eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animals
<ol>
	<li>Divide 24 Sprague-Dawley rats (4-8 weeks old) into two groups. Produce complete Freund&#39;s adjuvant (CFA) model by intraplantar injection of 100 &#956;L of CFA in one group of 14 rats and another group of 10 rats by treatment with saline. 
	NOTE: All of the animals were acquired from the Animal Center of the Fourth Military Medical University. Adult male and female Sprague-Dawley rats (150-200 g) were used for all procedures, and rats were randomly assigned to cages. Two rats were housed per cage under a 12-/12-hour light/dark cycle at a constant temperature (25 &#177; 1 &#176;C) with free access to food and water.</li>
</ol>
2. In Vivo Single-fiber Recording
<ol>
	<li>Prepare and disinfect all surgical instruments (scalpel, tweezers, ophthalmic scissors, shearing scissors, glass separating needle, suture needle, bone rongeur) prior to surgery. Prepare 1 L or 2 L of normal Ringer&#39;s extracellular solution (in mM: NaCl 124, KCl 3, MgSO4 1.3, CaCl2 2, NaHCO3 26, NaH2PO4 1.25, glucose 15; pH 7.4 and 305 mOsm). Store at 4 &#176;C until use.</li>
	<li>Anesthetize rats. On days 3 to 7 after CFA injection, use intraperitoneal injection of a mixed solution (1% chloralose and 17% urethane, 5 mL/kg body weight) to keep the animals in a stable aesthetic condition during the experiment. Apply supplementary injection of anesthetics, if necessary, after checking pupils and the response to pain stimulation. Monitor and maintain a body temperature near 37 &#176;C.</li>
	<li>Exposure of sciatic nerve trunk for recording
	<ol>
		<li>Cut open the skin and muscle on the dorsal part of the thigh. Perform a blunt dissection along femoral biceps. Carefully isolate the sciatic nerve trunk using ophthalmic scissors and a glass separation needle. Keep the tissue wet using Ringer&#39;s solution.</li>
		<li>Fix the animal on a homemade metal hoop (3 cm long, 2 mm wide metal hoop with an iron wire 1 mm in diameter) via sewing the skin into the slot around it. Pull the skin up slightly so as to establish a fluid bath.</li>
		<li>Expose 1 cm of sciatic nerve trunk at the proximal side. Place a small brown platform under the nerve trunk to enhance the contrast and observe the fine nerve trunk clearly. Heat liquid paraffin in a water bath to 37 &#176;C and drop it on the top of the nerve trunk to prevent drying of the surface of the fiber. Remove the pia mater spinalis and dura mater around the sciatic nerve.</li>
	</ol>
	</li>
	<li>Recording session
	<ol>
		<li>Select a platinum filament (29 &#956;m in diameter) as the recording electrode. Heat over for easier molding and create a small hook at the very end. Attach the electrode to a micromanipulator to move the electrode as required.</li>
		<li>In the bath, place a reference electrode in adjacent subcutaneous tissue. Split the spinal dura and the pia mater. Separate the sciatic nerve into a single fiber (15-20 &#956;m in diameter) in the recording pool. Then, pick up a fine fascicle of axon and suspend the proximal end of the axon on the hook of the recording electrode under a stereoscope at 25x magnification. 
		NOTE: The just-dissected filaments tend to be thicker and require further separation until a single unit may be recorded.</li>
		<li>Identify the receptive field of a single nociceptive C-fiber using a mechanical stimulus (Von Frey hairs) and thermal stimulus (small cotton ball with 50-55 &#176;C water). Briefly, if the firing of nerve fiber responds to the mechanical stimuli and hot water, then consider it as a polymodal nociceptive C-fiber. Next, insert two needle stimulus electrodes (2 mm interval) into the skin of the identified field for the delivery of electrical stimuli.</li>
		<li>Display the waveform of an action potential on oscilloscope and employ a computer analog-to-digital (A/D) board with a signal sampling rate of 20 kHz to amplify and record the spikes.</li>
		<li>Collect data using data acquisition software (Table of Materials). Save data on a computer and analyze it later with professional software (Table of Materials).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Instruments and software used in single fiber recording</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Nihon kohden</td>
			<td>MEZ-8201</td>
			<td>Amplification of the electrophysiological signals</td>
		</tr>
		<tr>
			<td>Bioelectric amplifier monitor</td>
			<td>ShangHai JiaLong Teaching instrument factory</td>
			<td>SZF-1</td>
			<td>Monitor firing process via sound which is transformed from physiological discharge signal</td>
		</tr>
		<tr>
			<td>Data acquisition and analysis system</td>
			<td>CED</td>
			<td>Spike-2</td>
			<td>Software for data acquisition and analysis</td>
		</tr>
		<tr>
			<td>Electrode manipulator</td>
			<td>Narishige</td>
			<td>SM-21</td>
			<td>Contro the movement of the electrode as required</td>
		</tr>
		<tr>
			<td>Hairspring tweezers</td>
			<td>A.Dumont</td>
			<td>5#</td>
			<td>Separate the single fiber</td>
		</tr>
		<tr>
			<td>Isolator</td>
			<td>Nihon kohden</td>
			<td>SS-220J</td>
			<td></td>
		</tr>
		<tr>
			<td>Memory oscilloscope</td>
			<td>Nihon kohden</td>
			<td>VC-9</td>
			<td>Display recorded discharge during</td>
		</tr>
		<tr>
			<td>experiment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>ZEISS</td>
			<td>SV-11</td>
			<td>Have clear observation when separate the local tissue and single fiber</td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Nihon kohden</td>
			<td>SEZ-7203</td>
			<td>Delivery of the electrical stimuli</td>
		</tr>
		<tr>
			<td>Von Frey Hair</td>
			<td>Stoelting accompany</td>
			<td></td>
			<td>Delivery of the mechanical stimuli</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Scientz biotechnology Co., Ltd.</td>
			<td>SC-15</td>
			<td>Heating paroline to maintain at 37oC</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>Origin lab</td>
			<td>Origin 8</td>
			<td>Software for drawing picture</td>
		</tr>
		<tr>
			<td>Other instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Sartorius</td>
			<td>BS 124S</td>
			<td>Weighing reagent</td>
		</tr>
		<tr>
			<td>pH Modulator</td>
			<td>Denver Instrument</td>
			<td>UB7</td>
			<td>Adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>Solutions/perfusion/chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Chloralose</td>
			<td>Shanghai Meryer Chemical Technology Co., Ltd.</td>
			<td>M07752</td>
			<td>Mixed solution for Anesthesia</td>
		</tr>
		<tr>
			<td>D (+) Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Liquid Paraffin</td>
			<td>TianJin HongYan Reagent Co., Ltd.</td>
			<td></td>
			<td>Maintain fiber wetting</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich</td>
			<td>62H0351</td>
			<td>Enzyme used for clearing the surface of DRG</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5671</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S0751</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>Extracellular solution</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td>Mixed solution for Anesthesia</td>
		</tr>
	</tbody>
</table>

in vivo single-fiber recording of sciatic nerve c-fibers in rats

Source: Mao, H., et al. <a href="https://app.jove.com/v/59234">Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure.</a> J. Vis. Exp. (2019).
This video demonstrates a single-fiber recording of C-fiber nerve conduction in rats. An anesthetized rat is incised to expose the sciatic nerve, which provides sensory and motor supply to the lower limbs. The individual C-fibers are then exposed. Electrical stimulation is provided using stimulus electrodes implanted in the foot, and a response is recorded from a single nerve fiber.

In Vivo Single-Fiber Recording of Sciatic Nerve C-Fibers in Rats

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

Take an anesthetized rat and make an incision on its thigh to expose the sciatic nerve, a peripheral nerve providing sensory and motor supply to the lower limbs.
Apply a physiological solution to keep the exposed tissue moistened.
Suture the incised skin to a metal hoop, creating a chamber for electrophysiological measurements.
Place a platform under the nerve to enhance the visibility of the nerve. Apply liquid paraffin to prevent the nerve from drying.
Place a reference electrode subcutaneously to establish a baseline electrical activity.
Remove the membranous nerve covering to expose the C-fibers. Suspend a single fiber over a recording electrode.
Apply mechanical and thermal stimuli to the foot and observe the fiber&#39;s response to both, confirming it as a polymodal C-fiber.
Implant stimulus electrodes on the foot for targeted single-fiber stimulation.
The stimulation generates action potentials that travel along the fiber and are monitored by the recording electrode.

in-vivo-single-fiber-recording-of-sciatic-nerve-c-fibers-in-rats

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

127 ビュー. 出典:Mao, H., et al. In vivo 単繊維記録と坐骨神経が付着した無傷の後根神経節を使用して、伝導不全のメカニズムを調べます。J. Vis. Exp.(2019年)。 このビデオは、ラットのC線維神経伝導の単線維記録を示しています。麻酔をかけたラットを切開して坐骨神経を露出させ、下肢に感覚と運動の供給を提供します。その後、個々のCファイバーが露出します。足に埋め込まれた刺激...

ビデオ: ラットの坐骨神経C線維のin vivo単線維記録 - 概念

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

糖尿病モデルラットにおける神経伝導の超音波ツール ブロックします。

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

JoVE Journal

生物工学

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

超音波検査と神経伝導検査を使用して手根管症候群における軸索変性のスクリーニング

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

神経科学

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Ex Vivo神経生理学のためのラット坐骨神経の調製

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...