The authors would like to thank the Canadian Institutes of Health Research (MOP53116) and the Alberta Heritage Foundation for Medical Research for the operating grant and establishment fund, respectively, to support the development of this experimental model.

1. Anesthesia
<ol>
	<li>Set the flow rate of the anesthetic machine at 2L/min. Connect the exhaust to vacuum suction.</li>
	<li>Charge face mask with anesthetic gas (Isoflurane) at 5% (&tilde;3 min).</li>
	<li>Newborn piglets will be induced with inhaled Isoflurane 5% in 100% oxygen (&tilde;3 min).</li>
	<li>Maintain anesthesia at 2-3% of Isoflurane. Fine adjustment of Isoflurane by 0.5% as appropriate, however, it may range from 0.5 to 5% depending on the condition of piglets.</li>
	<li>Once the vascular access has been established, the inhalational anesthesia can be switched to intravenous anesthesia using fentanyl (5-50 mcg/kg/h) and midazolam (200-500 mcg/kg/h) infusions. Pancuronium (50-100 mcg/kg/h) may be required to control excessive muscle movements during the surgery, whilst the ability to observe animal's state is preserved for the adjustment of anesthetic medications.</li>
	<li>The piglet is monitored by pulse oximetry (percutaneous oxygen saturation at 95-100%) and ECG (heart rate at 130-170 beats/min).</li>
	<li>The piglet's rectal temperature is maintained at 38-40&deg;C with heating blanket and radiant warmer.</li>
	<li>The anesthetic state of piglet is being regularly evaluated throughout the experimental period using neurological (pupil size, tearing, body movements), behavioral (agitation), cardiovascular (tachycardia and hypertension) and respiratory (tachypnoea) parameters as appropriate. Minimal paralysis is given. Previous experience of anesthesia in piglets with and without paralysis would be useful for evaluation.</li>
	<li>The protocol is a non-survival procedure with euthanization of the animal at the end of experiment with an overdose of pentobarbital (100 mg/kg) intravenously.</li>
</ol>
2. Surgical placement of vascular catheters at the groin (Figure 1)
<ol>
	<li>Make a long 2-3cm incision in the right groin.</li>
	<li>Dissect 1cm of the right femoral venous and 1cm right femoral artery. Put two 3-0 strings around each vessel.</li>
	<li>Right femoral venous catheterization: Ligate the distal of the vein. Insert an Argyle catheter (3.5 or 5 French, double- lumen)(Covidien, Mansfield, MA) to 15cm and this will place at the right atrium. Tie both strings to secure the catheter. The catheter can be used for maintenance fluid and medications infusion (secondary port) and central venous/right atrial pressure measurement (primary port).</li>
	<li>Right femoral arterial catheterization: Ligate the distal of the artery. Lift up the proximal string to stop the blood flow. Insert an Argyle catheter (3.5 or 5 French, single-lumen) to 5cm. This will place the arterial catheter at the infra-renal aorta for continuous mean arterial pressure measurement and blood sampling. Tie both strings to secure the catheter.</li>
	<li>Close the skin.</li>
</ol>
3. Establish mechanical ventilation (Figure 2)
<ol>
	<li>Make a long 2-3cm horizontal incision in the neck.</li>
	<li>Dissect and expose 1cm of the trachea. Put two 1-0 strings around the trachea.</li>
	<li>Insert an endotracheal tube (3.0 or 3.5) at 1cm into the trachea. Connect to a ventilator and commence mechanical ventilation. Secure the endotracheal tube.</li>
	<li>Dissect and expose the common carotid artery. Encircle the vessel with a transit time ultrasound flow probe (2SB or 2RB, Transonic Systems Inc., Ithica, NY) to continuously measure the blood flow.</li>
</ol>
4. Placement of flow probes at superior mesenteric (Figure 3) and left renal (Figure 4) arteries
<ol>
	<li>Extra doses of fentanyl (5-10 mcg/kg) and acepromazine (0.01-0.02 mg/kg) are required prior to skin incision.</li>
	<li>Make a long subcostal-flank incision and carefully dissect muscle layers.</li>
	<li>Expose the abdominal aorta.</li>
	<li>Minimize vascular handling (vasospasm) and lymphatic injury.</li>
	<li>Dissect 0.5-1cm superior mesenteric artery and put a Transonic flow probe (3SB) around it.</li>
	<li>Dissect 0.5-1cm left renal artery and put a Transonic flow probe (2SB) around it.</li>
	<li>Close the skin and secure the flow probe.</li>
</ol>
5. Placement of pulmonary artery catheter (Figure 5) and flow probe (Figure 6)
<ol>
	<li>Extra doses of fentanyl (5-10 mcg/kg) and acepromazine (0.01-0.02 mg/kg) are required prior to skin incision.</li>
	<li>Lie the animal at the right lateral position.</li>
	<li>Thoracotomy at the left 4th intercostal space.</li>
	<li>Watch out for the internal mammary artery and vein, ligate if needed.</li>
	<li>Use a dental swab to press down the left lung and increase oxygen as needed.</li>
	<li>Open the pericardium.</li>
	<li>Identify the ductus arteriosus which runs from the pulmonary artery to the aorta.</li>
	<li>Ductus arteriosus may be ligated by placing a clip or by a thick "3-O silk" tie at its origin.</li>
	<li>Free the main pulmonary artery and pass a vascular sling using a thick "0" tie.</li>
	<li>Perform a purse string (5-0 prolene) suture at the base for the placement of pulmonary artery catheter.</li>
	<li>Insert a 20G Angiocath (with 3 side holes at less 1 cm from the tip of the catheter) through the purse string to a maximum of 1 cm.</li>
	<li>Check for free flow of venous blood.</li>
	<li>Connect to pressure transducer, check for pulmonary artery pressure and waveform.</li>
	<li>Tighten the purse string and secure the pulmonary catheter.</li>
	<li>Place a Transonic flow probe (6SB) around the main pulmonary artery.</li>
	<li>Place ultrasonic gel between the flow probe and artery to allow for optimal signal transduction.</li>
	<li>Cover the wound with moist saline gauze.</li>
</ol>
6. Hypoxia and reoxygenation protocol
<ol>
	<li>Decrease the inspired oxygen concentration to 10% by increasing the concentration of inhaled nitrogen gas to induce hypoxemia.</li>
	<li>Adjust the inspired oxygen concentration between 10% and 15% to obtain a PaO2 of 20-40 mmHg or SaO2 of 30-40% for 2h.</li>
	<li>Perform arterial blood analysis to assess PaCO2 and adjust ventilator rate accordingly.</li>
	<li>With the induction of hypoxemia, the first hour is dedicated to steadily inducing a tachycardic (and cardiac output) response.</li>
	<li>Continue to monitor for changes in blood flow at the common carotid, superior mesenteric and left renal arteries.</li>
	<li>During the second hour of hypoxia, the hypoxic stress is increased to steadily lower cardiac output to 30-40% of baseline, mean arterial pressure to 30-35 mmHg and arterial pH 6.95-7.05.</li>
	<li>Hypoxic stress may be prematurely terminated or extended by 15 min as appropriate.</li>
	<li>Increase inspired oxygen concentration abruptly to 100% abruptly by discontinuing nitrogen gas, while continuing pure oxygen.</li>
	<li>Monitor cardiac output, mean arterial pressure and other hemodynamic parameters for rapid recovery.</li>
	<li>Resuscitation with 100% oxygen can be continued for 0.5h. Following this time period, reduce the inspired oxygen concentration quickly to 21%.</li>
	<li>Continue reoxygenation with 21% oxygen for the remaining period of experiment. The inspired oxygen concentration can be titrated to 25% if needed.</li>
	<li>Fluid boluses of 10 ml/kg Ringer's lactate solution may be needed as appropriate during the experimental period. Its use has to be protocolized.</li>
</ol>
7. Representative Results:
The induction of hypoxemia in the newborn piglet over the first hour of hypoxia should increase the cardiac output (pulmonary arterial flow) to 120%-130% of baseline (Figure 7A) and heart rate (Figure 7B). Typically, cardiac output should reach its peak compensation between the first 0.5h and 1h of hypoxia. Further, blood flow should become centralized resulting in decreased mesenteric and renal perfusion but a preserved or increased common carotid arterial flow (Figure 8). During the second hour of hypoxia, there is a steady decrease of cardiac output, development of hypotension (Figure 9A), slowing of heart rate with or without arrhythmia occurred. Hypoxia should induce pulmonary hypertension with increased pulmonary artery pressure (Figure 9B), which may sometimes lower in the final 30 min of hypoxia as the cardiac output decreases.
Upon resuscitation, all hemodynamic parameters will immediately recover to normoxic baseline, except for the renal blood flow which gradually recovers over the first hour of reoxygenation. However, the hemodynamic parameters especially for the cardiac output and mean arterial pressure will gradually deteriorate over the first 2 hour of the reoxygenation to about 70-75% of normoxic baseline and 35-45 mmHg, respectively. This cardiovascular dysfunction is at least in part for myocardial stunning and warrants cardiovascular supportive therapies such as vasoactive and inotropic agents.
<img alt="Figure 1" src="/files/ftp_upload/3166/3166fig1.jpg" /> 
Figure 1: Groin incision with the placement of femoral arterial and venous catheters
<img alt="Figure 2" src="/files/ftp_upload/3166/3166fig2.jpg" /> 
Figure 2: Neck incision with the placement of an endotracheal tube and a flow probe around the common carotid artery
<img alt="Figure 3" src="/files/ftp_upload/3166/3166fig3.jpg" /> 
Figure 3: Flank incision with the isolation of superior mesenteric artery
<img alt="Figure 4" src="/files/ftp_upload/3166/3166fig4.jpg" /> 
Figure 4: Flank incision with the isolation of left renal artery
<img alt="Figure 5" src="/files/ftp_upload/3166/3166fig5.jpg" /> 
Figure 5: Thoracotomy with the placement of pulmonary artery catheter
<img alt="Figure 6" src="/files/ftp_upload/3166/3166fig6.jpg" /> 
Figure 6: Thoracotomy with the placement of a Transonic flow probe around main pulmonary artery
<img alt="Figure 7" src="/files/ftp_upload/3166/3166fig7.jpg" /> 
Figure 7: Temporal changes in (A) cardiac output (pulmonary arterial flow) and (B) heart rate during hypoxia and reoxygenation
<img alt="Figure 8" src="/files/ftp_upload/3166/3166fig8.jpg" /> 
Figure 8: Temporal changes in blood flow at (A) common carotid, (B) superior mesenteric and (C) left renal arteries during hypoxia and reoxygenation
<img alt="Figure 9" src="/files/ftp_upload/3166/3166fig9.jpg" /> 
Figure 9: Temporal changes in (A) mean arterial pressure and (B) pulmonary artery pressure during hypoxia and reoxygenation

<ol>
	<li>Borke, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myocardial+matrix+metalloproteinases+in+hypoxic+newborn+pigs+during+resuscitation:+effects+of+oxygen+and+carbon+dioxide.">Increased myocardial matrix metalloproteinases in hypoxic newborn pigs during resuscitation: effects of oxygen and carbon dioxide.</a> Eur. J. Clin. Invest. 34, 459-466 (2004).</li><li>Munkeby, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitation+of+hypoxic+piglets+with+100%+O2+increases+pulmonary+metalloproteinases+and+IL-8.">Resuscitation of hypoxic piglets with 100% O2 increases pulmonary metalloproteinases and IL-8.</a> Pediatr. Res. 58, 542-548 (2005).</li><li>Haaland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posthypoxic+hypothermia+in+newborn+piglets.">Posthypoxic hypothermia in newborn piglets.</a> Pediatr. Res. 41, 505-512 (1997).</li><li>Haase, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitation+with+100%+oxygen+causes+intestinal+glutathione+oxidation+and+reoxygenation+injury+in+asphyxiated+newborn+piglets.">Resuscitation with 100% oxygen causes intestinal glutathione oxidation and reoxygenation injury in asphyxiated newborn piglets.</a> Ann. Surg. 240, 364-373 (2004).</li><li>Stevens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitation+with+21%+or+100%+oxygen+is+equally+effective+in+restoring+perfusion+and+oxygen+metabolism+in+hypoxic+newborn+piglet+liver.">Resuscitation with 21% or 100% oxygen is equally effective in restoring perfusion and oxygen metabolism in hypoxic newborn piglet liver.</a> Shock. 27, 657-662 (2007).</li><li>Johnson, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylcysteine+improves+the+hemodynamics+and+oxidative+stress+in+hypoxic+newborn+pigs+reoxygenated+with+100%+oxygen.">N-acetylcysteine improves the hemodynamics and oxidative stress in hypoxic newborn pigs reoxygenated with 100% oxygen.</a> Shock. 28, 484-490 (2007).</li><li>Chapados, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cortisol+response+to+ACTH+challenge+in+hypoxic+newborn+piglets+resuscitated+with+21%+and+100%+oxygen.">Plasma cortisol response to ACTH challenge in hypoxic newborn piglets resuscitated with 21% and 100% oxygen.</a> Shock. 33, 519-525 (2010).</li><li>Cheung, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+dysfunction+in+asphyxiated+newborn+piglets+resuscitated+with+21%+and+100%+oxygen.">Platelet dysfunction in asphyxiated newborn piglets resuscitated with 21% and 100% oxygen.</a> Pediatr. Res. 59, 636-640 (2006).</li><li>Martin-Ancel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+organ+involvement+in+perinatal+asphyxia.">Multiple organ involvement in perinatal asphyxia.</a> J. Pediatr. 127, 786-793 (1995).</li><li>Swindle, M. M., Smith, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+and+physiology+of+the+pig.">Comparative anatomy and physiology of the pig.</a> Scan. J. Lab. Anim. Sci. Suppl. 25, 11-22 (1998).</li><li>Chapados, I., Cheung, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+all+models+are+created+equal:+Animal+models+to+study+hypoxic-ischemic+encephalopathy+of+the+newborn.">Not all models are created equal: Animal models to study hypoxic-ischemic encephalopathy of the newborn.</a> Neonatology. 94, 300-303 (2008).</li><li>Liu, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+post-resuscitation+treatment+with+N-acetylcysteine+on+cardiac+recovery+in+hypoxia-injured+newborn+pigs.">Effects of post-resuscitation treatment with N-acetylcysteine on cardiac recovery in hypoxia-injured newborn pigs.</a> PLoS ONE. 5, e15322-e15322 (2010).</li><li>Cheung, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardio-renal+recovery+of+hypoxic+newborn+pigs+after+18%,+21%+and+100%+reoxygenation.">Cardio-renal recovery of hypoxic newborn pigs after 18%, 21% and 100% reoxygenation.</a> Intensive Care Med. 34, 1114-1121 (2008).</li><li>Temesvari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+blood-brain+barrier+permeability+in+neonatal+cytotoxic+brain+edema:+laboratory+and+morphological+findings+obtained+on+newborn+piglets+with+experimental+pneumothorax.">Modulation of the blood-brain barrier permeability in neonatal cytotoxic brain edema: laboratory and morphological findings obtained on newborn piglets with experimental pneumothorax.</a> Biol. Neonate. 46, 198-208 (1984).</li><li>Domoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reventilation+with+room+air+or+100%+oxygen+after+asphyxia+differentially+affects+cerebral+neuropathology+in+newborn+pigs.">Reventilation with room air or 100% oxygen after asphyxia differentially affects cerebral neuropathology in newborn pigs.</a> Acta. Paediatr. 95, 1109-1115 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+15:+Neonatal+resuscitation:+2010+American+Heart+Association+guidelines+for+cardiopulmonary+resuscitation+and+emergency+cardiovascular+care.">Part 15: Neonatal resuscitation: 2010 American Heart Association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care.</a> Circulation. 122, S909-S919 (2010).</li></ol>

No conflicts of interest declared.

The current experimental protocol has an advantage to examine the systemic and regional hemodynamic changes in neonatal subjects during the hypoxia and reoxygenation process. We can also examine the respective effect of interventions used to improve the cardiovascular function during recovery. We and others have reported the experience and findings in the study of neonatal asphyxia regarding the effects in cardiovascular1, pulmonary2, neurologic3, gastrointestinal4, hepatic<sup...

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.

Watch this Scientific Journal Video about A Swine Model of Neonatal Asphyxia at JoVE.com

A Swine Model of Neonatal Asphyxia

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, ...

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14.2K Views.  University of Alberta. 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.

Video: A Swine Model of Neonatal Asphyxia

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

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, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

To closely mimic the mode of burn injuries requires the interplay between clinical observation and studies in animal models. In this study, a swine model of severe burn injury was established to assess an experimental dressing in physiological and pathophysiological settings.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...