Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

The overall goal of this procedure is to infuse a drug of interest through the carotid artery to analyze the pharmacokinetic profile of the drug. This is accomplished by first fashioning a custom catheter from polyethylene tubing and then securing the catheter within the mouse carotid artery. Next, the mouse is attached to an automated infusion pump via the carotid artery catheter.

In the final step, the drug is infused into the mouse at a steady rate, and blood samples are collected at regular intervals. Ultimately, liquid chromatography, mass spectrometry is used to determine the concentration of the drug within the tissue and plasma samples. The main advantage of this technique over existing methods such as bolus, intraperitoneal, or tail vein injection or jugular infusion, are that it's representative of clinical infusion rates, as well as generally easier to perform than jugular insertion.

This method can answer key questions in pharmacology, such as what is the in vivo distribution of a drug, or we can determine if an inhibitor or modulator impacts the function of the protein. To prepare a catheter for the experiment, first, establish a low steady flame on a bunsen burner. Then hold a piece of tubing close to the flame to soften the polyethylene.

When the tubing begins to melt, slowly pull apart the two ends to create a thin section of tubing, approximately 0.25 millimeters in diameter. Next, cut a beveled end in the tubing approximately 0.75 centimeters along the thin section, and then pass the end quickly through the flame to cause it to become slightly rounded and enlarged. The tip will hook back slightly, which will help anchor the tubing during the insertion into the artery.

Cut the tubing 6.0 centimeters from the point where it begins to thin to make a catheter, and then fill a syringe equipped with a blunted needle with 200 microliters of heparin solution. Insert the needle into the wide end of the catheter and fill the catheter with heparin. Taking care to avoid bubbles.

Then store the needle and catheter in a sterile location to isolate the carotid artery. Begin by making a one centimeter longitudinal cut slightly to the right of the midline of the animal's neck, and then use forceps to separate the fat and muscle and expose the trachea. The carotid artery runs parallel to the trachea.

Next, carefully separate the fascia overlying the artery, and then lightly pull the VAs nerve aside. Insert the forceps into the space between the carotid artery and the VAs nerve, and gently open the forceps to create a gap in the facia. Carefully pull the nerve away from the artery, clearing a space from the fork in the anterior end near the larynx to the farthest exposed posterior end, isolating at least a three millimeter length of artery.

Then use forceps to draw a silk suture thread under the artery and tie a secure knot to close the artery off as far toward the anterior end as possible. After placing a second thread, tie a retractable knot to temporarily close off the artery as far toward the posterior end as possible. Then use a third thread to tie a very loose knot between the first two sutures to be used to quickly secure the catheter after placement.

To insert the catheter, grab the lower suture knot to pull the artery slightly taut. Then nick the artery above and very close to the anterior suture. Taking care not to cut too deeply.

Check the slit to make sure the opening is unobstructed. Remove the heparin filled catheter from the syringe needle, and then taking care not to create large pockets of air. At either end, manipulate the catheter to position the beveled end at a comfortable angle downward and slightly toward the dominant hand.

Next, while holding onto the suture, to keep the artery slightly taut, gently insert the catheter into the slit. Then use the forceps to grab the anterior suture knot and gently pull the artery down over the catheter. Carefully release the catheter and the anterior suture.

Then tighten the middle knot to secure the catheter close to the entrance of the catheter into the artery while taking care not to obstruct the flow through the catheter. Make a tight triple knot with the middle suture, and then use the anterior suture to secure the catheter in place below the entrance to the artery. Now use a connector plug to attach a saline lead to the catheter taking care not to create bubbles.

Then grasp the ends of the posterior suture and gently pull it to release the knot, maneuver the suture down the artery and over the end of the catheter without removing the thread. Blood should flow into the catheter when the flow appears unobstructed. Use the thread from the posterior suture to tie an additional knot slightly above the middle suture.

Then flush the catheter of blood and use a hemostat to clamp the end of the catheter close to the connector plug. Replace the connector with a port plug to seal off the end of the catheter and remove the hemostat. Then use one pair of forceps to hold onto the catheter just below the anterior suture, and another forceps to press a kink into the catheter so it will easily bend to the side after creating a second kink.

In the same way, turn the mouse onto its left side and clean the area below and right behind the animal's ear with 70%ethanol and povidone iodine. When the area has been disinfected, make a small four millimeter incision in the skin. Then use forceps to hold open the flap of tissue and work a blunt hollow probe under the skin to create a channel through the cheek to the cavity at the neck.

Use the forceps to carefully free a space for the probe to exit, and then gently thread the port plug and catheter through the probe to the exit hole. Taking care not to crush or constrict any blood vessels or organs after the animal has been allowed to recover for 30 to 60 minutes, attach a blunted needle to one end of a 40 centimeter piece of polyethylene tubing and a connector port to the other. Then draw the drug of interest into a syringe with a known inner diameter.

Attach the needle to the syringe and load the drug through the needle and tubing. After situating the syringe into the pump, according to the manufacturer's directions, prime the pump so that the drug flows smoothly out of the connector. Plug then to connect the animal to the pump, hold the mouse steady and use the hemostat to clamp the catheter close to the port plug.

Finally, replace the plug with a connector attached to the syringe and tubing. Quickly administer a fast pump to clear the volume of the catheter, and then immediately switch to the desired infusion rate. In this first figure, a comparison of the plasma concentration of Paclitaxel following jugular vein infusion and carotid artery infusion is shown.

The drug concentration drops quickly in the first 15 minutes following an initial high volume infusion and then levels off over the next 150 minutes. By comparison, the paclitaxel levels in a poor infusion start off relatively low and hover up and down throughout the assay, most likely due to a blockage in the line early in the infusion. Here, the relative levels of paclitaxel in the liver, brain, and blood plasma at the end of a three hour infusion are shown As expected, the animal exhibits a high plasma protein binding, coupled with low concentrations of drug in the brain tissue Once mastered, this technique should take 45 to 60 minutes if performed properly.

After watching the video, you should have a good understanding of how to isolate a carotid artery, insert a catheter, and perform an infusion.