Recherche dans mon laboratoire est soutenu par le Département WKU des fonds de démarrage de biologie, WKU Fondation de la recherche de la CRPA-je accorder # 11-8032 et par une subvention KBRIN-ZONE financé grâce à une subvention de parent de l&#39;Institut national de sciences médicales générales des National Institutes de la Santé sous le numéro d&#39;attribution 5P20GM103436-13. Je tiens également à remercier le Dr Tian Xu dans le laboratoire duquel j&#39;ai été présenté à cette technique et le Dr Raymond Pagliarini qui le premier a créé cette technique dans le laboratoire Xu.

Une. L&#39;induction de tumeurs non invasives bénignes <ol><li> Utiliser les stocks énumérés dans le tableau 2 pour l&#39;induction de tumeurs bénignes. </li><li> Préparer un levain pour la &quot;tester&quot; le bilan de la génotype suivante: y, w, ey-FLP1; Tub-GAL80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Préparer un levain pour la &quot;testé&quot; le bilan de la génotype suivant: w; FRT40A, UAS-Ras V12 / CYO </li><li> Recueillir dix filles vierges du &quot;testeur&quot; actions et dix mâles de la souche &quot;testé&quot;. </li><li> Traverser les hommes et les femmes de la &quot;tester&quot; et le stock &quot;testé&quot; en les plaçant à la fois dans un flacon avec de la nourriture à la mouche. </li><li> Placez la croix dans un incubateur à 25 ° C et laisser les mâles et les femelles pour s&#39;accoupler et pondre des œufs pendant 24-48 h. </li><li> Vérifiez les flacons pour la ponte suffisante et la présence de larves de premier stade de s&#39;assurer que la culture n&#39;est pas en train de s&#39;assécher. <ol><li> Ajouter quelques gouttes d&#39;autoclavel&#39;eau distillée à la culture pour le garder humide (si la culture est en train de s&#39;assécher). </li><li> Continuer à surveiller la culture et répétez l&#39;étape 1.7.1 si nécessaire. </li></ol></li><li> Respecter errant troisième stade larvaire sous un stéréomicroscope de fluorescence, comme indiqué à l&#39;article 3. </li></ol> 2. L&#39;induction de tumeurs invasives <ol><li> Utiliser les stocks énumérés dans le tableau 2 pour l&#39;induction de tumeurs invasives. </li><li> Utilisez la culture de départ de l&#39;étape 1.2 ci-dessus du génotype suivante: y, w, ey-FLP1; Tub-GAL80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Préparer un levain pour la &quot;testé&quot; le bilan de la génotype suivant: w; lgl 4, FRT40A, UAS-Ras V12 / CYO </li><li> Recueillir dix filles vierges du &quot;testeur&quot; actions et dix mâles de la souche &quot;testé&quot;. </li><li> Traverser les hommes et les femmes de la &quot;tester&quot; et le stock &quot;testé&quot; en les plaçant à la fois dans un flacon à la mouche food. </li><li> Placez la croix dans un incubateur à 25 ° C et laisser les mâles et les femelles pour s&#39;accoupler et pondre des œufs pendant 24-48 h. </li><li> Vérifiez les flacons pour la ponte suffisante et la présence de larves de premier stade de s&#39;assurer que la culture n&#39;est pas en train de s&#39;assécher. <ol><li> Ajouter quelques gouttes d&#39;eau distillée à l&#39;autoclave à la culture pour le garder humide (si la culture est en train de s&#39;assécher). </li><li> Continuer à surveiller la culture de la sécheresse et répétez l&#39;étape 2.7.1 si nécessaire. </li></ol></li><li> Respecter troisième stade larvaire sous un stéréomicroscope de fluorescence, comme indiqué à l&#39;article 3. </li></ol> 3. Observation de tumeurs bénignes et envahissantes <ol><li> Utilisez troisième stade larvaire pour l&#39;isolement de tumeur bénigne et la visualisation. <ol><li> Mouillez un pinceau dans de l&#39;eau distillée, et l&#39;utilisation de ce pinceau gratter un peu de larves de troisième stade de la paroi du flacon de culture. </li><li> Placer les larves dans une lame de dépression avec du PBS 1X, puis en utilisant une peinture humide brosse ferraillee de tout produit alimentaire de l&#39;épiderme larvaire. </li><li> Placer les larves dans une boîte de Petri et laisser au congélateur à -20 ° C pendant 30 min pour immobiliser la larve. <ol><li> Autre méthode pour immobiliser les larves à l&#39;aide FLYNAP. </li><li> Placez une baguette de FLYNAP trempé dans FLYNAP le flacon de 3.1.3.2 et après avoir branché le let flacon flacon bouché avec larves reposer pendant 30 à 40 min. </li></ol></li><li> Placez la larve immobilisé sur une lame de verre, ajouter une goutte d&#39;huile d&#39;halocarbures lumière, couvrir avec un couvercle en verre et observer sous un stéréomicroscope de fluorescence avec la possibilité de visualiser la protéine fluorescente verte (GFP). </li><li> Les larves de palier de tumeur bénigne va émettre une fluorescence verte dans la région antérieure, où le complexe est présent céphalique. </li></ol></li><li> Sélectionnez jour 10 larves (après induction) de la culture définie à l&#39;étape 2 ci-dessus pour l&#39;observation de tumeurs invasives. Jour 10 larves sont choisis parce que les larves d&#39;appui de tumeur invasive ont un l prolongéestade Arval et généralement ne parviennent pas à pupariate. </li><li> Suivez les étapes 3.1.1 à 3.1.4, sauf qu&#39;au lieu de la troisième journée stade de l&#39;utilisation des larves de 10 larves. <ol><li> Le jour 10 larves de palier de la tumeur invasive une fluorescence verte dans la région antérieure, où le complexe est présent céphalique. </li></ol></li><li> Si le microscope à fluorescence est équipé d&#39;un appareil photo numérique, puis prendre des photos des larves d&#39;appui de tumeur bénigne et invasive fluorescent. </li></ol> 4. Dissection de l&#39;Observation complexe et plus céphalique de tumeurs bénignes et envahissantes L&#39;épiderme translucides de la chenille, il est difficile d&#39;observer l&#39;étendue de l&#39;invasion des tumeurs. Ainsi, l&#39;étendue de l&#39;invasion est mieux visualisé par la dissection du complexe céphalique de la larve. Les étapes suivantes devraient être utilisés pour disséquer le complexe céphalique. <ol><li> L&#39;utilisation d&#39;un pinceau humide sélectionner troisième stade larvaire (pour les tumeurs bénignes) et le jour 10 larves (pour les tumeurs invasives) felon les flacons de culture respectives. </li><li> Placer les larves dans un puits d&#39;une plaque de dissection contenant du PBS 1X froid. Utilisez un pinceau pour racler les particules de nourriture de l&#39;épiderme larvaire. <ol><li> Transfert larves propre pour un nouveau puits de la boîte de dissection contenant du PBS 1X froid. </li><li> Vérification de la présence de fluorescence de la GFP en utilisant un microscope stéréoscopique capable de détecter la fluorescence. Jeter les larves de roulement non-GFP. </li></ol></li><li> Ajouter 1,0 ml de PBS 1X froid à un nouveau puits sur le plat de dissection et l&#39;aide d&#39;une paire de pinces transférer une larve portant soit des tumeurs bénignes ou envahissantes (le protocole de dissection reste le même pour ces deux types de tumeurs). </li><li> Maintenir la chenille vers le bas en utilisant une paire de forceps environ 2/3rds de l&#39;extrémité antérieure. <ol><li> Utilisez l&#39;autre paire de pinces et intelligemment séparer le 1/3ème postérieure de la larve et jetez-le. </li><li> Lâchez antérieure 2/3rds de la larve. Comme la pression à l&#39;intérieur de la chenille est libérée du contenu de lalarve suinter hors de la cavité du corps. </li><li> Utiliser une paire de pinces pour retirer le tube digestif, le corps gras et les autres composants internes de la larve qui ont suinté de la cavité du corps. </li><li> Utilisez une paire de pinces pour tenir le crochet de la bouche de la larve et le pousser dans l&#39;épiderme larvaire et en utilisant l&#39;autre paire de pinces inverser la larve complètement. </li><li> Utilisez la pince pour enlever délicatement et soigneusement le corps gras, les glandes salivaires, l&#39;intestin, le complexe de disque de l&#39;aile et des tubes de la trachée. Le complexe céphalique, devrait maintenant être visible attaché à l&#39;hameçon de la bouche de la larve à l&#39;envers. Les hémisphères du cerveau et la corde nerveuse ventrale (VNC) seraient toujours liée à l&#39;épiderme larvaire par les nerfs émanant de la VNC. </li><li> Utilisez une paire de pinces pour briser doucement les connexions entre les nerfs céphaliques et l&#39;épiderme larvaire. </li><li> Comme les connexions nerveuses entre la VNC et l&#39;épiderme larvaires sont brisées et le corps de l&#39;excès de graisse et d&#39;autres tissus retirés, la co céphaliquemplex deviendrait clairement visible et sera fixé à l&#39;épiderme larvaire seulement à l&#39;embouchure crochets. </li><li> Le complexe céphalique peut être laissée dans l&#39;épiderme larvaire si le complexe doit être fixé pour les applications en aval comme la coloration des anticorps ou pour la visualisation plus tard. </li><li> Si aucune autre applications en aval seraient effectuées alors le complexe céphalique peut être détaché de l&#39;épiderme larvaire et placé dans une goutte de glycérine à base de milieu de montage pour observation et l&#39;analyse. Utilisez Vectashield que les médias de montage. </li></ol></li></ol>

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C., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Ras(V12)+and+scribbled+clones+induces+tumour+growth+and+invasion.">Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion.</a> Nature. 463, 545-548 (2010).</li></ol>

Je n&#39;ai rien à divulguer.

Le cancer est une maladie complexe avec une bien meilleure compréhension aujourd&#39;hui que dans le passé. Cependant, il reste encore beaucoup à apprendre et expliqué avant que nous ayons une image complète des mécanismes sous-jacents. Le protocole simple présenté ici, il est possible d&#39;induire des tumeurs bénignes et génétiquement invasives dans un organisme entier, puis l&#39;étude de la biologie associée à la progression des tumeurs dans ce modèle. La plupart des techniques existantes de Droso...

La progression des tumeurs d&#39;une bénigne à un état ​​métastatique est un processus de sage étape qui se caractérise par l&#39;évasion de mécanismes protecteurs présents dans le corps 8. Par exemple les cellules tumorales dans le corps doivent être en mesure d&#39;échapper à l&#39;apoptose et le système immunitaire, la percée de la matrice extracellulaire spécialisée (ECM) appelée membrane basale, et de surmonter tous les contrôles sociaux imposés par les cellules environnantes 8. C&#39;est grâce à une progression pas à pas sage que les cellules cancéreuses acquièrent la capacité de migrer et de coloniser des sites distants dans un processus appelé métastase. Notre compréhension de la façon dont la cellule tumorale surmonte les barrières imposées par le corps est encore à ses balbutiements, cependant, l&#39;image qui émerge de la recherche effectuée points ainsi loin d&#39;une utilisation répétée des processus normaux de développement et des voies de signalisation par les cellules cancéreuses 11-13. La mouche des fruits Drosophila melanogaster a énormément contribué à notre understanding du développement normal et la maladie par l&#39;utilisation de techniques génétiques sophistiquées développées au cours des dernières décennies 14-17. En utilisant la mutagenèse et les outils de surexpression nous sommes arrivés à une meilleure compréhension des différents oncogènes et gènes suppresseurs de tumeurs 18-22. Cependant, les métastases de la tumeur est le résultat d&#39;une coopération entre plusieurs lésions génétiques qui ont été étudiés principalement dans des modèles de culture de cellules 23,24, ainsi que différents modèles de xénogreffe 25-27. Ces modèles quoique puissante ont leurs limites car ils ne reproduisent pas entièrement aux conditions énoncées dans un organisme vivant. En outre, les modèles transgéniques disponibles chez la souris sont encombrants et peu propice à l&#39;analyse génétique de comportement invasif 28,29. Plusieurs études ont tenté de comprendre l&#39;invasion des cellules tumorales chez la drosophile 30,31. Ces techniques utilisent principalement la transplantation de tumeurs primaires aux hôtes et reposent sur le suivi de la tratumeurs nsplanted pour invasion des tissus voisins 32,33. Une technique puissante appelée marcm 10 a été adapté par Pagliarini et Xu pour modéliser l&#39;invasion tumorale chez la drosophile 9. Cette modélisation élégante génétique de l&#39;invasion tumorale a exploité la coopération entre un oncogène activé et la perte de la polarité cellulaire. La puissance de cette modélisation réside dans le fait que les tumeurs invasives sont créés dans un organisme intact contournant ainsi le besoin d&#39;une transplantation de tissus. Pour provoquer la coopération oncogène, un oncogène Ras activé comme V12 est exprimée dans les clones de cellules dans le disque d&#39;eye-antennaire larvaire. En raison de la technique de marcm ces clones sont également marquées avec la protéine fluorescente verte (GFP) pour une visualisation facile et sont publiés homozygotes pour les mutants de la polarité de la cellule tels que les larves létale géant, bourrée, et des disques de grandes. Résulte GFP étiqueté tumeurs invasives dans l&#39; complexe céphalique. Dans ce rapport, jedémontrer comment provoquer, et de visualiser ces tumeurs invasives à la fois dans le cadre d&#39;une larve intact et dans disséquée complexe céphalique. L&#39;induction de tumeurs présentée ici utilise des réactifs sur le deuxième chromosome de Drosophila. Dans le tableau 2, je fournis une liste de stocks sur X et 3 e chromosomes qui peuvent être utilisés dans le même but. Je crois que ce protocole simplifié fera cette technique facilement accessibles aux chercheurs qui s&#39;intéressent à la compréhension de la base moléculaire de la progression tumorale.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

Drosophila a éclairé notre compréhension de la base génétique du développement normal et la maladie au cours des dernières décennies et aujourd&#39;hui, il continue de contribuer énormément à notre compréhension des maladies complexes 1-7. La progression des tumeurs d&#39;une bénigne à un état ​​métastatique est un événement complexe 8 et a été modélisée chez la drosophile pour nous aider à mieux comprendre la base génétique de cette maladie 9. Ici, je présente un protocole simple pour induire génétiquement, observer et ensuite analyser la progression des tumeurs chez les larves de drosophile. La technique d&#39;induction des tumeurs est basé sur le système de marcm 10 et exploite la coopération entre un oncogène activé, Ras V12 et la perte de gènes de polarité de la cellule (bourrée, et des disques à grande létale larves de géant) pour générer des tumeurs invasives 9. Je démontre comment ces tumeurs peuvent être visualisées dans les larves intactes etcomment ceux-ci peuvent être disséqués pour une analyse ultérieure. Le protocole simplifié présenté ici devrait permettre à cette technique pour être utilisé par les chercheurs intéressés à comprendre le rôle d&#39;un gène dans l&#39;invasion tumorale.

Par suite du protocole présenté ici, l&#39;utilisateur sera en mesure d&#39;induire des tumeurs bénignes par la surexpression d&#39;un oncogène activé dans l&#39;oeil disque imaginai antennaire larvaire. L&#39;utilisateur sera également en mesure d&#39;induire des tumeurs invasives dans le disque antennaire de l&#39;oeil en surexprimant un oncogène activé dans les clones de cellules mutantes également pour un gène de la polarité cellulaire. Les tumeurs peuvent être facilement visualisés à l&#39;aide d&#39;...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

La coopération entre un oncogène RAS activé comme V12 et des mutations dans les gènes de polarité cellulaire comme bourrée, à provoquer une croissance de la tumeur chez la drosophile, où les cellules tumorales présentent également des comportements invasives. Voici un protocole simple pour l&#39;induction et l&#39;observation des tumeurs bénignes et envahissantes est présenté.

Un protocole d&#39;induction génétique et visualisation des tumeurs bénignes et envahissantes dans céphalique Complexes de<em&gt; Drosophila melanogaster</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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Research

JoVE Journal

Medicine

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K vues.  Western Kentucky University.  La coopération entre un oncogène RAS activé comme V12 et des mutations dans les gènes de polarité cellulaire comme bourrée, à provoquer une croissance de la tumeur chez la drosophile, où les cellules tumorales présentent également des comportements invasives. Voici un protocole simple pour l'induction et l'observation des tumeurs bénignes et envahissantes est présenté. 

Vidéo: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Création et propagation des tumeurs de l&#39;homme en rétinoblastome immunitaire chez les souris déficientes

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Recherche

Médecine

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Non-invasive de mesure optique du métabolisme cérébral et hémodynamique chez les nourrissons

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Rénale Capsule xénogreffe sous-cutanée et Pellet implantation pour l&#39;évaluation de la carcinogenèse prostatique et l&#39;hyperplasie prostatique bénigne

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induction de l’athérosclérose accélérée chez les souris : le modèle « Wire-Injury »

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Microscopie intravitale et thrombus induction dans le lobe de l&#39;oreille d&#39;une souris sans poils

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Technique mini-Invasive transversal constriction aortique chez les souris pour l’Induction de l’hypertrophie ventriculaire gauche

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Évaluation non invasif de la fonction musculaire de dorsifléchisseurs chez la souris

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualisation des complexes mitophatonus endogènes in situ dans les cellules bêta pancréatiques humaines utilisant l'analyse de la ligation de proximité

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modèle préclinique de rat pour l'étude des dommages d'ischémie-réperfusion dans la microchirurgie reconstructive

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...