Rabbit Intraductal Injection: Localized Delivery of Solution of Interest into the Rabbit Mammary Gland

Overview

In this video we describe a technique of administering a solution of interest into the mammary duct of a rabbit and confirm the delivery by high-resolution ultrasound imaging of contrast agents.

Protocol

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.

Materials

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.
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
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-PulseOximeter.html
Warm Water Pump	Gaymar	TP700
Warm Water Blanket	Animal Health International	21232696	Maxi-Therm Lite Warming Pads