Intraductal Delivery to the Rabbit Mammary Gland

Summary

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Abstract

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects^{1,2,3,4,5,6,7}. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Introduction

The intraductal delivery of therapeutic agents has been studied in rodent models and in early stage human trials^{3,4,5,6,11,12}. A recent Phase I study demonstrated the safety and feasibility of intraductal carboplatin or intraductal pegylated liposomal doxorubicin in women awaiting mastectomy for the treatment of invasive cancer².

Previous protocols for intraductal delivery have been developed for mouse and rat mammary glands^6,7,8,9. For research purposes, intraductal tumor cell injections and the lentiviral vector delivery of oncogenes have also been performed in rodent models^13,14,15,16. However, an ideal in vivo model of the intraductal delivery process should permit the development of novel classes of therapeutic compounds and facilitate preclinical assessment. Anatomical differences between rodents and humans have complicated the translation of these studies.

Unlike mice, in which each duct ends at a separate teat, the human breast consists of 5 to 9 independent ductal systems, each with a separate opening ending at the teat. Rabbit mammary glands harbor four independent ductal systems, each separately accessible through one of four orifices in a single teat. A rabbit model more closely matches the human anatomy and permits the study of intraductal drug delivery in a more relevant context.

Here, we use two techniques to assess intraductal delivery. The co-administration of a vital dye permits visualization through the skin and provides a simple and rapid confirmation of the method. For some applications, higher resolution mapping of the ducts may be preferred. We present here a protocol for ultrasound imaging of the ducts through the intraductal delivery of a non-targeted contrast reagent.

Protocol

Procedures using animal subjects have been approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin.

1. Preoperative Preparation

1. Record the body weight of each rabbit. As with all preclinical studies, monitor animal weights regularly to assess potential toxicity.
2. Prior to anesthetizing the rabbit, fill a 50 mL conical tube with commercially available ultrasound gel and spin at 500 x g for 30 s; there should be no visible bubbles in the gel upon completion of the centrifugation.
3. Administer glycopyrrolate subcutaneously at a dose of 0.1 mg/kg and acepromazine intramuscularly at a dose of 0.75 mg/kg. Wait 15-20 min for the sedative to take effect while monitoring vital signs and behavior.
   NOTE: Glycopyrrolate is an anti-cholinergic agent that prevents bradycardia and reduces respiratory and GI secretions. Acepromazine is a sedative that serves as a premedication for anesthesia.
4. Administer 35 mg/kg of ketamine and 5 mg/kg of xylazine subcutaneously as an anesthetic. However, as the operator becomes more experienced and can accomplish the intraductal delivery more rapidly, decrease the drug dosages to 15 mg/kg of ketamine and 3 mg/kg of xylazine subcutaneously; this will shorten the anesthesia time and the time required for the animal to recover from anesthesia.
5. Check and document the heart rate, SPO_2, temperature, respiratory rate, and mucus membrane color every 15 min. Apply eye lubricant to both eyes.
   NOTE: Only personnel who have undergone appropriate training and have been approved by their institution's IACUC should administer or monitor anesthesia. The use of a veterinary anesthesia monitor can aid in acquiring vital signs and is recommended. The veterinary anesthesia monitor, however, does not replace the need to manually check the animal every 15 min. See the manufacturer's instructions for the proper use of a veterinary monitor.
6. Verify the onset of anesthesia by a gentle toe pinch; the rabbit should be non-responsive before continuing.
7. Carefully shave the caudal abdomen of the rabbit in the area around the third and fourth pairs of inguinal teats.
8. With the majority of the hair removed, apply an over-the-counter hair removal cream to the shaved area. Remove the cream 10 min after application using damp paper towels wetted with warm water.
   NOTE: Rabbit skin is very fragile/sensitive. The hair removal cream should be used for no more than 10 min. In fact, it is safer to try a “test spot” at 5 min and only leave on longer if test spot indicates a longer time period is needed.
9. Wipe the area with alcohol-soaked gauze pads to clean the injection site.
10. Place the rabbit on its dorsum in a v-shaped trough lined with a recirculating warm water blanket and an absorbent pad.

2. Preparation of the Contrast Agent

1. Reconstitute the non-targeted contrast reagent according to the manufacturer's instructions. Pipette up and down gently to mix.
   NOTE: The contrast agent used in this protocol is stable at room temperature for 4 - 6 h after reconstitution. Gently rock the vial between each retrieval.
   NOTE: The volume of solution required will depend on the number of ducts to be injected. The rabbit has 4 ductal openings per teat, and 0.2 mL of solution is sufficient to fill one ductal tree of an adult New Zealand White Rabbit (Oryctolaguscuniculus). Thus, a total volume of 0.8 mL may be delivered to one mammary gland.

3. Intraductal Delivery

1. Locate the appropriate teats to be injected; the 3^rd and 4^th pairs of inguinal teats are recommended, as they are easily visualized when the animal is positioned on its dorsum.
2. Load 0.2 mL of sterile 0.9% saline into a 1 mL Luer-lock tuberculin syringe with a 22 G needle. Properly dispose of the 22 G needle once the saline is in the syringe and replace it with a sterile 25-gauge needle. Gently wipe the area with 85% isopropyl alcohol on a gauze pad.
3. With the bevel of the needle up and the syringe parallel to the body of the animal, insert the bevel of the needle into the side of the teat and slowly inject 0.1-0.2 mL of saline; this will allow for better visualization of the ductal openings.
4. Load 0.2 mL of injection solution into a 1 mL Luer-lock tuberculin syringe.
5. Hold the teat gently with the thumb and index finger and lift it slightly to position it for the intraductal injection; a wearable ocular loupe may aid in visualizing the ductal openings.
6. While maintaining the lifted position of the teat, carefully cannulate the duct of interest using a 25 G blunt-tip needle.
7. After cannulation, gently twist the Luer-lock syringe on the hub of the blunt-tip infusion needle until it is locked in place.
8. Lift the teat and inject the solution slowly to minimize potential damage caused by rapidly moving fluid within the duct; at no time should there be resistance when injecting the solution.

4. Ultrasound Imaging

1. 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.
2. Set the imaging depth to 6 mm. Place the 21-MHz 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.
   NOTE: These settings have been developed for use with a specific photoacoustic ultrasound machine. Refer to the Table of Materials for more details. It may be necessary to adjust the transmit power and imaging depth of other imaging systems to optimize mammary gland visualization.
3. Remove the ultrasound gel from the animal's skin with a gauze pad.

5. Postoperative Care

1. Observe the injection site: there should be no signs of trauma to the teat region or to the surrounding tissue, and swelling in the area surrounding the teat likely indicates a mammary fat pad injection rather than a successful intraductal injection.
2. Place the rabbit in a sternal position. If needed, give 0.2 mg/kg of yohimbine intravenously to the marginal ear vein; this will reverse the effect of the xylazine and allow the animal to recover more quickly from anesthesia.
3. Monitor the rabbit every 15 min throughout the recovery period until the animal is alert, responsive, and maintains a sternal position.
   NOTE: This procedure should not result in tissue damage or swelling. If redness or swelling are observed, administer a dose of meloxicam 0.1-0.2 mg/kg PO once the animal is alert and able to take medication orally. Contact the institution's veterinary staff for further guidance.

Results

Here, we show that the intraductal delivery of contrast reagents to the mammary ducts of a rabbit can be achieved without trauma to the tissue (Figure 2). In rabbits, four separate ductal systems converge at one teat and thus may be accessed and imaged individually using this method. Individual ductal openings are easily visualized; note the arrowhead marking a second ductal opening adjacent to the cannulated duct in Figure 2B.

Discussion

This method of intraductal delivery to the rabbit mammary gland may be used for ultrasound contrast reagents and many other aqueous solutions, including vital dyes and therapeutics. Previous studies have demonstrated the intraductal delivery of hormones^17,18,19. In rodent models, the intraductal delivery of nucleic acids⁸, chemotherapeutics^6,7, an...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MicroMarker non-targeted contrast reagent	VisualSonics	VS-11694
Luer Lock 1 mL Syringes	BD	309628
Glycopyrrolate 0.2 mg/mL	Wedgewood Compounding Pharmacy	GLYCOP-INJ013VC	6 month shelf life, supply may be limited.
Atropine Sulfate 0.5 mg/mL	Animal Health International	15320764	If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.
Ketamine HCL 100 mg/mL	Animal Health International	21250699	http://www.animalhealthinternational.com/
Acepromazine 10 mg/mL	Animal Health International	17640541
Xylazine 20 mg/mL	Animal Health International	20101547
Yohimbine 0.2 mg/mL	Animal Health International	14588965
Hair Removing Cream	Veet		Sensitive skin solution. Available through local retailers.
Blunt tip infusion needles	Sai Infusion Technology	B14-50	http://www.sai-infusion.com/collections/blunt-needles
Veterinary Pulse Oximeter	EdanUSA	VE-H100B	http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html
Warm Water Pump	Gaymar	TP700
Warm Water Blanket	Animal Health International	21232696	Maxi-Therm Lite Warming Pads
Ultrasound system	VisualSonics	Vevo 2100