The overall goal of this procedure is to deliver aqueous solutions to the rabbit's mammary duct and visualize the intraductal delivery by ultrasound imaging. This method can help answer key questions in the breast biology field, such as how do therapeutics interact differently with the mammary ductal environment compared with other sites of administration. The main advantage of this technique is that the imaging contrast reagent or any potential therapeutics to be evaluated can be administered locally to the tissue where breasts lesions arise.

Demonstrating the technique will be Amelia Clark, a technician in my laboratory. To begin this procedure, carefully shave the caudal abdomen of an anesthetized rabbit in the area around the third and fourth pairs of inguinal teats. With the majority of the hair removed, apply the hair removal cream to the shaved areas and 10 minutes after application, remove the cream using a damp paper towel wetted with warm water.

Then wipe the area with alcohol-soaked gauze pads to clean the injection site. Place the rabbit on it's dorsum in a V-shaped trough lined with recirculating warm water blanket in an absorbent pad. To prepare the contrast agent reconstitute it according to the manufacturer's instructions.

Then gently rock the vial to mix. The contrast agent used here is stable at room temperature for four to six hours after reconstitution. In this procedure locate the third and fourth pairs of inguinal teats to be injected.

Then load 0.2 milliliters of sterile 0.9 saline into a one milliliter luer lock tuberculin syringe with a 22 gauge needle. Dispose of the 22 gauge needle once the saline is in the syringe and replace it with a sterile 25 gauge needle. Next gently wipe the area with 85%isopropyl alcohol on a guaze pad.

With the bevel of the needle up and the syringe parallel to the body of the animal, insert the needle into the side of the teat and slowly inject 0.1 to 0.2 milliliters of saline to allow for better visualization of the ductal openings. Afterward load 0.2 milliliters of injection solution into a one milliliter luer lock tuberculin syringe. Hold the teat gently with a thumb and index finger and lift it slightly for the intraductal injection.

While maintaining the lifted position of the teat, carefully cannulate the duct of interest using a 25 gauge blunt tip needle. After cannulation gently twist the luer lock syringe on the hub of the blunt tip infusion needle until it is locked in place. Lift the teat and inject the solution slowly to minimize potential damage caused by rapidly moving fluids within the duct.

Now apply a liberal amount of centrifuged ultrasound gel to the skin of the area of interest. Ensure that there are no bubbles in the gel as these will compromise the image quality. Next set the imaging depth to six millimeters.

Place the 21 megahertz transducer in contact with the gel and scan the area of interest in B mode. Observe the contrast medium in the scanned region, including the ductal opening and throughout the duct to confirm successful intraductal delivery. Then capture and save the image.

Remove the ultrasound gel from the animal's skin with gauze pads. Following that inspect the injection site and make sure that there are no signs of trauma to the teat region or to the surrounding tissue. Swelling in the area surrounding the teat likely indicates a mammary fat pad injection rather than a successful intraductal injection.

The contrast reagent is localized immediately after delivery and is visualized 30 minuted post-delivery and 45 minutes post-delivery. The persistence of the reagent inside the mammary duct allows visualization throughout the duration of this process. This image shows the external appearance after the intraductal injection of 0.2 milliliters of Evans blue solution.

Upon opening the skin Evans blue permits the visualization of the entire mammary ductal tree and confirms the intact ductal structure. And this image shows the whole mount specimen of a region of inguinal mammary gland after fixation and staining with carmine alum. Once mastered this technique can be done in approximately 15 minutes, excluding the time required to allow anesthesia to take effect.

While attempting this procedure, it's important to remember to lift the teat to straighten the ducts during intraductal delivery. At all times follow the animal welfare guidelines of your institution. Following this procedure, other methods like advanced imaging or endpoint tissue analyses such as a lizo or immunohistochemistry can be performed to answer additional questions about the pharmacodynamics or tissue-level morphological changes induced by the delivery of therapeutic agent.

After its development, this technique paves the way for researchers in mammary biology to explore the delivery of imaging agents as well as therapeutics in the mammary duct in the future. After watching this video, you should have a good understanding of how to perform intraductal delivery to the rabbit mammary gland.