The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.

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
<ol>
	<li>Record the body weight of each rabbit. As with all preclinical studies, monitor animal weights regularly to assess potential toxicity.</li>
	<li>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 ...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Feasibility+Study+of+the+Intraductal+Administration+of+Chemotherapy.">A Feasibility Study of the Intraductal Administration of Chemotherapy.</a> Cancer Prevention Research. 6 (1), 51-58 (2013).</li><li>Stearns, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+Clinical+Evaluation+of+Intraductally+Administered+Agents+in+Early+Breast+Cancer.">Preclinical and Clinical Evaluation of Intraductally Administered Agents in Early Breast Cancer.</a> Sci Transl Med. 3 (106), 106ra108 (2011).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Safety+Parameters+of+the+Study+on+Intraductal+Cytotoxic+Agent+Delivery+to+the+Breast+before+Mastectomy.">The Safety Parameters of the Study on Intraductal Cytotoxic Agent Delivery to the Breast before Mastectomy.</a> Chin J Cancer Res. 26 (5), 579-587 (2014).</li><li>Mahoney, M. E., Gordon, E. J., Rao, J. Y., Jin, Y., Hylton, N., Love, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+Therapy+of+Ductal+Carcinoma+In+Situ:+a+Presurgery+Study.">Intraductal Therapy of Ductal Carcinoma In Situ: a Presurgery Study.</a> Clin Breast Cancer. 13 (4), 280-286 (2013).</li><li>Murata, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+Access+for+Prevention+and+Therapy+of+Mammary+Tumors.">Ductal Access for Prevention and Therapy of Mammary Tumors.</a> Cancer Res. 66 (2), 638-645 (2006).</li><li>Chun, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductally+Administered+Pegylated+Liposomal+Doxorubicin+Reduces+Mammary+Stem+Cell+Function+In+the+Mammary+Gland+but+in+the+Long+Term,+Induces+Malignant+Tumors.">Intraductally Administered Pegylated Liposomal Doxorubicin Reduces Mammary Stem Cell Function In the Mammary Gland but in the Long Term, Induces Malignant Tumors.</a> Breast Cancer Res Treat. 135 (1), 201-208 (2012).</li><li>Brock, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+HoxA1+by+Intraductal+Injection+of+siRNA+Lipidoid+Nanoparticles+Prevents+Mammary+Tumor+Progression+in+Mice.">Silencing HoxA1 by Intraductal Injection of siRNA Lipidoid Nanoparticles Prevents Mammary Tumor Progression in Mice.</a> Sci Trans Med. 6 (217), 2172a2 (2014).</li><li>Krause, S., Brock, A., Ingber, D. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+Carcinoma+In+Situ+of+the+Breast.">Ductal Carcinoma In Situ of the Breast.</a> Annu. Rev Med. 51, 17-32 (2000).</li><li>Love, S. M., Barsky, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+Nipple+And+Breast+Ducts+Revisited.">Anatomy of the Nipple And Breast Ducts Revisited.</a> Cancer. 101 (9), 1947-1957 (2004).</li><li>Virnig, B. A., Shamliyan, T., Tuttle, T. M., Kane, R. L., Wilt, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+Management+of+Ductal+Carcinoma+In+Situ+(DCIS).">Diagnosis and Management of Ductal Carcinoma In Situ (DCIS).</a> Evidence Report/Technology Assessment. , 185 (2009).</li><li>Mills, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Duct+Physiology+in+the+Human+Mammary+Gland.">Examination of Duct Physiology in the Human Mammary Gland.</a> PLoS One. 11 (4), e0150653 (2016).</li><li>King, B. L., Love, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Intraductal+Administration+Of+Prolactin,+Actinomycin+D+and+Cycloheximide+on+Lipoprotein+Lipase+Activity+in+the+Mammary+Glands+of+Pseudopregnant+Rabbits.">Effects of Intraductal Administration Of Prolactin, Actinomycin D and Cycloheximide on Lipoprotein Lipase Activity in the Mammary Glands of Pseudopregnant Rabbits.</a> Biochim Biophys Acta. 218 (3), 508-514 (1970).</li><li>Singh, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Molecular+Size+on+the+Retention+of+Polymeric+Nanocarrier+Diagnostic+Agents+in+Breast+Ducts.">Influence of Molecular Size on the Retention of Polymeric Nanocarrier Diagnostic Agents in Breast Ducts.</a> Pharm Res. 29 (9), 2377-2388 (2012).</li><li>Jain, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+Ductal+Hyperplasia:+Interobserver+and+Intraobserver+Variability.">Atypical Ductal Hyperplasia: Interobserver and Intraobserver Variability.</a> Mod Pathol. 24 (7), 917-923 (2011).</li><li>Betsill, W. 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Long-term Follow-up After Treatment by Biopsy Alone.</a> JAMA. 239 (18), 1863-1867 (1978).</li><li>Eusebi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Follow-up+of+In+Situ+Carcinoma+of+the+Breast.">Long-term Follow-up of In Situ Carcinoma of the Breast.</a> Semin Diagn Pathol. 11 (3), 223-235 (1994).</li><li>Sanders, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+Of+Low-Grade+Ductal+Carcinoma+In+Situ+Of+The+Breast+In+Women+Treated+By+Biopsy+Only+Revealed+Over+30+Years+Of+Long-Term+Follow-Up.">The Natural History Of Low-Grade Ductal Carcinoma In Situ Of The Breast In Women Treated By Biopsy Only Revealed Over 30 Years Of Long-Term Follow-Up.</a> Cancer. 103 (12), 2481-2484 (2005).</li><li>Esserman, L. 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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 hormones17,18,19. In rodent models, the intraductal delivery of nucleic acids8, chemotherapeutics6,7, an...

The intraductal delivery of therapeutic agents has been studied in rodent models and in early stage human trials3,4,5,6,11,12. 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 glands6,7,8,9. For research purposes, intraductal tumor cell injections and the lentiviral vector delivery of oncogenes have also been performed in rodent models13,14,15,16. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MicroMarker non-targeted contrast reagent</td>
			<td>VisualSonics</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock 1mL Syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate 0.2mg/mL</td>
			<td>Wedgewood Compounding Pharmacy</td>
			<td>GLYCOP-INJ013VC</td>
			<td>6 month shelf life, supply may be limited.&#160;</td>
		</tr>
		<tr>
			<td>Atropine Sulfate 0.5 mg/mL</td>
			<td>Animal Health International</td>
			<td>15320764</td>
			<td>If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.</td>
		</tr>
		<tr>
			<td>Ketamine HCL 100mg/mL</td>
			<td>Animal Health International</td>
			<td>21250699</td>
			<td><a href="http://www.animalhealthinternational.com/">http://www.animalhealthinternational.com/</a></td>
		</tr>
		<tr>
			<td>Acepromazine 10mg/mL</td>
			<td>Animal Health International</td>
			<td>17640541</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20mg/mL</td>
			<td>Animal Health International</td>
			<td>20101547</td>
			<td></td>
		</tr>
		<tr>
			<td>Yohimbine 0.2mg/mL</td>
			<td>Animal Health International</td>
			<td>14588965</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removing Cream</td>
			<td>Veet</td>
			<td></td>
			<td>Sensitive skin solution. Available through local retailers.</td>
		</tr>
		<tr>
			<td>Blunt tip infusion needles</td>
			<td>Sai Infusion Technology</td>
			<td>B14-50</td>
			<td><a href="http://www.sai-infusion.com/collections/blunt-needles">http://www.sai-infusion.com/collections/blunt-needles</a></td>
		</tr>
		<tr>
			<td>Veterinary Pulse Oximeter</td>
			<td>EdanUSA</td>
			<td>VE-H100B</td>
			<td><a href="http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html">http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html</a></td>
		</tr>
		<tr>
			<td>Warm Water Pump</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Blanket</td>
			<td>Animal Health International</td>
			<td>21232696</td>
			<td>Maxi-Therm Lite Warming Pads</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraductal delivery to the rabbit mammary gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. 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.

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.
<p class="jove_content"...

Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

Intraductal Delivery to the Rabbit Mammary Gland

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.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

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.

intraductal-delivery-to-the-rabbit-mammary-gland

Research

JoVE Journal

Bioengineering

9.2K visualizações.  The University of Texas at Austin. 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 administere...