Previously, the chief method to examine the effect of chemicals on arterial contractility was by ex vivo wire myography in organ baths. The significance of this technique is that the effects of chemicals and gene manipulation on a vessel's segment in vivo can be examined. The main advantage of this technique is that chronic exposure and long-term evaluation of drugs'effect on arterial contractility and blood flow can be conducted.
This is not possible in isolated vessels in organ baths. To begin exteriorizing the fetus, ensure an adequate depth of anesthesia in the ewe. Perform a 10 centimeter standard laparotomy incision using a number 20 scalpel blade over the linear alba from the umbilicus to the cranial portion of the udder.
Then make a small incision through the midline of the body wall beneath the skin incision, and open the abdominal cavity using Metzenbaum scissors. Exteriorize the uterus containing the fetus through the abdominal wall, while placing sterile surgical towels underneath between the maternal abdomen and uterus. Then palpate the uterus to determine the fetal position and caudal lead-ins.
Using a cautery, make a 10 centimeter incision through the uterine wall with a large curvature over the dorsum of the head, avoiding any visible blood vessels and placentomes. Next, with four Babcock clamps, 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 head with a sterile non-latex glove filled with warm sterile saline at a temperature of 37 degrees Celsius to prevent the initiation of breathing.
To perform instrumentation of the carotid artery perivascular catheter, with the neck of the fetus exposed, make a three to 3.5 centimeter oblique skin incision along the anterior border of the sternocleidomastoid or SCM muscle on one side of the neck in the middle region. Then separate the fascia with mosquito forceps. Divide the platysma, and dissect along the medial border of the SCM muscle from its tendon superiorly, and to the level of the omohyoid muscle inferiorly.
Retract the skin with Babcock clamps. Perform a blunt dissection to free the carotid artery from the surrounding tissue and the carotid sheet. Next, take a three millimeter flow probe from the sterile pack, unscrew the backing plate, and slide it open to expose the L bracket.
Using forceps, close the flow probe bracket by gently sliding the backing plate to a closed position. Then secure the flow probe bracket by tightening the backing screw of the flow probe. Place a preflushed perivascular catheter near the carotid artery proximal to the flow probe with a 3.0 nonabsorbable silk suture.
Secure the proximal and distal lengths of the perivascular system and the flow probe to the nearby interstitial tissue. Using a continuous stitch, close the incision site and the fetal skin with a 3.0 nonabsorbable suture. Secure the catheters to the skin by wrapping the suture around the catheter three times.
After removing the glove, place the fetal head back into the uterus. To perform fetal limb catheterization, exteriorize the hind leg of the fetus, hold the leg, and turn it sideways to visualize the inner thigh area. After cleaning the area with sterile gauze, perform a two centimeter incision and expose the femoral artery.
Place and secure the flow probe as previously demonstrated for carotid, and close the incision. Next, make a two centimeter incision along the medial aspect of the tibia, approximately 0.5 centimeters distal to the knee. Expose the thick-walled posterior tibial artery and the thin-walled saphenous vein.
To insert polyvinyl catheters, using a standard cut cutdown technique, free the vessel of interest with blunt dissection. Then ligate the distal portion of the vessel with a 3.0 silk suture, and without a needle, using a square knot with three throws. Preplace a second silk free tie at the proximal aspect of the vessel, but leave the ligature untied.
Using Castroviejo scissors, make a small transverse cut in the vessel two millimeters proximal to the distal ligature. Restrict the vessel blood flow by gently pulling up on the proximal untied suture. Then fill the catheter with sterile heparinized saline.
Insert the beveled end of the catheter, and advance the tip 20 centimeters into the fetal vessel. 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 two millimeters from the insertion site with three throws.
Then tie the distal ligature proximal to the proximal tie, securing the vessel to the catheter. Close the skin incision using a 3.0 nonabsorbable suture, using a continuous pattern. Ensure the sutures are tied around the catheters to avoid restricting the blood flow if pulled.
Place a preflushed catheter in the uterus, and secure it to the fetus by a suture using a 3.0 nonabsorbable suture. Place the fetus back into the uterus. Suture the fetal membranes using 3.0 nonabsorbable sutures with a continuous locking pattern, and then close the uterine muscular layer using a 3.0 nonabsorbable suture.
Insert an 18 inch stainless steel surgical rod subcutaneously along the abdominal wall up to the paracostal region. Attach the catheters to the distal end of the surgical rod, having an assistant feed the catheters and flow probe cable through the paracostal exit site by pushing the rod fully through the paracostal opening. Next, secure all the catheters and flow probe cables at the paracostal incision site.
After placing the waterproof tape, suture the catheters to the ewe's skin. Suture a plastic mesh pouch to the exterior of the ewe over the catheters and probe to store the catheters. Using a 1.0 monofilament synthetic absorbable suture material, secure the linea alba with a continuous pattern, and secure the skin layer with surgical staples.
To measure the blood flow, attach the flow probes inserted into the fetus with the perivascular flow module to PowerLab and an attached computer. Attach the arterial and amniotic catheters to the bridge amplifier attached to an analog to digital converter. Administer a one milliliter bolus of 10 micromolar phenylephrine to the fetus intravenously, and measure the carotid and femoral flow for 15 minutes.
Then wait for 30 minutes, or until the blood flow returns to baseline. Next, infuse one milliliter of 10 micromolar phenylephrine into the perivascular catheter, and measure the blood flow for 15 minutes. Wash out the phenylephrine by administering five milliliters of warm saline through the perivascular catheter.
Then wait for 30 minutes, or until the blood flow returns to baseline. This study examined the localized effects of blood flow manipulation in near-term and preterm fetuses and sheep. Upon administering one milliliter of 10 micromolar phenylephrine, an alpha-1 AR agonist, into the perivascular space of the carotid artery, a significant reduction in carotid blood flow was observed in both near-term fetal sheep and the preterm fetus without any effect on systemic blood pressure.
Conversely, administering phenylephrine intravenously caused a significant increase in the systemic blood flow in both near-term fetal sheep and the preterm fetus without affecting the carotid blood flow. The results revealed that preterm fetuses are not responsive to phenylephrine mediated carotid blood flow regulation, however, the response is mature in near-term fetuses. Notably, the intravenous administration of phenylephrine increased the carotid blood flow only in preterm fetuses, with no significant effect in near-term fetuses.
However, the intravenous administration of phenylephrine raised the systemic blood pressure in both preterm and near-term fetuses. The results also demonstrate that the perivascular installation of phenylephrine did not affect systemic blood pressure. Utmost care must be taken while performing a blunt dissection to identify vessels and nicking them to insert the beveled end of the catheter.
The nick should be small and avoid cutting the whole vessel. This method can measure vessel contractility and blood flow of any vessel. Also it can be used to deliver chemicals and vectors near the vicinity of the organ or tissue to be targeted.
This technique can be utilized to determine the changes in cerebral blood flow autoregulation in utero in a developing fetus, and target any gene to upregulate or silence the transcription.
