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

Summary

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.

Abstract

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.

Introduction

Birth causes stress to the fetus, and there is a considerable increase in the levels of catecholamine, the major stress hormone^1,2. 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 rupture^3,4,5. 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 fetuses^4,5.

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 contractility^6,7. 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.

Protocol

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

1. Obtain animals from any sheep ranch.
2. Transport the ewes to the laboratory at 105 days ± 5 days to 137 days ± 5 days of gestational age (dGA). Maintain the sheep at a temperature of 22 °C ± 1 °C in ambient humidity. Provide alfalfa pellets (see Table of Materials), salts, and water ad libitum.

2. Material preparation

1. Construct the perivascular catheter system.
   1. 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.
   2. Make a small slit in the MPT so that the liquid/agents can come out into the perivascular space.
2. Sterilize the flow probes (see Table of Materials), catheters, and a small screwdriver using the gas sterilization method.

3. Presurgical animal preparation

1. Obtain approval for animal experiments from the Institutional Animal Care and Use Committee.
2. 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'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.
   1. Place an intravenous (IV) catheter in the ewe's jugular vein, and secure it to the skin with waterproof tape and wound clips (see Table of Materials).
3. Sedate 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).
4. 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.
5. Confirm adequate sedation (the sheep is expected to be lethargic and sleepy), 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.
   1. Secure the ewe'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.
6. Attach a pulse oximeter probe (see Table of Materials) to the ewe's tongue/ear to continuously monitor the oxyhemoglobin saturation and heart rate. Place a thermometer under the ewe's tongue to monitor the temperature.
   1. Connect the endotracheal tube to the respiratory circuit of the anesthesia machine, and initiate mechanical ventilation while monitoring the expired CO₂.
7. 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.
8. 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.
   1. 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.
9. Warm saline in a sterile container, and bring it to 37 °C. Keep this near the surgical table. Connect the cautery (see Table of Materials).
10. 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.
11. Drape the sterilized sheep's abdominal area with sterile towels.

4. Surgical procedure

1. Exteriorizing the fetus
   1. 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).
      1. 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).
   2. 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.
   3. 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 °C) to prevent the initiation of breathing.
2. Instrumentation of the carotid artery perivascular catheter
   1. 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.
      1. 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.
      2. Retract the skin with Babcock clamps, and perform a blunt dissection to free the carotid artery from the surrounding tissue and the carotid sheet.
   2. Take the 3 mm flow probe (see Table of Materials) from the sterile pack, unscrew the probe'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.
      1. 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.
   3. 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.
      1. 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.
3. Fetal limb catheterization
   1. 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.
   2. 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:
      1. 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's diameter.
      2. 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.
      3. 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.
   3. 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.

5. Placing the fetus back and closing the wound

1. 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.
2. 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.
   1. 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.
3. 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.
   1. Using a 1-0 monofilament synthetic absorbable suture material, secure the linea alba with a continuous pattern. Secure the skin layer with surgical staples.
4. 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.
5. 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).

6. Post-operative in vivo experiments

1. 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.
2. 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.
3. 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.

Representative Results

To examine the localized in vivo manipulation of blood flow, 1 mL of phenylephrine (10 µM), an α₁-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. Figure 1B shows the same data for a preterm fetus. Administering 1 mL of PHE by the IV route increased the systemic blood flow without affecting the carotid blood flow in near-term fetal sheep (Figure 1C). Figure 1D shows the same data for a preterm fetus. In contrast, the administration of PHE by a perivascular catheter did not have any effect in preterm sheep; however, administration by the intravenous route caused a significant increase in both the carotid blood flow and systemic BP. This experiment demonstrates a fully functional perivascular sleeve that can regulate blood flow in the carotid artery in utero without affecting the systemic BP. The results show that preterm fetuses are not responsive to phenylephrine-mediated carotid blood flow regulation; however, the response is mature in near-term fetuses (Figure 1E). Importantly, the IV administration of PHE increased the carotid blood flow only in preterm fetuses, with no significant effect in near-term fetuses (Figure 1G). However, the IV administration of PHE raised the systemic blood pressure in both preterm and near-term fetuses (Figure 1H). The results also demonstrate that the perivascular instillation of phenylephrine had no effect on the systemic blood pressure (Figure 1F).

Figure 1: In vivo manipulation of blood flow. An exemplary trace of systemic blood pressure and carotid artery blood flow baseline measurements and changes following the administration of phenylephrine (PHE) through the perivascular catheter from (A) an in utero near-term fetus and (B) an in utero preterm fetus. An exemplary trace of systemic blood pressure and carotid artery blood flow baseline measurements and changes following the intravenous administration of phenylephrine (PHE) from (C) an in utero near-term fetus and (D) an in utero preterm fetus. The changes in the (E) percentage of carotid blood flow and (F) systemic blood pressure via the perivascular catheter delivery system for near-term and preterm sheep are shown. The changes in the (G) percentage of carotid blood flow and (H) systemic blood pressure via systemic administration for near-term and preterm sheep are shown. The error bars demonstrate the standard error of the mean. N = 4 in each group. *P < 0.05 by a Student's t-test. Please click here to view a larger version of this figure.

Discussion

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 methods provide a way to conduct in vivo manipulations of arterial segments by introducing chemicals or vectors to modify gene expression. Furthermore, this method has a minimal effect on systemic circulation because of the local delivery of the agents.

The current experiments demonstrate that the administration of phenylephrine results in the constriction of the carotid artery with a reduction in blood flow. The above investigation elucidates the role of alpha-adrenergic receptors in regulating the carotid artery blood flow to the brain. This technique can be used to examine the effect of different pharmacological compounds on blood flow in real-time in live fetuses. The perivascular catheter can also be used to instill lentivirus in the perivascular space, which is taken up by the vasculature, to result in the knockdown or overexpression of the desired signaling protein or receptor.

For decades, organ and tissue baths have provided useful data regarding vessel contractility^6,8,9. However, these studies are ex vivo, which raises questions regarding the reproducibility in vivo and means continuous measurements cannot be performed. To overcome this limitation, this innovative approach examines carotid artery blood flow in vivo. An additional advancement in this methodology will include the adoption of genetic modulation using virus delivery approaches, which will allow arterial segments to be genetically altered to upregulate and downregulate gene expression by delivering shRNA or CRISPR/Cas9.

The critical step in the protocol is placing the perivascular catheter parallel to the vessel in close proximity. For this to work, one needs to know the diameter of the targeted artery. Additionally, developing a proper sleeve is important. One may place the sleeve adjacent to the artery to be modulated instead of surrounding it. This will also provide local delivery of the chemicals and targeting agents.

The limitation of the method is that it only regulates a segment of the artery, and the results regarding organ or tissue blood flow should be interpreted carefully. One may be required to change the length of the sleeve and the amount of chemicals to achieve the desired effect. The method has wide applications in modulating gene regulation in live fetuses. This can be adapted to modulate gene function and expression in a portion of any tissue. Additionally, the method can be applied to modulate gene expression in an adult organism.

Although there are other methods to measure in vivo blood flow, such as using transonic flow probes¹⁰, laser Doppler¹¹, and microspheres¹², none of those methods allow for examining the local effect of the drugs on the blood flow in the arterial segment as opposed to the systemic effects of the intervention. Thus, the current method is unique, as it can measure and modulate the local blood flow without any systemic effects.

Materials

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