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>
	<li>Chauhan, S. P., Blackwell, S. B., Ananth, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+brachial+plexus+palsy:+Incidence,+prevalence,+and+temporal+trends.">Neonatal brachial plexus palsy: Incidence, prevalence, and temporal trends.</a> Seminars in Perinatology. 38 (4), 210-218 (2014).</li><li>Foad, S. L., Mehlman, C. T., Ying, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+neonatal+brachial+plexus+palsy+in+the+United+States.">The epidemiology of neonatal brachial plexus palsy in the United States.</a> Journal of Bone and Joint Surgery - Series A. 90 (60), 1258-1264 (2008).</li><li>Garc&#237;a Cena, C. E., 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=Skeletal+modeling,+analysis+and+simulation+of+upper+limb+of+human+shoulder+under+brachial+plexus+injury.">Skeletal modeling, analysis and simulation of upper limb of human shoulder under brachial plexus injury.</a> Advances in Intelligent Systems and Computing. 252, 195-207 (2014).</li><li>Marani, E., van Leeuwen, J. L., Spoor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tensile+testing+machine+applied+in+the+study+of+human+nerve+rupture:+a+preliminary+study.">The tensile testing machine applied in the study of human nerve rupture: a preliminary study.</a> Clinical Neurology and Neurosurgery. 95, S33-S35 (1993).</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., Shaji, S., Delivoria-Papadopoulos, M., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+Responses+of+Neonatal+Brachial+Plexus+to+Mechanical+Stretch.">Biomechanical Responses of Neonatal Brachial Plexus to Mechanical Stretch.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 13 (1), e8-e14 (2018).</li><li>Driscoll, P. 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=An+in+vivo+study+of+peripheral+nerves+in+continuity:+biomechanical+and+physiological+responses+to+elongation.">An in vivo study of peripheral nerves in continuity: biomechanical and physiological responses to elongation.</a> Journal of Orthopaedic Research. 20 (2), 370-375 (2002).</li><li>Zapalowicz, 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=Experimental+investigations+of+traction+injury+of+the+brachial+plexus.+Model+and+results.">Experimental investigations of traction injury of the brachial plexus. Model and results.</a> Annales Academiae Medicae Stetinensis. 51 (2), 11-14 (2005).</li><li>Ma, Z., 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=In+vitro+and+in+vivo+mechanical+properties+of+human+ulnar+and+median+nerves.">In vitro and in vivo mechanical properties of human ulnar and median nerves.</a> Journal of Biomedical Materials Research - Part A. 101 (9), 2718-2725 (2013).</li><li>Rydevik, B. 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+in+vitro+mechanical+and+histological+study+of+acute+stretching+on+rabbit+tibial+nerve.">An in vitro mechanical and histological study of acute stretching on rabbit tibial nerve.</a> Journal of Orthopaedic Research. 8 (5), 694-701 (1990).</li><li>Kwan, M. K., Wall, E. J., Massie, J., Garfin, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain,+stress+and+stretch+of+peripheral+nerve+rabbit+experiments+in+vitro+and+in+vivo.">Strain, stress and stretch of peripheral nerve rabbit experiments in vitro and in vivo.</a> Acta Orthopaedica. 63 (3), 267-272 (1992).</li><li>Takai, S., 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=In+situ+strain+and+stress+of+nerve+conduction+blocking+in+the+brachial+plexus.">In situ strain and stress of nerve conduction blocking in the brachial plexus.</a> Journal of Orthopaedic Research. 20 (6), 1311-1314 (2002).</li><li>Zhe, S., Feng, T., Sun, C., Ma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+mechanical+properties+of+the+brachial+plexus+of+experimental+animals.">Tensile mechanical properties of the brachial plexus of experimental animals.</a> Journal of Clinical Rehabilitative Tissue Engineering Research. 14 (20), 3730-3733 (2010).</li><li>Alexander, M. 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. E., Gonik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+clinician-applied+maneuvers+on+brachial+plexus+stretch+during+a+shoulder+dystocia+event:+Investigation+using+a+computer+simulation+model.">Effect of clinician-applied maneuvers on brachial plexus stretch during a shoulder dystocia event: Investigation using a computer simulation model.</a> Obstetrical and Gynecological Survey. 203 (4), (2011).</li><li>Kawai, 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=Stretching+of+the+brachial+plexus+in+rabbits.">Stretching of the brachial plexus in rabbits.</a> Acta Orthopaedica. 60 (6), 635-638 (1989).</li><li>Narakas, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesions+found+when+operating+traction+injuries+of+the+brachial+plexus.">Lesions found when operating traction injuries of the brachial plexus.</a> Clinical Neurology and Neurosurgery. 95, S56-S64 (1993).</li><li>Kleinrensink, G. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+traumatic+axonal+injury+to+determine+the+effects+of+strain+and+displacement+rates.">A new model of traumatic axonal injury to determine the effects of strain and displacement rates.</a> Stapp Car Crash Journal. 50, 601-623 (2006).</li><li>Gonik, B., 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+timing+of+congenital+brachial+plexus+injury:+A+study+of+electromyography+findings+in+the+newborn+piglet.">The timing of congenital brachial plexus injury: A study of electromyography findings in the newborn piglet.</a> American Journal of Obstetrics and Gynecology. 178 (4), 688-695 (1998).</li></ol>

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.

methods-for-vivo-biomechanical-testing-on-brachial-plexus-neonatal

Research

JoVE Journal

Bioengineering

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...