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

Forskningen i denne publikasjonen ble støttet av det nasjonale Institutt for Dental og Craniofacial forskning (NIDCR) og National Cancer Institute (NCI) av National Institutes of Health under prisen nummer R56 DE025098, UG3 DE027695 og F30 CA206296. Innholdet er ansvar forfattere og representerer ikke nødvendigvis den offisielle synet til National Institutes of Health. Dette arbeidet ble også støttet av NSF DMR 1206219 og IADR-innovasjon i muntlig bekymre Award (2016).
Vi vil gjerne takke Jayne Gavrity for henne hjelp i å utføre IVIS eksperimenter. Vi vil gjerne takke Karen Bentley for hennes inngang og hjelp i å utføre EM. Vi vil gjerne takke Pei-Lun Weng for hans hjelp med IHC. Vi vil gjerne takke Matthew Ingalls for hans hjelp figur forberedelse. Vi vil gjerne takke Dr. Elaine Smolock og Emily Wu for kritisk lesning av dette manuskriptet.

Alle i vivo prosedyrer skissert nedenfor ble godkjent av universitetet dyr ressurser ved University of Rochester, Rochester, NY.
1. forberedelse
<ol><li>Bruker 32G intrakranielt kateter rør med wire senket, kuttet 3 cm slangen til vinklet, ca 45° lange aksen. Kontroller at kabelen er minst 1 cm lengre enn slangen.</li>
	<li>Laste 50 µL PSMA hydrogenion løsning (figur 1) eller annet injeksjon materiale, Hamilton sprøyter til. For å redusere sannsyn...

<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. 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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. 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Retroductal injeksjon er avgjørende for lokaliserte narkotika-leveranser til det salivary kjortelen. Denne teknikken har programmer i screening terapeutiske agenter for betingelser inkludert Sjøgrens syndrom og RIH9,10,28. Direkte narkotika-leveranser i SMG via retroductal injeksjon gir en viktig fordel over systemisk administrasjon i dens potensial for å redusere off-målet effekter, inkludert immun aktivisering<sup class="x...

Salivary kjortelen dysfunksjon har mange etiologies, inkludert Sjögrens syndrom, en autoimmun mediert tap av funksjonelle sekretoriske vev og stråling indusert hyposalivation (RIH), et felles sequella hode og nakke kreft strålebehandling1. Tap av salivary funksjon på grunn av forholdene predisponerer personer til muntlig og systemisk, tannråte, fordøyelsessystemet og svelge dysfunction, talefeil og store depresjonen1,2,3. Som et resultat, lider livskvalitet betraktelig, med intervensjonene begrenset til palliation av symptomer heller enn kur4. Undersøke romanen terapi i vivo, er det interessant å administrere bioaktive forbindelser direkte til det salivary kjortelen.
Retroductal injeksjon er en nyttig metode for å levere bioaktive forbindelser direkte til spyttkjertler og teste effekten i sykdom, skade, eller under normalt vev homeostase. Tre store spyttkjertler er Parodisk (PG), submandibular (SMG) og sublingvaltabletter (SLG), alle som Tom i munnhulen gjennom excretory kanaler. Anatomien i murint SMG tillater direkte tilgang gjennom cannulation av Wharton's duct, i gulvet i munnen under tungen5. Etter den cannulation, solvated legemidler kan administreres direkte til SMG. Etter retroductal levering, ekstra kjertel Diffusjon er begrenset av omkringliggende vev kapsel som regulerer utveksling av materiale med omkringliggende strukturer6. SMG sine rør er tilsvarende strukturert hos mennesker og rutinemessig tilgang under SMG kirurgi og sialoendoscopy7. Hos mennesker og mus er PG også tilgjengelig via Stensens rør i bukkal mucosa8.
I murine modeller av RIH, er SMG retroductal injeksjon brukt til å levere therapeutics inkludert vekstfaktorer, Primærceller, adenoviral vektorer, cytokiner og antioksidant forbindelser å modulere cellulær respons til skade og redusere den resulterende vev skade5,9,10,11,12,13,14,15,16. Mest bemerkelsesverdige klinisk suksess retroductal injeksjon er administrasjon av adenoviral vektor til direkte uttrykk for en vann kanal (Aquaporin 1; AQP1) hos pasienter etter stråling for hode og nakke kreft17.
Vi har tidligere utviklet og vises effekten av en retroductally injisert polymere hydrogenion-siRNA system å beskytte salivary kjortelen funksjonen RIH11,18,19,20. Som en forlengelse av vår siste arbeid, her viser vi vår protokoll for retroductal SMG injeksjon bruker en fluorescently merket hydrogenion (NP) lasting og levere ellers dårlig løselig narkotika21,22, 23.
Vi har syntetisert NP fra en diblock kopolymer består av poly (styren-alt-maleic anhydride)-b-poly(styrene) (PSMA) gjennom reversibel tillegg kjeden fragmentering (FLÅTEN) polymerization, som beskrevet tidligere21. Gjennom løsemiddel utveksling samle disse polymerer spontant selv inn micelle NP strukturer med en hydrofobe interiør og hydrofile utvendig21. NPs er merket med Texas-rød fluorophore å tillate kontroll av NP leveres til kjertler uten å ofre dyr. Live dyr bildebehandling og SMG immunohistochemistry vises på 1 h og 1 dag etter injeksjon.
Dette oppdatert og reproduserbar cannulation protokollen bør aktivere andre til å oppnå retroductal injeksjon. Vi forventer at denne raffinerte teknikken blir avgjørende for i vivo studier og terapeutiske utvikling24,25.

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			<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>
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		<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>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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retroductal nanoparticle injection to the murine submandibular gland

To felles mål av salivary kjortelen therapeutics er forebygging og helbredelse av vev dysfunksjon etter enten autoimmune eller stråling skader. Ved å levere lokalt bioaktive forbindelser til spyttkjertler, kan større vev konsentrasjoner trygt oppnås versus systemisk administrasjon. Videre av vevet kan effekter fra ekstra kjertel opphopning av materiale dramatisk nedsatte. I denne forbindelse er retroductal injeksjon en brukte metoden for å undersøke både salivary kjortelen biologi og patofysiologi. Retroductal administrasjonen av vekstfaktorer, Primærceller, adenoviral vektorer og små molekyl narkotika har vist seg å støtte kjertel-funksjonen i innstillingen for skade. Vi har tidligere vist effekten av en retroductally injisert hydrogenion-siRNA strategi for å opprettholde kjertel funksjon etter bestråling. Her, en svært effektiv og reproduserbar metode administrere nanomaterialer murine submandibular kjertel gjennom Wharton's duct er detaljert (figur 1). Vi beskriver tilgang til munnhulen og skissere trinnene nødvendig å cannulate Wharton ledningskanal, med ytterligere observasjoner som kvalitetskontroller i hele denne prosedyren.

Retroductal injeksjon kan brukes til å administrere NPs murine SMG (figur 1). Her, leverer vi 50 µg PSMA NPs merket med Texas røde fluorophore.
Riktig plassering av musen gir lettvint tilgang og visualisering av gulvet i munnen (figur 2A-B). De submandibular papillae identifiseres som to kjøttfulle utstikkende deler under tungen. Etter cannulation (...

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

Lokale narkotika-leveranser til submandibular kjertler er av interesse i forståelse salivary kjortelen biologi og utviklingen av romanen therapeutics. Vi presenterer en oppdatert og detaljert retroductal injeksjon protokoll, utformet for å forbedre levering nøyaktighet og eksperimentelle reproduserbarhet. Programmet presenteres her er levering av polymere nanopartikler.

Retroductal Nanoparticle injeksjon til Murine Submandibular kjertel

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

Research

JoVE Journal

Bioengineering

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

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 ...

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 ...

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

Et hormon-responsive 3D Culture Model of Human melkekjertlene Epitel

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

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Microfluidic Buffer Exchange for Forstyrrelsesfri Micro / partikler Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

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

Fastsettelse av Zeta Potensielle via nanopartikkel trans Hastigheter gjennom en fleksibel membran nanopore: Ved hjelp av DNA-modifisert Partikler som et eksempel

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

Intraductal Levering til kanin melkekjertlene

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

Et integrert System eksternt utløse intracellulær signaltransduksjon av Upconversion Nanoparticle-mediert Kinase Photoactivation

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

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Lipidico Injection Protocol for Serial Crystallography Målinger på den australske Synchrotron

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

Transplantasjon av en 3D Bioprinted Patch i en Murine Modell av hjerteinfarkt

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

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

Injiserbare supramolecular polymer-nanopartikkel hydrogeler for celle- og legemiddelleveringsapplikasjoner

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...