Name: retroductal nanoparticle injection to the murine submandibular gland
Uploaded: 2018-05-03T14:15:00
Description: Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Investigación en esta publicación fue apoyada por el Instituto Nacional de odontología y el Instituto Nacional de cáncer (NCI) de los institutos nacionales de salud Premio número R56 DE025098, DE027695 UG3 y F30 CA206296 Craniofacial Research (NIDCR). El contenido es responsabilidad exclusiva de los autores y no representan necesariamente las opiniones oficiales de los institutos nacionales de salud. Este trabajo también fue apoyado por la NSF DMR 1206219 y la innovación de la IADR en Premio de Cuidado Oral (2016).
Nos gustaría agradecer a Jayne Gavrity por su ayuda en la realización de experimentos IVIS. Nos gustaría agradecer a Karen Bentley por su entrada y asistencia en la realización de EM. Nos gustaría agradecer su ayuda con IHC Weng Pei-Lun. Nos gustaría agradecer a Matthew Ingalls por su ayuda en la preparación de la figura. Nos gustaría agradecer al Dr. Elaine Smolock y Emily Wu lectura crítica de este manuscrito.

Todo en vivo los procedimientos descritos a continuación fueron aprobados por el Comité de la Universidad sobre los recursos de la Universidad de Rochester, Rochester, NY.
1. preparación
<ol><li>Con tubo de 32G catéter intracraneal con inserción de alambre, corte 3 cm del tubo para formar un extremo biselado, aproximadamente 45° al eje longitudinal. Confirman que el cable es más largo que el tubo de al menos 1 cm.</li>
	<li>Coloque 50 μl de solución de nanopartículas PSMA ...

<ol>
	<li>Miranda-Rius, J., Brunet-Llobet, L., Lahor-Soler, E., Farre, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+Secretory+Disorders,+Inducing+Drugs,+and+Clinical+Management.">Salivary Secretory Disorders, Inducing Drugs, and Clinical Management.</a> International Journal Of Medical Sciences. 12 (10), 811-824 (2015).</li><li>Acauan, M. D., Figueiredo, M. A. Z., Cherubini, K., Gomes, A. P. N., Salum, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy-induced+salivary+dysfunction:+Structural+changes,+pathogenetic+mechanisms+and+therapies.">Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies.</a> Archives of Oral Biology. 60 (12), 1802-1810 (2015).</li><li>Dirix, P., Nuyts, S., Vander Poorten, V., Delaere, P., Van den Bogaert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+xerostomia+after+radiotherapy+on+quality+of+life.">The influence of xerostomia after radiotherapy on quality of life.</a> Supportive Care in Cancer. 16 (2), 171-179 (2008).</li><li>Vissink, 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=Clinical+management+of+salivary+gland+hypofunction+and+xerostomia+in+head-and-neck+cancer+patients:+successes+and+barriers.">Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers.</a> International Journal of Radiation Oncology Biology Physics. 78 (4), 983-991 (2010).</li><li>Delporte, C., 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=Increased+fluid+secretion+after+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+to+irradiated+rat+salivary+glands.">Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands.</a> Proceedings of the National Academy of Sciences. 94 (7), 3268-3273 (1997).</li><li>Samuni, Y., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+salivary+glands:+from+the+bench+to+the+clinic.">Gene delivery in salivary glands: from the bench to the clinic.</a> Biochimica et Biophysica Acta. 1812 (11), 1515-1521 (2011).</li><li>Beahm, D. 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=Surgical+approaches+to+the+submandibular+gland:+A+review+of+literature.">Surgical approaches to the submandibular gland: A review of literature.</a> International Journal of Surgery. 7 (6), 503-509 (2009).</li><li>Zheng, C., Shinomiya, T., Goldsmith, C. M., Di Pasquale, G., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+and+reproducible+in+vivo+gene+transfer+to+mouse+parotid+glands.">Convenient and reproducible in vivo gene transfer to mouse parotid glands.</a> Oral diseases. 17 (1), 77-82 (2011).</li><li>Zheng, C., 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=Prevention+of+Radiation-Induced+Salivary+Hypofunction+Following+hKGF+Gene+Delivery+to+Murine+Submandibular+Glands.">Prevention of Radiation-Induced Salivary Hypofunction Following hKGF Gene Delivery to Murine Submandibular Glands.</a> Clinical Cancer Research. 17 (9), 2842-2851 (2011).</li><li>Okazaki, Y., 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=Acceleration+of+rat+salivary+gland+tissue+repair+by+basic+fibroblast+growth+factor.">Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor.</a> Archives of Oral Biology. 45 (10), 911-919 (2000).</li><li>Arany, S., Benoit, D. S., Dewhurst, S., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+gene+silencing+confers+radioprotection+to+salivary+glands+in+vivo.">Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo.</a> Molecular Therapy. 21 (6), 1182-1194 (2013).</li><li>Cotrim, A. P., Sowers, A., Mitchell, J. B., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+irradiation-induced+salivary+hypofunction+by+microvessel+protection+in+mouse+salivary+glands.">Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.</a> Molecular Therapy. 15 (12), 2101-2106 (2007).</li><li>Redman, R. S., Ball, W. D., Mezey, E., Key, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+donor+salivary+gland+cells+are+widely+distributed+in+the+recipient+gland+when+infused+up+the+ductal+tree.">Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.</a> Biotechnic &amp; Histochemistry. 84 (6), 253-260 (2009).</li><li>Grundmann, O., Fillinger, J. L., Victory, K. R., Burd, R., Limesand, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+radiation+therapy-induced+salivary+gland+dysfunction+in+mice+by+post+therapy+IGF-1+administration.">Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration.</a> BMC Cancer. 10, 417-417 (2010).</li><li>Limesand, K. H., 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=Insulin-Like+Growth+Factor-1+Preserves+Salivary+Gland+Function+After+Fractionated+Radiation.">Insulin-Like Growth Factor-1 Preserves Salivary Gland Function After Fractionated Radiation.</a> International Journal of Radiation Oncology Biology Physics. 78 (2), 579-586 (2010).</li><li>Marmary, Y., 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=Radiation-induced+loss+of+salivary+gland+function+is+driven+by+cellular+senescence+and+prevented+by+IL-6+modulation.">Radiation-induced loss of salivary gland function is driven by cellular senescence and prevented by IL-6 modulation.</a> Cancer Research. , (2016).</li><li>Baum, B. 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=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (47), 19403-19407 (2012).</li><li>Arany, 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=Pro-apoptotic+gene+knockdown+mediated+by+nanocomplexed+siRNA+reduces+radiation+damage+in+primary+salivary+gland+cultures.">Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.</a> Journal of Cellular Biochemistry. 113 (6), 1955-1965 (2012).</li><li>Benoit, D. S. W., Henry, S. M., Shubin, A. D., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-responsive+polymeric+siRNA+carriers+sensitize+multidrug+resistant+ovarian+cancer+cells+to+doxorubicin+via+knockdown+of+polo-like+kinase+1.">pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.</a> Molecular pharmaceutics. 7 (2), 442-455 (2010).</li><li>Malcolm, D. W., Varghese, J. J., Sorrells, J. E., Ovitt, C. E., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Biological+Fluids+on+Colloidal+Stability+and+siRNA+Delivery+of+a+pH-Responsive+Micellar+Nanoparticle+Delivery+System.">The Effects of Biological Fluids on Colloidal Stability and siRNA Delivery of a pH-Responsive Micellar Nanoparticle Delivery System.</a> ACS Nano. , (2017).</li><li>Baranello, M. P., Bauer, L., Benoit, D. 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Retroductal inyección es crítica para la entrega de la droga localizada en la glándula salival. Esta técnica tiene aplicaciones en la detección de agentes terapéuticos para las condiciones incluyendo el síndrome de Sjögren y RIH9,10,28. Directa del fármaco en el SMG vía la inyección de retroductal ofrece una ventaja clave sobre la administración sistémica en su potencial para reducir los efectos off-target, incluyen...

Disfunción de las glándulas salivales tiene muchas etiologías, incluyendo el síndrome de Sjögren, una pérdida mediada autoinmune del tejido secretor funcional e hiposalivación inducida por la radiación (ROG), una secuela común de cabeza y cuello cáncer radioterapia1. Pérdida de la función salival debido a cualquier condición predispone a los individuos a oral y sistémica de la infección, caries dental, disfunción digestiva y deglución, debilitación de discurso y depresión mayor1,2,3. Como resultado, calidad de vida sufre significativamente, con las intervenciones que se limita a la paliación de los síntomas más que curar4. Para investigar nuevas terapias en vivo, es de interés para administrar compuestos bioactivos directamente a la glándula salival.
Retroductal inyectable es un método valioso para entregar compuestos bioactivos directamente a las glándulas salivales y comprobar la eficacia en la enfermedad, lesión, o en la homeostasis del tejido normal. Las tres glándulas salivales mayores son la parótida (PG), el submaxilar (SMG) y sublingual (SLG), todos de que está vacía en la cavidad bucal a través de conductos excretores. La anatomía de la murine SMG permite acceso directo a través de la canulación del conducto de Wharton, ubicada en el piso de la boca bajo la lengua5. Tras la canulación, fármacos solvatados pueden administrarse directamente en el SMG. Después del parto de retroductal, difusión extra glandular está limitada por la cápsula de tejido circundante que regula el intercambio de material con el que rodea las estructuras6. El SMG y su conducto están estructurados de manera similar en los seres humanos y habitualmente se acceden durante el SMG cirugía y sialoendoscopy7. En humanos y ratones, la PG es además accesible a través del conducto de Stensen en la mucosa bucal8.
En modelos murinos de RIH, inyección de retroductal SMG se ha utilizado para ofrecer terapéutica incluyendo factores de crecimiento, células primarias, vectores adenoviral, citoquinas y compuestos antioxidantes modulan la respuesta celular a la lesión, y reducir el tejido daño5,9,10,11,12,13,14,15,16. El más notable éxito clínico de retroductal inyectable es la administración de un vector adenoviral para dirigir la expresión de un canal de agua (Acuaporina 1; AQP1) en pacientes después de la radiación para cáncer de cabeza y cuello17.
Previamente, hemos desarrollado y demostrado la eficacia de un sistema de nanopartículas poliméricas-siRNA retroductally inyectada para proteger la función de la glándula salival de RIH11,18,19,20. Como una extensión de nuestro trabajo en el pasado, aquí, demostramos nuestro protocolo para la inyección de SMG de retroductal usando una nanopartícula fluorescente etiquetada (NP) capaz de cargar y entregar de lo contrario poco solubles drogas21,22, 23.
Se ha sintetizado lo NP de un copolímero del diblock compuesto de poli (estireno-alt-maleico anhydride)-b-poly(styrene) (PSMA) a través de la adición reversible cadena fragmentación (balsa) polimerización, tal como se describe anteriormente21. A través del intercambio de solvente, estos polímeros montan espontáneamente uno en estructuras de micelas NP con una hidrofóbica interior e hidrofílico exterior21. El NPs están marcado con rojo Texas fluoróforo para permitir la verificación de la entrega de la NP en las glándulas sin sacrificar el animal. Imágenes de animales en vivo y immunohistochemistry SMG se muestra en 1 h y 1 día después de la inyección.
Este actualizado y protocolo reproducible de la canalización debe permitir que otras personas lograr la inyección de retroductal. Esperamos que esta refinada técnica será fundamental para en vivo estudios y desarrollo terapéutico24,25.

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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

retroductal nanoparticle injection to the murine submandibular gland

Dos objetivos comunes de la terapéutica de la glándula salival son prevención y cura de la disfunción del tejido siguiendo ya sea autoinmune o lesión por radiación. Por entregar localmente compuestos bioactivos a las glándulas salivales, mayores concentraciones de tejido pueden lograrse con seguridad frente a la administración sistémica. Además, de tejido de la blanco los efectos de la acumulación extra glandular de material pueden dramáticamente reducidos. En este sentido, retroductal la inyección es un método ampliamente utilizado para la investigación de la glándula salival biología y Fisiopatología. Retroductal administración de factores de crecimiento, células primarias, vectores adenoviral y fármacos de molécula pequeña se ha demostrado para apoyar la función de la glándula en el entorno de la lesión. Previamente hemos demostrado la eficacia de una estrategia de nanopartículas-siRNA de retroductally inyectado para mantener la función de la glándula después de la irradiación. Aquí se detalla un método altamente eficaz y reproducible para administrar nanomateriales a la glándula submaxilar murina a través del conducto de Wharton (figura 1). Describir el acceso a la cavidad bucal y delinear los pasos necesarios al conducto de Wharton cannulate, con más observaciones que sirven como controles de calidad durante todo el procedimiento.

Inyección de Retroductal puede utilizarse para administrar NPs a SMG murino (figura 1). Aquí entregamos 50 μg PSMA NPs marcado con rojo Texas fluoróforo.
La colocación correcta del mouse permite de fácil acceso y visualización del piso de la boca (figura 2A-B). Las papilas submaxilares son identificadas como dos protuberancias carnosas debajo de ...

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Entrega local de la droga a las glándulas submaxilares es de interés en biología de la glándula salival de entendimiento y para el desarrollo de nuevas terapias. Presentamos un protocolo de inyección de retroductal actualizada y detallada, diseñado para mejorar entrega exactitud y reproducibilidad experimental. La aplicación presentada en este documento es el de nanopartículas poliméricas.

Inyección de nanopartículas de Retroductal a la glándula submaxilar de ratón

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

The overall goal of this procedure is to administer nanomaterials directly to the murine submandibular gland. The main advantage of this technique is that it achieves direct delivery of compounds to the submandibular gland while bypassing the systemic circulation. The implications of this technique extend toward therapy of salivary gland disfunction following radiation treatment for head and neck malignancy.Generally, individuals new to this method may be challenged by locating and cannulating the submandibular duct while the animal is sedated. To begin this procedure, cut three centimeters of a 32 gauge intracranial catheter tubing with wire inset to form a beveled end approximately 45 degrees to the long axis. Confirm that the wire is at least one centimeter longer than the tubing.Creating a beveled edge to the tubing will ease ductal cannulation. If the tubing gets bent during the procedure, a fresh beveled edge should be cut. Next, load 50 microliters of PSMA nanoparticle solution or other injection material into a Hamilton syringe.To reduce the probability of burrow trauma during injection, attach the catheter tubing to the syringe and expel dead volume. Inspect the injection solution to ensure the nanoparticle is fully solvated to prevent ductal obstruction. Then prepare atropine solution at 0.1 milligram per milliliter.To visualize the ductal entry point, place an anesthetized mouse in the prone position on a custom stage. Then apply ointment to the eyes to prevent dryness during the procedure. Afterward, secure the maxillary incisors over a metal beam and use an elastic band to apply downward tension behind the mandibular incisors.Align the mouse beneath the dissecting microscope to visualize the base of the jaw. To widen the mouth, use a custom curved steel retractor to apply tension to the buccal mucosa bilaterally. To visualize the submandibular papilla, grasp and gently lift the tongue from the floor of the mouth using blunt forceps.Place a piece of cotton between the tongue and the buccal mucosa to enable further manipulation within the oral cavity. To perform ductal cannulation and line placement, grasp the catheter tubing with a wire inset. For optimum manual control during cannulation, align the tubing with the curvature of the forceps.Using the dissecting microscope, move the forceps and wire into the field of view. Using the wire inset, gently apply pressure into the base of one submandibular papilla to produce a small superficial mucosal puncture that will facilitate later entry of the catheter tubing. If resistance is encountered, cut fresh beveled tips on both the tubing and wire inset with sharp dissecting scissors.Following the entry, withdraw the stylet, then, using the dissecting microscope, confirm the presence of saliva at the puncture site. Subsequently, retract the stylet further within the tubing. Avoid forceful or sudden movement of the stylet that may cause bleeding or compromise ductal integrity.To ensure that injection tubing will fit into the Wharton's duct opening, insert tubing containing the stylet as a rigid guide into the previously made puncture. To prevent back pressure from prolonged to ductal obstruction, withdraw the tubing. Under the microscope, ensure that an opening can be seen in the submandibular papilla.If visible bleeding occurs, remove the stylet and reattempt the procedures on the opposing submandibular papilla. Next, without moving the mouse, administer one milligram per kilogram atropine solution intraperitoneally to reduce elevation during the procedure. Then, wait for five to 10 minutes.Under the dissecting microscope, insert the syringe tubing into the orifice. If resistance is encountered, cut a fresh beveled end on the tubing and reattempt it. Once the tubing is in place within the submandibular papilla, slowly advance three to five millimeters into the duct.Then, release the tubing from the forceps. To improve the seal between the tubing and the submandibular papilla, dry the interface by gently blotting with gauze for one minute. Inspect to confirm that the position of the tubing has not shifted during drying.In this procedure, inject the material at a rate of 10 microliters per minute. Ensure that the mouse remains sedated and is not showing signs of distress. Following the injection, maintain syringe pressure for five minutes to improve the retention of material within the Wharton's duct and the submandibular gland.Inspect the submandibular papilla periodically to ensure that tubing does not exit the ductal orifice. Maintaining injection pressure will enhance the effectiveness of retroductal delivery. Using fine forceps, grasp and gently and withdraw the tubing from the submandibular papilla.Remove the retractor and cotton from the oral cavity before moving the mouse away from the stage. To verify nanoparticle delivery, in vivo imaging system shows the lateralization of red fluorescent signal to the treated side of the mouse one hour post injection. And this image shows that the nanoparticle signal has decreased significantly 24 hours after injection.To confirm nanoparticle persistence in the submandibular glands 24 hours following injection, they were sectioned and viewed by fluorescent imaging. Shown here are the uninjected control submandibular glands stained for aquaporin 5 and keritin five marking the secretory acini and ductal cells respectively. In retroductal nanoparticle injected submandibular glands, aquaporin five and keritin five stains show normal gland morphology and nanoparticles taken up in both acini and ducts.Once mastered this technique can be done in 20 minutes if it is performed properly. While attempting this procedure, it's important to remember to avoid any extraneous movements that could dislodge the cannula or disrupt the seal made between the tubing and the submandibular papilla. Following the procedure, other methods like immunostaining, RNA extraction, or flow cytometry can be performed in order to evaluate gene expression or nanomaterial localization in the submandibular gland.After its development, this technique paved the way for salivary gland researchers to screen and test compounds for xerostomia prevention. After watching this video, you should have a good understanding of how to administer nanomaterials directly to the murine submandibular gland via cannulation of Wharton's duct.

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Research

JoVE Journal

Bioengineering

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.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

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

Investigación

Bioingeniería

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Síntesis, de la Asamblea, y caracterización de una sola capa de nanopartículas de oro protegidas Films de Electroquímica de proteínas monocapa

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

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.

Un 3D Cultura Modelo sensibles a hormonas de la glándula mamaria humana epitelio

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

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinación del Potencial Zeta a través de nanopartículas de translocación velocidades a través de un sintonizable nanoporos: El uso de partículas de ADN modificado como ejemplo

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

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 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.

Intraductal La entrega al conejo Glándula Mamaria

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

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Un sistema integrado para activar remotamente Transduction de la señal intracelular por fotoactivación de la cinasa mediada por nanopartículas de conversión ascendente

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Hoja de luz fluorescencia microscopía para el estudio del corazón murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Protocolo de inyección lipídica para mediciones de cristalografía en serie en el sincrotrón australiano

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Trasplante de un parche bioimpreso 3D en un modelo murino de infarto de miocardio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Mejorar la reproducibilidad para cumplir con la información mínima para estudios de vesículas extracelulares 2018 Directrices en el análisis de seguimiento de nanopartículas

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...