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

프로토콜

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

1. Animals

1. Divide 24 Sprague-Dawley rats (4-8 weeks old) into two groups. Produce complete Freund's adjuvant (CFA) model by intraplantar injection of 100 μL 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 ± 1 °C) with free access to food and water.

2. In Vivo Single-fiber Recording

1. 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'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 °C until use.
2. 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 °C.
3. Exposure of sciatic nerve trunk for recording
   1. 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's solution.
   2. 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.
   3. 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 °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.
4. Recording session
   1. Select a platinum filament (29 μm 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.
   2. 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 μm 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.
   3. 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 °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.
   4. 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.
   5. Collect data using data acquisition software (Table of Materials). Save data on a computer and analyze it later with professional software (Table of Materials).

자료

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