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

Summary

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).

Abstract

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.

Introduction

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 systems^2,3. To define the neurobiological pathways leading to autonomic dysfunction, studies in rodents are importa....

Protocol

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 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 connecto....

Results

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 sympathoinhibition^4,6. To quantify RSNA, the raw RSNA was rectified and averaged for non-overlapping 10 s segments; the noise estimate was subtracted from each segment.

Discussion

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.......

Materials

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