Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Summary

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.

Abstract

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.

Introduction

The current understanding of fundamental nerve function as modeled in silico is lacking in several aspects, notably with respect to the effects of nerve tissue compartmentalization outside of the soma, axon, and dendrites. Axon-myelin interactions are still poorly understood as evidenced by the fact that even detailed computational nerve models such as MRG¹ (for mammalian nerves) that adequately capture conventional electrical stimulation response, do not capture other experimentally observed behaviors such as high-frequency block carryover² or secondary onset response³.

This protocol provides a method to efficiently investigate neurophysiological processes at the nerve level in an acute small laboratory animal model, using a standardized preparation protocol to isolate the nerve, control its environment, and remove it from an in vivo context to an ex vivo context. This will prevent other body processes or anesthetics used by in vivo nerve stimulation protocols to alter nerve behavior and confound measured results or their interpretation^4,5. This enables the development of more realistic models focusing solely on effects specific to nerve tissues that are poorly understood. This protocol is also useful as a testbed for new nerve stimulation and recording electrode materials and geometries, as well as new stimulation paradigms such as high-frequency block^2,3. Variations of this technique have been used previously to study nerve physiology in tightly controlled conditions⁶, for example, to measure ion channel dynamics and properties or the effects of local anesthetics⁷.

This technique provides several advantages compared to alternatives such as acute in vivo small animal experimentation⁸. The technique obviates the need to maintain anesthesia depth as the tissue has been extracted from the body, reducing the amount of required equipment such as an anesthetic diffuser, oxygen concentrator, and heating pad. This simplifies the experimental protocol, reducing the risk of mistakes. As anesthetics can potentially alter nerve function⁴, this technique ensures that measures will not be confounded by side effects from these anesthetic compounds. Finally, this technique is more appropriate than acute in vivo experiments when studying the effects of neurotoxic compounds such as tetrodotoxin, which would kill an anesthetized animal by paralysis.

Peripheral nerve sections are a unique ex vivo system since there is a high chance that the fibers responsible for recorded neural signals do not contain any soma. As these would normally be located, for motor neurons, in the spine, and for sensory neurons in the dorsal root ganglia next to the spine, the preparation of a section of the mammalian nerve can be roughly modeled as a collection of tubular membranes with ion channels, open at both ends⁹. Metabolism is maintained by the mitochondria located in the axon at the time of tissue dissection¹⁰. Suturing of the open ends of the axolemma is encouraged after extraction to close them and thereby help maintain existing ionic gradients across the membrane, which are essential for normal nerve function.

To maintain tissue homeostasis outside the body, several environmental variables must be tightly controlled. These are temperature¹¹, oxygenation¹², osmolarity, pH^13,14, and access to glucose to maintain metabolism. For this protocol, the approach is to use a modified Krebs-Henseleit buffer^15,16 (mKHB) continuously aerated with a mixture of oxygen and carbon dioxide. The mKHB is in the family of cardioplegic buffers^6,17 used to preserve dissected tissues outside of the body, for example, in ex vivo experiments. These buffers do not contain any hemoglobin, antibiotics, or antifungals and are, therefore, only suitable for preparations involving small amounts of tissue for a limited time. pH control was achieved with the carbonate and carbon dioxide redox pair, requiring constant aeration of the buffer with carbon dioxide to maintain pH equilibrium. This is to avoid using other common buffering agents such as HEPES, which can modify nerve cell function¹⁸. To oxygenate the buffer and provide pH control, a mixture of 5% carbon dioxide in oxygen called carbogen (95% O₂, 5% CO₂) was used. A heating stirrer was used for temperature control of a buffer container, and the buffer was perfused through a nerve bath, and then recirculated to the starting container. A typical experiment would last 6-8 h before the nerve loses its viability and no longer responds sufficiently to stimulation for measures to be representative of healthy tissue.

To optimize the signal-to-noise ratio, silver-chloride electrodes were used for recording, which were prepared according to previously described methods¹⁹. For stimulation, a combination of commercial off-the-shelf platinum cuff electrodes and custom-made conductive polymer cuff electrodes can be used. Conductive polymer cuff electrodes have notably higher charge capacities, which are useful when stimulating the nerve using high amplitude waveforms²⁰.

The stimulator used in this protocol has been previously described²⁰. Documentation, design files, and software scripts to use it are publicly available²¹. Other stimulators can be used to execute this protocol; however, the custom stimulator is also capable of high-frequency alternative current (HFAC) block^2,20, which enables a wider range of neurophysiology experiments. To use HFAC block, conductive elastomer cuffs are recommended to avoid damage to the nerve. Conductive elastomer nerve cuffs are soft and fully polymeric electrode arrays produced from conductive elastomers as the conductive component and polydimethylsiloxane as the insulation²². Devices were manufactured in a bipolar configuration using conventional laser microfabrication techniques.

Protocol

All animal care and procedures were performed under appropriate licenses issued by the UK Home office under the Animals (Scientific Procedures) Act (1986) and were approved by the Animal Welfare and Ethical Review Board of Imperial College London. 1. Preparation of buffers NOTE: This part of the protocol can be carried out well in advance of the rest of the protocol, except for the final steps involving the preparation of modified Krebs-Henseleit Buff…

Representative Results

Representative results that can be obtained with this protocol are the consistent compound action potentials from A-type nerve fibers within the sciatic nerve. These action potentials typically have a peak-to-peak amplitude of approximately 1 mV at the electrode and therefore 100 mV once amplified (Figure 2). Similar stimulation amplitudes and pulse widths should yield similar CAP amplitudes. Conductive elastomer cuff electrodes will generally require slightly higher stimulation amplitudes i…

Discussion

In this work, we described a protocol to prepare rat sciatic nerves for ex vivo neurophysiology. Tissue extraction takes approximately 30 min, including animal handling, anesthesia, culling, and dissection, while nerve cleaning, placement in the bath, and electrode implantation should require an additional 30 min before recording can be started. Buffer preparation can be carried out in 30 min, though this can be done ahead of the rest of the experiment. This type of preparation and experiment has been used and d…

Materials

1 L Glass bottle	VWR International Ltd	215-1595	Borosilicate glass
1 L Glass graduated flask	VWR International Ltd	612-3626	Borosilicate glass
2 L Glass bottle	VWR International Ltd	215-1596	Borosilicate glass
2 L Glass graduated flask	VWR International Ltd	BRND937254	Borosilicate glass
Adaptor, pneumatic, 8 mm to 1/4 NPT	RS UK	536-2599	push-to-fit straight adaptor between oxygen hose and gas dispersion tube
Alkoxy conformal coating	Farnell	1971829	ACC15 Alkoxy conformal coating for dissection petri dish preparation
Anesthetic	Chanelle	N/A	Isoflurane inhalation anesthetic, 250 mL bottle
Beaker, 2 L	VWR International Ltd	213-0469	Borosilicate glass
Bipolar nerve cuff	Cortec GMBH	N/A	800 micron inner diameter, perpendicular lead out, no connector termination
Bossheads	N/A	N/A	Standard wet laboratory bossheads for attaching grippers to rods
Calcium Chloride dihydrate	Sigma Aldrich	C7902-500g	500 g in plastic bottle
Carbogen canister	BOC	N/A	F-size canister
Centrifuge Tubes, 15 mL volume	VWR International Ltd	734-0451	Falcon tubes
Conductive elastomer nerve cuff	N/A	N/A	high charge capacity nerve cuff for stimulation, see protocol for fabrication reference
Connector, Termimate	Mouser UK	538-505073-1100-LP	These should be soldered to wire terminated with crocodile clips (see entry 11)
Crocodile clip connectors	RS UK	212-1203	These should be soldered to wire terminated with TermiMate connectors (see entry 10)
Deionized Water	N/A	N/A	Obtained from deionized water dispenser
Forceps angled 45 degrees	InterFocus Ltd	91110-10	Fine forceps, student range
Forceps standard Dumont #7	InterFocus Ltd	91197-00	Student range forceps
Gas Disperson Tube, Porosity 3	Merck	12547866	N/A
Glucose anhydrous, powder	VWR International Ltd	101174Y	500 g in plastic bottle
Grippers	N/A	N/A	Standard wet laboratory rod-mounted grippers
Heating Stirrer	RS UK	768-9672	Stuart US152
Hemostats	N/A	N/A	Any hemostat >12 cm in length is suitable
Insect Pins, stainless steel, size 2	InterFocus Ltd	26001-45	N/A
Laptop computer	N/A	N/A	Any laboratory-safe portable computer with at least 2 unused USB ports is suitable
Line Noise Filter	Digitimer	N/A	Humbug noise eliminator (50 Hz line noise filter)
Low-Noise Preamplifier, SR560	Stanford Research Systems	SR560	Low-noise voltage preamplifier
Magnesium Sulphate salt	VWR International Ltd	291184P	500g in plastic bottle
MATLAB scripts	Github	https://github.com/Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch	Initialization, calibration and stimulation scripts for the custom stimulator
MATLAB software	Mathworks	N/A	Standard package
Microscope Light, PL-2000	Photonic	N/A	Light source with swan necks. Product may be obtained from third party supplier
Microscope, SMZ 745	Nikon	SM745	Stereoscopic Microscope
Mineral oil, non-toxic	VWR International Ltd	31911.A1	Oil for nerve bath
Nerve Bath	N/A	N/A	Plexiglas machined nerve bath, see protocol for details.
Oscilloscope	LeCroy	N/A	434 Wavesurfer. Product may be obtained from 3rd party suppliers
Oxygen Hose, 1 meter	BOC	N/A	1/4" NPT terminations
Oxygen Regulator	BOC	C106X/2B:3.5BAR-BS3-1/4"NPTF 230Bar	N/A
Peristaltic Pump P-1	Pharmacia Biotech	N/A	Product may be obtained from third party supplier
Petri Dish, Glass	VWR International Ltd	391-0580	N/A
Potassium Chloride salt	Sigma Aldrich	P5405-250g	250 g in plastic bottle
Potassium Dihydrogen Sulphate salt	Merck	1.04873.0250	250 g in plastic bottle
Rat	Charles River Laboratories	N/A	Sprague Dawley, 250-330 grams, female
Reference electrode, ET072	eDaQ (Australia)	ET072-1	Silver silver-chloride reference electrode
Rod	N/A	N/A	Standard wet laboratory rods with fittings for stands
Scale	Sartorius	N/A	M-Power scale, for weighing powders. Product may be obtained from third-party suppliers
Scissors straight 12 cm edge	InterFocus Ltd	91400-12	blunt-blunt termination, student range
Signal Acquisition Device	Cambridge Electronic Design	Micro3-1401	Micro3-1401 Multichannel ADC
Silicone grease, non-toxic	Farnell	3821559	for sealing of bath partition
Silicone tubing, 2 mm inner diameter	N/A	N/A	N/A
Silicone tubing, 5 mm inner diameter	N/A	N/A	N/A
Silver wire	Alfa Aesar	41390	0.5 mm, annealed
Sodium Bicarbonate salt	Sigma Aldrich	S5761-500g	500 g in plastic bottle
Sodium Chloride salt	VWR International Ltd	27810.295	1 kg in plastic bottle
Spring scissors angled 2 mm edge	InterFocus Ltd	15010-09	N/A
Stand	N/A	N/A	Standard wet laboratory stands with sockets for rods
Stimulator	Digitimer	DS3	DS3 or Custom Stimulator (see references)
Stirring flea	VWR International Ltd	442-0270	For use with the heating stirrer
Syringe tip, blunt, 1 mm diameter	N/A	N/A	N/A
Syringe tip, blunt, 2 mm diameter	N/A	N/A	N/A
Syringe, plastic, 10 mL volume	N/A	N/A	syringe should have luer lock fitting
Tape, water-resistant	N/A	N/A	For securing tubing and wiring to workbench
Thermometer	VWR International Ltd	620-0806	glass thermometer
USB Power Bank	RS UK	135-1000	Custom Stimulator power supply, fully charge before experiment. Not needed if using DS3
Valve, Leuer Lock, 3-Way	VWR International Ltd	229-7440	For attaching syringe to bath feed tube and priming siphon