Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs

Summary

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.

Abstract

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.

Introduction

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 space^1,2,3,4,5. 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 trachea^{5,6,7,8,9,10,11,12,13}. 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 birth^15,16,17. 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.

Protocol

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 °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

1. Ewe preparation
   1. At ~80 days gestation, premedicate the ewe with an intramuscular injection of acepromazine (0.04 mg/Kg) and morphine (0.3 mg/Kg).
   2. Induce anesthesia intravenously with ketamine (5 mg/Kg) and midazolam (0.25 mg/Kg).
   3. 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).
   4. 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.
   5. Place the ewe supine on the gurney. Loop ropes around the ewe's legs and gently secure to the table.
   6. Shear wool from the ewe's abdomen and flank and then clip it close to the skin.
   7. Remove dirt and lanolin from the ewe's abdomen, flank, and groin with soap and water.
   8. Give cefazolin (20 mg/Kg) intravenously every 90 min during surgery.
   9. Transfer the ewe to the surgical theatre on a gurney.
   10. Move the ewe from the transport gurney to the surgical table.
   11. Place ewe in dorsal recumbency position on the surgical table.
   12. Secure all limbs to the surgical table with soft rope ties.
   13. Aseptic preparation of the exposed abdomen: Wash 3 times with 4% chlorhexidine surgical scrub, removing contaminated solution between each wash with 70% alcohol.
   14. Complete sterile preparation by lightly spraying 10% iodine solution on the abdomen, flank, and groins.
   15. 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.
2. Surgical approach
   1. 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.
   2. Expose the uterus by a para-midline abdominal incision (8-11 cm) through the ewe's skin, subcutaneous tissue, and linea alba made with a size 10 surgical blade with cautery as required to minimize incisional bleeding.
3. Diaphragmatic hernia creation
   1. Locate the fetal head/thorax via palpation.
   2. Cover the uterus with sterile plastic wrap.
   3. Use surgical scissors to make a window (6 cm x 4 cm) in the plastic wrap.
   4. Make a 5-7 cm uterine incision through the uterus and amniotic sac with cautery cutting, avoiding uterine blood vessels and placental cotyledons.
   5. Exteriorize the fetal head and forelimbs.
   6. Use Babcock uterine clamps to seal the uterine incision edges and sterile plastic wrap tightly around the fetal abdomen to prevent amniotic fluid loss.
   7. 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 9^th and 10^th ribs on the left and cut a hole in the plastic wrap with surgical scissors to access the intended fetal incision site.
   8. 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 9^th intercostal space immediately adjacent to the upper border of the 10^th rib to avoid subcostal vessels.
   9. Use a 60 mL sterile syringe to bathe the exposed fetal skin with warm Hartmann's solution (5-10 mL every 5-10 min) to keep the fetal skin moist throughout subsequent steps.
   10. 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.
   11. Use mosquito hemostats to bluntly dissect the subcutaneous tissue, intercostal muscles, and pleural membrane to access the diaphragm.
   12. 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.
   13. Make a 1 cm incision between the two hemostats using small angled scissors.
   14. Release the hemostats holding the diaphragm.
   15. Use atraumatic Potts forceps to pull the stomach superiorly through the hole in the diaphragm.
   16. Use a single tie of absorbable 5-0 polydioxanone monofilament (RB-1 taper point needle) to carefully envelop the 9^th and 10^th ribs to oppose the fetal ribs, avoiding the underlying lung.
   17. Close thoracotomy incision with a mattress suture (absorbable 5-0 polydioxanone monofilament).
4. Surgical closure
   1. Return the fetus to the amniotic sac.
   2. Administer intra-operative meloxicam (0.25 mg/Kg; subcutaneously) to the ewe for postoperative analgesia.
   3. Replace lost amniotic fluid using warmed Hartmann's solution (~50 mL) along with intra-amniotic Piperacillin/Tazobactam (1000 mg/125 mg).
   4. 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.
   5. Close the ewe's linea alba, subcutaneous tissue, and skin (absorbable polydioxanone monofilament 1, absorbable poliglecaprone 25 monofilament 3-0, and nonabsorbable polypropylene monofilament 2-0 respectively).
5. Ewe recovery and monitoring
   1. 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.
   2. Apply a transdermal fentanyl patch (100 µg/h) to the groin for additional postoperative analgesia.
   3. Recover the ewe and extubate when breathing spontaneously, then allow it to wake.
   4. Monitor ewes daily until delivery for fetal intervention surgery.
   5. Remove sutures after 14 days.

2. Placement of the fetal intervention and pressure monitoring device

1. Ewe preparation for fetal intervention surgery at 101 days of gestation
   1. Inject medroxyprogesterone (1 mL) intramuscularly at ~85 days of gestation (term ~147 days) to reduce the risk of preterm birth.
   2. Use the same anesthetic and surgical preparation procedure used for the initial diaphragm creation surgery.
      1. Follow the surgical approach in steps 2.1.2.2 and 2.1.2.3, similar to steps 1.2.1-1.2.2.
      2. 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.
      3. 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's skin, subcutaneous tissue, and linea alba made with a size 10 surgical blade with cautery as required to minimize incisional bleeding.
   3. Additionally, administer oxytetracycline (20 mg/Kg) intramuscularly to the ewe to provide fetal antimicrobial protection against the chronically implanted monitoring device.
2. Device activation
   1. Activate pressure measurement device¹⁹.
   2. Clamp pressure measurement tubing with hemostats.
   3. 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.
   4. 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.
3. Surgical approach and fetal intervention
   1. 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).
   2. Keep the exteriorized fetal head and neck wrapped in the sterile plastic drape to minimize loss of temperature and fluid.
   3. Instrument the trachea with a catheter for pressure measurements (Figure 1).
      1. For unoccluded trachea, follow steps 2.3.3.2-2.3.3.3.
      2. 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's mouth.
      3. 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.
      4. For cccluded trachea, follow steps 2.3.3.5-2.3.3.6.
      5. 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's mouth.
      6. 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.
   4. 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.
   5. Suture the catheter at the corner of the lamb's mouth and neck (nonabsorbable nylon monofilament 4-0).
   6. Secure the pressure measurement device to the lamb's skin on the chest (nonabsorbable nylon monofilament 4-0) (Figure 2).
4. Close the fetal and ewe surgical incisions as per initial diaphragmatic hernia creation surgery (steps 1.4- 1.5).
5. Test device
   1. Test if the device is transmitting signal¹⁹.
6. Ewe recovery and monitoring
   1. Recover the ewe as per the initial surgery.
   2. Monitor ewes daily until delivery.
   3. Give a second dose of medroxyprogesterone (0.5 mL) intramuscularly at ~120 days of gestation.
   4. Give betamethasone (5.7 mg) intramuscularly 48 h and 24 h before cesarean delivery to promote fetal maturation for postnatal care.
7. Lamb delivery
   1. 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).
   2. 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.
   3. Proceed with surgical delivery of the fetus via hysterotomy as per previous surgeries, with ewe in dorsal recumbency (steps 1.1 - 2.2).
   4. Remove the monitoring device. Clean the device with water and 70% ethanol.
   5. Humanely kill the ewe with an intravenous injection of 160 mg/Kg of pentobarbitone sodium solution.
   6. Resuscitate and ventilate the lamb for 2-3 h and then humanely kill (160 mg/Kg of pentobarbitone sodium solution, intravenous injection).
8. Data
   1. 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.

Results

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...

Discussion

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 hypoplasia^20,21,22,23. Tracheal occlus...

Materials

List of materials used in this article

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