In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Summary

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Abstract

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques "by threshold tracking" expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.

The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.

Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

Introduction

The use of electrophysiological techniques is an essential tool for the in vivo investigation of peripheral nerve function in neurological disorders. Conventional nerve conduction methods utilize supramaximal stimuli to record motor action potential amplitude and latency. These techniques therefore provide useful information on the number of conducting fibers and on the conduction velocity of the fastest fibers. A valuable complementary tool is that of axonal excitability testing. This technique uses sophisticated electrophysiological stimulation patterns to indirectly assess the biophysical properties of peripheral nerves, such as the activity of ion channel....

Protocol

All experimental procedures described here complied with the Animal Care and Ethics Committee of UNSW Sydney and were performed in accordance with the National Health and Medical Research Council (NHMRC) of Australia regulations for animal experimentation.

1. Experimental Set Up

NOTE: 12 week old, female Long-Evans rats were used in this procedure.

1. Anesthetize the rat in an induction chamber using 4% isoflurane and 1 L per min O₂ flow rate. Confirm sufficient anesthesia by testing for the righting reflex and ensure its absence before removing the animal from the induction chamber. Note that....

Results

Electrophysiological measures of the rat ulnar nerve were obtained with the present protocol. Figure 3 demonstrates a representative recording from the left ulnar nerve of a 12 week-old female Long Evans rat. Compound muscle action potential relates to the number of conducting fibers that are simultaneously activated. The supramaximal peak response (mV) (Figure 3A) demonstrates the peak response achieved when incrementally increa.......

Discussion

The described procedure demonstrates a simple and reliable, minimally-invasive technique that allows assessment of the biophysical properties and membrane potential of the axon in a short period of time. Compared with other more invasive techniques, which require the exposure of the nerve, the present method of axonal excitability testing induces minimal tissue damage thus enabling in vivo assessment that preserves the physiological conditions of the nerve of interest and allows for repeated measurements.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
QTracS Program	Digitimer Ltd.		Axonal excitability program
AM-Systems 2200, Analog Stimulus Isolator, 2200V/50Hz	SDR Scientific	850005	Stimulator
High Performance AC Amplifier Model LP511	Grass Technologies		Amplifier
Humbug 50/60Hz Noise eliminator	Quest Scientific Instruments	726310	Noise eliminator
Low Impedance Platinum Monopolar Subdermal Needle Electrodes	Grass Technologies	F-E2-24	Recording electrodes, 10 mm length, 30 gauge
Low Impedance Platinum Electroencephalography Needle Electrodes	Cephalon	9013L0702	Stimulating electrodes, 10 mm length, 30 gauge
Multifunction I/O Device Model USB-6341	National Instruments		Multifunction input/output device
Iron Base Plate IP	Narishige Scientific Instrument Laboratory		Used for holding stimulating needle electrode in place
Rotating X-block X-4	Narishige Scientific Instrument Laboratory		Used for holding stimulating needle electrode in place
Magnetic Stand GJ-8	Narishige Scientific Instrument Laboratory		Used for holding stimulating needle electrode in place
Micromanipulator M-3333	Narishige Scientific Instrument Laboratory		Used for holding stimulating needle electrode in place