KF und KO sind Co ersten Autoren zusammen mit KS Diese Arbeit wurde zum Teil durch die AHPBA Fellowship (KS), NIH K23 CA148964-01 (LZ), Johns Hopkins School of Medicine Clinical Scientist Award (LZ), Viragh Stiftung und das Überspringen Viragh Bauchspeicheldrüsenkrebszentrum an der Johns Hopkins (EMJ und unterstützt LZ), die National Foundation Pankreas (LZ), die Lefkofsky Family Foundation (LZ), das NCI SPORE in Magen-Darm-Krebs P50 CA062924 (EMJ, LZ), der Lustgarten-Stiftung (LZ) und die Sol Goldman Bauchspeicheldrüsenkrebszentrum Zuschüsse (KS , BHE, LZ).

Alle Tierversuche entsprach den Richtlinien der Animal Care und Verwenden Ausschuss der Johns Hopkins University, und die Tiere wurden in Übereinstimmung mit den Richtlinien der Gesellschaft für Evaluierung und Akkreditierung von Laborbetreuung (AAALAC) gehalten. Das chirurgische Verfahren wird unter sterilen Bedingungen im Operationssaal, die ein Minimum von fünfzehn Luftwechsel pro Stunde unterzogen geführt. Alle Instrumente werden durch Autoklavieren vor der Prozedur und erneut mit 70% Ethanol in zwischen jeder Maus während des Verfahrens sterilisiert. Alle Mäuse nicht erwartet, dass das chirurgische Verfahren überleben werden unter CO 2 für 3 min, gefolgt durch Genickbruch noch unter Narkose eingeschläfert. 1. Zellpräparation <ol><li> Resuspendieren kultiviert Panc02 Zellen in phosphatgepufferter Salzlösung mit 2 x 10 7 Zellen / ml, und auf Eis halten. </li></ol> 2. Maus Vorbereitung <ol><li> Verwalten KetaminE (100 mg / kg) mit Xylazin (10 mg / kg) gemischt intraperitoneal 8-12 Wochen alte weibliche C57BL / 6 Mäuse in geteilten Dosen. </li><li> Überprüfen Sie, ob der Zeh Prise Rückzug Reflex wird abgeschafft, um sicherzustellen, dass die Maus vollständig betäubt wird. </li><li> Verwaltung zusätzlicher Dosen von Ketamin (100 mg / kg) mit Xylazin (10 mg / kg) nach Bedarf, um die Mäuse vollständig betäuben gemischt. </li><li> Anwenden Puralube Vet Salbe auf die Augen der Mäuse um das Trocknen zu verhindern, während die Mäuse unter Anästhesie. </li><li> Rasur das Operationsgebiet der Mäuse zu einer Kontamination der Wunde zu vermeiden. </li><li> Bereiten Sie die linke subkostalen Bereich der Schnittführung mit 70% Ethanol und dann Iod. </li><li> Legen Sie ein Operationstuch um den Bauch, um die Sterilität zu erhalten. </li></ol> 3. Laparotomie <ol><li> Sie links subkostalen Einschnitt im Einklang mit dem linken Ohr durch die Haut und durch das Peritoneum mit einem Skalpell. </li><li> Ausdruck der Milz durch den Einschnitt durch die Anwendung gleichzeitiger digitaler pruck entlang der kranialen und kaudalen Aspekte des Einschnitts. </li><li> Suchen Sie die Milz Blutgefäße am unteren Ende der Milz. </li><li> Teilen Sie die Milz, indem zwei Horizon mittlerer Größe Liga Clips in der Mitte der Milz vorsichtig, um eine Beschädigung der Milz-Blutgefäße an der Milz Pole. </li><li> Legen Sie den oberen Pol der Milz zurück in die Bauchhöhle, um Verunreinigungen zu vermeiden. </li></ol> 4. Tumorinjektion <ol><li> Auszuarbeiten 150 ul Phosphat-gepufferter Kochsalzlösung in einer 26 G x 8.5 &quot;Spritze. </li><li> Auszuarbeiten 100 ul Panc02 (2 x10 6 Zellen) in die gleiche Spritze Aufrechterhaltung der Spritze in einer aufrechten Position zu jedem Zeitpunkt während der Injektion. </li><li> Tauchen Sie die Nadel in einem 70% igen Ethanollösung. Die Nadel wird in 70% Ethanol eingetaucht, um Sterilität zu gewährleisten. </li><li> Injizieren die Zellen langsam in die freigelegte hemispleen wobei Sie die Spritze aufrecht zu allen Zeiten gehalten. Die Abschrägung der syringe zu allen Zeiten durch die Milz Kapsel beobachtet werden, um zu vermeiden Injektion der Zellen in der Milz. Eine Weißfärbung der Milz und Blutgefäße sollte bei Injektion beobachtet werden. </li><li> Erhöhen die Milz und gelten ein Medium Horizont Clip unter der Milz, die Milz Blutgefäße verschließen. </li><li> Wenden Sie einen kleinen Horizont Clip auf die meisten distalen Aspekt der Bauchspeicheldrüse und Milz Gefäße. </li><li> Ligieren die Bauchspeicheldrüse und Milz Schiffe aus der hemispleen durch Schneiden über dem Horizont Clips. </li><li> Platzieren Sie die Maus auf seiner &#39;Seite, und bewässern den Schnitt mit destilliertem Wasser. </li></ol> 5. Bauchdeckenverschluss und aus der Narkose <ol><li> Schließen Sie das Bauchfell mit einem 4-0 Lauf Stich. </li><li> Gelten zwei vor drei Hautklammern, um den Hautschnitt zu schließen. </li><li> Verwalten 0,1 mg / kg Buprenorphin oder 5-10 mg / kg Carprofen subkutan postoperative Schmerzen zu lindern. </li><li> Platzieren Sie die Maus auf einem Heizkissen für recOvery, bis Handy und die Maus zeigt, regelmäßige Atmung. Tiere werden nur in der Gesellschaft von anderen Tieren, nach völlig von der Operation erholt zurückgekehrt. </li></ol> 6. Necropsy Prüfung und Ernten der Leber <ol><li> Zwischen 30 bis 60 Tage nach der Operation, wird Mäusen beginnen, klinische Symptome der Metastasierung zeigen und Euthanasie verlangen. Anzeichen von Metastasen erfordern humane Sterbehilfe sind vergrößerte Bauch und die Entwicklung von Aszites. Euthanize Mäuse durch Inhalation von CO 2. </li><li> Nach Einatmen, führen Genickbruch. </li><li> Nachdem das Tier festgestellt worden, die auf nicht sein, einen Einschnitt durch das Peritoneum, um den Bauch mit einer Schere aus. </li><li> Mit einer Pinzette und Schere vorsichtig schneiden die Leber aus dem Bauch. </li><li> Legen Sie die Leber in OCT-Verbindung und Flash mit flüssigem Stickstoff einfrieren. Bewahren Sie die Leber bei -80 ° C. Alternativ kann das Liver kann in 16% neutral gepuffertem Formalin bei RT für 24 h und in Paraffin eingebetteten fixiert werden. </li></ol>

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Facts &amp; Figures. , (2013).</li><li>Clark, C. E., Beatty, G. L., Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosurveillance+of+pancreatic+adenocarcinoma:+insights+from+genetically+engineered+mouse+models+of+cancer.">Immunosurveillance of pancreatic adenocarcinoma: insights from genetically engineered mouse models of cancer.</a> Cancer letters. 279, 1-7 (2009).</li><li>Rhim, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+depletion+of+regulatory+T+cells+allows+the+recruitment+of+mesothelin-specific+CD8+T+cells+to+the+antitumor+immune+response+against+a+mesothelin-expressing+mouse+pancreatic+adenocarcinoma.">Effective depletion of regulatory T cells allows the recruitment of mesothelin-specific CD8 T cells to the antitumor immune response against a mesothelin-expressing mouse pancreatic adenocarcinoma.</a> Clinical and translational science. 1, 228-239 (2008).</li><li>Jain, 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=Synergistic+effect+of+a+granulocyte-macrophage+colony-stimulating+factor-transduced+tumor+vaccine+and+systemic+interleukin-2+in+the+treatment+of+murine+colorectal+cancer+hepatic+metastases.">Synergistic effect of a granulocyte-macrophage colony-stimulating factor-transduced tumor vaccine and systemic interleukin-2 in the treatment of murine colorectal cancer hepatic metastases.</a> Annals of surgical oncology. 10, 810-820 (2003).</li><li>Yoshimura, K., 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=Selective+targeting+of+antitumor+immune+responses+with+engineered+live-attenuated+Listeria+monocytogenes.">Selective targeting of antitumor immune responses with engineered live-attenuated Listeria monocytogenes.</a> Cancer research. 66, 1096-1104 (2006).</li><li>Yoshimura, K., 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=Live+attenuated+Listeria+monocytogenes+effectively+treats+hepatic+colorectal+cancer+metastases+and+is+strongly+enhanced+by+depletion+of+regulatory+T+cells.">Live attenuated Listeria monocytogenes effectively treats hepatic colorectal cancer metastases and is strongly enhanced by depletion of regulatory T cells.</a> Cancer research. 67, 10058-10066 (2007).</li><li>Olino, K., 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=Tumor-associated+antigen+expressing+Listeria+monocytogenes+induces+effective+primary+and+memory+T-cell+responses+against+hepatic+colorectal+cancer+metastases.">Tumor-associated antigen expressing Listeria monocytogenes induces effective primary and memory T-cell responses against hepatic colorectal cancer metastases.</a> Annals of surgical oncology. 19, 597-607 (2012).</li><li>Hingorani, S. 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=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer cell. 7, 469-483 (2005).</li><li>Marion, A., Aoudi, W., Basarab, A., Delachartre, P., Vray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+evaluation+in+high-frequency,+40+MHz+imaging:+a+comparative+study+of+four+vector+velocity+estimation+methods.">Blood flow evaluation in high-frequency, 40 MHz imaging: a comparative study of four vector velocity estimation methods.</a> Ultrasonics. 50, 683-690 (2010).</li><li>Zheng, 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=Tyrosine+23+phosphorylation-dependent+cell-surface+localization+of+annexin+A2+is+required+for+invasion+and+metastases+of+pancreatic+cancer.">Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer.</a> PloS one. 6, e19390 (2011).</li></ol>

Die Autoren haben keine relevanten Interessenkonflikte offen zu legen.

Diese präklinischen Mausmodell für Bauchspeicheldrüsenkrebs Metastasierung in die Leber über eine hemispleen Injektionstechnik ist das erste Modell beschrieben, die viele der klinischen und immunologischen Bedingungen der Patienten mit metastatischer Erkrankung der Leber spiegelt. Dieses Modell bietet mehrere Vorteile gegenüber anderen Mausmodellen. Erstens, anders als transgene Modelle von Pankreaskrebs, bei dem nur 30% der Mäuse entwickeln Metastasen in der Leber und dem Zeitpunkt der Metastasenbildung ist sehr ...

Duktalen Adenokarzinom des Pankreas (PDA) ist die vierthäufigste Ursache von Krebs Todesfälle in den Vereinigten Staaten 1. Der fünfjährige Überlebensrate für Patienten mit metastasiertem PDA ist 2-12% 1. Obwohl Adjuvans und neoadjuvante Chemotherapie bei Bauchspeicheldrüsenkrebs ist heute effektiver als je zuvor, bleibt es immer noch einen verzweifelten Bedarf an neuen Therapien. Die Entwicklung neuartiger Therapeutika erfordert zuverlässige präklinischen Tiermodellen. Obwohl subkutanen Tumormodellen sind einfach zu bedienen, sind Nachteile, die Unfähigkeit dieser Tumoren, Metastasen zu bilden und imitieren die Tumorprogression und Mikroumgebungen in menschlichen Krebserkrankungen gesehen. Gentechnisch veränderte Modelle, wie der gentechnisch veränderte Maus enthalten, Kras und p53-Knock-in onkogene Mutationen (die KPC-Modell) 2 und die KPC-Modell mit einem YFP-Linie Tracer (die PKCY Modell) 3 ermöglichen die Untersuchung von natürlich vorkommenden Tumoren in immunocompetent Mäusen. Zusätzlich ist der Tumor-Mikroumgebung unverändert und ähnelt mehr der in menschlichen Tumorprogression angesehen. Leider ist die Zeitsteuerung der Tumorentwicklung Variable innerhalb dieser Modelle, was es schwierig experimentelle Therapien zu studieren. Zusätzlich Rückkreuzung dieser Mäuse erfordert eine erhebliche Menge an Zeit und finanziellen Engagement, das nicht machbar ist immer. Schließlich implantierten Xenograft PDA Mausmodelle erforderlich machen immungeschwächten Mäusen, die verändert wurden oder nicht vorhanden Tumormikroumgebungen. Als ein Ergebnis wird die Wirksamkeit von experimentellen Behandlungen in diesen Tiermodellen gezeigt werden oft in klinischen Studien am Menschen 4 nicht reproduzierbar. Diese Hemisplenektomie Mausmodell verwendet Panc02 Tumorzellen, die ein hoch tumorigenen, Methylcholanthren induzierten Pankreastumorzelllinie aus C57BL / 6 Mäusen, 5, 6 ableiten. Zusätzlich anderen murinen PDA Zelllinien aus der KPC mic abgeleitete kann in diesem Modell verwendet werden. Schulick et al. Zuvor entwickelte eine Technik Hemisplenektomie und schuf eine syngenen Mausmodell von Darmkrebs Metastasen 7. Diese Hemisplenektomie Modell (Abbildung 1) bietet konsistente und gleich Tumorimpfung bei immunkompetenten Mäusen Kredit sich auf die Untersuchung von neuen therapeutischen Mittel 7-10.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture media and components</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">will vary depending on cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:197px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 mL centrifigure tubes</td>
			<td style="width:197px;">Corning</td>
			<td style="width:119px;">430052</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved Operating Scissor</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-6835</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micro dissecting Graefe Forceps</td>
			<td style="width:197px;">Roboz Surgical Store</td>
			<td style="width:119px;">RS-5130</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle holder (regular)</td>
			<td style="width:197px;">World Precision Instruments, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3-0 or 4-0 suture</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">courtesy of dept of surgery, Johns Hopkins</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, small, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">137085</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon Open Ligating Clip Applier, medium, angled</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">237115</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon medium ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">002200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Weck Horizon small ligating clips</td>
			<td style="width:197px;">Teleflex</td>
			<td style="width:119px;">001200</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 3/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309625</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe, 1 cc, tb syringe, 5/8&#34; slip tip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">309597</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip Applier</td>
			<td style="width:197px;">Mikron</td>
			<td style="width:119px;">427630</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9 mm Autoclip</td>
			<td style="width:197px;">Becton Dickinson</td>
			<td style="width:119px;">427631</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:197px;">Sunbeam</td>
			<td style="width:119px;">CAT93</td>
			<td style="width:181px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:197px;"></td>
			<td style="width:119px;"></td>
			<td style="width:181px;">sold by numerous vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride&#160;</td>
			<td style="width:197px;">Hospira</td>
			<td style="width:119px;">22395DD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine hydrochloride</td>
			<td style="width:197px;">Sigma</td>
			<td style="width:119px;">X1251</td>
			<td></td>
		</tr>
	</tbody>
</table>

a preclinical murine model of hepatic metastases

Zahlreiche murine Modelle wurden entwickelt, um menschliche Karzinome zu studieren und zu fördern, das Verständnis der Krebsbehandlung und Entwicklung. Hier, ein präklinischer, murinen Pankreastumormodell von Lebermetastasen über eine Injektion von syngenen hemispleen murinen Pankreastumorzellen beschrieben. Dieses Modell imitiert viele der klinischen Bedingungen bei Patienten mit Metastasen in die Leber. Mäuse konsequent entwickeln Metastasen in der Leber so dass für die Untersuchung des metastatischen Prozess, experimentelle Therapie Testen und Tumorimmunologie Forschung.

Panc02 Tumorzellen in das hemispleen spritzt (Figur 1) bilden Lebermetastasen bei 100% der Mäuse, während die Lunge und Peritonealmetastasen werden nicht beobachtet, wenn das Verfahren vollständig durchgeführt wird. Diese Technik hat zu über einhundert Mäusen mit Panc02 Zellen in mehrere, unabhängige Experimente durchgeführt worden, und reproduzierbar, sterben alle unbehandelten Mäuse mit Lebermetastasen zwischen 30 und 60 Tagen nach der Hemisplenektomie Verfahren (Figur 2). Um...

Watch this Scientific Journal Video about A Preclinical Murine Model of Hepatic Metastases at JoVE.com

A Preclinical Murine Model of Hepatic Metastases

Eine präklinische, Mausmodell der Lebermetastasen über eine hemispleen Injektionstechnik durchgeführt.

Einer präklinischen Mausmodell der Lebermetastasen

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

a-preclinical-murine-model-of-hepatic-metastases

Research

JoVE Journal

Medicine

28.4K Aufrufe.  The Johns Hopkins University School of Medicine.  Eine präklinische, Mausmodell der Lebermetastasen über eine hemispleen Injektionstechnik durchgeführt. 

Serie: A Preclinical Murine Model of Hepatic Metastases

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murine Bioluminescent Leber Tumor-Modell

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Forschung

Medizin

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Gentransfer bei ischämischen Herzinsuffizienz in einem vorklinischen Modell

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Mausmodell der Wundheilung

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Einem Mausmodell der Subarachnoidalblutung

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Mausmodell für nicht-invasive Bildgebungs zu erfassen und zu überwachen Eierstockkrebs Rezidiv

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex-vivo-Ansprechen auf die Behandlung von Primärtumoren und / oder assoziierten Metastasen für die präklinische und klinische Entwicklung von Therapeutika

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolierte Leberperfusion als Behandlung für Lebermetastasen von Aderhautmelanom

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

Die perkutane Leberperfusion (PHP) mit Melphalan als Behandlung für inoperablen Metastasen auf die Leber beschränkt

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Kontraktionen der iPSC abgeleitete Cardiomyocyte Syncytia gemessen mit einem Ca-sensitiven Fluoreszenzfarbstoff in temperaturgesteuerten 384-Well-Platten

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Präklinische Modell von Hind Limb Ischemia in diabetischen Kaninchen

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...