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>

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<li>Varvarousi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asphyxial+cardiac+arrest%2C+resuscitation+and+neurological+outcome+in+a+Landrace%2FLarge-White+swine+model%5BTitle%5D)+AND+%22Laboratory+animals%22%5BJournal%5D)">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/pubmed?term=((Activity+of+tyrosine+hydroxylase+in+the+striatum+of+newborn+piglets+in+response+to+hypocapnic+hypoxia%5BTitle%5D)+AND+%22Journal+of+neurochemistry%22%5BJournal%5D)">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/pubmed?term=((Age-dependent+metabolic+effects+of+repeated+hypoxemia+in+piglets%5BTitle%5D)+AND+%22Canadian+journal+of+physiology+and+pharmacology%22%5BJournal%5D)">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/pubmed?term=((Assessing+the+severity+of+perinatal+hypoxia-ischemia+in+piglets+using+near-infrared+spectroscopy+to+measure+the+cerebral+metabolic+rate+of+oxygen%5BTitle%5D)+AND+%22Pediatric+research%22%5BJournal%5D)">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/pubmed?term=((Anesthetic+regimen+effects+on+a+pediatric+porcine+model+of+asphyxial+arrest%5BTitle%5D)+AND+%22Resuscitation%22%5BJournal%5D)">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/pubmed?term=((Xenon+offers+stable+haemodynamics+independent+of+induced+hypothermia+after+hypoxia-ischaemia+in+newborn+pigs%5BTitle%5D)+AND+%22Intensive+care+medicine%22%5BJournal%5D)">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/pubmed?term=((A+piglet+model+for+detection+of+hypoxic-ischemic+brain+injury+with+magnetic+resonance+imaging%5BTitle%5D)+AND+%22Acta+radiologica%22%5BJournal%5D)">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/pubmed?term=((Newborn+piglets+exposed+to+hypoxia+after+nicotine+or+saline+pretreatment%3A+long-term+effects+on+brain+and+heart%5BTitle%5D)+AND+%22J+Matern+Fetal+Neonatal+Med%22%5BJournal%5D)">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/pubmed?term=((Resuscitation+with+100%25+O2+increases+cerebral+injury+in+hypoxemic+piglets%5BTitle%5D)+AND+%22Pediatric+research%22%5BJournal%5D)">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><tbody><tr><td>Warm-touch-pediatric blanket</td><td>Covidien</td><td>5030840</td><td></td></tr><tr><td>Adhesive Apertrue Drape</td><td>Barrier</td><td>915447</td><td></td></tr><tr><td>Utility Drape (sterile) 75x80 cm</td><td>Barrier</td><td>800530</td><td></td></tr><tr><td>Neoflon</td><td>BD - Luer</td><td>391350</td><td></td></tr><tr><td>Laryngoscope</td><td>Miller</td><td>85-0045</td><td></td></tr><tr><td>Endotracheal tube 2.5 mm&amp;nbsp;</td><td>Covidien</td><td>111-25</td><td></td></tr><tr><td>Endotracheal tube 3.0 mm with cuff</td><td>Unomedical</td><td>MM61110030</td><td></td></tr><tr><td>Endotracheal tube 3.5 mm with cuff</td><td>Unomedical</td><td>MM61110035</td><td></td></tr><tr><td>Anesthesia machine</td><td>GE Healthcare</td><td>1009-9002-000</td><td></td></tr><tr><td>EEG - electrodes/disposable subdermal needle electrode</td><td>Cephalon</td><td>ACCE120550</td><td></td></tr><tr><td>ECG - electrodes</td><td>medtronic</td><td>3010107-003</td><td></td></tr><tr><td>ECG-electrodes for MR</td><td>philips</td><td>ACCE120550</td><td></td></tr><tr><td>Arterial blood sampler - aspirator</td><td>Radiometer medical ApS</td><td>956552</td><td></td></tr><tr><td>Polyurethane Umbilical vein catheter (5 Fr/Ch)</td><td>Covidien</td><td>8888160341</td><td></td></tr><tr><td>Polyurethane Umbilical vein catheter (3,5 Fr/ch)</td><td>Covidien</td><td>8888160333</td><td></td></tr><tr><td>Suture set (size 3-0)</td><td>Covidien</td><td>8886 623341</td><td></td></tr><tr><td>BD Spinal needle 0.7x38mm</td><td>BD needles</td><td>405254</td><td></td></tr><tr><td>Gas with 96% Nitrogen / 4% oxygen</td><td>Air Liquide</td><td>made on order</td><td></td></tr><tr><td>NeuroMonitor (CFM) system</td><td>Natus Medical Incorporated</td><td>OBM70002</td><td></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

19.6K 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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Neuroscience

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, ...

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

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 ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying neonatal seizures and hypoxia ischemia. This manuscript describes a method for continuous video electroencephalogram (EEG) monitoring in neonatal mice to detect seizures and analyze EEG background during hypoxia ischemia. Use of video and EEG in conjunction allows description of seizure semiology and confirmation of seizures. This method also allows analysis of power spectrograms and EEG background pattern trends over the experimental time period. In this hypoxia ischemia model, the method allows EEG recording prior to injury to obtain a normative baseline and during injury and recovery. Total monitoring time is limited by the inability to separate pups from the mother for longer than four hours. Although, we have used a model of hypoxic-ischemic seizures in this manuscript, this method for neonatal video EEG monitoring could be applied to diverse disease and seizure models in rodents.

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

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

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

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

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

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

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