All protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) by the State University of New York Buffalo. An illustration of the methodology depicting invasive and non-invasive monitoring is shown in Figure 1.
1. Animals
<ol>
	<li>Use time-dated, Q-fever seronegative, pregnant ewes (mixed-breed, Suffolk-Dorset-Katahdin breed) with fetal lambs at 127&#8211;143 days gestation. 
	NOTE: Sheep term gestation is 145 days and 127 gestation lambs behave like extreme premature infants.</li>
</ol>
2. Pre-surgical Preparation
<ol>
	<li>Withhold food from the ewe 12 h prior to the surgery.</li>
	<li>Sedate the ewe with intravenous (IV) diazepam (0.25&#8211;1.5 mg/kg) and ketamine (4 mg/kg).</li>
	<li>Guide the ewe into lateral recumbency on to a lift cart.</li>
	<li>Move the ewe to dorsal recumbency onto a V-top surgical table.</li>
</ol>
3. Anesthesia
NOTE: Prior to the caesarean section, the ewe is sedated with diazepam and ketamine and intubated followed by continuous inhalation of isoflurane (1&#8211;4%). Adequacy of anesthesia is monitored by evaluation of muscle tone and eye reflexes along with continuous monitoring of blood oxygen level with a pulse oximeter and the heart rate. During the instrumentation, lambs will be under the influence of maternal anesthesia and will also receive local anesthetic to sites of instrumentation.
<ol>
	<li>Intubate the ewe with a 10.0 mm cuffed endotracheal tube (ETT). Advance the ETT through the vocal cords 1&#8211;2 cm beyond the cuff. Inflate the ETT cuff and tie the ETT around the ewe&#39;s jaw to secure it in place.
	<ol>
		<li>When the palpebral response is absent, adjust the isoflurane (2&#8211;3% is usually sufficient).</li>
		<li>Provide artificial breaths by conventional mechanical ventilation (CMV) with a fraction of inspired oxygen (FIO2) of 0.21. Set the ventilator to provide tidal volumes of 10&#8211;15 mL/kg.</li>
		<li>Monitor the oxyhemoglobin saturation continuously by a pulse oximeter placed on the ewe&#39;s tongue.</li>
		<li>Measure the end-tidal carbon dioxide (EtCO2) with a capnograph.</li>
		<li>Adjust the FIO2 and CMV to maintain oxyhemoglobin saturation between 90 - 97% and exhaled CO2 between 35&#8211;45 mm Hg, respectively.</li>
	</ol>
	</li>
	<li>Secure the ewe&#39;s limbs to the surgical table with surgical tie-downs.
	<ol>
		<li>Adjust the surgical table to reverse Trendelenburg position to relieve pressure on the fetus.</li>
	</ol>
	</li>
	<li>Place an orogastric tube to decompress and drain gastric contents.</li>
	<li>Supplement IV fluid (normal saline or Lactated Ringers solution) at 10 mL/kg/h.</li>
	<li>Place a rectal temperature probe to obtain continuous core temperature.</li>
</ol>
4. Surgery
NOTE: The cesarean section and fetal procedures are considered acute surgeries in which the animals are euthanized by administering sodium pentobarbital 100 mg/kg IV. Ewes are euthanized following the delivery of the lambs and the lambs are euthanized after the completion of the experiments. Euthanasia is confirmed by asystole. A secondary method of bilateral thoracotomy or exsanguination is also employed. In this case, the aseptic technique is not practiced during the surgery. Personnel still need to wear protective attire during animal contact.
<ol>
	<li>Shave the wool from the ventral abdomen.</li>
	<li>Use the cautery to make a 15&#8211;18 cm abdominal skin-deep incision over the linea alba to expose the fascia.
	<ol>
		<li>Create a small opening into the abdomen using pressure with a blunt tipped hemostat.</li>
		<li>Maintain this opening while sliding a blunt tipped scissor into the abdominal wall.</li>
		<li>Use the scissors to complete the incision of the linea alba.</li>
	</ol>
	</li>
	<li>Locate and exteriorize the fetal head within the uterus from the abdomen.
	<ol>
		<li>Hold the fetal head with one hand and use the cautery to make a 10 cm incision through the uterine wall (over the lamb&#39;s forehead). Ensure to avoid any cotyledons.</li>
		<li>Use Babcock forceps to clamp the layers of the uterus and placenta together at four opposing corners.</li>
		<li>Expose the lamb&#39;s head through the uterus and outside of the ewe&#39;s abdomen.</li>
		<li>Leave the Babcock forceps laying outside of the abdomen during the fetal surgery.</li>
	</ol>
	</li>
</ol>
5. Fetal Airway
<ol>
	<li>Intubate the partially exposed fetal lamb with a 3.5&#8211;4.5 mm cuffed ETT. Advance the ETT through the vocal cords 1 cm beyond the cuff.
	<ol>
		<li>Inflate the cuff and secure the ETT by tying an umbilical tape around the tube and then around the head.</li>
		<li>Allow excess fetal lung fluid in the ETT to drain passively by tilting the head to the side. This simulates egress of lung liquid during labor.</li>
		<li>Occlude the ETT to prevent gas exchange during gasping in the asphyxial period.</li>
	</ol>
	</li>
</ol>
6. Neck Vessel Instrumentation
<ol>
	<li>Shave the wool and infiltrate 1&#8211;2 mL of subcutaneous bupivacaine hydrochloride (0.25%) at all incision sites.
	<ol>
		<li>Make two 3 cm skin incisions on each side of the neck (approximately 6 cm distal to the head) adjacent to the trachea. The incisions should transverse the neck.</li>
		<li>Perform the incision using the cautery while tenting the skin, to avoid lacerating the deep tissue.</li>
	</ol>
	</li>
	<li>Separate the fascia using curved Kelly or mosquito forceps.</li>
	<li>Isolate the right internal jugular vein and right common carotid artery.
	<ol>
		<li>Place two 20 cm, 0 silk sutures under both vessels.</li>
		<li>Allow a (proximal to distal) space of 1 cm between the silk sutures.</li>
		<li>Gently lift each suture that is around the vessel, superficially, to restrict blood flow and make a 1&#8211;2 mm transverse cut into the vessel using Iris surgical scissors.</li>
	</ol>
	</li>
	<li>For the carotid artery, insert one pre-flushed&#160;catheter (15&#8211;17 G) in the right carotid artery caudally towards the aortic arch for blood pressure monitoring and blood sampling.
	<ol>
		<li>Use the proximal tie to ligate the artery completely.</li>
		<li>Repeat steps 6.3.2&#160;- 6.3.3.</li>
		<li>Insert the pre-flushed carotid arterial catheter and use the distal silk suture to tie around both the artery and catheter with 3 knots.</li>
	</ol>
	</li>
	<li>Use a similar technique, to insert one pre-flushed catheter (14&#8211;16 G) into the jugular vein, repeat steps 6.3.2&#8211;6.3.3, advance 7&#8211;10 cm caudally to rest within the thoracic inlet (towards the right atrium). This is used for fluid and medication administration as well as venous pressure measurements.
	<ol>
		<li>Check the catheter for patency with heparinized saline and then complete the suture tie around the vessel and catheter (with the proximal suture only) using&#160;2&#8211;3 knots.</li>
		<li>Insert the second catheter (14&#8211;16 G) approximately 5 cm rostrally to collect blood from the cerebral circulation. Use the same cut for the placement of both catheters.</li>
		<li>Repeat step 6.5.1 as it pertains to the distal suture and catheter and then tie the silk sutures around the opposing catheter lines to secure them in place to prevent twisting.</li>
	</ol>
	</li>
	<li>Extend the previously made incision into a T-shape (1&#8211;2 cm) on the left side.
	<ol>
		<li>Elevate the left carotid artery using opened curved hemostats.</li>
		<li>Place a 2 mm perivascular flow-probe around the vessel to measure the blood flow.</li>
		<li>Cover the flow-probe with a 1-cm flexible polymer sleeve to stabilize the probe in position.</li>
		<li>Place the flow-probe cable line into the T-shaped incision, allowing the cable to run parallel with the vessel.</li>
		<li>Close the skin incision. Tie around the cable, and around a loop of the cable, to avoid restricting the flow if pulled.</li>
	</ol>
	</li>
</ol>
7. Great Vessel Instrumentation
<ol>
	<li>Extract the fetal lamb to expose the chest, and cover the exposed skin with polyethylene wrap to prevent heat loss.</li>
	<li>Place the lamb in right lateral recumbency.</li>
	<li>Position the left forelimb around the neck to expose the surgical site.</li>
	<li>Infiltrate 3 mL of bupivacaine hydrochloride (0.25%) along the 4th intercostal space and make a 6 cm skin incision using the cautery.
	<ol>
		<li>Carefully pierce the intercostal muscle with blunt curved forceps, circle under the 3rd rib, and come out between the next intercostal space. Make sure not to injure the lung or the heart.</li>
		<li>Open the clamp for a pre-cut piece of 0 silk and bring it through to encircle the rib.</li>
		<li>Repeat steps 7.4.1&#8211;7.4.2 to secure the suture under the 4th rib.</li>
		<li>Clamp the ties around each rib. Gently pull up and insert a cotton-tipped applicator into the chest under the 4th intercostal space.</li>
		<li>Use the applicator stick to protect the chest contents while using the cautery to open the intercostal space in 1 cm for a final opening of 6 - 8 cm. Ensure that the lung is not caught in the rib ties.</li>
		<li>Place a rib spreader into the chest-opening. Opening the chest will reveal the left upper lobes of the lung, the main pulmonary artery (PA) and the ductus arteriosus.</li>
	</ol>
	</li>
	<li>Use 2 x 2 inch gauzes to envelop the lung and gently push any exposed tissue away from the surgical area.
	<ol>
		<li>Use a small manual retractor to improve the field of view (if necessary).</li>
		<li>Place an umbilical tape (1/4 inch width, 6 inches length) pre-soaked in amnion around the great vessels. Soaking the umbilical tape decreases friction (and injury) onto the vessel walls.</li>
	</ol>
	</li>
	<li>Using forceps, lift the pericardium and cut along the main pulmonary artery, taking care not to cut the vagal nerve. Use an applicator stick to keep the right atrium from interfering with a lateral cut of the pericardium along the vagal nerve.</li>
	<li>Using Gemini forceps, move the forceps slowly from the ductal/PA notch around and behind the left PA to &#34;encircle&#34; and emerge on the other side of the left PA. Many delicate tissues run under this area and proceed only if there is no resistance.
	<ol>
		<li>When the tip of the forceps is observed, open only far enough to grab one end of the pre-moistened umbilical tape. Clamp with only 1 position clicked in place. It is possible that surrounding tissue could be caught in the forceps too, and if tightly clamped that tissue might be caught with the umbilical tape resulting in vessel injury.</li>
		<li>Gently back out the forceps to pull the tape into position. This serves to open a space for the flow probe and helps to direct the insertion of the flow probes (Figure 2). Care to protect the notch portion of any vessel is imperative as this is the most fragile part of the tissue.</li>
	</ol>
	</li>
	<li>Place a 4&#8211;6 mm perivascular flow-probe by gently lifting the umbilical tape and guiding the (L-bracket) metal portion of the probe around the vessel along the established opening.
	<ol>
		<li>Direct the tape toward the back of the probe to help seat the vessel in place and to visualize the end of the L-bracket thus allowing the sliding closure to be secured.</li>
		<li>Make sure the vessel is &#62; 75% of the probe size to ensure a tight fit and accurate flow measurements.</li>
		<li>Carefully cut the end of the umbilical tape close to the probe and gently pull the other end to remove the tape from the vessel. The tape will interfere with measurements if not removed.</li>
		<li>Apply ultrasound gel and adjust the probe and cable position to improve signal intensity and quality.</li>
	</ol>
	</li>
	<li>Repeat steps 7.6&#8211;7.7.4 as it pertains to the placement of a 6 - 8 mm perivascular flow-probe around the ductus arteriosus.</li>
	<li>Remove any protective 2 x 2 gauze from the chest and allow the lung to re-position.</li>
	<li>Place a new 2 x 2 gauze around each metal bracket of the flow probe to protect the lung.</li>
	<li>Close the chest in layers using a tapered needle and 2.0 synthetic monofilament suture. Secure the cables of the flow-probes to the outer skin with a loop using a cutting needle and 2.0 silk suture.</li>
</ol>
8. Non-invasive Measurement
<ol>
	<li>Attach EKG leads at the right axilla, left axilla, and right inguinal area (three-lead EKG). Pre- and post-ductal arterial oxyhemoglobin saturation (SpO2) is monitored with a pulse oximeter placed on the right forelimb and either hindlimb, respectively. Secure a near-infrared spectroscope (NIRS) monitor over the forehead with sutures and an opaque dressing.</li>
</ol>
9. Data Collection
<ol>
	<li>Collect and record data using a data acquisition software. Figure 1 illustrates invasive and non-invasive parameters including: arterial and central venous blood pressure, left common carotid, left pulmonary and ductal arterial blood flow, EKG, SpO2, NIRS, ETCO2. 
	NOTE: The software and equipment set-up for data collection can vary and is beyond the scope of this article.</li>
</ol>
10. Experimental Protocol
<ol>
	<li>Occlude and cut the umbilical cord, and move the lamb from the maternal abdomen to a radiant warmer.</li>
	<li>During the asphyxial period, insert an umbilical venous catheter for epinephrine administration (0.01&#8211;0.03 mg/kg IV, per NRP recommendations).</li>
	<li>Insert an umbilical arterial catheter to collect post-ductal arterial blood gases.</li>
	<li>Monitor the lamb for impending asystole, which can be defined as the absence of detected carotid flow and pressure.</li>
	<li>Ensure that adequate personnel is available and dedicate each person to the following tasks: (1) manage ventilation and the airway, (2) perform chest compressions, (3) administer medication, (4) draw blood samples, (5) lead the code.</li>
	<li>Begin resuscitation by providing positive pressure ventilation (PPV) with a T-piece resuscitator at pressures of 35/5 cm H2O.&#160;Initiate the 20 min timer for experimental resuscitation protocol.</li>
	<li>After 30 s of ventilation, begin chest compressions by placing the thumb and fingers of one hand on opposite sides of the chest.
	<ol>
		<li>Provide chest compressions by squeezing the fingers and thumb together to a depth of approximately 1/3.</li>
	</ol>
	</li>
	<li>Administer epinephrine at 3-min intervals.</li>
	<li>Continue resuscitation efforts until return of spontaneous circulation or up to 20 min.</li>
	<li>At return of spontaneous circulation, continuous IV fluids (5 cc/kg/hr) and fentanyl (0.5-2.0 mcg/kg/hr; titrated to effect), are given through the previously established jugular venous catheter. Following the 20 min resuscitation period, the animal is euthanized. If lambs do not achieve ROSC by 20 min, proceed with euthanasia.&#160;</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligating+the+ductus+arteriosus+before+birth+causes+persistent+pulmonary+hypertension+in+the+newborn+lamb.">Ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb.</a> Pediatr Res. 25 (3), 245-250 (1989).</li><li>Morin, F. C., Egan, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hemodynamics+in+fetal+lambs+during+development+at+normal+and+increased+oxygen+tension.">Pulmonary hemodynamics in fetal lambs during development at normal and increased oxygen tension.</a> J Appl Physiol (1985). 73 (1), 213-218 (1992).</li><li>Morin, F. C., Egan, E. A., Ferguson, W., Lundgren, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pulmonary+vascular+response+to+oxygen.">Development of pulmonary vascular response to oxygen.</a> Am J Physiol. 254 (3 Pt 2), H542-H546 (1988).</li><li>Rawat, 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=Neonatal+resuscitation+adhering+to+oxygen+saturation+guidelines+in+asphyxiated+lambs+with+meconium+aspiration.">Neonatal resuscitation adhering to oxygen saturation guidelines in asphyxiated lambs with meconium aspiration.</a> Pediatr Res. , (2015).</li><li>Chandrasekharan, P. K., 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=Continuous+End-Tidal+Carbon+Dioxide+Monitoring+during+Resuscitation+of+Asphyxiated+Term+Lambs.">Continuous End-Tidal Carbon Dioxide Monitoring during Resuscitation of Asphyxiated Term Lambs.</a> Neonatology. 109 (4), 265-273 (2016).</li><li>Lakshminrusimha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+suctioning+improves+gas+exchange+but+not+hemodynamics+in+asphyxiated+lambs+with+meconium+aspiration.">Tracheal suctioning improves gas exchange but not hemodynamics in asphyxiated lambs with meconium aspiration.</a> Pediatr Res. 77 (2), 347-355 (2015).</li><li>Meuli, 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=In+utero+surgery+rescues+neurological+function+at+birth+in+sheep+with+spina+bifida.">In utero surgery rescues neurological function at birth in sheep with spina bifida.</a> Nat Med. 1 (4), 342-347 (1995).</li><li>Meuli, 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=Creation+of+myelomeningocele+in+utero:+a+model+of+functional+damage+from+spinal+cord+exposure+in+fetal+sheep.">Creation of myelomeningocele in utero: a model of functional damage from spinal cord exposure in fetal sheep.</a> J Pediatr Surg. 30 (7), 1028-1032 (1995).</li><li>Adzick, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+congenital+diaphragmatic+hernia+in+utero.+IV.+An+early+gestational+fetal+lamb+model+for+pulmonary+vascular+morphometric+analysis.">Correction of congenital diaphragmatic hernia in utero. IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis.</a> J Pediatr Surg. 20 (6), 673-680 (1985).</li><li>Harrison, M. R., Bressack, M. A., Churg, A. M., de Lorimier, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+congenital+diaphragmatic+hernia+in+utero.+II.+Simulated+correction+permits+fetal+lung+growth+with+survival+at+birth.">Correction of congenital diaphragmatic hernia in utero. II. Simulated correction permits fetal lung growth with survival at birth.</a> Surgery. 88 (2), 260-268 (1980).</li><li>Turley, K., 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=Intrauterine+cardiothoracic+surgery:+the+fetal+lamb+model.">Intrauterine cardiothoracic surgery: the fetal lamb model.</a> Ann Thorac Surg. 34 (4), 422-426 (1982).</li><li>Reddy, V. 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=In+utero+placement+of+aortopulmonary+shunts.+A+model+of+postnatal+pulmonary+hypertension+with+increased+pulmonary+blood+flow+in+lambs.">In utero placement of aortopulmonary shunts. A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs.</a> Circulation. 92 (3), 606-613 (1995).</li><li>Hemway, R. J., Christman, C., Perlman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3:1+is+superior+to+a+15:2+ratio+in+a+newborn+manikin+model+in+terms+of+quality+of+chest+compressions+and+number+of+ventilations.">The 3:1 is superior to a 15:2 ratio in a newborn manikin model in terms of quality of chest compressions and number of ventilations.</a> Arch Dis Child Fetal Neonatal Ed. 98 (1), F42-F45 (2013).</li><li>Solev&#229;g, A. L., Schm&#246;lzer, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Chest+Compression+Rate+and+Compression+to+Ventilation+Ratio+in+Delivery+Room+Resuscitation:+Evidence+from+Newborn+Piglets+and+Neonatal+Manikins.">Optimal Chest Compression Rate and Compression to Ventilation Ratio in Delivery Room Resuscitation: Evidence from Newborn Piglets and Neonatal Manikins.</a> Frontiers in Pediatrics. 5 (3), (2017).</li><li>Solev&#229;g, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chest+compressions+in+newborn+animal+models:+A+review.">Chest compressions in newborn animal models: A review.</a> Resuscitation. 96, 151-155 (2015).</li></ol>

The authors have no conflicts of interest to declare

The lamb model is comparable in size to human newborns and allows easy instrumentation to measure invasive hemodynamic variables. The fetal/newborn lamb model has been an invaluable research tool that has richly contributed to understanding the transitioning circulation, as well as the newborn&#39;s pulmonary and cardiovascular physiology. Several experimental lamb models have been established over the years to study optimal ventilation strategies in preterm lambs13,14...

Perinatal asphyxia occurs in roughly 4 per 1,000 term births in the United States and is responsible for approximately 25% of the 4 million neonatal deaths worldwide1,2. Throughout the fetus&#39;s natural development, several adaptations must take place during labor and at birth to allow for a seamless transition from the intra- to the extrauterine environment when the lungs take on the role of the placenta as the organ of gas exchange. Any failure of the newborn to adequately transition at birth further compromises resuscitative efforts. Instances when fetal lung clearance is incomplete or delayed3,4, and circumstances that result in a persistent high pulmonary vascular resistance (PVR)5 impact the efficacy of ventilation, which remains the most important intervention in the resuscitation of the asphyxiated newborn6. In addition, immediate clamping of the umbilical cord and removal of the low-resistance placenta can lead to abrupt changes in cardiac output that may cause myocardial dysfunction7,8.
Owing to the infrequent need for aggressive resuscitation (need for chest compressions and/or epinephrine administration)1,9, there is a lack of strong evidence from large randomized clinical trials to support the current neonatal resuscitation program (NRP) guidelines. Many translational research studies in neonatal resuscitation are conducted using postnatal animal models (particularly piglets) that fail to adequately depict the transitioning fetal circulation and fluid-filled lungs inherent to the newborn in the delivery room. Given the unique challenges related to transition from fetal circulation to neonatal circulation, the perinatal asphyxiated cardiac arrest fetal lamb model is ideal to study newborn resuscitative physiology.
The studies by Joseph Barcroft on fetal lambs, as early as the 1930&#39;s, laid the foundation for fetal and neonatal physiology10. In the second half of the 20th century, Geoffrey Dawes&#39; innovative and meticulous experiments on fetal lamb models, and later those by Abraham Rudolph have tremendously contributed to the knowledge of cardiovascular and pulmonary physiology in the fetus11,12. In recent years, studies on fetal/neonatal lamb models have provided a better understanding of the impact of ventilation on hemodynamics13,14, the effects of oxygenation on PVR15,16, as well as the circulatory changes that occur during cord clamping7,17. Finally, in the past year, the newborn lamb has served as a novel model to study the hemodynamic effects during resuscitation18,19,20. A step-by-step narrative of what is involved in conducting a lamb experiment, as well as a detailed description of the surgical instrumentations and the experimental methodology will be presented.

<table><tbody><tr><td>Babcock forceps</td><td>Miltex</td><td>16-44</td><td></td></tr><tr><td>Blood pressure transducer</td><td>Becton Dickinson</td><td>P23XL-1</td><td>Used with saline filled diaphragm domes</td></tr><tr><td>Blunt tipped scissors</td><td>Miltex</td><td>98SCS50-56</td><td></td></tr><tr><td>Capnograph</td><td>Philips</td><td>7900</td><td>Used with Neonatal Flow Sensors</td></tr><tr><td>Cautery pencil</td><td>Valley Lab</td><td>287879</td><td></td></tr><tr><td>Cautery unit</td><td>Valley Lab</td><td>SSE2K</td><td></td></tr><tr><td>Curved Forceps</td><td>Everost</td><td>711714</td><td></td></tr><tr><td>Data acquisition software</td><td>Biopac Systems Inc.</td><td>ACK100W</td><td></td></tr><tr><td>EKG</td><td>Biopac Systems Inc.</td><td>ECG100C</td><td></td></tr><tr><td>Endotracheal tube -cuffed</td><td>Rusch</td><td>111780035</td><td></td></tr><tr><td>Flow modulator</td><td>Transonic Systems Inc.&amp;nbsp;</td><td>T403</td><td></td></tr><tr><td>Flow-probe</td><td>Transonic Systems Inc.&amp;nbsp;</td><td>MC4PSS-LS-WC100-CM4B-GA</td><td></td></tr><tr><td>Gastric tube</td><td>Jorgensen Labs Inc.</td><td>J0106LE</td><td>To decompress and drain ewe stomach</td></tr><tr><td>Hair clipper</td><td>Andis Company</td><td>65340</td><td>&amp;nbsp;# 40 Clipper Blade</td></tr><tr><td>Infant radiant warmer</td><td>GE healthcare</td><td>7810</td><td></td></tr><tr><td>Intravenous catheters</td><td>Becton Dickinson</td><td>381234</td><td></td></tr><tr><td>Iris surgical scissors</td><td>Patterson</td><td>510585</td><td></td></tr><tr><td>Kelly Foreceps</td><td>Patterson</td><td>510535</td><td></td></tr><tr><td>Mosquito Forceps</td><td>RICA Surgical Products INC</td><td>1-74</td><td></td></tr><tr><td>Near-infrared spectroscopy</td><td>Nonin Medical Inc.&amp;nbsp;</td><td>X-100M</td><td>Sensmart Equanox &amp;amp; PureSAT</td></tr><tr><td>RSO2 Sensor</td><td>Nonin Medical Inc.&amp;nbsp;</td><td>8004CB-NA</td><td>Neonatal&amp;nbsp;</td></tr><tr><td>Scalpel handle and blade</td><td>Everost</td><td>707203</td><td></td></tr><tr><td>Sutures -silk 2-0</td><td>Covidien</td><td>SS-695</td><td>Used for tying catheters to vessels</td></tr><tr><td>Sutures -vicryl&amp;nbsp; 2-0</td><td>Ethicon</td><td>J269H</td><td>Used for closing thoracotomy&amp;nbsp;</td></tr><tr><td>T-piece resuscitator</td><td>Neo-Tee</td><td>MCM1050812</td><td></td></tr><tr><td>Umbilical ties</td><td>Jorgensen Labs Inc.</td><td>J0025UA</td><td></td></tr><tr><td>Weitlander Retractor</td><td>Miltex</td><td>11-625</td><td></td></tr></tbody></table>

the perinatal asphyxiated lamb model: a model for newborn resuscitation

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. Many aspects of the current neonatal resuscitation guidelines remain controversial given the difficulties in conducting randomized clinical trials owing to the infrequent and often unpredictable need for extensive resuscitation. Most studies on neonatal resuscitation stem from manikin models that fail to truly reflect physiologic changes or piglet models that have cleared their lung fluid and that have completed the transition from fetal to neonatal circulation. The present protocol provides a detailed step-by-step description on how to create a perinatal asphyxiated fetal lamb model. The proposed model has a transitioning circulation and fluid-filled lungs, which mimics human newborns following delivery, and is, therefore, an excellent animal model to study newborn physiology. An important limitation to lamb experiments is the higher associated cost.

Following instrumentation of the fetal lambs, hemodynamic variables can be recorded (Figure 3 and Figure 4), then analyzed and interpreted (Figure 5). Frequent blood samples can be collected, and Figure 6 shows the pH and PaCO2 data from one of the experiments. Occasionally, catheters or flow probes may malfunction or get dislodged and data can, therefore, not...

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The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation

Invasive instrumentation of the fetal lamb provides accurate physiologic measurements of the transitioning circulation in a model that closely mimics the newly born infant.

Birth asphyxia accounts for nearly one million deaths worldwide each year, and is one of the primary causes of early neonatal morbidity and mortality. ...

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10.8K Views.  University of California Davis. Invasieve instrumentatie van het foetale lam biedt nauwkeurige fysiologische metingen van de kopstoot circulatie in een model dat nauw samen van de Nabootsers van het pasgeboren kind.

Video: The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

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

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

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

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

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

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

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 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 ...

Extracorporeal Cardiopulmonary Resuscitation in Animal Model After Cardiac Arrest

Pediatric Animal Model of Extracorporeal Cardiopulmonary Resuscitation After Prolonged Circulatory Arrest

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive investigation of this disease requires appropriate and validated animal models. Piglets are commonly used for translational research due to their analogous anatomy and physiology. This work aimed to describe and validate a neonatal piglet model of cardiopulmonary bypass (CPB) with circulatory and cardiac arrest (CA) as a tool for studying severe brain damage and other complications of cardiac surgery. In addition to including a list of materials, this work provides a roadmap for other investigators to plan and execute this protocol. After experienced practitioners performed several trials, the representative results of the model demonstrated a 92% success rate, with failures attributed to small piglet size and variant vessel anatomy. Furthermore, the model allowed practitioners to select from a wide variety of experimental conditions, including varying times in CA, temperature alterations, and pharmacologic interventions. In summary, this method uses materials readily available in most hospital settings, is reliable and reproducible, and can be widely employed to enhance translational research in children undergoing heart surgery.

Author Spotlight: Simulating Pediatric Cardiac Surgery Using a Neonatal Piglet Model

This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB.

Congenital heart disease (CHD) is the most prevalent congenital malformation, with about one million births impacted worldwide per year. Comprehensive ...

Laryngeal Mask Airway (LMA) Placement in a Neonatal Patient Simulator Using a Non-Inflatable Supraglottic Airway (SGA)

The effective delivery of positive pressure ventilation (PPV) can be challenging during neonatal resuscitation. Achieving a patent airway through an appropriate interface during neonatal resuscitation is critical for avoiding airway obstruction and leakage and optimizing access to PPV. Due to the complexity of face mask ventilation, providers have explored corrective steps. However, these methods are difficult to master and thus may present a risk for ventilation delay and/or interruptions at the critical time of resuscitation and the development of complications. In addition, neonatal endotracheal intubation is an invasive procedure that requires significant practice and training. The supraglottic airway (SGA) is a useful laryngeal mask airway (LMA) interface that decreases the time required to achieve a secure airway and reduces the need for endotracheal intubation. Despite the available evidence regarding its effectiveness, insufficient training and awareness limit SGA use in the real world, and frontline providers report low confidence in SGA placement. Here we provide a detailed description of SGA placement, the instruction of which requires only minimal training and leads to a short time to proficiency. Briefly, after the administration of initial ventilatory corrective steps in a neonatal manikin, a provider inserts a non-inflatable SGA into the larynx. This method allows for a single individual to provide effective delivery of PPV in a noninvasive manner without the need for expensive equipment such as video laryngoscopy. Instructors can easily teach this technique with ease and little cost in any clinical and research setting. This is also true for different income settings, including high-, middle-, and low-income countries.

Laryngeal Mask Airway LMA Placement in a Neonatal Patient Simulator Using a Non-Inflatable Supraglottic Airway SGA

This protocol is designed to describe a method to place and establish a supraglottic (alternative) airway during neonatal resuscitation. It can be used in situations where frontline providers cannot ventilate or endotracheally intubate neonates requiring resuscitation.

The effective delivery of positive pressure ventilation (PPV) can be challenging during neonatal resuscitation. Achieving a patent airway through an ...

The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation

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