Name: methods for in vivo biomechanical testing on brachial plexus in neonatal piglets
Uploaded: 2019-12-19T12:00:00
Description: Watch this Scientific Journal Video about Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets at JoVE.com

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and by the National Science Foundation CAREER Award Number 1752513.

Institutional Animal Care and Use Committee at Drexel University approved all procedures (#20704).
1. Animal Arrival and Acclimation
<ol>
	<li>Quarantine 1&#8211;2 day-old piglets for at least 24 h after arrival.</li>
	<li>House piglets in clean and sanitized stainless-steel cages (36 in x 48 in x 36 in) on aspen chip bedding and feed ad libitum with pig milk replacer.</li>
	<li>Maintain the room temperature at 85 &#176;F to ensure a thermo-neutral environment.</li>
</ol>
<p class="jove_titl...

<ol>
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J., Barkmeier-Kraemer, J. M., Geest, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vande+Biomechanical+properties+of+recurrent+laryngeal+nerve+in+the+piglet.">Vande Biomechanical properties of recurrent laryngeal nerve in the piglet.</a> Annals of Biomedical Engineering. 38 (8), 2553-2562 (2010).</li><li>Zilic, L., 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=An+anatomical+study+of+porcine+peripheral+nerve+and+its+potential+use+in+nerve+tissue+engineering.">An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering.</a> Journal of Anatomy. 227 (3), 302-314 (2015).</li><li>Gonik, B., Zhang, N., Grimm, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+brachial+plexus+stretching+during+shoulder+dystocia+using+a+computer+simulation+model.">Prediction of brachial plexus stretching during shoulder dystocia using a computer simulation model.</a> American Journal of Obstetrics and Gynecology. 189 (4), 1168-1172 (2003).</li><li>Grimm, M. J., Costello, R. 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J., 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=Upper+limb+tension+tests+as+tools+in+the+diagnosis+of+nerve+and+plexus+lesions+-+Anatomical+and+biomechanical+aspects.">Upper limb tension tests as tools in the diagnosis of nerve and plexus lesions - Anatomical and biomechanical aspects.</a> Clinical Biomechanics. 15 (1), 9-14 (2000).</li><li>Zapa&#322;owicz, K., Radek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+human+brachial+plexus.">Mechanical properties of the human brachial plexus.</a> Neurologia, i Neurochirurgia Polska. 34 (6), 89-93 (2000).</li><li>Singh, A., Lu, Y., Chen, C., Cavanaugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+spinal+nerve+roots+subjected+to+tension+at+different+strain+rates.">Mechanical properties of spinal nerve roots subjected to tension at different strain rates.</a> Journal of Biomechanics. 39 (9), 1669-1676 (2006).</li><li>Singh, A., Lu, Y., Chen, C., Kallakuri, S., Cavanaugh, J. 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Available literature on the biomechanical responses of stretch on the BP tissue exhibit a wide range of threshold values as well as methodological discrepancies4,6,8,18,19,20,21,22,23. Variations in published results could...

Despite recent advances in obstetrics, the problem of NBPP caused by stretch injury to the BP complex continues to be a global health burden, with an incidence of 1.5 cases per 1,000 live births1,2. Associated risk factors can be maternal (i.e., excessive weight, maternal diabetes, uterine abnormalities, history of BP paralysis), fetal (i.e., fetal macrosomia), or birth-related (i.e., shoulder dystocia, prolonged labor, assisted delivery with forceps or vacuum extractors, breech presentation3). While these complications are unavoidable in certain circumstances, prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of the neonatal BP when subjected to stretch.
Reported biomechanical studies on the BP have used adult animals and human cadaveric tissue and show significant discrepancies4,5,6,7,8,9,10,11,12,13,14,15. Clinical relevancy of biomechanical properties of the complex BP tissue warrants a neonatal animal model as well as an in vivo biomechanical testing approach. Furthermore, limitations with studying BP stretch injury in complicated real-world delivery scenarios increases the reliance on computer models that provide methods that allows investigation of the effects of various delivery complications and techniques. The key to clinical relevance of these models is their biofidelity (human-like response). Available computational models by Gonik et al.16 and Grimm et al.17 rely on rabbit and rat nerve tissue but not neonatal BP tissue. Performing in vivo biomechanical testing in a clinically relevant neonatal animal model can fill the critical gap of unavailable neonatal BP data.
The current study describes an in vivo mechanical testing device and procedure to conduct biomechanical testing in 3-5 day-old male Yorkshire neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads during failure. The camera system also allows monitoring of the failure location during rupture. Overall, the system allows for detailed biomechanical characterization of the neonatal BP when subjected to stretch, thereby providing the BP's threshold strains and stresses for mechanical failure in vivo. The data obtained can further improve human-like behavior (biofidelity) of the existing computational models that are designed to investigate the effects of exogenous and endogenous forces on BP stretch in delivery scenarios associated with NBPP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Omega Subminature Tension &#38; Compression Load Cell</td>
			<td>Omega</td>
			<td>LCM201-200N</td>
			<td>200N load cell</td>
		</tr>
		<tr>
			<td>Basler acA640-120uc camera</td>
			<td>Basler</td>
			<td>acA640-120uc</td>
			<td></td>
		</tr>
		<tr>
			<td>Feedback Linear Actuator</td>
			<td>Progressive Automations</td>
			<td>PA-14P</td>
			<td>10&#34; stroke, 150lb force, 15mm/s speed</td>
		</tr>
		<tr>
			<td>Motion Tracking Software</td>
			<td>Kinovea</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>Proramming Software - MATLAB</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>version 2018A</td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>11006-12 and 11027-12 or 11506-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fine Science Tools Inc</td>
			<td>13009-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>14094-11 or 14060-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for in vivo biomechanical testing on brachial plexus in neonatal piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

A representative load-time plot and strains from four segments of BP plexus (between four markers) are shown in Figure 5 and Figure 6, respectively. The obtained failure load of 8.3 N at 35% average failure strain reports the biomechanical responses of neonatal BP when subjected to stretch. Some regions of the nerve undergo higher strains than others, indicative of non-uniform injury along the length of the nerve. The camera data...

Watch this Scientific Journal Video about Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets at JoVE.com

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

This technique replicates in vivo biomechanical stretch testing of the brachial plexus in a piglet, serving as a highly relevant clinical large neonatal animal model. These methods can not only help in understanding stretch injury mechanisms but can also report the injury threshold values for functional and structural deficits within the neonatal brachial plexus. Demonstrating the procedure will be Rachel Magee, a graduate student from my lab.After confirming a lack of palpebral and withdrawal reflexes, place the anesthetized pig in the supine position on the operating table, with the upper limb in abduction to expose to axillary region. Place a drape over the animal and use a number 10 scalpel blade to make incisions over the marked skin. Midline incision is overlying the trachea down to the upper third of the sternum, exposing the brachial plexus complex on both sides of the spine.To expose one side of the animal's brachial plexus, a superior incision is made from the upper end of the midline incision, corresponding to C3, to the upper arm, and an inferior incision is made from the lower end of the midline incision, corresponding to T3, to the upper arm. Use forceps on each side of the incision to separate tissue from the suprasternal notch along the edge of the clavicle to the upper arm while sparing the cephalic and basilic veins. Using scissors, forceps, and blunt dissection, release the superior flap to access the cervical region of the brachial plexus, and the inferior flap to access the thoracic region of the brachial plexus.Perform blunt dissection on superficial muscles to expose the brachial plexus. Then examine the plexus carefully to locate bifurcations of the divisions and identify the brachial plexus regions below the bifurcations, closer to the arm, as the cord and the nerve and the regions above the bifurcations, closer to the spine, as the root or trunk. To set up the biomechanical testing device, attach the base of the device to a cart and use large C-clamps to attach the electromechanical actuator to the base.Attach a 200-newton load cell to the actuator and screw in a clamp with padded plexiglass to prevent stress concentration at the clamping site. Using a tripod, attach a camera that can record up to 100 frames per second at a 658 by 492-pixel resolution and attach USB cables from the camera, actuator, and load cell to the computer to integrate and synchronize all of the components of the setup. Then plug the computer, actuator, and load cell into a power source.To calibrate the load cell before recording the applied loads, use the adjustable handle to set the actuator at a 90-degree angle such that it is aligned vertically and check the angle with a protractor. Open the load cell software and click Start to show a live readout of the voltage. Next hang zero to 1, 000-gram weights from the clamp in 100-gram increments, recording the measured voltages at each load.When the voltages have been recorded for all 10 weights, calculate the slope and intercept to determine the linear equation of the voltages and weights. For biomechanical testing of the isolated brachial plexus nerve, use fine scissors to cut the nerve and use a custom clamp to clamp the cut side of the nerve. Label the clamped nerve segment with black acrylic paint and place a one-centimeter ruler flat within the animal to set the scale for the data analysis.In the camera software, place the camera field of view directly over the tested segments to allow monitoring of the motion and/or displacement of the markers and to determine the actual tissue strain at a specific time point. Record the baseline measurements such as the height at which the nerve inserts into the body from the table, the height of the clamp from the table, the angle of the actuator, and the full length of the tissue. Open the programming software and click Run.Enter the file name and displacement and click Initialize and TARE. Click Start to stretch the brachial plexus segment. The tissue will be pulled at an assigned rate of 500 millimeters per minute until complete failure occurs in any segment of the nerve tissue.Then save a video file, the applied tensile load, the displacement of the tissue, and the duration of the test, and record the failure site as the segment at which the tissue ruptures. In this representative test of four brachial plexus segments, the obtained failure load was 8.3 newtons and the average strain failure was 35%for the neonatal nerve tissue samples when subjected to stretch. Some regions of the nerve underwent a higher strain than others, indicating a nonuniform injury along the length of the nerve.The camera data allowed identification of the location of failure in this experiment as proximal to the foramen. It is important to ensure that first, the animal is deeply anesthetized and shows no sign of pain or discomfort, second, to calibrate the load cell, and third, to clamp the tissue firmly. This in vivo animal model can be used to study functional and histological changes in the brachial plexus tissue after varying degrees of stretches and to study complicated birthing scenarios.

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