X-ray Visualization of Intraductal Ethanol-based Ablative Infusion for Prevention of Breast Cancer in Rabbit Models

Breast cancer is the second leading cancer-related cause of death. While there are few proactive interventions for average-risk women, prophylactic mastectomy and hormone therapy are available options for high-risk women. However, due to the severe negative side effects of these preventative options, many high-risk women choose not to take them.

Therefore, a new strategy of prevention for high-risk individuals is needed. We have developed a intraductal delivery technique for ablating the memory epithelial cells from which breast cancer arise as a local, less invasive alternative to mastectomy. Our previous study has shown the effectiveness of single intraductal infusion of 70%ethanol for preventing breast cancer formation in an aggressive mouse model.

We were able to scale up this procedure to a rat model and confirm by micro CT the success of the infusion. Although we have demonstrated the filling and in vivo X-ray imaging the ductal tree in both mice and rat models, these models only have a single ductal tree per mammary gland. Therefore, in this procedure, we will be addressing the scalability of this technique in a rabbit model with a multi-duct tree system.

Rabbits provide a practical and relevant intermediate large-animal model for translational application of this ID ablative procedure to humans. Briefly, we began by preparing the animal for the procedure by removing the fur surrounding the teats to be injected, then intraductal infusions are performed and, following each infusion, an X-ray fluoroscopy image is taken to ensure the ductal tree has been filled. Lastly, tissue analysis is done to determine the success rate of the ablative solution.

Sedate the rabbit 20 minutes before isoflurane administration by injecting anesthesia intramuscularly. We used ketamine and xylazine. Inject an analgesia after clinical signs of sedation are shown.

We used ketoprofen. Place the rabbit on the imaging table lined with a recirculating warm water blanket and intubate the rabbit using the appropriately sized intubation tube that is then attached to an isoflurane machine. The size of the tube depends on the weight of the rabbit, but it is not always accurate, so it is good practice to have multiple sizes on hand.

A mask can be used in place of the intubation tube if it can't get a proper seal. Insert a 25-gauge venous catheter into the ear of the rabbit to allow for emergency drug administration. Once the rabbit has been fully anesthetized, shave the caudal abdomen around the second and third pairs of teats.

A cordless vacuum can aid in maintaining a clean infusion site. Once most of the hair is removed, apply hair removal cream around the teat area and remove it with wetted gauze after 15 seconds of application. Confirm good visibility and access to the teat area and repeat if necessary.

Wipe the area with chlorhexidine gauze pads to clean the injection site before cannulation. Rabbits can have a plug protruding from the ductal opening that can prevent successful cannulation of the teat if not removed. Gently remove the layer of skin over the ductal openings using fine-pointed forceps.

Insert a 28-gauge needle, beveled side up, into the side of the teat and slowly infuse 0.2 milliliters of sterile 0.9%saline. Some saline may eject out of the ductal openings. This will allow for a better visualization of the ductal openings during cannulation.

Prepare the 12-inch extension line for cannulation using aseptic technique. Clean the bench surface with ethanol and carefully put on sterile gloves. Remove the protective caps at both ends of the extension line.

Aspirate one milliliter of prepared ablative solution using a one-milliliter Luer-Lok syringe. Carefully attach the syringe to the female winged end, then carefully attach a 27-gauge blunt-tip needle to the opposite end. Slowly push the solution through the line until a steady stream of solution comes out of the needle.

Be careful not to tip the syringe, as this will cause air bubbles to form. Hold the teat gently with a thumb and index finger and lift it slightly for the intraductal infusion. Carefully cannulate the duct of interest using a 27-gauge blunt-tip needle.

A 10X magnifying lamp can aid in visualization of the ductal openings. Slowly infuse the solution to minimize potential damage caused by rapidly moving fluids within the duct. Typically, there is one researcher cannulating and holding the needle while a second researcher holds the syringe and pushes the plunger at the desired rate.

Omnipaque is an FDA-approved iodine-based contrast agent that allows us to see the ductal tree under x-ray fluoroscopy imaging. This allows us to see a ductal tree being filled with ablative solution in real time and cease delivery when the solution reaches the ductal tree ends. Fluoroscopy imaging also provides a quick and easy confirmation of successful filling of the entire ductal tree right after infusion.

In this live session of an infusion into the third duct, you can see Ducts 1 and 2 have already been infused. This live imaging guides to maximize filling of the ductal tree. Once the procedure is completed, begin to recover rhe rabbit by administering a reversal agent intramuscularly.

In our case, we used atipamezole. Signs of recovery include chewing and nose twitching. Be sure the rabbit can hold themselves upright before letting them recover on their own.

Administer ketoprofen subcutaneously for at least three days post procedure to help reduce inflammation and minimize scarring. Hematoxylin and eosin stain, or more commonly known as H&E stain, is the most used stain in histology. With this, we can see the four ductal openings and the ductal pathways within the mammary gland.

In our experiments, we tested a range of solutions with 10 to 70%ethanol concentration. Based on this range of concentrations, we can see that an ablative solution with less than 10%ethanol can still efficiently ablate the ductal tree compared to higher percentage of ethanol. These results indicate that this lower 10%ethanol ablative solution should be prioritized for further study.

We describe a procedure for image-guided infusion into the ductal tree system of the rabbit mammary gland. We demonstrate controlled infusion of x-ray contrast agent containing ethanol-based ablative solution into all teat openings by fluoroscopy real-time imaging and confirm rate of epithelial cell ablation and tissue damage by histological analysis. This procedure provides a step towards scalability of a less invasive method for primary prevention of breast cancer as an alternative to prophylactic mastectomy.

This protocol uses instruments, reagents, and materials for fluoroscopy-guided infusion of Omnipaque-containing ethanol-based ablative solution that are compatible with current clinical practice for visualization of ductal trees. Thus, the described procedure could be readily implemented and evaluated in first in-human clinical trials.