Name: Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies
Uploaded: 2024-04-12T14:50:00
Description: Read this Scientific Article Protocol about Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies at JoVE.com

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01AR081235 (to L. K. Lepley). The authors thank the following individuals for their contribution to the fabrication and testing of our biocompatible electrode: Joel Pingel, Grant Gueller, Akhil Ramesh, Joe Letner, Jacky Tian, and Ross Brancati.

The in vivo procedure was conducted under the approval of the Institutional Animal Care &#38; Use Committee at the University of Michigan (IACUC approval #PRO00010765) and in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals.
1. Electrode sourcing and fabrication
NOTE: Figure 1 provides a high-level summary of all key fabrication steps with a QR link that provides addit...

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

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Online. 8, 11-35 (2006).</li><li>Tankisi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standards+of+instrumentation+of+EMG.">Standards of instrumentation of EMG.</a> Clin. Neurophysiol. 131 (1), 243-258 (2020).</li><li>Kumar, A., Accorsi, A., Younghwa, R., Mahasweta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J. Vis. Exp. JoVE. (99), e52793 (2015).</li><li>Wang, C., Yue, F., Kuang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+histology+characterization+using+H&amp;E+staining+and+muscle+fiber+type+classification+using+immunofluorescence+staining.">Muscle histology characterization using H&amp;E staining and muscle fiber type classification using immunofluorescence staining.</a> Bio-Protoc. 7 (10), e2279 (2017).</li><li>Kreifeldt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+versus+noise+characteristics+of+filtered+EMG+used+as+a+control+source.">Signal versus noise characteristics of filtered EMG used as a control source.</a> IEEE Trans. Biomed. Eng. BME-18 (1), 16-22 (1971).</li><li>Farina, D., Yoshida, K., Stieglitz, T., Koch, K. 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Neural Eng. 16 (1), 016010 (2018).</li><li>Guo, L., Guvanasen, G., Tuthill, C., Nichols, T., Deweerth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of a Stretchable Multielectrode Array for Epimysial Recording. , 694 (2011).</li><li>Zwarts, M. J., Stegeman, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+surface+EMG:+basic+aspects+and+clinical+utility.">Multichannel surface EMG: basic aspects and clinical utility.</a> Muscle Nerve. 28 (1), 1-17 (2003).</li><li>Koch, K. P., Leinenbach, C., Stieglitz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+test+of+robust+spherical+epimysial+electrodes+for+lower+limb+stimulation.">Fabrication and test of robust spherical epimysial electrodes for lower limb stimulation.</a> Aalb. Den. , (2000).</li><li>Uhlir, J. P., Triolo, R. J., Davis, J. A., Bieri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+epimysial+stimulating+electrodes+in+the+lower+extremities+of+individuals+with+spinal+cord+injury.">Performance of epimysial stimulating electrodes in the lower extremities of individuals with spinal cord injury.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 12 (2), 279-287 (2004).</li><li>Deer, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+appropriate+use+of+neurostimulation:+new+and+evolving+neurostimulation+therapies+and+applicable+treatment+for+chronic+pain+and+selected+disease+states.">The appropriate use of neurostimulation: new and evolving neurostimulation therapies and applicable treatment for chronic pain and selected disease states.</a> Neuromodulation Technol. Neural Interface. 17 (6), 599-615 (2014).</li><li>Ortiz-Catalan, M., Br&#229;nemark, R., H&#229;kansson, B., Delbeke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+viability+of+implantable+electrodes+for+the+natural+control+of+artificial+limbs:+Review+and+discussion.">On the viability of implantable electrodes for the natural control of artificial limbs: Review and discussion.</a> Biomed. Eng. OnLine. 11, 33 (2012).</li><li>Sando, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+peripheral+nerve+interface+for+prostheses+control:+electrode+comparison.">Regenerative peripheral nerve interface for prostheses control: electrode comparison.</a> J. Reconstr. Microsurg. 32 (3), 194-199 (2016).</li></ol>

Our objective was to streamline the EMG fabrication process, enabling broader adoption and implementation of epimysial electrode designs, thus promoting accessibility, and advancing neuromuscular research. To this end, we present a user-friendly guide for sourcing, fabricating, and testing low-cost epimysial electrodes in-house. In hopes of supporting other research groups, we also provide supplemental 3D printing templates to facilitate the production of in-house epimysial electrodes for their research endeavors.
<p...

Electromyography (EMG) is a powerful tool for studying the electrical activity of muscle. EMG recordings can be especially useful in preclinical animal models to assess the effectiveness of interventions to treat neuromuscular dysfunction. In these models, implantable biocompatible electrodes are commonly used to assess the neurophysiological interface between motor neurons and muscle fibers. These implantable electrodes can provide localized measurements of muscle excitation and can be diverse in terms of their configuration, shape, and material, with the optimal design ultimately dictated by the location and intended use.
Despite their suitability for assessing muscle excitation in preclinical models, the use of epimysial electrodes can be limited by cost. As a result, many investigators use custom-fabricated epimysial electrodes that are produced in-house. Although resources exist detailing the fundamental considerations of electrode fabrication, testing, and use1,2, there is a need for an updated instructional guide detailing the sourcing, fabrication, and validation of epimysial electrodes using modern methods. Informed by the foundational works of Loeb and Gans3 and others in electrode theory, we present modern instructions on the sourcing and fabrication of low-cost epimysial electrodes and test their performance in a series of ex vivo and in vivo experiments. The aim is to offer a user-friendly guide for others in the scientific community to source, fabricate, and test in-house low-cost epimysial electrodes for animal use, enabling the broader quantification of muscle excitation in preclinical models.
In this protocol, we provide an instructional guide to the sourcing, fabrication, and testing of epimysial electrodes for animal use in the modern electrophysiology laboratory. Electrode parameters chosen for fabrication, such as the shape, dimensions, contact surface area, interelectrode distance, lead length, etc., were selected to suit our experimental needs and were comparable to a commercially available industry standard epimysial electrode (see the Table of Materials). We encourage other groups to modify these parameters to suit their needs in addition to selecting a reliable industry standard electrode that matches their use case.
In an effort to give readers a relatively quick sense of electrode performance, we also provide an example of an ex vivo testing protocol with the option of measuring electrode impedance. Additionally, we give an example assessment of electrode performance in vivo. The ex vivo experiment compared the custom-fabricated electrode to an industry standard in a saline bath to mimic stable physiological conditions. Impedance was also assessed ex vivo via electrochemical impedance spectroscopy (EIS). The in vivo experiment consisted of the surgical implantation of the custom-fabricated electrode into the vastus lateralis (VL) muscle of a 16-week-old female Long Evans rat (HsdBlu: LE, Envigo) to measure the EMG signal during conditions known to elicit a high or low signal (uphill, downhill walking). To assess the reliability of the custom-fabricated electrode, EMG signaling was acquired during level walking following full surgical recovery and prior to sacrifice (14 days and 56 days post implantation, respectively). Hematoxylin-eosin (H&#38;E) staining was conducted on the instrumented muscle to assess the biocompatibility of the custom-fabricated electrode.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrode Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Contact surface</td>
			<td>Prince and Izant PT90/IR10 1.25 mm x 5 mm foil</td>
			<td>Catalog #1040055&#160;</td>
			<td>2 per electrode 
			$7.50 per foil 
			$15.00 per electrode</td>
		</tr>
		<tr>
			<td>PFA coated stainless-steel electrode lead wire&#160;</td>
			<td>A-M Systems Multi-Stranded PFA-Coated Stainless Steel Wire 50.8 &#181;m strand diameter&#160;</td>
			<td>Catalog #793500</td>
			<td>Dependent on desired lead length (e.g., 9 inch lead wires x2) 
			$128 per 25 ft spool 
			$5.12 per foot 
			$0.42 per inch (x18) 
			$7.68 per electrode</td>
		</tr>
		<tr>
			<td>Folding jig&#160;</td>
			<td>3D printed 
			(see .gcode file)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Sealant for electrode body</td>
			<td>Nusil Med-1137 liquid silicone</td>
			<td>Catalog #MED-1137</td>
			<td>1 gram 
			$344.66 per 2 oz. (59.15 mL) 
			$5.83 per electrode</td>
		</tr>
		<tr>
			<td>Silicone base</td>
			<td>Implantech Alliedsil Silicone Sheeting-Reinforced, Long Term Implantable (8&#8221; x 6&#8221;) .007 thick</td>
			<td>Catalog #701-07&#160;</td>
			<td>10mm x 5mm sheet 
			$225.00 per 8 x 6 inch 
			$0.36 per electrode (10 mm x 5 mm)</td>
		</tr>
		<tr>
			<td>&#160;Thinner for sealant mixture</td>
			<td>Toluene 99.5% ACS Reagent 500mL or Xylene ACS 99.5%</td>
			<td>Catalog #179418-500 ML</td>
			<td>0.75 mL 
			$25.53 per 500 mL 
			$0.38 per electrode</td>
		</tr>
		<tr>
			<td>Template for perforating silicone base</td>
			<td>Cutting jig &#8211; 3D printed 
			(see CAD file)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Custom-fabricated electrode: $29.25</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Industry standard electrode (EP105 EMG Patch Electrode, 2 contacts, single-sided, 7mm x 4mm, MicroProbe for Life Science): $305.00</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Additional Fabrication Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;&#160;</td>
			<td>&#160; &#160;&#160;</td>
			<td>&#160; &#160;&#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>3D printing software&#160;</td>
			<td>Solidworks (Solidworks, 2022)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Tig welder&#160;</td>
			<td>Micro-Tig Welder (CD1000SPM, Single Pulse Research and Light Production Resistance Spot Welder, Sunstone)</td>
			<td>SKU&#160;301010</td>
			<td>$3,500</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Ultrasonic bath (CPX Series Ultrasonic Bath, Fisherbrand).&#160;</td>
			<td>15-337-403</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Ex Vivo Testing Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Data acquisition platform and software</td>
			<td>DigitalLynx 4sX Base Cheetah version 6.0 (Neuralynx Inc.)&#160;</td>
			<td>NA</td>
			<td>EMG acquisition hardware and software</td>
		</tr>
		<tr>
			<td>Electrode interface board (EIB)</td>
			<td>EIB, EIB16-QC, Neuralynx Inc.</td>
			<td>31-0603-0007</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>5 MHz Function Generator, B&#38;K Precision&#160;</td>
			<td>&#160;4005DDS220V</td>
			<td>$387.46</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>PGSTAT1 potentiostat (EcoChemie, Utrecht, Netherlands)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Stainless steel screw</td>
			<td>Fine Science Tools</td>
			<td>19010-00</td>
			<td>$98</td>
		</tr>
		<tr>
			<td>Ex Vivo Testing Materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity &#38; price per electrode</td>
		</tr>
		<tr>
			<td>Rodent treadmill&#160;</td>
			<td>Exer 3/6 Open Treadmill, Columbus Instruments</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Excel Formula&#174; Pourable Dental Material, St. George Technology Inc.</td>
			<td>#24211</td>
			<td>$125.60</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Keyence BZ-X800, Keyence Corporation, Osaka, Japan&#160;</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>Optitrack Color Camera, Optitrack, NaturalPoint Inc.</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Peak detection algorithm</td>
			<td>&#8220;SciPy.signal.find_peaks - SciPy v1.8.1 Manual&#8221;, 2022</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Python software</td>
			<td>Python Software Foundation. Python Language Reference, version 3.9. Available at http://www.python.org</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>HsdBlu: LE, Envigo</td>
			<td>140</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Statistical sotware</td>
			<td>GraphPad Prism version 10.0.0 (GraphPad Software, Boston, Massachusetts USA)</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

development of a low-cost epimysial electromyography electrode: a simplified workflow for fabrication and testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

We seek to understand the neuromuscular and electrical-physiological consequences of ACL injury using a preclinical rodent model. Our current experiments use implantable epimysial EMG electrodes to investigate how non-invasive ACL injury alters quadriceps activation. Although fundamental resources exist detailing the considerations for electrode fabrication and testing, we aim to provide an updated, easy-to-follow guide to epimysial electrode fabrication and testing for the modern electrophysiology laboratory.Our goal was to streamline the epimysial EMG fabrication and testing process, facilitating the broader use of epimysial electrodes in the advancement of neuromuscular research.

Ex vivo performance 
ICCs revealed high levels of agreement between the custom-fabricated and industry standard electrodes across all waveforms (sine [ICC = 0.993], square [ICC = 0.995], triangle [ICC = 0.958]; p &#60; .001). Bland-Altman plots also revealed a high degree of signal agreement between electrodes. Bland Altman plots and Pearson correlations are summarized in Figure 3 with strong positive correlations between the custom-fabricated and industry stan...

Read this Scientific Article Protocol about Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies at JoVE.com

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

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JoVE Journal

Neuroscience

Fabrication of Epimysial Electrodes for Electromyography Recordings 

To begin, use tape to secure the silicone base on the cutting jig. Perforate the silicone with cutting jig guides and an X-ACTO knife replacement of the contact foils. Using the folding jig, bend the biocompatible precut platinum or radium contact foils into a U-shape.Insert the foils into the perforated slots in the silicone base to construct the contact surfaces of the electrode, ensuring proper spacing between electrodes. Place a piece of surgical tape over the contact surfaces to hold the foils in place. Remove the silicone base from the cutting jig.Flip the silicone base to expose U-shaped foil arms. Hold one arm of each U-shaped foil flush with the silicone base. Cut the perfluoroalkoxy-coated stainless steel wire to a desired length.Using a lighter, denude one end of the wire. Position the denuded end of the wire on the inside of the unfolded arm of the foil and weld it using a micro TIG welder. To test the wire foil connection, apply tension to the stainless steel wire.If the connection holds, fold the foil arm down flush with the silicone base, and remove the tape bordering of the silicone sheet. Next, mix biocompatible liquid silicone with toluene until desired thinner or syrup-like consistency is achieved. Drop the mixture using an 18 gauge blunt tipped syringe and apply it to the back of the welded electrodes to seal it.After 72 hours, use scissors to cut the silicone base into individual electrodes.

Ex Vivo Testing of Epimysial Electrodes in a Saline Bath 

After electrode fabrication, pin each electrode lead wire to a channel on an electrode interface board. Connect the industry-standard electrode to the same board for comparison. Now connect the electrode interface board to a data-acquisition platform via a magnetic tethered cable system.Denude one end of a perfluoroalkoxy coated stainless steel wire and spot weld it to a grounding source. Pin the ground lead wire to the designated ground position on the board. In a 250 milliliter glass beaker, add 180 milliliters of 0.9%sterile saline.Submerge both custom fabricated and industry standard epimysial electrodes in the saline bath and secure them in a stable position. Then place the grounding source in the saline bath and secure its position. Submerge two stimulating needle electrodes in the saline bath and connect them to the signal generator.On a signal generator, set the voltage to 0.1 volts and frequency to five hertz. Deliver repeated waveforms into the saline bath to compare recorded signals between the custom fabricated and industry standard electrodes. Assess performance informally and visually in real time to evaluate signal variation between electrodes.The intraclass correlations revealed high levels of agreement between the custom fabricated and industry standard electrodes across all waveforms. The sine waveforms showed an intraclass correlation of 0.993, square waveforms of 0.995 and triangle waveforms of 0.958. Bland-Altman plots also indicated a high degree of signal agreement between the electrodes.Similarly, Pearson correlations demonstrated strong positive correlations between the custom fabricated and industry standard electrodes.