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>

<ol>
	<li>Wyckoff, M. H., Berg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+chest+compressions+during+delivery-room+resuscitation.">Optimizing chest compressions during delivery-room resuscitation.</a> Semin Fetal Neonatal Med. 13 (6), 410-415 (2008).</li><li>Black, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+causes+of+child+mortality+in+2008:+a+systematic+analysis.">Global, regional, and national causes of child mortality in 2008: a systematic analysis.</a> Lancet. 375 (9730), 1969-1987 (2010).</li><li>Guglani, L., Lakshminrusimha, S., Ryan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+tachypnea+of+the+newborn.">Transient tachypnea of the newborn.</a> Pediatr Rev. 29 (11), e59-e65 (2008).</li><li>Brown, M. J., Olver, R. E., Ramsden, C. A., Strang, L. B., Walters, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+adrenaline+and+of+spontaneous+labour+on+the+secretion+and+absorption+of+lung+liquid+in+the+fetal+lamb.">Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb.</a> J Physiol. 344, 137-152 (1983).</li><li>Lakshminrusimha, S., Saugstad, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fetal+circulation,+pathophysiology+of+hypoxemic+respiratory+failure+and+pulmonary+hypertension+in+neonates,+and+the+role+of+oxygen+therapy.">The fetal circulation, pathophysiology of hypoxemic respiratory failure and pulmonary hypertension in neonates, and the role of oxygen therapy.</a> J Perinatol. 36 Suppl 2, S3-S11 (2016).</li><li>Wyckoff, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+13:+Neonatal+Resuscitation:+2015+American+Heart+Association+Guidelines+Update+for+Cardiopulmonary+Resuscitation+and+Emergency+Cardiovascular+Care.">Part 13: Neonatal Resuscitation: 2015 American Heart Association Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care.</a> Circulation. 132 (18 suppl 2), S543-S560 (2015).</li><li>Bhatt, 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=Delaying+cord+clamping+until+ventilation+onset+improves+cardiovascular+function+at+birth+in+preterm+lambs.">Delaying cord clamping until ventilation onset improves cardiovascular function at birth in preterm lambs.</a> J Physiol. 591 (Pt 8), 2113-2126 (2013).</li><li>Katheria, A. C., Brown, M. K., Rich, W., Arnell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+a+Placental+Transfusion+in+Newborns+Who+Need+Resuscitation).">Providing a Placental Transfusion in Newborns Who Need Resuscitation).</a> Front Pediatr. 5 (1), (2017).</li><li>Kapadia, V. S., Wyckoff, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+during+delivery+room+resuscitation--what,+when+and+why?.">Drugs during delivery room resuscitation--what, when and why?.</a> Semin Fetal Neonatal Med. 18 (6), 357-361 (2013).</li><li>Barcroft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Researches on Pre-natal Life. 1, 292 (1946).</li><li>Dawes, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Foetal and Neonatal Physiosolgy. A Comparative Study of the Changes at Birth. , (1968).</li><li>Rudolph, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Congenital Diseases of the Heart: Clinical-Physiological Considerations. , 1-24 (2009).</li><li>Sobotka, K. 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=An+initial+sustained+inflation+improves+the+respiratory+and+cardiovascular+transition+at+birth+in+preterm+lambs.">An initial sustained inflation improves the respiratory and cardiovascular transition at birth in preterm lambs.</a> Pediatr Res. 70 (1), 56-60 (2011).</li><li>Polglase, G. R., 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=Positive+end-expiratory+pressure+differentially+alters+pulmonary+hemodynamics+and+oxygenation+in+ventilated,+very+premature+lambs.">Positive end-expiratory pressure differentially alters pulmonary hemodynamics and oxygenation in ventilated, very premature lambs.</a> J Appl Physiol (1985). 99 (4), 1453-1461 (2005).</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=Oxygen+concentration+and+pulmonary+hemodynamics+in+newborn+lambs+with+pulmonary+hypertension.">Oxygen concentration and pulmonary hemodynamics in newborn lambs with pulmonary hypertension.</a> Pediatr Res. 66 (5), 539-544 (2009).</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=Pulmonary+hemodynamics+in+neonatal+lambs+resuscitated+with+21%,+50%+and+100%+oxygen.">Pulmonary hemodynamics in neonatal lambs resuscitated with 21%, 50% and 100% oxygen.</a> Pediatr Res. 62 (3), 313-318 (2007).</li><li>Hooper, S. B., 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=Cardiovascular+transition+at+birth:+a+physiological+sequence.">Cardiovascular transition at birth: a physiological sequence.</a> Pediatr Res. 77 (5), 608-614 (2015).</li><li>Vali, P., 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=Evaluation+of+Timing+and+Route+of+Epinephrine+in+a+Neonatal+Model+of+Asphyxial+Arrest.">Evaluation of Timing and Route of Epinephrine in a Neonatal Model of Asphyxial Arrest.</a> J Am Heart Assoc. 6 (2), (2017).</li><li>Vali, P., 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+Chest+Compressions+During+Sustained+Inflations+in+a+Perinatal+Asphyxial+Cardiac+Arrest+Lamb+Model.">Continuous Chest Compressions During Sustained Inflations in a Perinatal Asphyxial Cardiac Arrest Lamb Model.</a> Pediatr Crit Care Med. , (2017).</li><li>Vali, P., 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=Hemodynamics+and+gas+exchange+during+chest+compressions+in+neonatal+resuscitation.">Hemodynamics and gas exchange during chest compressions in neonatal resuscitation.</a> PLoS One. 12 (4), e0176478 (2017).</li><li>Tana, 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=Determination+of+Lung+Volume+and+Hemodynamic+Changes+During+High-Frequency+Ventilation+Recruitment+in+Preterm+Neonates+With+Respiratory+Distress+Syndrome.">Determination of Lung Volume and Hemodynamic Changes During High-Frequency Ventilation Recruitment in Preterm Neonates With Respiratory Distress Syndrome.</a> Crit Care Med. 43 (8), 1685-1691 (2015).</li><li>Morin, F. 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.

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		<tr height="20">
			<td height="20" style="height:20px;">Babcock forceps</td>
			<td>Miltex</td>
			<td>16-44</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Blood pressure transducer</td>
			<td style="width:192px;">Becton Dickinson</td>
			<td style="width:211px;">P23XL-1</td>
			<td style="width:200px;">Used with saline filled diaphragm domes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blunt tipped scissors</td>
			<td style="width:192px;">Miltex</td>
			<td style="width:211px;">98SCS50-56</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Capnograph</td>
			<td style="width:192px;">Philips</td>
			<td style="width:211px;">7900</td>
			<td style="width:200px;">Used with Neonatal Flow Sensors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cautery pencil</td>
			<td style="width:192px;">Valley Lab</td>
			<td style="width:211px;">287879</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cautery unit</td>
			<td style="width:192px;">Valley Lab</td>
			<td style="width:211px;">SSE2K</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved Forceps</td>
			<td style="width:192px;">Everost</td>
			<td style="width:211px;">711714</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Data acquisition software</td>
			<td style="width:192px;">Biopac Systems Inc.</td>
			<td style="width:211px;">ACK100W</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EKG</td>
			<td style="width:192px;">Biopac Systems Inc.</td>
			<td style="width:211px;">ECG100C</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Endotracheal tube -cuffed</td>
			<td style="width:192px;">Rusch</td>
			<td style="width:211px;">111780035</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow modulator</td>
			<td style="width:192px;">Transonic Systems Inc.&#160;</td>
			<td style="width:211px;">T403</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow-probe</td>
			<td style="width:192px;">Transonic Systems Inc.&#160;</td>
			<td style="width:211px;">MC4PSS-LS-WC100-CM4B-GA</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gastric tube</td>
			<td style="width:192px;">Jorgensen Labs Inc.</td>
			<td style="width:211px;">J0106LE</td>
			<td style="width:200px;">To decompress and drain ewe stomach</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hair clipper</td>
			<td style="width:192px;">Andis Company</td>
			<td style="width:211px;">65340</td>
			<td style="width:200px;">&#160;# 40 Clipper Blade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Infant radiant warmer</td>
			<td>GE healthcare</td>
			<td>7810</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Intravenous catheters</td>
			<td>Becton Dickinson</td>
			<td>381234</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iris surgical scissors</td>
			<td style="width:192px;">Patterson</td>
			<td style="width:211px;">510585</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kelly Foreceps</td>
			<td style="width:192px;">Patterson</td>
			<td style="width:211px;">510535</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mosquito Forceps</td>
			<td style="width:192px;">RICA Surgical Products INC</td>
			<td style="width:211px;">1-74</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Near-infrared spectroscopy</td>
			<td style="width:192px;">Nonin Medical Inc.&#160;</td>
			<td style="width:211px;">X-100M</td>
			<td style="width:200px;">Sensmart Equanox &#38; PureSAT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RSO2 Sensor</td>
			<td style="width:192px;">Nonin Medical Inc.&#160;</td>
			<td style="width:211px;">8004CB-NA</td>
			<td style="width:200px;">Neonatal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle and blade</td>
			<td style="width:192px;">Everost</td>
			<td>707203</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sutures -silk 2-0</td>
			<td style="width:192px;">Covidien</td>
			<td style="width:211px;">SS-695</td>
			<td style="width:200px;">Used for tying catheters to vessels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sutures -vicryl&#160; 2-0</td>
			<td style="width:192px;">Ethicon</td>
			<td style="width:211px;">J269H</td>
			<td style="width:200px;">Used for closing thoracotomy&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T-piece resuscitator</td>
			<td>Neo-Tee</td>
			<td>MCM1050812</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Umbilical ties</td>
			<td style="width:192px;">Jorgensen Labs Inc.</td>
			<td>J0025UA</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Weitlander Retractor</td>
			<td style="width:192px;">Miltex</td>
			<td style="width:211px;">11-625</td>
			<td style="width:200px;"></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...

Watch this Scientific Journal Video about The Perinatal Asphyxiated Lamb Model: A Model for Newborn Resuscitation at JoVE.com

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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10.7K Views.  University of California Davis. Invasive instrumentation of the fetal lamb provides accurate physiologic measurements of the transitioning circulation in a model that closely mimics the newly born infant. 

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 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 ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Sterile Pericarditis in Aachener Minipigs As a Model for Atrial Myopathy and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only target the electrical abnormalities and not the underlying atrial myopathy. For the development of novel therapies, a reproducible large animal model of atrial myopathy is necessary. This paper presents a model of sterile pericarditis-induced atrial myopathy in Aachener minipigs. Sterile pericarditis was induced by spraying sterile talcum and leaving a layer of sterile gauze over the atrial epicardial surface. This led to inflammation and fibrosis, two crucial components of the pathophysiology of atrial myopathy, making the atria susceptible to the induction of AF. Two pacemaker electrodes were positioned epicardially on each atrium and connected to two pacemakers from different manufacturers. This strategy allowed for repeated non-invasive atrial programmed stimulation to determine the inducibility of AF at specified time points after surgery. Different protocols to test AF inducibility were used. The advantages of this model are its clinical relevance, with AF inducibility and the rapid induction of inflammation and fibrosis-both present in atrial myopathy-and its reproducibility. The model will be useful in the development of novel therapies targeting atrial myopathy and AF.

We describe a sterile pericarditis model in minipigs to study atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only ...

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

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

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

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

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