Questo studio è stato finanziato dalla Fondazione Mcihael J. Fox per la ricerca 2011 Rapid Response Innovations di Parkinson Awards Program. I finanziatori hanno avuto alcun ruolo nel disegno dello studio, la raccolta e l&#39;analisi dei dati, la decisione di pubblicare, o preparazione del manoscritto.

Prima dell&#39;intervento assicurarsi tutte le procedure da fare sono approvati dalla IACUC e qualsiasi autorità etiche o giuridiche addizionali e utilizzare crudeli pratiche di trattamento degli animali. Questo include l&#39;utilizzo di condizioni di chirurgia sterili, anestetizzare il mouse utilizzando IACUC metodo approvato, lubrificanti occhi topi con vet unguento durante l&#39;intervento chirurgico, e fornire assistenza post-chirurgica. Non procedere con la chirurgia se c&#39;è qualche problema se aspetti della procedura sono approvati. Tutte le procedure eseguite nel presente documento sono state approvate dalla Università Institutional Animal Care ed uso comitato di Boston. 1. Preparazione degli animali e Forniture chirurgici <ol><li> Autoclavare tutto strumento chirurgico che verrà utilizzato durante l&#39;intervento chirurgico. </li><li> Assicurarsi che tutte le tecniche che devono essere eseguite sono approvati dalle agenzie di regolamentazione degli animali. </li></ol> 2. Raccolta della mucosa Graft <ol><li> Ha scelto un topo geneticamente identico di età simile comeil mouse sperimentale e eutanasia in un metodo approvato IACUC (qui: asfissia isoflurane seguita da dislocazione cervicale). </li><li> Utilizzando forbici chirurgiche, togliere la pelle intorno alla regione nasale della testa del mouse esporre il cranio. </li><li> Con un martello pneumatico, volume con tre linee di cui due laterali fiancheggiano la regione nasale e un terzo in linea con gli occhi che collega le due linee perpendicolarmente. </li><li> Drill-down ventralmente al fine di separare il setto nasale dal tessuto circostante. Un percorso più ampio eviterà danni alla membrana mucosa tuttavia sarà anche rendere più difficile isolare la membrana. Si raccomanda un taglio stretto vicino al linea mediana. </li><li> Usare le forbici per tagliare il setto libero da qualsiasi tessuto aderito ad essa e conservarla in una soluzione salina sterile. A questo punto l&#39;innesto può essere pulito per rimuovere qualsiasi tessuto collegato. La situazione ideale è avere membrane mucose integre esposti su entrambi i lati del setto cartilagine. Uno grpoppiera può fornire membrana per due topi disponibile la superficie della membrana è sufficiente a coprire i siti craniotomia. Si raccomanda che l&#39;innesto viene utilizzato il più rapidamente possibile e il ricercatore procede alla fase 3 non appena l&#39;innesto è isolato. </li></ol> 3. Impianto chirurgico di innesto mucoso <ol><li> Utilizzo di procedure standard, asettiche murini chirurgici, anestetizzare e montare un topo nella cornice stereologica. Utilizzare circa il 2% isoflurano in ossigeno puro utilizzando una macchina di anestesia roditore. </li><li> Immobilizzare il mouse in un apparato stereotassico con orecchio bar e un supporto naso. Applicare una pomata oftalmica per gli occhi e strofinare il cuoio capelluto con Betadine e il 75% di etanolo per tre turni. Utilizzando sia le forbici o un regolatore dei capelli, rimuovere il pelo sulla testa. Esporre il cranio con una lametta e livellare la testa. Eseguire una craniotomia sopra la posizione del cervello che verrà dosato. Per esempio, quando il targeting striato tagliare un 1,25 millimetridiametro del foro circolare nel cranio (centrato a AP: 1,00 mm, ML: 0,88 mm) utilizzando un martello pneumatico. Bagnare la zona forata con soluzione fisiologica sterile e utilizzare una lama di rasoio per rimuovere il cranio. </li><li> Rimuovere con cautela la dura con la punta di un ago. Inoltre, questo può essere realizzato applicando una quantità minima di tessuto adesivo alla superficie durale umida. Una volta che questo strato è indurito, il movimento laterale con una punta lametta può essere usato per rimuovere la membrana. </li><li> Posizionare la membrana mucosa al di sopra della superficie del cervello avendo cura estrema per mantenere il lato epiteliale rivolto lontano dalla ferita. Questo è fatto meglio trasferendo l&#39;intero setto sulla superficie del cranio adiacente al sito craniotomia con le pinzette. Con la punta di un paio di forbici chirurgiche, tirare fuori la membrana della cartilagine e sulla superficie del cranio e cervello. Non lasciate che la membrana asciugare o toccare con qualsiasi materiale assorbente. L&#39;innesto dovrebbe generosamente sovrapporsi tutti i bordi ossee del craniotomia site. </li><li> Coprire l&#39;innesto con un pezzo sterile di nitrile. Questo agisce per impedire l&#39;adesione della pelle per l&#39;innesto durante la guarigione. Il nitrile deve essere abbastanza grande da coprire l&#39;intera membrana mucosa. Tagliare eccessivo membrana se necessario. Evitare qualsiasi movimento del nitrile una volta entrato in contatto con l&#39;innesto. </li><li> Chiudere la pelle con un running 5-0 sutura sterile e lasciare il mouse recuperare per 3-7 giorni prima di procedere alla fase successiva. Fare attenzione a non perturbare la barriera nitrile o l&#39;innesto di mucosa durante la chiusura della pelle. </li></ol> 4. Amministrazione di dosaggio Soluzione <ol><li> Dopo aver assicurato il topo anestetizzato nel frame stereotassico, tagliare la sutura con le forbici e togliere la pelle in eccesso intorno al cranio. </li><li> Rimuovere la barriera nitrile e pulire la superficie del cranio. Usare tamponi salini e cotone sterile per pulire la zona fino a quando l&#39;innesto è visibile. Può essere necessario tagliare l&#39;innesto con un rasoio se ha assunto dimensioni desired superficie. </li><li> Se l&#39;esperimento sarà più lungo di un paio di giorni, è saggio impiantare almeno due viti cranio a rafforzare l&#39;impianto testa. </li><li> Posizionare il ben sopra l&#39;innesto in modo che i bordi sono in contatto con il cranio. Applicare l&#39;adesivo cianoacrilato all&#39;incrocio tra il bene e il cranio. Riempire il pozzetto con soluzione salina sterile e verificare che non vi siano perdite. Wells sono costituiti da aghi di siringa taglio. </li><li> Applicare cemento osseo sul cranio per fissare il bene in posizione. </li><li> Rimuovere la soluzione fisiologica dal pozzo con una pipetta. Lavare le ben diverse volte per verificare che l&#39;adesivo non è trapelato trovi Aggiungi la soluzione desiderata; in questo caso viene utilizzato 50 ml di destrano fluorescente. Si prevede che i composti idrosolubili di una polarità simile si comportano come destrano. Consegna di composti idrofobi o sospensione non è stato esplorato con questo metodo. </li><li> Chiudere la parte superiore del pozzo utilizzando un pezzo circolare di nitrile fissato altop con cianoacrilato. Controllare che l&#39;adesivo non venga a contatto con il contenuto dei pozzetti. </li></ol> 5. Analisi dei Transmucosal consegna <ol><li> Dopo la quantità desiderata di tempo è passato, anestetizzare il mouse e scambiare il contenuto dei pozzetti con una soluzione di colorante blu di Evan. Questo colorante è utilizzato per verificare che l&#39;innesto era intatto. </li><li> Dopo 30 minuti, anestetizzare il mouse pesantemente, rimuovere la soluzione colorante, e eutanasia mediante decapitazione. </li><li> Rimuovere manualmente l&#39;impianto e rimuovere il cervello con le forbici chirurgiche. Fare attenzione a tenere l&#39;innesto in posizione. </li><li> Una volta rimosso, Flash-congelare il cervello in una soluzione di isobutano raffreddata in un bagno di ghiaccio secco. </li><li> Incorporare il cervello in Ottimale taglio Temperatura soluzione (OCT) e la fetta a 50 micron. </li><li> Collocare le fette desiderati direttamente su un vetrino da microscopio. </li><li> Immagine della sezione utilizzando un microscopio a fluorescenza non appena la soluzione Ottobre è asciugato. </li></ol>

<ol>
	<li>Cardoso, F. L., 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=Looking+at+the+blood-brain+barrier:+Molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches.</a> Brain Res. Rev. 64, 328-363 (2010).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transport+across+the+blood-brain+barrier.">Drug transport across the blood-brain barrier.</a> J. Cereb. Blood Flow Metab. 32, 1959-1972 (2012).</li><li>Chen, Y., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+methods+for+delivery+of+drugs+across+the+blood-brain+barrier.">Modern methods for delivery of drugs across the blood-brain barrier.</a> Adv. Drug Deliv. Rev. 64, 640-665 (2012).</li><li>Cheng, F. -. 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=Glial+cell+line-derived+neurotrophic+factor+protects+against+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine+(MPTP)-induced+neurotoxicity+in+C57BL/6+mice.">Glial cell line-derived neurotrophic factor protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in C57BL/6 mice.</a> Neurosci. Lett. 252, 87-90 (1998).</li><li>Grondin, 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=controlled+GDNF+infusion+promotes+structural+and+functional+recovery+in+advanced+parkinsonian+monkeys.">controlled GDNF infusion promotes structural and functional recovery in advanced parkinsonian monkeys.</a> Brain. 125, 2191-2201 (2002).</li><li>Kirik, 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=Localized+striatal+delivery+of+GDNF+as+a+treatment+for+Parkinson+disease.">Localized striatal delivery of GDNF as a treatment for Parkinson disease.</a> Nat. Neurosci. 7, 105-110 (2004).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+and+gene+targeting+to+the+brain+with+molecular+trojan+horses.">Drug and gene targeting to the brain with molecular trojan horses.</a> Nat. Rev. Drug Discov. 1, 131-139 (2002).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+delivery+of+protein+and+non-viral+gene+therapeutics+with+molecular+Trojan+horses.">Blood-brain barrier delivery of protein and non-viral gene therapeutics with molecular Trojan horses.</a> J. Control. Release. 122, 345-348 (2007).</li><li>Bellavance, M. -. 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=Recent+advances+in+blood-brain+barrier+disruption+as+a+CNS+delivery+strategy.">Recent advances in blood-brain barrier disruption as a CNS delivery strategy.</a> AAPS J. 10, 166-177 (2008).</li><li>Merkus, F. H. M., Berg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+nasal+drug+delivery+bypass+the+blood-brain+barrier.">Can nasal drug delivery bypass the blood-brain barrier.</a> Drugs R. D. 8, 133-144 (2007).</li><li>Dhuria, S. 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=Intranasal+delivery+to+the+central+nervous+system:+Mechanisms+and+experimental+considerations.">Intranasal delivery to the central nervous system: Mechanisms and experimental considerations.</a> J. Pharm. Sci. 99, 1654-1673 (2010).</li><li>Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+drug+delivery-possibilities,+problems+and+solutions.">Nasal drug delivery-possibilities, problems and solutions.</a> J. Control. Release. 87, 187-198 (2003).</li><li>Craft, 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=Intranasal+insulin+therapy+for+alzheimer+disease+and+amnestic+mild+cognitive+impairment:+A+pilot+clinical+trial.">Intranasal insulin therapy for alzheimer disease and amnestic mild cognitive impairment: A pilot clinical trial.</a> Arch. Neurol. 69, 29-38 (2012).</li><li>Freiherr, 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=Intranasal+insulin+as+a+treatment+for+Alzheimer's+Disease:+A+review+of+basic+research+and+clinical+evidence.">Intranasal insulin as a treatment for Alzheimer's Disease: A review of basic research and clinical evidence.</a> CNS Drugs. 27, 505-514 (2013).</li><li>Gill, S. 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=Direct+brain+infusion+of+glial+cell+line-derived+neurotrophic+factor+in+Parkinson+disease.">Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease.</a> Nat. Med. 9, 589-595 (2003).</li><li>Love, 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=Glial+cell+line-derived+neurotrophic+factor+induces+neuronal+sprouting+in+human+brain.">Glial cell line-derived neurotrophic factor induces neuronal sprouting in human brain.</a> Nat. Med. 11, 703-704 (2005).</li><li>Antunes, M. 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=Murine+nasal+septa+for+respiratory+epithelial+air-liquid+interface+cultures.">Murine nasal septa for respiratory epithelial air-liquid interface cultures.</a> BioTechniques. 43, 195-204 (2007).</li><li>Bleier, B. 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=Permeabilization+of+the+blood-brain+barrier+via+mucosal+engrafting:+implications+for+drug+delivery+to+the+brain.">Permeabilization of the blood-brain barrier via mucosal engrafting: implications for drug delivery to the brain.</a> PLoS ONE. 8, (2013).</li><li>Bernal-Sprekelsen, 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=Closure+of+cerebrospinal+fluid+leaks+prevents+ascending+bacterial+meningitis.">Closure of cerebrospinal fluid leaks prevents ascending bacterial meningitis.</a> Rhinology. 43, 277-281 (2005).</li><li>Bleier, B. 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=Laser-assisted+cerebrospinal+fluid+leak+repair:+An+animal+model+to+test+feasibility.">Laser-assisted cerebrospinal fluid leak repair: An animal model to test feasibility.</a> Otolaryngol. Head Neck Surg. 137, 810-814 (2007).</li></ol>

Benjamin S. Bleier MD è inventore di piombo di metodi di copertura brevettuale provvisori di somministrazione di farmaci per il sistema nervoso centrale.

Il passo più difficile della procedura qui descritta è la riuscita del trasferimento di una membrana mucosa adeguate dimensioni sulla superficie del cervello. Questo passaggio è notevolmente semplificata se il setto nasale raccolto è abbastanza grande e ben puliti. Se la parte ventrale del setto viene troncato, un nuovo innesto deve essere ottenuta. L&#39;angolo di perforazione deve essere perpendicolare alla testa mouse per assicurare che la membrana mucosa non è danneggiato dal trapano. Se viene preso un percorso...

Il trattamento della malattia neurologica e psichiatrica è fortemente ostacolato dalla presenza della barriera ematoencefalica (BBB) ​​che impedisce oltre il 95% di tutti i potenziali agenti farmaceutici di raggiungere il sistema nervoso centrale 1-3. Ad esempio, derivazione gliale fattore neurotrofico (GDNF) ha dimostrato di essere efficace nel trattamento della malattia di Parkinson quando iniettato direttamente nel cervello, tuttavia è inefficace quando consegnato sistemica perché non può penetrare la BBB 4-6. Numerosi avvicinato sono stati sviluppati per cercare di aggirare questo problema. Miglioramento della consegna sistematica di neurotheraputics è stata dimostrata utilizzando coniugati contenenti anticorpi selettivi per le proteine ​​di trasporto localizzate sull&#39;endotelio capillare cervello; tuttavia questo metodo non ha dimostrato di essere applicabile per una vasta gamma di farmaci 7,8. Inoltre, l&#39;apertura osmotica della BBB è stato usato clinicaalleato, tuttavia questo metodo soffre di dosaggio di farmaci sistemici rispetto a una consegna più diretto alla regione del cervello di interesse 9. Notevole sforzo è stato messo in ottimizzazione consegna transnasale nella speranza di mira direttamente il cervello 10-12. Sebbene un certo successo è stato raggiunto, risultati conclusivi sono stati ottenuti solo per farmaci che posseggono recettori endogeni, come l&#39;insulina 13,14. Inoltre il meccanismo di consegna transnasale è stato controverso con prove che suggeriscono ingresso indiretto nel cervello attraverso olfattivo neurone assorbimento o attraverso il flusso sanguigno 11. , Consegna transcranica diretta utilizzando cateteri impiantabili è stato raggiunto, tuttavia questa procedura è altamente invasiva e associata a numerose complicanze 15,16. Ad oggi, non esiste un metodo generale, minimamente invasivo per fornire composti ad alto peso molecolare nel cervello. Qui presentata è una procedura chirurgica murinoche crea un&#39;interfaccia semipermeabile con il cervello. Questo si ottiene innestando un espianto membrana mucosa 17 su un difetto craniotomia chirurgica in un topo. Utilizzando questa procedura è stato dimostrato che i composti solubili fino a 500 kDa possono essere trasportati nel sistema nervoso centrale (direttamente nel parenchima cerebrale e nel liquido cerebrospinale) sia un tempo e peso molecolare di modo dipendente 18. Questo metodo di aggirare la BBB è un modello per la riparazione dei difetti di base del cranio negli esseri umani che utilizza innesti di mucosa vascolarizzati per riparare buchi nel cranio dopo la chirurgia endoscopica transnasale 19,20.

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			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:220px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">mice</td>
			<td style="width:220px;">Taconic</td>
			<td>C57BL/6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane</td>
			<td style="width:220px;">Piramal Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Fine Scissors</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91461-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">pneumatic drill</td>
			<td style="width:220px;">MTI Dental</td>
			<td style="width:119px;">333-CB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">drill bit</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">forcepts</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">0.9% Sodium Chloride Injection USP</td>
			<td style="width:220px;">Abbott Laboratories</td>
			<td>4925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Polystyrene Detri Dish</td>
			<td style="width:220px;">Fisher</td>
			<td>08-757-12</td>
			<td>for temporarily storing graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Sterilizer</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen/Isoflurane System</td>
			<td style="width:220px;">SurgiVet</td>
			<td>V720100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Control System</td>
			<td style="width:220px;">Physitemp</td>
			<td style="width:119px;">TCAT-2LV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Instrument</td>
			<td style="width:220px;">KOPF</td>
			<td style="width:119px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye Ointment</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Electric shaver</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Cotton-Tipped Applicators</td>
			<td style="width:220px;">Fisher</td>
			<td>23-400-106</td>
			<td></td>
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			<td height="21" style="height:21px;">7.5% Provadone Iodine</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>Betadine surgical scrub</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical Blade Stainless</td>
			<td style="width:220px;">Feather</td>
			<td style="width:119px;">2976#10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Scalpel Handle - #3</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% Hydogen Peroxide</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>for cleaning the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetbond Tissue Adhesive</td>
			<td style="width:220px;">3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">needles</td>
			<td>Becton, Dickinson and Company</td>
			<td>305176</td>
			<td>needle tip cut off and used as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">nitrile gloves</td>
			<td style="width:220px;">Denville Scientific Inc</td>
			<td>G4162</td>
			<td>for well closure and protection of graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Nylon Suture Thread</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Halsey Needle Holder</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cyanoacrylate adhesive</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>commecially available super glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Kit, 1 lb, Pink Opaque</td>
			<td style="width:220px;">Stoelting</td>
			<td>51458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isobutane (2-methylbutane)</td>
			<td style="width:220px;">Aldrich</td>
			<td>M32631</td>
			<td>for dry ice bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">dry ice</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

heterotopic mucosal engrafting procedure for direct drug delivery to the brain in mice

Consegna di terapeutica nel cervello è ostacolato dalla presenza della barriera ematoencefalica (BBB) ​​che limita il passaggio di composti polari e ad alto peso molecolare dal flusso sanguigno e nel tessuto cerebrale. Alcuni successi consegna diretta negli esseri umani è stato raggiunto tramite l&#39;impianto di cateteri transcraniali; Tuttavia questo metodo è altamente invasiva e associati con numerose complicazioni. Un&#39;alternativa meno invasiva sarebbe dosare il cervello attraverso una impiantato chirurgicamente, membrana semipermeabile come la mucosa nasale che viene utilizzato per riparare difetti base cranica seguente endoscopica transnasale rimozione chirurgica del tumore nell&#39;uomo. Trasferimento Drug se questa membrana potrebbe effettivamente bypassare la BBB e diffondere direttamente nel cervello e nel liquido cerebrospinale. Ispirato da questo approccio, un approccio chirurgico nei topi è stata sviluppata che utilizza una membrana mucosa del setto donatore innestato su un difetto extracranica BBB chirurgico. Questo modello è stato dimostrato efficaceconsentire il passaggio di composti ad alto peso molecolare nel cervello. Dal momento che numerosi farmaci candidati sono in grado di attraversare la BBB, questo modello è utile per l&#39;esecuzione di sperimentazione preclinica di nuove terapie per le malattie neurologiche e psichiatriche.

Ottenere un grande abbastanza setto nasale espianto è fondamentale per le fasi successive. Questo può essere realizzato mediante foratura nella posizione sul cranio del topo donatore mostrato in Figura 1a. Tagliando lungo questo percorso produrrà un espianto di sufficiente dimensione come mostrato in Figura 1b. Se la profondità non è abbastanza profonda, l&#39;innesto sarà troncata e sarà difficile ottenere una grande membrana sufficiente a coprire la superficie del cervello. Per...

Watch this Scientific Journal Video about Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice at JoVE.com

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Un modello murino di ricostruzione base cranica endoscopica umana sviluppato che crea un&#39;interfaccia semipermeabile tra il cervello e il naso utilizzando innesti mucose nasali. Questo metodo permette ai ricercatori di studiare consegna al sistema nervoso centrale di alto peso molecolare terapeutica che altrimenti esclusi dalla barriera emato-encefalica, se somministrati per via sistemica.

Eterotopico mucosa engrafting Procedura di Drug Delivery diretto al cervello nei topi

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

heterotopic-mucosal-engrafting-procedure-for-direct-drug-delivery-to

Research

JoVE Journal

Medicine

10.7K Views.  Boston University.  Un modello murino di ricostruzione base cranica endoscopica umana sviluppato che crea un'interfaccia semipermeabile tra il cervello e il naso utilizzando innesti mucose nasali. Questo metodo permette ai ricercatori di studiare consegna al sistema nervoso centrale di alto peso molecolare terapeutica che altrimenti esclusi dalla barriera emato-encefalica, se somministrati per via sistemica. 

Video: Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmotica Drug Delivery per arti posteriori ischemico e la perfusione dei vasi con Microfil per Micro-Tomografia Computerizzata Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Ricerca

Medicina

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Lento rilascio Drug Delivery attraverso Elvax 40W al Rat Retina: Implicazioni per il trattamento di condizioni croniche

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Diretta mouse Trauma / Burn modello di ossificazione eterotopica

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Eterotopico cervicale trapianto cardiaco nei topi

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminale Drug Delivery per il mouse fistola artero-venosa endotelio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Metodo ottimizzato di LC-MS/MS per l'analisi di High-throughput di campioni clinici di Ivacaftor, suoi principali metaboliti e Lumacaftor nei fluidi biologici di pazienti affetti da fibrosi cistica

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Veicolazione Trans-timpanico per il trattamento dell'ototossicità

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

In Vivo Photolabeling delle cellule del colon per valutare il potenziale migratorio delle cellule ematopoietiche in topi neonatali

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Metodi innovativi per la somministrazione intranasale in anestesia per inalazione per valutare il naso--cervello Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...