KF et KO sont co premiers auteurs avec KS Ce travail a été financé en partie par le AHPBA Fellowship (KS), NIH K23 CA148964-01 (LZ), Johns Hopkins School of Medicine Bourse de recherche clinique scientifique (LZ), Fondation Viragh et Cancer Center Passer Viragh pancréas à l&#39;Université Johns Hopkins (EMJ et LZ), le pancréas Fondation nationale (LZ), la Fondation de la famille Lefkofsky (LZ), la SPORE NCI dans Gastrointestinal Cancers CA062924 P50 (EMJ, LZ), la Fondation Lustgarten (LZ), et les subventions Sol Goldman pancréas Cancer Center (KS , BHE, LZ).

Toutes les expériences sur les animaux étaient conformes aux lignes directrices du soin et l&#39;utilisation des animaux Comité de l&#39;Université Johns Hopkins, et les animaux ont été maintenus en conformité avec les directives de l&#39;Association pour l&#39;évaluation et l&#39;accréditation des soins de laboratoire (AAALAC). La procédure chirurgicale est effectuée dans des conditions stériles dans une salle d&#39;opération qui est soumis à un minimum de quinze renouvellements d&#39;air par heure. Tous les instruments sont stérilisés par autoclavage avant la procédure et à nouveau avec de l&#39;éthanol à 70% entre chacune des souris au cours de la procédure. Toutes les souris qui ne devraient pas survivre à l&#39;intervention chirurgicale sont euthanasiés sous CO 2 pendant 3 min puis par dislocation cervicale tout en restant sous anesthésie. 1 Préparation des cellules <ol><li> Remettre en suspension les cellules cultivées Panc02 dans un tampon phosphate salin à 2 x10 7 cellules / ml, et garder sur la glace. </li></ol> 2 Préparation de la souris <ol><li> Administrer la kétaminee (100 mg / kg) en mélange avec la xylazine (10 mg / kg) par voie intrapéritonéale à 8-12 semaines les souris C57BL / 6 de souris femelles en doses fractionnées. </li><li> Vérifiez que la pointe retrait de pincement réflexe est aboli afin de s&#39;assurer que la souris est totalement anesthésié. </li><li> Administrer des doses supplémentaires de kétamine (100 mg / kg) en mélange avec xylazine (10 mg / kg) comme nécessaire pour anesthésier les souris complètement. </li><li> Appliquer Puralube Vet onguent pour les yeux des souris pour éviter le dessèchement tandis que les souris sont sous anesthésie. </li><li> Raser la région chirurgicale de la souris afin d&#39;éviter la contamination de la plaie. </li><li> Prep la région sous-costale gauche de l&#39;incision avec 70% d&#39;éthanol et de l&#39;iode. </li><li> Placez un drap chirurgical autour de l&#39;abdomen pour maintenir la stérilité. </li></ol> 3. laparotomie <ol><li> Faire une incision sous-costale gauche en ligne avec l&#39;oreille gauche à travers la peau et à travers le péritoine à l&#39;aide d&#39;un scalpel. </li><li> Exprimez la rate à travers l&#39;incision en appliquant p numérique simultanéeression long des aspects crânienne et caudale de l&#39;incision. </li><li> Localiser les vaisseaux sanguins de la rate, à la fin inférieure de la rate. </li><li> Divisez la rate en plaçant deux Horizon taille moyenne clips ligature dans le centre de la rate en prenant soin de ne pas endommager les vaisseaux sanguins spléniques aux pôles de la rate. </li><li> Placer le pôle supérieur de la rate de nouveau dans le péritoine pour éviter la contamination. </li></ol> 4. tumorale par injection <ol><li> Dresser 150 pi de tampon phosphate salin dans un 26 G x 5/8 &quot;seringue. </li><li> Dresser 100 pi de Panc02 (2 x 10 6 cellules) dans la même seringue en maintenant la seringue en position verticale pendant toute la durée de l&#39;injection. </li><li> Tremper l&#39;aiguille dans une solution d&#39;éthanol à 70%. L&#39;aiguille est immergée dans de l&#39;éthanol à 70% pour assurer la stérilité. </li><li> Injecter les cellules lentement dans le hemispleen exposée étant que la seringue est maintenu en position verticale en tout temps. Le biseau de la Syringe doit être respecté en toutes circonstances par la capsule splénique pour éviter d&#39;injecter les cellules dans la rate. Un blanchiment de la rate et les vaisseaux sanguins doit être observée lors de l&#39;injection. </li><li> Élever la rate et d&#39;appliquer un milieu Horizon pince sous la rate pour obturer les vaisseaux sanguins spléniques. </li><li> Appliquer une petite pince Horizon à l&#39;aspect le plus distale du pancréas et vaisseaux spléniques. </li><li> Ligaturer le pancréas et les vaisseaux spléniques de la hemispleen en coupant au-dessus des clips Horizon. </li><li> Placez la souris sur son «côté, et irriguer l&#39;incision avec de l&#39;eau distillée. </li></ol> 5. abdominale fermeture et récupération de l&#39;anesthésie <ol><li> Fermez le péritoine avec un point 4-0 de course. </li><li> Appliquer deux à trois clips de la peau pour fermer l&#39;incision de la peau. </li><li> Administrer 0,1 mg / kg de buprénorphine ou 5-10 mg / kg par voie sous cutanée Carprofen pour soulager la douleur post-opératoire. </li><li> Placez la souris sur un coussin chauffant pour recovery jusqu&#39;à mobile et la souris montre les modes de respiration réguliers. Animaux ne sont retournés à la compagnie d&#39;autres animaux après avoir récupéré complètement de l&#39;intervention chirurgicale. </li></ol> 6 L&#39;autopsie examen et récolte du foie <ol><li> Entre 30 à 60 jours après l&#39;opération, les souris vont commencer à montrer des symptômes cliniques de métastases et nécessiteront l&#39;euthanasie. Les signes de la maladie métastatique nécessitant euthanasie comprennent abdomen élargie et le développement de l&#39;ascite. Euthanasier les souris par inhalation de CO 2. </li><li> En cas d&#39;inhalation, effectuer dislocation cervicale. </li><li> Après que l&#39;animal a été déterminée comme étant non recevable, faire une incision à travers le péritoine d&#39;exposer l&#39;abdomen avec des ciseaux. </li><li> En utilisant des pinces et ciseaux, couper délicatement le foie de l&#39;abdomen. </li><li> Placer le foie dans le composé PTOM, et le flash geler l&#39;aide d&#39;azote liquide. Conservez le foie à -80 ° C. Alternativement, le directr peut être fixé à 16% du formol tamponné neutre à la température ambiante pendant 24 heures et inclus en paraffine. </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. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+chemotherapeutic+response+of+two+transplantable+ductal+adenocarcinomas+of+the+pancreas+in+C57BL/6+mice.">Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice.</a> Cancer research. 44, 717-726 (1984).</li><li>Leao, I. C., Ganesan, P., Armstrong, T. D., Jaffee, E. 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>

Les auteurs n&#39;ont aucun conflit d&#39;intérêt pertinent de divulguer.

Ce modèle murin préclinique de la métastase du cancer du pancréas dans le foie via une technique d&#39;injection de hemispleen est le premier modèle qui reflète décrit un grand nombre de conditions cliniques et immunologiques de patients ayant une maladie métastatique au foie. Ce modèle offre plusieurs avantages par rapport à d&#39;autres modèles murins. Tout d&#39;abord, à la différence des modèles transgéniques de cancer du pancréas, dans lequel seulement 30% des souris développent des métastases ver...

Canalaire pancréatique adénocarcinome (PDA) est la quatrième cause de décès liés au cancer aux États-Unis 1. La survie à cinq ans pour les patients atteints de PDA métastatique est 2-12% 1. Bien que la chimiothérapie adjuvante et néoadjuvante pour cancer du pancréas est plus efficace que jamais, il reste encore un besoin désespéré de nouvelles thérapies. Le développement de nouveaux produits thérapeutiques requiert des modèles animaux précliniques fiables. Bien que les modèles de tumeurs sous-cutanées sont simples à utiliser, les inconvénients comprennent l&#39;incapacité de ces tumeurs et de métastases imiter la progression de la tumeur et des microenvironnements vu dans les cancers humains. Des modèles génétiquement modifiés, tels que la souris transgénique contenant Kras et p53 knock-in mutations oncogéniques (le modèle KPC) 2 et le modèle KPC avec une lignée traceur YFP (le modèle de PKCY) 3 permettent l&#39;étude des tumeurs d&#39;origine naturelle dans immunocompetesouris NT. En outre, le micro-environnement de la tumeur n&#39;est pas modifiée et ressemble plus étroitement à celle observée dans la progression tumorale humaine. Malheureusement, le calendrier de développement de la tumeur est variable dans ces modèles, ce qui rend difficile l&#39;étude de thérapies expérimentales. En outre, le rétrocroisement de ces souris nécessite une quantité importante de temps et d&#39;engagement financier, qui n&#39;est pas toujours possible. Enfin, les modèles de xénogreffe de souris implantées PDA nécessitent du souris, qui ont modifié ou micro-environnements tumoraux absents immunodéprimés. Par conséquent, l&#39;efficacité des thérapies expérimentales démontré dans plusieurs modèles précliniques sont souvent pas reproductible dans quatre essais cliniques humains. Ce modèle de souris de hemisplenectomy utilise des cellules tumorales qui sont Panc02, une lignée cellulaire de tumeur hautement tumorigène induit par le méthylcholanthrène pancréatique provenant de souris C57BL / 6 5, 6. En outre, d&#39;autres lignées cellulaires murines PDA dérivées de la micro KPCe peut être utilisé dans ce modèle. Schulick et al. Ont déjà mis au point une technique de hemisplenectomy et créé un modèle de souris syngéniques de métastases du cancer du côlon 7. Ce modèle de hemisplenectomy (figure 1) offre inoculation de la tumeur cohérent et égal chez les souris immunocompétentes se prêtant à la recherche de nouveaux agents thérapeutiques 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

De nombreux modèles murins ont été développées pour étudier les cancers humains et de faire progresser la compréhension du traitement du cancer et le développement. Ici, un modèle préclinique murin pancréas tumeur des métastases hépatiques par une injection de cellules syngéniques de hemispleen tumorales pancréatiques de souris est décrit. Ce modèle imite un grand nombre de conditions cliniques chez les patients ayant une maladie métastatique au foie. Souris développent constamment des métastases dans le foie qui permet d&#39;observer le processus métastatique, les essais de thérapie expérimentale, et la recherche en immunologie tumorale.

Cellules tumorales Panc02 injectés dans le hemispleen (figure 1) des métastases hépatiques en forme 100% des souris, alors que pulmonaires et péritonéales métastases ne sont pas respectées, même si la technique est réalisée correctement. Cette technique a été effectuée sur plus d&#39;une centaine de souris en utilisant des cellules Panc02 dans plusieurs expériences indépendantes, et reproductible, toutes les souris non traitées meurent de métastases hépatiques entre 30 et 60 jours apr?...

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

A Preclinical Murine Model of Hepatic Metastases

Un modèle murin de métastases hépatiques préclinique réalisée par une technique d&#39;injection de hemispleen.

Un préclinique modèle murin de métastases hépatiques

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 vues.  The Johns Hopkins University School of Medicine.  Un modèle murin de métastases hépatiques préclinique réalisée par une technique d'injection de hemispleen. 

Vidéo: 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.

Modèle murin Bioluminescent tumeurs hépatiques

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

Recherche

Médecine

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.

Transfert de gènes pour insuffisance cardiaque ischémique dans un modèle préclinique

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.

Modèle murin de la cicatrisation

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 modèle murin d&#39;hémorragie sous-arachnoïdienne

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.

Modèle murin pour l&#39;imagerie non invasive pour détecter et surveiller l&#39;ovaire récurrence du cancer

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.

La réponse au traitement ex vivo de tumeurs primaires et / ou métastases associées préclinique et développement clinique de la thérapeutique

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.

Perfusion hépatique isolée comme un traitement pour les métastases hépatiques de mélanome uvéal

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.

Percutanée hépatique Perfusion (PHP) avec le melphalan comme un traitement pour inopérables Métastases Confiné au foie

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.

Modèle préclinique d’ischémie des membres postérieurs chez les lapins diabétiques

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