Nous sommes reconnaissants au laboratoire du Dr Robert S. Kerbel (Université de Toronto, l&#39;Institut de recherche Sunnybrook, Toronto, Canada) pour l&#39;aide technique et l&#39;expertise dans le développement de cette procédure. Nous tenons également à remercier le Dr Sandra Sexton et le Roswell Park Cancer Institute Département de Laboratory Animal Resources. Ce travail a été soutenu par une bourse de la Fondation Alliance Roswell Park (à JMLE).

1. Orthotopique tumeur du rein Implantation <ol><li> Culture cellulaire <ol><li> Avant l&#39;implantation orthotopique, cultiver des cellules de souris RENCA LUC comme une monocouche à 75% de confluence. </li><li> À la suite de traitement à la trypsine et de la remise en suspension dans des milieux contenant du FBS à 5%, les cellules de centrifugation à 1000 tpm, 4 ° C pendant 5 minutes, en répétant trois fois pour laver dans du PBS. Puis remettre en suspension les cellules dans des milieux sans sérum à une concentration de 5 x 10 4 RENCA LUC / 5 médias sans sérum ul. Remarque: Selon la variante ou lignée cellulaire utilisée, les cellules peuvent être remises en suspension dans un rapport de 1:1 de matrigel et des milieux sans sérum pour ralentir la croissance très agressif de cellules de souris et de la diffusion après l&#39;implantation. </li></ol></li><li> Chirurgie et cellule implantation Remarque: Toutes les études animales qui y sont décrits, y compris l&#39;entretien et la détermination des paramètres expérimentaux, ont été réalisées selon le Comité de protection et d&#39;utilisation des animaux institutionnels (IACUC) protocol approuvé par Roswell Park Cancer Institute. <ol><li> Anesthésier une souris Balb / C en utilisant l&#39;isoflurane (2-3%). Pincez pied et vérifier réflexe pour assurer une anesthésie suffisante. Appliquer vétérinaire pommade pour les yeux pour éviter le dessèchement. Placez la souris en décubitus latéral droit et gauche se raser entre avant et des membres postérieurs. Appliquer de l&#39;alcool et de l&#39;iode long dorsal région lombaire pour préparer de façon aseptique zone pour l&#39;incision. </li><li> Utilisez des ciseaux chirurgicaux pour lancer une incision cutanée de 1 cm dans une direction longitudinale entre la dernière côte et la hanche. Desserrer tissu conjonctif sous la peau à l&#39;aide d&#39;un ciseau dissection émoussée. Utiliser des ciseaux chirurgicaux pour effectuer une incision de 0,5 cm dans une direction longitudinale dans la paroi abdominale. Utilisez une pince courbes pour pousser doucement vers le bas autour plaie ouverte - ce sera d&#39;extérioriser les reins et permettre l&#39;immobilisation douce d&#39;organes avant l&#39;injection. </li><li> Charger la seringue Hamilton avec 5 pl du mélange de cellules préparé. Pour implan sous-capsulairestions, deux méthodes d&#39;implantation peuvent être utilisés: <ol><li> Superficiel: Parallèlement à l&#39;rein, l&#39;aiguille d&#39;insertion orientée longitudinalement sous la capsule rénale (mais au-dessus du parenchyme) avec le bord biseauté vers le haut. Une fois l&#39;aiguille est en place, injecter toutes les cellules jusqu&#39;à ce que de petites formes de bulles blanches. Retirer lentement l&#39;aiguille de la capsule et éponger immédiatement à l&#39;injection avec un coton applicateur incliné stérile pour absorber toute fuite et empêcher la diffusion cellulaire. </li><li> Interne: Avec bord biseauté vers le haut et à partir du côté opposé de rein finale de site d&#39;implantation, à insérer l&#39;aiguille à travers l&#39;intérieur du rein jusqu&#39;à ce que l&#39;aiguille est visible (pas de perforation) l&#39;espace sous-capsulaire. Injecter toutes les cellules jusqu&#39;à ce que les formes de bulles blanches et écouvillon site d&#39;injection pour éviter toute fuite. </li></ol></li><li> Retourner doucement le rein à la cavité du corps. Fermez la paroi abdominale du corps en utilisant résorbable Vicryl 5-0. Une fois terminé, retirez la couche de peau ensemble et placer 2-3 wplaies clips. S&#39;assurer que les attaches sont fermes et régulièrement espacées afin de s&#39;assurer qu&#39;aucun exposition de la plaie. Avant d&#39;administrer le recouvrement 500 ul de NaCl à 0,9% et 100 ul de la buprénorphine (0,01 mg / ml) par voie sous cutanée au moyen d&#39;une aiguille G 25. Surveiller régulièrement le placement et la stabilité clip et retirer après 10 jours. </li></ol></li></ol> 2. Néphrectomie / ablation de la tumeur primaire <ol><li> Suivez protocole identique pour l&#39;anesthésie de la souris, l&#39;immobilisation, l&#39;incision, et l&#39;exposition de rein comme décrit dans les sections 1.2.1 et 1.2.2 (ci-dessus). </li><li> Utiliser une deuxième paire de pinces d&#39;isoler les reins en retirant doucement le tissu adipeux de liaison à partir de l&#39;extrémité caudale et la glande surrénale de l&#39;extrémité crânienne du rein. Tout en saisissant doucement le rein, utilisez résorbable Vicryl 5-0 pour faire un double noeud autour de l&#39;uretère, l&#39;artère rénale, et la veine. Lentement sécurisé puis couper au-dessus du nœud à retirer le rein. Si aucun signe de perte de sang immédiatement, ou il ya un risque de prise insuffisantede suture sur l&#39;uretère, l&#39;artère et la veine, puis utilisez cauterizer au lieu de ciseaux pour couper au-dessus noeud. </li><li> Une fois le rein est retiré, vérifiez soigneusement pour des saignements de l&#39;artère liée et utiliser cauterizer si nécessaire. Fermez la paroi abdominale du corps en utilisant 5-0 Vicryl. Tirez la couche de peau ensemble et placer 2-3 des agrafes suivant mêmes précautions que décrit dans 1.2.4. Suivre de près l&#39;animal tous les jours après la chirurgie pour des signes de détresse, des saignements ou à mobilité réduite. Suivez les directives institutionnelles pour animaux en toute sécurité. </li></ol> 3. Surveillance métastatique Progression et localisation à Endpoint <ol><li> Quantification de la maladie métastatique: surveillance bioluminescentes <ol><li> Voir méthode décrite précédemment dans JOVE pour surveiller les métastases en utilisant des cellules transfectées avec la luciférase et quantifiés à l&#39;aide du système d&#39;imagerie IVIS 11,12 Xenogen. </li></ol></li><li> Guide visuel de l&#39;autopsie d&#39;évaluation comparative de la distribution métastatiquetion <ol><li> Sacrifier des animaux selon les directives institutionnelles en utilisant des paramètres, y compris des signes de détresse, respiration laborieuse, perte de poids, etc Remarque: Une surveillance étroite des animaux est essentiel que la progression métastatique spontanée peut être très variable et rapide. </li><li> Utilisez des ciseaux chirurgicaux pour lancer une incision au-dessus de l&#39;urètre qui se poursuit jusqu&#39;à la cavité thoracique de la mandibule inférieure. Utilisez des ciseaux à bouts francs à séparer la peau du muscle de la paroi abdominale. </li><li> Initier incisions le long bras et les jambes. Peel peau loin. Examiner de près l&#39;aponévrose de la peau et la paroi abdominale des nodules, puis les ganglions lymphatiques superficiels: inguinal, brachial, axillaires et cervicales superficielle. </li><li> Faire une incision dans la paroi abdominale et couper le long des côtés latéraux gauche et droit jusqu&#39;à la membrane. Avec une coupe horizontale enlever la partie avant de la paroi abdominale pour mettre à nu les viscères. </li><li> Examiner l&#39;aspect intact des viscères fou grossièrement tumeurs notables. A noter également le contenu de l&#39;estomac et des intestins; si elles sont vides, la souris peut ne pas avoir été capable de manger. Si le contenu est sombre, cela peut indiquer une hémorragie supérieure / inférieure gastro-intestinal. </li><li> Marquer visuellement chaque organe de la présence ou de l&#39;absence de la tumeur, l&#39;attribution d&#39;un score total pour chaque groupe de souris (quatre animaux par groupe est utilisé comme exemple dans la figure 1D-i). Notes: 1) l&#39;élargissement des ganglions lymphatiques ne signifie pas nécessairement qu&#39;il s&#39;agit d&#39;une croissance de la tumeur; si elle est grandement taille, très ferme, et une couleur blanchâtre, il est probable un nodule métastatique. 2) laiteux excessive ou liquide trouble dans l&#39;abdomen indique ascite. L&#39;ascite peuvent inclure des accumulations de solides sur les organes abdominaux, en particulier entre les lobes du foie et de l&#39;estomac. 3) pour des organes tels que les poumons, de multiples nodules peuvent être présents et compté pour les comparaisons entre les animaux (voir 13 par exemple). </li><li> Identifier des tumeurs ou des anomalies flagrantes des organes abdominaux individuels: foie, la rate, les reins et le pancréas par soigneusement enlever et laver avec du PBS d&#39;un vaporisateur. </li><li> Après avoir retiré les organes abdominaux, inspecter les ganglions lymphatiques abdominaux, y compris la région lombaire, sacrale, rénal, et le ganglion lymphatique mésentérique. </li><li> Évaluer diaphragme pour preuve de la dissémination métastatique. Remarque: propager largement sur ​​la membrane peut lui faire perdre sa contractilité, conduisant à travaillé ou perte de la respiration. </li><li> Retirer la membrane et faire deux incisions latérales dans la cavité thoracique, retirer la partie frontale pour exposer les poumons et le cœur. </li><li> Retirer les poumons et le cœur en saisissant délicatement et en soulevant l&#39;œsophage et la trachée coupe au-dessus du cœur. Évaluer les tissus des nodules. Pressez coeur. Manque de cohérence élastique peut indiquer une tumeur présente. </li><li> Utilisez des ciseaux chirurgicaux pour couper linéairement par crâne. Retirez délicatement les petits morceaux de crâne jusqu&#39;à ce que til tout le cerveau est exposé. Rechercher dans tout le cerveau des nodules visibles ou des anomalies des décolorations. </li></ol></li></ol>

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Les auteurs n&#39;ont rien à révéler.

Le but de ce protocole est d&#39;évaluer la maladie métastatique spontanée cliniquement pertinente en utilisant un modèle de souris de tumeur syngénique à décrire les techniques d&#39;implantation / de résection. Actuellement, la majorité des études précliniques évaluant de nouveaux traitements expérimentaux ne comprend pas l&#39;étude de la maladie métastatique et seulement quelques-uns récapituler les étapes de la croissance de la tumeur primaire, la résection chirurgicale, et une éventuelle dissém...

La principale cause de mortalité chez les patients atteints de carcinome à cellules rénales (RCC) est une maladie métastatique systémique qui se produit généralement après l&#39;élimination chirurgicale d&#39;une tumeur primaire de plus en plus dans le rein. Cependant, très peu de modèles précliniques de tumeurs évaluation thérapeutique expérimentale chez la souris incluent la maladie métastatique, et encore moins récapitulent fidèlement les stades cliniques de croissance localisée, la chirurgie et l&#39;initiation et la progression 1-3 micro-métastatique spontanée. Cet écart dans les tests est devenu de plus en plus important dans l&#39;évaluation de nouvelles thérapies car parfois saisissants effets anti-tumeurs observés chez des modèles animaux ne sont pas toujours traduits dans le traitement similaire de patients avec succès 4. Ces différences de résultats peuvent provenir d&#39;efficacités de drogue différentiels entre les modèles extra-utérines ou orthotopiques localisées tumeur primaire et à un stade avancé métastatique 5-7. Dans le cas de RCC, seules quelques études ont établi un employéNimal protocoles qui incluent la maladie récurrente spontanée qui imite les patients qui ont eu généralement reins porteuses de tumeurs retirées totalement ou partiellement de 2,3. Les raisons de cette pénurie dans les tests de modèle de souris varient. Tout d&#39;abord, il est le coût élevé des animaux et de la variabilité inhérente à la sélection de cellules de tumeur et son potentiel métastatique. Par exemple, des lignées de cellules de rein humain ont tendance à former des métastases rarement, et doivent être choisis sur des cycles multiples de l&#39;implantation primaire orthotopique et sélection métastatique de dériver des variants qui diffusent de manière cohérente et forment des lésions éloignés (voir la description d&#39;un tel dérivé de lignée cellulaire humaine 8-10) . A l&#39;inverse, des cellules de souris dans des modèles immunocompétents ont tendance à se comporter de manière agressive, et un faible nombre de cellules doivent être injectés avec matrigel à réduire la propagation systémique immédiate 3. Deuxièmement, des difficultés techniques dans l&#39;exécution de l&#39;implantation proprement dite, la résection chirurgicale (néphrectomie), et suivi (et quantification) metastati spontanéela croissance c peut être difficile et plusieurs variables critiques besoin considération lors de l&#39;utilisation de cette technique (voir discussion pour plus de détails). Le but de ce protocole est de décrire les étapes essentielles (ainsi que les pièges potentiels) d&#39;implantation orthotopique, la résection (néphrectomie), et la surveillance de la maladie spontanée RCC métastatique et d&#39;offrir une ligne directrice pour standardisée (et plus largement) utiliser les laboratoires scientifiques qui évaluent l&#39;efficacité des traitements expérimentaux.

<table border="0" cellpadding="0" cellspacing="0" width="830">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

L&#39;un des principaux obstacles à l&#39;amélioration de l&#39;essai de nouveaux traitements expérimentaux dans le carcinome à cellules rénales (RCC) est le développement de modèles qui récapitulent fidèlement la progression de la maladie métastatique précoce et à un stade avancé. Des modèles d&#39;implantation de la tumeur typiques utilisent implantation de la tumeur primaire ectopique ou orthotopique, mais peu comprennent la maladie métastatique spontanée systémique qui imite l&#39;environnement clinique. Ce protocole décrit les principales étapes pour développer la progression de la maladie RCC met en scène similaire aux patients. En premier lieu, il utilise une lignée de cellules de tumeur de souris hautement métastatique dans un modèle orthotopique syngénique pour montrer l&#39;implantation des cellules tumorales. Les méthodes comprennent l&#39;implantation superficielle et interne dans l&#39;espace sous-capsulaire avec des cellules associées à matrigel pour éviter les fuites et la propagation rapide. Ensuite, il décrit les procédures pour l&#39;excision de la tumeur portant rein (néphrectomie), les soins de la souris pré-et post-opératoire critique. Enfin, il décrit les étapes nécessaires pour surveiller et évaluermicro-et macro progression de la maladie métastatique, y compris l&#39;imagerie de bioluminescence ainsi fournit un guide détaillé de l&#39;autopsie visuelle de marquer distribution de la maladie systémique. Le but de cette description du protocole est de faciliter l&#39;utilisation généralisée de modèles de CCR métastatiques cliniquement pertinentes pour améliorer la valeur prédictive du test thérapeutique futur.

La figure 1A montre un schéma décrivant les procédures décrites dans ce résumé du protocole. Plusieurs facteurs importants doivent être pris en considération à chaque étape. Par exemple, dans l&#39;étape 1, on montre deux méthodes pour l&#39;implantation des cellules tumorales sous-capsulaire dans le rein. Les cellules tumorales peuvent être implantés dans l&#39;espace sous-capsulaire avec un petit blanc-bulle confirmant le placement localisée des cellules avec fuite empêché par l&#39;?...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Les modèles de métastases de carcinome à cellules rénales (RCC), la progression de la maladie spontanée peuvent être utilisées pour l&#39;évaluation des traitements dans un cadre clinique. Ce protocole illustre différentes procédures d&#39;implantation orthotopique de cellules de tumeur du rein, une néphrectomie proprement dit, et présente enfin un guide de notation visuelle de l&#39;autopsie et bioluminescent de charge métastatique et de la localisation.

Modélisation spontanée carcinome rénal métastatique (MRCC) chez des souris après néphrectomie

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

modeling-spontaneous-metastatic-renal-cell-carcinoma-mrcc-mice

Research

JoVE Journal

Medicine

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

16.4K vues.  Roswell Park Cancer Institute.  Les modèles de métastases de carcinome à cellules rénales (RCC), la progression de la maladie spontanée peuvent être utilisées pour l'évaluation des traitements dans un cadre clinique. Ce protocole illustre différentes procédures d'implantation orthotopique de cellules de tumeur du rein, une néphrectomie proprement dit, et présente enfin un guide de notation visuelle de l'autopsie et bioluminescent de charge métastatique et de la localisation. 

Vidéo: Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

Simple néphrectomie Port

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

Recherche

Médecine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Course de modélisation chez la souris - Moyen-occlusion artérielle cérébrale avec le modèle filamentaire

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Transplantation mammaires de cellules stromales et les cellules de carcinome chez les souris C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Hémorragie intracérébrale chez la souris Modélisation: L&#39;injection de sang autologue ou collagénase bactérienne

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Génération de sous-cutanée et intra-hépatique droits de carcinome hépatocellulaire xénogreffes chez la souris immunodéficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Induction du envahissantes Transitional cancer de la vessie cellulaire dans immunitaire humain MUC1 souris transgéniques intactes: un modèle pour le développement d&#39;immunothérapie

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation de carcinome du sein métastatique en ciblant de l&#39;épithélium canalaire avec adénovirus Cre: un modèle de souris transgénique roman du cancer du sein

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modélisation des maladies chez les souris: Coagulation permanente de l&#39;artère cérébrale moyenne distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.