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 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.
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 interpretation4,5. 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 block2,3. 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, pH13,14, and access to glucose to maintain metabolism. For this protocol, the approach is to use a modified Krebs-Henseleit buffer15,16 (mKHB) continuously aerated with a mixture of oxygen and carbon dioxide. The mKHB is in the family of cardioplegic buffers6,17 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) block2,20, 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.
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…
The authors have nothing to disclose.
The authors acknowledge Dr. Gerald Hunsberger of GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA, and Galvani Bioelectronics (Stevenage, UK) for sharing their original nerve preparation technique with us. The authors acknowledge Robert Toth for the Dual-Chamber nerve bath design. The authors acknowledge funding from the Healthcare Technologies Challenge Awards (HTCA) grant of the Engineering and Physical Sciences Research Council (EPSRC). The authors acknowledge the High Performance Embedded and Distributed Systems Centre for Doctoral Training (HiPEDS CDT) of Imperial College London for funding Adrien Rapeaux (EP/L016796/1 ). Adrien Rapeaux is currently funded by the UK Dementia Research Institute, Care Research and Technology Centre. The authors gratefully acknowledge Zack Bailey of Imperial College, in the Department of Bioengineering, for help with experiments and access to animal tissues during the production of the JoVE video article.
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 |