The authors acknowledge the surgical assistance of Jane Choi (University of Western Australia), Ellen Williams (University of Western Australia), and Veena Kurup (University of Western Australia), as well as the husbandry care of the Animal Care Services staff at the University of Western Australia. This study was supported by the Telethon Perth Children&#39;s Research Fund, National Health and Medical Research Council RF 1077691 (JJP), Metropolitan Health and Medical Research Infrastructure Fund (West Australian Government), and Australian International Research Training Program (MD).

The protocol adhered to the Australian National Health and Medical Research Council Australian code for the care and use of animals for scientific purposes18. The University of Western Australia Animal Ethics Committee prospectively approved the protocol (RA3/100/1596). Sheep were sourced from the University of Western Australia (UWA) Ridgefield farm (2018-2020). The sheep were introduced to the AAALAC-accredited UWA Large Animal Facility 2-3 weeks prior to surgical intervention. Sheep were initially housed indoors in shared raised floor pens, with single pens used in the peri-operative period. Sheep were fed pellets, oaten chaff, and lupins with a mineral mix calculated on body weight. Rooms were controlled for temperature (20.5-21.5 &#176;C) and maintained on a 12:12 h light: dark cycle. Pain and well-being were evaluated in the postoperative period twice daily for 7 days, then daily until study completion. Monitored postoperative parameters included signs of pain, orbital tightening, respiratory effort, temperature, heart rate, the surgical site, appetite and water intake, activity, lameness, coat condition, fecal consistency, preterm labor, demeanor, and signs of pregnancy toxemia. The scores attributed to each parameter triggered an intervention where necessary.
NOTE: Two recovery surgical procedures were required: ewe preparation, surgical approach and closure, and ewe recovery are the same for both surgical procedures. The first procedure is the surgical creation of the diaphragmatic hernia in the fetal lamb. The second procedure is for placement of the fetal intervention and pressure monitoring device. The study design necessitated two survival surgeries to allow for the abdominal contents to move into the chest cavity after the creation of the diaphragmatic hernia so that lung hypoplasia could develop before the subsequent intervention surgery.
1. Surgical creation of the diaphragmatic hernia in the fetal lamb
<ol>
	<li>Ewe preparation
	<ol>
		<li>At ~80 days gestation, premedicate the ewe with an intramuscular injection of acepromazine (0.04 mg/Kg) and morphine (0.3 mg/Kg).</li>
		<li>Induce anesthesia intravenously with ketamine (5 mg/Kg) and midazolam (0.25 mg/Kg).</li>
		<li>Intubate the ewe trachea with an endotracheal tube (8 mm inner diameter [ID]). Maintain anesthesia with isoflurane (1%-3% in oxygen) delivered by positive pressure ventilation (10-12 breaths/min, tidal volume 8-10 mL/kg).</li>
		<li>Establish ewe monitoring with an electrocardiogram (ECG), invasive arterial blood pressure via an ear arterial catheter, expired CO2, peripheral oxygen saturation, and temperature during surgery.</li>
		<li>Place the ewe supine on the gurney. Loop ropes around the ewe&#39;s legs and gently secure to the table.</li>
		<li>Shear wool from the ewe&#39;s abdomen and flank and then clip it close to the skin.</li>
		<li>Remove dirt and lanolin from the ewe&#39;s abdomen, flank, and groin with soap and water.</li>
		<li>Give cefazolin (20 mg/Kg) intravenously every 90 min during surgery.</li>
		<li>Transfer the ewe to the surgical theatre on a gurney.</li>
		<li>Move the ewe from the transport gurney to the surgical table.</li>
		<li>Place ewe in dorsal recumbency position on the surgical table.</li>
		<li>Secure all limbs to the surgical table with soft rope ties.</li>
		<li>Aseptic preparation of the exposed abdomen: Wash 3 times with 4% chlorhexidine surgical scrub, removing contaminated solution between each wash with 70% alcohol.</li>
		<li>Complete sterile preparation by lightly spraying 10% iodine solution on the abdomen, flank, and groins.</li>
		<li>Cover the ewe with a sterile fenestrated surgical drape so that only the abdominal incision site is exposed. Ensure that all surgical instruments, catheters, syringes, and solutions used during surgery are sterile.</li>
	</ol>
	</li>
	<li>Surgical approach
	<ol>
		<li>Establish line-block local anesthesia by local subcutaneous and muscular infiltration of bupivacaine (5%; 9.5 mL) with a 10 mL syringe and 22 G needle.</li>
		<li>Expose the uterus by a para-midline abdominal incision (8-11 cm) through the ewe&#39;s skin, subcutaneous tissue, and linea alba made with a size 10 surgical blade with cautery as required to minimize incisional bleeding.</li>
	</ol>
	</li>
	<li>Diaphragmatic hernia creation
	<ol>
		<li>Locate the fetal head/thorax via palpation.</li>
		<li>Cover the uterus with sterile plastic wrap.</li>
		<li>Use surgical scissors to make a window (6 cm x 4 cm) in the plastic wrap.</li>
		<li>Make a 5-7 cm uterine incision through the uterus and amniotic sac with cautery cutting, avoiding uterine blood vessels and placental cotyledons.</li>
		<li>Exteriorize the fetal head and forelimbs.</li>
		<li>Use Babcock uterine clamps to seal the uterine incision edges and sterile plastic wrap tightly around the fetal abdomen to prevent amniotic fluid loss.</li>
		<li>Position the fetus on its right side and secure a second sterile plastic wrap over the exteriorized fetus to reduce loss of heat and moisture. Identify the intercostal space between the 9th and 10th ribs on the left and cut a hole in the plastic wrap with surgical scissors to access the intended fetal incision site.</li>
		<li>Establish line-block local anesthesia in the fetus by local subcutaneous and muscular infiltration of bupivacaine (5%; 0.25 mL diluted to 0.5 mL with sterile saline; 1 mL sterile syringe and 27 G needle) in the 9th intercostal space immediately adjacent to the upper border of the 10th rib to avoid subcostal vessels.</li>
		<li>Use a 60 mL sterile syringe to bathe the exposed fetal skin with warm Hartmann&#39;s solution (5-10 mL every 5-10 min) to keep the fetal skin moist throughout subsequent steps.</li>
		<li>Create a left posterolateral thoracotomy by making a 2-4 cm skin incision with electrocautery in the 9th intercostal space immediately adjacent to the cranial border of the 10th rib.</li>
		<li>Use mosquito hemostats to bluntly dissect the subcutaneous tissue, intercostal muscles, and pleural membrane to access the diaphragm.</li>
		<li>Use two small mosquito hemostats to pick up the diaphragm at each end of the intended 1 cm long hernia site and cranially elevate the diaphragm to keep it away from the underlying stomach, liver, and spleen.</li>
		<li>Make a 1 cm incision between the two hemostats using small angled scissors.</li>
		<li>Release the hemostats holding the diaphragm.</li>
		<li>Use atraumatic Potts forceps to pull the stomach superiorly through the hole in the diaphragm.</li>
		<li>Use a single tie of absorbable 5-0 polydioxanone monofilament (RB-1 taper point needle) to carefully envelop the 9th and 10th ribs to oppose the fetal ribs, avoiding the underlying lung.</li>
		<li>Close thoracotomy incision with a mattress suture (absorbable 5-0 polydioxanone monofilament).</li>
	</ol>
	</li>
	<li>Surgical closure
	<ol>
		<li>Return the fetus to the amniotic sac.</li>
		<li>Administer intra-operative meloxicam (0.25 mg/Kg; subcutaneously) to the ewe for postoperative analgesia.</li>
		<li>Replace lost amniotic fluid using warmed Hartmann&#39;s solution (~50 mL) along with intra-amniotic Piperacillin/Tazobactam (1000 mg/125 mg).</li>
		<li>Appose the opposing surfaces of the chorioamnion and adjacent uterine walls and use an absorbable polyglactin 910 braided 2-0 suture with an inverted mattress suture to close the uterus and amniotic sac together.</li>
		<li>Close the ewe&#39;s linea alba, subcutaneous tissue, and skin (absorbable polydioxanone monofilament 1, absorbable poliglecaprone 25 monofilament 3-0, and nonabsorbable polypropylene monofilament 2-0 respectively).</li>
	</ol>
	</li>
	<li>Ewe recovery and monitoring
	<ol>
		<li>Spray the abdominal wound post-operatively with waterproof dressing retention tape and cover it with a dressing to reduce the risk of postoperative wound infection.</li>
		<li>Apply a transdermal fentanyl patch (100 &#181;g/h) to the groin for additional postoperative analgesia.</li>
		<li>Recover the ewe and extubate when breathing spontaneously, then allow it to wake.</li>
		<li>Monitor ewes daily until delivery for fetal intervention surgery.</li>
		<li>Remove sutures after 14 days.</li>
	</ol>
	</li>
</ol>
2. Placement of the fetal intervention and pressure monitoring device
<ol>
	<li>Ewe preparation for fetal intervention surgery at 101 days of gestation
	<ol>
		<li>Inject medroxyprogesterone (1 mL) intramuscularly at ~85 days of gestation (term ~147 days) to reduce the risk of preterm birth.</li>
		<li>Use the same anesthetic and surgical preparation procedure used for the initial diaphragm creation surgery.
		<ol>
			<li>Follow the surgical approach in steps 2.1.2.2 and 2.1.2.3, similar to steps 1.2.1-1.2.2.</li>
			<li>Establish line-block local anesthesia by local subcutaneous and muscular infiltration of bupivacaine (5 %; 9.5 mL) with a 10 mL syringe and 22 G needle.</li>
			<li>With a para-midline abdominal incision (8 - 11 cm) adjacent to the abdominal incision site used for the diaphragm creation surgery, expose the uterus with a through the ewe&#39;s skin, subcutaneous tissue, and linea alba made with a size 10 surgical blade with cautery as required to minimize incisional bleeding.</li>
		</ol>
		</li>
		<li>Additionally, administer oxytetracycline (20 mg/Kg) intramuscularly to the ewe to provide fetal antimicrobial protection against the chronically implanted monitoring device.</li>
	</ol>
	</li>
	<li>Device activation
	<ol>
		<li>Activate pressure measurement device19.</li>
		<li>Clamp pressure measurement tubing with hemostats.</li>
		<li>Place the entire device into a sterile bowl containing a mixture of 0.08% peracetic acid and 1% hydrogen peroxide for at least 1 h, ensuring the entire device is covered.</li>
		<li>Using a sterile clamp, move the device to a sterile bowl containing sterile water for at least 10 min prior to fetal implantation. Ensure the entire device is covered with sterile water.</li>
	</ol>
	</li>
	<li>Surgical approach and fetal intervention
	<ol>
		<li>Exteriorize the fetal head by hysterotomy using the same surgical approach used for the initial surgery but with adjacent uterine incisions (steps 1.1-2.2).</li>
		<li>Keep the exteriorized fetal head and neck wrapped in the sterile plastic drape to minimize loss of temperature and fluid.</li>
		<li>Instrument the trachea with a catheter for pressure measurements (Figure 1).
		<ol>
			<li>For unoccluded trachea, follow steps 2.3.3.2-2.3.3.3.</li>
			<li>Intubate the fetal lamb with a 5 French (1.67 mm ID) suction catheter with the suction connector removed to a depth of 14 cm at the corner of the lamb&#39;s mouth.</li>
			<li>Connect the external end of the catheter with a 1.59 mm (outside diameter, OD) to 3.18 mm (OD) connector to 30 cm long 3.18 mm (ID) silicone tubing.</li>
			<li>For cccluded trachea, follow steps 2.3.3.5-2.3.3.6.</li>
			<li>Intubate the fetal lamb with a 3.0 microcuff endotracheal tube with the connector removed to a depth of 14 cm to a corner of the lamb&#39;s mouth.</li>
			<li>Connect the external end of the catheter with a 3.18 mm (OD) to 3.18 mm (OD) connector to 30 cm long 3.18 mm (ID) silicone tubing.</li>
		</ol>
		</li>
		<li>Connect the other end of the 30 cm long 3.18 mm (ID) silicone tubing to the pressure measurement device by a 3.18 mm (OD) to 1.59 mm (OD) connector on the pressure measurement device.</li>
		<li>Suture the catheter at the corner of the lamb&#39;s mouth and neck (nonabsorbable nylon monofilament 4-0).</li>
		<li>Secure the pressure measurement device to the lamb&#39;s skin on the chest (nonabsorbable nylon monofilament 4-0) (Figure 2).</li>
	</ol>
	</li>
	<li>Close the fetal and ewe surgical incisions as per initial diaphragmatic hernia creation surgery (steps 1.4- 1.5).</li>
	<li>Test device
	<ol>
		<li>Test if the device is transmitting signal19.</li>
	</ol>
	</li>
	<li>Ewe recovery and monitoring
	<ol>
		<li>Recover the ewe as per the initial surgery.</li>
		<li>Monitor ewes daily until delivery.</li>
		<li>Give a second dose of medroxyprogesterone (0.5 mL) intramuscularly at ~120 days of gestation.</li>
		<li>Give betamethasone (5.7 mg) intramuscularly 48 h and 24 h before cesarean delivery to promote fetal maturation for postnatal care.</li>
	</ol>
	</li>
	<li>Lamb delivery
	<ol>
		<li>At ~142 days of gestation (term ~147-150 days), pre-medicate ewe by intramuscular injection of acepromazine (0.03 mg/Kg) and morphine (0.3 mg/Kg).</li>
		<li>Induce anesthesia intravenously with thiopental (10-15 mg/Kg), intubate the ewe with an endotracheal tube (size 8.5 mm), and maintain anesthesia by isofluorane with appropriate monitoring per earlier anesthetics.</li>
		<li>Proceed with surgical delivery of the fetus via hysterotomy as per previous surgeries, with ewe in dorsal recumbency (steps 1.1 - 2.2).</li>
		<li>Remove the monitoring device. Clean the device with water and 70% ethanol.</li>
		<li>Humanely kill the ewe with an intravenous injection of 160 mg/Kg of pentobarbitone sodium solution.</li>
		<li>Resuscitate and ventilate the lamb for 2-3 h and then humanely kill (160 mg/Kg of pentobarbitone sodium solution, intravenous injection).</li>
	</ol>
	</li>
	<li>Data
	<ol>
		<li>Download data to a laptop and analyze19. Filter the pressure data using a 6-h window. Compute the difference in pressure between amniotic and tracheal pressures.</li>
	</ol>
	</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+tracheal+obstruction+in+the+fetal+sheep:+effects+on+tracheal+fluid+pressure+and+lung+growth.">Reversible tracheal obstruction in the fetal sheep: effects on tracheal fluid pressure and lung growth.</a> J Pediatr Surg. 30 (8), 1172-1177 (1995).</li><li>Hellmeyer, 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=Telemetric+monitoring+of+tracheal+pressure+after+tracheal+occlusion+for+treatment+of+severe+congenital+diaphragmatic+hernia.">Telemetric monitoring of tracheal pressure after tracheal occlusion for treatment of severe congenital diaphragmatic hernia.</a> Arch Gynecol Obstet. 275 (4), 245-248 (2007).</li></ol>

The authors declare that no conflicts of interest exist.

Fetal lung liquid fills the potential airspaces during gestation and is vital for normal lung development1. Altering the normal lung liquid amount and pressure affects fetal lung growth: narrowing or constriction of the fetal trachea leads to lung hyperplasia; conversely, oligohydramnios and chronic drainage of lung fluid cause lung hypoplasia20,21,22,23. Tracheal occlusion is effective in improving lung hypoplasia15,16,17 Animal and human studies suggest that occluding the trachea earlier in gestation is of greater benefit due to the longer period of expansion and higher chest wall compliance of a younger fetus17,24,25,26. However, the timing of placement and duration of the tracheal occlusion needs further refinement. Additionally, understanding the pressure changes generated by tracheal occlusion over time is essential for improving in utero treatments.
Early methods for in utero intrathoracic pressure measurements used tracheotomy and externalized tracheal loops with periodic measurements1,2,4,5,6,7. While reporting increased lung pressure with occlusion, these early methods ligated the trachea. Occlusion of an intact trachea and measurement of intra-tracheal pressure with a Swan-Ganz catheter increased pulmonary pressure27. Previous studies measuring pressure with a Swan-Ganz catheter obtained measurements weekly. Telemetric measurement of tracheal pressure with a modified urinary catheter has collected data over 2 min three times a day28.
The innovative telemetric approach advances previous methods used to monitor fetal tracheal pressures. Firstly, no externalized catheters were used: internal catheters provide the opportunity to measure pressures without restricting the movements of the ewe, while externalized catheters are a potential source of infection and can dampen the pressure signal. Secondly, the approach facilitated continuous measurement of tracheal pressure regardless of ewe position and sleep/wake cycle. Fetal breathing and associated pressure fluctuation are essential in normal lung development, and the effect of tracheal occlusion on these pressures can be understood better by measuring pressures continuously.
The protocol has three limitations. Firstly, the lamb quadruples in size from fetal intervention to delivery, so securing the device is challenging. Initially, the device was attached to the fetus using elastic band netting. However, the mesh caused thickened and reduced amniotic fluid. Creating several attachment points on the device and suturing the fetal skin with extra sutures reduced the risk of detachment without causing an inflammatory response. Secondly, the battery life of the device limited the duration of measurement recordings. Thirdly, the fluid-filled amniotic sac environment presented challenges for maintaining the device&#39;s impermeability to water and maintaining the integrity of the device&#39;s electronics.
In conclusion, the innovative telemetric measurement protocol allows continuous monitoring of fetal intratracheal and amniotic pressure in unrestrained pregnant animals. Additionally, with improved device battery life, the continuous measurement of fetal intratracheal and amniotic pressure will provide a complete understanding of how these pressures change throughout gestation.

Normal lung development and growth rely upon the expansion of the potential airspaces with fetal lung liquid and the controlled efflux of the lung liquid into the amniotic space1,2,3,4,5. Fetal lung liquid production and the resistance of the upper airways create an in utero intrathoracic pressure1. Historically, in utero measurements of fetal airway pressures were obtained using external pressure sensors via catheters tunneled through the abdominal wall to the fetal trachea5,6,7,8,9,10,11,12,13. The use of these catheters and external sensors (distal to the measurement site) may dampen the pressure signal and necessitate restricted movement of the ewe for continuous measurement or measurements obtained at intervals across gestation. This study aimed to develop a method that allows continuous monitoring of fetal intratracheal and amniotic pressure in unrestrained pregnant animals. The continuous measurements of fetal intratracheal and amniotic pressure will provide a complete understanding of how these pressures change throughout the day over the course of gestation.
Human fetuses with congenital diaphragmatic hernia (CDH) have lung hypoplasia due to herniation of the stomach, the bowel, and the liver (in the most severe cases) into the chest cavity. Lung hyperplasia in infants with tracheal atresia (narrowing of the trachea) highlighted the potential of therapeutic tracheal occlusion for antenatal treatment of CDH14. Tracheal occlusion using an intra-tracheal balloon reduces the severity of lung hypoplasia in fetuses with CDH but at the cost of an increased risk of premature birth15,16,17. An additional risk of aspiration or suffocation exists if the balloon is not removed before birth. Consequently, current tracheal occlusion protocols require a second fetal procedure to remove the balloon occlusion before birth17. The optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is unknown, but this knowledge is vital for the optimization of in utero corrective treatments for CDH.
We tested the method using fetal lambs with a surgically created diaphragmatic hernia with and without an occluded trachea.

<table><tbody><tr><td>1.59 mm (outside diameter, OD) to 3.18 mm (OD) connector</td><td>Qosina</td><td>11913</td><td></td></tr><tr><td>3.18 mm (OD) to 3.18 mm (OD) connector</td><td>Qosina</td><td>11684</td><td></td></tr><tr><td>70 % Alcohol</td><td>Henry Schein</td><td>1127067</td><td></td></tr><tr><td>Absorbable poliglecaprone 25 monofilament 3-0</td><td>Riverpoint Medical</td><td>Q316</td><td></td></tr><tr><td>Absorbable polydioxanone monofilament 1</td><td>Riverpoint Medical</td><td>D879</td><td></td></tr><tr><td>Absorbable polyglactin 910 braided 2-0</td><td>Riverpoint Medical</td><td>V317</td><td></td></tr><tr><td>Absorbable polydioxanone monofilament 5-0</td><td>Riverpoint Medical</td><td>D303</td><td></td></tr><tr><td>Acepromazine</td><td>Ceva Animal Health</td><td>APVMA No: 36680</td><td></td></tr><tr><td>Babcock, uterine forceps 6.25 inch</td><td>Roboz</td><td>RS-8022</td><td></td></tr><tr><td>Betamethasone</td><td>Merck Sharp &amp;amp; Dohme</td><td>Aust R 18777</td><td></td></tr><tr><td>Blade, size 10</td><td>Becton Dickinson</td><td>371110</td><td></td></tr><tr><td>Blade, size 15</td><td>Becton Dickinson</td><td>371115</td><td></td></tr><tr><td>Bupivacaine</td><td>Pfizer Australia Pty Ltd</td><td>AUST R 11312</td><td></td></tr><tr><td>Cefazolin</td><td>AFT pharmaceuticals</td><td>AUST R 171582</td><td></td></tr><tr><td>Chlorhexidine</td><td>Henry Schein</td><td>0404-0175-02</td><td></td></tr><tr><td>Endotracheal tube (size 8.0)</td><td>&amp;nbsp;Jorgen Kruuse</td><td>272411</td><td></td></tr><tr><td>Forceps, Potts-Smith</td><td>Roboz</td><td>RS-5314</td><td></td></tr><tr><td>Iodine solution (10 %)</td><td>Henry Schein</td><td>6907281</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Critical Care&amp;nbsp;</td><td>APVMA No: 53120/112272&lt;br/&gt; M. L. No.:220/AP/MD/96/B&amp;amp;F/R</td><td></td></tr><tr><td>Ketamine</td><td>Ceva Animal Health</td><td>APVMA 37711/58317&lt;br/&gt; KETALAB04</td><td></td></tr><tr><td>Hartmanns Solution</td><td>Baxter&amp;nbsp;</td><td>AUST R 48510</td><td></td></tr><tr><td>Hemostats, Mosquito forceps curved delicate</td><td>Roboz</td><td>RS-7271</td><td></td></tr><tr><td>Medroxyprogesterone acetate</td><td>Pfizer Australia Pty Ltd</td><td>AUST R 12300</td><td></td></tr><tr><td>Meloxicam</td><td>Ilium</td><td>APVMA Approval No.: 62535/127884&lt;br/&gt; LI0119V1</td><td></td></tr><tr><td>Methocel</td><td>Colorcon&amp;nbsp;</td><td>ID34435</td><td></td></tr><tr><td>Microcuff endotracheal tube (3.0)</td><td>Halyard</td><td>35111</td><td></td></tr><tr><td>Midazolam</td><td>Mylan</td><td>AUST R 160205</td><td></td></tr><tr><td>Morphine</td><td>Pfizer Australia Pty Ltd</td><td>AUST R 101240</td><td></td></tr><tr><td>Needle, 22 G</td><td>Becton Dickinson</td><td>305155</td><td></td></tr><tr><td>Needle, 27 G</td><td>Becton Dickinson</td><td>305109</td><td></td></tr><tr><td>Nonabsorbable nylon monofilament 4-0</td><td>Riverpoint Medical</td><td>662BL</td><td></td></tr><tr><td>Nonabsorbable polypropylene monofilament 2-0</td><td>Riverpoint Medical</td><td>P8411</td><td></td></tr><tr><td>OpSite Transparent Film</td><td>Smith and Nephew</td><td>66000040</td><td></td></tr><tr><td>Oxytetracycline</td><td>Norbrook</td><td>APVMA Approval No: 53087/49616</td><td></td></tr><tr><td>Pentobarbitone sodium 300 mg/mL</td><td>Jurox</td><td>APVMA 36208</td><td></td></tr><tr><td>Peracetic acid/hydrogen peroxide</td><td>Medivators Inc&amp;nbsp;</td><td>ref: 78401-649</td><td></td></tr><tr><td>Piperacillin/Tazobactam&amp;nbsp;</td><td>Sandoz Pty Ltd</td><td>AUST R 140840</td><td></td></tr><tr><td>Scissors, Metzenbaum Surgical 7 inch straight</td><td>Roboz</td><td>RS-6955SC</td><td></td></tr><tr><td>Scissors, Vannas 0.15 mm tip width</td><td>Roboz</td><td>RS-5618</td><td></td></tr><tr><td>Silicone tubing (1.59 mm inside diameter)</td><td>Qosina</td><td>T2013</td><td></td></tr><tr><td>Suction catheter (5 French)</td><td>Covidien</td><td>30500</td><td></td></tr><tr><td>Syringe, 1 mL</td><td>Becton Dickinson</td><td>309659</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>Becton Dickinson</td><td>309604</td><td></td></tr><tr><td>Syringe, 60 mL</td><td>Becton Dickinson</td><td>309654</td><td></td></tr><tr><td>Telemetry device</td><td>Politecnico di Milano</td><td>-</td><td>Not commercially available</td></tr><tr><td>Thiopentone sodium</td><td>Jurox Pty Ltd</td><td>APVMA No. 51520/5g/0809</td><td></td></tr><tr><td>Transdermal fentanyl patch</td><td>Janssen-Cilag Pty Ltd</td><td>AUST R 112371</td><td></td></tr></tbody></table>

continuous telemetric in utero tracheal pressure measurements in fetal lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Congenital diaphragmatic hernia creation and pressure measurement device insertion were performed in 28 fetal lambs (14 unoccluded and 14 balloon occluded). Fifteen fetal lambs (6 unoccluded and 9 balloon occluded) survived to near term (142 days gestation; term ~147 days gestation). Pressure measurement recordings of 14 days were successful in 8 fetal lambs (4 unoccluded and 4 balloon occluded).
Pressure measurements were analyzed from the two groups of fetal lambs following the completion of the surgical protocol. The two pressure sensors in the device allow continuous and simultaneous sampling of the amniotic and fetal intratracheal pressures. Measurement of both amniotic and intra-tracheal pressures allows compensation for changes in fetal lung pressure due to ewe movement and abdominal/uterine pressure. The difference between the fetal intratracheal and amniotic pressures is the transrespiratory pressure. Occluding the fetal trachea led to higher transrespiratory pressure compared to the unoccluded trachea animal (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64767/64767fig01.jpg" /> 
Figure 1: Device setup. (A) Setup for the balloon unoccluded fetal lamb. (B) Setup for the occluded fetal lamb. <a href="https://www.jove.com/files/ftp_upload/64767/64767fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64767/64767fig02.jpg" /> 
Figure 2: Device after insertion of pressure measurement catheter. <a href="https://www.jove.com/files/ftp_upload/64767/64767fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64767/64767fig03.jpg" /> 
Figure 3: Representative fetal in utero pressure measurements over 18 days of gestation. Transrespiratory pressure represents the actual intraluminal pressure of the fetal lung. Pressure in the balloon-occluded lamb (grey line) is greater than that of the unoccluded (black line) lamb. <a href="https://www.jove.com/files/ftp_upload/64767/64767fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs at JoVE.com

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants ...

continuous-telemetric-utero-tracheal-pressure-measurements-fetal

Nanyang Technological University

Research

JoVE Journal

Medicine

245 Views.  The University of Western Australia. Optimization of occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improve in utero corrective treatments for congenital diaphragmatic hernia (CDH). This study reports a new method for continuous measurement of the tracheal pressure in an occluded and unoccluded fetal lamb surgical model of CDH.

Video: Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as endotoxins, bacteria, umbilical cord occlusions, hypoxia and various pharmacological treatments. The life-saving clinical practices of glucocorticoid treatment in fetuses at risk of premature birth and the therapeutic hypothermia have been developed in this model. This is due to the unique amenability of the non-anesthetized fetal sheep to the surgical placement and maintenance of catheters and electrodes, allowing repetitive blood sampling, substance injection, recording of bioelectrical activity, application of electric stimulation and in vivo organ imaging. Here we describe the surgical instrumentation procedure required to achieve a stable chronically instrumented non-anesthetized fetal sheep model including characterization of the post-operative recovery from blood gas, metabolic and inflammation standpoints.

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

The chronically instrumented pregnant sheep has been used as a model of human fetal development and responses to pathophysiologic stimuli such as ...

Developmental Biology

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Bioengineering

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Transuterine Fetal Tracheal Occlusion Model in Mice

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Various animal models of congenital diaphragmatic hernia and fetal tracheal occlusion present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and availability of genetic tools. This model provides a new tool to study the impact of both tracheal occlusion and increased luminal pressure on lung development.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as cerebral autoregulation (CAR), which occurs in the carotid artery. In contrast to full-term neonates, preterm neonates are unable to reduce the cerebral blood flow (CBF) in response to increased systemic BP. In preterm neonates, this exposes the fragile cerebral vessels to high perfusion pressures, leading to their rupture and brain damage. Ex vivo studies using wire myography have demonstrated that carotid arteries from near-term fetuses constrict in response to the activation of adrenergic alpha1 receptors. This response is blunted in the preterm fetus. Thus, to examine the role of alpha1-AR in vivo, presented here is an innovative approach to determine the effects of drugs on a carotid arterial segment in vivo in an ovine fetus during the developmental progression of gestation. The presented data demonstrate the simultaneous measurement of fetal blood flow and blood pressure. The perivascular delivery system can be used to conduct a long-term study over several days. Additional applications for this method could include viral delivery systems to alter the expression of genes in a segment of the carotid artery. These methods could be applied to other blood vessels in the growing organism in utero as well as in adult organisms.

In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus

The present protocol describes a method to deliver drugs and gene expression-modifying agents perivascularly in an in utero developing fetus. Importantly, the effect of drugs/agents on blood flow can be measured with the progression of pregnancy.

The ability of an organism to maintain a constant blood flow to the brain in response to sudden surges in systemic blood pressure (BP) is known as ...

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.

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. ...

Blind Endotracheal Intubation in Neonatal Rabbits

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical characteristics, which is further complicated in the case of the newborn. Of the different methods of advanced airway management, endotracheal intubation is less aggressive than tracheostomy, and is more feasible than supraglottic management given the lack of supraglottic devices of such a small size. As direct glottis visualization is very difficult in animals this size, this blind intubation model is presented as an effective alternative, especially for experiments requiring prolonged anesthesia. Using this method, we performed blind intubations with a 90% success rate.

We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube.

The newborn rabbit is a useful animal model for various pathologies and procedures. Airway management of the rabbit is complex due to its anatomical ...

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

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Blind Endotracheal Intubation in Neonatal Rabbits