KF y KO son co primeros autores, junto con KS Este trabajo fue apoyado en parte por el AHPBA Fellowship (KS), NIH K23 CA148964-01 (LZ), la Escuela de Medicina Johns Hopkins premio Clinical Scientist (LZ), Fundación Viragh y el Centro de Cáncer Skip Viragh pancreático en la Universidad Johns Hopkins (EMJ y LZ), la Fundación Nacional de Páncreas (LZ), la Fundación Lefkofsky Familia (LZ), el NCI SPORE en Gastrointestinal Cánceres P50 CA062924 (EMJ, LZ), la Fundación Lustgarten (LZ), y las subvenciones del Centro de Cáncer Sol Goldman pancreático (KS , BHE, LZ).

Todos los experimentos con animales se ajustaban a las directrices del Comité para el Cuidado y Uso de Animales de la Universidad Johns Hopkins, y los animales fueron mantenidos de acuerdo con las directrices de la Asociación para la Evaluación y Acreditación de Cuidado de Laboratorio (AAALAC). El procedimiento quirúrgico se realiza bajo condiciones estériles en un quirófano que sufre un mínimo de quince cambios de aire por hora. Todos los instrumentos son esterilizados en autoclave antes del procedimiento y de nuevo con etanol al 70% entre cada ratón durante el procedimiento. Todos los ratones que no llegará a la intervención quirúrgica son sacrificados bajo CO 2 durante 3 minutos seguido por dislocación cervical, mientras que todavía en la anestesia. 1. Preparación de la célula <ol><li> Resuspender las células cultivadas en Panc02 fosfato salino tamponado a 2 x10 7 células / ml, y mantener en hielo. </li></ol> 2. Preparación Ratón <ol><li> Administrar ketaminae (100 mg / kg) mezclado con xilazina (10 mg / kg) por vía intraperitoneal a 8-12 semanas de edad C57BL / 6 ratones hembra en dosis divididas. </li><li> Compruebe que la punta retirada pizca reflejo se abolió para asegurarse de que el ratón esté totalmente anestesiado. </li><li> Administrar dosis adicionales de ketamina (100 mg / kg) mezclado con xilazina (10 mg / kg) según sea necesario para anestesiar totalmente los ratones. </li><li> Aplicar Puralube Vet Ungüento para los ojos de los ratones para que no se seque mientras que los ratones están bajo anestesia. </li><li> Afeitar el área quirúrgica de los ratones para evitar la contaminación de la herida. </li><li> Preparar el área subcostal izquierdo de la incisión con etanol al 70% y luego yodo. </li><li> Coloque un paño quirúrgico alrededor del abdomen para mantener la esterilidad. </li></ol> 3. laparotomía <ol><li> Hacer una incisión subcostal izquierda en línea con el oído izquierdo a través de la piel y a través del peritoneo usando un escalpelo. </li><li> Expresar el bazo a través de la incisión aplicando p digitales simultánearesión a lo largo de los aspectos craneal y caudal de la incisión. </li><li> Localizar los vasos sanguíneos esplénicos en el extremo inferior del bazo. </li><li> Divida el bazo mediante la colocación de dos medianas clips de ligadura Horizon en el centro del bazo teniendo cuidado de no dañar los vasos sanguíneos del bazo en los polos del bazo. </li><li> Coloque el polo superior del bazo de nuevo en el peritoneo para evitar la contaminación. </li></ol> Inyección 4. Tumor <ol><li> Elaborar 150 l de solución salina tamponada con fosfato en un 26 T x 5/8 &quot;de la jeringa. </li><li> Elaborar 100 l de Panc02 (2 x10 6 células) en la misma jeringa manteniendo la jeringa en posición vertical en todo momento durante la inyección. </li><li> Sumergir la aguja en una solución de etanol al 70%. La aguja se sumerge en etanol al 70% para asegurar la esterilidad. </li><li> Inyectar las células lentamente en el hemispleen expuesta asegurándose la jeringa se mantiene en posición vertical en todo momento. El bisel de la Syringe debe observarse en todo momento a través de la cápsula esplénica para evitar la inyección de las células en el bazo. Un blanqueamiento del bazo y los vasos sanguíneos se debe observar después de la inyección. </li><li> Elevar el bazo y aplicar un medio Horizonte clip debajo del bazo para ocluir los vasos sanguíneos esplénicos. </li><li> Aplicar un pequeño clip Horizonte al aspecto más distal del páncreas y vasos esplénicos. </li><li> Ligar el páncreas y los vasos esplénicos de la hemispleen cortando por encima de los clips Horizon. </li><li> Coloca el ratón en su &#39;lado, y el riego de la incisión con agua destilada. </li></ol> 5. abdominal Clausura y recuperación de la anestesia <ol><li> Cierre del peritoneo con una puntada 4-0 consecutivo. </li><li> Aplicar una a dos clips de la piel para cerrar la incisión de la piel. </li><li> Administrar 0,1 mg / kg de buprenorfina o 5-10 mg / kg por vía subcutánea Carprofeno para aliviar el dolor post quirúrgico. </li><li> Coloca el ratón sobre una almohadilla térmica para recovery hasta móvil y el ratón demuestra patrones de respiración regulares. Los animales sólo se devuelven a la compañía de otros animales después de recuperarse plenamente de la intervención quirúrgica. </li></ol> 6. Necropsia Examen y cosecha del Hígado <ol><li> Entre 30 y 60 días después de la cirugía, los ratones comienzan a mostrar síntomas clínicos de metástasis y requerirán la eutanasia. Los signos de la enfermedad metastásica que requieren eutanasia humanitaria incluyen abdómenes agrandados y el desarrollo de ascitis. La eutanasia a los ratones por inhalación de CO 2. </li><li> Después de la inhalación, realice dislocación cervical. </li><li> Después de que el animal ha sido determinado que no responde, hacer una incisión a través del peritoneo para exponer el abdomen usando tijeras. </li><li> El uso de pinzas y tijeras, corte suavemente el hígado fuera del abdomen. </li><li> Coloque el hígado en compuesto OCT, y flash congelar con nitrógeno líquido. Guarde el hígado a -80 ° C. Alternativamente, el vivor puede fijarse en 16% neutros formalina tamponada a temperatura ambiente durante 24 horas y embebidos en parafina. </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. 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=EMT+and+dissemination+precede+pancreatic+tumor+formation.">EMT and dissemination precede pancreatic tumor formation.</a> Cell. 148, 349-361 (2012).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nature reviews. Cancer. 7, 645-658 (2007).</li><li>Corbett, T. 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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>

Los autores no tienen conflicto de interés relevante para revelar.

Este modelo murino preclínica de la metástasis del cáncer de páncreas al hígado a través de una técnica de inyección hemispleen es el primer modelo descrito que refleja muchas de las condiciones clínicas e inmunológicas de los pacientes con enfermedad metastásica en el hígado. Este modelo ofrece varias ventajas sobre otros modelos murinos. En primer lugar, a diferencia de modelos transgénicos de cáncer de páncreas en la que sólo el 30% de los ratones desarrollan metástasis en el hígado y el momento de ...

Adenocarcinoma ductal pancreático (PDA) es la cuarta causa principal de muertes relacionadas con el cáncer en los Estados Unidos 1. La supervivencia de cinco años para los pacientes con PDA metastásico es 2-12% 1. Aunque la quimioterapia adyuvante y neoadyuvante para el cáncer de páncreas es hoy más eficaz que nunca, sigue existiendo una necesidad desesperada de nuevas terapias. El desarrollo de nuevas terapias requiere modelos animales preclínicos fiables. Aunque los modelos de tumores subcutáneos son sencillos de usar, inconvenientes incluyen la incapacidad de estos tumores y metástasis a imitar la progresión tumoral y microambientes visto en los cánceres humanos. Modelos de ingeniería genética, tales como el ratón ingeniería genética que contiene Kras y p53 knock-in mutaciones oncogénicas (el modelo KPC) 2 y el modelo KPC con un trazador de linaje YFP (el modelo PKCY 3) permiten el estudio de tumores que ocurren naturalmente en immunocompeteratones NT. Además, el microambiente tumoral se altera y se asemeja más estrechamente a la observada en la progresión tumoral humano. Por desgracia, el calendario de desarrollo de tumores es variable dentro de estos modelos, lo que hace difícil el estudio de terapias experimentales. Además, el retrocruzamiento de estos ratones requiere una cantidad significativa de tiempo y compromiso financiero, que no siempre es factible. Por último, los modelos de PDA xenoinjerto de ratón implantadas, requieran ratones, que han alterado o microambientes tumorales ausentes inmunocomprometidos. Como resultado, la eficacia de terapias experimentales demostrado en estos modelos preclínicos a menudo no son reproducibles en ensayos clínicos humanos 4. Este modelo de ratón hemisplenectomy utiliza células tumorales Panc02, que son una, inducida por metilcolantreno de páncreas línea celular de tumor altamente tumorigénico derivado de ratones C57BL / 6 5, 6. Además, otras líneas celulares PDA murinos derivan del micrófono KPCe se puede utilizar en este modelo. Schulick et al. Han desarrollado previamente una técnica hemisplenectomy y ha creado un modelo de ratón singénico de metástasis de cáncer de colon 7. Este modelo hemisplenectomy (Figura 1) ofrece la inoculación del tumor consistente e iguales en ratones inmunocompetentes se presta a la investigación de nuevos agentes terapéuticos 7-10.

<table border="0" cellpadding="0" cellspacing="0" width="713">
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			<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>
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			<td height="21" style="height:21px;width:216px;">Curved Operating Scissor</td>
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			<td style="width:119px;">RS-6835</td>
			<td style="width:181px;"></td>
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			<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>
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			<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

Se han desarrollado numerosos modelos murinos para estudiar los cánceres humanos y avanzar en la comprensión de tratamiento y desarrollo del cáncer. Aquí, se describe un modelo preclínico, de páncreas murino tumoral de las metástasis hepáticas por medio de una inyección hemispleen de las células tumorales pancreáticas murinos singénicos. Este modelo imita muchas de las condiciones clínicas en pacientes con enfermedad metastásica en el hígado. Los ratones desarrollan consistentemente metástasis en el hígado permitiendo para la investigación del proceso metastásico, las pruebas de terapia experimental, la investigación y la inmunología tumoral.

Panc02 células tumorales inyectadas en el hemispleen (Figura 1) metástasis de hígado en forma 100% de los ratones, mientras que de pulmón y las metástasis peritoneales no se observan si la técnica se realiza correctamente. Esta técnica se ha realizado en más de un centenar de ratones usando células Panc02 en múltiples experimentos independientes, y reproducible, todos los ratones no tratados mueren con metástasis hepáticas de entre 30 y 60 días después del procedimiento hemisplenectomy <st...

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

A Preclinical Murine Model of Hepatic Metastases

Un modelo preclínico, murino de metástasis hepáticas realiza a través de una técnica de inyección hemispleen.

A preclínicos modelo murino de metástasis hepática

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

28.3K vistas.  The Johns Hopkins University School of Medicine.  Un modelo preclínico, murino de metástasis hepáticas realiza a través de una técnica de inyección hemispleen. 

Video: 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.

Murino hepática Bioluminiscente modelo tumoral

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

Investigación

Medicina

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.

Transferencia génica para la insuficiencia cardiaca isquémica en un modelo preclínico

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.

Modelo murino de Curación de Heridas

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.

Un modelo murino de hemorragia subaracnoidea

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.

Modelo murino de imagen no invasivas para detectar y vigilar cáncer ovárico de Recurrencia

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 respuesta al tratamiento de los tumores primarios y / o Asociados Metástasis de Preclínica y Desarrollo Clínico de Terapéutica

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.

Aislado hepática de perfusión como un tratamiento para las metástasis hepáticas de melanoma uveal

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.

Percutánea hepática perfusión (PHP) con melfalán como un tratamiento para la metástasis no resecables limitado al hígado

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

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Método de la entrega directa segmentaria intra-hepática utilizando un modelo de hígado de rata hiliar abrazadera

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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.

Modelo preclínico de isquemia de extremidades traseras en conejos diabéticos

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