The authors would like to thank John Kristensen and S&#248;ren Braad Andersen from the Department of Communication, Aarhus University Hospital, Denmark, for their exceptional help with filming and editing. Animal technician Diana Gyldenl&#248;ve and veterinarian Birgitte Kousgaard, Institute of Clinical Medicine Aarhus University Hospital, Denmark for assisting with animal care. This study was supported by the Lundbeck Foundation, the Laerdal Foundation for Acute Medicine, Central Denmark Region&#8217;s Research Foundation, Augustinus Foundation, Aase and Ejnar Danielsens Foundation, the Institute of Clinical Medicine Aarhus University Hospital, Br&#248;drene Hartmanns Foundation, Karen Elise Jensens Foundation, Fonden til L&#230;gevidenskabens Fremme, and Marie Dorthea og Holger From, Haderslevs Fond.

The present protocol was approved by the Danish Animal Experiments Inspectorate. All test animals were anesthetized throughout the procedures. Reproduction of this protocol must be carried out in accordance with national ethics and animal welfare guidelines, and approved by local ethics committees.
1. Animals
<ol>
	<li>Danish Landrace piglets &#60; 24 hr old weighing approximately 1,500 - 2,000 g.</li>
</ol>
2. Anesthesia and Maintenance Fluids
<ol>
	<li>Prepare the equipment needed for anesthesia (Figure 1): A mask for sevoflurane administration, alcohol swabs, peripheral intravenous catheter, rubber band, syringes with saline, propofol (5 mg/kg), fentanyl (10 &#181;g/kg), and procaine benzylpenicillin (15,000 IU/kg).</li>
	<li>Induce anesthesia by delivering 1-2% sevoflurane through a breathing mask.
	<ol>
		<li>Assess the depth of anesthesia by assessing for palpebral and withdrawal reflexes. When certain that the piglet is deeply anesthetized, percutaneously insert a peripheral intravenous catheter into the ear vein.</li>
		<li>To confirm patency of the peripheral intravenous catheter, flush the catheter with 1-2 ml of sterile 0.9% saline. Administer bolus injections of propofol (5 mg/kg) and fentanyl (10 &#181;g/kg). Subsequent to bolus injection administration flush the intravenous catheter a second time using 1-2 ml of sterile 0.9% saline.</li>
		<li>Place syringes with propofol (10 mg/ml) and fentanyl (10 &#181;g/ml) in two separate syringe infusion pumps. Connect i.v. tubes from the two syringe pumps to a three-way stopcock joining infusions into a single line that is connected to the intravenous catheter. Start continuous i.v. infusion of propofol (4-12 mg/kg/hr) and fentanyl (10 &#956;g/kg/hr). Once continuous infusions are running, do not give further bolus injections of propofol and fentanyl.</li>
		<li>Discontinue administration of the sevoflurane gas anesthesia.</li>
		<li>Inject procaine benzylpenicillin (15,000 IU/kg) subcutaneously or intramuscularly as per local guidelines for antibiotic prophylaxis. Repeat daily. The video shows s.c. administration, which in piglets has been shown to result in higher plasma concentration and longer half-life when compared to i.m. administration.</li>
		<li>Apply eye lubricant ointment to prevent drying of the eyes. Pull the lower eyelid down gently to form a pocket-like opening. Squeeze a small amount of ointment inside the pocket. Close eye to distribute ointment. Check for dryness hourly and reapply as needed.</li>
		<li>Initiate a continuous rate infusion of 5% dextrose/0.45% NaCl i.v. at 10 ml/kg/hr. Reduce rate to 5 ml/kg/hr during and after hypoxia. Adjust infusion rate to maintain blood glucose levels between 2-10 mmol/L.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52454/52454fig1.jpg" /> 
Figure 1. Equipment for anesthesia and intubation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Intubation and Ventilation
<ol>
	<li>Prepare the equipment needed for intubation (Figure 1): rocuronium bromide (1 mg/kg) for muscle relaxation, a rope sling (tie both ends of the same 4 mm nylon rope together to form a circle as depicted in Figure 1) for opening the mouth, a cotton tipped swab and veterinary laryngoscope with straight blade, xylocaine spray (100 mg/ml), various sized endotracheal tubes with cuff (sizes 3.0 mm, 2.5 mm, 2.0 mm), 500 ml self-inflating bag (bag valve mask) for ventilation, 2 ml syringe for cuff inflation, and stethoscope.</li>
	<li>Intubate by following the steps below:
	<ol>
		<li>Place piglet in the supine position supporting the neck from each side to secure a straight laryngeal passage for intubation.</li>
		<li>Estimate the length of the endotracheal tube by measuring from the tip of the snout to the sternal notch (typically around 13 cm).</li>
		<li>Place rope sling around the upper jaw (facing down) and hold lower jaw and tongue upward to keep the mouth open.</li>
		<li>Administer rocuronium bromide (1 mg/kg) i.v. to induce muscle relaxation.</li>
		<li>Use laryngoscope to lift the tongue upwards.</li>
		<li>Use the cotton tipped swab to free the piglet&#8217;s long epiglottis, which may be either retroverted into the esophagus or caught behind the soft palate.</li>
		<li>Advance laryngoscope to keep the epiglottis lifted against the tongue base enabling full view of the arytenoid cartilages and vocal cords.</li>
		<li>Apply xylocaine spray (100 mg/ml) topically in the larynx to prevent laryngeal spasms.</li>
		<li>Advance endotracheal tube through the vocal cords. Use a rotating movement to aid the passing of the narrow tracheal cartilages. Advance tube according to the premeasured distance and connect to a self-inflating bag (bag valve mask) for manual ventilation. To help reduce friction during intubation spray the distal 1/3 of the endotracheal tube with xylocaine spray.</li>
		<li>To confirm correct endotracheal tube placement: observe for any indication of difficulty breathing, ausculate the chest listening for bilateral air entry into both lungs, visually confirm the presence of condensation in the proximal aspect of the endotracheal tube, and verify the presence of end tidal carbon dioxide with a colorimetric carbon dioxide detector or by expiratory CO2 reading on the mechanical ventilator if available. Normal end tidal carbon dioxide is around 5%. A value above 2% together with a normal-appearing waveform will confirm that the endotracheal tube is in the trachea.</li>
		<li>Inflate the endotracheal tube cuff to prevent aspiration. Cuff inflation pressure should be less than 25 cm H2O to avoid ischemic damage to the surrounding tissue. Holding the endotracheal tube in place, wind a piece of tape around the tube, close the jaw and continue taping around the snout to secure the tube in place. Gently pull on the tube to ensure that it stays in place.</li>
		<li>Connect endotracheal tube to mechanical ventilator.</li>
	</ol>
	</li>
	<li>Adjust ventilator settings: volume-controlled ventilation, tidal volume (TV): 10 ml/kg [or peak inspiratory pressure (PIP) 15 cm with pressure controlled ventilation]. Positive end expiratory pressure (PEEP): 5 cm. I:E ratio 1:2. Respiratory rate: 35 (adjust rate to maintain end-tidal CO2 between 4.5-5.5 kPa).</li>
</ol>
4. Monitoring and Body Fluid Sampling
<img alt="Figure 2" src="/files/ftp_upload/52454/52454fig2.jpg" /> 
Figure 2. Equipment for monitoring. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Prepare the equipment needed for monitoring (Figure 2): adhesive tape, sterile lubricant, probe for continuous rectal temperature measurement, electrocardiogram (ECG) electrodes, pulse oximeter, razor, and EEG electrodes.
	<ol>
		<li>Place saturation probe on a hind leg, lubricate rectal temperature probe and insert 6 cm into the rectum, place overhead radiant heater and/or heated inflatable air mattress to maintain the rectal temperature at the physiologic level of 38.5-39 &#176;C, and place ECG electrodes.</li>
		<li>Place piglet in the prone position to shave areas of 1 cm x 1 cm for placement of subcutaneous needle EEG electrodes; one in front of each ear, and the reference electrode in the midline just behind the eyes. Clean the electrode site with an alcohol swab, then insert the needle electrode subcutaneously. Secure electrodes with adhesive tape and return piglet to the supine position.</li>
		<li>Turn on aEEG monitor. 
		NOTE: The amplitude integrated EEG consists of a dense trace with upper and lower margins. Lower and upper margins in this model are typically 15-50 &#956;V, often higher than seen in infants. During hypoxia it is important to note, that artifact from the ECG may falsely elevate the aEEG trace. Bolus medications (propofol or fentanyl) may also transiently suppress the aEEG trace and should be avoided if possible during the experiment. Administration of drugs and clinical events should be marked to facilitate interpretation of the aEEG trace.</li>
	</ol>
	</li>
	<li>Prepare the equipment needed for placing umbilical catheters for central arterial blood pressure monitoring and blood sampling (Figure 3): sterile gloves, sterile draping, scalpel, alcohol swabs, sterile wipes, umbilical vein catheter (5 Fr), umbilical artery catheter (3.5 Fr), suture set with forceps, curved micro forceps, scissors, needle holder and suture (e.g., size 3-0), transparent adhesive patch, 5 ml syringes for blood sampling.</li>
</ol>
	<img alt="Figure 3" src="/files/ftp_upload/52454/52454fig3.jpg" /> 
	Figure 3. Equipment for umbilical lines and blood sampling. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
	<ol start="3">
		<li>Sterilize and drape the area around the umbilicus. Use a scalpel to cut the umbilical cord as close to the skin as possible. If this does not expose the umbilical vessels in the anaesthetized piglet, cut the umbilical skin 2 mm below the umbilical cord to expose the vessels. Identify the two small umbilical arteries and the one larger umbilical vein (Figure 4).</li>
		<li>Use curved micro forceps to dilate the artery (Figure 4) and insert the umbilical arterial catheter (3.5 Fr). Estimate insertion length in cm to 3 x weight (kg) + 10 (empirical formula based on correct placement in descending aorta above renal arteries on autopsy).</li>
		<li>Place a second 5 Fr catheter 5 cm into the umbilical vein (Figure 4). Verify intravascular catheter placement by blood return. Secure catheters by placing a purse-string suture around the umbilicus, pass the suture ends around each of the umbilical catheters and tie a knot. Cover catheters with transparent adhesive dressing.</li>
		<li>Collect blood samples at pre-specified time points: 1) immediately before hypoxia, 2) 30 min into hypoxic insult 3) at the end of the 45 min hypoxic insult 4) 2 hr after hypoxic insult. Depending on the purpose of the experiment other time points for blood samples may be chosen.</li>
		<li>Use arterial blood gas analysis from blood samples taken during hypoxia to verify blood gas changes caused by hypoxia-ischemia (Table 1). Note that piglets have low hemoglobin at birth (around 8 g/dL) and may become anemic from frequent blood sampling. Withdraw less than 2 ml blood/kg bodyweight per draw, less than 5 ml blood/kg bodyweight within 24 h and adhere to local guidelines for blood sampling. Signs of anemia include decreasing hematocrit and tachycardia.</li>
		<li>Connect arterial line to a monitor for continues intra-arterial blood pressure monitoring (MABP). Use venous line for fluid and drug administration.</li>
	</ol>
<img alt="Figure 4" src="/files/ftp_upload/52454/52454fig4.jpg" /> 
Figure 4. Umbilical vessels. Umbilical vein (right) and one of two umbilical arteries (left). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Hypoxia
<ol>
	<li>Wait 60 min after completing monitoring. Induce hypoxia by switching to a 4% O2/96% N2 gas mixture. Monitor vital parameters and aEEG closely and continue hypoxia for 45 min.</li>
	<li>Once the aEEG trace is flat (upper margin &#60;7 &#181;V), adjust oxygenation by altering the inspired oxygen fraction (FiO2) and mean airway pressure to the highest FiO2 level maintaining a flat trace aEEG according to the flowchart shown in Figure 5. Aim for MABP below 70% of baseline for at least 10 min and if needed lower FiO2 to ensure hypotension and ischemia. The described level of hypotension has previously been shown by others19,13,30 to produce relevant hypoxic-ischemic brain injury.</li>
	<li>In case of severe hypotension (MABP &#60;25 mm Hg) treat stepwise as follows: briefly increase FiO2 by 1-5%, saline bolus (10 ml/kg), infusion of dopamine (5-20 &#181;g/kg/min), and infusion of noradrenaline (20 ng-1 &#181;g/kg/min).</li>
	<li>In case of seizures lasting &#62;10 min (clonic, tonic or myoclonic seizures, typically focal or as indicated by sudden changes in aEEG amplitude) treat stepwise with (allowing 30 min before proceeding to the next injection): slow bolus of phenobarbital i.v. 20 mg/kg, repeat phenobarbital i.v. 20 mg/kg, and midazolam i.v. 0.5 mg/kg.
	<ol>
		<li>If seizures develop and are nonresponsive to drug therapy euthanasia is indicated.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52454/52454fig5.jpg" /> 
Figure 5. Hypoxia-ischemia flowchart. Flowchart showing adjustments in oxygenation (FiO2 and Paw) according to aEEG response. Mean airway pressure (Paw) was adjusted by changing PIP/TV (lower PIP gives lower Paw) and respiratory rate (lower RR gives lower Paw). Target aEEG = upper margin of trace &#60; 5 &#181;V and lower margin of trace &#62; 3 &#181;V (average 4 &#181;V). Target heart rate (HR) = &#62; 80. Target mean arterial blood pressure (MABP) = MABP &#62; 25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. 72 hr survival
<ol>
	<li>Monitor the piglet closely after the hypoxic insult, and gradually reduce the rates of infusion of propofol and fentanyl. Extubate at the point at which the animal is breathing voluntarily.</li>
	<li>During survival period, keep piglets at an animal facility with 24 hr watch by personnel trained in intensive care and monitoring. Administer i.v. glucose infusions 20 ml/kg 2-3 hourly. At 26-48 hr after hypoxia-ischemia partial bottle feeding may be initiated. Aspiration to the trachea and lungs may occur by bottle feeding if the gag reflex is not present.</li>
	<li>If relevant to the experiment, evaluate neurologic status using scoring system developed by Thoresen et al., described in detail in the original publication17.</li>
</ol>
7. Outcome Evaluation
<ol>
	<li>After 72 hr, anesthetize and ventilate again as described in section 2 and 3. Perform MRI scan with relevant imaging modalities as described by Munkeby et al31 and others2.</li>
	<li>At the end of the experiment euthanize piglets with a lethal dose of pentobarbital (5 g/kg i.v.).</li>
	<li>Prepare the brain for examination according to the goal of the experiment.
	<ol>
		<li>For brain histology on formalin fixed tissue: use cardiac perfusion with 4% paraformaldehyde in PBS, followed by dissection and post-fixing in paraformaldehyde as described by Robertson et al.2 , Chakkarapani et al1 or Liu et al.18 or remove the brain and immerse in 4% paraformaldehyde as described by Andresen et al.32</li>
		<li>For analysis requiring snap-frozen tissue (e.g., RNA analysis or enzyme activity assays33): Remove the brain, dissect out brain regions of interest and snap-freeze tissue blocks of maximum 1 cm x 1 cm in liquid nitrogen as described by Munkeby et al.33</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chakkarapani, 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=Xenon+enhances+hypothermic+neuroprotection+in+asphyxiated+newborn+pigs.">Xenon enhances hypothermic neuroprotection in asphyxiated newborn pigs.</a> Annals of. 68, 330-341 (2010).</li><li>Robertson, N. 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=Melatonin+augments+hypothermic+neuroprotection+in+a+perinatal+asphyxia+model.">Melatonin augments hypothermic neuroprotection in a perinatal asphyxia model.</a> Brain : a journal of neurology. 136, 90-105 (2013).</li><li>Lawn, J. 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=Every+Newborn:+progress,+priorities,+and+potential+beyond+survival.">Every Newborn: progress, priorities, and potential beyond survival.</a> Lancet. 384, 189-205 (2014).</li><li>Lee, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapartum-related+neonatal+encephalopathy+incidence+and+impairment+at+regional+and+global+levels+for+2010+with+trends+from+1990.">Intrapartum-related neonatal encephalopathy incidence and impairment at regional and global levels for 2010 with trends from 1990.</a> Pediatric research. 74, 50-72 (2013).</li><li>Kurinczuk, J. J., White-Koning, M., Badawi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+neonatal+encephalopathy+and+hypoxic-ischaemic+encephalopathy.">Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy.</a> Early human development. 86, 329-338 (2010).</li><li>Shankaran, S., Woldt, E., Koepke, T., Bedard, M. P., Nandyal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+neonatal+morbidity+and+long-term+central+nervous+system+sequelae+of+perinatal+asphyxia+in+term+infants.">Acute neonatal morbidity and long-term central nervous system sequelae of perinatal asphyxia in term infants.</a> Early human development. 25, 135-148 (1991).</li><li>Robertson, C. M., Finer, N. N., Grace, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=School+performance+of+survivors+of+neonatal+encephalopathy+associated+with+birth+asphyxia+at+term.">School performance of survivors of neonatal encephalopathy associated with birth asphyxia at term.</a> The Journal of pediatrics. 114, 753-760 (1989).</li><li>Bennet, L., Booth, L., Gunn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+biomarkers+for+hypoxic-ischemic+encephalopathy.">Potential biomarkers for hypoxic-ischemic encephalopathy.</a> Seminars in feta., &amp; neonatal medicine. 15, 253-260 (2010).</li><li>Yager, J. Y., Ashwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+perinatal+hypoxic-ischemic+brain+damage.">Animal models of perinatal hypoxic-ischemic brain damage.</a> Pediatric neurology. 40, 156-167 (2009).</li><li>Buckley, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+circulatory+system+in+three+mammalian+models+of+human+development.">Maturation of circulatory system in three mammalian models of human development.</a> Comparative biochemistry and physiology. A, Comparative. 83, 1-7 (1986).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+the+brain+growth+spurt.">Comparative aspects of the brain growth spurt.</a> Early human development. 3, 79-83 (1979).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+growth+and+development+of+human+brain.">Quantitative growth and development of human brain.</a> Archives of disease in childhood. 48, 757-767 (1973).</li><li>Foster, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+survival+model+of+hypoxia/ischaemia+in+the+piglet+suitable+for+neuroprotection+studies.">An improved survival model of hypoxia/ischaemia in the piglet suitable for neuroprotection studies.</a> Brain research. 919, 122-131 (2001).</li><li>LeBlanc, M. H., Li, X. Q., Huang, M., Patel, D. M., Smith, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+antagonist+LY293558+does+not+affect+the+severity+of+hypoxic-ischemic+injury+in+newborn+pigs.">AMPA antagonist LY293558 does not affect the severity of hypoxic-ischemic injury in newborn pigs.</a> Stroke; a journal of cerebral circulation. 26, 1908-1914 (1995).</li><li>Andresen, J. 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=Nicotine+affects+the+expression+of+brain-derived+neurotrophic+factor+mRNA+and+protein+in+the+hippocampus+of+hypoxic+newborn+piglets.">Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets.</a> J Perinat Med. 37, 553-560 (2009).</li><li>Cheung, P. Y., Gill, R. S., Bigam, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+swine+model+of+neonatal+asphyxia.">A swine model of neonatal asphyxia.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Thoresen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piglet+survival+model+of+posthypoxic+encephalopathy.">A piglet survival model of posthypoxic encephalopathy.</a> Pediatric research. 40, 738-748 (1996).</li><li>Liu, X., Tooley, J., Loberg, E. M., Suleiman, M. S., Thoresen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+hypothermia+reduces+cardiac+troponin+I+after+hypoxic-ischemic+encephalopathy+in+newborn+pigs.">Immediate hypothermia reduces cardiac troponin I after hypoxic-ischemic encephalopathy in newborn pigs.</a> Pediatric research. 70, 352-356 (2011).</li><li>Bjorkman, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic/Ischemic+models+in+newborn+piglet:+comparison+of+constant+FiO2+versus+variable+FiO2+delivery.">Hypoxic/Ischemic models in newborn piglet: comparison of constant FiO2 versus variable FiO2 delivery.</a> Brain research. 1100, 110-117 (2006).</li><li>Robertson, N. 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=Which+neuroprotective+agents+are+ready+for+bench+to+bedside+translation+in+the+newborn+infant.">Which neuroprotective agents are ready for bench to bedside translation in the newborn infant.</a> The Journal of pediatrics. 160, 544-552 (2012).</li><li>Jacobs, S. 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=Cooling+for+newborns+with+hypoxic+ischaemic+encephalopathy.">Cooling for newborns with hypoxic ischaemic encephalopathy.</a> Cochrane Database Syst Rev. 1, CD003311 (2013).</li><li>Andresen, J. 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=Resuscitation+with+21+or+100%+oxygen+in+hypoxic+nicotine-pretreated+newborn+piglets:+possible+neuroprotective+effects+of+nicotine.">Resuscitation with 21 or 100% oxygen in hypoxic nicotine-pretreated newborn piglets: possible neuroprotective effects of nicotine.</a> Neonatology. 93, 36-44 (2008).</li><li>Karlsson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+hypothermia+as+selective+head+cooling+or+whole+body+cooling+does+not+protect+brain+or+body+in+newborn+pig+subjected+to+hypoxia-ischemia.">Delayed hypothermia as selective head cooling or whole body cooling does not protect brain or body in newborn pig subjected to hypoxia-ischemia.</a> Pediatric research. 64, 74-78 (2008).</li><li>Kovacs, 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=Asphyxia-induced+release+of+alpha-melanocyte-stimulating+hormone+in+newborn+pigs.">Asphyxia-induced release of alpha-melanocyte-stimulating hormone in newborn pigs.</a> Peptides. 22, 1049-1053 (2001).</li><li>Varvarousi, G., 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=Asphyxial+cardiac+arrest,+resuscitation+and+neurological+outcome+in+a+Landrace/Large-White+swine+model.">Asphyxial cardiac arrest, resuscitation and neurological outcome in a Landrace/Large-White swine model.</a> Laboratory animals. 45, 184-190 (2011).</li><li>Tammela, O., Pastuszko, A., Lajevardi, N. S., Delivoria-Papadopoulos, M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+tyrosine+hydroxylase+in+the+striatum+of+newborn+piglets+in+response+to+hypocapnic+hypoxia.">Activity of tyrosine hydroxylase in the striatum of newborn piglets in response to hypocapnic hypoxia.</a> Journal of neurochemistry. 60, 1399-1406 (1993).</li><li>Cote, A., Barter, J., Meehan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+metabolic+effects+of+repeated+hypoxemia+in+piglets.">Age-dependent metabolic effects of repeated hypoxemia in piglets.</a> Canadian journal of physiology and pharmacology. 78, 321-328 (2000).</li><li>Tichauer, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+severity+of+perinatal+hypoxia-ischemia+in+piglets+using+near-infrared+spectroscopy+to+measure+the+cerebral+metabolic+rate+of+oxygen.">Assessing the severity of perinatal hypoxia-ischemia in piglets using near-infrared spectroscopy to measure the cerebral metabolic rate of oxygen.</a> Pediatric research. 65, 301-306 (2009).</li><li>Jasani, M. S., Salzman, S. K., Tice, L. L., Ginn, A., Nadkarni, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+regimen+effects+on+a+pediatric+porcine+model+of+asphyxial+arrest.">Anesthetic regimen effects on a pediatric porcine model of asphyxial arrest.</a> Resuscitation. 35, 69-75 (1997).</li><li>Chakkarapani, E., Thoresen, M., Liu, X., Walloe, L., Dingley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenon+offers+stable+haemodynamics+independent+of+induced+hypothermia+after+hypoxia-ischaemia+in+newborn+pigs.">Xenon offers stable haemodynamics independent of induced hypothermia after hypoxia-ischaemia in newborn pigs.</a> Intensive care medicine. 38, 316-323 (2012).</li><li>Munkeby, B. 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=A+piglet+model+for+detection+of+hypoxic-ischemic+brain+injury+with+magnetic+resonance+imaging.">A piglet model for detection of hypoxic-ischemic brain injury with magnetic resonance imaging.</a> Acta radiologica. 49, 1049-1057 (2008).</li><li>Andresen, J. 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=Newborn+piglets+exposed+to+hypoxia+after+nicotine+or+saline+pretreatment:+long-term+effects+on+brain+and+heart.">Newborn piglets exposed to hypoxia after nicotine or saline pretreatment: long-term effects on brain and heart.</a> J Matern Fetal Neonatal Med. 22, 161-168 (2009).</li><li>Munkeby, B. 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=Resuscitation+with+100%+O2+increases+cerebral+injury+in+hypoxemic+piglets.">Resuscitation with 100% O2 increases cerebral injury in hypoxemic piglets.</a> Pediatric research. 56, 783-790 (2004).</li></ol>

The authors have nothing to disclose.

Due to its complexity, the described model can only be implemented in facilities accredited and experienced in animal research. Approval by local ethics committees must be obtained prior to initiation of the experiments, and optimal animal welfare must be ensured at all times. As the model is based on the survival of the test animals, it is important that a sterile environment is maintained during invasive procedures to prevent infections.
The choice of anesthesia is important as most if not all anesthetic drugs have potential neuroprotective properties. Differences in outcome between previously published models may have been due in part to differences in anesthetics used, such as halothane or propofol17,19. This is a point that is rarely touched upon in the literature, as anesthesia cannot be omitted and there is a paucity of evidence which would be applicable to this model. Our pragmatic approach is to use as few different anesthetics as possible, that is sevoflurane is only used briefly, then on propofol and fentanyl are infused continuously. In our experiments medication boluses affect the aEEG transiently, while staying within the specified limits for continuous infusion does not. When testing neuroprotective treatments bias is avoided by using a randomized trial design.
The most critical step in the model is establishment of the hypoxic-ischemic insult, as the primary goal is to induce a moderate to severe hypoxic-ischemic insult whilst at the same time ensuring animal survival. The unique quality of this HIE model is the ability to accommodate the different biological responses to hypoxia from animal to animal. Titration of oxygen delivery to the highest possible fraction resulting in a depressed aEEG (&#60;7 &#181;V), allows for adjustments according to each individual piglet&#180;s tolerance to hypoxia, thus ensuring a high survival rate combined with maximum neurological injury19. The duration of flat trace aEEG during the 45 min of hypoxia should be compared with the degree of histopathologic injury for evaluation and optimization of injury severity. Other researchers using the model, have similarly graded the hypoxic-ischemic insult from mild to severe depending on the duration of aEEG suppression. As seizures in this model have previously been shown to correlate with severity of brain injury on histopathology17 with little treatment potential, we recommend a level of hypoxia-ischemia severity not producing seizures.
Most studies using the piglet HIE model include 10 min of low MABP (MABP &#60;70% of baseline)19. Bj&#246;rkman et al. found that if oxygen delivery varied during hypoxia, the degree of brain injury was independent of the period of time MABP was below 35 mm Hg19. Pilot experiments performed when establishing the present HIE model, revealed that sustained hypotension where MABP was lower than 25 mm Hg was associated with severe brain injury and permanently flat trace aEEG. Thus, immediate response to severe hypotension must be ensured to avoid extensive brain ischemia followed by brain-death.
The post-hypoxic period is also critical. Many previous studies have reoxygenated study animals by use of 100% oxygen, which is known to exacerbate brain injury.

Perinatal asphyxia is an acute and frequently unpredicted condition associated with hypoxic-ischemic encephalopathy (HIE). The overall goal of this protocol is to demonstrate a piglet survival model of perinatal hypoxic-ischemic encephalopathy. This model can be used to investigate the effect of various degrees of hypoxia-ischemia on the neonatal brain and of experimental treatments on neuropathology, magnetic resonance imaging and spectroscopy (MRI and MRS) and biomarkers in body fluids such as blood, cerebrospinal fluid and urine. The model has also proven useful for investigating the cardiovascular system, respiratory system, kidney and liver, all of which are affected in global hypoxia-ischemia.
Perinatal asphyxia is the result of compromised oxygen supply intrapartum or in the immediate post-partum period. Intrapartum hypoxic events account for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths in 20123. In 2010 1.15 million babies were estimated to have developed neonatal encephalopathy following birth asphyxia4. HIE defined as encephalopathy in infants born after 34 weeks of gestation occurs in 1-3/1,000 live births5 in the industrialized world and up to 8.5/1,000 live births in developing countries4. The risk of death is 10-60%, and the risk of neurological handicap in survivors 30-100%6,7. 50.2 million disability adjusted life years (DALYs) are attributed to intrapartum hypoxic events4. Presently the only treatment other than supportive for HIE is post-hypoxic hypothermia. Thus, advancements in diagnostic procedures and treatment strategies are essential to improve the management of HIE8.
Improvements in the prognosis after perinatal asphyxia and management of neonatal brain injury are based on expanding knowledge of underlying disease mechanisms and possible treatments. Animal models of HIE are particularly useful as different clinical events may lead to HIE and the incidence in any single birth center is low5. An experimental setup in which the influence of biological variation can be minimized, is essential when testing new prognostic and diagnostic tools and treatment strategies. An animal model should approximate the clinical situation as closely as possible, thus contributing to the understanding of the pathological mechanisms underlying the induced injury and the dynamic process involved in the disease and it&#180;s outcome9. Animal models of neonatal HIE have included a number of species, including rodents, lamb, and swine. In comparison, the newborn piglet has higher resemblance to a human newborn with respect to size, cardiovascular system10 and brain maturity at the time of delivery11,12. Monitoring, instrumentation and outcome evaluation in the piglet model is similar to that used in the clinical care of infants with HIE. Accordingly, there is a high degree of translation into newborn care from this model.
Piglets models of perinatal hypoxia and HIE are used by many groups and vary in a number of areas13. According to the purpose of the experiment, careful attention must be paid to the choice of medications, method of inducing hypoxia-ischemia, method of controlling insult duration and severity, post-insult resuscitation and care, and outcome evaluation. To avoid bias a randomized trial design should always be used in interventional studies.
The method applied when inducing hypoxic-ischemic injury is important. Global hypoxia leading to HIE often results in multi-organ failure involving brain, heart, lungs, kidney and liver. Depending on the outcomes evaluated, models of HIE should be based on global hypoxia and ischemia rather than rely on focal ischemia, e.g., by ligation of carotid arteries14. A recent paper applied a combination of hypoxia (FiO2 12%) and carotid artery compression while maintaining mean arterial blood pressure &#62; 40 mm Hg2. Another group induced global hypoxia by 8% O2 until negative base excess &#62; 20 mmol/L or mean arterial blood pressure (MABP) &#60; 15 mm Hg, and sacrificed the animals at 4 hr15. Hypoxia has also been titrated by cardiac output (to 30-40% of baseline), MABP (to 30-35 mm Hg) and arterial pH (6.95-7.05)16.
Models of global hypoxia-ischemia titrated by aEEG suppression similar to the one presented in this report, have demonstrated encephalopathy that is clinically, electrophysiologically, and neuropathologically comparable to the condition found in the asphyxiated term infant17,18.
The degree of HIE induced is essential. A useful animal model of HIE must also allow for testing of new diagnostic procedures and treatment options. To enable this, the models should induce moderate HIE where there is a treatment potential as severe brain injury with little or no treatment potential would be less relevant when evaluating new treatments. Tolerance to hypoxia varies considerably between test animals. Previous studies have shown that a more consistent brain injury can be achieved and that more animals survive17,19 by individualizing the induced hypoxia according to each piglet&#8217;s cerebral response evaluated by amplitude integrated electroencephalography (aEEG) rather than using a set FiO2 value throughout the hypoxic event. The duration of aEEG suppression correlates to the degree of brain injury, with few histopathologic changes at &#60; 20 min aEEG suppression and severe seizures increasing at &#62; 45 min aEEG suppression. A recent review of neuroprotective treatments for HIE identified the need for survival models enabling behavioral outcome measures in animal models20.
There are numerous advantages of the presented HIE piglet model. It is based on a species where results are highly likely to translate to the human physiology. Global hypoxia-ischemia models multi-organ failure and titration of hypoxia-ischemia by aEEG induces a consistent degree of brain injury with survival clinical relevant outcomes such that biomarkers, MRI and behavior may be evaluated at relevant time points.
Piglet models of perinatal asphyxia and HIE have not only contributed significantly to current insight into HIE pathophysiology, but have also successfully preceded clinical trials, ultimately resulting in new treatments in humans. Piglet model studies played a key role in establishing hypothermia as a treatment for HIE21, and are used in neonatal resuscitation research22. Various groups have used piglet models when performing research within asphyxia and HIE, and studies include hypothermia23, alpha-melanocyte-stimulating hormone24, cardiac arrest25, tyrosine hydroxylase activity26, repeated hypoxic exposure27, NMDA receptor activity14, and near-infrared spectroscopy28.
The piglet HIE model presented in this report is technically challenging to work with, as minor adjustments during the course of the procedure may result in either too mild or too severe brain injury29,2. We found that the existing literature lacked sufficient detail to reproduce previously published models. Thus, we here demonstrate each step of the technical procedures necessary for the establishment of a piglet 72 hr survival model in this report, enabling researchers to establish this advanced model for the study of HIE.

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Warm-touch-pediatric blanket</td>
			<td style="width:211px;">Covidien</td>
			<td style="width:176px;">5030840</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Apertrue Drape</td>
			<td>Barrier</td>
			<td>915447</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Utility Drape (sterile) 75x80 cm</td>
			<td>Barrier</td>
			<td>800530</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neoflon</td>
			<td>BD - Luer</td>
			<td>391350</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Laryngoscope</td>
			<td style="width:211px;">Miller</td>
			<td style="width:176px;">85-0045</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 2.5 mm&#160;</td>
			<td>Covidien</td>
			<td>111-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 3.0 mm with cuff</td>
			<td>Unomedical</td>
			<td>MM61110030</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endotracheal tube 3.5 mm with cuff</td>
			<td>Unomedical</td>
			<td>MM61110035</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Anesthesia machine</td>
			<td style="width:211px;">GE Healthcare</td>
			<td style="width:176px;">1009-9002-000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EEG - electrodes/disposable subdermal needle electrode</td>
			<td>Cephalon</td>
			<td>ACCE120550</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG - electrodes</td>
			<td>medtronic</td>
			<td>3010107-003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG-electrodes for MR</td>
			<td>philips</td>
			<td>ACCE120550</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Arterial blood sampler - aspirator</td>
			<td>Radiometer medical ApS</td>
			<td>956552</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyurethane Umbilical vein catheter (5 Fr/Ch)</td>
			<td>Covidien</td>
			<td>8888160341</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyurethane Umbilical vein catheter (3,5 Fr/ch)</td>
			<td>Covidien</td>
			<td>8888160333</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture set (size 3-0)</td>
			<td>Covidien</td>
			<td>8886 623341</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Spinal needle 0.7x38mm</td>
			<td>BD needles</td>
			<td>405254</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">Gas with 96% Nitrogen / 4% oxygen</td>
			<td style="width:211px;">Air Liquide</td>
			<td style="width:176px;">made on order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:647px;">NeuroMonitor (CFM) system</td>
			<td>Natus Medical Incorporated</td>
			<td>OBM70002</td>
		</tr>
	</tbody>
</table>

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.

The effects of hypoxia-ischemia on the brain that occur during the induced insult are documented by recording the aEEG trace. A representative aEEG trace is shown in Figure 6.
<img alt="Figure 6" src="/files/ftp_upload/52454/52454fig6.jpg" /> 
Figure 6. Representative aEEG trace. Low amplitude due to hypoxia-ischemia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The extreme values of pH, base excess, and lactate during hypoxia are recorded along with heart rate and blood pressure. Clinical neurology scoring is carried out17. Data from an experimental animal with a duration of 38 min of low amplitude aEEG and an insult leading to HIE with low pH and relative hypotension, are shown in table 1.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>aEEG &#60; 7uV (min)</td>
			<td>pH low during insult</td>
			<td>Base excess low during insult</td>
			<td>Lactate high during insult</td>
			<td>MABP baseline</td>
			<td>MABP high</td>
			<td>MABP low</td>
			<td>HR high</td>
			<td>HR low</td>
		</tr>
		<tr>
			<td>37.5</td>
			<td>6.93</td>
			<td>-19.80</td>
			<td>19</td>
			<td>48</td>
			<td>72</td>
			<td>33</td>
			<td>219</td>
			<td>162</td>
		</tr>
	</tbody>
</table>
Table 1. Representative physiologic data. For a piglet with 38 min of aEEG &#60; 7 &#181;V the extreme values of pH, base excess, lactate, heart rate (HR) and blood pressure (MABP) within the 45 min of hypoxia are listed.
MRI at 72 hr may reveal injury depending on the modalities used. Figure 7 shows a T2 weighted MRI image with hypoxic-ischemic injury.
<img alt="Figure 7" src="/files/ftp_upload/52454/52454fig7.jpg" /> 
Figure 7. Representative T2 weighted MRI image. T2-wighted image in axial plane, 72 hr post-hypoxia-ischemia. Normal signal intensity within the mesencephalon, pons and medulla oblongata. Diffuse abnormal signal intensity throughout the white matter in both hemispheres. The T2-weighted MRI shows lesions corresponding to diffuse ischemic infarction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
H&#38;E staining shows neuronal injury as seen on hematoxylin and eosin stain. Different groups have published various scoring systems for quantifying the degree of histologic injury on either a 0.5 interval scale of 0-417 or a scale of 0-9 for 5 different brain regions with a maximum injury score of 4513.
<img alt="Figure 8" src="/files/ftp_upload/52454/52454fig8.jpg" /> 
Figure 8. Representative histopathology. Light microscope image with hematoxylin and eosin stain. Damaged neurons are eosinophilic with a small nucleus (arrows). The image was captured at 200x magnification. Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52454/52454fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Expected outcome from model: Previously published studies using comparable models have resulted in approximately 20% dead piglets, 10% with no injury and 70% surviving with brain injury19. These studies correlate well with our findings. Multi organ injury manifests by 1) lactate acidosis 2) anuria (kidney injury) 3) hypotension (ischemic cardiac injury) and may be verified by visual inspection and histopathology of organs post mortem. In this model, cardiac dysfunction has been demonstrated by increase in cardiac troponin T and ischemic lesions on cardiac examination18. Clinical signs of encephalopathy may be quantified by neurological scoring and include changes in respiration, consciousness, orientation, walking, tone, activity level, sucking, vocalization and the presence of pathological movements as described by others17.

Watch this Scientific Journal Video about A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy at JoVE.com

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

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-piglet-model-of-neonatal-hypoxic-ischemic-encephalopathy

Nanyang Technological University

Research

JoVE Journal

Medicine

19.5K Views.  Institute of Clinical Medicine, Aarhus University Hospital. 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. 

Video: A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

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, ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under 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 ...

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 ...

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 ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...