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 occlus...

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.

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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...

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 ...

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Research

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239 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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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 ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

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. ...

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...