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.
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 Buffer (mKHB) at 1x concentration.
2. Pre-dissection preparations
NOTE: This step starts the experiment. The below steps must be carried out on the same day, in this order.
3. Animal anesthesia and euthanization
NOTE: Female rats between 250 and 330 g (Table of Materials) were used for the studies.
4. Dissection protocol
NOTE: Place the animal with its belly down on the dissection table. Repeat the following steps for both legs. Typically, the right leg is dissected first.
5. Nerve cleaning procedure
6. Equipment setup
NOTE: The equipment setup used to carry out experiments is illustrated in Figure 1. Briefly, it consists of a dual-compartment nerve bath, a 2 L bottle placed on a heating stirrer, a source of carbogen for buffer aeration, and tubing to allow the buffer to flow from the bottle to the bath, and back to the bottle using a peristaltic pump. The bath can be machined out of plexiglass or 3D-printed from watertight materials. It has a depth of approximately 2 cm, and the partition separating the two chambers of the bath features a 1.5 mm diameter hole to allow the threading of a peripheral nerve across both chambers. One chamber is large, must be at least 4 or 5 cm long, and will be filled with buffer. The other chamber should be at least 3 cm long and will be filled with silicone or mineral oil. The bath must not be made too large as this will degrade control of perfusion, temperature, and pH. Different bath sizes may be required depending on the size of the nerve tissue being studied.
7. Electrode implantation on the nerve in the bath
8. Stimulation and recording
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 in order to obtain the same CAP amplitude compared to commercially available platinum cuff electrodes. This difference is generally small compared to the variation in stimulation amplitude required to stimulate nerves coming from different animals. This is because small differences in nerve size and cuff fit have a large effect on the required stimulation amplitude to obtain a specific CAP amplitude, regardless of the cuff material. This can be used to test the effects of different buffer compositions, such as different ion concentrations or the addition of nerve excitatory or inhibitory substances such as tetrodotoxin. If the buffer waste pipe is routed to an extra container, the addition of nerve excitability-altering substances can be made temporary for the experiment, with wash-out rates dependent on the rate of buffer inflow.
The minimum current density, calculated as the stimulation amplitude divided by the surface area of the stimulation electrodes, required to activate the A-type fibers, and obtain an observable compound action potential of the oscilloscope was plotted versus pulse width in Figure 3. The results shown in Figure 3 represent typical nerve excitability for both commercially available standard platinum nerve cuffs and custom-made conductive elastomer nerve cuffs.
The extracted nerves should remain viable for approximately 6 h after extraction and, therefore, experiments must fit within this time window. Loss of nerve viability leads to a progressive decline in CAP amplitude and conduction speed. After action potential amplitude declines below 50% of initial amplitude (at the start of recording), the nerve should be considered no longer viable as results will be significantly skewed. Representative results with respect to nerve longevity are shown in Figure 4. The right and left sciatic nerves were extracted from one animal between 10:00 AM and 11:00 AM on a given day. Initial CAPs were obtained from the right sciatic nerve during initial tests before experiments, and standard CAPs were obtained from both left and right sciatic nerves, which had been kept alive using this protocol, at the end of the experiments. Minimal CAP amplitude reduction was observed with the right sciatic nerve, while the left sciatic nerve CAP amplitude at approximately 3 mV was similar to that of the right sciatic nerve at the start of the experiment more than 6 h after nerve extraction.
Figure 1: Schematic representation of the experimental setup used in the protocol. This figure has been modified from Rapeaux, A. et al. (2020)20. Please click here to view a larger version of this figure.
Figure 2: Representative CAPs obtained ex vivo following stimulation by metallic and conductive elastomer nerve cuff arrays. Reproduced with modifications from Cuttaz, E. A. et al. (2021)22. Please click here to view a larger version of this figure.
Figure 3: A-fibre activation threshold of metallic and conductive elastomer cuff arrays. Error bars represent ±1 standard deviation from the mean. Reproduced with modifications from Cuttaz, E. A. et al. (2021)22. Please click here to view a larger version of this figure.
Figure 4: Representative CAPs obtained ex vivo over a day of experiments and using both sciatic nerves from one animal. (A) A-type fiber CAP obtained near midday from the right sciatic nerve. (B) A-type fiber CAP obtained mid-afternoon from the left sciatic nerve. (C) A-type fiber CAP obtained at the end of experiments with the same left sciatic nerve in (B). The x-axis corresponds to the time of day at which the recordings were taken. Please click here to view a larger version of this figure.
Supplementary File. Please click here to download this File.
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 described in the past7,12, using similar buffers and for the same tissue type. To the authors’ knowledge, however, this is the first time a description of the buffer preparation, dissection, equipment set up, and subsequent recording has been given in the same document.
This protocol can enable a wide variety of experiments in neurophysiology that would not be possible in either in vitro or in vivo contexts. For example, an advantage of ex vivo preparations is that they preserve the macro and micro-structure of the extracted tissue while isolating this tissue from the rest of the body. This results in a simpler setup as anesthesia does not have to be maintained, which is otherwise a requirement in in vivo experiments. In terms of enabling experiments, ex vivo preparations allow the use of substances such as tetrodotoxin, which are difficult to justify in an in vivo context23 as they carry a high risk for the animal. When the use of such substances benefit investigation, they are easier to use in ex vivo preparations. The use of the custom stimulator20 enables experiments using HFAC block for neuromodulation using this experimental setup.
The most critical step in the protocol is the dissection step, because even a small mistake using the dissection scissors can damage the nerve if enough care is not taken. The speed at this stage is also essential as the tissue must be rapidly extracted from the body and placed in a chilled buffer to maximize viability at the start of the recording. After tissue extraction, while care should be taken when cleaning the nerve and implanting any electrodes, the protocol is more flexible with respect to time, and the risk of operator error is, therefore, lower. As nerve diameters and placement of fascicles within the nerves will vary from animal to animal, some variability in results should be expected even if using the same electrodes and stimulation protocol. The effect of this variability can be seen in the error bars for stimulation thresholds in Figure 3. It is important not to pinch the nerve at any stage of the preparation as this can cause irreversible damage to the tissue. Handle the nerve only by its ends using forceps and with great care not to pull the nerve taut.
Several aspects of the protocol in its current form can be improved by increasing the amount of equipment and setup time. To help with the diagnosis of potential issues with this experimental setup, automated measurements of pH and dissolved oxygen in the bath could be useful but have not been implemented here. Both measurements can be achieved using amperometric or potentiometric methods12,19. Equipment that will require regular maintenance is the tubing and glassware, which accumulates salt deposits over time. The AgCl recording hooks will also require regular re-coating or replacement, along with the AgCl reference. Stimulation electrodes should be cleaned after each use but will generally not require replacement for many experiments.
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 |