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>
2. Day of Experiment
<ol>
	<li>Remove the feed 2 h prior to the experiment.</li>
	<li>Inject piglets with an intramuscular injection of ketamine (10&#8211;40 mg/kg)/xylazine (1.5&#8211;3.0 mg/kg IM) and transport via a transport cage to the surgical space.</li>
</ol>
3. Induction and Maintenance of Anesthesia
<ol>
	<li>Administer 4% isoflurane inhalation anesthetic mixed in oxygen by nose cone and confirm that the animal is deeply anesthetized by assessing the absence of palpebral and withdrawal reflexes.</li>
	<li>Intubate the animal by placing it in the supine position and use a laryngoscope (straight blade) to help guide the intubation tube (diameter 2.5&#8211;2 mm) into the trachea.</li>
	<li>Place the animal on the ventilator once intubation tube is secured.</li>
	<li>Ensure that piglets receive a mix of isoflurane (0.25%&#8211;3% maintenance), oxygen, and nitrous oxide.</li>
	<li>Provide a dose of fentanyl (10 &#956;g/kg) and continue giving a dose every 1&#8211;2 h to ensure continued adequate depth of analgesia and sedation and to avoid motion artifacts that could risk dislodgment of the endotracheal tube.</li>
	<li>Establish intravenous (IV) access in the subcutaneous abdominal vein or any other peripheral vein.</li>
	<li>Establish the arterial line through the femoral artery. This can be done non-invasively or by performing a cut-down.</li>
</ol>
4. Monitoring and Care
<ol>
	<li>Monitor the depth of anesthesia by confirming the absence of canthal reflex and absence of withdrawal response to toe pinch.</li>
	<li>Perform continuous monitoring of physiological parameters during anesthesia and throughout the experiment, which includes arterial blood pressure, electrocardiography (ECG), end-tidal CO2, pulse oximetry, and body temperature.</li>
	<li>Monitor blood gases and blood sugars every 0.5&#8211;1 h and give intravenous fluids (50% dextrose and 50% normal saline) to animals anesthetized longer than 1 h at ~100 cc/kg/day, as needed, to ensure euglycemia.</li>
	<li>Monitor the animal&#39;s anesthetic plane closely and frequently. Provide analgesia and/or increase inhalant anesthesia.</li>
	<li>Maintain the animal at normal oxygen tension by controlling the ventilator parameters and drug dosages as needed to ensure normoxia, then place the animal on a temperature-regulated circulating water blanket such that normal body temperature is maintained at 39 &#176;C for the duration of the experiment.</li>
</ol>
5. Brachial Plexus Surgery
<ol>
	<li>Place the animal in a supine position on the operating table after proper anesthesia as described in section 3, with the upper limb in abduction, exposing the axillary region.</li>
	<li>Use any surgical drape to cover the animal. Use clean but non-sterile techniques.</li>
	<li>Expose the brachial plexus complex on both sides of the spine by making a midline incision (using a #10 blade) over the skin and fascia overlying the trachea, down to the upper third of the sternum, corresponding to spine levels between C3&#8211;T3.</li>
	<li>Extrapolate the incision using the forceps and hemostat horizontally on each side from the suprasternal notch along the edge of the clavicle to the upper arm, while sparing the cephalic and basilic veins.</li>
	<li>Release the superior and inferior flaps by blunt dissection using scissors and forceps, allowing access to the cervical and thoracic regions of the brachial plexus, respectively.</li>
	<li>Identify the axis (C2) and first rib at the T1. Using these landmarks, identify the lower three cervical (C6&#8211;C8) and first thoracic (T1) spinal vertebral foramen, then examine the plexus carefully to locate bifurcations of the divisions (M shape) to achieve exposure.</li>
	<li>Label (using nerve loops) the brachial plexus regions above these bifurcations closer to the spine as root/trunk and label those below these bifurcations as chord followed by the nerve, which are located closer to the arm.</li>
</ol>
6. Biomechanical Testing
<ol>
	<li>Set-up of the biomechanical testing device 
	NOTE: A custom-built mechanical testing device was designed and fabricated to perform in vivo stretch of the BP (Figure 1).
	<ol>
		<li>Attach the base of the set-up to a cart.</li>
		<li>Attach the electromechanical actuator onto the base using large C-clamps. The actuator is capable of providing 150 lb of force, 10&#34; stroke, and speed of 15 mm/s. The speed can be reduced to 0.2 mm/s and still function as desired.</li>
		<li>Attach the 200 N load cell to actuator.</li>
		<li>Attach (screw-in) a clamp to the load cell that consists of padded plexiglass, which prevents the stress concentration at the clamping site.</li>
		<li>Attach a camera to a tripod. Ensure that the camera has the capability of recording up to 120 f/s at a resolution of 658 x 4926 pixels.</li>
		<li>Attach USB cables from the camera, actuator, and load cell to the computer to integrate and synchronize all components of the set-up.</li>
		<li>Plug the computer, actuator, and load cell into a power source.</li>
	</ol>
	</li>
	<li>Calibrate the load cell prior to recording the applied loads. To do so, perform the steps below:
	<ol>
		<li>Set the actuator at a 90&#176; angle using the adjustable handle and checking the angle with a protractor.</li>
		<li>Open the software that works with the load cell (Table of Materials). Press the Start button to show a live readout of voltage.</li>
		<li>Hang weights from the clamp ranging from 0&#8211;1,000 g in increments of 100 g from the setup and record the measured voltages.</li>
		<li>Calculate the linear equation of the voltages and weights by finding the slope (m) and intercept (b). This is done using a spreadsheet program and the included slope function to calculate b from the Equation 1 below. Insert Equation 2 shown below into the mechanical set-up code. 
		Equation 1: b = y - mx 
		Where: y is the weight, x is the voltage, m is the slope, and b is the intercept (constant). 
		Equation 2: y = mx + b 
		Where: y is the weight, x is the voltage, m is the slope, and b is the constant.</li>
	</ol>
	</li>
	<li>Testing: the BP nerve is cut and anchored to the testing set-up by custom-built clamp.
	<ol>
		<li>Cut the BP nerve using fine scissors.</li>
		<li>Clamp the cut side of the BP nerve in the custom-built clamp as shown in Figure 1.</li>
		<li>Manually place black acrylic paint or India ink on the clamped BP segment (Figure 2).</li>
		<li>Place a calibration grid, which is a 1 cm ruler, flat within the animal to set the scale for data analysis.</li>
		<li>Use the camera&#39;s software to view the camera&#39;s placement directly over the tested segments, thereby allowing the monitoring of the motion/displacement of the markers and determining the actual tissue strain at any timepoint.</li>
		<li>Record initial measurements such as the height at which the nerve inserts into the body from the table and the height of the clamp from the table, the angle of the actuator, and the full length of the tissue.</li>
		<li>Open the programming software (table containing the graphical user interface [GUI] as shown in Figure 3).</li>
		<li>Run the GUI by pressing the Run button.</li>
		<li>Initialize the system by pressing the Initialize button.</li>
		<li>Tare the system by pressing the Tare button.</li>
		<li>Stretch the BP segment by pressing the start test button. This pulls the tissue at an assigned rate of 500 mm/min until complete failure occurs in any segment of the BP. This stretch rate is selected based on the available literature4,8,18. The program also saves a video file, the applied tensile load, displacement of the tissue, and duration of the test.</li>
		<li>Record the failure site, which is the point at which the tissue ruptures.</li>
	</ol>
	</li>
	<li>Euthanasia: euthanize piglets at the end of the experiment with a lethal dose of pentobarbital (120 mg/kg i.v.).</li>
	<li>Data analysis: use motion tracking software for the analysis of the videos acquired during testing.
	<ol>
		<li>Open the video file from the experiment within the motion tracking software by selecting File | Open Video File.</li>
		<li>Use the calibration grid to setup the scale in the motion tracking software using the Line tool, right-clicking on the line after it is drawn, selecting Calibrate Measure, and entering a known value in centimeters (Figure 4).</li>
		<li>Track the markers on the tissue within the motion tracking software by right-clicking on the video and selecting Track Path and aligning the center of the marker with the marker on the tissue and tacking it until rupture.</li>
		<li>Export the x- and y-coordinates from the markers by selecting File Export to Spreadsheet so that it can be used to calculate the strains.</li>
		<li>Import the data into a programming software to calculate the distance between the x- and y-coordinates over time to calculate the strains.</li>
		<li>Calculate strain values at each timepoint by dividing the change in distance by the original distance after accounting for changes in inclination during stretch. The actual strain values are determined between each pair of adjacent markers at each timepoint. The average of these strains is also calculated.</li>
	</ol>
	</li>
</ol>

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<li>Singh, A., Lu, Y., Chen, C., Cavanaugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+properties+of+spinal+nerve+roots+subjected+to+tension+at+different+strain+rates%5BTitle%5D)+AND+%22Journal+of+Biomechanics%22%5BJournal%5D)">Mechanical properties of spinal nerve roots subjected to tension at different strain rates.</a> Journal of Biomechanics. 39 (9), 1669-1676 (2006).</li>
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</ol>

The authors have nothing to disclose.

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 be due to differences in the tissue processing (e.g., fixed vs. unfixed tissue), methodological differences in measuring elongation, and differences in species used. Moreover, these data are obtained from adult animals or human cadavers and not neonates. Ethical reasons make it difficult to obtain mechanical data from live human neonates, so large animal models that have anatomical similarities to humans may be used instead. Piglets serve as an animal model that has already been used in BP-related studies6,24.
The proposed methods and set-up allow for measuring the in vivo biomechanical response of neonatal BP in a large animal model, offering an understanding of injury mechanism during BP stretch. While the testing protocol and set-up is robust, it offers some limitations (i.e., slips occurring during mechanical testing, loss of marker visibility during testing, movement of the entire body when testing until failure occurs). While slips occur during testing, ensuring proper clamping can minimize slippage. Adding padding can further secure the tissue and avoid slips. Clamps can also be easily substituted with other different types of clamps as needed. Loss of marker visibility occurs in less than 2% cases and are inevitable. Securing the animal torso while testing may require a securing rig. Since the set-up allows tracking of the insertion movement through a camera system, it accounts for any animal movements during testing. An additional limitation of the system is its ability to provide a camera view live through a separate program, thereby limiting live camera view during testing. This can be improved in the future by integrating a live camera view into the program that is currently used to run the test.
In summary, NBPP is a significant injury with life-long sequelae for many individuals. Unfortunately, over the last three decades there has not been a decrease in the rate of its occurrence, despite increased technological development and training of obstetricians. This lack of a decrease in occurrence may directly be attributed to the limitations in developing preventative strategies that minimize the occurrence of NBPP. Preventative strategies cannot be explored until a detailed understanding of the injury mechanism at all levels (i.e., mechanical, functional, and histological) becomes available. No method to date has been reported to measure in vivo BP strains in a neonatal large animal model, and the current study is the first to offer a protocol that further explores physiological and functional changes in neonatal BP tissue post-stretch. By performing tests at various strains, injury threshold values for functional and structural injuries in the neonatal brachial plexus can be reported.

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><tbody><tr><td>Omega Subminature Tension &amp;amp; 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&quot; 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 allows reporting the location of failure being proximal to the foramen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59860/59860fig1.jpg" /> 
Figure 1: Details of in vivo mechanical testing device including the actuator, load cell, and clamp. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59860/59860fig2.jpg" /> 
Figure 2: Markers placed over the BP segments to record strains sustained by the tissue during stretch. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59860/59860fig3.jpg" /> 
Figure 3: Steps for data acquisition using graphical user interface. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59860/59860fig4.jpg" /> 
Figure 4: Marker tracking and strain analysis details. Test videos saved in AVI format are imported in the tracking software. Strain between each marker and the first and last markers are obtained as detailed. An average of between markers strains is used to report the failure strains. An example of nerve stretch with three markers and the calculated average strain-time plot are shown here, with reported failure strains of 43%. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59860/59860fig5.jpg" /> 
Figure 5: Maximum load reported during failure. Load cell attached to the actuator acquires the load data during stretch. The data are used to obtain a load-time plot as shown. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59860/59860fig6.jpg" /> 
Figure 6: Strains reported in four different segments of the stretched plexus. Strains are calculated between each marker and compared against average strains obtained from all four segments (between each of the two adjacent markers). Some regions of the nerve undergo higher strains than others and the average strains indicative of non-uniform injury along the length of the nerve. <a href="https://www.jove.com/files/ftp_upload/59860/59860fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad Científica del Sur

Research

JoVE Journal

Bioengineering

6.0K Views.  Widener University. Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

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

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Medicine

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for TBI. Unlike rodent models for TBI, there are few behavioral tests that have been standardized. A larger animal requires more invasive handling in test areas than rodents, which potentially adds stress and variation to the animals&#39; responses. Here, the human approach test (HAT) is described, which was developed to be performed in front of laboratory pigs&#39; home pen. It is noninvasive, but flexible enough that it allows for differences in housing set-ups.
During the HAT, three behavioral ethograms were developed and then a formula was applied to create an approach index (AI). Results indicate that the HAT and its index, AI, are sensitive enough to detect mild and temporary alterations in pigs&#39; behavior after a mild TBI (mTBI). In addition, although specific behavior outcomes are housing-dependent, the use of an AI reduces variation and allows for consistent measurements across laboratories. This test is reliable and valid; HAT can be used across many laboratories and for various types of porcine models of injury, sickness, and distress. This test was developed for an optimized manual timestamping method such that the observer consistently spends no more than 9 min on each sample.

This protocol describes a new behavioral test&#8212;the human approach test in the pigs&#39; home pen&#8212;to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for ...

Behavior

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Biomechanical Analysis of Peripheral Nerves Using DLT-Calibrated Stereo-Imaging

Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while undergoing surgical repair. Understanding the biomechanical response of peripheral nerves to stretch is critical to the understanding of their response to different loading conditions and thus, to optimizing treatment strategies and surgical interventions. This protocol describes in detail the calibration process of the stereo-imaging camera system via direct linear transformation and the tracking of the three-dimensional in-situ tissue displacement of peripheral nerves during stretch, obtained from three-dimensional coordinates of the video files captured by the calibrated stereo-imaging camera system.
From the obtained three-dimensional coordinates, the nerve length, change in the nerve length, and percent strain with respect to time can be calculated for a stretched peripheral nerve. Using a stereo-imaging camera system provides a non-invasive method for capturing three-dimensional displacements of peripheral nerves when stretched. Direct linear transformation enables three-dimensional reconstructions of peripheral nerve length during stretch to measure strain. Currently, no methodology exists to study the in-situ strain of stretched peripheral nerves using a stereo-imaging camera system calibrated via direct linear transformation. Capturing the in-situ strain of peripheral nerves when stretched can not only aid clinicians in understanding underlying injury mechanisms of nerve damage when overstretched but also help optimize treatment strategies that rely on stretch-induced interventions. The methodology described in the paper has the potential to enhance our understanding of peripheral nerve biomechanics in response to stretch to improve patient outcomes in the field of nerve injury management and rehabilitation.

Stereo-Imaging System DLT Calibration to Capture 3D In Situ Displacements of Stretched Peripheral Nerves

This protocol implements a stereo-imaging camera system calibrated using direct linear transformation to capture three-dimensional in-situ displacements of stretched peripheral nerves. By capturing these displacements, strain induced at varying degrees of stretch can be determined informing the stretch injury thresholds that can advance the science of stretch-dependent nerve repair.

Peripheral nerves undergo physiological and non-physiological stretch during development, normal joint movement, injury, and more recently while ...

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation

Neonatal piglets have been extensively used as translational models for perinatal asphyxia. In 2007, we adapted a well-established piglet asphyxia model by introducing cardiac arrest. This enabled us to study the impact of severe asphyxia on key outcomes, including the time taken for the return of spontaneous circulation (ROSC), as well as the effect of chest compressions according to alternative protocols for cardiopulmonary resuscitation. Due to the anatomical and physiological similarities between piglets and human neonates, piglets serve as good models in studies of cardiopulmonary resuscitation and hemodynamic monitoring. In fact, this cardiac arrest model has provided evidence for guideline development through research on resuscitation protocols, pathophysiology, biomarkers, and novel methods for hemodynamic monitoring. Notably, the incidental finding that a substantial fraction of piglets have pulseless electrical activity (PEA) during cardiac arrest may increase the applicability of the model (i.e., it may be used to study pathophysiology extending beyond the perinatal period). However, the model generation is technically challenging and requires various skill sets, dedicated personnel, and a fine balance of the measures, including the surgical protocols and the use of sedatives/analgesics, to ensure a reasonable rate of survival. In this paper, the protocol is described in detail, as well as experiences with adaptations to the protocol over the years.

This piglet model involves surgical instrumentation, asphyxiation until the cardiac arrest, resuscitation, and post-resuscitation observation. The model allows for multiple sampling per animal, and by using continuous invasive arterial blood pressure, ECG, and non-invasive cardiac output monitoring, it provides knowledge about hemodynamics and cardiac pathophysiology in perinatal asphyxia and neonatal cardiopulmonary resuscitation.

Neonatal piglets have been extensively used as translational models for perinatal asphyxia. In 2007, we adapted a well-established piglet asphyxia model ...

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...

Biology

Measuring Brain Glutamate Levels Under Anesthesia in Neonatal Piglets Using a Microelectrode Array

Source: Geyer, E. D., et al. <a href="https://app.jove.com/v/57391">Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain.</a> J. Vis. Exp. (2018).
This video demonstrates the measurement of glutamate levels in the brain of an anesthetized neonatal piglet using a microelectrode array (MEA). The procedure involves creating a craniotomy window in the pig skull and inserting the MEA into the brain region of interest. The glutamate-oxidase-coated recording sites of the MEA convert extracellular brain glutamate to hydrogen peroxide, which generates a measurable current. This current is measured and normalized against background noise to determine brain glutamate levels under anesthesia.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

Exposing Brachial Plexus: A Surgical Technique to Visualize Brachial Plexus in Neonatal Piglet

This video describes an in vivo surgical procedure to expose the brachial plexus, a neural network that supplies cutaneous and muscular interventions to the upper limb. This surgery helps locate the plexus and eventually assess the extent of nerve injury involving the upper limb.

This video describes an in vivo surgical procedure to expose the brachial plexus, a neural network that supplies cutaneous and muscular interventions to ...

Large Animal Models

Porcine

In Vivo Biomechanical Testing of Nerve: A Procedure for Biomechanical Analysis of Brachial Plexus Nerve Injury in a Neonatal Pig Model

Source: Singh, A., et al. <a href="https://www.jove.com/v/59860">Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets.</a> J. Vis. Exp. (2019).
In this video, we demonstrate the biomechanical testing of the brachial plexus nerve in a pig model to measure the threshold tensile strength of the nerve when subjected to stretching. This technique helps in determining the structural and mechanical properties of the nerve.

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

Summary

Explore More Videos

A Swine Model of Neonatal Asphyxia

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Direct Linear Transformation for the Measurement of In-Situ Peripheral Nerve Strain During Stretching

A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Measuring Brain Glutamate Levels Under Anesthesia in Neonatal Piglets Using a Microelectrode Array

Exposing Brachial Plexus: A Surgical Technique to Visualize Brachial Plexus in Neonatal Piglet

In Vivo Biomechanical Testing of Nerve: A Procedure for Biomechanical Analysis of Brachial Plexus Nerve Injury in a Neonatal Pig Model