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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aaron Bovie Electrosurgical Cautery</td>
			<td>Henry Schein, Inc</td>
			<td>5905974&#160;</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&#160;</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&#160;</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&#160;</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&#228;ger&#160;</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&#160;</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&#160;</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&#160;</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 ...

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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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JoVE Journal

Developmental Biology

249 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

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

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

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Chapters in this video

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice