Name: preparation of peripheral nerve stimulation electrodes for chronic implantation in rats
Uploaded: 2020-07-14T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats at JoVE.com

This work was funded by the University of Texas at Dallas and the UT Board of Regents. We thank Solomon Golding, Bilaal Hassan, Marghi Jani, and Ching-Tzu Tseng for technical assistance.

All procedures described in this protocol are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.
1. Stimulating cuff electrode fabrication
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	<li>Prepare the cuff tubing.
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		<li>Using a razor blade, cut a piece of polymer tubing 2.5 mm in length. Insert forceps tips or a paper clip through the tubing and use the blade to make a slit lengthwis...

<ol>
	<li>Koopman, F. A., 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=Vagus+nerve+stimulation+inhibits+cytokine+production+and+attenuates+disease+severity+in+rheumatoid+arthritis.">Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2016).</li><li>Levine, Y. A., 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=Neurostimulation+of+the+cholinergic+anti-inflammatory+pathway+ameliorates+disease+in+rat+collagen-induced+arthritis.">Neurostimulation of the cholinergic anti-inflammatory pathway ameliorates disease in rat collagen-induced arthritis.</a> PLoS One. , (2014).</li><li>Zhang, Y., 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=Chronic+vagus+nerve+stimulation+improves+autonomic+control+and+attenuates+systemic+inflammation+and+heart+failure+progression+in+a+canine+high-rate+pacing+model.">Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model.</a> Circulation: Heart Failure. , (2009).</li><li>Ganzer, P. D., 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=Closed-loop+neuromodulation+restores+network+connectivity+and+motor+control+after+spinal+cord+injury.">Closed-loop neuromodulation restores network connectivity and motor control after spinal cord injury.</a> Elife. , (2018).</li><li>Hays, S. A., 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=Vagus+nerve+stimulation+during+rehabilitative+training+enhances+recovery+of+forelimb+function+after+ischemic+stroke+in+aged+rats.">Vagus nerve stimulation during rehabilitative training enhances recovery of forelimb function after ischemic stroke in aged rats.</a> Neurobiology of Aging. , (2016).</li><li>Khodaparast, N., 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=Vagus+nerve+stimulation+delivered+during+motor+rehabilitation+improves+recovery+in+a+rat+model+of+stroke.">Vagus nerve stimulation delivered during motor rehabilitation improves recovery in a rat model of stroke.</a> Neurorehabilitation and Neural Repair. , (2014).</li><li>Meyers, E. 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=Vagus+nerve+stimulation+enhances+stable+plasticity+and+generalization+of+stroke+recovery.">Vagus nerve stimulation enhances stable plasticity and generalization of stroke recovery.</a> Stroke. , (2018).</li><li>Hays, S. A., 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=Vagus+nerve+stimulation+during+rehabilitative+training+improves+functional+recovery+after+intracerebral+hemorrhage.">Vagus nerve stimulation during rehabilitative training improves functional recovery after intracerebral hemorrhage.</a> Stroke. , (2014).</li><li>Farrand, A., 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=Vagus+nerve+stimulation+improves+locomotion+and+neuronal+populations+in+a+model+of+Parkinson's+disease.">Vagus nerve stimulation improves locomotion and neuronal populations in a model of Parkinson's disease.</a> Brain Stimulationation. , (2017).</li><li>Souza, R. 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=Vagus+nerve+stimulation+reverses+the+extinction+impairments+in+a+model+of+PTSD+with+prolonged+and+repeated+trauma.">Vagus nerve stimulation reverses the extinction impairments in a model of PTSD with prolonged and repeated trauma.</a> Stress. , (2019).</li><li>Noble, L. J., Souza, R. R., McIntyre, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+as+a+tool+for+enhancing+extinction+in+exposure-based+therapies.">Vagus nerve stimulation as a tool for enhancing extinction in exposure-based therapies.</a> Psychopharmacology. , (2019).</li><li>Childs, J. E., Kim, S., Driskill, C. M., Hsiu, E., Kroener, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+during+extinction+learning+reduces+conditioned+place+preference+and+context-induced+reinstatement+of+cocaine+seeking.">Vagus nerve stimulation during extinction learning reduces conditioned place preference and context-induced reinstatement of cocaine seeking.</a> Brain Stimulationation. , (2019).</li><li>Pe&#241;a, D. F., Engineer, N. D., McIntyre, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+remission+of+conditioned+fear+expression+with+extinction+training+paired+with+vagus+nerve+stimulation.">Rapid remission of conditioned fear expression with extinction training paired with vagus nerve stimulation.</a> Biological Psychiatry. , (2013).</li><li>Childs, J. E., DeLeon, J., Nickel, E., Kroener, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+reduces+cocaine+seeking+and+alters+plasticity+in+the+extinction+network.">Vagus nerve stimulation reduces cocaine seeking and alters plasticity in the extinction network.</a> Learning &amp; Memory. , (2017).</li><li>Engineer, C. T., 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=Temporal+plasticity+in+auditory+cortex+improves+neural+discrimination+of+speech+sounds.">Temporal plasticity in auditory cortex improves neural discrimination of speech sounds.</a> Brain Stimulationation. , (2017).</li><li>Rios, M., 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=Protocol+for+Construction+of+Rat+Nerve+Stimulation+Cuff+Electrodes.">Protocol for Construction of Rat Nerve Stimulation Cuff Electrodes.</a> Methods Protoc. , (2019).</li><li>Childs, J. 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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=Norepinephrine+and+serotonin+are+required+for+vagus+nerve+stimulation+directed+cortical+plasticity.">Norepinephrine and serotonin are required for vagus nerve stimulation directed cortical plasticity.</a> Exp. Neurol. , (2019).</li><li>Hulsey, D. 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=Reorganization+of+Motor+Cortex+by+Vagus+Nerve+Stimulation+Requires+Cholinergic+Innervation.">Reorganization of Motor Cortex by Vagus Nerve Stimulation Requires Cholinergic Innervation.</a> Brain Stimulation. 9, 174-181 (2016).</li><li>Bouverot, P., Crance, J. 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A., 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=Thin+Film+Multi-Electrode+Softening+Cuffs+for+Selective+Neuromodulation.">Thin Film Multi-Electrode Softening Cuffs for Selective Neuromodulation.</a> Scientific Reports. , (2018).</li><li>Thakur, R., Nair, A. R., Jin, A., Fridman, G. 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Here we describe a simple, low-cost approach for assembly of chronically implantable stimulating cuff electrodes for use in rodents, facilitating preclinical investigations of this emerging therapy. This simplified method requires no specialized training or equipment, and uses a small number of tools and supplies that are easily accessible to most research labs, reducing both the monetary and labor costs of device manufacture compared to other approaches16,26<sup...

Recently, the demand for chronically implantable cuff electrodes for stimulation of peripheral nerves has grown, as studies increasingly demonstrate the preclinical usefulness of this technique for the treatment of numerous inflammatory diseases1,2,3 and neurological disorders4,5,6,7,8,9,10,11,12,13,14,15. Chronic VNS, for example, has been shown to enhance neocortical plasticity in a variety of learning contexts, improving motor rehabilitation4,5,6,7,8, extinction learning10,11,12,13,14, and sensory discrimination15. Commercially available peripheral nerve cuff electrodes are often associated with extended times for order fulfillment and relatively high costs, which can limit their accessibility. Alternatively, protocols for &#8220;in-house&#8221; fabrication of chronically implantable cuff electrodes remain limited, and rodent anatomy presents particular challenges due to their small size. Current protocols for constructing cuff electrodes for chronic rodent experiments often require the use of complex equipment and techniques, as well as extensively trained personnel. In this protocol, we demonstrate a simplified approach to cuff electrode fabrication based on previously published and widely used methods16,17. We validate the functionality of our chronically implanted electrodes in rats by demonstrating that, at the time of cuff implantation around the left cervical vagus nerve, stimulation applied to the cuff electrodes successfully produced a cessation of breathing and drop in SpO2. Stimulation of afferent pulmonary receptor vagal fibers is known to engage the Hering-Breuer reflex, in which the inhibition of several respiratory nuclei in the brainstem results in the suppression inspiration18. Thus, cessation of breathing consistent with the Hering-Breuer reflex, and the resulting drop in SpO2, provide a straightforward test for proper electrode implantation and cuff function in anesthetized rats. To validate the long-term functionality of chronically implanted cuff electrodes, reflex responses were measured at the time of implantation and compared to the responses obtained in the same animals six weeks after implantation. A second group of rats was implanted with VNS cuff electrodes after behavioral training on a lever pressing task. In these rats, VNS paired with correct task performance produced reorganization of the cortical motor map, consistent with previously published studies19,20,21,22. At the time of motor cortical mapping under anesthesia, which occurred 5&#8211;10 weeks after device implantation, we further validated cuff function in VNS-treated animals by confirming that VNS successfully induced a cessation of breathing and a greater than 5% drop in SpO2.
The recently published protocols from Childs et al.17 and Rios et al.16 provide a well-validated starting point for a simplified cuff electrode fabrication approach, as this popular method has been utilized by multiple labs conducting chronic VNS studies in rodents1,2,3,4,5,6,7,8,9,10,11. The original method involves several high-precision steps for manipulating the fine microwires such that cuff electrode fabrication takes over an hour to complete, and extensive training to perform reliably. The simplified approach described here requires significantly fewer materials and tools and can be completed in under one hour by minimally trained personnel.

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		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Biocompatible polyurethane-based polymer tubing, 0.080&#34; OD x 0.040&#34; ID</td>
			<td style="width:178px;">Braintree Scientific</td>
			<td style="width:174px;">MRE080 36 FT</td>
			<td style="width:542px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td style="width:178px;">AM Scopes</td>
			<td style="width:174px;">#SM-6T-FRL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Serrated Scissors, straight, 22mm cutting edge</td>
			<td style="width:178px;">Fine Science Tools</td>
			<td style="width:174px;">#14058-09</td>
			<td>for cutting Pt/Ir wire and suture thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps, #5 Dumont forceps, straight, 11 cm, 0.1 x 0.06 mm tip</td>
			<td style="width:178px;">Fine Science Tools</td>
			<td style="width:174px;">#11626-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps, ceramic tipped forceps, 0.3 mm x 30 mm tips</td>
			<td style="width:178px;">Electron Microscopy Sciences</td>
			<td style="width:174px;">#78127-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Pins, PCB Press Fit Socket</td>
			<td style="width:178px;">Mill-Max</td>
			<td style="width:174px;">#1001-0-15-15-30-27-04-0</td>
			<td>or similar small pins for connecting cuff leads to headcap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isobutane lighter</td>
			<td style="width:178px;">BIC</td>
			<td style="width:174px;">#LCP21-AST</td>
			<td>for de-insulating Pt/Ir wire</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Micro strip connector with latch, 4-pin</td>
			<td style="width:178px;">Omnetics</td>
			<td style="width:174px;">A24002-004 / PS1-04-SS-LT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette tip, 10 uL</td>
			<td style="width:178px;">VWR</td>
			<td>89079-464</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum-Iridium (90/10%) Wire, 0.001&#34; (diameter) x 9 strands, PTFE insulated</td>
			<td style="width:178px;">Sigmund Cohn</td>
			<td style="width:174px;">10IR9/49T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor Blade, Single Edge, Surgical Carbon Steel No.9</td>
			<td style="width:178px;">VWR</td>
			<td style="width:174px;">#55411-050</td>
			<td>for cutting MicroRenathane tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sewing needle, ca. 4.0 cm length x 0.7 mm diameter (size 6-7)</td>
			<td style="width:178px;">Singer</td>
			<td style="width:174px;">00276</td>
			<td>Smaller needle for threading Pt/Ir wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sewing needle, ca. 4.5 cm length x 0.8 mm diameter (size 2-3)</td>
			<td style="width:178px;">Singer</td>
			<td style="width:174px;">00276</td>
			<td>Larger needle for pinning cuff during assembly and for threading suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small foam board</td>
			<td style="width:178px;">Juvo+/Amazon</td>
			<td>B07C9637SJ</td>
			<td>for fabrication platform; our dimensions are ca. 2.5&#34; x 3.5&#34; x 1&#34; (L x W x H)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solder, multicore lead-free, 0.38mm diameter</td>
			<td style="width:178px;">Loctite/Multicore</td>
			<td style="width:174px;">#796037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering station</td>
			<td style="width:178px;">Weller</td>
			<td style="width:174px;">WES51</td>
			<td>or similar soldering iron compatible with long conical tips (this part has been discontinued)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering tip, long conical, 0.01&#34; / 0.4 mm</td>
			<td style="width:178px;">Weller</td>
			<td style="width:174px;">1UNF8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture, nonabsorbable braided silk ,size 6/0</td>
			<td style="width:178px;">Fine Science tools</td>
			<td style="width:174px;">#18020-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV (405 nm) spot light</td>
			<td style="width:178px;">Henkel/Loctite</td>
			<td style="width:174px;">#2182207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Light Cure Adhesive 25 ml</td>
			<td style="width:178px;">Henkel/Loctite</td>
			<td style="width:174px;">AA 3106</td>
			<td>or similar biocompatible UV cure adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wire wrapping wire, 30 AWG</td>
			<td style="width:178px;">Digikey</td>
			<td style="width:174px;">K396-ND</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Vagus nerve cuff electrodes and headcaps were chronically implanted in rats according to previously published surgical procedures17,19,20,21,22. Prior to implantation, impedance at 1 kHz was measured across the cuff leads with the cuff tubing submerged in saline (impedance = 1.2 &#177; 0.17 k&#937; [mean &#177; std]; N = 9). Only cuffs with impedances less tha...

Watch this Scientific Journal Video about Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats at JoVE.com

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

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. ...

Peripheral nerve stimulation is increasingly under investigation as a treatment for a variety of diseases including inflammatory diseases and neural disorders. Construction of chronically implantable peripheral nerve cuff electrodes for small laboratory animals can be time consuming and expensive. This protocol describes a simple, low cost approach for constructing chronically implantable peripheral nerve stimulating electrodes for use in rats.The method requires very few and easily accessible materials and supplies and a reduced number of hazardous or technically demanded steps compared to similar techniques which reduces both the cost of the device as well as the time required to train personnel and device assembly. This technique was designed to enable preclinical studies that test the effectiveness of chronic Vegas nerve stimulation during stroke recovery or other types of Metro dysfunction. Begin by preparing the cuff tubing.Use a razor blade to cut a 2.5 millimeter piece of polymer tubing. Then insert a paperclip or force tips through the tubing and make a lengthwise slip through the wall of the tubing on one side of the cuff. Remove the forceps from the tubing and insert a large sewing needle through the midline of the cuff.Place the needle into the foam board to pin the cuff in place during the remaining assembly steps. To place a suture for securing cuff closure during implantation insert a small sewing needle through the wall of the cuff on the midline approximately point five millimeters from the top slit on one side. Then insert two centimeters of a six zero suture through the eye of the needle and pull the needle through the wall of the tubing to thread the suture into the cuff.Puncture a second hole through the tubing wall approximately point five millimeters below the first hole along the midline of the cuff. Insert the suture through the eye of the needle and pull the needle through the tubing wall. Apply a small amount of UV cure adhesive to the short end of the suture extending from the lower hole and pull the longer suture end until the lower tail is nearly flush with the exterior wall of the tubing.Then cure the adhesive with a UV wand. Repeat this process to place another suture on the opposite side of the cuff. Next place the Platinum Iridium wire leads.Use the small sewing needle to make four holes in the cuff wall. Then insert the sewing needle again, this time working from exterior to interior through the first lead hole. Insert approximately point five centimeters of a 7.5 centimeter Platinum Iridium wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff wall.Adjust the wire so that approximately 4.5 centimeters extends on the exterior side of the cuff. Insert the needle through the first lead hole again then through the second lead hole. Insert point five centimeters of the shorter end of the wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff walls.Repeat the process to thread the wire through the third and fourth lead holes. Using a butane lighter, carefully remove the insulation from five to six millimeters of the wires extending from the second and fourth lead holes Then gently pull on the end of the wire extending from the first hole until the uninsulated portion of the wire is flush with the hole. Repeat this with the other lead to align the uninsulated end of the wire threaded through the third and fourth lead holes.Apply a small amount of UV cure adhesive to the wire loops on the exterior side of the cuff at the first and third lead holes. Then use the UV wand to cure of the adhesive and secure the leads in place. Use a small pipette tip to push the uninsulated wire leads against the interior wall of the cuff.Pull the one millimeter tails of the wire flat against the exterior surface of the cuff, taking care not to short them together. Then apply a small amount of UV adhesive to just cover the two tails and cure it. Remove the large needle with the cuff assembly from the foam board.Insert a three centimeter length of a six zero suture through the eye of the needle and pull the needle through the tubing to thread the suture through the bottom of the cuff at the midpoint. Insert the small sewing needle through the same midline hole working from interior to exterior to avoid deformation, the tubing and the wire leads. Then insert the exterior tail of the suture through the eye of the needle and pull the needle through the cuff wall to create a loop of suture around the edge of the cuff.Create the second loop around the opposite end of the cuff by tying the ends of the suture and a half knot on the exterior side of the cuff. While holding the knot flat against the tubing, apply a small amount of UV adhesive to the half knot and cure it. Carefully cut the ends of the suture thread as close to the knot as possible.Then, use a butane lighter to remove the insulation from approximately three millimeters at the end of each of the Platinum Iridium wire leads. Solder the cup side of a gold pin to the uninsulated end of each lead. Before proceeding with implantation test the impedance of the assembled device to verify that the assembled cuff has an impedance of less than two kilo ohm at one kilohertz.After assembling the head cap and implanting the cuff electrodes according to manuscript directions, stimulate the vagus nerve while the rat is awake. Connect the rat to a stimulus generator via the implanted head cap and adjust the stimulation settings. For VNS induced reorganization of the motor cortical map, deliver a single train of 15 biphasic pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere at a frequency of 30 hertz.A stimulation train is delivered immediately after the detection of each successful lever press throughout 10, 30 minute training sessions. Use an oscilloscope to monitor successful delivery of current stimulation. This protocol, was used to chronically implant vagus nerve cuff electrodes and head caps in rats.During implantation, functional validation of all cuffs was performed by testing for a VNS induced drop in blood oxygen saturation. To evoke this response, a 10 second train of 30 hertz pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere was delivered across the cuff leads. For all cuffs, we observed a VNS induced cessation of breathing for the duration of the 10 seconds stimulation, which was accompanied by a drop in blood oxygen saturation of at least 5%confirming cuff function and proper implantation.To test the long term functionality of the devices, rats were anesthetized six weeks after implantation, and VNS was again applied. For seven out of nine devices, the magnitude of stimulation evoked change in blood oxygen saturation, did not differ from that observed at initial implantation, suggesting continued excellent performance. The remaining two devices, increasing the stimulation amplitude to 1.6 milliampere was sufficient to evoke a reliable reduction in blood oxygen saturation of at least 5%suggesting that these devices continued to function.But the changes in impedance, nerve damage, or cuff orientation may have resulted in reduced performance. To further test the long term functionality of the chronically implanted devices, a second group of rats was trained on a simplified version of a skilled reaching lever press task. The rats were required to reach two centimeters outside the training booth to fully depress the lever and then to release it within two seconds.Consistent with prior studies demonstrating that vagus nerve stimulation drives expansion of task relevant motor map representations, VNS treated rats exhibited significantly larger proximal forelimb representations than Sham treated rats. This protocol can be easily adapted to accommodate chronic stimulation experiments in other laboratory species or at other peripheral nerve sites, including sciatic or sacral nerves. For example.

preparation-peripheral-nerve-stimulation-electrodes-for-chronic

Research

JoVE Journal

Neuroscience

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 ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

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.

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 ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter TMS

Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable tool to probe the procedural and declarative influence over sensorimotor integration during skilled motor actions. This manuscript describes the methodology and contributions of short-latency afferent inhibition (SAI) for understanding sensorimotor integration. SAI quantifies the effect of a convergent afferent volley on the corticospinal motor output evoked by transcranial magnetic stimulation (TMS). The afferent volley is triggered by the electrical stimulation of a peripheral nerve. The TMS stimulus is delivered to a location over the primary motor cortex that elicits a reliable motor-evoked response in a muscle served by that afferent nerve. The extent of inhibition in the motor-evoked response reflects the magnitude of the afferent volley converging on the motor cortex and involves central GABAergic and cholinergic contributions. The cholinergic involvement in SAI makes SAI a possible marker of declarative-procedural interactions in sensorimotor performance and learning. More recently, studies have begun manipulating the TMS current direction in SAI to tease apart the functional significance of distinct sensorimotor circuits in the primary motor cortex for skilled motor actions. The ability to control additional pulse parameters (e.g., the pulse width) with state-of-the-art controllable pulse parameter TMS (cTMS) has enhanced the selectivity of the sensorimotor circuits probed by the TMS stimulus and provided an opportunity to create more refined models of sensorimotor control and learning. Therefore, the current manuscript focuses on SAI assessment using cTMS. However, the principles outlined here also apply to SAI assessed using conventional fixed pulse width TMS stimulators and other forms of afferent inhibition, such as long-latency afferent inhibition (LAI).

Author Spotlight: Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning  

Short-latency afferent inhibition (SAI) is a transcranial magnetic stimulation protocol to probe sensorimotor integration. This article describes how SAI can be used to study the convergent sensorimotor loops in the motor cortex during sensorimotor behavior.

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable ...

Abdominal VNS for Treating Inflammatory Conditions in Rat Models

Implantation Surgery for Abdominal Vagus Nerve Stimulation and Recording Studies in Awake Rats

Abdominal vagus nerve stimulation (VNS) can be applied to the subdiaphragmatic branch of the vagus nerve of rats. Due to its anatomical location, it does not have any respiratory and cardiac off-target effects commonly associated with cervical VNS. The lack of respiratory and cardiac off-target effects means that the intensity of stimulation does not need to be lowered to reduce side effects commonly experienced during cervical VNS. Few recent studies demonstrate the anti-inflammatory effects of abdominal VNS in rat models of inflammatory bowel disease, rheumatoid arthritis, and glycemia reduction in a rat model of type 2 diabetes. Rat is a great model to explore the potential of this technology because of the well-established anatomy of the vagus nerve, the large size of the nerve that allows easy handling, and the availability of many disease models. Here, we describe the methods for cleaning and sterilizing the abdominal VNS electrode array and surgical protocol in rats. We also describe the technology required for confirmation of suprathreshold stimulation by recording evoked compound action potentials. Abdominal VNS has the potential to offer selective, effective treatment for a variety of conditions, including inflammatory diseases, and the application is expected to expand similarly to cervical VNS.

Author Spotlight: Exploring Abdominal VNS for Inflammatory Conditions

The present protocol describes the surgical technique for implanting an electrode array onto the abdominal vagus nerve in rats, along with methods for chronic electrophysiology testing and stimulation using the implanted device.

Abdominal vagus nerve stimulation (VNS) can be applied to the subdiaphragmatic branch of the vagus nerve of rats. Due to its anatomical location, it does ...