This work was sponsored by the Defense Advanced Research Projects Agency (DARPA) MTO under the auspices of Dr. Jack Judy and Dr. Doug Weber through the Space and Naval Warfare Systems Center, Pacific Grant/Contract No.N66001-12-C-4173. We would like to thank Thomas Eggers for his help in the fabrication process, and Ronald Triolo, Matthew Schiefer, Lee Fisher and Max Freeburg for their contribution in the development of the composite nerve cuff design.

1. Electrode Components Preparation
<ol>
	<li>Gather four electrode components that require precision cut (laser-cut was used, Please refer to the Materials List) prior to the manufacturing process. These components are (Figure 1): 
	Contacts array frame: This frame is made out of 125 &#181;m thick Polyether ether ketone (PEEK) sheet. It covers the entire width of the cuff and holds the middle contacts and has serpentine-shaped edges (Figure 1B). The middle contacts are wrapped in the guide channels; hence the exposed width of the contacts is limited by the width of the channels and the spacing is determined by the spacing between the channels. 
	The middle contacts strips: The middle contacts are formed by wrapping these strips around the contacts array frame (Figure 1B). Cut the strips out of the Platinum/10% Iridium sheet to the width of the guiding channels and add extra length to allow them to be fully folded around the frame. Spot-weld the contact&#39;s lead at 0&#186; angle with the strip&#39;s major axis. 
	The reference contacts: Four references are needed. The long dimension of these contacts is slightly shorter than the cuff width to fully contain them inside the cuff. Spot weld each reference contact to a lead at 90&#186; angle with the contact&#39;s major axis. 
	PEEK spacers: Spacers are used to create thinner region on the electrode to allow bending and closing (Figure 1C). All the spacers are made from PEEK (other material could be used) and cut to the length of the electrode. The width of the middle space is equal to the height of the electrode.</li>
</ol>
2. Contacts Array Preparation
<ol>
	<li>Clean the components made in step 1 by sonication in ethanol for 2 min at 40 KHz and room temperature, then 2 min in distilled deionized water under the same sonication parameters. Let dry.</li>
	<li>Visually inspect the contacts for any defects like laser-cut residuals or surface deformations.</li>
	<li>Position the contacts one by one under the microscope with the welding spot facing up. Hold the contact with tweezers at approximately 1/3 of length starting from the free end. Elevate the lead to a 45&#186; angle while holding the contact to make the first bend.</li>
	<li>Place the pre-bent contact underneath the array frame with the weld facing up. Hold the frame down with tweezers and elevate the lead to a 45&#186; angle to make a second bend. While Continuing holding the frame down, grab the free end of the contact with tweezers and bend at a 180&#186; angle (fold toward the middle line of the frame).</li>
	<li>Straighten and pull the contact toward operator and then bend at 180&#186; angle (fold to the middle line). The spot welding point should now be enclosed in between the two bent ends.</li>
	<li>Repeat steps 2.3 - 2.5 for the remaining contacts. Make as tight as possible. Alternate the contact leads on each side of the array frame.</li>
</ol>
3. Cuff Layout Guide
<ol>
	<li>Create a 2D diagram of the cuff in flat open position. 
	NOTE: Use any CAD software to produce a true-scale diagram. This diagram will determine the dimensions of the electrode and the placement site for the various electrode components.</li>
	<li>Print the 2D diagram on regular printing paper to scale using ordinary printing machine, and then cut out a 5 cm by 5 cm square piece with the drawing located in the center.</li>
	<li>Cut out 5 cm by 5 cm square piece of the thermal transparency sheet (T1) with a scalpel.</li>
	<li>Place the transparency piece T1 on top of the diagram paper, and then place both layers on the base plate with the diagram facing up. Tape them down to the base plate with adhesive tape.</li>
</ol>
4. Electrode Base Layer and Reference Contacts Placement
<ol>
	<li>Cut out 5 cm by 5 cm silicone sheet with a scalpel (S1), and then place it on the transparency layer. Start by dropping one corner then slowly lower the rest of the sheet to avoid trapping air bubbles in between T1 and S1 sheets (Figure 2A).</li>
	<li>Mix approximately 2 g of uncured silicone as directed on manufacturer data sheet. Rigorously stir the two parts together with sterilized wooden stirring stick. Place the mix in a vacuum chamber for 3 min. Cycle the vacuum to eliminate the bubbles as they rise to surface. Preheat the isotemp oven at 130 &#186;C. 
	Note: Latex gloves can inhibit the curing process of the silicone. Latex gloves also contain sulfur which can leave contaminants on the working surfaces. Using nitrile gloves instead is recommended.</li>
	<li>Using the dental pick tool, apply a thin line of uncured silicone along the middle of the spacer segments where they are located on the guiding diagram.</li>
	<li>Place the spacers onto the designated regions, and then press them down against the silicone sheet S1.</li>
	<li>Partially cure the silicone in the isotemp oven for 30 min, let it cool down for 10 min.</li>
	<li>Place the reference contacts onto designated areas. Ensure that the weld points are facing up and contact leads are routed towards the midline of the cuff to exit at the far end. After ensuring correct positioning, press the contacts down onto the silicone layer S1. Deposit uncured silicone into the through-holes.</li>
	<li>Tape down the leads and then fully cure the silicone at 130 &#186;C for 90 min, or overnight at room temperature (Figure 2B).</li>
</ol>
5. Center Contacts Array Placement
<ol>
	<li>Cut out 1.5 cm by 5 cm transparency piece with a scalpel (T2). Tape down the reference leads away from the middle region to prevent them from running underneath the contacts array during the next step.</li>
	<li>Place the contact arrays on the dedicated location with the leads side facing up. Deposit uncured silicone to tack the array in place.</li>
	<li>Place the piece from 5.1 (T2) across the midline of the electrode and over the arrays to hold them down, and then tape the ends while pressing down on the arrays. Manually align the array with the dedicated position. Tape down the leads outside the cuff&#39;s perimeter.</li>
	<li>Place the small fixture bar across the center of the electrode and over the transparency segment T2. Clamp it down to the base plate with moderate pressure to press the middle contacts against the base silicone layer S1.</li>
	<li>Fully cure the silicone for 90 min at 130 &#186;C, or overnight at RT.</li>
</ol>
6. Embedding the Electrode Components
<ol>
	<li>Remove the small fixture bar and gently remove the transparent sheet T2 to expose the middle contact arrays. Remove all the tapes that hold the leads for both references and middle contacts (Figure 2C).</li>
	<li>Cut a square piece of the transparency sheet with a scalpel to the same width of the electrode and 5 cm in length (T3), and then cut a square piece of silicone sheet to cover the entire electrode surface (S2).</li>
	<li>Lay the silicone sheet (S2) on top of the transparency piece (T3) and stretch it to remove any waves or irregularities and to eliminate air bubbles from being trapped in between.</li>
	<li>Cut four pieces of silicone tubing; 5 cm long each. Place them on the exit site of the leads as assigned on the guiding diagram. Leave a 2 mm space between the electrode edge and the tubes&#39; edges. While holding down each pair of tubes with tweezers, tape down the tubes starting at 1 mm away from the tube end. Repeat for the other pair.</li>
	<li>Arrange and leads of the middle contacts and the references into bundles, and then pass them through the corresponding tube near the exit sites. Repeat for the other three tubes. (Figure 2D).</li>
	<li>Deposit generous amount of uncured silicone over the entire electrode body. 
	NOTE: Avoid forming air bubbles during this step by either slowly pouring the uncured silicone from the vacuumed mixing container or injecting it with a syringe.</li>
	<li>Place the structure from 6.3 on top of the deposited uncured silicone with the silicone sheet S2 facing down. Align the transparency piece T3 with the electrode while keeping the silicone sheet S2 adhered to it.</li>
	<li>Tape down the transparency piece T3 and then apply pressure to channel out any trapped air bubbles. Place the large fixture bar across the center of the electrode and over the transparency segment T3. Then clamp it down to the base plate with moderate pressure. Fully cure the silicone for 90 min at 130 &#186;C, or overnight at RT.</li>
</ol>
7. Shielding Layer Placement (Recommended for Recording Cuffs)
<ol>
	<li>Remove the large fixture bar and delaminate the transparency piece (T3) with tweezers. Place the shielding sheet in the center of each face of the electrode and apply slight pressure to press them into the electrode. Deposit uncured silicone into the through-holes.</li>
	<li>Partially cure the silicone for 30 min at 130 &#186;C, and then let it cool completely to room temperature. Place adhesive tape over the outer ends of the electrode and over the closing flanges to prevent adding extra uncured silicone to these segments.</li>
	<li>Repeat steps 6.6 through 6.8.</li>
</ol>
8. Cutting out the Finished Electrode
<ol>
	<li>Peel off and cut the excess silicone on top of the adhesive tape added in step 7.2 using scalpel blade, then carefully remove the adhesive tape.</li>
	<li>Cut out windows through the silicone to expose the spacer segments through the S2 layer. Extract the embedded spacer segments with tweezers. This step will leave voids and form flexible single silicone sheet at these regions (originally S1).</li>
	<li>Peel off the excess silicone on top of the adhesive tapes that cover the silicone tubes, and then cut it with scalpel blade to level the tubes with the electrode body.</li>
	<li>Cut around the perimeter of the electrode down to base plate.</li>
	<li>Cut out a triangle between each tubes pair completely through the base plate, and on the outer side following the guiding diagram to shape the leads&#39; exit sites. Remove all the silicone material that was detached from the electrode body during last steps.</li>
</ol>
9. Exposing Contacts and Shielding Layers
<ol>
	<li>Cut out windows through the silicone layer S2 that covers the shielding layer. Glide the polypropylene suture filament in between the electrode base (layer S1) and the transparent layer T1 on the base plate to delaminate the finished cuff electrode.</li>
	<li>Flip the electrode such that the center contacts and the Silicone layer S1 are facing up, and then expose them by cutting out windows through the base silicone layer S1. Repeat for the outer reference contacts exposing 1 mm wide segments along the center of the contacts. Ensure that the stabilizing through-holes at the sides of the reference contacts are fully embedded inside the electrode&#39;s body.</li>
</ol>
10. Soldering a Connector to the Leads
<ol>
	<li>Deposit soldering substance onto the leads and onto the connecter pins separately, and then heat and fuse both parts together with soldering iron. 
	Note: The DFT lead wires consist of silver core surrounded by an outer layer made out of the Nickel-Cobalt base alloy MP35N. Depositing the solder substance onto these wires requires the use of specialty flux to allow adhering to the wire (please refer to the Materials List).</li>
</ol>

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The authors declare that they have no competing financial interests. The suppliers listed in this manuscript are provided for reference only.

The manufacturing method described in this article requires dexterous and fine movements in order to ensure the quality of the final cuff. The recording contacts must be placed precisely in the middle of the two reference electrodes. This placement has been shown to significantly reduce interferences from surrounding muscles electrical activity27. Any imbalance in the relative position of the contact during the fabrication can degrade the rejection of common mode interfering signals generated outside the cuff....

Interfacing with the peripheral nervous system (PNS) provides access to highly-processed neural command signals as they travel to different structures within the body. These signals are generated by axons confined within fascicles and surrounded by tightly-jointed perineurium cells. The magnitude of the measurable potentials resulting from the neural activities is affected by the impedance of the various layers within the nerve such as the highly resistive perineurium layer that surrounds the fascicles. Consequently, two interface approaches have been explored depending on the recording location with respect to the perineurium layer, namely intrafascicular and extrafascicular approaches. Intra-fascicular approaches place the electrodes inside the fascicles. Examples of these approaches are the Utah array17, the Longitudinal Intra-fascicular Electrode (LIFE)18, and the transverse intra-fascicular multichannel electrode (TIME)32. These techniques can record selectively from the nerve but have not been shown to reliably retain functionality for long periods of time in vivo, likely due to the size and the compliance of the electrode12.
Extra-fascicular approaches place the contacts around the nerve. The cuff electrodes used in these approaches do not compromise the perineurium nor the epineurium and have been shown to be both a safe and robust means of recording from the peripheral nervous system12. However, extra-fascicular approaches lack the ability to measure single unit activity&#160;&#8212; compared to intra-fascicular designs. Neuroprosthetic applications that utilize nerve cuff electrodes include activation of the lower extremity, the bladder, the diaphragm, treatment of chronic pain, block of neural conduction, sensory feedback, and recording electroneurograms1. Potential applications to utilize peripheral nerve interfacing include restoring movement to victims of paralysis with functional electrical stimulation, recording motor neuron activity from residual nerves to control powered limb prostheses in amputees, and interfacing with the autonomic nervous system to deliver bio-electronic medicines20.
A design implementation of the cuff electrode is the flat-interface nerve electrode (FINE)21. This design reshapes the nerve into a flat-cross section with larger circumference compared to a round shape. The advantages of this design are increased number of contacts that can be placed on the nerve, and the close proximity of the contacts with the rearranged internal fascicles for selective recording and stimulation. Furthermore, upper and lower extremity nerves in large animals and human can take various shapes and the reshaping generated by the FINE does not distort the natural geometry of the nerve. Recent trials have shown that FINE is capable of restoring sensation in the upper extremity16 and restoring movement in the lower extremity22 with functional electrical stimulation in humans.
The basic structure of a cuff electrode consists of placing several metal contacts on the surface of a nerve segment, and then insulating these contacts along with the nerve segment within a nonconductive cuff. To achieve this basic structure, several designs have been proposed in previous studies including:
(1) Metal contacts embedded into a Dacron mesh. The mesh is then wrapped around the nerve and the resulting cuff shape follows the nerve geometry4, 5.
(2) Split-cylinder designs which use pre-shaped rigid and non-conductive cylinders to fix the contacts around the nerve. The nerve segment that receives this cuff is reshaped into the cuff&#39;s internal geometry6-8.
(3) Self-coiling designs where the contacts are enclosed between two insulation layers. The internal layer is fused while stretched with an external un-stretched layer. With different natural resting lengths for the two bonded layers causes the final structure to form a flexible spiral that wraps itself around the nerve. The material used for these layers have typically been polyethylene9 polyimide10, and silicone rubber1.
(4) Uninsulated segments of the lead wires placed against the nerve to serve as the electrode contacts. These leads are either woven into silicone tubing11 or molded in silicone nested cylinders12. A similar principle was used to construct FINEs by arranging and fusing insulated wires to form an array, and then an opening through the insulation is made by stripping a small segment through the middle of these joined wires13. These designs assume a round nerve cross section and conform to this assumed nerve geometry.
(5) Flexible polyimide based electrodes33 with contacts formed by micromachining polyimide structure, and then integrating into stretched silicone sheets to form self-coiling cuffs. This design also assumes a round nerve cross section.
Cuff electrodes should be flexible and self-sizing in order to avoid stretching and compressing the nerve that can cause nerve damage3. Some of the known mechanisms by which cuff electrodes can induce these effects are the transmission of forces from adjacent muscles to the cuff and hence to the nerve, mismatch between the cuff&#39;s and nerve&#39;s mechanical properties, and the undue tension in the cuff&#39;s leads. These safety issues lead to specific set of design constraints on the mechanical flexibility, geometric configuration, and size1. These criteria are particularly challenging in the case of a high contact count FINE because the cuff must be at the same time stiff in the transverse direction to reshape the nerve and flexible in the longitudinal direction to prevent damage as well as accommodating multiple contacts. Self-sizing spiral designs can accommodate multiple contacts cuff14, but the resulting cuff is somewhat rigid. Flexible polyimide design can accommodate a high number of contacts but are prone to delamination. The wire array design13 produces a FINE with flat cross section, but in order to retain this geometry the wires are fused together along the length of the cuff producing stiff faces and sharp edges making then unsuitable for long term implants.
The fabrication technique described in this article produces a high contact density FINE with flexible structure that can be made by hand with consistently high precision. It uses a rigid polymer (Polyether ether ketone (PEEK)) to allow precise placement of the contacts. The PEEK segment maintains a flat cross section at the center of the electrode while remaining flexible in the longitudinal direction along the nerve. This design also minimizes the overall thickness and stiffness of the cuff since the electrode body does not have to be rigid in order to flatten the nerve or secure the contacts.

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			<td style="width:279px;">MED 4765</td>
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			<td height="21" style="height:21px;width:241px;">Soldering Flux</td>
			<td style="width:223px;">SRA Soldering Products</td>
			<td style="width:279px;">FLS71</td>
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			<td height="21" style="height:21px;width:241px;">Tape</td>
			<td style="width:223px;">3M Healthcare</td>
			<td style="width:279px;">1535-0 (SKUMMM15350H)</td>
			<td>Paper, hypoallergenic surgical tape</td>
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			<td height="42" style="height:42px;width:241px;">Spot welding machine</td>
			<td style="width:223px;">Unitek</td>
			<td style="width:279px;">125 Power Supply with 101F Welding Head</td>
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			<td height="21" style="height:21px;width:241px;">Laser cutting platform</td>
			<td style="width:223px;">Universal Laser Systems</td>
			<td style="width:279px;">PLS6.150D</td>
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fabrication of high contact-density, flat-interface nerve electrodes for recording and stimulation applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

Recording neural activity was performed with a customized pre-amplifier using super-&#946; input instrumentation amplifier (700 Hz - 7 kHz bandwidth and total gain of 2,000). An example of the fabricated FINE electrode with the presented protocol is shown in Figure 3. Implanting the FINE around the nerve is done by suturing the two free edges together. A demonstration of the cuff&#39;s flexibility (Figure 3B) indicates that the cuff flattens the nerve while retaining flexibility in the l...

Watch this Scientific Journal Video about Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications at JoVE.com

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

fabrication-high-contact-density-flat-interface-nerve-electrodes-for

Research

JoVE Journal

Neuroscience

9.6K Views.  Case Western Reserve University. This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Video: Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...