Die in diesem Artikel beschriebene Arbeiten wurde durch ein NCI RO1 CA166907, NIDCD RO1-DC 002396 und RO3 DC011621 unterstützt.

Männliche Wistar Ratten in Übereinstimmung mit den National Institutes of Health Tier behandelt wurden verwenden Richtlinien und ein Protokoll vom südlichen Illinois Universität Schule von Medizin Laboratory Animal Care and verwenden Committee genehmigt. Auditory Brainstem Response (ABR) wurde an Ratten während der Narkose vor der Medikamentengabe und 72 h nach durchgeführt, um zu überprüfen, die Wirkung von Trans-Trommelfells Drug-Delivery.
1. auditory Brainstem Response (ABR)
Hinweis: ABR-Messungen wurden mit dem auditiven Test-Equipment und Software gesammelt. ABR steht für evozierte Potentiale oder Hochfrequenz-Wellen erzeugt durch den achten Hirnnerven (wave I und II) und andere höhere auditive Hirnstamm Strukturen einschließlich Anteroventral cochlear Nucleus (Welle III), seitlicher Lemniscus (Welle IV) und minderwertig Colliculus (Welle V). Diese Wellen unterscheiden sich je nach der Latenz. ABR Messungen wurden bereits12beschrieben.
<ol><li>Ratten mit einer Mischung aus 90 mg/kg Ketamin und 17 mg/kg Xylazin über intraperitoneale Injektion zu betäuben. Bestätigen Sie die Tiefe der Narkose über Zeh-Prise Reflex. Wenden Sie ophthalmologische Schmierung (oder Mineralöl Tropfen an) auf beiden Augen, die Augen vor dem Austrocknen zu verhindern und zu vermeiden Geschwürbildung während der Narkose</li>
	<li>Legen Sie die Ratte in Bauchlage auf ein Heizkissen (37 ° C) innerhalb einer kontrollierten akustischen Stand. Audiologie Prüfeinrichtungen befindet sich außerhalb und neben dem Stand.</li>
	<li>Edelstahl-Elektroden entsprechend einfügen: der Masseelektrode im hinteren Flanke, die positive Elektrode zwischen den beiden Ohren direkt oben auf dem Schädel und der negativen Elektroden unterhalb der Pinna jedes Ohr.</li>
	<li>Gelten Sie akustische Reize mit Hochfrequenz-Wandler als ein 5 ms Ton Burst auf 8, 16 und 32 kHz. Stimulus Intensitäten als Dezibel Schalldruck (dB SPL) zu bestimmen. Dies beginnt bei 10 dB SPL und 90 dB SPL mit einer Schrittweite von 10 dB erreicht. Die soliden Intensitäten zu einer maximalen Leistung von 90 dB SPL mit einer Impuls Präzision-Schallpegelmesser zu kalibrieren. Hinweis: Das Testergebnis gibt Aufschluss über die Integrität der periphere Hörorgan, der Cochlea und die vorgenannten auditiven Strukturen. Die Wellenformen werden innerhalb von 15 ms Impuls Eingabe generiert und die Latenz der Wellen hängt Wärmeleitung Zeit, die wiederum von drei kritischen Faktoren geregelt ist: Volumen des Gehirns, die Intensität und die Frequenz des Tones. Akustische evozierte Potentialen verzeichneten mit vom Hersteller mitgelieferten Software.</li>
	<li>Betrachten Sie die minimale Intensität, die zwei verschiedene Wellenformen (II und III) von 0,5 µV Amplitude prominent als Schwellenwert hervorrufen kann. Hinweis: Verschiebung der Schwelle entspricht der Differenz in der Schwelle nach Behandlung im Vergleich mit dem Schwellwert erhalten vor der Behandlung gemessen.</li>
</ol>2. Trans-Trommelfells Injektionen
<ol><li>Um das Medikament Trans-tympanically zu verwalten, legen Sie die Ratte in der linken seitlichen Dekubitus-Position.<ol>
<li>Einweg-Ohrtrichter 2,5 mm in den Gehörgang einsetzen.</li>
		<li>Positionieren Sie mit dem Einsatz eines OP-Bereichs der Spekula so, dass das Trommelfell sichtbar ist.</li>
		<li>Mit einem 29 X ½, 0,5 mL Insulin Spritze, 50 µL [R] - N - Phenyl Isopropyl Adenosin aufzustellen (R-PIA) Lösung (1 µM), 8-Cyclopentyl-1,3-Dipropylxanthine (DPCPX)-Lösung (3 µM) oder SiRNA-Lösung (0,9 µg), (5 Stück) zu injizieren.</li>
		<li>Verwenden der Spekula, die Nadel auf die minderwertige Frontzahnbereich das Trommelfell.</li>
		<li>Poke ein Loch durch die Membran mit der Nadel und verabreichen der Medikamente in 1.2.3 erwähnt. Lassen Sie die Ratte in dieser Position für 15 min ruhen. Hinweis: 50 µL sollte ausreichend Volumen hinter dem Trommelfell zu passen. Nach der Verabreichung sollte keine Flüssigkeit im Gehörgang sein.</li>
		<li>Legen Sie die Ratte in der rechten seitlichen Dekubitus positionieren und wiederholen Sie die Schritte 1.2.1 bis 1.2.5 für die Verwaltung in das andere Ohr.</li>
	</ol>
</li>
	<li>Legen Sie für Ratten intraperitoneal Cisplatin empfangen die Ratte in der Rückenlage auf ein Heizkissen bei 37 ° C.<ol>
<li>Mit einer 21 X 3/4 Schmetterling Nadel (Länge Schlauch 12"), Cisplatin (11 mg/kg, 1 mg/mL Lösung in sterilen Phosphat Puffer Kochsalzlösung [PBS]) aufzustellen.</li>
		<li>Über eine Spritzenpumpe, verwalten Sie Cisplatin (1 mg/mL) über intraperitoneale Injektion über 30 min. Hinweis: Für eine 250-g-Ratte würde das Volumen 2,75 mL mit einer Rate von etwa 0,1 mL / min. betragen.</li>
	</ol>
</li>
	<li>Auch die Tiefe der Narkose während dieser Verfahren zu verfolgen. Wenn Cisplatin Verwaltung abgeschlossen ist, legen Sie die Ratte zurück in den Käfig in Bauchlage, sicherstellen, dass gibt es nichts, ihre Atmung behindern.</li>
	<li>Überwachen Sie die Ratte bis vollständig erholt.</li>
</ol>(3) Cochlea Dissection und Entkalkung
<ol><li>Im Anschluss an die letzte ABR (nach 72 h) die Ratte mit einer Mischung aus 90 mg/kg Ketamin und 17 mg/kg Xylazin über intraperitoneale Injektion zu betäuben. Anästhesie mit einem Zeh-Prise Reflex zu bestätigen. Die Ratte über Enthauptung einschläfern.</li>
	<li>Sezieren Sie, Felsenbein bereits13beschrieben.</li>
	<li>4 % Paraformaldehyd in 1 x PBS-Lösung in einem 7-mL-Glas funkeln Fläschchen (vollständig bedeckt die Cochlea) entgegenbringen Sie Cochlea. Im Kühlschrank über Nacht bei 4 ° C. Entfernen Sie Paraformaldehyd zu und waschen Sie mit 1 X PBS bei Raumtemperatur.</li>
	<li>Entfernen Sie PBS zu und füllen Sie das Rohr vollständig zu, mit einer 120-mM-Lösung von EDTA (pH 7,3). Legen Sie auf ein Rotator bei Raumtemperatur und ermöglichen Sie Entkalkung für 2 bis 3 Wochen, täglich wechselnden die EDTA-Lösung zu.</li>
</ol>(4) Kryoschneiden
<ol><li>Nach dem Abschluss der Entkalkung komplett zu halten der Cochlea in den folgenden Lösungen (7 mL) für 24 h bei 4 ° C: 10 % Saccharose, 20 % Saccharose, 1:1 Mischung aus 20 % Saccharose und optimale Arbeitstemperatur (OCT) zusammengesetzte einbetten.</li>
	<li>Legen Sie frische OCT Verbindung zu füllen und vollständig bedecken die Cochlea in ein 15 x 15 mm x 5 mm Einweg Form einbetten. Richten Sie die Cochlea auf seiner Seite, so dass es parallel zum Boden der einbettenden Form, wie von Whitlon Et al.beschrieben. 14
</li>
	<li>Legen Sie die Form sofort auf Trockeneis, das Office-Anpassungstool zu festigen und über Nacht bei-80 ° C lagern.</li>
	<li>Tauchen Objektträger in 0,01 % Poly-L-Lysin bei Raumtemperatur für 30 min. entfernen die Folien nicht ausspülen und über Nacht trocknen lassen. Verwenden Sie diese Folien für Cryosections.</li>
	<li>Entfernen der Cochlea im Oktober aus dem-80 ° C Gefrierschrank und legen Sie es auf Trockeneis. Mit einem scharfen Mikrotom Klinge (L x b: 80 mm x 8 mm, Stärke 0,25 mm, Schnittwinkel von 34°), Abschnitt die OCT-Blöcke bei 10 µm mit einem Kryostaten bei-30 ° C. Legen Sie zwei Abschnitte pro Folie.</li>
	<li>Kühlen Sie Folien bei 4 ° C, wenn Sie fertig sind.</li>
</ol>5. Immunohistochemistry
<ol><li>Legen Sie die Folien in einen Glas-Objektträger Färbung Gericht auf einem Dia-Gestell. Füllen Sie die Schale mit 350 mL 1 X PBS und waschen Sie der Objektträger für 5 min bei Raumtemperatur dreimal.</li>
	<li>Die Folien aus der Schale entfernen und das Gebiet rund um das Gewebe mit einem trockenen Tuch, wobei Sie nicht auf das Gewebe trocknen trocknen.<ol>
<li>Mit einem flüssigen blockierende Stift, zeichnen Sie einen Kreis um den Gewebe-Abschnitt.</li>
	</ol>
</li>
	<li>Blockieren Sie das Gewebe für 1 h bei Raumtemperatur durch Zugabe von 150 µL blocking Lösung mit 10 % Normal (Esel) Serum, 1 % nichtionische Reinigungsmittel und 1 % BSA mit 1 X PBS-Puffer.</li>
	<li>Tippen Sie auf Ausschalten überschüssige blockierende Lösung und inkubieren Sie das Gewebe über Nacht bei 4 ° C in eine feuchte Kammer mit 150 µL Serum 10 % Normal (Esel), 0,1 % nichtionische Reinigungsmittel und primären Antikörper (siehe Hinweis unten) mit 1 X PBS-Puffer. Hinweis: Für die folgenden Antikörper wurden diese Verdünnungen verwendet: für Abbildung 2 und 3, p-STAT1 Ser727 1: 300. Abbildung 4, p-STAT1 Ser727, 1: 100 und TRPV1 1: 100.</li>
	<li>Legen Sie die Folien in Färbung Gericht auf eine Folie Rack Glas-Objektträger. Füllen Sie die Schale mit 350 mL 1 X PBS und waschen Sie Folien für 5 min bei Raumtemperatur dreimal.</li>
	<li>Mit der Sekundärantikörper verdünnt in eine Lösung mit 0,01 % nichtionische Waschmittel und 10 % Normal (Esel) Serum mit 1 X PBS-Puffer für 2 bis 3 h bei Raumtemperatur in eine feuchte Kammer im Dunkeln inkubieren. Hinweis: Für die folgenden Sekundärantikörper wurden diese Verdünnungen verwendet: für Abbildung 2 und 3, Esel IgG Anti-Kaninchen-1:600. Abbildung 4Rhodamine (TRITC) Esel Anti-Kaninchen IgG 1: 500 und Esel Anti-Ziege IgG 1: 500.</li>
	<li>Legen Sie die Folien in Färbung Gericht auf eine Folie Rack Glas-Objektträger. Füllen Sie die Schale mit 350 mL 1 X PBS und waschen Sie Folien für 5 min bei Raumtemperatur dreimal.</li>
	<li>Tippen Sie auf überschüssige Flüssigkeit aus der Folie, und trocknen Sie die Folie mit einem trockenen Tuch auf das Gewebe. Montieren Sie Folien durch Zugabe von einem Tropfen des Montage-Agents mit DAPI direkt auf das Gewebe. Langsam oben, um sicherzustellen, dass keine Luftblasen, darunter entstehen legen Sie das Deckglas auf und lassen Sie die Folien über Nacht bei Raumtemperatur im Dunkeln zu heilen. Speichern von Folien bei 4 ° C.</li>
	<li>Bildfolien mit Hilfe der konfokalen Mikroskopie. Laser Imaging-wie folgt zu verwenden: UV-Laser für DAPI, 488 nm Laser für Esel IgG Anti-Kaninchen und 543 nm Laser für Rhodamin TRITC.</li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+adverse+effect+profiles+of+platinum+drugs.">Comparative adverse effect profiles of platinum drugs.</a> Drug Saf. 13 (4), 228-244 (1995).</li><li>Boulikas, T., Vougiouka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+and+platinum+drugs+at+the+molecular+level.">Cisplatin and platinum drugs at the molecular level.</a> Oncol Rep. 10 (6), 1663-1682 (2003).</li><li>Fouladi, 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=Phase+II+study+of+oxaliplatin+in+children+with+recurrent+or+refractory+medulloblastoma,+supratentorial+primitive+neuroectodermal+tumors,+and+atypical+teratoid+rhabdoid+tumors:+a+pediatric+brain+tumor+consortium+study.">Phase II study of oxaliplatin in children with recurrent or refractory medulloblastoma, supratentorial primitive neuroectodermal tumors, and atypical teratoid rhabdoid tumors: a pediatric brain tumor consortium study.</a> Cancer. 107 (9), 2291-2297 (2006).</li><li>Pasetto, L. 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P., Husain, K., Morris, C., Whitworth, C., Somani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+protective+agents+against+cisplatin+ototoxicity.">Effect of protective agents against cisplatin ototoxicity.</a> Am J Otol. 21 (4), 513-520 (2000).</li><li>Lee, J. E., 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=Role+of+reactive+radicals+in+degeneration+of+the+auditory+system+of+mice+following+cisplatin+treatment.">Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment.</a> Acta Otolaryngol. 124 (10), 1131-1135 (2004).</li><li>Lee, J. E., 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=Mechanisms+of+apoptosis+induced+by+cisplatin+in+marginal+cells+in+mouse+stria+vascularis.">Mechanisms of apoptosis induced by cisplatin in marginal cells in mouse stria vascularis.</a> ORL J Otorhinolaryngol Relat Spec. 66 (3), 111-118 (2004).</li><li>Lawenda, B. D., Kelly, K. M., Ladas, E. J., Sagar, S. M., Vickers, A., Blumberg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+supplemental+antioxidant+administration+be+avoided+during+chemotherapy+and+radiation+therapy.">Should supplemental antioxidant administration be avoided during chemotherapy and radiation therapy.</a> J Natl Cancer Inst. 100 (11), 773-783 (2008).</li><li>Akil, O., Oursler, A. E., Fan, K., Lustig, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+auditory+brainstem+response+testing.">Mouse auditory brainstem response testing.</a> Bio Protoc. 6 (6), 1768 (2016).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Mount+Dissection+and+Immunofluorescence+of+the+Adult+Mouse+Cochlea.">Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.</a> J. Vis. Exp. (107), e53561 (2016).</li><li>Whitlon, D. S., Szakaly, R., Greiner, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoembedding+and+sectioning+of+cochleas+for+immunocytochemistry+and+in+situ+hybridization.">Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.</a> Brain Res Brain Res Protoc. 6 (3), 159-166 (2001).</li><li>Londos, C., Cooper, D. M., Wolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclasses+of+external+adenosine+receptors.">Subclasses of external adenosine receptors.</a> Proc Natl Acad Sci. 77 (5), 2551-2554 (1980).</li><li>Lohse, M. J., Klotz, K. N., Lindenborn-Fotinos, J., Reddington, M., Schwabe, U., Olsson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-Cyclopentyl-1,3-dipropylxanthine+(DPCPX)--a+selective+high+affinity+antagonist+radioligand+for+A1+adenosine+receptors.">8-Cyclopentyl-1,3-dipropylxanthine (DPCPX)--a selective high affinity antagonist radioligand for A1 adenosine receptors.</a> Naunyn Schmiedebergs Arch Pharmacol. 336 (2), 204-210 (1987).</li><li>Rybak, L. P., Whitworth, C., Scott, V., Weberg, A. D., Bhardwaj, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+as+a+potential+model+for+hearing+loss+in+biotinidase+deficiency.">Rat as a potential model for hearing loss in biotinidase deficiency.</a> Ann Otol Rhinol Laryngol. 100 (4), 294-300 (1991).</li><li>Mukherjea, 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=NOX3+NADPH+oxidase+couples+transient+receptor+potential+vanilloid+1+to+signal+transducer+and+activator+of+transcription+1-mediated+inflammation+and+hearing+loss.">NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss.</a> Antioxid Redox Signal. 14 (6), 999-1010 (2011).</li><li>Kaur, T., 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=Adenosine+A1+receptor+protects+against+cisplatin+ototoxicity+by+suppressing+the+NOX3/STAT1+inflammatory+pathway+in+the+cochlea.">Adenosine A1 receptor protects against cisplatin ototoxicity by suppressing the NOX3/STAT1 inflammatory pathway in the cochlea.</a> J Neurosci. 36 (14), 3962-3977 (2016).</li><li>Kaur, T., Mukherjea, D., Sheehan, K., Jajoo, S., Rybak, L. P., Ramkumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+interfering+RNA+against+STAT1+attenuates+cisplatin-induced+ototoxicity+in+the+rat+by+suppressing+inflammation.">Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation.</a> Cell Death Dis. 2 (180), (2011).</li></ol>

Kein Interessenkonflikt erklärt.

Die Trans-Trommelfells Applikationsweges ermöglicht eine lokalisierte Lieferung von Arzneimitteln und sonstigen Erfüllungsgehilfen der Cochlea, die andernfalls erheblichen systemische Nebenwirkungen wenn systemisch verabreicht produzieren könnte. Diese Methode der Gesundheitsbehörde ermöglicht schnellen Zugriff von Medikamenten, um den Ort des Geschehens bei deutlich höheren Dosen, als durch die systemische Route erreicht werden würde. Ergebnisse hier vorgestellt und veröffentlicht zuvor gezeigt, dass Trans-Tromm...

Die peripheren Hörsystems ist exquisit empfindlich auf Medikamente wie Cisplatin und Aminoglykosid-Antibiotika. Cisplatin ist ein weit verbreitetes Chemotherapeutikum zur Behandlung von einer Vielzahl von soliden Tumoren, wie Eierstöcke, Hoden und Kopf und Hals-Tumoren. Ototoxizität erlebt mit der Verwendung dieser Droge ist Dosis-Begrenzung und durchaus üblich, 75-100 % der Patienten betroffen1behandelt. Andere Drogen, wie z. B. Carboplatin und Oxaliplatin, entstanden als Alternativen zu Cisplatin2,3,4,5, aber ihre Nützlichkeit ist auf wenige Krebsarten beschränkt.
Frühe Studien haben die kritische Rolle von reaktiven Sauerstoffspezies (ROS) gezeigt, bei der Vermittlung der Ototoxizität von Cisplatin und Aminoglykoside produziert. Nachfolgende Studien zeigten, dass die NOX3 Isoform von NADPH-Oxidase die primäre Quelle von ROS in der Cochlea ist und durch Cisplatin6,7aktiviert. Die Generation der ROS Kompromisse stieg das Antioxidans Pufferkapazität der Zellen, was zu Lipidperoxidation von Zellmembranen8. Cisplatin erhöht darüber hinaus die Produktion von Hydroxyl-radikale die hochgiftigen Aldehyd 4-Hydroxynonenal (4-HNE), Initiator der Zelle Tod9,10erzeugen. Aufbauend auf diesen Erkenntnissen, wurden mehrere Antioxidantien für die Behandlung von Cisplatin Ototoxizität untersucht. Dazu gehören D-Methionin, N-Acetyl-Cystein (NAC), Natriumthiosulfat (STS) und Amifostine. Ein Hauptanliegen der antioxidative Therapie ist jedoch, dass diese Antioxidantien Cisplatin chemotherapeutischen Wirksamkeit bei11 durch das Zusammenspiel von Cisplatin mit Thiol-Gruppen in den antioxidativen Molekülen systemisch verabreicht senken könnte.
Angesichts dieser Probleme mit antioxidative Therapie war das Ziel dieser Studie zu prüfen, die Trans-Trommelfell Weg der Bereitstellung von Antioxidantien und anderen Drogen zu der Cochlea, Verlust der Hörfähigkeit zu reduzieren. Der Trans-Trommelfell Weg von Drogen und kurze Einmischung (Si) RNA, wie unten beschrieben, scheint besonders Erfolg versprechend.

<table border="0" cellpadding="0" cellspacing="0" style="width:1025px;" width="1025">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:423px;">Ketathesia (100 mg/ml) 10 ml</td>
			<td style="width:192px;">Henry Schein</td>
			<td style="width:127px;">56344</td>
			<td style="width:284px;">Controlled substance&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">AnaSed Injection/Xylazine (20 mg/ml) 20 ml</td>
			<td>Henry Schein</td>
			<td>33197</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2.5 mm disposable ear specula</td>
			<td>Welch Allyn</td>
			<td>52432</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Surgical Scope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">29 G X 1/2 insulin syringe</td>
			<td>Fisher Scientific</td>
			<td>14-841-32&#160;</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cis-Diammineplatinum(II) dichloride</td>
			<td>Sigma Aldrich</td>
			<td>P4394</td>
			<td>TOXIC - wear proper PPE</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Harvard 50-7103 Homeothermic Blanket Control Unit</td>
			<td>Harvard Apparatus</td>
			<td>Series 863</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Excel International 21 G X 3/4 butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-34&#160;</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSP Single Speed Syringe Pump</td>
			<td>Brain Tree Sci, Inc</td>
			<td>BSP-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pulse Sound Measurement System</td>
			<td>Bruel &#38; Kjaer</td>
			<td>Pulse 13 software</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-Frequency Module</td>
			<td>Bruel &#38; Kjaer</td>
			<td>3560C</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">1/8&#8243; Pressure-field Microphone &#8212;-Type 4138</td>
			<td>Bruel &#38; Kjaer</td>
			<td>bp2030</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">High Frequency Transducer</td>
			<td>Intelligent Hearing System</td>
			<td>M014600</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Opti-Amp Power Transmitter</td>
			<td>Intelligent Hearing System</td>
			<td>M013010P</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SmartEP ABR System</td>
			<td>Intelligent Hearing System</td>
			<td>M011110</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Disposable Subdermal EEG Electrodes</td>
			<td>CareFusion</td>
			<td>019-409700</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">16% Formaldehyde, Methanol-free</td>
			<td>Fisher Scientific</td>
			<td>28908</td>
			<td>TOXIC - wear proper PPE&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">7 mL Borosilicate Glass Scintillation Vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-26</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP118-500</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tissue Plus OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CryoMolds (15 mm x 15 mm x 5mm)</td>
			<td>Fisher Scientific</td>
			<td>22-363-553</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microscope Slides (25mm x 75mm)</td>
			<td>MidSci</td>
			<td>1354W</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Coverslips (22 x 22 x 1)</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Poly-L-Lysine Solution (0.01%)</td>
			<td>EMD Millipore</td>
			<td>A-005-C</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HM525 NX Cryostat</td>
			<td>Thermo Fischer Scientific</td>
			<td>956640</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MX35 Premier Disposable Low-Profile Microtome Blades</td>
			<td>Thermo Fischer Scientific</td>
			<td>3052835</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wheaton&#8482;&#160;Glass 20-Slide Staining Dish with Removable Rack</td>
			<td>Fisher Scientific</td>
			<td>08-812</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Super Pap Pen Liquid Blocker</td>
			<td>Ted Pella, Inc.</td>
			<td>22309</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Normal Donkey Serum</td>
			<td>Jackson Immuno Research</td>
			<td>017-000-121</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">TritonX-100</td>
			<td>Acros</td>
			<td>21568</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Can be purchased through other vendors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phospho-Stat1 (Ser727) antibody</td>
			<td>Cell Signaling</td>
			<td>9177</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">VR1 Antibody (C-15)</td>
			<td>Santa Cruz</td>
			<td>sc-12503</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DyLight 488 Donkey anti Rabbit</td>
			<td>Jackson Immuno Research</td>
			<td>711-485-152</td>
			<td>Discontinued</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DyLight 488 Donkey anti Goat</td>
			<td>Jackson Immuno Research</td>
			<td>705-485-003</td>
			<td>Discontinued</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rhodamine (TRTIC) Donkey anti Rabbit</td>
			<td>Jackson Immuno Research</td>
			<td>711-025-152</td>
			<td>Discontinued</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ProLong&#174; Diamond Antifade Mountant w/ DAPI</td>
			<td>Thermo Fisher</td>
			<td>P36971</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">(&#8722;)-N6-(2-Phenylisopropyl)adenosine</td>
			<td>Sigma Aldrich</td>
			<td>P4532</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">8-Cyclopentyl-1,3-dipropylxanthine</td>
			<td>Sigma Aldrich</td>
			<td>C101</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">siRNA pSTAT1</td>
			<td>Qiagen</td>
			<td>Custome Made</td>
			<td>Kaur et al. 201120</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">siRNA NOX3</td>
			<td>Qiagen</td>
			<td>Custome Made</td>
			<td>Kaur et al. 201120</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Scrambled Negative Control siRNA</td>
			<td>Qiagen</td>
			<td>1022076</td>
			<td>Kaur et al. 201120</td>
		</tr>
	</tbody>
</table>

trans-tympanic drug delivery for the treatment of ototoxicity

Der systemischen Verabreichung der Schutzmittel, Medikamenten-induzierten Ototoxizität zu behandeln ist begrenzt durch die Möglichkeit, dass diese Schutzmittel mit der chemotherapeutischen Wirksamkeit der primären Medikamente stören könnten. Dies gilt insbesondere für das Medikament Cisplatin, deren Anti-Krebs-Aktionen durch Antioxidantien gedämpft, die ausreichenden Schutz vor Verlust der Hörfähigkeit zu bieten. Andere aktuelle oder potenzielle Otoprotective Agenten könnte ein ähnliches Problem aufwerfen, wenn systemisch verabreicht. Die Anwendung verschiedener Biologicals oder Schutzstoffe direkt an die Cochlea würde hohe dieser Wirkstoffe lokal mit begrenzten systemischen Nebenwirkungen ermöglichen. In diesem Bericht zeigen wir eine Trans-Trommelfells Methode der Lieferung von verschiedenen Drogen oder biologische Reagenzien der Cochlea, sollten grundlegende Wissenschaftsforschung auf der Cochlea und bieten eine einfache Möglichkeit, die Verwendung von Otoprotective Agenten in den Kliniken zu leiten. Dieser Bericht beschreibt eine Methode des Trans-Trommelfells Drug-Delivery und bietet Beispiele dafür, wie diese Technik erfolgreich bei Versuchstieren eingesetzt hat, um Cisplatin Ototoxizität zu behandeln.

ABR Reaktionen gemessen bei Ratten an drei Tagen nach Cisplatin-Verabreichung eine deutliche Erhöhung Schwellen zeigte. Höhe dieser Schwellen wurde deutlich reduziert, bei Ratten verabreicht mit Trans-Trommelfell [R] - N - Phenylisopropyladenosine (R-PIA), Adenosin1 Rezeptor Agonist15, vor Cisplatin. Die Spezifität des Handelns der R-PIA an der Adenosin-Rezeptor1 zeigte sich durch die Beobachtung, dass es durch 8-...

Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Wir präsentieren Ihnen eine Technik für lokalisierte Verabreichung von Medikamenten durch die Trans-Trommelfells Route in die Cochlea. Drug-Delivery über diesen Weg würden die Anti-Krebs-Wirksamkeit von Chemotherapeutika wie Cisplatin nicht beeinträchtigen.

Trans-Trommelfells Drug-Delivery für die Behandlung von Ototoxizität

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

trans-tympanic-drug-delivery-for-the-treatment-of-ototoxicity

Research

JoVE Journal

Medicine

9.3K Aufrufe.  Southern Illinois University School of Medicine. Wir präsentieren Ihnen eine Technik für lokalisierte Verabreichung von Medikamenten durch die Trans-Trommelfells Route in die Cochlea. Drug-Delivery über diesen Weg würden die Anti-Krebs-Wirksamkeit von Chemotherapeutika wie Cisplatin nicht beeinträchtigen.

Serie: Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Thermische Ablation zur Behandlung von Abdominaltumoren

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Forschung

Medizin

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.

Osmotic Drug Delivery zu ischämischen Hinterbeine und Perfusion von Blutgefäßen mit Microfil für Mikro-Computertomographie

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

The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis

Psoriasis is a chronic, immune-mediated inflammatory skin disease affecting approximately 2-3% of the population. The Goeckerman regimen consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). Goeckerman therapy is extremely effective and relatively safe for the treatment of psoriasis and for improving a patient's quality of life. In the following article, we present our protocol for the Goeckerman therapy that is utilized specifically at the University of California, San Francisco. This protocol details the preparation of supplies, administration of phototherapy and application of topical tar. This protocol also describes how to assess the patient daily, monitor for adverse effects (including pruritus and burning), and adjust the treatment based on the patient's response. Though it is one of the oldest therapies available for psoriasis, there is an absence of any published videos demonstrating the process in detail. The video is beneficial for healthcare providers who want to administer the therapy, for trainees who want to learn more about the process, and for prospective patients who want to undergo treatment for their cutaneous disease.

Psoriasis is a chronic, immune-mediated inflammatory skin disease. The Goeckerman regimen, formulated for the treatment of psoriasis, consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). The following protocol is for the administration of Goeckerman therapy for the treatment of moderate-to-severe psoriasis.

Die Goeckerman Regimen für die Behandlung von mittelschwerer bis schwerer Psoriasis

Psoriasis is a  chronic, immune-mediated inflammatory skin disease affecting approximately 2-3%  of the population. The Goeckerman regimen consists of ...

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

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 molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Heterotope Mucosal-Transplantationsverfahren für Direkt Drug Delivery zum Gehirn bei Mäusen

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 ...

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.

Slow-Release-Drug Delivery durch Elvax 40W, um die Ratte Retina: Implikationen für die Behandlung von chronischen Erkrankungen

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 ...

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 auf die Maus arteriovenöse Fistel Endothel

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

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Netzwerkanalyse von Foramen Ovale Elektrode Recordings in Resistente Temporallappen Epilepsie-Patienten

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Pankreasgang-Infusion: Eine effektive und selektive Methode der Medikamenten- und Virusabgabe

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

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.

Optimierte LC-MS/MS-Methode für die Hochdurchsatz-Analyse der klinischen Proben von Ivacaftor, seinen Hauptmetaboliten und Lumacaftor in biologischen Flüssigkeiten von Mukoviszidose-Patienten

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.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

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) ...