Name: intraductal delivery to the rabbit mammary gland
Uploaded: 2017-03-09T13:00:00
Description: Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

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>
	<li>Flanagan, M., Love, S., Hwang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+Intraductal+Therapy+for+Ductal+Carcinoma+in+Situ.">Status of Intraductal Therapy for Ductal Carcinoma in Situ.</a> Curr Breast Cancer Rep. 2 (2), 75-82 (2010).</li><li>Love, S. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+Injection+For+Localized+Drug+Delivery+To+The+Mouse+Mammary+Epithelium.">Intraductal Injection For Localized Drug Delivery To The Mouse Mammary Epithelium.</a> J Vis Exp. (80), e50692 (2013).</li><li>Brock, A., Goh, H. T., Yang, B., Yu, L., Li, H., Loh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Reprogramming:+A+New+Technology+Frontier+In+Pharmaceutical+Research.">Cellular Reprogramming: A New Technology Frontier In Pharmaceutical Research.</a> Pharm Res. 29 (1), 35-52 (2012).</li><li>Silverstein, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Intraductal+Approach+to+the+Breast:+Raison+d'Etre.">The Intraductal Approach to the Breast: Raison d'Etre.</a> Breast Cancer Res. 8 (2), 206 (2006).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo+.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo .</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Lyons, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Direct+Mammotrophic+Action+of+Lactogenic+Hormone.">The Direct Mammotrophic Action of Lactogenic Hormone.</a> Proc. Soc. Exp. Bio. Med. 51 (2), 308-311 (1942).</li><li>Fiddler, T. J., Birkinshaw, M., Falconer, I. R. <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+Prolactin+On+Some+Aspects+of+the+Ultrastructure+and+Biochemistry+of+Mammary+Tissue+in+the+Pseudopregnant+Rabbit.">Effects Of Intraductal Prolactin On Some Aspects of the Ultrastructure and Biochemistry of Mammary Tissue in the Pseudopregnant Rabbit.</a> J Endocrinol. 49 (3), 459-469 (1971).</li><li>Falconer, I. R., Fiddler, T. 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. L., Rosen, P. P., Lieberman, P. H., Robbins, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+Carcinoma.+Long-term+Follow-up+After+Treatment+by+Biopsy+Alone.">Intraductal Carcinoma. 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. 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=Addressing+Overdiagnosis+and+Overtreatment+in+Cancer:+A+Prescription+For+Change.">Addressing Overdiagnosis and Overtreatment in Cancer: A Prescription For Change.</a> Lancet Oncol. 15 (6), e234-e242 (2014).</li></ol>

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

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...