Name: in vivo real-time study of drug effects on carotid blood flow in the ovine fetus
Uploaded: 2023-04-28T14:40:00.000+00:00
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Intramural funds from the University of Arizona were used for these studies.

For the present study, approval for the animal experiments was obtained from the Animal Care and Use Committee of the University of Arizona. One time-mated, pregnant Columbia-Rambouillet ewes between 2-4 years of age were used for the present study. The animals were obtained from the University of Arizona Sheep Unit.
1. Animal maintenance
<ol>
	<li>Obtain animals from any sheep ranch.</li>
	<li>Transport the ewes to the laboratory at 105 days &#177; 5 days to 137 days &#177; 5 days of gestational age (dGA). Maintain the sheep at a temperature of 22 &#176;C &#177; 1 &#176;C in ambient humidity. Provide alfalfa pellets (see Table of Materials), salts, and water ad libitum.</li>
</ol>
2. Material preparation
<ol>
	<li>Construct the perivascular catheter system.
	<ol>
		<li>Join one end of 4 ft of Tygon tubing to a 2 cm manifold pump tubing (MPT) (see Table of Materials). Attach the other end of the 2 cm MPT to another 4 ft of Tygon tube.</li>
		<li>Make a small slit in the MPT so that the liquid/agents can come out into the perivascular space.</li>
	</ol>
	</li>
	<li>Sterilize the flow probes (see Table of Materials), catheters, and a small screwdriver using the gas sterilization method.</li>
</ol>
3. Presurgical animal preparation
<ol>
	<li>Obtain approval for animal experiments from the Institutional Animal Care and Use Committee.</li>
	<li>Prior to surgery, keep the ewes on nil per os (NPO) food for 24 h and NPO water for 16 h. On the day of surgery, wrap the ewe&#39;s face with a pad to protect her eyes. Shave the left side of the neck to expose the jugular vein, and clean the skin using povidone-iodine and 70% ethanol.
	<ol>
		<li>Place an intravenous (IV) catheter in the ewe&#39;s jugular vein, and secure it to the skin with waterproof tape and wound clips (see Table of Materials).</li>
	</ol>
	</li>
	<li>Anesthetize the ewes with an IV administration of diazepam (0.15 mg/kg) and ketamine hydrochloride (16 mg/kg). Administer an IM injection of penicillin G procaine suspension (25,000 I/kg) and IV ketoprofen (2.2 mg/kg) (see Table of Materials).</li>
	<li>Shave the incision site of the sheep and the surrounding areas (abdomen, flanks, and groin) with #10 blade clippers. To ensure the wool is completely removed, reshave the area with a #40 blade. Wash the shaved area with a germicidal cleanser (see Table of Materials) and water. Dry with a disposable pad.</li>
	<li>Confirm the depth of anesthesia (determined and maintained by response to skin pinch, corneal reflex, and assessment of jaw tone), and then intubate the ewe with a 6.5-7.5 mm inner diameter endotracheal tube (see Table of Materials), and secure the tube in place. Place the ewe on a lift table in the lateral recumbent position, and transfer to a V-top surgical table in the supine position.
	<ol>
		<li>Secure the ewe&#39;s limbs to the V-top surgical table with surgical tie-downs. Bring the ewe into the Trendelenburg position to alleviate pressure on the fetal-placental unit.</li>
	</ol>
	</li>
	<li>Attach a pulse oximeter probe (see Table of Materials) to the ewe&#39;s tongue/ear to continuously monitor the oxyhemoglobin saturation and heart rate. Place a thermometer under the ewe&#39;s tongue to monitor the temperature.
	<ol>
		<li>Connect the endotracheal tube to the respiratory circuit of the anesthesia machine, and initiate mechanical ventilation while monitoring the expired CO2.</li>
	</ol>
	</li>
	<li>Maintain the anesthesia by adjusting the isoflurane between 2.5%-4% throughout the surgery. Ensure the animal is adequately anesthetized by pinching the ear. Administer a balanced poly-ionic (saline 0.9% w/v) solution at 5 mL/kg/h using the jugular catheter placed during step 3.2.1.</li>
	<li>Perform a sterile scrub. Spray the abdominal area and the flanks with povidone solution (10% iodine solution). Scrub the area with an iodine-soaked gauze starting from the incision site and working outward, making sure not to go back to the center after scrubbing outward.
	<ol>
		<li>Next, spray the area with ethanol (70% ethanol w/v), and scrub with an ethanol-soaked gauze in a similar way to the scrubbing with povidone. Repeat the whole process three times. Spray the area with povidone solution.</li>
	</ol>
	</li>
	<li>Warm saline in a sterile container, and bring it to 37 &#176;C. Keep this near the surgical table. Connect the cautery (see Table of Materials).</li>
	<li>Have the surgical team members dress in caps, face masks, and shoe covers, wash their hands (surgical scrub), and put on sterilized surgical gowns and gloves. From this point onward, a strict sterile surgical practice must be followed.</li>
	<li>Drape the sterilized sheep&#39;s abdominal area with sterile towels.</li>
</ol>
4. Surgical procedure
<ol>
	<li>Exteriorizing the fetus
	<ol>
		<li>After ensuring an adequate depth of anesthesia, perform a 10 cm standard laparotomy incision using a scalpel (#20 blade) over the linea alba from the umbilicus to the cranial portion of the udder. Control the bleeding while making an incision with a cautery (power settings: 50 cut and 25 coagulation).
		<ol>
			<li>Make a small incision through the midline of the body wall beneath the skin incision, and open the abdominal cavity using Metzenbaum scissors (see Table of Materials).</li>
		</ol>
		</li>
		<li>Exteriorize the uterus containing the fetus through the abdominal wall while placing sterile surgical towels underneath (between the maternal abdomen and uterus). Palpate the uterus to determine the fetal position and cotyledons. Using a cautery, make a ~10 cm incision through the uterine wall with a large curvature over the dorsum of the head, avoiding any visible blood vessels and placentomes.</li>
		<li>Use four Babcock clamps (see Table of Materials) to secure the uterus and placental membranes, and pull the Babcock clamps at the four opposing corners to make the fetal head visible. Exteriorize the cranial half of the fetus through this incision, and cover the fetal head with a sterile, non-latex glove filled with warm, sterile saline (37 &#176;C) to prevent the initiation of breathing.</li>
	</ol>
	</li>
	<li>Instrumentation of the carotid artery perivascular catheter
	<ol>
		<li>When removing the fetal head from the uterus, have an assistant gently hold the Babcock forceps upright to minimize amniotic fluid loss. With the neck of the fetus exposed, perform a 3-3.5 cm oblique skin incision along the anterior border of the sternocleidomastoid (SCM) muscle on one side of the neck in the middle region, and separate the fascia with mosquito forceps.
		<ol>
			<li>Divide the platysma, and perform a dissection along the medial border of the SCM muscle from its tendon superiorly to the level of the omohyoid muscle inferiorly. Retracting the SCM will expose the carotid sheet, which superficially contains a thin-walled internal jugular vein, and below it will be the carotid artery as a thick-walled vessel.</li>
			<li>Retract the skin with Babcock clamps, and perform a blunt dissection to free the carotid artery from the surrounding tissue and the carotid sheet.</li>
		</ol>
		</li>
		<li>Take the 3 mm flow probe (see Table of Materials) from the sterile pack, unscrew the probe&#39;s backing plate, and slide it open to expose the L-bracket. Carefully lift the carotid artery, and gently hook the bracket underneath the vessel while avoiding contact with the vessel.
		<ol>
			<li>Use forceps to close the flow probe bracket by gently sliding the backing plate to a closed position. Secure the flow probe bracket by tightening the backing screw of the flow probe. To ease this process, gently grip the ends of the flow probe with forceps to stabilize the flow probe while the screw is being tightened.</li>
		</ol>
		</li>
		<li>Pre-flush the perivascular catheter, and place it in the close vicinity of the carotid artery proximal to the flow probe. Ensure that the open slit of the perivascular catheter is in close proximity to the carotid artery.
		<ol>
			<li>With a 3-0 silk nonabsorbable suture, secure the proximal and distal ends of the perivascular system and the flow probe to the nearby interstitial tissue. Close the incision site using a continuous stitch, close the fetal skin with a 3-0 silk nonabsorbable suture, and secure the catheters to the skin by wrapping the suture around the catheter three times. Remove the glove, and place the fetal head back in the uterus.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fetal limb catheterization
	<ol>
		<li>Exteriorize the hind leg of the fetus. Hold the leg, and turn it sideways to visualize the inner thigh area. Clean the area with sterile gauze, perform a 2 cm incision, and expose the femoral artery. Place and secure the flow probe following a similar procedure as done with the carotid, and then close the incision.</li>
		<li>Make a 2 cm incision along the medial aspect of the tibia ~0.5 cm distal to the knee. Expose the posterior tibial artery (thick-walled) and saphenous vein (thin-walled). Insert polyvinyl catheters (outer diameter: 1.4 mm and inner diameter: 0.9 mm) into the posterior tibial artery and saphenous vein using a standard cut-down technique, as mentioned below:
		<ol>
			<li>Free the vessel of interest with blunt dissection. Ligate the distal portion of the vessel with a 3-0 silk suture (no needle) using a square knot with three throws. Pre-place a second silk-free tie at the proximal aspect of the vessel (under the vessel), but leave the ligature untied. Using Castroviejo scissors (see Table of Materials), make a small, transverse cut in the vessel 2 mm proximal to the distal ligature. The length of the cut should be ~25% of the vessel&#39;s diameter.</li>
			<li>Restrict the vessel blood flow by gently pulling up on the proximal, untied suture. Fill the catheter with sterile heparinized saline. Insert the beveled end of the catheter, and advance the tip 20 cm into the fetal vessel.</li>
			<li>Hold the catheter in place with forceps while an assistant ties the proximal silk free-tie suture to secure the vessel to the catheter; ligate the vessel completely around the inserted catheter using square knots 2 mm from the insertion site with three throws. Tie the distal ligature proximal to the proximal tie securing the vessel to the catheter.</li>
		</ol>
		</li>
		<li>Close the skin incision using a 3-0 silk nonabsorbable suture using a continuous suture pattern. Ensure the sutures are tied around the catheters to avoid restricting the blood flow if pulled. Place a pre-flushed catheter in the uterus, and secure it to the fetus by a suture using a 3-0 nonabsorbable silk suture.</li>
	</ol>
	</li>
</ol>
5. Placing the fetus back and closing the wound
<ol>
	<li>Return the fetus to the uterus. Suture the fetal membranes using 3-0 nonabsorbable silk sutures with a continuous locking (Cushing) pattern. Close the uterine muscular layer using a 3-0 nonabsorbable silk suture.</li>
	<li>Insert an 18 in stainless steel surgical rod subcutaneously along the abdominal wall up to the paracostal region. Let the proximal end of the rod exit the paracostal site by performing a 1 cm incision.
	<ol>
		<li>Attach catheters to the distal end of the surgical rod, and have an assistant feed the catheters and flow probe cable through the paracostal exit site by pushing the rod fully through the paracostal opening.</li>
	</ol>
	</li>
	<li>Secure all the catheters and flow probe cables at the paracostal incision site. Place with waterproof tape, and suture the catheters to the skin of the ewe. Suture a plastic mesh pouch to the exterior of the ewe over the catheters and probe to store the catheters.
	<ol>
		<li>Using a 1-0 monofilament synthetic absorbable suture material, secure the linea alba with a continuous pattern. Secure the skin layer with surgical staples.</li>
	</ol>
	</li>
	<li>Stop the general anesthesia, and extubate the ewe once the laryngeal reflexes have returned to normal baseline. Do not leave the animal unattended until it has regained complete consciousness. Move the ewe to the metabolic cart once she is stable following general anesthesia. Return the animal to the post-operative experiment room after fully recovering from the anesthesia.</li>
	<li>Administer post-operative analgesics intravenously (10 mg/kg/day phenylbutazone) for 3 days. Flush the vascular catheters daily with heparinized saline solution (100 U/mL heparin in 0.9% NaCl solution).</li>
</ol>
6. Post-operative in vivo experiments
<ol>
	<li>Flush the catheters every day with heparinized saline (75 U/ml). Wait for 72 h before making any measurements. To measure the blood flow, attach the flow probes inserted into the fetus with the perivascular flow module to PowerLab and an attached computer. 
	NOTE: The recordings can be done on the PowerLab software (see Table of Materials) to measure the carotid and femoral blood flow. Take the baseline measurement for 30 min.</li>
	<li>Attach the arterial and amniotic catheters to the bridge amplifier attached to an analog-to-digital converter (see Table of Materials). Administer a 1 mL bolus of 10 uM phenylephrine to the fetus intravenously, and measure the carotid and femoral flow for 15 min. Then, wait for 30 min or until the blood flow returns to baseline.</li>
	<li>Infuse 1 mL of 10 uM phenylephrine into the perivascular catheter, and measure the blood flow for 15 min. Wash out the phenylephrine by administering 5 mL of warm saline through the perivascular catheter. Then, wait for 30 min or until the blood flow returns to baseline.</li>
</ol>

<ol>
	<li>Lagercrantz, H., Slotkin, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;stress&amp;#34;+of+being+born.">The &amp;#34;stress&amp;#34; of being born.</a> Scientific American. 254 (4), 100-107 (1986).</li><li>Ronca, A. E., Abel, R. A., Ronan, P. J., Renner, K. J., Alberts, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+labor+contractions+on+catecholamine+release+and+breathing+frequency+in+newborn+rats.">Effects of labor contractions on catecholamine release and breathing frequency in newborn rats.</a> Behavioral Neuroscience. 120 (6), 1308-1314 (2006).</li><li>Czynski, A., 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=Cerebral+autoregulation+is+minimally+influenced+by+the+superior+cervical+ganglion+in+two-+week-old+lambs,+and+absent+in+preterm+lambs+immediately+following+delivery.">Cerebral autoregulation is minimally influenced by the superior cervical ganglion in two- week-old lambs, and absent in preterm lambs immediately following delivery.</a> PLoS One. 8 (12), e82326 (2013).</li><li>Ballabh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+prevention+of+intraventricular+hemorrhage.">Pathogenesis and prevention of intraventricular hemorrhage.</a> Clinics in Perinatology. 41 (1), 47-67 (2014).</li><li>Ballabh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+hemorrhage+in+premature+infants:+Mechanism+of+disease.">Intraventricular hemorrhage in premature infants: Mechanism of disease.</a> Pediatric Research. 67 (1), 1-8 (2010).</li><li>Goyal, R., Goyal, D., Chu, N., Van Wickle, J., Longo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+artery+alpha-1+AR+subtypes:+High+altitude+long-term+acclimatization+responses.">Cerebral artery alpha-1 AR subtypes: High altitude long-term acclimatization responses.</a> PLoS One. 9 (11), e112784 (2014).</li><li>Goyal, R., Mittal, A., Chu, N., Zhang, L., Longo, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha(1)-Adrenergic+receptor+subtype+function+in+fetal+and+adult+cerebral+arteries.">alpha(1)-Adrenergic receptor subtype function in fetal and adult cerebral arteries.</a> American Journal of Physiology - Heart and Circulatory Physiology. 298 (1), H1797-H1806 (2010).</li><li>Goyal, D., Goyal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+maturation+and+alpha-1+adrenergic+receptors-mediated+gene+expression+changes+in+ovine+middle+cerebral+arteries.">Developmental maturation and alpha-1 adrenergic receptors-mediated gene expression changes in ovine middle cerebral arteries.</a> Scientific Reports. 8 (1), 1772 (2018).</li><li>Goyal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+the+role+of+PKC-mediated+contractility+in+ovine+cerebral+arteries.">Maturation and the role of PKC-mediated contractility in ovine cerebral arteries.</a> American Journal of Physiology - Heart and Circulatory Physiology. 297 (6), H2242-H2252 (2009).</li><li>Gratton, R., Carmichael, L., Homan, J., Richardson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+arterial+blood+flow+in+the+ovine+fetus+as+a+continuous+measure+of+cerebral+blood+flow.">Carotid arterial blood flow in the ovine fetus as a continuous measure of cerebral blood flow.</a> Journal of the Society for Gynecologic Investigation. 3 (2), 60-65 (1996).</li><li>Bishai, J. M., Blood, A. B., Hunter, C. J., Longo, L. D., Power, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+lamb+cerebral+blood+flow+(CBF)+and+oxygen+tensions+during+hypoxia:+a+comparison+of+laser+Doppler+and+microsphere+measurements+of+CBF.">Fetal lamb cerebral blood flow (CBF) and oxygen tensions during hypoxia: a comparison of laser Doppler and microsphere measurements of CBF.</a> Journal of Physiology. 546, 869-878 (2003).</li><li>Ashwal, S., Dale, P. S., Longo, L. 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The authors have no disclosures.

Currently, no method exists to examine vessel contractility and dilatation in vivo in response to drug compounds and gene manipulation. As a standard in the field, in vivo blood flow is measured by Doppler flow probes, microspheres, and radioactive molecules such as tritiated water. However, to manipulate the receptors' functions or downstream signaling, the animals are sacrificed, and experiments are conducted in vitro in organ baths following the isolation of arterial segments. The current me...

Birth causes stress to the fetus, and there is a considerable increase in the levels of catecholamine, the major stress hormone1,2. This raises the systemic BP, and if this pressure is transmitted to the fragile brain capillaries via the carotid arteries, this can lead to their rupture3,4,5. Surges in systemic BP are prevented from reaching the brain by the constriction of the carotid arteries in the full-term fetus. However, this mechanism is not developed in the preterm fetus, and this is responsible for the significantly higher likelihood of brain damage in preterm fetuses4,5.
Currently, no suitable method exists to examine the maturation of the pathways involved in regulating the carotid blood flow with developing fetuses. These studies on carotid blood flow and vasoresponsiveness are crucial from both basic science and clinical perspectives. Currently, to determine the molecular pathways involved in the regulation of arterial contractility, the standard method involves isolating the arterial segments postmortem. Then, the experiments are conducted using wire myography to determine the vasocontractility of different pharmacological molecules that define the regulatory pathways involved in arterial contractility6,7. Of note, the ex vivo findings are not able to fully replicate the in vivo environment because of the blood flow regulation upstream and downstream of the carotid artery. Thus, the present study aimed to develop a technique that can determine the effects of vasoresponsive chemicals or agents on blood flow in an artery in vivo.
The perivascular delivery methodology described in this article provides an in vivo approach to study the effect of the pharmacological or genetic manipulation of signaling pathways on different arterial segments. Using this method, one can manipulate the fetal blood pressure and carotid blood flow. Additionally, experiments with sheep fetuses are demonstrated for studying the effects of signaling molecules in a developing fetus. Hopefully, the detailed methodology provided will lead to new investigations in the field of blood flow studies, especially in relation to fetal physiology and pathology.

<table><tbody><tr><td>Aaron Bovie Electrosurgical Cautery</td><td>Henry Schein, Inc</td><td>5905974&amp;nbsp;</td><td></td></tr><tr><td>Aaron Bovie Electrosurgical Generator</td><td>Henry Schein, Inc</td><td>1229913</td><td></td></tr><tr><td>Alfalfa Pellets</td><td>Sacate Pellet Mills, Inc. Maricopa AZ</td><td>100-80&amp;nbsp;</td><td></td></tr><tr><td>Analog to Digital Converter</td><td>ADI Instruments</td><td>Powerlab</td><td></td></tr><tr><td>Babcock forceps</td><td>Roboz Surgicals</td><td>RS8020</td><td></td></tr><tr><td>Bridge Amplifier</td><td>ADI Instruments</td><td>Bridge Amplifier</td><td></td></tr><tr><td>Castroviejo scissors</td><td>Roboz Surgicals</td><td>RS5650SC</td><td></td></tr><tr><td>Diazepam</td><td>Henry Schein, Inc</td><td>1278188</td><td></td></tr><tr><td>Endotracheal Tube</td><td>Henry Schein, Inc</td><td>7020408&amp;nbsp;</td><td></td></tr><tr><td>Flow Probes</td><td>Transonic Systems Inc.</td><td>MC2PSS-JS-WC100-CRS10-GC, MC3PSS-LS-WC100-CRS10-GC</td><td></td></tr><tr><td>Heparin</td><td>Henry Schein, Inc</td><td>1162406&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein, Inc</td><td>1182097</td><td></td></tr><tr><td>Ketamine</td><td>Henry Schein, Inc</td><td>1273383</td><td></td></tr><tr><td>Ketoprofen</td><td>Zoetis Inc., Kalamazoo, MI</td><td>Ketofen</td><td></td></tr><tr><td>Manifold Pump Tubing</td><td>Fisher Scientific</td><td>14-190-508</td><td></td></tr><tr><td>Metzenbaum scissors</td><td>Roboz Surgicals</td><td>RS6010</td><td></td></tr><tr><td>Narkomed 4 Anesthesia Machine</td><td>North American Dr&amp;auml;ger&amp;nbsp;</td><td>Narkomed 4</td><td></td></tr><tr><td>Normal Saline</td><td>Fisher Scientific</td><td>Z1376</td><td></td></tr><tr><td>penicillin G procaine suspension&amp;nbsp;</td><td>Henry Schein, Inc</td><td>7455874</td><td></td></tr><tr><td>phenylbutazone</td><td>VetOne Boise, ID</td><td>510226</td><td></td></tr><tr><td>Phenylephrine</td><td>Sigma Aldrich Inc.</td><td>P1240000</td><td></td></tr><tr><td>Pivodine Scrub</td><td>VetOne&amp;nbsp;</td><td>510094</td><td>Germicidal cleanser</td></tr><tr><td>PowerLab</td><td>ADInstruments</td><td></td><td>Data acquisition hardware device</td></tr><tr><td>Pulse Oximeter</td><td>Amazon Inc.</td><td>UT100V&amp;nbsp;</td><td></td></tr><tr><td>Tygon Tubing</td><td>Fisher Scientific</td><td>ND-100-80</td><td></td></tr><tr><td>V-Top Surgical Table</td><td>VetLine Veterinary Classic Surgery</td><td>TSP-4010</td><td></td></tr><tr><td>Wound Clips</td><td>Fisher Scientific</td><td>10-001-024</td><td></td></tr></tbody></table>

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.

To examine the localized in vivo manipulation of blood flow, 1 mL of phenylephrine (10 &#181;M), an &#945;1-AR agonist, was administered into the perivascular space of the carotid artery by an exteriorized infusion catheter to determine the effect on the local carotid blood flow and the effect on the systemic blood pressure. Figure 1A demonstrates a significant reduction in carotid blood flow without any effect on systemic blood pressure in near-term fetal sheep. <strong ...

Watch this Scientific Journal Video about In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus at JoVE.com

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.

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 ...

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Developmental Biology

250 Views.  University of Arizona, Tucson. 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.

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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

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 ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Medicine

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

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 ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

3D Angiography for Venous Compliance Assessment in Sheep

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native vessel into which they are implanted. Compliance matching with the native vessel is emerging as a key property for graft success. The current gold standard for assessing vessel compliance involves the vessel's excision and ex vivo biaxial mechanical testing. We developed an in vivo method to assess venous compliance and distensibility that better reflects natural physiology and takes into consideration the impact of a pressure change caused by flowing blood and by any morphologic changes present. 
This method is designed as a survival procedure, facilitating longitudinal studies while potentially reducing the need for animal use. Our method involves injecting a 20 mL/kg saline bolus into the venous vasculature, followed by the acquisition of pre and post bolus 3D angiograms to observe alterations induced by the bolus, concurrently with intravascular pressure measurements in target regions. We are then able to measure the circumference and the cross-sectional area of the vessel pre and post bolus. 
With these data and the intravascular pressure, we are able to calculate the compliance and distensibility with specific equations. This method was used to compare the inferior vena cava's compliance and distensibility in native unoperated sheep to the conduit of sheep implanted with a long-term expanded polytetrafluorethylene (PTFE) graft. The native vessel was found to be more compliant and distensible than the PTFE graft at all measured locations. We conclude that this method safely provides in vivo measurements of vein compliance and distensibility.

Author Spotlight: Improved Imaging for Neovascular Development in Congenital Heart Disease Research

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native ...

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

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Chapters in this video

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

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

A Swine Model of Neonatal Asphyxia

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

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

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

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

Ultrasonography in Experimental Reproductive Investigations on Rats

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography