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Protocol

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Materials

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Neuroscience

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

Published: September 11th, 2018

DOI:

10.3791/58205

1Department of Biobehavioral Health Science, College of Nursing, University of Illinois at Chicago, 2Department of Anesthesiology, Medical College of Wisconsin and Zablocki VA Medical Center

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 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.

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 importa....

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All methods described were approved by the Institutional Animal Care Committee at the University of Illinois at Chicago.

1. Create Bipolar RSNA Electrodes

  1. 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.
  2. Remove the insulation from the ends of the wires; trim the wires leav.......

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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.

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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 troublesh.......

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

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

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