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

Forskning rapporteret i denne publikation blev støttet af den nationale Dental-Institut og Craniofacial forskning (NIDCR) og National Cancer Institute (NCI) af National Institutes of Health under Award antallet R56 DE025098, UG3 DE027695 og F30 CA206296. Indholdet er udelukkende ansvarlig for forfattere og repræsenterer ikke nødvendigvis de officielle synspunkter af National Institutes of Health. Dette arbejde blev også støttet af NSF DMR 1206219 IADR Innovation i Oral Care Award (2016).
Vi vil gerne takke Jayne Gavrity for hendes hjælp i udfører IVIS eksperimenter. Vi vil gerne takke Karen Bentley for hendes input og bistand i udførelsen af EM. Vi vil gerne takke Pei-Lun Weng for hans hjælp med IHC. Vi vil gerne takke Matthew Ingalls for sin bistand i figur forberedelse. Vi vil gerne takke Dr. Elaine Smolock og Emily Wu for kritisk læsning af dette manuskript.

Alle i vivo procedurer skitseret nedenfor blev godkendt af universitetet Udvalget om dyret ressourcer på University of Rochester, Rochester, NY.
1. forberedelse
<ol><li>Brug 32G intrakraniel kateter slanger med wire inset, skåret 3 cm af slangen til at danne en skrå ende, ca 45° med den lange akse. Bekræfte, at ledningen er mindst 1 cm længere end slangen.</li>
	<li>Indlæse 50 µL af PSMA nanopartikler løsning (figur 1), eller andet materiale, i...

<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. 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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 injektion er kritisk for lokaliserede medicinafgivelse til spytkirtel. Denne teknik har applikationer i screening terapeutiske agenter for betingelser herunder Sjögrens syndrom og RIH9,10,28. Direkte medicinafgivelse i SMG via retroductal injektion giver en afgørende fordel frem for systemisk administration i dens potentiale til at nedbringe ud-target effekter, herunder immun aktivering11. E...

Spytkirtel dysfunktion har mange etiologies, herunder Sjögrens syndrom, en autoimmun medieret tab af funktionelle sekretoriske væv og stråling induceret hyposalivation (RIH), en fælles sequella på hoved og hals kræft strålebehandling1. Tab af spyt funktion på grund af enten tilstand disponerer enkeltpersoner til mundtlige og systemisk infektion, huller i tænderne, fordøjelsessystemet og synke dysfunktion, tale værdiforringelse og store depression1,2,3. Som et resultat, lider livskvalitet væsentligt, med indgreb begrænset til palliation af symptomer i stedet kur4. For at undersøge nye behandlingsformer i vivo, er det af interesse at administrere bioaktive stoffer direkte til spytkirtel.
Retroductal injektion er en værdifuld metode til at levere bioaktive stoffer direkte til spytkirtlerne og teste effekten i sygdom, skade, eller under normale væv homøostase. De tre store spytkirtler er parotideale (PG), de submandibulære (SMG) og sublinguale (SLG), alle som Tom ind i mundhulen gennem ekskretionsorganerne kanaler. Anatomi af murin SMG tillader direkte adgang gennem cannulation af Whartons kanalen, ligger i gulvet i munden under tungen5. Efter cannulation, solvated medicin kan administreres direkte til SMG. Efter retroductal levering, ekstra glandulær diffusion er begrænset af den omkringliggende væv kapsel, som regulerer udvekslingen af materiale med omgivende strukturer6. SMG og dens kanalen er ligeledes struktureret i mennesker, og rutinemæssigt adgang til under SMG kirurgi og sialoendoscopy7. I mennesker og mus er PG ligeledes tilgængelige via Stensens kanal i buccale slimhinde8.
I murine modeller af RIH, er SMG retroductal injektion blevet brugt til at levere therapeutics herunder vækstfaktorer, primærelementer, adenoviral vektorer, cytokiner og antioxidant forbindelser at modulere den cellulære reaktion på skade, og reducere den resulterende væv skader5,9,10,11,12,13,14,15,16. Den mest bemærkelsesværdige kliniske succes af retroductal injektion er administrationen af adenoviral vektor til direkte udtryk for en vand kanal (Aquaporin 1; AQP1) hos patienter efter stråling for hoved og hals kræft17.
Tidligere har vi udviklet og vist effekten af en retroductally indsprøjtet polymere nanopartikel-siRNA system at beskytte spytkirtel funktion fra RIH11,18,19,20. Som en forlængelse af vores tidligere arbejde, her, vise vi vores protokol for retroductal SMG injektion ved hjælp af en fluorescently mærket nanopartikel (NP), som er i stand til at indlæse og levere ellers tungtopløselige stoffer21,22, 23.
Vi har syntetiseret NP fra diblock copolymer bestående af poly (styren-alt-maleinsyre anhydride)-b-poly(styrene) (PSMA) via reversible tilføjelse kæde fragmentering (TØMMERFLÅDE) polymerisering, som tidligere beskrevet21. Gennem udveksling af opløsningsmidler samles disse polymerer spontant selvstændige til micelle NP strukturer med en hydrofobe interior og hydrofile udvendige21. De nationale parlamenter er mærket med Texas-rød fluorophore at tillade kontrol af NP levering i kirtler uden at ofre dyr. Levende dyr imaging og SMG Immunhistokemi er vist på 1 t og 1 dag efter injektionen.
Denne opdaterede og reproducerbare cannulation protokol skal gøre det muligt for andre at nå retroductal injektion. Vi forventer, at denne raffineret teknik vil blive afgørende for i vivo undersøgelser og terapeutiske udvikling24,25.

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			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
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			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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retroductal nanoparticle injection to the murine submandibular gland

To fælles mål af spytkirtel therapeutics er forebyggelse og helbredelse af væv dysfunktion efter enten autoimmun eller stråling skader. Ved at levere lokalt bioaktive stoffer til spytkirtlerne, kan større væv koncentrationer opnås sikkert versus systemisk administration. Derudover off målvæv kan effekter fra ekstra glandulær ophobning af materiale drastisk reduceres. I denne henseende er retroductal injektion en almindeligt anvendt metode for at undersøge både spytkirtel biologi og patofysiologi. Retroductal administration af vækstfaktorer, primærelementer, adenoviral vektorer og lille molekyle narkotika har vist sig at støtte kirtel funktion i fastsættelsen af skade. Vi har tidligere vist effekten af en retroductally indsprøjtet nanopartikel-siRNA strategi at opretholde kirtel funktion efter bestråling. Her, en yderst effektiv og reproducerbar metode til at administrere nanomaterialer til murine submandibulære kirtel gennem Whartons kanalen er detaljeret (figur 1). Vi beskriver adgang til mundhulen og skitsere de nødvendige skridt til at cannulate Wharton kanalen, med yderligere observationer tjener som kvalitetskontrol under hele proceduren.

Retroductal injektion kan bruges til at administrere NPs til den murine SMG (figur 1). Her leverer vi 50 µg PSMA NPs mærket med Texas rød fluorophore.
Korrekt placering af musen tillader facile adgang og visualisering af gulvet i munden (figur 2A-B). De submandibulære papiller er identificeret som to kødfulde fremspring under tungen. Efter cannulat...

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 medicinafgivelse til de submandibulære kirtler er af interesse i forståelse spytkirtel biologi og for udviklingen af nye lægemidler. Vi præsenterer en opdateret og detaljerede retroductal injektion protokol, designet til at forbedre leveringen nøjagtighed og eksperimenterende reproducerbarhed. Programmet præsenteres heri er levering af polymert nanopartikler.

Retroductal nanopartikel injektion til Murine submandibulære kirtel

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

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Bioengineering

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