision using sharp scissors and passing the gavage needle through so the tip is visible through the wall of the aorta (but does should not reach the aortic arch). Use a surgical or electrical clip to secure the needle in place.</li>
	<li>Using an infusion pump, administer 0.9% normal saline (room temperature). Immediately create a 2-3 mm incision in the right atrium to create an outlet for the saline rinse. Do not cut the descending aorta. Continue the saline rinse until the liver changes color from red/brown to pale yellow, an infusion of approximately 400 mL over 2-3 min.</li>
	<li>Stop the pump. Switch the perfusate to the fixative (e.g., 10% formalin or 4% paraformaldehyde); infuse 400 mL over 2-3 min. Remove the brain and store the specimen in fixative solution overnight at 4 &#176;C before transferring the tissue to 30% sucrose (30 mL of sucrose dissolved in 100 mL of 0.1 M phosphate-buffered saline) for at least 3 days or until the brain sinks, for cryoprotection before cryogenetic sectioning18.</li>
</ol>
9. Analyze Data
<ol>
	<li>Full-wave rectify the raw RSNA to obtain absolute values. Full-wave rectify a 10 s segment of the raw noise signal. It is important to exclude any studies that were affected by a low signal-to-noise ratio. In studies quantifying RSNA, investigators applied a priori criteria such as requiring signal to noise ratios to exceed 2:1 to 6:117,19,20.</li>
	<li>Calculate the mean rectified RSNA for non-overlapping segments (&#181;V) and subtract the noise estimate (&#181;V). Depending on the aims of the experiment, investigators may select intervals such as 10 s (Figure 1) or 1 s. Calculate means by using waveform analysis options in the physiologic software or exporting the data into spreadsheets to calculate averages for selected time intervals.</li>
	<li>To normalize across different animals, express values for further analysis as the percent change from baseline. Use parametric statistics to conduct group comparisons.</li>
</ol>

<ol><li>Mancia, G., Grassi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+autonomic+nervous+system+and+hypertension%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">The autonomic nervous system and hypertension.</a> Circulation Research. 114 (11), 1804-1814 (2014).</li>
<li>Kannan, A., Medina, R. I., Nagajothi, N., Balamuthusamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+sympathetic+nervous+system+and+the+effects+of+denervation+on+renal+arteries%5BTitle%5D)+AND+%22World+Journal+of+Cardiology%22%5BJournal%5D)">Renal sympathetic nervous system and the effects of denervation on renal arteries.</a> World Journal of Cardiology. 6 (8), 814-823 (2014).</li>
<li>Johns, E. J., Kopp, U. C., DiBona, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+control+of+renal+function%5BTitle%5D)+AND+%22Comprehensive+Physiology%22%5BJournal%5D)">Neural control of renal function.</a> Comprehensive Physiology. 1 (2), 767(2011).</li>
<li>Fink, A. M., Dean, C., Piano, M. R., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pedunculopontine+tegmentum+controls+renal+sympathetic+nerve+activity+and+cardiorespiratory+activities+in+Nembutal-anesthetized+rats%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The pedunculopontine tegmentum controls renal sympathetic nerve activity and cardiorespiratory activities in Nembutal-anesthetized rats.</a> PLoS One. 12 (11), e0187956(2017).</li>
<li>Dean, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocannabinoid+modulation+of+sympathetic+and+cardiovascular+responses+to+acute+stress+in+the+periaqueductal+gray+of+the+rat%5BTitle%5D)+AND+%22American+Journal+of+Physiology%2C+Regulatory%2C+Integrative+and+Comparative+Physiology%22%5BJournal%5D)">Endocannabinoid modulation of sympathetic and cardiovascular responses to acute stress in the periaqueductal gray of the rat.</a> American Journal of Physiology, Regulatory, Integrative and Comparative Physiology. 300 (3), R771-R779 (2011).</li>
<li>Seagard, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anandamide+content+and+interaction+of+endocannabinoid%2FGABA+modulatory+effects+in+the+NTS+on+baroreflex-evoked+sympathoinhibition%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition.</a> American Journal of Physiology-Heart and Circulatory Physiology. 286 (3), H992-H1000 (2004).</li>
<li>Ferreira, C. B., Cravo, S. L., Stocker, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Airway+obstruction+produces+widespread+sympathoexcitation%3A+Role+of+hypoxia%2C+carotid+chemoreceptors%2C+and+NTS+neurotransmission%5BTitle%5D)+AND+%22Physiological+Reports%22%5BJournal%5D)">Airway obstruction produces widespread sympathoexcitation: Role of hypoxia, carotid chemoreceptors, and NTS neurotransmission.</a> Physiological Reports. 6 (3), (2018).</li>
<li>Stocker, S. D., Muntzel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recording+sympathetic+nerve+activity+chronically+in+rats%3A+Surgery+techniques%2C+assessment+of+nerve+activity%2C+and+quantification%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Recording sympathetic nerve activity chronically in rats: Surgery techniques, assessment of nerve activity, and quantification.</a> American Journal of Physiology-Heart and Circulatory Physiology. 305 (10), H1407-H1416 (2013).</li>
<li>Miki, K., Kosho, A., Hayashida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+continuous+measurements+of+renal+sympathetic+nerve+activity+and+cardiovascular+function+during+exercise+in+rats%5BTitle%5D)+AND+%22Experimental+Physiology%22%5BJournal%5D)">Method for continuous measurements of renal sympathetic nerve activity and cardiovascular function during exercise in rats.</a> Experimental Physiology. 87 (1), 33-39 (2002).</li>
<li>Huetteman, D. A., Bogie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+blood+pressure+monitoring+in+laboratory+rodents+via+implantable+radio+telemetry%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Direct blood pressure monitoring in laboratory rodents via implantable radio telemetry.</a> Methods in Molecular Biology. 573, 57-73 (2009).</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+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> Journal of Visualized Experiments. (59), 3496(2012).</li>
<li>Paxinos, G., Watson, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+rat+brain+in+stereotaxic+coordinates%22">The rat brain in stereotaxic coordinates</a>. , Academic Press. Sydney/New York. (2014).</li>
<li>Scislo, T. J., Augustyniak, R. A., O'Leary, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+arterial+baroreflex+regulation+of+renal%2C+lumbar%2C+and+adrenal+sympathetic+nerve+activity+in+the+rat%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">Differential arterial baroreflex regulation of renal, lumbar, and adrenal sympathetic nerve activity in the rat.</a> American Journal of Physiology. 275, R995-R1002 (1998).</li>
<li>Kopp, U. C., Jones, S. Y., DiBona, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Afferent+renal+denervation+impairs+baroreflex+control+of+efferent+renal+sympathetic+nerve+activity%5BTitle%5D)+AND+%22American+Journal+of+Physiology%2C+Regulatory%2C+Integrative+and+Comparative+Physiology%22%5BJournal%5D)">Afferent renal denervation impairs baroreflex control of efferent renal sympathetic nerve activity.</a> American Journal of Physiology, Regulatory, Integrative and Comparative Physiology. 295 (6), R1882-R1890 (2008).</li>
<li>Guild, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+sympathetic+nerve+activity%3A+problems%2C+pitfalls+and+the+need+for+standardization%5BTitle%5D)+AND+%22Experimental+Physiology%22%5BJournal%5D)">Quantifying sympathetic nerve activity: problems, pitfalls and the need for standardization.</a> Experimental Physiology. 95 (1), 41-50 (2010).</li>
<li>Stocker, S. D., Hunwick, K. J., Toney, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypothalamic+paraventricular+nucleus+differentially+supports+lumbar+and+renal+sympathetic+outflow+in+water-deprived+rats%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water-deprived rats.</a> Journal of Physiology. 15 (563 Pt 1), 249-263 (2005).</li>
<li>Stocker, S. D., Gordon, K. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutamate+receptors+in+the+hypothalamic+paraventricular+nucleus+contribute+to+insulin-induced+sympathoexcitation%5BTitle%5D)+AND+%22Neurophysiology%22%5BJournal%5D)">Glutamate receptors in the hypothalamic paraventricular nucleus contribute to insulin-induced sympathoexcitation.</a> Neurophysiology. 113 (5), 1302-1309 (2015).</li>
<li>Saponjic, J., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Injection+of+glutamate+into+the+pedunculopontine+tegmental+nuclei+of+anesthetized+rat+causes+respiratory+dysrhythmia+and+alters+EEG+and+EMG+power%5BTitle%5D)+AND+%22Sleep+and+Breathing%22%5BJournal%5D)">Injection of glutamate into the pedunculopontine tegmental nuclei of anesthetized rat causes respiratory dysrhythmia and alters EEG and EMG power.</a> Sleep and Breathing. 9 (2), 82-91 (2005).</li>
<li>DiBona, G. F., Jones, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+analysis+of+renal+nerve+activity+responses+to+baroreceptor+denervation+in+hypertensive+rats%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Dynamic analysis of renal nerve activity responses to baroreceptor denervation in hypertensive rats.</a> Hypertension. 37 (4), 1153-1163 (2001).</li>
<li>Kunitake, T., Kannan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discharge+pattern+of+renal+sympathetic+nerve+activity+in+the+conscious+rat%3A+spectral+analysis+of+integrated+activity%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Discharge pattern of renal sympathetic nerve activity in the conscious rat: spectral analysis of integrated activity.</a> Journal of Neurophysiology. 84 (6), 2859-2867 (2000).</li>
<li>Machado, B. H., Bonagamba, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+L-glutamate+into+the+nucleus+tractus+solitarii+increases+arterial+pressure+in+conscious+rats%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats.</a> Brain Research. 576 (1), 131-138 (1992).</li>
<li>Murakami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhalation+anesthesia+is+preferable+for+recording+rat+cardiac+function+using+an+electrocardiogram%5BTitle%5D)+AND+%22Biological+and+Pharmaceutical+Bulletin%22%5BJournal%5D)">Inhalation anesthesia is preferable for recording rat cardiac function using an electrocardiogram.</a> Biological and Pharmaceutical Bulletin. 37 (5), 834-839 (2014).</li>
<li>Nakamura, T., Tanida, M., Niijima, A., Hibino, H., Shen, J., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auditory+stimulation+affects+renal+sympathetic+nerve+activity+and+blood+pressure+in+rats%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Auditory stimulation affects renal sympathetic nerve activity and blood pressure in rats.</a> Neuroscience Letters. 416 (2), 107-112 (2007).</li>
<li>Bardgett, M. E., McCarthy, J. J., Stocker, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutamatergic+receptor+activation+in+the+rostral+ventrolateral+medulla+mediates+the+sympathoexcitatory+response+to+hyperinsulinemia%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Glutamatergic receptor activation in the rostral ventrolateral medulla mediates the sympathoexcitatory response to hyperinsulinemia.</a> Hypertension. 55 (2), 284-290 (2010).</li>
<li>Brozoski, D. T., Dean, C., Hopp, F. A., Seagard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uptake+blockade+of+endocannabinoids+in+the+NTS+modulates+baroreflex-evoked+sympathoinhibition%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.</a> Brain Research. 1059 (2), 197-202 (2005).</li>
<li>Barman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+can+we+learn+about+neural+control+of+the+cardiovascular+system+by+studying+rhythms+in+sympathetic+nerve+activity%3F%5BTitle%5D)+AND+%22International+Journal+of+Psychophysiology%22%5BJournal%5D)">What can we learn about neural control of the cardiovascular system by studying rhythms in sympathetic nerve activity?</a> International Journal of Psychophysiology. 103, 69-78 (2016).</li>
<li>Charkoudian, N., Wallin, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sympathetic+neural+activity+to+the+cardiovascular+system%3A+integrator+of+systemic+physiology+and+interindividual+characteristics%5BTitle%5D)+AND+%22Comprehensive+Physiology%22%5BJournal%5D)">Sympathetic neural activity to the cardiovascular system: integrator of systemic physiology and interindividual characteristics.</a> Comprehensive Physiology. 4 (2), 825-850 (2014).</li>
<li>Malpas, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sympathetic+nervous+system+overactivity+and+its+role+in+the+development+of+cardiovascular+disease%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Sympathetic nervous system overactivity and its role in the development of cardiovascular disease.</a> Physiological Reviews. 90 (2), 513-557 (2010).</li>
<li>Chen, W. W., Xiong, X. Q., Chen, Q., Li, Y. H., Kang, Y. M., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+sympathetic+afferent+reflex+and+its+implications+for+sympathetic+activation+in+chronic+heart+failure+and+hypertension%5BTitle%5D)+AND+%22Acta+Physiologica%22%5BJournal%5D)">Cardiac sympathetic afferent reflex and its implications for sympathetic activation in chronic heart failure and hypertension.</a> Acta Physiologica. 213 (4), 778-794 (2015).</li>
<li>Linz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+renal+sympathetic+innervation%3A+Recent+insights+beyond+blood+pressure+control%5BTitle%5D)+AND+%22Clinical+Autonomic+Research%22%5BJournal%5D)">Modulation of renal sympathetic innervation: Recent insights beyond blood pressure control.</a> Clinical Autonomic Research. , Epub ahead of print (2018).</li>
</ol>

Anne M. Fink is a member of the Customer Advisory Board for Data Sciences International.

Critical steps for measuring RSNA include: (1) avoiding stretching of the renal artery and nerves when separating the kidney from the paraspinal muscle and when placing the nerve segment on the recording electrodes, (2) carefully dissecting the renal nerve fibers from the surrounding tissue/vessel, (3) ensuring that the electrode wires are free of tissue, blood, or lymph fluid, and (4) preventing the nerve from drying out by applying mineral oil to the renal nerve and silica gel to the nerve-electrode unit. For troubleshooting, it is important to ensure that the recording system is adequately grounded. To obtain a clear RSNA signal, the position of the electrode can be carefully adjusted while visualizing and listening to the raw RSNA signal, prior to embedding in silica gel. Successful completion of the surgery results in a RSNA signal that can be modulated by CNS manipulations for the experiments lasting several hours.
When interpreting the results, the investigators should consider the influence of anesthesia on mean arterial pressure and RSNA. This protocol uses barbiturate anesthesia (pentobarbital sodium), which can reduce mean arterial pressure and modify autonomic responses21. Depending on the experiment aims, other injectable formulations or inhalation anesthesia (via nose-cone or tracheostomy) can be used22. Researchers may consider the alternatives such as urethane23 and alpha-chloralose24. These agents have less impact on blunting cardiovascular reflexes but can pose potential health hazards to the investigator.
In addition to the methods described in this protocol, alternative approaches have been employed by other laboratories for recording and fabricating electrodes. RSNA can be recorded using stainless steel4,9, silver25, or platinum26 wire. In addition to lifting the exposed nerve segment onto the electrode wire, scientists have successfully recorded monophasic RSNA at the central ends of cut renal sympathetic nerves26. Flexibility differs based on the tensile strength of the wire (measured with kPSI units). Higher kPSI wire is more brittle but retains its shape; low kPSI wire is more flexible and less likely to break when bent, repetitively. For RSNA recordings, it is important to select a wire that can be easily bent and repositioned during recordings. The wire should not be too flexible, making it difficult to create hooks to position under the nerve, but not too stiff. The latter increases the risk of stretching and damaging the nerves. Our laboratory uses stainless steel wire with 155-185 kPSI.
Many approaches for RSNA analysis are available. Rather than quantifying the averages for 10 s recording segments and calculating the differences as the percent change, RSNA can be determined by quantifying burst frequency4,26,27. This approach may be preferred when the baseline levels and magnitudes of RSNA responses differ among rats in a study15,26. Another approach involves the rectification and integration of the RSNA signal; the RSNA amplitude (measured in mV) is summed over a selected interval of time (e.g., 20 ms)15,26. An integrator applies a low-phase filter and provides the average discharge amplitude during bursts of activity exceeding the time constant (e.g., &#62; 20 s)15,27. Integrated signals are useful for examining the amplitude and phase of the RSNA, but this approach does not provide information about oscillatory changes. Frequency domain and time domain methods have been applied when researchers examine RSNA oscillations. The approach frequently used for RSNA is the fast Fourier transformation (FFT), which categorizes a signal into its sinusoidal oscillations, each with a distinct amplitude and phase20,26. FFT is a useful approach for examining the low- and high-frequency bursts in the RSNA and for studying respiratory and cardiac modulation of the RSNA signal.
The methods in this protocol are important for addressing hypotheses about the functional significance of CNS nuclei. Renal sympathetic nerves direct neural communication between the CNS and kidney, and therefore, acute changes in RSNA represent an important variable in cardiovascular research. Defining CNS mechanisms regulating sympathetic outflow is a priority research area, considering that renal sympathoexcitation contributes to the pathophysiology and clinical presentation of many diseases (e.g., chronic kidney disease, heart failure, arrhythmias, diabetes mellitus, and obstructive sleep apnea)28,29. Indirect measures of sympathetic nerve activity (e.g., BP, heart rate variability, catecholamine levels) are not always suitable for the studies on the functional significance of CNS nuclei. Therefore, the direct measurement of RSNA and mean arterial pressure in anesthetized rats represents a valuable method for functionally, anatomically defining the sources of aberrant renal sympathetic function.

Pre-clinical discoveries about autonomic control of the cardiovascular system inform strategies for managing disorders such as hypertension, heart failure, and chronic kidney disease. Over-activity of the sympathetic nervous system and reduced vagal cardiac tone contribute to elevated blood pressure (BP)1. Chronically elevated renal sympathetic outflow enhances catecholamine secretion and decreases renal blood flow, with deleterious consequences to the cardiovascular/renal systems2,3. To define the neurobiological pathways leading to autonomic dysfunction, studies in rodents are important for determining how central nervous system (CNS) neurons regulate sympathetic parameters. The purpose of this protocol is to provide technical information about measuring renal sympathetic nerve activity (RSNA) and BP and to outline the techniques for quantifying acute sympathetic changes in response to CNS manipulations in anesthetized rats.
Acute (non-survival) RSNA measurements (lasting min to h) are useful when scientists will probe the CNS pharmacologically, electrically, or optogenetically in anesthetized rats to determine the functions of specific nuclei. Using these methods, structures such as the solitary nucleus, periaqueductal gray, pedunculopontine tegmentum, and rostral ventrolateral medulla have been investigated to define neurobiological pathways regulating sympathetic parameters4,5,6,7. This approach is important for identifying CNS targets to be investigated further in chronic models of autonomic dysfunction8,9. To complete these experiments, the laboratory requires a soldering iron, surgical microscope, stereotaxic frame, microelectrode amplifier, and audio monitor. Depending on the factors present in the laboratory that contribute to electrical noise, the surgical/recording area may require a Faraday cage/grounding strap to reduce electrical noise in the RSNA recording. If brain analyses will require tissue fixation, a perfusion pump and fume hood are required. Data can be digitized and recorded using multiple physiologic software/data acquisition (analogue-digital converter) units4,5, with different analysis options and compatibilities for incorporating telemetric signals.

<table><tbody><tr><td>Stainless steel wire</td><td>A-M Systems; Sequim, WA</td><td>791000</td><td>RSNA electrode</td></tr><tr><td>Polyethylene (PE-50) tubing</td><td>VWR; Radnor, PA</td><td>63019-048</td><td>RSNA electrode; vessel cannulation</td></tr><tr><td>Miniature pin connector</td><td>A-M Systems; Sequim, WA</td><td>520200</td><td>RSNA electrode</td></tr><tr><td>Crimping tool</td><td>Daniels Manufacturing Corp.; Orlando, FL</td><td>M22520</td><td>RSNA electrode</td></tr><tr><td>Connector strip</td><td>Amphenol; Clinton Township, MI</td><td>221-2653</td><td>RSNA electrode</td></tr><tr><td>J-B Kwik Epoxy</td><td>J-B Weld, Sulphur Springs, TX</td><td>8270</td><td>RSNA electrode</td></tr><tr><td>Silicone</td><td>Permatex; Hartford, CT</td><td>2222</td><td>RSNA electrode</td></tr><tr><td>Heparin sodium; Injectable (10 mL vial, 1000 U/mL)</td><td>KV Veterinary Supply; David City, NE</td><td>P03466</td><td>Venous line patency</td></tr><tr><td>Phenylephrine HCl; Injectable (1 mL vial; 10 mg/mL)</td><td>ACE Surgical Supply; Brockton, MA</td><td>950-6312</td><td>Testing renal sympathoinhibition</td></tr><tr><td>Single-hook elastic surgical stays</td><td>Harvard Apparatus; Holliston, MA</td><td>72-2595</td><td>Incision</td></tr><tr><td>Silk surgical tape</td><td>3M, Minneapolis, MN</td><td>1538-0</td><td>Secure surgical stays</td></tr><tr><td>Needles, 20 G</td><td>Sigma-Aldrich; St. Louis, MO</td><td>Z192554-100EA</td><td>Vessel cannulation</td></tr><tr><td>Dumont #7 curved forceps</td><td>Fine Science Tools; Foster City, CA</td><td>11274-20</td><td>Vessel cannulation</td></tr><tr><td>5-0 silk suture ties</td><td>Braintree Scientific; Braintree, MA</td><td>SUT-S 106</td><td>Vessel cannulation</td></tr><tr><td>Delicate hemostatic forceps</td><td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td><td>RS-7117</td><td>Vessel cannulation and RSNA surgery</td></tr><tr><td>Crile Hemostatic forceps</td><td>Fine Science Tools; Foster City, CA</td><td>13004-14</td><td>Needle bending</td></tr><tr><td>Telemetry transmitter</td><td>Data Sciences International; Minneapolis, MN</td><td>PA-10</td><td>Mean arterial pressure monitoring (telemetry)</td></tr><tr><td>Re-gel syringe</td><td>Data Sciences International; Minneapolis, MN</td><td>276-0038-001</td><td>Transmitter reuse (telemetry)</td></tr><tr><td>Disposable pressure transducer</td><td>Transpac; San Clemente, CA</td><td>MI-1224</td><td>Mean arterial pressure monitoring</td></tr><tr><td>Clear-Cuff pressure infuser</td><td>MILA International Inc.; Florence, KY</td><td>2281339</td><td>Mean arterial pressure monitoring</td></tr><tr><td>Vessel cannulation forceps</td><td>Fine Science Tools; Foster City, CA</td><td>00574-11</td><td>Catheter insertion</td></tr><tr><td>Black monofilament nylon 4-0 suture on reverse cutting needle</td><td>McKesson Medical-Surgical; San Francisco, CA</td><td>S661GX</td><td>Secure telemetry transmitter</td></tr><tr><td>Telemetry receiver</td><td>Data Sciences International; Minneapolis, MN</td><td>RPC-1</td><td>Mean arterial pressure monitoring (telemetry)</td></tr><tr><td>LabChart Pro (software), PowerLab (acquisition hardware)</td><td>AD Instruments; Colorado Springs, CO</td><td>ML846, MX2 matrix 2.0 (Compatible with Data Science International telemetry)</td><td>3 options for software/acquisition hardware</td></tr><tr><td>SciWorks (software), DataWave (acquisition hardware)</td><td>DataWave Technologies, Loveland, CO</td><td>N/A</td><td></td></tr><tr><td>Spike 2 (software), Micro1401-3</td><td>Cambridge Electronic Design Ltd., London UK</td><td>1401-3</td><td></td></tr><tr><td>Micro-drill</td><td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td><td>RS-6300</td><td>CNS surgery</td></tr><tr><td>Stereotaxic surgery frame</td><td>Stoelting; Wood Dale, IL</td><td>51600</td><td>CNS surgery</td></tr><tr><td>Microelectrode amplifier with 10X pre-amplifier</td><td>A-M Systems; Sequim, WA</td><td>1800-2</td><td>RSNA recording</td></tr><tr><td>Retractors</td><td>Fine Science Tools; Foster City, CA</td><td>17009-07</td><td>RSNA surgery</td></tr><tr><td>Micro-dissecting tweezers</td><td>Fine Science Tools; Foster City, CA</td><td>11251-10</td><td>RSNA surgery</td></tr><tr><td>Micro-hook</td><td>Fine Science Tools; Foster City, CA</td><td>10064-14</td><td>RSNA surgery</td></tr><tr><td>Mineral oil</td><td>Fisher Scientific; Waltham, MA</td><td>8042-47-5</td><td>RSNA surgery</td></tr><tr><td>Audio monitor</td><td>A-M Systems; Sequim, WA</td><td>3300</td><td>RSNA surgery</td></tr><tr><td>Silica gel</td><td>Wacker, Munchen; Germany</td><td>RT601A-B</td><td>RSNA surgery</td></tr><tr><td>Electrical clips</td><td>Tyco Electronics; Schaffhausen, Switzerland</td><td>EB0283-000</td><td>Grounding or securing perfusion needle</td></tr><tr><td>Bonn scissors, straight/sharp points</td><td>Roboz Surgical Instrument Co; Gaithersburg, MD</td><td>RS-5840</td><td>Perfusion</td></tr><tr><td>Gavage needle</td><td>Harvard Apparatus; Holliston, MA</td><td>75-0286</td><td>Perfusion</td></tr><tr><td>Masterflex perfusion pump</td><td>Cole-Parmer; Vernon Hills, IL</td><td>7524-10</td><td>Perfusion</td></tr><tr><td>Masterflex platinum-cured silicone tubing</td><td>Cole-Parmer; Vernon Hills, IL</td><td>96410-15</td><td>Perfusion</td></tr><tr><td>Formalin (10% buffered solution; 4 L)</td><td>Sigma-Aldrich; St. Louis, MO</td><td>HT501128</td><td>Perfusion</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich; St. Louis, MO</td><td>S0389</td><td>Cryoprotection</td></tr></tbody></table>

quantifying acute changes in renal sympathetic nerve activity in response to central nervous system manipulations in anesthetized rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Figure 1 illustrates a sample RSNA and BP recording from a Nembutal-anesthetized rat. An intravenous injection of phenylephrine was used to induce an increase in mean arterial pressure and to evoke the baroreflex and transient sympathoinhibition4,6. To quantify RSNA, the raw RSNA was rectified and averaged for non-overlapping 10 s segments; the noise estimate was subtracted from each segment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58205/58205fig1.jpg" /> 
Figure 1: RSNA and BP in response to phenylephrine injection IV. The raw RSNA (A) was full-wave rectified (B); rectified crushed &#34;zero&#34; RSNA is shown in inset C. Non-overlapping 10 s averages (minus noise) were calculated (D). To evoke the baroreflex, 0.1 mL of phenylephrine (1 &#181;g/mL) was injected intravenously (at arrow). The bolus infusion elicited an abrupt increase in BP and transient inhibition of RSNA. This figure was adapted from Fink AM, Dean C, Piano MR, Carley DW. The pedunculopontine tegmentum controls renal sympathetic nerve activity and cardiorespiratory activities in Nembutal-anesthetized rats. PLoS One. 2017;12(11):e01879564. <a href="https://www.jove.com/files/ftp_upload/58205/58205fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats at JoVE.com

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

quantifying-acute-changes-renal-sympathetic-nerve-activity-response

Universidad Científica del Sur

Research

JoVE Journal

Neuroscience

7.7K Views.  University of Illinois at Chicago. Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Video: Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for erectile dysfunction. However, the methods used vary between laboratories, and pitfalls still exist. The goal of this study was to describe a surgical technique that would provide a reliable and reproducible model. By exposing the ischiocavernosus muscle at its point of insertion on the ischial tuberosity, the penile crus could be cannulated with minimal dissection and injury to the structures involved in erectile function. Repeated stimulation of the CN, without the need for lifting and drying, was achieved by using a 125 &#181;m bipolar silver electrode and biocompatible silicon glue to isolate the electrode-nerve complex. This method prevents neuropraxia by reducing stretching and drying the nerve and provides complete isolation of the nerve, negating electrical leakage and preventing stimulation of alternative pathways.

This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement.

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the regulatory mechanism of RDN on hypertension remains elusive. Thus, it&#39;s essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&#38;E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model

Source: Wang, M., et al., <a href="https://app.jove.com/v/63719">Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion.</a> J. Vis. Exp. (2022)
This video demonstrates a procedure for renal artery sympathetic denervation in an angiotensin II-induced hypertensive mouse model, using phenol to disrupt sympathetic nerve function and reduce blood pressure.

Source: Wang, M., et al., Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion. J. Vis. Exp. (2022)
This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Recording Changes in Renal Sympathetic Nerve Activity in Response to Arterial Pressure in Rats

Source: Fink, A. M., et al. <a href="https://app.jove.com/v/58205">Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats.</a> J. Vis. Exp. (2018).
This video demonstrates a technique of studying the activity of renal sympathetic nerves (RSNs) in response to changes in arterial pressure in rats. Upon attaching a pressure-sensing catheter to the femoral artery, the renal sympathetic nerves are exposed. The nerves are then lifted onto a recording electrode, and their electrical activity is recorded. Next, a vasoconstricting drug is injected to elevate blood pressure, and corresponding changes in RSN activity are monitored.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Create ...