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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: February 14th, 2018

DOI:

10.3791/54120

1Department of Pediatrics, University of Alberta, 2Department of Physiology & Biophysics, University of Mississippi Medical Center, 3Mississippi Center for Obesity Research

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 (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±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.

Interest in using mice in several areas of biomedical research continues to expand with the development of countless genetically engineered models. For the most part, technical advances have kept pace with the increased use of mice in physiology and there is now an impressive selection of miniaturized devices developed specifically for measuring important physiological parameters in mice. Although telemetric devices for direct measurement of autonomic nervous tone in the conscious rat have been available for over a decade, miniaturized devices for assessing nerve activity in conscious mice are currently not available. Investigators typically circumvent this limitation....

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All of the experimental procedures are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

1. Animals and Housing

  1. House mice (24 - 35 g) upon arrival in the institutional laboratory animal facility.
  2. Offer mice standard rodent chow and tap water ad libitum at all stages of the experimental protocol in a temperature and humidity .......

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Following the described protocol, survival rate was 100% - all mice instrumented in this study survived and recovered well following the surgical procedure. Within 24 hours of surgical preparation, all mice behaved normally, exhibiting typical eating, grooming and exploratory behaviors. No animals showed any sign of pain or distress at this time. 48 hours following surgery, a verifiable and clear RSNA signal was recorded in 10 out of the 12 mice. This signal was maintained in these mice 7.......

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Herein we have outlined, demonstrated and validated a novel method for targeted evaluation of RSNA in conscious mice, free to move and rest comfortably in their home cages. Following surgical implantation of an arterial pressure radiotelemeter, an indwelling intravenous infusion catheter and a custom-designed bipolar RSNA electrode, mice recovered from surgery and were left undisturbed for 48 to 72 hours. Mice remained comfortably settled in their home cage at all times (including experimental periods) with unrestricted .......

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S.M.H. was supported by postdoctoral fellowships from the Canadian Institutes for Health Research (CIHR), Heart & Stroke Foundation of Canada (HSFC) and Alberta Innovates Health Solutions (AiHS); J.E.H. is supported by a grant from the National Heart, Lung and Blood Institute PO1HL-51971.

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Name Company Catalog Number Comments
Teflon-coated stainless steel multiple stranded wire A-M Systems 793200 0.001in diameter bare; 0.0055in diameter coated
#11 Scalpel Blade Fisher Scientific ALMM9011
Soldering Iron and solder Any make or model suitable
Male miniature pin connectors A-M Systems 520200 Brass with gold plating
Female miniature pin connectors A-M Systems 520100 Brass with gold plating
Heat Shrink tubing Radio Shack Model #: 278-1610 | Catalog #: 2781610 1.6 mm diameter
Polyethylene 90 (PE90) tubing VWR CA-63018-703 0.86mm inner diameter; 1.27mm outer diameter
Dissecting microscope Leica Microsystems Leica M80 Any make or model also suitable
Polyethylene 10 (PE10) tubing Braintree Scientific PE10 50 FT 0.28mm inner diameter; 0.61mm outer diameter
Super Glue Liquid Loctite n/a Liquid Formula; any brand suitable
Super Glue Gel Loctite n/a Gel Formula; any brand suitable
Polyethylene tubing Scientific Commodities BB31695-PE/13 For pedestal 2.7mm inner diameter; 4.0mm outer diameter
Hospital Sterilization Services & Ozone Sterilization packets Contact local hospital sterilization services
Isoflurane anesthesia Abbott 05260-05
Deltaphase isothermal heat pads & surgical table Braintree Scientific 39OP Keep heat pads warm in a 37°C water bath; Corresponding surgical table essential
Glycopyrrolate Amdipharm Mercury Company Limited n/a
Isoflurane vaporizer system & flow gauge Braintree Scientific VP I Include medical grade oxygen supply
Tissue scissors Fine Science Tools 14173-12
Fine spring scissors Fine Science Tools 15006-09
Small cotton-tipped applicators Fisher Scientific 23400100
Fine Straight Forceps Fine Science Tools 11254-20 #5, FST by Dumont Biologie Tip
Angled Forceps Fine Science Tools 11251-35 #5/45 FST by Dumont
Small Absorbent Spears Fine Science Tools 18105-03
Parafilm Sigma Aldrich BR701605 ALDRICH
Kwik-Sil 2 component Silicone Polymer World Precision Instruments (WPI) KWIK-SIL Purchase extra specialized tips from WPI
5-0 Polysorb Suture Tyco Healthcare n/a
6-0 Silk Suture Braintree Scientific SUT-S 104 Deknatel brand, spool
Radiotelemetry Probe Data Sciences International (DSI) TA11-PAC10
Radiotelemetry Receiver Data Sciences International (DSI) PhysioTel RPC-1
Ambient Pressure Reference Data Sciences International (DSI) Apr-01
Pressure Output Adapter Data Sciences International (DSI) R11CPA
Rena Pulse Tubing Braintree Scientific RPT-040
Infusion Swivel Instech Solomon 375/D/22
Swivel Support Arm & Mount Instech Solomon SMCLA
Polysulfone button  Instech Solomon LW62S/6
Stainless steel spring Instech Solomon PS62
Vetbond surgical adhesive 3M n/a
Triple Antibiotic Ointment Fougera n/a
PowerLab 8 Channel Data Acquisition System & Software ADInstruments PowerLab 8/35
PVC Insulated Cable Belden PVC Audio Connection Cable 32 AWG
Preamplification Headstage Dagan Corporation Model 4002
Differential Amplifier Dagan Corporation EX4-400
Sodium Nitroprusside Sigma Aldrich 71778-25G
Phenylephrine Sigma Aldrich P6126-5G
Sterile Physiological Saline 0.9% NaCl Beckton Dickinson Contact local hospital supplier
hexamethonium Sigma Aldrich H0879-5G
Stainless Steel top anti vibration table n/a n/a Custom designed in-house; Solid steel plate on a benchtop is also suitable
Faraday cage n/a n/a Custom designed and constructed in-house
Small animal hair trimmer n/a n/a Drugstore, men's beard trimmer suitable
Dipilatory Cream n/a n/a Veet brand, sensitive skin formula
10% Povidone Iodine Purdue Products Betadiene
70% Ethanol n/a n/a
Steel microretractors n/a n/a Made in-house. Bend a steel paper clip & loop 4-0 silk to form a retractor
Hemostats Fine Science Tools 13011-12
Heat Gun Fisher Scientific 09-201-27

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